Investigating the Role of Cytokinin and Beta-1,3-Glucanases in Potato Tuber Dormancy and Sprouting

Untersuchungen zur Rolle von Cytokinin und Beta-1,3- Glucanasen bei der Dormanz und Keimung von Kartoffelknollen

Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades Dr. rer. nat.

vorgelegt von Anja Hartmann

aus Merseburg a. d. Saale

Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Friedrich- Alexander-Universität Erlangen-Nürnberg

Tag der mündlichen Prüfung: 29.06.2012

Vorsitzender der Promotionskommission: Prof. Dr. Rainer Fink Erstberichterstatter: Prof. Dr. Uwe Sonnewald Zweitberichterstatter: Dr. Mark Taylor

Darin besteht das Wesen der Wissenschaft: Zuerst denkt man an etwas, das wahr sein könnte. Dann sieht man nach, ob es der Fall ist und im Allgemeinen ist es nicht der Fall. Bertrand Russell (1872-1970)

Table of Contents

Table of Contents Table of Contents ______i 1 Summary / Zusammenfassung ______1 1.1 Summary ______1 1.2 Zusammenfassung ______3 2 Introduction ______6 2.1 The potato - an important crop plant ______6 2.1.1 Use and impact of the potato tuber in nutrition and industry ______6 2.1.2 The potato plant and its life cycle ______6 2.2 Dormancy of potato tubers ______7 2.2.1 Physiological changes during dormancy break and sprouting and their impact on nutritional value and industrial processing ______8 2.2.2 Factors influencing potato tuber dormancy ______8 2.3 Phytohormones involved in dormancy, bud breakage and sprout growth _____10 2.3.1 Abscisic acid (ABA) ______11 2.3.2 Auxin (AUX) ______12 2.3.3 Gibberellin (GA) ______13 2.3.4 Cytokinin (CK) ______15 2.3.4.1 CK metabolism ______15 2.3.4.2 CK transport ______18 2.3.4.3 CK signalling ______18 2.3.4.4 CK functions ______21 2.4 Plasmodesmata, callose and tuber bud meristems ______21 2.4.1 Plasmodesmata - structure and function ______22 2.4.2 Callose and its deposition at plasmodesmata ______23 2.4.3 Plasmodesmata and dormancy ______23 2.5 Aims of this thesis ______24 3 Material and Methods ______25 3.1 Chemicals, enzymes and consumables ______25 3.2 Bacterial strains ______25 3.3 Vectors ______25 3.4 Oligonucleotides and sequencing ______26 3.5 Growth and transformation of bacteria ______27 3.6 Plant cultivation ______28 3.6.1 Cultivation of Solanum tuberosum cv. Solara ______28 3.6.2 Cultivation of Nicotiana benthamiana ______28 3.7 Plant transformation ______28 3.7.1 Stable transformation of Solanum tuberosum ______28 3.7.2 Transient transformation of Nicotiana benthamiana ______29 3.8 Sprout Release Assay ______29 3.9 Microscopy and histological methods ______29 3.9.1 Embedding of plant tissues ______30 3.9.2 Staining of plant tissues ______30 3.9.3 Immunolocalization of callose ______31 3.10 Biomolecular methods ______31 3.10.1 Standard cloning procedures ______31 3.10.2 Isolation of plant genomic DNA ______31

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3.10.3 Isolation of RNA and Northern Blot ______32 3.10.4 DNAse treatment of RNA and cDNA synthesis ______32 3.10.5 Quantitative real-time PCR ______32 3.10.6 Microarray hybridisation and scanning ______33 3.10.7 Microarray data analysis ______33 3.10.8 Protein Extraction and Western Blot ______33 3.11 Bioinformatic analysis______34 4 Results ______35 4.1 Alteration of endogenous cytokinin content in transgenic potato plants ______35 4.1.1 Constitutive overexpression of bacterial IPT and AtCKX1 in potato ______35 4.1.1.1 Phenotypic characterization of 35S:IPT and 35S:CKX1 overexpressing potato plants 36 4.1.1.2 Natural and phytohormone-induced sprouting of IPT-6 and 35S:CKX1-overexpressing tubers ______38 4.1.1.3 Transcriptional analysis of wild-type, IPT-6 and CKX1-4 tubers after GA3-induced sprouting ______41 4.1.1.3.1 Functional categorization of differentially expressed genes ______44 3.1.1.3.2 Comparison of cell cycle and selected phytohormone pathways in the three genotypes ______46 4.1.2 Expression of bacterial IPT and AtCKX1 in potato under control of the meristem-specific UFO promoter ______58 4.1.2.1 Phenotypic characterization of UFO:IPT and UFO:CKX1 overexpressing potato plants ______59 4.1.2.2 Characterization of sprouting behaviour of UFO:IPT and UFO:CKX1-expressing potato tubers ______60 4.2 In silico analysis of cytokinin signalling and metabolism genes of potato _____63 4.2.1 Availability of potato genome sequences ______63 4.2.2 Developing a method for annotation of genes in the potato genome ______64 4.2.2.1 Gene annotation by POCI EST sequences ______64 4.2.2.2 Employing BLAST searches of known sequences from other species ______66 4.2.2.3 Gene identification via functional domains ______67 4.2.3 Cytokinin metabolism genes ______68 4.2.3.1 Identification of adenylate IPT and tRNA IPT genes ______68 4.2.3.2 Identification of cytokinin oxidase/ dehydrogenase genes ______71 4.2.3.3 Identification of zeatin glycosyl- and xylosyltransferases ______73 4.2.4 Cytokinin signalling genes ______75 4.2.4.1 Identification of cytokinin receptors ______75 4.2.4.2 Identification of A-type and B-type response regulators ______78 4.2.4.3 Identification of purine transporters (PUP) ______81 4.3 Beta-1,3-glucanases ______83 4.3.1 Identification of potato beta-1,3-glucanases in the potato genome ______83 4.3.1.1 EST sequences representing beta-1,3-glucanases ______83 4.3.1.2 Identification of full-length beta-1,3-glucanases ______84 4.3.1.3 Clustering of identified beta-1,3-glucanases according to expression data ______86 4.3.2 Putative PD-associated beta-1,3-glucanases ______87 4.3.2.1 GFP-Fusions of StPdB13G_1 and StPdB13G_2 ______88 4.3.2.2 Generation of stably transformed PdB13G_RNAi and 35S:AtBG_PAP potato plants 91 4.3.3 Callose turnover during GA3-induced sprouting of wild-type tubers ______94 5 Discussion ______97 5.1 Alteration of endogenous cytokinin content ______97 5.1.1 Transcriptional analysis confirmed that cytokinin is needed for bud break, but not sufficient for sprout growth ______99 5.1.2 Ectopic expression of IPT changed meristem fate ______103 5.2 Analysis of the potato genome reveals sequences of cytokinin metabolism and signalling genes ______104 5.2.1 Isopentenyltransferase genes ______105 5.2.2. Cytokinin oxidase/ dehydrogenase genes ______106

ii Table of Contents

5.2.3. Cytokinin glycosyl- and xylosyltransferase genes ______106 5.2.4. Cytokinin receptors ______107 5.2.5. Response regulators type A and B ______107 5.2.6. PUP purine permeases ______108 5.3 The role of beta-1,3-glucanases in tuber dormancy and sprouting ______109 5.3.1 Pd gating by callose deposition and removal might not be a regulatory mechanism in tuber dormancy release ______109 5.3.2 Tuber sprouting is accompanied by de novo formation of vascular tissue ______111 5.4 Future prospects ______114 6 References ______115 7 Abbreviations ______I 8 Appendix ______III List of publications ______LXIII Conference contributions ______LXIII Acknowledgement ______LXIV CV______LXV

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Summary / Zusammenfassung

1 Summary / Zusammenfassung 1.1 Summary

Potato tubers are mainly consumed fresh, generating a constant demand all year round that can only be met by long-term storage of tubers after harvest. Sprouting of tubers is an important factor for quality loss during storage, with tubers shrivelling from remobilization of storage substances and water loss. Unravelling the molecular mechanisms underlying potato tuber dormancy and sprouting is therefore of vital interest for both breeders and processing industries and may enable directed manipulation of dormancy length in the future.

Phytohormone measurements and treatment experiments have credited cytokinins (CKs) with a role in dormancy break. To further elucidate the role of this phytohormone, transgenic plants with altered CK biosynthesis were generated by expressing either an isopentenyltransferase (IPT) from Agrobacterium tumefaciens or an Arabidopsis thaliana CK oxidase/ dehydrogenase (CKX1) gene and analysed in detail. Strong expression of IPT, the rate-limiting enzyme of CK biosynthesis, led to a typical CK-responsive phenotype with bushy growth, small leaflets, inhibition of root growth and limited or absent tuber formation. However, one weakly expressing line (IPT-6), which resembled wild-type (WT) concerning plant growth and tuber yield, formed thicker and longer tuber sprouts compared to WT. In an in vitro sprouting assay (SRA), IPT-6 tubers were also shown to sprout earlier than the WT. In contrast, when CK content was lowered by expression of catabolic enzyme CKX1, transgenic plants showed reduced shoot growth, an increased number of fragile side shoots and a lower number of leaflets per leaf. CKX1 transcript abundance was inversely correlated to tuber yield and onset of sprouting. This effect was most pronounced in the strongest line (CKX1-4) which sprouted up to eight weeks later than the WT. Moreover, sprouts of CKX1-4 tubers remained diminutive after bud break and failed to initiate proper growth. In an in vitro sprouting assay using WT and CKX1-4 tubers, GA3 treatment did not initiate sprouting in the transgenic tubers. Additional transcriptional analysis of WT, IPT-6 and CKX1-4 tubers revealed that many transcripts up-regulated during GA3-mediated sprouting in WT and IPT-6 are not induced in CKX1-4 tubers. Together, these results clearly demonstrated that the presence of cytokinins is an essential requirement for the induction of tuber sprouting.

1

In 2009, release of the first draft version of the potato genome disclosed new prospects for the study of potato dormancy and sprouting. Due to lack of annotations in the genomic sequences, a toolbox involving alignments of potato EST and genomic sequences, search for conserved domains in different translation frames of genomic sequences and comparison of potato sequences with sequences of closely or distantly related species, like tomato and tobacco or Arabidopsis, was developed to identify genomic sequences and exon/ intron structure of genes of interest (GOI). This toolbox was then employed to discover genes of CK metabolism and signalling, thus providing useful sequence information for future investigations of the mechanism of CK action during bud break.

Experiments with fluorescent dyes and markers have shown that meristematic cells in potato tubers are symplasmically isolated from the surrounding tissue during dormancy. In other species, symplasmic isolation was shown to result from callose deposition at plasmodesmata (PD), symplasmic connections between adjacent plant cells. To investigate whether callose deposition is an important factor of dormancy control, 34 members of the beta-1,3-glucanase (B13G) gene family encoding callose-degrading enzymes were identified in the potato genome. Comparison of available microarray data revealed several candidate genes which were up-regulated shortly before or at sprouting. Two of these candidates, termed StPdB13G_1 and StPdB13G_2, were shown to have high protein sequence similarity to a PD-located beta-1,3-glucanase of Arabidopsis thaliana (AtBG_PAP). Transgenic plants carrying an RNAi construct to silence both genes showed no phenotypical differences to the WT and during post-harvest storage, their tubers sprouted slightly, but not significantly later than WT tubers. To investigate the effects of the over-expression of a PD-located glucanase, AtBG_PAP was expressed in transgenic potatoes. This resulted in a WT-like plant phenotype and a delay in sprouting of about one week. Taken together, de-regulation of PD-glucanases seemed not to contribute greatly to tuber dormancy release.

Additional evidence for this was provided by immunolocalisation of callose during GA3- induced sprouting of WT tubers which showed no differences in callose distribution before, during and after onset of sprouting. Instead, massive formation of new vascular tissue was observed and subsequent analysis of transcription data revealed up- regulation of auxin biosynthesis and signalling pathway genes as well as markers for vascular development. These findings indicate that de novo formation of vascular bundles may support carbon allocation to meet the energy demand of (meristematic) cells in the newly forming sprout.

2 Summary / Zusammenfassung

1.2 Zusammenfassung

Kartoffelknollen werden hauptsächlich frisch verzehrt, so dass das ganze Jahr über ein Bedarf an Knollen besteht, der aufgrund der Wachstumsansprüche der Kartoffelpflanze nur durch Langzeitlagerung nach der Ernte gewährleistet werden kann. Knollenkeimung während der Lagerung ist ein Hauptfaktor für Qualitätsverlust, da die Kartoffeln durch Remobilisierung von Speicherstoffen und Wasserverlust zusammenschrumpfen. Das Verständnis der molekularen Prozesse, die dem Keimungsprozess zugrunde liegen, ist daher sowohl für Züchter als auch für die verarbeitenden Industriezweige von entscheidender Bedeutung und könnte in Zukunft die gezielte Manipulation der Dormanzlänge ermöglichen.

Bisherige Forschungsergebnisse haben Cytokininen (CK) eine Rolle bei der Brechung der Keimruhe zugeschrieben. Um die Rolle von CK näher zu untersuchen wurden transgene Pflanzen mit erhöhtem oder verringertem CK- Gehalt analysiert. Starke Expression einer Isopentenyltransferase (IPT), dem Schlüsselenzym der CK Biosynthese, führte zu dem „typischen“ CK-responsiven Phänotyp mit buschigem Wuchs, stark verkleinerten Blättchen, einem gehemmten Wurzelwachstum und limitierter bis ausbleibender Knollenbildung. Knollen einer schwach exprimierenden Linie (ITP-6), die in Wachstum und Ertrag dem Wildtyp (WT) glich, konnten weiter analysiert werden. IPT-6 Knollen bildeten im Vergleich zum WT dickere und längere Keime und keimten einem in vitro Keimungsexperiment (SRA) früher als WT Knollen. Im Gegensatz dazu führte die Verringerung des CK-Gehalts in transgenen Kartoffelpflanzen durch Expression des katabolischen Enzyms CKX1 (Cytokinin Oxidase/ Dehydrogenase1) zu reduziertem Wuchs, Verringerung der apikalen Dominanz und einer geringeren Anzahl an Fiederblättchen in den Blättern. Die CKX1 Transkriptmenge korrelierte zudem negativ mit Knollenertrag und einer Verzögerung der Keimung, die in der stärksten Linie (CKX1-4) bis zu acht Wochen später einsetzte als in WT Knollen. Ferner blieben die Keime der CKX1-4 Knollen nach Beginn der Keimung winzig und ein weiteres Wachstum blieb aus Die Keimung der transgenen CKX1-4

Knollen konnte im Gegensatz zu WT Knollen nicht durch GA3-Behandlung induziert werden. Die transkriptionelle Analyse der CKX1-4, IPT-6 und WT Knollen offenbarte, dass viele Transkripte, die während der GA3-induzierten Keimung im WT und in IPT-6 hochreguliert werden, in den CKX1-4 Knollen nicht induziert werden. Zusammen genommen belegen diese Ergebnisse, dass das Vorhandensein von CK eine notwendige Voraussetzung für das Einsetzen der Keimung ist.

3

Als im Jahr 2009 eine Vorab-Version des Kartoffelgenoms öffentlich zugänglich gemacht wurde, eröffnete dies neue Perspektiven für die Untersuchung der Dormanz und Keimung von Kartoffelknollen. Den genomischen Sequenzen fehlten jedoch Annotationen, was die Identifizierung von Genen erschwerte. Es wurden daher im Rahmen dieser Arbeit zahlreiche Instrumente der in silico Sequenzanalyse, zum Beispiel Sequenzabgleich zwischen genomischen und EST- Sequenzen, Suche nach konservierten Domänen in allen möglichen Leserastern einer genomischen Sequenz, oder Sequenzvergleiche mit nah oder weiter entfernt verwandten Spezies, wie etwa Tomate und Tabak oder Arabidopsis, zusammengefasst und daraus ein Repertoire zur Identifizierung von Intron-Exon-Strukturen und den genomischen Sequenzen zu untersuchender Gene entwickelt. Die hierbei entwickelte Methode wurde anschließend angewendet, um Gene des CK-Metabolismus und -Signalweges im Kartoffelgenom zu identifizieren. Diese Sequenzen stellen einen wertvollen Ausgangspunkt für zukünftige Untersuchungen zu den molekularen Mechanismen der CK-Wirkung bei der Brechung der Keimruhe dar.

Experimente mit Fluoreszenz-Farbstoffen und -Markern zeigten, dass die Knollenmeristeme während der Dormanz vom umgebenden Gewebe symplastisch isoliert sind. Wie aus anderen Spezies bekannt, resultiert diese Isolation meist aus Callose-Ablagerungen an den Plasmodesmata, den pflanzlichen Zell-Zell-Verbindungen. Um die Funktion der Callose während der Keimruhe näher zu bestimmen, wurde die Genfamilie der Beta-1,3-Glucanasen (B13G), die Callose- abbauende Enzyme kodieren, in Kartoffel näher charakterisiert. 34 Gene dieser Genfamilie wurden im Kartoffelgenom identifiziert und durch den Vergleich von vorhandenen Microarray-Daten konnten mehrere Kandidaten eingegrenzt werden, die kurz vor oder bei Einsetzen der Keimung hochreguliert waren. Für zwei dieser Kandidaten, StPdB13G_1 und StPdB13G_2, konnte gezeigt werden, dass sie auf Proteinebene eine hohe Sequenzähnlichkeit zu einer PD-assoziierten β-1,3- Glucanase aus Arabidopsis thaliana (AtBG_PAP) aufweisen. Transgene Pflanzen, die ein RNAi-Konstrukt zur Herunterregulation beider Gene trugen, unterschieden sich phänotypisch nicht vom WT, aber nach der Ernte keimten die Knollen geringfügig, aber nicht signifikant, später als die des Wildtyps. Um den Effekte der Überexpression einer PD-lokalisierten Glucanase zu untersuchen, wurde AtBG_PAP in transgenen Kartoffeln exprimiert. Dies führte zu einem WT-ähnlichem Phänotyp und einem um etwa eine Woche verzögerten Einsetzen der Keimung. Zusammengenommen scheint die

4 Summary / Zusammenfassung

Veränderung der Expression von PD-Glucanasen nicht im vermuteten Maße Einfluss auf die Länge der Keimruhe zu nehmen. Einen zusätzlichen Hinweis dafür lieferte die Immunolokalisation von Callose in WT Knollen während eines Keimungsexperiments. Über einen Zeitraum von mehreren

Tagen nach GA3- Behandlung wurden keine Unterschiede in der Callose-Verteilung vor, während und nach Einsetzen der Keimung beobachtet. Stattdessen konnte im selben Zeitraum die massive Bildung von neuem vaskulärem Gewebe beobachtet werden und die Analyse von Transkriptdaten offenbarte, dass Gene des Auxin-Metabolismus und - Signalweges sowie Marker für vaskuläre Entwicklung während der Keimung hochreguliert werden. Diese Erkenntnisse deuten darauf hin, dass de novo Bildung von Leitgewebe die Nährstoff-Umverteilung zur Deckung des Energiebedarfs der (meristematischen) Zellen im neu gebildeten Keim unterstützt.

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2 Introduction 2.1 The potato - an important crop plant

The potato plant (Solanum tuberosum L.), together with other agricultural crops like tobacco, tomato, bell pepper and eggplant, belongs to the Solanaceae family. Its domestication as a food crop dates back several thousand years, presumably in the central and southern Peru area (Spooner et al., 2005). The Andean region still constitutes a rich gene pool with an estimated 3900 cultivated and wild potato varieties (www.potato2008.org/en/potato/biodiversity.html).

2.1.1 Use and impact of the potato tuber in nutrition and industry

Potato is the fourth most important agricultural crop and the most important among non- grain foods. Its annual production worldwide regularly exceeds 300 million tons (2010: 324,2 million tons; http://faostat.fao.org) and is only surpassed by maize (844,4 million tons), rice (672,0 million tons) and wheat (650,9 million tons).

Being rich in high molecular carbohydrates, proteins, vitamins B1, B2 and C as well as sodium, potassium, magnesium and trace elements, potato tubers are an excellent staple food. Due to both its nutritional value and its relatively easy cultivation, potatoes become more and more important in fighting hunger in third world countries. Indeed, potato production in developing countries has nearly doubled in the last two decades (www.potato2008.org/en/world/index.html). About 50% to 60% of the annual potato production is used for human consumption (Sonnewald, 2001), either directly as fresh tubers or in an industrially processed form: as potato chips and French fries, in thickeners, hot dog sausages and instant soups or even turned into vodka. Starch from potato tubers is also a raw product used in many technical processes, for example in the textile industry, for paper production and as an adhesive.

2.1.2 The potato plant and its life cycle

Although potato plants can propagate vegetatively as well as generatively, non-sexual proliferation via tubers is the main way of breeding and propagation. Seed potatoes are planted in the ground from which sprouts emerge and elongate to reach the soil surface. During this elongation process the emerging sprout subsists on carbohydrates and amino acids provided by its mother tuber. This net import of assimilates marks the sprout as a sink organ during this stage of its development. Organs with a net export, in this case the mother tuber, are termed source organs (Bresinsky et al., 2008). When the

6 Introduction sprout reaches the surface, it forms leaves and becomes fully autotrophic as a photosynthetically active plant. Below ground, the plant produces both roots and diageotropically growing stem organs called stolons. Approximately around the time when the plant starts flowering, tuber formation is initiated by swelling of the stolon tips. This process requires the stop of diageotropical growth and a change from transverse to longitudinal cell division in pith and cortex (Xu et al., 1998). The bulk of tuber tissue is subsequently formed by cell expansion, randomly oriented cell division (Jackson, 1999) and massive depositing of C- and N-assimilates like starch and patatin (Visser et al., 1994; Shewry, 2003), making the tuber a strong storage sink (Sonnewald and Willmitzer, 1992; Fernie and Willmitzer, 2001). As a stem-derived organ, the tuber retains a node- internode structure with the nodes turned into tuber eyes with a leaf scar. Simultaneous to the swelling of the stolon, meristematic activity in the stolon apex and the nodes ceases completely (Ewing, 1995). While the above ground plant dies, the tuber enters a period of dormancy in which no visible growth occurs, even under favourable conditions (Burton, 1989). After a time of storage, dormancy is lost, sprouting occurs and the potato life cycle starts anew.

2.2 Dormancy of potato tubers

Many plant tissues and life stages like seeds, tubers or buds of perennial plants undergo a period of dormancy as a survival strategy for unfavourable environmental conditions. Dormancy is therefore defined in broad terms as ‘the absence of visible growth of any plant structure containing a meristem’ (Lang et al., 1987). However, this description does not suitably describe proceedings at the cellular and molecular level. Thus, Rohde and Bhalerao (2007) argued for a more precise definition of dormancy as ‘the inability to initiate growth from meristems and other organs and cells with the capacity to resume growth under favourable conditions’. In potato tubers, dormancy occurs only in the tuber eyes which contain a meristem, the rest of the tuber is still metabolically active. Dormancy of the eye meristems is established during tuber formation (Burton, 1989; Ewing, 1995). According to definition by Lang et al. (1987), two phases of dormancy are found in potato: endodormancy and ecodormancy. During endodormancy, growth inhibition is caused by factors in the affected meristem itself whereas in ecodormancy, it is brought about by limiting environmental conditions (Lang et al., 1987).

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2.2.1 Physiological changes during dormancy break and sprouting and their impact on nutritional value and industrial processing

When potato tubers start sprouting, they turn from a storage sink into a source organ for the growing sprout. During this transition, rates of DNA, RNA and protein biosynthesis increase (Suttle, 1996), enzymatic activity rises (Merlo et al., 1993) and starch and storage proteins are remobilized and transported into the sprout (Davies and Ross, 1984). Additionally, the sprout meets its water requirements by depleting the tuber until roots are formed. Thus, both dehydration and reduction of storage substances lead to a quality loss in stored tubers. Although maintaining quality during tuber storage presents a challenge to the processing industry, long-term storage cannot be completely avoided since the crop’s climate requirements do not allow year-round cultivation (Sonnewald, 2001). Commonly, tubers are either stored at low temperatures or treated with sprouting inhibitors like chloropropham (CIPC) and maleic hydrazide to delay sprouting. Both procedures, however, have disadvantages: Inhibitor treatment needs to be carefully handled as they are usually potent inhibitors of cell division. Untimely application of maleic hydrazide limits tuber size and yield. Residues of CIPC aerosols can remain in buildings for up to one year after application, requiring intensive cleaning and venting before storages can be used for other crops (www.umaine.edu/umext/potatoprogram/ pest_control_guide.htm). In addition, inhibitor treatment may trigger environmental and consumer concerns. Cold storage of tubers, although easy to arrange, often leads to degradation of tuber starch to reducing sugars in a process called cold-induced sweetening. This not only results in a sweet taste of the tubers, but also presents a major problem for starch- processing industries because of unwanted ‘browning’ during frying (Sonnewald, 2001). Recent approaches to silence vacuolar acid invertase genes in potato showed successful prevention of cold-induced sweetening (Bhaskar et al., 2010; Liu et al., 2011), but did not address the problem of undesired tuber sprouting. A deeper understanding of processes occurring during potato tuber sprouting at the cellular and molecular level could have direct impact on the growing and processing industry by providing tools for enhanced tuber storage and plants with improved storage properties.

2.2.2 Factors influencing potato tuber dormancy

Although dormancy is largely under genetic control and differs between cultivars (Hemberg, 1985; Wiltshire and Cobb, 1996), numerous environmental and endogenous factors can modulate dormancy length.

8 Introduction

Environmental factors influencing plant growth, such as light period, temperature and water and nutrient supply, are also reflected in dormancy length as tuber formation directly depends on the plant’s performance. Early research reported a direct correlation between day length and dormancy: tubers of plants grown under short day conditions have a significantly shorter dormancy period than those of plants grown under long day conditions (Emilsson, 1949; Hemberg, 1985). Temperature, on the other hand, has the adverse effect: high temperatures along with water deficit in the soil lead to shortened dormancy (Burton, 1989). Additionally, water stress and repetitive cycles of high and low nitrogen during tuberization lead to the formation of secondary tubers. In this chain- tuberization, dormancy cannot be established, the stolon tip resumes its growth and forms a second tuber when nitrogen concentrations decrease (Krauss, 1985; Jackson, 1999). Beside environmental conditions, dormancy length also depends on endogenous changes of phytohormone levels and intensive research has been dedicated to unravel the roles played by the different phytohormone classes. In general, dormancy and dormancy break are thought to be controlled by an intricate interplay of inhibiting and promoting phytohormones. Four phytohormones whose roles have been studied widely, abscisic acid (ABA), auxin (AUX), gibberellin (GA) and cytokinin (CK), will be outlined in greater detail in the following chapter. For other phytohormones data remains largely unclear: In accordance with the biphasic model of ethylene action in which the hormone exerts growth-promoting functions at low concentrations and acts as an inhibitor at high levels (Pierik et al., 2006), exogenous ethylene application has been described as both sprout-promoting (Claassens et al., 2005) and inhibitory (Rylski et al., 1974; Prange et al., 1998), depending on its concentration. Nevertheless, further experiments suggest that ethylene is not involved in dormancy break (Suttle, 2009), but rather acts in concert with ABA during the initiation of dormancy (Suttle, 1998; 2004b). Although a promoting role during tuber induction has been described for jasmonic acid (Pelacho and Mingo-Castel, 1991), measurements of endogenous jasmonates and treatment experiments render participation of this class of hormones in dormancy break unlikely (Suttle et al., 2011). Convincing evidence for brassinosteroid involvement in dormancy and sprouting is lacking, considering that there is only one report in which data on exogenous application of the hormone was presented (Korableva et al., 2002). This study suggested an inhibitory role for brassinosteriods in dormancy release.

9

Strigolactones, a novel class of hormones first found in the root parasite Striga lutea (Cook et al., 1966), have not been analysed in terms of their role during dormancy break. Transgenic tubers down-regulated in expression of carotenoid cleavage dioxygenase4 (CCD4), encoding an enzyme of carotenoid cleavage which might act up-stream of strigolactone (and ABA) biosynthesis, showed a distinct sprouting and developmental phenotype (Campbell et al., 2010). However, the authors accredited this phenotype and sprouting behaviour to an involvement of CCD4 in tuber heat responses rather than changes in phytohormone levels.

One aspect that hampers research into phytohormone function during dormancy could lie within the tuber’s metabolism. It has been proposed that potato tubers need to attain a certain metabolic competence before they can start to sprout (Sonnewald, 2001). The sink-to-source transition of the tuber during sprouting is attended by starch and storage protein degradation and subsequent transport of sucrose and amino acids to the sprout via the phloem (Hajirezaei et al., 2003). However, the initial growth of the sprout seems to be supported by preformed sucrose, as a significant increase in starch breakdown can only be detected after sprout growth has started (Sonnewald, 2001; Hajirezaei et al., 2003). Transgenic potato tubers over-expressing a bacterial pyrophosphatase under control of the patatin promoter or expressing a cytosolic yeast invertase in the phloem are in accordance with this hypothesis: An increase in sucrose content due to pyrophosphatase activity led to earlier sprouting of transgenic tubers (Farre et al., 2001) whereas low sucrose supply caused by invertase activity in the phloem resulted in dormancy break, but ensuing sprout growth failed (Hajirezaei et al., 2003). Similar results were also obtained for expression of other enzymes of carbohydrate utilization, implying that carbohydrate status has a regulatory role in controlling dormancy length (Lytovchenko et al., 2005).

2.3 Phytohormones involved in dormancy, bud breakage and sprout growth

Four major classes of phytohormones, abscisic acid, auxin, gibberellin and cytokinin, are considered important for tuber dormancy and sprouting, although their individual roles are often not completely understood. The current state of knowledge concerning their metabolism, signalling and function during dormancy and dormancy break is presented in this chapter.

10 Introduction

2.3.1 Abscisic acid (ABA)

Very early in dormancy research, the existence of endogenous growth inhibitors regulating dormancy was hypothesized by Hemberg (1949) and in the following years dormancy-inducing and growth-inhibiting substances were isolated from etiolated tissues of different plant species (‘inhibitor-β complex’ (Bennet-Clark and Kefford, 1953)), young cotton fruit (‘Abscisin II’ (Ohkuma et al., 1963)) and perennial woody plants (‘dormins’ (Eagles and Wareing, 1963)). Determination of the chemical structure of these substances showed that they are identical, containing a monocyclic sesquiterpene which was given the trivial name abscisic acid (Addicott and Lyon, 1969). The ABA biosynthetic pathway has been elucidated by the isolation of auxotrophic mutants, which was reviewed recently by Nambara and Marion-Poll (2005). ABA levels within the plant seem to be regulated by a finely tuned balance of biosynthesis and catabolism, with biosynthesis regulated transcriptionally via expression levels of nine-cis- epoxycarotenoid dioxygenase (NCED) genes (Qin and Zeevaart, 1999) and, to a lesser extent, via expression levels of zeaxanthin epoxidase (ZEP) genes (Frey et al., 1999). ABA catabolism seems to be predominantly controlled by expression of ABA 8’ hydroxylases, cytochrome P450 monooxygenases of the CYP707A family (Kushiro et al., 2004). Great effort has been undertaken to unravel the ABA signalling network, but due to genetic redundancy, the PYR/PYL/RCAR family of ABA receptors could only very recently be identified (Ma et al., 2009; Park et al., 2009; Klingler et al., 2010) and their structure be determined (Santiago et al., 2011), triggering a burst of research activity and the emergence of a core signalling pathway. In this core pathway, PYR/PYL/RCAR ABA receptors are not bound to type 2 C protein phosphatases (PP2C) in the absence of ABA, allowing high PP2C activity which prevents activation of Snf1-related protein kinase (SnRK2). When ABA is present, PYR/PYL/RCARs bind PP2Cs, leading to their inhibition and subsequent accumulation of phosphorylated SnRK2. SnRK2s, in turn, phosphorylate ABA-responsive element binding factors (ABFs) which leads to expression of ABA- responsive genes (Cutler et al., 2010; Melcher et al., 2010; Weiner et al., 2010; Antoni et al., 2011; Liu, 2011). As to its function, ABA plays a major role in the response to biotic and abiotic stresses (Zhu, 2002; Fujita et al., 2006). In tissues undergoing a dormancy period, ABA is required to initiate and maintain the dormant state (Nambara and Marion-Poll, 2005). In potato tubers in particular, it could be shown that exogenously applied ABA inhibits sprouting (El-Antably et al., 1967; Suttle and Hultstrand, 1994) and that tuber ABA content decreases continuously after harvest, being highest when tubers are deeply 11

dormant (Coleman and King, 1984; Suttle, 1995; Biemelt et al., 2000). Experiments with inhibitors of ABA biosynthesis and monitoring of ABA biosynthetic and catabolic gene expression during storage demonstrated that constant ABA production is required for dormancy continuation (Suttle and Hultstrand, 1994) and that decreases of ABA content correlate with decreased expression of NCED and higher expression of ABA 8’ hydroxylases (Destefano-Beltrán et al., 2006). ABA decline, however, does not directly correlate with onset of sprouting, as no particular threshold concentration could be determined below which dormancy is broken (Suttle, 2004b).

2.3.2 Auxin (AUX)

In his book “The Power of Movement in Plants”, Charles Darwin described, after observing the bending of Phalaris canariensis coleoptiles towards the light, ‘the presence of some matter in the upper part […] which transmits its effects to the lower part’ (Darwin, 1880). In 1926, Went succeeded in isolating this growth-stimulating substance from Avena sativa coleoptiles (Went, 1926) and later this class of substances was termed Auxins, from the greek word ‘αυξειν’, meaning ‘to grow’ (Kögl and Smit, 1931). Naturally occurring auxins are indole-3-acetic acid (IAA), phenylacetic acid (PAA), 4-chloroindole- 3-acetic acid (4-Cl-IAA) and indole-3-butyric acid (IBA), but the latter three seem to play only a marginal role as they are not found in all plant species (Bresinsky et al., 2008). Despite recent discoveries of several genes of auxin biosynthesis, no complete pathway for de novo synthesis of IAA has been elucidated so far. Several tryptophan-dependent and tryptophan-independent pathways have been described which contribute to auxin biosynthesis. Beside these complex pathways, IAA production and transport are tightly regulated temporally and spatially, ensuring the formation and upkeep of local auxin gradients. For extensive review, see Normanly (2010) and Zhao (2010). Auxin signalling occurs through a novel mechanism involving protein degradation via SCFTIR1 ubiquitin-ligase-mediated ubiquitination. Without IAA, auxin response factor (ARF) transcription factors bind to auxin response elements (AREs) in the promotors of auxin-responsive genes. Interaction of ARFs with Aux/IAA proteins and the transcriptional repressor TOPLESS (TPL) prevents gene expression (Szemenyei et al., 2008). The F-Box protein TIR1, a subunit of the SCFTIR1 complex, acts as an auxin receptor (Dharmasiri et al., 2005; Kepinski and Leyser, 2005). Upon auxin binding, interaction of Aux/IAA proteins and the SCFTIR1 complex is stabilized which leads to Aux/IAA protein degradation via the ubiquitin proteasome pathway (Gray et al., 1999). In this way, ARF transcription factors are released from their inhibition, allowing expression

12 Introduction of auxin-responsive genes (Teale et al., 2006; Mockaitis and Estelle, 2008; Wu et al., 2011). Auxin is a key regulator of many steps of plant development and growth, like vascular patterning of shoots, roots and leaves (Aloni et al., 2006; Scarpella et al., 2006; Ilegems et al., 2010), reproductive development, fruit set and fruit growth (De Jong et al., 2009; Sundberg and Østergaard, 2009), apical dominance (Ongaro and Leyser, 2008; Shimizu- Sato et al., 2009), lateral root formation (Dubrovsky et al., 2008; Farquharson, 2010), cell expansion, cell division and differentiation (Schenck et al., 2010). Reports on the role of auxin during potato tuber dormancy differ. Several groups describe an inhibition of sprouting by application of high concentrations of IAA or a synthetic auxin, 1-naphthaleneacetic acid (NAA), as summarised by Bradley and Dean (1949). Low auxin concentrations, however, failed to induce sprouting of dormant tubers and only promoted sprout growth after dormancy had terminated (Hemberg, 1949). Bioassay data and HPLC-coupled fluorometric detection of auxins also suggested an increase in hormone levels after bud break had occurred (Hemberg, 1949; Sukhova et al., 1993). Conflicting results have been published by Sorce et al. (2000), claiming ‘a significant increase of the free hormone concentration from harvest to the end of dormancy’ and later stating that auxin concentrations were highest at early stages of tuber dormancy and falling until dormancy ended (Sorce et al., 2009). In summary, auxin seems to be involved in processes accompanying sprouting rather than in dormancy control itself (Suttle, 2004b).

2.3.3 Gibberellin (GA)

Gibberellins (GA) are growth-promoting phytohormones first isolated from the fungus Gibberella fujikuroi, the causal agent of bakanae disease (‘foolish seedlings’) in rice. Chemical structures of GA were first described in the 1950s by groups from Japan, the USA and Britain (http://www.plant-hormones.info/gibberellinhistory.htm). To date, structures of more than 120 GA-species have been described, the abundance requiring a simple numbering: GA1 to GAn in order of their discovery. Only four of these compounds are biologically active, GA1, GA3, GA4 and GA7 (Hedden and Phillips, 2000). Chemically, GA are tetracyclic diterpenes comprised of four isoprene units. GA biosynthesis starts in the plastids, intermediate products are further converted in the endoplasmic reticulum (ER) and the final steps of GA biosynthesis take place in the cytosol. Here, two different compounds, GA12 and GA53, which differ only in their functional group at the C13 atom, are converted to either GA4 and GA7 or to GA3 and

GA1 as bioactive GA-species, respectively (Yamaguchi, 2008). It has been shown that

13

preference for one of the two pathways depends on species, developmental stage and plant tissue. For example, in Arabidopsis thaliana, predominantly GA4 is found (Sponsel et al., 1997) whereas in potato the main bioactive GA is GA1 (van den Berg et al., 1995).

Young plants of Picea sitchensis contain mostly GA4 whereas in mature and flowering plants GA1 and GA3 are present (Moritz et al., 1989). In rice, vegetative tissues produce

GA1 and reproductive tissues contain GA4 (Takahashi and Kobayashi, 1990). GA concentration is mainly controlled by a feedback mechanism regulating expression of biosynthesis genes GA20-oxidase (GA20-ox) and GA3-β-hydroxylase (GA3-ox) as well as by a feedforward regulation of GA2-oxidase (GA2-ox) expression, coding for an inactivating enzyme (Yamaguchi, 2008). The GA signalling pathway has been extensively reviewed recently (Gao et al., 2011; Sun, 2011; Wang and Deng, 2011). In brief: In the absence of GA, DELLA proteins, which were named after their conserved N-terminal DELLA domain, act as nuclear transcriptional repressors to inhibit GA signalling and transcription of GA-responsive genes (Sun, 2011). Bioactive GA present in the cell is bound by soluble GA receptors like GIBBERELLIN INSENSITIVE DWARF1 (GID1) with high affinity (Ueguchi-Tanaka et al., 2005). Upon binding of the phytohormone, GA-GID1 directly interacts with DELLA proteins (Ueguchi-Tanaka et al., 2007; Willige et al., 2007) and a conformational change in the DELLA-GA-GID1 complex stimulates its recognition by the SCFSLY/GID2/SNZ complex to target DELLA proteins for proteasome-dependent degradation (Fu et al., 2002; Dill et al., 2004; Ariizumi et al., 2011). DELLA proteins lack a DNA-binding motif and seem to exert their function by interaction with transcription factors, for example blocking DNA binding activity of transcription factors PIF3 and PIF4 (Davière et al., 2008; de Lucas et al., 2008; Feng et al., 2008). Lifting of DELLA repression through degradation then allows transcription of GA-responsive genes. Based on observations in rice and Arabidopsis, an additional, proteolysis-independent model was recently proposed in which ‘relief of repression’ by DELLA proteins is achieved by inactivation without degradation (Gao et al., 2011). As growth-promoting phytohormones, GAs regulate numerous physiological processes, including stem and hypocotyl elongation (Sun and Gubler, 2004), induction of flowering (Blázquez et al., 1998; Levy and Dean, 1998) and termination of seed dormancy (Finkelstein et al., 2008). Examination of GA function during potato tuber dormancy and sprouting started soon after discovery of these hormones. Several groups showed independently that exogenous application can induce sprouting of dormant tubers (Brian et al., 1955; Rappaport et al., 1957; Hemberg, 1985; Suttle, 2004a). Measurements of endogenous

14 Introduction

gibberellins seemed to support this finding at first, indicating a constant increase of GA1 concentration until dormancy ended and sprouting began (Smith and Rappaport, 1961). Improved detection methods, however, showed no significant difference between deeply dormant tubers and stored non-sprouted tubers, and GA concentrations only increased significantly after sprouting had commenced (Suttle, 2004a). Transgenic potato tubers over-expressing GA biosynthesis gene StGA20-ox1 showed earlier sprouting and strongly elongated sprouts (Carrera et al., 2000), arguing for a regulatory role of gibberellins in dormancy release. In contrast, there was no difference in sprouting after down-regulation of GA biosynthesis, neither by antisense expression of StGA20-ox1 nor by over-expression of GA-inactivating enzyme StGA2-ox1 (Carrera et al., 1999; Kloosterman et al., 2007). Taken together, the exact role of gibberellins in dormancy and sprouting remains elusive, but results suggest that GA regulates sprout growth rather than triggering its initiation.

2.3.4 Cytokinin (CK)

In the search for growth-promoting substances for plant tissue culture, 6- furfurylaminopurine, also known as kinetin, was identified and isolated from autoclaved herring sperm DNA (Miller et al., 1955; Miller et al., 1956). Isolation of zeatin from maize followed (Letham, 1963) and the new class of phytohormones was termed cytokinins (CK) (Skoog et al., 1965). Naturally occurring CKs are adenine derivates carrying an aromatic or isoprenoid side chain at the N6 atom. In plants, the nucleobases N6-(Δ2- isopentenyl) adenine (iP), trans-zeatin (tZ), cis-zeatin (cZ) and dihydrozeatin (DZ) are the predominant forms, but nucleotides, nucleosides, glucosides and sugar conjugates can also be found (Mok and Mok, 2001). The nucleobases show the highest CK activity and their nucleosides, isopentenyladenine riboside (iPR), trans-zeatin riboside (tZR), cis- zeatin riboside (cZR) and dihydrozeatin riboside (DZR) are also weakly active (Sakakibara, 2006). Since the role of CK was investigated in this study, aspects of CK metabolism, transport, signalling and function will be described in great detail in the following sections.

2.3.4.1 CK metabolism

A finely tuned CK homeostasis is vital to plant development and growth. CK activity is therefore regulated at various steps such as biosynthesis, conjugation, interconversion and degradation. The central metabolic pathways of CKs are summarized in Fig.1. De novo biosynthesis of CKs starts with transfer of an isoprenoid moiety from dimethylallyl pyrophosphate (DMAPP) to adenine, either in its nucleotide form e.g. ADP or AMP

15

(Kakimoto, 2001; Sakakibara, 2006) or tRNA-bound (Takei et al., 2001a). This is the rate-limiting step of the biosynthesis pathway (Morris, 1995). Both reactions are catalysed by isopentenyltransferases (IPT) specific for one or the other substrate and result in formation of the iP nucleotide, in the case of adenylate IPT, or the cis-zeatin nucleotide by tRNA-specific IPT (Miyawaki et al., 2006). Hydroxylation of the side chain of the iP nucleotide by a cytochrome P450 monooxygenase (CYP735A) gives trans- zeatin nucleotide (Takei et al., 2004). Dihydrozeatin nucleotide, nucleoside and the free nucleobase are produced by reduction of tZRMP, tZR or tZ, respectively, through the action of a zeatin reductase (Martin et al., 1989; Gaudinová et al., 2005).

DMAPP + ATP/ADP DMAPP + tRNA

adenylate IPT tRNA IPT

iPRDP/iPRTP CYP735A tZRDP/tZRTP prenyl-tRNA

dephosphorylation dephosphorylation

iPRMP CYP735A tZRMP ZRED DZRMP cZRMP nucleotides

iPR LOG tZR ZRED DZR LOG cZR nucleosides

iP tZ ZRED DZ cZ nucleobases

CKX O-Glycosylation Degradation N-Glycosylation Figure 1: Overview of cytokinin biosynthesis, degradation and conversion pathways. For a detailed description of the pathways, see text. Orange arrows indicate reactions where enzymes and genes have been identified, black arrows mark reactions where enzymes and/ or genes have not been discovered so far. Orange boxes denote genes important for CK homeostasis. Blue boxes frame all compounds belonging CK nucleotides, CK nucleoside or CK nucleobases, respectively. Grey boxes represent known enzymatic steps which do not seem to be important for CK homeostasis. Adenylate IPT, adenosine phosphate-isopentenyltransferase; ADP, adenosine diphosphate; ATP, adenosine triphosphate; cZ, cis-zeatin; cZR, cis-zeatin riboside; cZRMP, cZR 5’ monophosphate; DMAPP, dimethylallyl pyrophosphate; DZ, dihydrozeatin; DZR, dihydrozeatin riboside; DZRMP, DZR 5’ monophosphate; iP, N6-(Δ2-isopentenyl)adenine; iPR, -(Δ2-isopentenyl)adenine riboside; iPRMP, iPR 5’ monophosphate; iPRDP, iPR 5’ diphosphate; iPRTP, iPR 5’ triphosphate; LOG, LONELY GUY; tRNA-IPT, tRNA-isopentenyltransferase; tZ, trans-zeatin; tZR, trans-zeatin riboside; tZRMP, tZR 5’ monophosphate; tZRDP, tZR 5’ diphosphate; tZRTP, tZR 5’ triphosphate; ZRED, zeatin reductase.

For generation of bioactive nucleobases from their corresponding nucleotides, both a one-step and a two-step ‘cytokinin-activation pathway’ have been proposed (Sakakibara, 2006; Kurakawa et al., 2007). In the one-step pathway, CK riboside 5’- monophosphate phosphoribohydrolase, encoded by the LONELY GUY (LOG) gene first identified in rice, catalyses direct conversion of CK riboside monophosphates (iPRMP, tZRMP, DZRMP, cZRMP) to the equivalent free bases (Kurakawa et al., 2007). In Arabidopsis, LOG genes

16 Introduction were found to be encoded by a small gene family whose members are differentially expressed in various tissues, indicating a vital role in regulating CK activity (Kuroha et al., 2009). In the postulated two-step pathway, CK nucleotides are first converted into nucleosides by a nucleotidase and then into nucleobases by a nucleosidase (Sakakibara, 2006). Enzymes for these two steps were partially purified from wheat (Chen and Kristopeit, 1981a; 1981b), but the corresponding genes have not been identified so far (Tokunaga et al., 2011). Although this does not rule out a contribution of the two-step pathway to CK biosynthesis, recent analysis of higher order log mutants suggests that the LOG-dependent single step pathway attributes to the bulk of bioactive cytokinins (Tokunaga et al., 2011). Inactivation of active CKs can occur via degradation or glycosylation. Degradation is catalysed by cytokinin oxidase/ dehydrogenase (CKX) enzymes which irreversibly cleave off the side chain forming adenine (Ade) or adenosine (Ado) and an unsaturated aldehyde (Pačes et al., 1971; Mok and Mok, 2001). It was first reported that this reaction required oxygen (Whitty and Hall, 1974) leading to the name ‘cytokinin oxidase’, but highly purified CKX from wheat showed no such requirement and behaved more like a dehydrogenase (Galuszka et al., 2001), hence the re-classification and double name of the enzyme. CKX enzymes are flavoproteins which covalently bind their cofactor FAD (Malito et al., 2004). CKX genes were first cloned from maize (Houba-Hérin et al., 1999; Morris et al., 1999) and found to constitute small gene families of varying size in many plant species (Werner et al., 2006; Galuszka et al., 2007). Recent work by several groups illustrates that degradation is a crucial mechanism of CK homeostasis (Werner et al., 2001; Ashikari et al., 2005). CKs can be glycosylated at the N3, N7 and N9 positions of the purine ring and at the hydroxyl group of the side chain. Glycosylation reactions are catalysed by glucosyltransferases (GT) specific for either N- or O-glycosylation (Sakakibara, 2006). N- glycosylation is commonly regarded as an irreversible inactivation as the reaction products show very little to no CK activity in bioassays and, so far, no plant β- glucosidases that release free CKs from N-glucosides have been identified (Frébort et al., 2011). Zeatins, which carry a hydroxyl group on their side chain, can be O- glycosylated and both enzymes and genes of zeatin-O-glycosyltransferases have been characterized in different species (Martin et al., 1999b, 1999a; Veach et al., 2003; Meek et al., 2008). Some of these enzymes distinguish between the cis- and trans-isomer of zeatin (Martin et al., 2001) and some are capable of also transferring xylosyl instead of glycosyl moieties (Turner et al., 1987; Martin et al., 1999a). CK-O-glycosides are considered to be storage forms, for two reasons: They cannot be degraded by CKX

17

(Laloue and Pethe, 1982) and they can be cleaved by β-glucosidases to release active cytokinins (Brzobohaty et al., 1993). Another enzyme implied in CK metabolism is an adenosine kinase (ADK), an enzyme of the purine salvage pathway that normally catalyses phosphorylation of adenosine to AMP in order to maintain intracellular purine nucleotide pools (Moffatt et al., 2000). Novel analyses of ADK-deficient Arabidopsis plants suggest involvement of the enzyme in the conversion of CK ribosides to their corresponding ribotides in vivo, thus contributing to CK homeostasis (Schoor et al., 2011). There have also been reports on the interconversion of trans- and cis-zeatin via the activity of a cis-trans isomerase. An enzyme with this activity was partially purified from Phaseolus vulgaris (Bassil et al., 1993), but its activity has seldom been shown in vivo, e.g. in potato where radioactively labelled cis-zeatin was applied to tubers and about 10% of recovered label was found to be trans-zeatin (Suttle and Banowetz, 2000).

2.3.4.2 CK transport

CKs are mobile hormones that can act both locally and at distant sites (Kudo et al., 2010), requiring transport mechanisms within the plant and molecules at the cell membrane to coordinate CK import and export. Two proteins families have been suggested to mediate CK transport across the plasma membrane: the purine permease (PUP) family and the equilibrative nucleoside transporter (ENT) family (Hirose et al., 2008). PUP transporters seemed to preferably transport iP and tZ (Gillissen et al., 2000; Bürkle et al., 2003) whereas ENT proteins were found to transport iPR and tZR (Hirose et al., 2005). Research into this area has not advanced very far yet and further studies will probably elucidate PUP and ENT function. Long-distance transport of cytokinins occurs in both xylem and phloem, but distribution of iP-type and tZ-type CKs in these tissues differs. In xylem sap, tZR is most abundant (Beveridge et al., 1997; Takei et al., 2001b) while in phloem sap, iPR and iP nucleotides predominate (Corbesier et al., 2003). This differential allocation indicates that the two CK types deliver different messages, but components of this transport system, its regulatory mechanisms and precise functions remain to be elucidated (Kudo et al., 2010).

2.3.4.3 CK signalling

Although CKs have been known since the 1950s, details on their perception and signalling cascades remained elusive for a long time. The first breakthrough in the search for a CK receptor was the discovery of the CYTOKININ INDEPENDENT1 (CKI1) gene in Arabidopsis mutants that showed CK responses in tissue culture in the absence

18 Introduction of cytokinin and the protein turned out to be an integral membrane protein similar to histidine two-component regulators found in prokaryotes (Kakimoto, 1996). The suspected similarity of cytokinin signalling to known bacterial two-component signalling pathways and the genetic tools developed with the sequencing of the Arabidopsis genome allowed a rapid elucidation of the CK signalling pathway (Kieber and Schaller, 2010). An overview of this pathway is given in Fig.2.

iP tZ cZ

PM / ER membrane

P AHK

P AHP6 AHP P feedback inhibition

P A-type ARR cytosol nucleus

P

target gene transcription B-type ARR

Figure 2: Overview of the cytokinin signalling pathway For a detailed description, see text. In brief, perception of CK by the CHASE domain of CK receptors leads to receptor autophosphorylation. In a phosphorelay system, the phosphorylation signal is transmitted via histidine phosphotransmitter proteins to A- and B-type response regulators in the nucleus which in turn coordinate target gene transcription and feedback modulation of the signalling cascade. AHK, Arabidopsis histidine kinase; AHP, Arabidopsis histidine-containing phosphotransfer protein; ARR, Arabidopsis response regulator; cZ, cis-zeatin; ER, endoplasmatic reticulum; iP, N6-(Δ2-isopentenyl)adenine; PM, plasma membrane; tZ, trans-zeatin

A genuine CK receptor was first identified in the Arabidopsis cytokinin resistant1 (cre1) mutant (Inoue et al., 2001), carrying a loss-of-funtion allele of the CRE1 gene which encodes a sensor histidine kinase identical to ARABIDOPSIS HISTIDINE KINASE4 (AHK4), one of 11 histidine sensor kinases predicted in the Arabidopsis genome (Imamura et al., 1999; Suzuki et al., 2001). CRE1/AHK4 is also known as WOL (WOODEN LEG), since the WOL locus in the wooden leg mutant of Arabidopsis, which is defective in vascular development (Scheres et al., 1995), was mapped to AHK4 (Mähönen et al., 2000). Beside CRE1/AHK4/WOL, there are two other CK receptors encoded in the Arabidopsis genome, AHK2 and AHK3 (Suzuki et al., 2001; Higuchi et al.,

19

2004), with both overlapping and specific activities (Riefler et al., 2006; Heyl et al., 2011). CKs are perceived by the N-terminal CHASE (cyclases/histidine kinases-associated sensory extracellular) domain which occurs in extracellular or periplasmic locations and is always flanked by transmembran regions on either side (Anantharaman and Aravind, 2001). For a long time CK receptors were thought to reside in the plasma membrane, but recent localization studies suggest that they are located mainly to the ER membrane (Wulfetange et al., 2011). The intracellular tail of CK receptors contains a histidine kinase (HisKA) domain, a Histidine kinase-like ATPase (HATPase_c) domain and a signal receiver (REC) domain (Inoue et al., 2001). Upon binding of CKs, the receptors autophosphorylate at a conserved histidine residue in the HisKA domain, starting the multi-step phosphorelay: Next, the phosphate is transferred to a conserved aspartate residue in the REC domain. From there the phosphate group is transferred to a His residue of an ARABIDOPSIS HISTIDIN-CONTAINING PHOSPHOTRANSFER (AHP) protein. Phosphorylated AHPs translocate to the nucleus where they in turn phosphorylate A- and B-type ARABIDOPSIS RESPONSE REGULATOR (ARR) proteins (Ferreira and Kieber, 2005). The Arabidopsis genome encodes six AHP proteins (AHP1-6) most of which have been shown to transfer the phosphorelay signal (Mähönen et al., 2006). AHP6, however, lacks the conserved His residue and acts as an inhibitor of CK signalling, probably by interfering at different steps of the phosphorelay (Mähönen et al., 2006; Perilli et al., 2010). Novel studies report that AHP proteins are distributed evenly among nucleus and cytosol and that they are actively transported in and out of the nucleus in a constant cycling process, independent of their phosphorylation state and unaffected by application of exogenous CK. This questions the current translocation model that assumes accumulation of phosphorylated AHPs in the nucleus, but the possible backflow of phosphorylated AHP from the nucleus into the cytosol might serve as a level of CK signalling regulation (Punwani et al., 2010). The B-type ARRs are transcription factors (Sakai et al., 2001) encoded by a small gene family with eleven members in Arabidopsis. They directly initiate transcriptional cascades leading to CK response and act, in part, redundantly (Mason et al., 2005; Argyros et al., 2008; Ishida et al., 2008). Type-A ARRs are major negative regulators of CK signalling, providing an efficient instrument to modulate, dampen or shut down CK output in response to a change in the signal input or through crosstalk with other pathways (Kieber and Schaller, 2010). They are direct targets of B-type ARR transcription factors (Hwang and Sheen, 2001) and require phosphorylation by AHPs to be stabilized (To et al., 2007). Although their role in

20 Introduction the regulation of CK signalling is clear, not much is known about their target genes/ proteins and their mode of action. A small subset of AP2/ERF transcription factor genes termed Cytokinin Response Factors (CRF) have also been implied in the signal transduction pathway as the proteins accumulate in the nucleus in an AHP-dependent manner. Analysis of arr1,12 mutants as well as microarray data suggest that they act downstream of B-type ARRs (Rashotte et al., 2006; Argueso et al., 2010).

2.3.4.4 CK functions

CKs influence numerous physiological and developmental processes in a positive or negative manner. They were first discovered as factors promoting cell division, but they also stimulate formation, maintenance and activity of shoot apical meristems (SAM), incite chloroplast development even in the absence of light, impede leaf senescence and control root growth and shoot branching. In roots, they also take part in specification and maintenance of the root vascular meristem (Riefler et al., 2006; Sakakibara, 2006; Bresinsky et al., 2008; Werner and Schmülling, 2009; Perilli et al., 2010). Most of these functions are attributed to trans-zeatin- and iP-type cytokinins. Research into specific functions of cis-zeatin is still in its infancy, but current bioassay data suggests that cis- zeatin is associated with developmental stages in which growth is limited (Gajdošová et al., 2011). A quite intriguing finding, since cis-zeatin and its riboside have been reported as the predominant cytokinin species in many plants, at certain developmental stages or in specialized tissues, among them potato stolons and tubers (Mauk and Langille, 1978; Frébort et al., 2011). There have been several reports on the effect and concentration of CKs during potato tuber dormancy and sprouting. Application of either trans-zeatin or kinetin, a synthetic cytokinin, led to premature sprouting of dormant tubers, but only at the very beginning and towards the end of the dormancy period (Hemberg, 1970), probably explained by a changed sensitivity of the tissue (Turnbull and Hanke, 1985b; Suttle, 2001). Endogenous CK levels rise close to the end of dormancy (Turnbull and Hanke, 1985b; Sukhova et al., 1993; Suttle and Banowetz, 2000). This, together with the observation that exogenous CK cannot further stimulate sprout growth once dormancy has broken, led to the assumption that CK has a role in dormancy break (Turnbull and Hanke, 1985a).

2.4 Plasmodesmata, callose and tuber bud meristems

Multicellular eukaryotic species rely on direct cell-to-cell communication to exchange positional information and ensure proper development and growth. Due to their rigid cell

21

wall which prevents membrane contact of neighbouring cells, plants have evolved a system of membrane-lined channels through the cell wall, named plasmodesmata (PD), to link adjacent cells. In the following sections PD structure and function, callose cycling at PD and how both is involved in dormancy will be discussed.

2.4.1 Plasmodesmata - structure and function

PD join the protoplasts of adjacent cells to form a symplasmic continuum. They are commonly distinguished by their origin and by their structure. Primary PD always originate during cell division, when parts of the ER are trapped by deposition of cell wall material in the cell plate. Secondary PD are formed post-cytokinesis by yet unknown mechanisms. Both primary and secondary PD can occur as simple or branched forms, very complex branched PD are even composed of several channels and a central cavity (see Ehlers and Kollmann (2001) for a comprehensive review of primary and secondary PD). The occurrence of simple and branched PD is strongly associated with plant development. Young and immature sink tissues mostly contain simple, unbranched PD whereas in mature source tissues, branched and complex PD predominate (Imlau et al., 1999; Oparka, 2005). Simple and branched PD also differ in their size exclusion limit (SEL), which describes the maximum size of molecules that can pass through PD. For example, during sink-to-source transition of tobacco leaves, SEL decreases, indicating that cell-to-cell movement is more stringently regulated in mature tissues with complex PD (Oparka et al., 1999; Crawford and Zambryski, 2001) Simple PD are composed of three major structural components: a central membranous rod of appressed ER called desmotubule (DT), the plasma membrane (PM) that is shared by the two adjoining cells and the cell wall that constitutes the physical the boundaries of the PD. These structures provide defined spaces for cell-to-cell communication: the cytoplasmic sleeve between PM and DT, the ER lumen and the apoplast (reviewed by Maule et al. (2011)). Despite recent advances in identifying potential PD proteins, many details of PD structure are still unresolved. For a thorough overview of candidate PD components and models of PD trafficking, see Lucas et al. (2009) and Maule et al. (2011). PD constitute the plant’s network for intercellular transport of nutrients, hormones, regulatory proteins and RNA (Hyun et al., 2011; Maule et al., 2011). As such, PD play a crucial role in plant defense against fungal, bacterial and viral pathogens (Lee and Lu, 2011) and in development by establishing symplastic domains (Lucas et al., 2009; Burch-Smith et al., 2011).

22 Introduction

2.4.2 Callose and its deposition at plasmodesmata

Since PD are important for many physiological, developmental and stress responses, regulation of PD permeability is a vital factor in the plant’s performance. One of the major mechanisms of this regulation is decreasing or increasing the SEL of PD. The SEL is highly variable and actively modified by the plant by deposition and degradation of callose at the neck regions of PD (Rinne et al., 2001). Callose is a polysaccharide composed of β-1,3-linked glucose units and has been shown to be involved in may biological processes in the plant (Chen and Kim, 2009). Its turnover is controlled by an interplay of callose synthases, enzymes mediating callose biosynthesis, and β-1,3- glucanases (B13G), degrading enzymes that hydrolyse callose. In Arabidopsis, both types of enzymes are encoded in gene families with specific members responsible for callose synthesis and degradation at PD and are subject to transcriptional and post- translational regulation (Zavaliev et al., 2011).

2.4.3 Plasmodesmata and dormancy

Symplasmic isolation of meristems through closure of PD has been shown to be involved in many developmental processes in which meristems undergo reorganization, such as the transition from vegetative growth to flowering in the apex of Arabidopsis. Here, the symplasmic trafficking of fluorescent tracers ceased in the shoot apical meristem prior to the onset of flowering and resumed after inflorescences had formed (Gisel et al., 1999). In perennial plants, shoot apices are transformed into dormant, freezing-tolerant buds for overwintering. This process requires shutdown of all symplasmic pathways through massive callose depostition at PD. Callose needs to be removed in order to re-establish symplastic connections and resume meristem activity in spring (Rinne et al., 2001). This callose removal is most likely achieved by degradation mediated by β-1,3-glucanases, as has been shown in poplar recently (Rinne et al., 2011). A similar role of callose deposition and restriction of PD traffic during dormancy periods has also been shown for other perennial trees and for seed dormancy (Leubner-Metzger, 2003). Interestingly, it has been shown for demonstrated that tuber bud meristems are also symplasmically isolated from the surrounding parenchyma during dormancy and it was postulated that this isolation needs to be removed prior to onset of sprouting (Viola et al., 2007).

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2.5 Aims of this thesis

This thesis aimed at identifying regulatory mechanisms of potato tuber dormancy and sprouting. Main focus herein lay on the roles of the phytohormone CK and callose- degrading beta-1,3-glucanases (B13G) which control PD SEL.

In order to investigate the role of CKs, transgenic plants over-expressing key genes of CK biosynthesis and CK degradation were characterised. The sprouting behaviour of their tubers was assessed in an in vitro assay and transcriptional differences to the wild- type were studied by microarray analysis.

Beside this, the identification of genes of CK biosynthesis and signalling in the potato genome was an important issue. To this end, a toolbox using alignments with potato EST sequences, known sequences of closely related species like tomato or tobacco and protein sequences of Arabidopsis thaliana was developed and employed.

The third part of this thesis strove at identifying B13G genes which may play a role in the regulation of bud breakage. To this end, members of the B13G gene family were identified in the potato genome using bioinformatic tools. Available microarray data was analysed to select candidate genes possibly involved in dormancy termination. Suitable candidates were subsequently cloned and their localisation examined by transient expression in Nicotiana benthamiana. For exploration of their influence on tuber dormancy, the candidates were down-regulated in an RNAi approach and an ortholog from Arabidopsis thaliana was overexpressed in transgenic potato plants. Tubers of these transgenic plants were then analysed for changes in sprouting behaviour.

24 Material and Methods

3 Material and Methods 3.1 Chemicals, enzymes and consumables

Unless stated otherwise, chemicals, enzymes and consumables were purchased from Carl Roth GmbH (Karlsruhe), Sigma-Aldrich (St. Louis, USA), Fermentas (St. Leon-Rot), Roche Diagnostics GmbH (Mannheim) und VWR (Darmstadt). Materials and soil for plant cultivation were procured from Bayerische Gärtnereigenossenschaft e.G. Nürnberg. Kits for clean-up of plasmids, PCR products and DNA fragments were obtained from Qiagen (Hilden) and radioactive chemicals were bought from Hartmann Analytic (Braunschweig).

3.2 Bacterial strains

The following bacterial strains were used for general cloning procedures and plant transformation.

Table 1: Bacterial strains used in this thesis

Strain Genotype Origin/Reference

E. coli XL1 Blue recA1 endA1 gyrA96 thi-1 hsdR17 Bullock et al. (1987) supE44 relA1 lac [F´proAB lacIqZΔ M15 Tn10 (TetR)] E. coli TOP10F´ F-(TetR) mcrA D(mrr-hsdRMS-mcrBC) Invitrogen f80lacZDM15 DlacX74 deoR recA1 araD139 D(ara-leu)7697 galU galK rspL (StrR) end A1 nupG Agrobacterium RifR; with helper plasmid pGV2260 AmpR van Larebeke et al. tumefaciens (Deblaere et al., 1985) (1974) C58C1

3.3 Vectors

Vectors used for general cloning procedures and plant transformation are listed in the following table.

Table 2: Vectors used in this thesis

Name Usage and Resistance Marker Origin/Reference pBinAR binary vektor, KanR Höfgen and Willmitzer (1990) UFO-BinAR binary vektor, KanR PhD thesis M. Senning (2010) pK7GWIWG2(II) binary vektor, SmR/ SpR Invitrogen (Karlsbad, USA) pK7FWG2 binary vektor, SmR/ SpR Invitrogen (Karlsbad, USA)

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pENTR/ D-TOPO® E. coli cloning vector, KanR Invitrogen (Karlsbad, USA) pCR-Blunt E. coli cloning vector, AmpR Invitrogen (Karlsbad, USA) pGEM-T® Easy E. coli cloning vector, AmpR Promega (Madison, USA)

3.4 Oligonucleotides and sequencing

PCR Primers used in this thesis were obtained from Metabion (Martinsried) and are listed in table 3. Primers for quantitative real-time PCR analysis were deduced with the aid of Primer3Plus software (Untergasser et al., 2007) which is available online (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi). Plasmid sequencing was done by GATC Biotech AG (Konstanz).

Table 3: Primers used in this thesis Restriction sites and other artificial sequences added to the primer are given in capital letters. No. Name Target gene Sequence

AH5 IPT 5’ Asp718 IPT GGTACCatggacctgcatctaattttc AH6 IPT 3’ SalI IPT GTCGACctaatacattccgaacgg AH7 AtCKX1 5’ BamHI AtCKX1 GGATCCatgggattgacctcatccttacgg AH8 AtCKX1 3’ SalI AtCKX1 GTCGACttatacagttctaggtttcggcag AH85 M.2181.C1 BamHI 5’ StPDB13G_2 GGATCCatgatggaacccaatcccaatcc AH86 M.2181.C1 SalI 3’ StPDB13G_2 GTCGACttacaagtggtagtagttgtcaag AH87 M.1430.C2 EcoRI 5’ StPDB13G_1 GAATTCatggattccatcattcatatcaactac AH88 M.1430.C2 SalI 3’ StPDB13G_1 GTCGACttacagttgagaaaatagcagtaaaattg AH93 AtBG_PAPP 5’ GW AtBG_PAP CACCatggcttcttcttctctgcagtc AH97 M.2181.C1 GW 5’ StPDB13G_2 CACCatgatggaacccaatcccaatcc AH98 M.1430.C2 GW 5’ StPDB13G_1 CACCatggattccatcattcatatcaactac AH99 PD-B13G_RNAi 5’ StPDB13G_1/2 CACCctccggagaaagtagttccattag AH100 PD-B13G_RNAi 3’ StPDB13G_1/2 ggtacgaagtctcgagaatggc AH126 M.7538.C2 3’ RACE StB13G_01 ctcaagtggggtttaatggaaactaac AH127 M.6397.C1 3’ RACE StB13G_02 atggcaagaggatttagtatcatatttg AH132 M.2181.C1 3’ w/o stop StPDB13G_2 caagtggtagtagttgtcaag AH133 M.1430.C2 3’ w/o stop StPDB13G_1 cagttgagaaaatagcagtaaaattg AH134 AtBG_PAP 3’ w/o stop AtBG_PAP caacggaagcttgatgatgcaaag AH141 bf_susp55c06 qPCR 5’ StB13G_07 cggaaaccaccactcaaga AH142 bf_susp55c06 qPCR 3’ StB13G_07 tcacaaaaaccccacaatca AH143 POAD094TV qPCR 5’ StB13G_03 ggtccagcatcagagagga AH144 POAD094TV qPCR 3’ StB13G_03 cagcgttcaaaagtgtatcaaga

26 Material and Methods

AH145 STMHT66TV qPCR 5’ B13G_04 tcaggaaaacgaccagcaa AH146 STMHT66TV qPCR 3’ StB13G_04 cacattgacagggttagttgt AH147 STMHC29TV qPCR 5’ StB13G_01 ggtttggattgggcttgc AH148 STMHC29TV qPCR 3’ StB13G_01 tggattagttgtggtgattgt AH149 MICRO.1763.C2 qPCR 5’ StB13G_08 tgaagtggagatgtggttgttt AH150 MICRO.1763.C2 qPCR 3’ StB13G_08 cagagatggtgagtagggtagca AH155 MICRO.6397.C1 qPCR 5’ StB13G_02 gacttgctggcgttttctc AH156 MICRO.6397.C1 qPCR 3’ StB13G_02 gtatttggggcttccgtga AH157 MICRO.1592.C2 qPCR 5’ StB13G_05 cgtcttcctcttcgctcttt AH158 MICRO.1592.C2 qPCR 3’ StB13G_05 acttttcgctcattcgggta AH159 SSBN003C06u qPCR 5’ StB13G_06 gacttgctggcgttttctc AH160 SSBN003C06u qPCR 3’ StB13G_06 gtatttggggcttccgtga AH309 AtBG_PAP OE 5’ Acc65I AtBG_PAP GGTACCatggcttcttcttctctgcagtc AH310 AtBG_PAP OE 3’ BamHI AtBG_PAP GGATCCttacaaccgaagcttgatgatgca AH331 M1430C2 qPCR 5’ StPDB13G_1 tgcggttcactctgctttag AH332 M1430C2 qPCR 3’ StPDB13G_1 tcatcggcgtctcctttc AH251 M2181C1 qPCR 5’ StPDB13G_2 ttcctatccgccatctgc AH252 M2181C1 qPCR 3’ StPDB13G_2 agcacgaaatcaagggagac MS56 5’ St-ACT PoAc97 gene gtctgtgacaatggaac (X55751) for actin MS57 3’ St-ACT PoAc97 gene gtgaggatcttcatc (X55751) for actin MS201 5´-ubi3 ubi3 (L22576) ttccgacaccatcgacaatgt (Kloosterman et al., 2005) MS202 3´-ubi3 ubi3 (L22576) cgaccatcctcaagctgctt (Kloosterman et al., 2005)

3.5 Growth and transformation of bacteria

E. coli bacteria were cultured at 37° C in liquid or on solid LB medium containing appropriate antibiotics. Agrobacterium tumefaciens strains were grown at 28° C in liquid or on solid YEB medium supplemented with suitable antibiotics. The following antibiotic concentrations were employed for bacteria selection: ampicillin: 100 μg/ ml, kanamycin: 50 μg/ ml, rifampicin: 50 μg/ ml, spectinomycin: 100 μg/ ml, streptomycin: 40 μg/ ml. For blue/ white screening, IPTG and X-Gal were added to a final concentration of 40 µM (IPTG) and 40 µg/ ml (X-Gal). All antibiotics and additives were dissolved in water and sterile-filtered, except rifampicin and X-Gal which were dissolved

27

in dimethyl sulfoxide (DMSO) and dimethylformamide (DMF), respectively. Media were autoclaved and antibiotics and other additives were added thereafter.

Transformation of E.coli via heat shock followed standard or, for purchased competent cells, manufacturer’s protocols. Positive transformands were selected by adding the appropriate antibiotic to the medium. Transformation of Agrobacterium tumefaciens was carried out according to protocol by Höfgen and Willmitzer (1990).

3.6 Plant cultivation

3.6.1 Cultivation of Solanum tuberosum cv. Solara

Potato plants (Solanum tuberosum L. cultivar Solara) were obtained from Bioplant (Ebstorf) and cultivated in tissue culture at 21° C and 50 % humidity on Murashige Skoog (MS) medium supplemented with 2% (w/v) sucrose, appropriate phytohormones and antibiotics under a 16 h light/ 8 h dark regime (Murashige and Skoog, 1962). For tuber production, plants were transferred to individual pots (Ø 20 cm, 15.5 cm height) in the greenhouse and grown at 21°C during a 16 h light phase with additional illumination (250 to 300 mmol quanta m-2 s-1) and at 18°C during 8 h of darkness. Relative humidity was 50% at all times. About three months after transfer to the greenhouse, when leaf senescence began, watering of the plants was stopped and tubers were harvested three to five days later. Subsequently, tubers were stored in darkness at room temperature (RT) until sampling or until sprouting commenced.

3.6.2 Cultivation of Nicotiana benthamiana

Tobacco plants (Nicotiana benthamiana) for microscopic analyses were obtained from Vereinigte Saatzuchten EG (Ebstorf) and grown in the greenhouse at 25° C during a 16 h light phase with additional illumination (250 - 300 mmol quanta m-2 s-1) and at 20° C during 8 h of darkness. Relative humidity was 40 % at all times.

3.7 Plant transformation

3.7.1 Stable transformation of Solanum tuberosum

Plant transformation was carried out using Agrobacterium tumefaciens-mediated gene transfer. The Agrobacterium strain C58C1 carrying helper plasmid pGV2260 (Deblaere et al., 1985) was utilised and transformed with binary vector constructs according to protocol by Höfgen and Willmitzer (1990). Transformation of potato plants was performed according to protocol by Rocha-Sosa et al. (1989).

28 Material and Methods

3.7.2 Transient transformation of Nicotiana benthamiana

For transient plant transformation, sterile-filtered 2-(N-morpholino)ethanesulfonic acid (MES) pH 5.6 and acetosyringone, dissolved in DMSO, were added to 50 ml cultures of transformed agrobacteria to final concentrations of 10 mM (MES) and 20 µM (acetosyringone). Bacteria were grown over night at 28° C and cells were then harvested by centrifugation at 4000 rpm for 15 minutes. After washing with sterile water, the pellet was dissolved in buffer containing 10 mM MgCl2, 10 mM MES pH 5.6 and 100 µM acetosyringone until the suspension had an optical density (oD600) of 0.8 to 1.0. After 2-3 h incubation at RT, bacteria were infiltrated into the lower side of growing leaves using a blunt-ended syringe.

3.8 Sprout Release Assay

The sprout release assay (SRA) is an in vitro sprouting experimental assay using excised potato tuber discs containing a single bud. Discs of 5 mm height were excised using a korkbohrer size 4 (Ø 8 mm) and subsequently washed three times for 15 min in sterile- filtered buffer containing 20mM MES, 300mM D-mannitol, and 5 mM ascorbic acid, pH

6.5. Discs were then incubated with 50 µM GA3, 50 µM 6-benyzlaminopurine (BAP), or sterile water for 5 min and placed in petri dishes lined with moist filter paper. Petri dishes were sealed with Leukopor tape (BSN medical, Hamburg) and stored in darkness under tissue culture conditions. The filter paper was regularly moistened by adding sterile water. Sprouting behaviour of tuber discs was scored daily. For sample taking at specific time points, four to eight discs were removed from the petri dish, excess parenchyma was removed by excising the bud with a size 2 korkbohrer (Ø 4 mm) and cutting off the lower part of the disc. Samples were immediately frozen in liquid nitrogen and stored at -80° C until further usage.

3.9 Microscopy and histological methods

Images of tuber disc cross sections were captured with a Leica DFC480 digital camera on a Leica MZ16F stereomicroscope. Images were processed with Adobe Photoshop software. For confocal imaging, leaf sections were applied to microscope slides using double-sided adhesive tape and covered with water immediately. If necessary, counterstaining of cell walls with propidium iodine (PI) was carried out (see 3.9.2 for procedure). Confocal analysis was performed on a Leica TCS SP2 confocal laser scanning microscope (Leica, Bensheim) using a 488 nm 20 mW argon laser for excitation of fluorescence. GFP

29

fluorescence was detected between 497 and 526 nm, PI fluorescence was detected between 598 and 650 nm and autofluorescence was detected between 682 and 730 nm. Confocal images were processed using Leica Confocal Software 2.5 and Adobe Photoshop CS3 software.

3.9.1 Embedding of plant tissues

For very thin cross sections, tuber tissue was embedded in wax. Samples were first infiltrated 3 x 10 min with 4 % (w/v) paraformaldehyde in 1x PEM buffer (0.1 M PIPES,

1mM EGTA, 1 mM MgSO4), then dehydrated in an ethanol series (1h 70% EtOH, 1 h 80% EtOH, 2 h 90% EtOH, 2 x 1 h 100%, 1 x 2 h 100%) and cleared twice in 100 % xylol for 1 h before embedding in Paramat Extra wax. Except infiltration, all embedding steps were performed using a Leica TP 1020 tissue processor. Using Leica embedding station EG 1150, wax-infiltrated samples were placed in standardized biopsy cassettes to produce paraffin blocks for microtome sectioning. Samples were cut into sections of 8 to 12 µm thickness using a Leica rotary microtome RM2265. Sections were allowed to stretch in a 50° C water bath, moved onto polylysin-coated glass slides and dried at 55° C over night. Wax was removed from the sections by histoclear treatment (2 x 30 min) and tissue was rehydrated in an ethanol series (2 mins each of 100% EtOH, 95% EtOH, 85% EtOH, 70% EtOH and 40% EtOH) and equilibrated in 1 x PBS before being used for staining or immunolocalization studies.

3.9.2 Staining of plant tissues

Counterstaining of cell walls for confocal imaging was performed by immersion of leaf material in a saturated propidium iodine solution for 5-10 minutes. Excess staining solution was removed in five washing steps with water.

For staining of xylem vessels, tissue sections were incubated in 0.1 % phloroglucinol for 40 min and examined under the microscope immediately. Alternatively, sections were incubated first in 10% phloroglucinol in 95% EtOH for 30 min, then in concentrated hydrochloric acid (HCl) for 2-3 min. After a washing step with water, sections were examined under the microscope.

Ruthenium red staining of cell walls was done by incubation of sections in 0.05% aqueous ruthenium red solution for 1 to 2 mins and subsequent washing with 95% ethanol. Sections were dried completely, mounted with DPX mounting medium (Fluka) and examined under the microscope.

30 Material and Methods

3.9.3 Immunolocalization of callose

For immunolocalization of callose, thin cross sections of tuber tissue were prepared as described in 3.9.1. and then incubated in blocking buffer (50 mM TrisHCl pH 7.5, 150 mM NaCl, 1% powdered milk) for 30 minutes. Incubation with the first, callose-specific antibody (Biosupplies) diluted 1:30 in blocking buffer occurred over night at 4°C in a humid chamber. Following eight washing steps with blocking buffer, the slides were incubated with secondary antibody Cy2 (1:300 dilution) for 1 hour at room temperature. After washing steps with blocking buffer (8x) and TBS (1x), detection of Cy2 was carried out under a confocal laser scanning microscope (CLSM).

3.10 Biomolecular methods

3.10.1 Standard cloning procedures

Basic techniques of nucleic acid handling such as amplification of DNA via PCR, DNA cleaving by restriction enzymes, DNA ligation, clean-up of DNA fragments, agarose gel electrophoresis, transfer of nucleic acids from gel onto membranes, plasmid preparation and radioactive labelling of DNA fragments to be used as hybridisation probes was carried out according to manufacturer’s instructions or standard protocols described in ‘Molecular Cloning’ (Sambrook et al., 1989).

3.10.2 Isolation of plant genomic DNA

For isolation of genomic DNA, about 6 g of leaf material were homogenised in liquid nitrogen and, still frozen, transferred to a 50 ml tube for suspending in 15 ml DNA extraction buffer (500 mM NaCl, 100 mM Tris-HCl pH 8, 50 mM EDTA, 10 mM β- mercaptoethanol). After 1 min, 1 ml of 20% SDS was added and the suspension was incubated at 65°C for 10 min. Following addition of 5 ml 5 M potassium acetate, thorough mixing and incubation on ice for 30 min, the suspension was centrifuged at 12000 rpm for 30 min. The supernatant was filtered through two layers of Miracloth (Calbiochem) and DNA was precipitated by addition of 10 ml isopropanol. After incubation at -20°C for 20 min, DNA was pelleted by centrifugation at 10000 rpm for 10 min. The pellet was dried at RT and resuspended in 700 µl buffer (50 mM Tris-HCl pH 8; 10 mM EDTA). Subsequently, 75 µl 3 M sodium acetate were added and the suspension was centrifuged at 13000 rpm for 15 min. DNA in the resulting supernatant was precipitated with 500 µl isopropanol and incubation at RT for 5 min. DNA was pelleted by centrifugation at 13000 rpm for 10 min. The pellet was dried and resuspended in 200 µl TE buffer (10 mM Tris- HCl pH 8; 1 mM EDTA).

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3.10.3 Isolation of RNA and Northern Blot

RNA extraction from plant tissues essentially followed the protocol of Logemann et al. (1987). RNA concentrations were determined using the ND-1000 spectrophotometer (NanoDrop Technologies). After a denaturing step, 20 to 30 µg of total RNA were separated on a 1. % (w/v) formaldehyde agarose gel and then transferred onto a nylon membrane (GeneScreen, NEN, Boston, USA) over night. RNA was crosslinked to the membrane by UV light and stored at RT until the hybridisation. For Norhern Blot hybridisation, the membrane was prehybridised in CHURCH buffer (Church and Gilbert, 1984) for 2 hours at 65°C and DNA fragments for detection of the desired mRNA were labelled radioactively using 40 µCi [α32P]dCTP and High Prime Mix (Roche) according to manufacturer’s instructions. Probes were denatured at 95°C for 2 min and added to the CHURCH buffer for hybridisation over night at 65°C. After washing with 6x SSC buffer with 0.2% SDS to remove unspecifically bound probe, signals were detected by exposing the membrane to radiographic film (Kodak). All solutions used for RNA handling were made using DEPC-treated water.

3.10.4 DNAse treatment of RNA and cDNA synthesis

In order to remove DNA contamination from extracted RNA, total RNA was subjected to DNAseI (Fermentas) treatment according to manufacturer’s instructions. DNAseI-treated RNA was transcribed into cDNA using RevertAid™ H Minus M-MuLV Reverse Transkriptase (Fermentas) following manufacturer’s protocols. Unless stated otherwise, oligo(dT)30 was used as primer for cDNA synthesis.

3.10.5 Quantitative real-time PCR

For quantitative detection of transcripts, real-time PCR analyses were conducted on a Mx3000P qPCR system (Stratagene) using Brilliant II SYBR Green QPCR master mix (Stratagene). cDNA was diluted 1:10 in RNAse-free water and used as template. Expression values were normalised to expression of ubiquitin3 as an internal control (primers no. MS201 und MS202; Kloosterman et al., 2005). Temperature cycle was as follows: 10 min 95°C followed by 45 cycles of 20 s 95°C, 20 s 60°C and 20 s 72°C. Primer efficiency was tested beforehand using a serial dilution of cDNA. Relative expression of transcripts was calculated by MxPro v4.10 software (Stratagene) considering previously determined primer efficiencies.

32 Material and Methods

3.10.6 Microarray hybridisation and scanning

For transcript profiling, total RNA was isolated as described above and purified using RNeasy Mini Spin Columns (Qiagen) following the manufacturer’s protocol. RNA quantity was measured with the ND-100 spectrophotometer v3.3.0 (NanoDrop Technologies). RNA integrity was verified using an Agilent RNA 6000 Nano Chip on an Agilent 2100 BioAnalyzer (version B.02.03 BSI307) as recommended by the manufacturer’s protocol (Agilent RNA 6000 Nano Assay Protocol2). Sample labelling and preparation for microarray hybridization were performed as described in the one-colour microarray-based gene expression analysis protocol provided by Agilent including the one-colour RNA spike-in kit (Agilent Technologies). Slides were scanned on the Agilent Microarray Scanner with extended dynamic range at high resolution. Data sets were extracted by the feature extraction software package (version 9.5.3.1; Agilent Technologies) using a standard protocol.

3.10.7 Microarray data analysis

For data analysis, text files generated by the feature extraction software were imported into GeneSpring GX 11.5.1 (Silicon Genetics). Data was normalized per chip (normalization to the 75th percentile) and per gene (the signal of each feature was normalized to the median of its value across the data set). Features passing the quality check (flags present) and showing changes in expression levels equal or more than 2- fold were selected for further analysis. A volcano plot was applied to identify statistically significant (P ≤ 0.05; equal variances assumed), more than 2-fold differentially expressed genes between two conditions including the Benjamini-Hochberg multiple test correction (Benjamini and Hochberg, 1995). Annotations were taken from the POCI online tool (http://pgrc.ipk-gatersleben.de/poci). Functional assignment was done based on annotations, Gene Ontology terms, or by homology search against the Arabidopsis genome. Heat maps for selected pathways were generated using the MAPMAN tool (https://www.gabipd.org/projects/MapMan/).

3.10.8 Protein Extraction and Western Blot For protein detection by Western Blot, total protein was extracted from WT and transgenic potato leaf discs (2x Ø 5 mm) homogenized in 100 µl 2x LÄMMLI buffer. Samples were separated on 12,5% (v/v) SDS-PAGE and blotted onto nitrocellulose membranes (Macherey-Nagel, Düren, Germany). Membranes were blocked for 2 h in 5% skimmed milk/TBST (20 mM Tris, pH 7; 500 mM NaCl; and 0.1% Tween 20), and incubated overnight at 4°C with mouse antibody against GFP (diluted 1:1000 in 1%

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skimmed milk/TBST; Roche Diagnostics, Mannheim). ECL immunodetection using rabbit anti-mouse antibodies (1:20000 diluted in 1% skimmed milk/ TBST; Sigma, Germany) was performed according to standard procedures.

3.11 Bioinformatic analysis

All bioinformatic analyses, editing and manipulation of DNA, RNA and protein sequences, pairwise and multiple alignments, phylogenetic tree building and assembly of DNA sequences were carried out using the Geneious Pro 5.4.6 software (Drummond et al., 2010). If not stated otherwise, pairwise and multiple DNA alignments were done using the ‘global alignment with free end gaps’ alignment type and ‘93% similarity (5.0/-9.026168)’ cost matrix for potato sequences or ‘65% similarity (5.0/-4.0)’ cost matrix for alignments involving different species, e.g. potato and Arabidopsis. Phylogenetic trees were built using default settings of the Geneious Tree Builder Tool within Geneious Pro 5.4.6. These settings were: ‘global alignment with free end gaps’, ‘Jukes-Cantor’ genetic distance model, ‘neighbour-joining’ tree build method, no outgroup and, as cost matrix, ‘93% similarity (5.0/-9.026168)’ for DNA sequences or ‘BLOSUM62’ for protein sequences.

34 Results

4 Results 4.1 Alteration of endogenous cytokinin content in transgenic potato plants

Both, reports on the sprout-inducing activity of CK when applied to dormant tubers (Hemberg, 1970) and measurements of endogenous CK levels (Turnbull and Hanke, 1985b; Sukhova et al., 1993) suggested an involvement of the phytohormone in controlling dormancy termination and sprouting. CK biosynthesis is a multistep process starting with the transfer of an isoprenoid moiety to adenine catalyzed by isopentenyltransferases (IPT) and followed by dephosphorylation, phosphoribohydrolysis and/or hydroxylation (comp. Figure 1). Bioactive CKs, namely adenine nucleosides and adenine nucleobases, are inactivated through irreversible degradation by cytokinin oxidases/ dehydrogenases (CKX). IPT as well as CKX play major roles in the regulation of CK homeostasis, the former as the rate-limiting enzyme of CK biosynthesis (Morris, 1995), the latter as the hormone’s key inactivating enzyme (Werner et al., 2001), rendering them suitable targets for manipulation of CK biosynthesis in plants. In order to determine the effect of an elevated or lowered CK content on plant development and tuber sprouting of potato, transgenic potato plants carrying either the Agrobacterium tumefaciens IPT gene or the Arabidopsis thaliana CKX1 gene (AtCKX1) under control of the CaMV 35S promoter were generated. Constructs in which the transgenes were under control of the Arabidopsis thaliana UFO promoter (UNUSUAL FLORAL ORGANS) were also generated and stably transformed in potato. The UFO transcript encodes an F-Box protein and is expressed in ‘a subset of cells in all meristems’ (Samach et al., 1999). Thus, expression of either transgene under the 3.7 kb long UFO promoter allows analysis of localized, meristem-specific changes of CK content. Transgenic potato plants expressing IPT or AtCKX1 driven by either CaMV 35S or UFO promoter were analyzed in detail in the present thesis.

4.1.1 Constitutive overexpression of bacterial IPT and AtCKX1 in potato

For constitutive overexpression of IPT (accession no. AF242881) and AtCKX1 (TAIR ID AT2G41510; accession no. NM_129714), both genes were amplified by PCR, from either a pUC9 vector carrying a subclone of the Agrobacterium tumefaciens Ti plasmid pTi5955 (kindly provided by Prof. T. Schmülling, FU Berlin) or Arabidopsis thaliana leaf cDNA, and cloned into the BinAR vector (Höfgen and Willmitzer, 1990) containing the CaMV 35

35S promoter and the octopine synthase polyadenylation signal. The binary constructs (Fig. 3) were stably transformed in potato and transformants were analysed.

EcoRI Acc65I BamHI EcoRV SalI HindIII EcoRI BamHI NcoI EcoRI XbaI SalI HindIII

CaMV 35S IPT OCS CaMV 35S AtCKX1 OCS 200 bp 200 bp

Figure 3: Schematic representation of IPT and AtCKX1 binary constructs Full-length coding sequences of bacterial IPT and Arabidopsis thaliana AtCKX1 were amplified by PCR and cloned into binary vector pBinAR via Acc65I/SalI or BamHI/SalI restriction sites, respectively, to create the 35:IPT and 35S:CKX1 constructs.

4.1.1.1 Phenotypic characterization of 35S:IPT and 35S:CKX1 overexpressing potato plants

Out of two rounds of transformation of the 35S:IPT construct, 15 transgenic plants could be regenerated. Expression of the transgene was verified by RT-PCR (Fig. 4A) and four positive lines (IPT-6 to IPT-9) were selected for further analysis. Transformation of the 35S:AtCKX1 construct yielded 12 transgenic plants, six of which expressed the transgene (CKX1-2, CKX1-4, CKX1-6 and CKX1-9 to CKX1-11), as confirmed by Northern Blot analysis. Expression levels varied and three lines with different expression levels, CKX1-4, CKX1-10 and CKX1-11 (Fig. 4B), were chosen for further analysis.

Of the four positive IPT-overexpressing lines, three already showed a characteristic CK- responsive phenotype in tissue culture, with bushy growth, small leaflets and inhibited root growth (Fig. 4C). Two of these lines did not survive transfer to soil. The third one, IPT-9, displayed strongly reduced growth, a loss of apical dominance and smaller leaf size (Fig. 4C). Another positive line, IPT-6, in which the transgene was less strongly expressed, resembled wild-type (WT) plants concerning growth and tuber yield, but was unable to form flowers (Fig.4E). Tuber yield did not significantly differ from WT in line IPT-6, but in the stronger expressing line IPT-9, tuber yield was reduced by 94% (Table 4), because about 30% of the plants did not produce tubers at all and tubers formed remained very small.

The three transgenic CKX1-expressing lines showed a reduced shoot growth, and the degree of retardation in final shoot size corresponded with the expression of the transgene (Fig. 4, B and D; Table 4). Moreover, the plants produced a high number of fragile side shoots and had a lower number of leaflets per leaf. The strongest line, CKX1- 4, formed only single lanceolate leaves instead of the typical compound leaves (Fig. 4F). With increasing CKX1 expression, tuber yield was reduced severely and only a few,

36 Results small, drop-shaped tubers were formed (Table 4; Fig. 4F). Additionally, stolon length was severely reduced in CKX1-4 and plants occasionally formed aerial tubers with small leaflets.

A IPT B CKX1 WT 6 7 8 9 C WT 4 10 11

IPT At CKX1

Ubi RbcS

C IPT D CKX1

WT WT 4 10 11

E WT IPT-6 F WT CKX1 WT CKX1-4

10 11 4 Figure 4: Constitutive overexpression of IPT and AtCKX1 in potato plants (A) RT-PCR analysis of IPT-expressing potato plants (lines 6, 7, 8, and 9). Five micrograms of total RNA isolated from leaves was reverse transcribed using oligo(dT) primers and RevertAid H2 reverse transcriptase. An aliquot of first-strand cDNA was amplified with gene-specific primers for IPT or ubi3. (B) Northern Blot analysis of CKX1-expressing (lines 4, 10, and 11) potato plants. Thirty micrograms of total RNA isolated from leaves was loaded per lane and probed with AtCKX1. Hybridization with the small subunit of Rubisco (RbcS) was used as a loading control. (C) Phenotypic alteration of transgenic 35S:IPT potato plants. From left to right: wild-type (WT) and IPT lines 6, 7, 8, and 9. Photographs were taken 8 weeks after transfer to the greenhouse (WT and IPT lines 6 and 9) and from plants propagated in tissue culture (IPT lines 7 and 8) six weeks after cutting. (D) Phenotypic alteration of transgenic 35S:CKX1 potato plants approximately 8 weeks after transfer into the greenhouse. From left to right: two wild-type plants (WT) and CKX1 lines 4, 10, and 11. (E) Low expression of IPT in line IPT-6 impairs flower development. (F) Effect of CKX1 expression on leaf and tuber morphology.

37

Table 4: Phenotypic characteristics of transgenic potato plants expressing either the CKX1 gene from Arabidopsis or an IPT gene from Agrobacterium under control of the CaMV35S promoter Parameters were determined from soil-grown plants at harvest. Results are shown for three independent experiments and represent means ± SE of 25 to 40 plants. Statistically significant differences from the WT were determined using one-tailed t tests assuming unequal variance and are indicated by asterisks (P≤ 0.05).

Plant Stem Height Tuber Yield No. of Tubers per Plant

cm g plant -1

Wild-type (Solara) 56.7 ± 2.0 176.7 ± 10.4 7.6 ± 0.4

CKX1 CKX1-4 39.2 ± 0.8* 20.0 ± 0.7* 6.5 ± 0.6 CKX1-10 46.5 ± 1.2* 136.1 ± 5.1 3.9 ± 0.4* CKX1-11 55.3 ± 1.3 168.0 ± 2.7 5.8 ± 0.3*

IPT IPT-6 54.2 ± 1.1 170.2 ± 3.9 7.2 ± 0.2 IPT-9 11.1 ± 0.9* 10.7 ± 3.4* 2.8 ± 0.6*

4.1.1.2 Natural and phytohormone-induced sprouting of IPT-6 and 35S:CKX1- overexpressing tubers

In order to find out if constitutive overexpression of IPT or AtCKX1 in potato affect tuber dormancy and sprouting, tubers of the selected CKX1 lines and of IPT-6 were stored at room temperature after harvest and sprouting was scored regularly over the course of several months (Fig. 5). Tubers formed by line IPT-9 were too few and too small to be included in this analysis. A B

weeks after harvest weeks after harvest Figure 5: Natural sprouting of IPT- and AtCKX1-expressing transgenic tubers (A) Sprouting behavior of IPT-expressing line 6 compared to wild-type. Tubers were stored at room temperature in darkness and monitored for 14 weeks after harvest until 100% sprouting was reached. The graph shows the mean values ± SD of two independent harvests (n=25-36 tubers). (B) Sprouting behaviour of AtCKX1-expressing lines monitored for 24 weeks after harvest until 100% sprouting was reached. Tubers were stored at room temperature in darkness (n=25-48 tubers). Diamonds – wild-type; triangles – CKX1-11; closed circles – CKX1-10 ; squares – CKX1-4.

38 Results

For up to five weeks after harvest, tubers of both WT and transgenic lines were completely dormant. WT tubers started sprouting approximately eight to nine weeks after harvest and around twelve to thirteen weeks after harvest, more than 90% of tubers had visibly sprouted. Sprouting of IPT-6 tubers set in at six weeks after harvest and the percentage of sprouted tubers remained a little higher than in the WT until 100% sprouting was reached (Fig. 5A), but the difference was not statistically significant. Onset of sprouting of stored CKX1 tubers was visible after fourteen to seventeen weeks after harvest which corresponds to a delay of five to eight weeks compared with the wild-type. Moreover, the length of this delay is equivalent to CKX1 transcript abundance (Fig. 4B, 5B). Phenotypically, sprouts formed by CKX1-10 and CKX1-11 did not differ from the WT and developed normally after bud breakage. IPT-6 and CKX1-4, however, were dissimilar: IPT-6 tubers formed thicker and longer sprouts, as highlighted by cross sections through sprouting tuber buds (Fig. 6A). Size differences of both sprout length and sprout diameter were statistically significant (Fig.6B).

A WT IPT-6 B 4,5 WT 4,0 * IPT-6 3,5 Harvest 3,0 * 2,5 WT [mm] 2,0 1,5 1,0 16 weeks 0,5 0,0 sprout length sprout diameter

Figure 6: Phenotype of IPT-expressing transgenic sprouts (A) Tuber cross sections at harvest and after 16 weeks of storage. Bud complexes of freshly harvested tubers and sprouts of 16-week old tubers stored at room temperature were sectioned by hand and imaged on a stereomicroscope. From top left to bottom right: the wild-type at harvest, IPT-6 at harvest, the wild-type at 16 weeks, and IPT-6 at 16 weeks. Black bars = 500 µm, and white bars = 1 mm. (B) Length and diameter of hand-sectioned sprouts of wild-type and IPT line 6 were determined 16 weeks after harvest and the mean value ± SE of five samples calculated. Statistically significant differences from the wild-type were determined using one-tailed t-tests assuming unequal variance and are indicated by asterisks (p ≤ 0.05).

In CKX1-4 tubers, bud break occurred and petite sprouts were formed, but even through long-term storage of up to nine months they remained diminutive and failed to achieve proper growth (Fig. 7 top panel). Additional cross sections through dormant tuber buds revealed that the size of the meristem of CKX1-4 tubers is much smaller than that of wild-type tubers and that the two main vascular bundles were in close proximity to each other (Fig. 7 bottom panel). 39

Figure 7: Phenotype of CKX1-4 transgenic tuber buds and sprouts Upper panel: sprouted tubers of WT and line CKX1-4 imaged at different time points of storage. From left to right: WT at 18 weeks of storage, CKX1-4 at 18 weeks of storage WT CKX1-4 CKX1-4 and CKX1-4 stored for 9 months. Arrowheads indicate sprouts. WT CKX1-4 Lower panel: Cross sections of wild-type and CKX1-4 tubers at harvest. Freshly harvested tubers were hand- sectioned and imaged on a stereomicroscope. Arrowheads indicate vascular strands. The black bar represents 1 mm.

To further investigate whether IPT or AtCKX1 expression leads to altered sprouting, IPT- 6 and CKX1-4 tubers were tested for their sprouting behavior in an in vitro sprout release assay (SRA). In this assay, excised dormant tuber buds were treated with 50 µM GA3 to achieve near-synchronous tuber sprouting. First WT sprouts emerged in 40% to 50% of discs three days after treatment and 100% sprouting was reached within five to seven days after treatment. In contrast, transgenic IPT-6 tubers responded one day earlier to the treatment, as shown in Figure 8. About 25% of tuber discs had already started sprouting after two days, which significantly differed from the WT.

dat

Figure 8: GA3-induced sprouting of WT and IPT-6 In vitro sprout assay of WT and IPT-6 tubers. Tuber discs containing one bud complex were incubated with 50µM

GA3 or sterile water and monitored for visible sprouting. The number of sprouted discs was recorded daily. dat – day(s) after treatment The graph shows the mean values ± SE of two independent experiments (n=20-30). Closed diamonds – wild-type treated with GA3, open diamonds – wild-type treated with water, closed triangles – IPT line 6 treated with GA3, open triangles – IPT line 6 treated with water. * statistically significant differences from wild-type (P ≤ 0.05).

When the assay was performed with WT and CKX1-4 tubers, sprouting could be induced in the WT as before, but CKX1-4 tubers showed no signs of sprouting upon GA3 treatment, even when observed for a longer time (nine days, Fig. 9). To confirm that this 40 Results phenotype is due to a reduced level of CKs, the SRA was repeated with 50 µM BAP (6- benzylaminopurine, a synthetic CK). As seen in Figure 9, BAP treatment of CKX1- expressing tubers restored the WT phenotype.

+MQ +50 µM GA3 + 50 µM BAP 6d 6d 9d 5d WT WT WT

CKX1-4 CKX1-4 CKX1-4 CKX1-4

Figure 9: Phytohormone-induced sprouting of WT and CKX1-4 Cross sections of tuber discs from wild-type (upper panel) and CKX1-4 (lower panel) treated with water or phytohormones, respectively. Samples were hand-sectioned and imaged on a stereo microscope at the indicated days after treatment. The black bar represents 1 mm.

4.1.1.3 Transcriptional analysis of wild-type, IPT-6 and CKX1-4 tubers after GA3- induced sprouting

In order to explore the cause for the altered sprouting phenotype of IPT-6 and CKX1-4 tubers after GA3 application, a transcript analysis was conducted using microarrays. To this end, dormant wild-type, IPT-6 and CKX-4 tuber buds were subjected to a SRA and transcript profiles were obtained from samples taken immediately (‘0d’) and three days after GA3 (‘3d’) treatment using the Agilent 4x44K array (Kloosterman et al., 2008). This microarray compiles a collection of 42034 60mer oligonucleotides representing potato EST sequences from different tissues, treatments and cultivars. Each spot on the chip specifically represents one potato EST sequence. ESTs will also be referred to as ‘entities’ or ‘transcripts’ in the following analysis. Due to a certain level of redundancy among the ESTs, two or more EST sequences might represent the same gene.

During the SRA, wild-type tubers started sprouting three days after GA3 treatment and in about 30% of the samples, little sprouts were already visible, indicating meristem reactivation and the beginning of sprout outgrowth. In contrast to this, 20% of IPT-6 tuber discs had already sprouted after two days and CKX1-4 tuber discs showed no sprouts at all. For sample taking, discs were re-excised with a size 2 korkbohrer (Ø 4 mm), excess

41

parenchyma was removed and four discs were pooled into a single replicate. For each genotype, at least three replicates were hybridized. Data analysis was performed as described in “Materials and Methods” using the GeneSpring GX11.5.1 software. A condition tree based on hierarchical clustering of expression values of all entities (Fig.10) showed that replicate grouping is strongly influenced by phytohormone treatment, with all ‘3d GA’ and all ‘0d’ samples clustering together. Within these clusters, CKX1-4 samples formed an isolated branch whereas IPT-6 samples did not separate clearly from WT samples, probably due to the low expression of the IPT transgene.

IPT WT IPT IPT WT WT WT CKX CKX CKX IPT WT WT WT WT IPT IPT CKX CKX CKX Genotype 0d 3d Treatment

Figure 10: Condition tree based on hierarchical clustering of expression data of WT, IPT-6 and CKX1-4 replicates Hierarchical clustering on samples with ‘Pearson centered’ as distance metric and ‘average’ linkage rule. Coloured boxes at the bottom indicate phenotypes and treatments. Black – wild-type; blue – CKX1-4; orange – IPT-6; green – ‘0d’, samples taken immediately after treatment; red - ‘3d’, samples taken three days after GA3 treatment.

42 Results

In the subsequent analysis, 36037 entities passed the ‘Filter for flags’ because they were marked as ‘present’ in at least 3 out of 20 samples. Filtering for entities expressed between -0.5 and 0.5 removed 1457 entities which were less than two-fold differentially regulated. The remaining 34580 features were subjected to volcano plot analysis in which both expression level and statistical significance are considered. Table 5 lists the numbers of statistically significant entities for ‘3d versus 0d’ comparisons of each genotype which were identified employing an unpaired t-test with unequal variance, p- value ≤ 0.05, fold change ≥ 2.0 and Benjamini-Hochberg (1995) false discovery rate (BH- FDR). These lists were further divided into up- and down-regulated entities (Tab.5), with

‘up-regulated’ meaning that an entity is higher expressed at three days after GA3 treatment than at the ‘0d’ time-point and ‘down-regulated’ designating entities with a lower expression three days after GA3-treatment compared to ‘0d’. Strikingly, the total number of differentially expressed entities in the CKX1-4 ‘3d versus 0d’ comparison is 26.3% higher than in the WT comparison and 17.9% higher than in the IPT comparison.

Table 5: Statistically significant, differentially expressed entities in WT, CKX1-4 and IPT-6 potato tuber buds 3d after GA3 treatment Statistically significant entities with at least two-fold changes in expression between ‘3d’ and ‘0d’ were identified by volcano plot analysis and divided into up- and down-regulated.

Comparison Total no. of entities Up-regulated Down-regulated

WT 3d vs WT 0d 7209 4349 2860

CKX1-4 3d vs CKX1-4 0d 9103 4878 4225

IPT-6 3d vs IPT-6 0d 7720 4331 3389

This increase is mainly due to a high number of down-regulated entities – an increase of 47.7% and 24.7% compared to WT and IPT-6, respectively. This singularity of line CKX1-4 was further illustrated by VENN diagram comparison of the three genotypes (Fig.11). Approximately half of the entities expressed in the ‘CKX1-4 3d versus 0d’ comparison (48.1% of up-regulated and 52.3% of down-regulated entities) are uniquely expressed in this genotype. In contrast, in WT and IPT-6, uniquely expressed entities only amount to about one sixth (WT) and one third (IPT-6) of the total number of regulated transcripts. IPT-6 shares nearly two thirds of its up-regulated and more than half of its down-regulated entities with the WT. Of the entities differentially regulated in CKX1-4, on the other hand, about 43% can also be found up- or down-regulated in the WT, respectively. For exact percentages, see table A1 in the appendix.

43

A Comparison of up-regulated entities B Comparison of down-regulated entities

WT 3d versus 0d CKX1-4 3d versus 0d WT 3d versus 0d CKX1-4 3d versus 0d (4349 entities) (4878 entities) (2860 entities) (4225 entities)

755 624 809 2346 419 2210

1440 1115 1345 337 702 276

1209 1296 (4331 entities) (3389 entities) IPT-6 3d versus 0d IPT-6 3d versus 0d Figure 11: VENN diagrams of up- and downregulated entities of WT, IPT-6 and CKX1-4 Comparison of at least 2-fold up-regulated (A) or down-regulated (B), statistically significant entities identified earlier by volcano plot analysis.

4.1.1.3.1 Functional categorization of differentially expressed genes

For further analysis, differentially expressed entities were categorized into 16 functional groups based on their GO-term assignments. Two additional categories group entities without annotation (‘Unknown’) and entities which could not be classified into any of the other categories (‘Unclassified’). Together, these two categories comprise between 49.6% and 55.6% of all entities in a list (compare table A2 in the appendix) and were therefore left out in the graphical representation of the functional groups shown in Figure 12.

Down-regulated Entities Up-regulated Entities

WT

CKX1-4

IPT-6

Cell Cycle/ Replication/ Chromatin-associated Protein fate Storage protein Transport DNA-/ RNA-associated Cell wall biosynthesis/ modification Signaling Metabolism Transcription/ Translation Cytosceleton Photosynthesis Development/ Senescence Transcription factors Stress/ Defense Phytohormones Electron Transport/ Redox Figure 12: Functional assignment of up- and down-regulated entities of WT, IPT-6 and CKX1-4 Classification of entities found to be differentially and statistically significant in ‘3d versus 0d’ comparisons of WT, IPT-6 and CKX1-4. The colour code for each category is given in the legend below. Data shown represent percentages calculated from the number of entities in a category in relation to the total number of entities in the corresponding list after substracting ‘Unclassified’ and ‘Unknown’ entities. A list of the corresponding numbers can be found in the appendix, table A3.

44 Results

As seen before, IPT-6 deviates only to a certain extend from the WT: For most categories such as cell wall biosynthesis and modification, transcription/ translation or transport, IPT-6 shows similar percentages of entities as the WT. Some categories, however, differed in the transgenic line: A higher number of entities for transcription factors and storage proteins were found to be up-regulated in IPT-6 than in the WT. Among the down-regulated entities, ESTs encoding cell cycle components were less represented in IPT-6 whereas a higher fraction of ESTs for stress/ defense genes were present in the transgenic line than in the WT. Noticeably, percentages of both up- and down-regulated entities encoding metabolism genes also differ from the WT, with less entities up-regulated in IPT-6 and more entities down-regulated in the transgenic line. Exact percentages are listed in table A3 in the appendix.

By contrast, CKX1-4 is more dissimilar to the WT: Among the up-regulated entities, five categories, cell cycle/ replication, cell wall biosynthesis/ modification, cytoskeleton, storage proteins and transport, are represented to a lesser degree in CKX1-4, correlating with the non-sprouting phenotype of this transgenic line. Interestingly, more entities of the transcription/ translation and phytohormones category could be found up-regulated in CKX1-4 than in the WT. Among the down-regulated entities, cell cycle/ replication and transcription/ translation made up a smaller percentage in the CK-deficient line, whereas more entities representing transcription factors, protein fate, cell wall biosynthesis/ modification, cytoskeleton, storage proteins, signalling, phytohormones and transport proteins were down-regulated in CKX1-4 than in the WT (see table A3 in the appendix for exact numbers).

As the previous in vitro and natural sprouting experiments had shown, phytohormones have a strong effect on dormancy and sprouting of potato tubers. Therefore this functional category was further divided into sub-categories for each of the nine phytohormone classes. Figure 13 shows the functional assignment of differentially regulated, phytohormone-related entities to the different phytohormone classes. No entities representing strigolactone signalling or metabolism are statistically significant, differentially expressed in any of the genotypes. Entities corresponding to salicylate and jasmonate remain largely unchanged between the three genotypes. Among the up- regulated entities, line CKX1-4 is characterized by a high number of entities in the ethylene-, cytokinin- and brassinosteroid-related subcategories and a decrease in the auxin- and gibberellin-related groups. In comparison to the WT, down-regulated entities of CKX1-4 are distinguished by a decrease in the CK-related and an increase in ethylene-related subcategory. In transgenic line IPT-6, on the other hand, similar

45

differences to the WT can be observed, but increases and decreases are less pronounced than in CKX1-4. Strikingly, IPT-6 shows a marked increase of the subcategories ‘ethylene-related’ and ‘brassinosteroid-related’ when comparing its down- regulated entities to the WT.

Down-regulated Entities Up-regulated Entities 100% 80% 60% 40% 20% 0% 0% 20% 40% 60% 80% 100% 100 90 80 70 60 50 40 30 20 10 0 0 10 20 30 40 50 60 70 80 90 100

WT WT WT

CKX1-4CKX1-4CKX1-4

IPT-6 IPT-6 IPT-6

Auxin ABA Brassinosteroids

Ethylene Jasmonate Salicylate

Gibberellin Cytokinin

Figure 13: Functional allocation of phytohormone-related entities of WT, IPT-6 and CKX1-4 Classification of entities from the ‘phytohormone-related’ category into nine sub-categories. The colour code for each category is given in the legend below. Data shown represent percentages calculated from the number of entities in a subcategory in relation to the total number of entities in the ‘phytohormone-related’ category. A list of the corresponding numbers can be found in the appendix, table A4.

3.1.1.3.2 Comparison of cell cycle and selected phytohormone pathways in the three genotypes

As the analysis of functional categories has shown, the two transgenic lines CKX1-4 and IPT-6 displayed considerable differences to the WT. In order to illustrate these differences in higher detail, lists of ‘overlapping’ and ‘uniquely expressed’ entities had been generated from the VENN diagrams comparisons (Fig.11). These lists were now compared in depth, focusing mainly on five categories: ‘cell cycle/ replication/ chromatin- associated’ was chosen because it might explain differences between the sprouting and non-sprouting genotypes. Four phytohormone-related subcategories, ‘auxin’, ‘cytokinin’, ‘ethylene’ and ‘gibberellin’, were selected because they showed strong differences between the three phenotypes and/ or have been implicated to regulate tuber dormancy and sprouting.

46 Results

The two overlap lists of all three genotypes, which contain 1440 up- and 1115 down- regulated entities, contain 21 entities (17 up-, 4 down-regulated, see tables A5 and A6 in the appendix) of the cell cycle/ replication and chromatin-associated category. Eight of the 17 up-regulated entities are among the top 30 most up-regulated entities in the complete list, being up-regulated up to 403-fold and 367-fold in the WT and IPT-6 (Tab.6), respectively. Among others, these strongly up-regulated ESTs showed homology to a B-type cyclin, to cyclins A1 and A2, the cytokininesis-specific KNOLLE gene (Lukowitz et al., 1996) and histones H4 and H3. Notably, induction of cell cycle- related transcripts, which reflects increasing meristematic activity during onset of sprouting, is much lower in CKX1-4, corresponding with the non-sprouting phenotype of this line. For example, expression of a deoxyuridine 5'-triphosphate nucleotide hydrolase (dUTPase) gene, an early marker for the meristem’s transition from the dormant to the sprouting state (Senning et al., 2010) is up-regulated 30.56-fold in the WT, 10.89-fold in IPT-6, but only 2.18-fold in CKX1-4. Of the 2047 entities shared by IPT-6 and the WT in total (compare Fig.11), 63 additional cell cycle/ replication-related entities (see tables A7 and A10) could be found. These transcripts commonly regulated in the two sprouting genotypes, represent, for example, D-type cyclins and histone H2B. Two ESTs were even regulated in an opposing way in the sprouting and non-sprouting phenotypes: SDBN004G07u.scf_725, corresponding to CDC45, a component of the replication machinery, and MICRO.12318.C1_677, showing homology to a G2/ mitotic-specific B-type cyclin, were up-regulated in WT and IPT-6, but down-regulated in CKX1-4 (table A15). In contrast, CKX1-4 overlaps with either WT or IPT-6 are impoverished in cell cycle/ replication-related entities, with only 15 out of 1379 ESTs in the two WT/ CKX1-4 overlaps (tables A9 and A12) and 3 out of 613 in the two IPT-6/ CKX1-4 overlaps (tables A8 and A11) belonging to this category.

Table 6: Top 30 most up-regulated WT entities from the overlap of all three genotypes and their corresponding fold changes in the two transgenic lines From the VENN diagram shown in Fig.11A, an ‘overlap up-regulated in all three genotypes’ list containing 1440 entities was generated. The list was ordered by fold change value of the WT ‘3d vs 0d’ comparison and the top 30 most up-regulated entities with their corresponding fold changes in WT, CKX1-4 and IPT-6 are shown. Abbreviations: CC - cell cycle; CW - cell wall biosynthesis/ modification; ET/ R - electron transport/ redox- associated; H_GA - gibberellin-associated; M - metabolism; S - signalling; T - transport; U - unknown; UC – unclassified.

47

category T CHR H_GA U CC CC M CHR CHR U UC RA U M CHR U CC U U U U CW UC RA CHR S CW U M U

group)] -

iana] tiva (japonica cultivar

lycopersicum]

Description, according to BLAST hit glucanase precursor glucanase precursor - - related protein [Ipomoea nil] -

beta beta

- -

1,4 1,4 - -

type cyclin [Nicotiana tabacum] - Sugar transporter superfamily [Medicago truncatula] Histone H4 GAST1 protein precursor unknown [Solanum B Knolle [Capsicum annuum] phytocyanin Histones H3 and H4 (ISS) [Ostreococcus tauri] PREDICTED: similar to histone 1, H2ai (predicted) [Canis familiaris] unknown [Arabidopsis thal T25K16.10 [Arabidopsis thaliana] peroxidase unknown protein [Arabidopsis thaliana] Glycoside hydrolase, family 1 [Medicago truncatula] histone 3 unknown protein [Arabidopsis thaliana] DNA topoisomerase II [Nicotiana tabacum] NA Os04g0474800 [Oryza sa NA NA endo Polyphenol oxidase A, chloroplast precursor (PPO) (Catechol oxidase) peroxidase [Arabidopsis thaliana] histone 3 Protein kinase [Medicago truncatula] endo unknown protein [Arabidopsis thaliana] wsus [Citrullus lanatus] unknown [Olimarabidopsis pumila]

6 - 03 IPT 89.88 98.93 96.54 64.50 54.21 71. 34.68 40.66 10.00 46.08 46.48 33.77 10.00 56.67 486.93 367.04 222.97 633.62 413.29 382.97 834.62 194.53 136.22 125.45 133.18 161.82 103.74 154.47 408.22 164.59

4

- 9.26 3.27 7.87 6.30 9.89 3.45 7.31 9.84 4.17 8.51 6.91 3.93 3.43 6.37 2.45 7.29 4.78 4.95 7.89 2.83 6.29 16.91 35.76 22.55 12.42 15.39 62.33 12.52 12.38 155.86 CKX1

.31 WT 99.58 97.97 96.36 82.56 76.34 76.04 73.19 491.41 403 331.58 325.17 315.36 283.30 239.87 213.36 211.05 203.01 182.18 179.33 173.91 172.20 157.35 151.72 145.49 142.83 121.86 107.64 105.61 104.03 100.66 FCAbsolute [3d] versus [0d]

RO.2385.C2_1100 ICRO.10271.C2_629 POCI ID STMIC16TV_404 MICRO.5000.C3_344 MICRO.11045.C2_125 MIC SDBN001F10u.scf_564 MICRO.15499.C1_637 MICRO.16548.C1_521 MICRO.1026.C2_455 MICRO.1026.C5_493 MICRO.934.C1_877 MICRO.9977.C1_856 MICRO.499.C1_1039 MICRO.7351.C1_787 MICRO.960.C2_1012 MICRO.2825.C1_425 MICRO.15345.C1_557 MICRO.15342.C1_660 M MICRO.960.C1_679 MICRO.15980.C2_31 STMEA61TV_300 MICRO.14756.C1_447 MICRO.646.C2_1609 MICRO.1405.C1_1290 BF_TUBSXXXX_0056B12_T3M.SCF_425 STMEJ08TV_315 MICRO.14756.C2_213 MICRO.1635.C1_324 MICRO.15082.C1_1400 MICRO.17411.C1_468

48 Results

3 CDKA 2 CDKB proteolysis of ICK 1

0 P -1 CyclinA -2 CDKA CAK (CDKD;1) CDKA ICK P -3 CYCD CYCD P P

RBR

CyclinB G1 RBR

EXIT mitosis: proteolysis of E2F DP1 CYCA/B by APC S E2F DP1 M CyclinD G CDC20 2 Activation of genes CCS52B required for S phase CDC20 entry/ DNA- P P replication CCS52B WEE1 WEE1 CDKA/B CDKA/B

E2F CYCA/B CAK CYCA/B 0d 3d 0d 3d 0d 3d CDC25 WT CKX1-4 IPT-6

Figure 14: Expression of cell cycle genes in wild-type, CKX1-4 and IPT-6 tubers after GA3- mediated tuber sprouting. Expression data of differentially expressed entities representing genes of the cell cycle are shown in the left-hand panel and a schematic overview of the mitotic cell cycle, modified after Inzé and De Veylder (2006) by M. Senning, is shown on the right. Colour boxes as displayed by the MAPMAN tool were copied to and aligned in Microsoft PowerPoint. The colour scale next to the panel indicates transcript levels, with red representing an increase and blue representing a decrease in transcript levels. The colours saturate at 8-fold change. A list of the POCI identifiers shown can be found in the appendix, table A16. At the G1/S-phase transition, CDK inhibitory protein ICK dissociates from CDKA-CYCD complexes and is degraded via the proteasome pathway. Phosphorylation of CDKA then activates the CDKA-CYCD complex, which initiates phosphorylation of the retinoblastoma-related protein (RBR), releasing the E2F-DP1 complex. This complex promotes the transcription of genes required for progression into and through S-phase, where DNA replication takes place. The G2/M-phase transition is accompanied bya marked increase in transcription of both A- and B-type CDKs and A- and B-type cyclins. CDKA/B-CYCA/B complexes are activated by removal of the inhibitory phosphate by CDC25 and phosphorylation by CDK-activating kinase (CAK), both enzymes acting on the CDK part of the complex. The reverse reaction, inhibitory phosphorylation of CDK, is catalysed by kinase WEE1 and promotes endoreduplication. During mitosis, CDC20- and CCS25-activated anaphase-promoting complex (APC) degrades cyclins A and B, leading to the transition from mitosis into G1-phase. CDC, cell division cycle; DP, docking protein.

For visualisation of the differences in cell cycle progression between the three genotypes, normalized microarray data from the GA3 treatment were illustrated using the

MAPMAN tool (Fig.14). With induction of sprouting in wild-type and IPT-6 tubers, ESTs representing cell cycle regulators, such as cyclins A, B, and D as well as CDKB, were strongly increased three days after GA3 treatment, correlating with the resumption of growth in both genotypes. Transcripts corresponding to CDC20 and CCS52B, both activators of the anaphase- promoting complex (APC) that are required for APC-dependent proteolysis of mitotic cyclins and transition into G1 phase, were exclusively up-regulated in the two genotypes that initiate sprouting. 49

Conversely, in the non-sprouting CKX1-4 tubers, these transcripts may be induced upon

GA3 treatment, but much less than in WT and IPT-6. Several ESTs, such as MICRO.4616.C1_818 and STMDB52TV_517, which are all annotated as ‘CyclinD3;3 [Solanum lycopersicum]’, even showed a weaker expression in CKX1-4 at the beginning of the treatment and decreased further after three days of GA3 induction (compare table A16 in the appendix). Apart from ESTs encoding cell cycle/ replication genes, transcriptional differences between the sprouting and non-sprouting genotypes were evident in many other functional groups: The expression of ‘cytoskeleton- associated’ and ‘cell wall biosynthesis/ modification- related’ ESTs such as transcripts coding for alpha and beta tubulin, cellulose synthases, or cell wall- loosening xyloglucan endotransglucosylase hydrolases was found to be increased with onset of sprouting in WT and IPT-6. In contrast, they were not or down-regulated in CKX1-4 tubers. Expression of transcription factors involved in meristem maintenance, such as the homeobox transcription factors Let6 (Janssen et al., 1998) and knotted-1 (Sinha et al., 1993) which are associated with formation and maintenance of the shoot apical meristem (SAM), were increased in all three genotypes. In contrast, transcription factors implicated with organ growth and differentiation processes like PHANTASTICA (Waites et al., 1998), GROWTH- REGULATING FACTOR3 (Kim et al., 2003) or OVATE (Hackbusch et al., 2005) were preferentially expressed in both WT and IPT-6 (compare table A24).

With respect to phytohormones, the most noticeable differences between the three genotypes occurred within the auxin and ethylene signalling pathways, as illustrated by

MAPMAN visualizations shown in figures 15 and 16. Entities encoding auxin biosynthesis enzymes flavin mononxygenase-like proteins, orthologs to YUCCA-like proteins in Arabidopsis, were up-regulated 3.2- to 4-fold in WT and 2.3 to 3-fold in IPT-6 (see table

A7 in the appendix) three days after GA3 treatment. They were not found to be differentially expressed in the CK-deficient CKX1-4 tubers. Consistent with an increased auxin biosynthesis, a substantial augmentation in the expression of various auxin- responsive SAUR (small auxin up-regulated RNAs) and SAUR-like as well as GH3-type protein genes was detected in WT and IPT-6 (compare Fig.15), but not in CKX1-4 tubers. Expression levels for auxin influx PGP1/PGP19 ABC transporters and PIN1-like auxin efflux carriers were almost identical in IPT-6 and the WT.

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51

Figure 15: Expression of auxin transport and signalling genes in wild-type, CKX1-4 and IPT-6 tubers after GA3 treatment An overview of auxin transport proteins and the auxin signalling pathway as derived from Arabidopsis thaliana (Mockaitis and Estelle, 2008; Weijers and Friml, 2009). is shown on the previous page. Expression data of differentially expressed entities representing genes of auxin transport and signalling are shown in the grey panel. Colour boxes as displayed by the MAPMAN Tool were copied to and aligned in Powerpoint. The colour scale above the panel indicates transcript levels, with red representing an increase and blue representing a decrease in transcript levels. The colours saturate at fourfold changes. Fold change and normalized values for all entities shown can be found in table A17 in the appendix. Pathway description: Auxin transport: IAA is the most abundantly occurring auxin in nature. It can enter the cell in its protonated form either by diffusion through the plasma membrane or facilitated by AUX1 permeases. Inside the cell, IAAH is deprotonated and trapped due to the change in pH. Auxin efflux is performed by two types of transporters, PGP1/PGP19 ABC transporters and PIN proteins. PGP1/19 usually have a nonpolar localization whereas PIN proteins show a polar localization which determines the orientation of the auxin flow within the cell. Auxin signalling: Without IAA, ARF transcription factors bound to AREs in the promoters of auxin-responsive genes and interact with Aux/IAA proteins which in turn recruit the transcriptional co-repressor TPL to prevent gene expression. When accumulating in the nucleus, IAA binds to Aux/IAA proteins and the TIR1 subunit of the SCFTIR1 complex, targeting Aux/IAA for degradation via the 26S proteasome and releasing ARF transcription factors from inhibition. The activity of the SCRTIR1 complex can be regulated by addition and removal of ubiquitin-like protein RUB1 through the action of conjugating (AXR1, RCE1 and ECR1) and de-conjugating (CSN) enzymes. ARFs released from inhibition act as monomers or dimers to promote expression of auxin-responsive genes, e.g. those coding for SAUR and GH3 proteins. Abbreviations: AUX1 – Auxin 1; ADP – adenosine diphosphate; ARE – Auxin Responsive Element; ARF – Auxin Response Factor; ATP – adenosine triphosphate Aux/IAA – auxin/indole-3-acetic acid protein; AXR1 – auxin resistant 1; CSN – COP9 Signalosome; ECR1 – E1 C-terminal Related 1; PGP – P-Glycoprotein; PIN – PIN- formed; PM – plasma membrane; RBX – RING-Box protein; RCE1 – RUB1-conjugating enzyme; RUB1 – Related to Ubiquitin; SAUR – Small Auxin-upregulated RNA; SCF complex – SKP1-Cullin-F-Box complex; SKP1 – S- phase kinase associated protein 1; TIR1 – Transport Inhibitor Response 1; TPL – TOPLESS; Ub - Ubiquitin

In line CKX1-4, however, expression of the same transcripts was already considerably lower at the beginning of the experiment than in WT and IPT-6 and these entities were not up-regulated upon GA3 treatment. Intriguingly, sprouting and non-sprouting genotypes also differ in their activation of ARF transcription factors. While ARF4, ARF5 and ARF9 were up-regulated in WT and IPT-6, in CKX1-4, ARF9, ARF10 and ARF19 were transcriptionally activated upon GA3-treatment. Entities with homology to ARF8 and ARF16, however, showed similar expression patterns in all three genotypes. Differences between WT, IPT-6 and CKX1-4 tubers were also observed in the ethylene signalling pathway (Fig.16). Apart from the key transcription factor ETHYLENE-

INSENSITIVE3 (EIN3), which is 6.3-fold down-regulated in CKX1-4 three days after GA3 treatment, the entire ethylene signalling pathway appeared to be activated more strongly in the CK-deficient line compared with WT and IPT-6. The decrease in EIN3 could be linked to the transcriptional activation of F-box proteins EIN3-BINDING F-BOX1 (EBF1) and EBF2, which are themselves controlled by the activity of EIN5, a 5’-3’ exoribonuclease. Entities with homology to EBF1, EBF2, and EIN5 were found to be 2- to 3.6-fold induced upon GA3 treatment of CKX1-4 tubers (appendix, table A18). Normalized expression values for the same entities were lower in the WT and IPT-6, but the calculated fold changes surpass those of CKX1-4. Especially the ethylene receptors ERS1, ETR2 and EIN4, the negative regulator of ETR1, RTE1, the EIN3 and EIN3-like

52 Results

(EIL) transcription factors as well as EBF1- representing entities showed a remarkably low expression in IPT-6 at the ‘0d’ time-point. Within the signalling pathway, only EBF2 and ETHYLENE RESPONSE FACTORs (ERFs) are directly activated by EIN3 and EIL, which probably constitutes a rapid feedback regulatory loop. ESTs with homology to

2 RTE1 1 ERS1 0 ETR2 without Ethylene with Ethylene -1 C2H2 ER EIN4 -2 RTE1 cytosol CTR1 Ethylene receptors Ethylene receptors ETR1 ETR2 ERS1 ERS2 EIN4 EIN3 active CTR1 inactive CTR1

EIL1 MKKK? MKK MKK9 EIN2 MKK9 unknown membrane MPK3/6 MPK

nucleus nucleus ERF1 P MKK9 MPK3/6 EIN2 EIN3/EIL1 unknown membrane EBF1/2 P Skp1 ERF5 Cullin EIN3/EIL1 Rbx E2

EIN5/XRN4 EBF1/2 ERF1

EBF1 Constant Transcription of degradation via Ethylene-responsive 26 S proteasome genes e.g. ERF5 EBF2

EIN5 0d 3d 0d 3d 0d 3d WT CKX1-4 IPT-6

Figure 16. Expression of ethylene signalling genes in WT, CKX1-4 and IPT-6 tubers after GA3- induced sprouting. Expression data of differentially expressed entities representing genes of the ethylene signalling pathway are shown in the left-hand panel and a schematic overview of this pathway, as assumed from knowledge in Arabidopsis (Kendrick and Chang, 2008; Yoo et al., 2009), is shown on the right. Colour boxes as displayed by the MAPMAN tool were copied to and aligned in Microsoft PowerPoint. The colour scale next to the panel indicates transcript levels, with red representing an increase and blue representing a decrease in transcript levels. The colours saturate at 4-fold change. Fold change and normalized values for all entities shown can be found in table A18 in the appendix. Pathway description: Without ethylene binding, the ethylene receptor complexes in the endoplasmic reticulum membrane activate CTR1. RTE1 acts as a negative regulator of ethylene response by regulating the ethylene receptor ETR1. CTR1 is proposed to be a MKKK that activates a cytosolic kinase signalling cascade, leading to phosphorylation of residues Thr-592 and Thr-546 of the transcription factors EIN3 and EIL1 in the nucleus as well as the inhibition of membrane-bound protein EIN2. Phosphorylated EIN3 and EIL1 interact with F-box proteins EBF1 and EBF2, resulting in constant degradation of the transcription factors and thus suppression of ethylene signalling. EIN5/XRN4 indirectly influences transcript levels of EBF1 and EBF2. Upon binding of ethylene, CTR1 is inactivated, which leads to inactivation of the CTR1-MAPK signalling and activation of another kinase signalling cascade involving MKK9, MPK3, and MPK6 and probably an as yet unknown MKKK. This cascade leads to the phosphorylation of residues Thr-176 and Thr-176 of EIN3 and EIL1, presumably resulting in increased stability because of a reduced interaction with EBF1 and EBF2. Membrane-bound protein EIN2 also positively influences EIN3 accumulation through a mechanism that has not been elucidated yet. EIN3/EIL1 activates ethylene responses both directly and indirectly through expression of the transcriptional activator ERF1. CTR1, Constitutive triple response 1; ERS, ethylene response sensor; MKK, mitogen-activated protein (MAP) kinase kinase; MKKK, MAP kinase kinase kinase; MPK, MAP protein kinase; RBX, RING box protein; RTE1, reversion to ethylene sensitivity 1; SCF complex, SKP1-cullin-F-box complex; SKP1, S-phase kinase-associated protein 1; XRN4, exoribonuclease 4.

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tomato or Arabidopsis ERF5 and ERF1 showed an increase in wild-type and, to a stronger extent, in CKX1-4 upon GA3 treatment, but were not (as for ERF1-representing entities) or adversely regulated in line IPT-6.

With respect to CK and GA biosynthetic and signalling pathways (Fig.17 and Fig.18), differences between the genotypes were less prominent. Overall, entities of the CK biosynthesis pathway seem to be weaker expressed in CKX1-4 than in the other two lines. Two ESTs, however, MICRO.11289.C1_201 and MICRO.3788.C4_980, both annotated as zeatin xylosyltransferase, an enzyme thought to be involved in nuclear- cytoplasmic CK transport (Martin et al., 1993), showed their highest fold change in CKX1-4 and were less strongly regulated in the other two genotypes. Cytokinins can be transported directly into a cell by purine permeases (PUPs) and individual PUP genes seemed to be affected in CKX1-4, showing a stronger induction than the wild-type three days after GA3 treatment. MICRO.5974.C1_1055 (‘AtPUP3

[Arabidopsis thaliana]’) which was up-regulated 7.6-fold in CKX1-4 tubers after GA3 treatment, showed the highest fold change of all PUP transcripts in this experiment. In parallel, two other PUP transcripts are markedly down-regulated three days after GA3 incubation, hinting at a tissue- or development- dependent expression of individual PUP genes. Within the CK signal transduction pathway, transcripts of IPT-6 essentially followed the wild-type’s expression pattern. Notably, expression of an EST encoding a CK receptor, MICRO.7190.C1, was 2.8-fold and 7.4-fold up-regulated in WT and CKX1, the two genotypes with ‘normal’ CK levels. In IPT-6 tubers which likely contain higher levels of the phytohormone, receptor induction was less pronounced. Further downstream in the signalling pathway, A-type response regulators displayed a markedly different expression in CKX1-4, with entities very weakly expressed both at the ‘0d’ and the ‘3d GA’ time- point. In fact, the three A-type response regulator transcripts that were visibly up- regulated in WT and IPT-6, also showed a higher expression three days after GA3 treatment than at the ‘0d’ time-point. Due to their very low expression, however, the difference is not visible on the given scale. The gibberellin biosynthesis pathway was marked by a decrease of expression levels of most entities, most prominent in line CKX1-4. Three days after treatment with GA3, ESTs representing the major biosynthetic genes, were down-regulated in all three genotypes, possibly as a feedback response to the applied phytohormone treatment. Additionally, expression of the soluble GA receptor GID1 also decreases in all three genotypes. GA signalling output, however, seemed not to be affected by this down-regulation as genes

54 Results

Cytokinin metabolism

CYP735A DMAPP + tRNA ATP iPATP tZRTP IPT CYP735A tRNA-IPT LOG DMAPP + ADP iPADP tZRDP AMP iPAMP tZRMP cZRMP LOG LOG LOG CKX Degradation iP tZ cZ Zeatin-GT Glycosyl Xylosyl transferase (GT) transferase (XT)

Glycosylation Xylosylation Zeatin-XT 2

1 0d 3d 0d 3d 0d 3d WT CKX1-4 IPT-6 0 Cytokinin signaling & transport iP tZ cZ iP tZ cZ -1 PM -2 P PUP AHK PUP

P AHP6 P AHP feedback inhibition AHK cytosol

nucleus AHP P

A-type ARR P

A-type ARR

B-type ARR target gene transcription B-type ARR 0d 3d 0d 3d 0d 3d WT CKX1-4 IPT-6 Figure 17: Cytokinin metabolism and signalling genes in wild-type, CKX1-4 and IPT-6 tubers after GA3 treatment A simplified overview of the CK metabolism and signalling pathways, modeled after recent reviews (Ferreira and Kieber, 2005; Sakakibara, 2006; Kamada-Nobusada and Sakakibara, 2009; Argueso et al., 2010) is shown on the left and corresponding expression data for differentially expressed entities is shown on the panel on the right. Colour boxes as displayed by the MAPMAN Tool were copied to and aligned in Powerpoint. The colour scale beside the panel indicates transcript levels, with red representing an increase and blue representing a decrease in transcript levels. The colours saturate at fourfold changes. Fold change and normalized values for all entities shown can be found in table A19 in the appendix. For a detailed description of the pathways, see 2.3.4 (p.15) in the introduction. Abbreviations: A-type ARR, Arabidopsis response regulator type A; ADP, adenosine diphosphate; AHK, Arabidopsis histidine kinase; AHP, Arabidopsis histidin phosphotransfer protein; AMP, adenosine monophosphate; ATP, adenosine triphosphate; B-type ARR, Arabidopsis response regulator type B; CKX, cytokinin oxidase; CYP735A, cytochrome P450 monooxygenase 735A; cZ, cis-zeatin; cZR, cis-zeatin riboside; cZRMP, cZR 5’ monophosphate; DMAPP, dimethylallyl pyrophosphate; iP, N6-(Δ2-isopentenyl)adenine; iPRMP, iPR 5’ monophosphate; iPRDP, iPR 5’ diphosphate; iPRTP, iPR 5’ triphosphate; IPT, isopentenyltransferase; LOG, LONELY GUY; PUP, purine permease; tRNA-IPT, tRNA-isopentenyltransferase; tZR, trans-zeatin riboside; tZRMP, tZR 5’ monophosphate; tZRDP, tZR 5’ diphosphate; tZRTP, tZR 5’ triphosphate.

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further downstream in the signalling pathway, like the PIF3/4 transcription factor, showed an increased expression three days after the treatment. Numerous GA-responsive genes were also induced in WT and IPT-6 tubers, in correspondence with the sprouting shown by these two genotypes. CKX1-4 tubers which do not sprout after GA3- treatment, showed no induction of GA-responsive genes.

Gibberellin metabolism

KO CPS KS KO GGDP ent-CDP ent-Kaurene ent-Kaurenoic acid

KAO KAO GA 2-ox GA 13-ox KAO GA GA -Aldehyde 97 (class III) GA53 GA12 12 GA 2-ox (class III) GA 20-ox GA 20-ox GA 20-ox GA 110 GA 3-ox GA 2-ox GA GA GA GA 2-ox GA 29 (class I&II) 20 9 (class I&II) 51 GA 3-ox GA 3-ox GA 2-ox

GA 2-ox GA 2-ox GA8 GA 0d 3d 0d 3d 0d 3d (class I&II) GA1 GA3 GA4 (class I&II) 34 WT CKX1-4 IPT-6 Gibberellin signaling 2

1 GID1

without GA with GA 0 -1

-2 GID1 RUB1-conjugation RUB1-removal by CSN DELLA by AXR1-ECR1 and proteins RCE1 RUB1 Ubi SPY Rbx E2 Cullin SKP1 SCFSLY1/GID2 PIF3/4 SLY1/ GID2 DELLA DELLA Degradation via PIF3/4 GID1 26S proteasome GA GA- responsive genes GARE PIF3/4 GARE

no transcription Transcription of GA-responsive genes 0d 3d 0d 3d 0d 3d WT CKX1-4 IPT-6

56 Results

Figure 18. Expression of gibberellin metabolism and signalling genes in WT, CKX1-4 and IPT-6 tubers after GA3-induced sprouting. Expression data of differentially expressed entities representing genes of gibberellin metabolism and signalling are shown on the right-hand panel and a schematic overview of these pathways, as assumed from knowledge in Arabidopsis (Yamaguchi, 2008; Chebotar and Chebotar, 2011), is shown on the left. Colour boxes as displayed by the MAPMAN tool were copied to and aligned in Microsoft PowerPoint. The colour scale next to the panel indicates transcript levels, with red representing an increase and blue representing a decrease in transcript levels. The colours saturate at 4-fold change. Fold change and normalized values for all entities shown can be found in table A20 in the appendix. Pathway description: GA biosynthesis: During the first step of GA biosynthesis in the plastids, trans-geranylgeranyl diphosphate (GGDP) is converted into ent-kaurene by ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS). A four-step oxidation process in the ER converts ent-kaurene to GA12-aldehyde, catalysed by ent-kaurene oxidase (KO) and ent-kaurenoic acid oxidase (KAO). Oxidation at the C7 atom by GA7-oxidase (GA7-ox) leads to formation of GA12 which can be further hydroxylated to GA53 by GA13-oxidase (GA13-ox). In the cytosol, GA12 and GA53 are successively oxidised by GA20-oxidase (GA20-ox) and GA3-oxidase (GA3-ox) to form bioactive GAs (black box).GA1 and GA4 can be degraded by GA2-oxidases (GA2-ox). GA Signalling: In the absence of GA, DELLA proteins act as nuclear transcriptional repressors to inhibit GA signalling and transcription of GA-responsive genes. Bioactive GA present in the cell is bound by soluble GA receptors like GID1. Upon binding of the phytohormone, GA-GID1 directly interacts with DELLA proteins and a conformational change in the DELLA-GA-GID1 complex stimulates its recognition by the SCFSLY/GID2/SNZ complex to target DELLA proteins for proteasome-dependent degradation. DELLA proteins lack a DNA-binding motif and seem to exert their function by interaction with transcription factors, for example blocking DNA binding activity of transcription factors PIF3 and PIF4. Lifting of DELLA repression through degradation then allows transcription of GA-responsive genes.

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4.1.2 Expression of bacterial IPT and AtCKX1 in potato under control of the meristem-specific UFO promoter

Isopentenyltransferases and cytokinin oxidases are key enzymes of the cytokinin metabolism pathway and their over-expression under control of a constitutively active promoter leads to severe defects in growth and development. For the examination of localized, tuber meristem-specific alteration of phytohormone levels, expression of either gene, IPT and AtCKX1 under control of the Arabidopsis thaliana UFO (UNUSUAL FLORAL ORGANS) promoter was selected. The UFO gene encodes an F-Box protein required for proper floral development and is expressed ‘at low levels in the center of the SAM and at higher levels towards the periphery of the SAM’ in vegetative and floral meristems (Long and Barton, 1998). Promoter activity in potato had been tested by UFO::GUS and UFO::GFP constructs and had revealed similar expression patterns as in Arabidopsis thaliana (S. Sonnewald and P. Laufs, unpublished).

BamHI EcoRV EcoRI Acc65I SalI HindIII

UFO IPT OCS

400 bp

EcoRI BamHI NcoI EcoRI XbaI SalI HindIII

UFO AtCKX1 OCS

400 bp

Figure 19: Schematic representation of IPT and AtCKX1 binary constructs for meristem- specific expression Full-length coding sequences of bacterial IPT and Arabidopsis thaliana AtCKX1 were amplified by PCR and cloned into binary vector UFO:BinAR containing the 3.7 kb long UFO promoter via Acc65I/SalI or BamHI/SalI restriction sites, respectively, to create the UFO:IPT and UFO:CKX1 constructs.

IPT (acc. no. AF242881) and AtCKX1 (acc. no. NM_129714) were amplified by PCR, from either a pUC9 vector (kindly provided by Prof. T. Schmülling, FU Berlin) or Arabidopsis thaliana leaf cDNA. After subcloning into pCRblunt, the IPT and CKX1 fragments were excised via artificially added Acc65I / SalI or BamHI / SalI restriction sites, respectively, and inserted into a BinAR vector containing the AtUFO promoter and the octopine synthase polyadenylation signal. The binary constructs (Fig. 19) were stably transformed in potato and transformants were analysed.

58 Results

4.1.2.1 Phenotypic characterization of UFO:IPT and UFO:CKX1 overexpressing potato plants

In a first round of transformation of the UFO:IPT construct, 10 primary transformants could be regenerated. Expression of the transgene was verified by RT-PCR (Fig. 19A) and three of the five positive lines (UFO:IPT-2, UFO:IPT-8 and UFO:IPT-10) were selected for further analysis because they displayed a retarded growth phenotype. Transformation of the UFO:CKX1 construct yielded 5 transgenic plants, all of which expressed the transgene, as confirmed by RT-PCR analysis (Fig. 19B). Expression levels varied and the three strongest expressors UFO:CKX1-2, UFO:CKX1-3 and UFO:CKX1-5 were chosen for further analysis.

A B UFO:IPT UFO:CKX1 2 3 7 8 10 WT + 1 2 3 4 5 WT + IPT CKX1 Actin Actin C D UFO:IPT UFO:CKX1

WT WT 2 8 10 WT WT 2 3 5

Figure 20: Expression of IPT and AtCKX1 in potato plants under control of the UFO promoter (A) RT-PCR analysis of UFO:IPT-expressing potato plants (lines 2, 3, 7, 8, and 10). Five micrograms of total RNA isolated from shoot tips containing the apical meristem were reverse transcribed using oligo(dT) primers and RevertAid H2 reverse transcriptase. An aliquot of first-strand cDNA was amplified with gene-specific primers for IPT or actin. The plasmid carrying the UFO:IPT construct was used as a positive control (‘+’). (B) RT-PCR analysis of UFO:CKX1-expressing potato plants (lines 1 to 5). Five micrograms of total RNA isolated from shoot tips containing an apical meristem were reverse transcribed using oligo(dT) primers and RevertAid H2 reverse transcriptase. An aliquot of first-strand cDNA was amplified with gene-specific primers for AtCKX1 or actin. The plasmid carrying the UFO:CKX1 construct was used as a positive control (‘+’). (C) Phenotypic alteration of transgenic UFO:IPT potato plants. From left to right: two wild-type (WT) plants and UFO:IPT lines 2, 8, and 10. Photographs were taken 8 weeks after transfer to the greenhouse. (D) Phenotypic alteration of transgenic UFO:CKX1 potato plants approximately 8 weeks after transfer into the greenhouse. From left to right: two wild-type plants (WT) and UFO:CKX lines 2, 3, and 5.

In the greenhouse, UFO:CKX1 plants showed a reduction in plant height (Fig. 20D, Table 7), but were not otherwise affected by expression of the transgene. Both tuber number and tuber yield of the transgenic plants did not differ from the wild-type (Table 7). Surprisingly, transgenic plants expressing IPT under control of the UFO promoter showed a peculiar phenotype: Alongside a reduction in plant height (Fig. 20C, Table 7), flower-like structures could be found at branching points in the plant’s inflorescence (Fig. 21A), at the tips of side branches (Fig. 21B) and side shoots formed by axillary meristems and even underground, at the tips of a few stolons (Fig. 21C). Hand sections

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through these structures confirmed that they represent flower rudiments (Fig. 21D). All three UFO:IPT lines exhibited a significant reduction in tuber yield and in UFO:IPT-2, presumed to be the strongest expressing line, this is accompanied by a reduction in tuber number (Table 7).

Table 7: Phenotypic characteristics of transgenic potato plants expressing either the CKX1 gene from Arabidopsis or an IPT gene from Agrobacterium under control of the AtUFO promoter Parameters were determined from soil-grown plants at harvest. Results are shown for two (UFO:CKX1) or three (UFO:IPT) independent experiments and represent means ± SD of 5 plants. Statistically significant differences from the WT were determined using one-tailed t tests assuming unequal variance and are indicated by asterisks (P≤ 0.05).

Plant Stem Height Tuber Yield No. of Tubers per Plant

cm g plant -1

Wild-type (Solara) 55.8 ± 2.5 188.9 ± 14.5 8.1 ± 1.6

UFO:CKX1 UFO:CKX1-2 49.8 ± 2.8* 186.0 ± 11.4 8.6 ± 1.2 UFO:CKX1-3 43.6 ± 3.2* 190.3 ± 11.4 8.3 ± 1.3 UFO:CKX1-5 49.7 ± 2.5* 184.2 ± 16.7 8.5 ± 0.8

UFO:IPT UFO:IPT-2 53.7 ± 1.9* 105.7 ± 35.6* 4.6 ± 1.2* UFO:IPT-8 52.2 ± 2.0* 138.3 ± 26.0* 8.0 ± 1.8 UFO:IPT-10 54.6 ± 1.8 122.8 ± 15.0* 7.5 ± 1.9

4.1.2.2 Characterization of sprouting behaviour of UFO:IPT and UFO:CKX1- expressing potato tubers

To find out whether local expression of IPT or CKX1 under control of the UFO promoter has an effect on tuber sprouting, harvested tubers were stored at room temperature and scored regularly for their sprouting behaviour.

Tubers of UFO:CKX1 plants started sprouting coevally with the wild-type, about eight to nine weeks after harvest, and reached 100% sprouting within the same time frame as the non-transformed control. Phenotypically, sprouts formed by UFO:CKX1 tubers resembled the wild-type (data not shown).

60 Results

A C

WT UFO:IPT-2 B D

WT UFO:IPT-2 WT UFO:IPT-2 E UFO:IPT

WT 2 8 10

Figure 21: Phenotype of UFO:IPT-expressing plants (A) Inflorescence and flowers of a WT and a UFO:IPT-2 plant. Rudimentary flowers at the branching points of the UFO:IPT-2 inflorescence are indicated by white arrowheads. (B) Bastard branches of WT and UFO:IPT-2. The WT apical meristem of the bastard branch produces leaves whereas in the transgenic line, a ‘rudimentary inflorescence’ is formed. (C) Stolons of UFO:IPT-2, photographed at harvest. A small number of stolons of the seemingly strongest expressing line, UFO:IPT-2, form rudimentary flowers at their tips. (D) Comparison of WT and UFO:IPT-2 bastard branch apices. The tips of bastard branches were hand sectioned and imaged on a stereomicroscope. (E) Cross sections through WT and UFO:IPT tubers at harvest.

Of the tubers from UFO:IPT lines, many had already broken dormancy while still attached to the plant (Fig. 21E). The effect was most pronounced in line UFO:IPT-2 (91.3% visible sprouts) and lesser in lines UFO:IPT-8 and -10, with 80.9% and 68.4% sprouted tubers at harvest, respectively. Phenotypically, the small sprouts formed prematurely on UFO:IPT tubers mostly resembled those of the wild-type. Up to 14.9 % of the tubers, however, formed sprouts that bear resemblance to the flower rudiments found in above-ground tissues and some stolon tips (Fig. 22). The highest percentage of these

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‘flowering’ tubers was found in UFO:IPT-2, in the other two lines, they represented only a small fraction of the total number of sprouted tubers.

100

80

60

40

20

0 WT UFO:IPT-2 UFO:IPT-8 UFO:IPT-10

Figure 22: Percentages of sprouted tubers expressing IPT under control of the UFO promoter at harvest At harvest, tubers of WT and UFO:IPT lines were scored for sprouting and phenotypical traits of the sprouts formed on the tubers. The graphs show the number of tubers sprouted at harvest (red) and the number of tuber s carrying sprouts with a ‘flower-like’ appearance (blue) as percentages of the total number of tubers ± SD of two independent harvests with five plants of each line and three to eleven tubers per plant. A representation of the phenotypes is given next to the graphs.

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4.2 In silico analysis of cytokinin signalling and metabolism genes of potato

The first complete genome sequence of a plant, Arabidopsis thaliana, which was published by the Arabidopsis Genome Initiative (2000), has started numerous projects aimed at identifying the function for all of the estimated 25,000 genes (Bevan and Walsh, 2005) and analysis of its organization has highlighted the importance of gene and segmental duplications to the evolution of a genome (Blanc et al., 2003; Zhou and Huang, 2008; Paterson et al., 2010). Thus, it became the foundation of and an indispensable tool for plant molecular biology. Just like the Arabidopsis genome, the potato genome sequence will present a boost to the field of crop plant research.

So far, genes of the cytokinin signalling and metabolism pathways have been identified primarily in Arabidopsis. In the present thesis, the potato genome sequence available from the homepage of the Potato Genome Sequencing Consortium (PGSC; http://potatogenomics.plantbiology.msu.edu/index.html) was utilized to identify full-length sequences of genes from the CK signalling and metabolism pathways, as these sequences are critical to further studies on gene expression and function.

4.2.1 Availability of potato genome sequences

The PGSC was initiated in 2006 and first aimed at sequencing the genome of a diploid potato variety, RH (RH89-039-16), for which an Ultra High Density (UHD) map with about 10,000 AFLP™ Markers was available. Progressing with RH was slow due to the variety’s heterozygosity so that sequencing of the doubled monoploid clone DM (S. tuberosum group Phureja DM1-3 516 R44) was started in parallel (http://www.potatogenome.net/index.php/introduction).

The first draft of the RH genome sequence was made available to the public in the fall of 2009 and contained 34440 ‘scaffold’ sequences ranging from 139 bp to over 5 million bp. The toolset to annotate potato genes was developed using this genome release. With release of the third version of the DM (DMv3) genome in the end of 2010, work was switched from RH to DM. The sequence analyses and comparisons described in the following chapters were mainly performed with the DMv3 genome release, as a newer version was only made available to the public in July 2011, alongside the publication of the genomic sequence in the journal ‘Nature’ (PGSC, 2011).

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4.2.2 Developing a method for annotation of genes in the potato genome

For potato transcript profiling as described in the previous section, the Agilent 4x44k POCI (Potato Oligo Chip Initiative) microarray chip has been widely used. The array contains 42,034 60mer oligo sequences derived from potato EST sequences and represents the most comprehensive collection of potato ESTs available today. A thorough description of how the array was developed can be found in Kloosterman et al. (2008). Despite extensive sequence analysis, the POCI collection likely contains several ESTs that represent one and the same gene. Therefore, primary intention for the development of an annotation method for the available potato genome was to compare - and, if possible, reduce - the redundancy of ESTs from the POCI collection.

Although the first annotations were done using the RH genomic draft sequence released in 2009, I will describe the annotation process based on the DM genome, version three (DMv3), because most sequences have been identified in this genome release and it allows easy comparison with the published DM genome version 3.4 (PGSC, 2011). DMv3 contained about 60000 Scaffold sequences, between 100 bp and 2.7 million bp in length, which were imported into the Geneious 5.4.6 software. While working with the RH genome, it had already been shown that all tested sequences of interest were located on scaffolds with at least 100,000 bp length. In order to reduce loading times in the Geneious software, DMv3 sequences were sorted by size and 1859 scaffold sequences larger than 100,000 bp were singled out and used as a database for homology searches. To allow searches on the amino acid level, for example for conserved protein domains, the 1859 large scaffolds were translated into all six possible reading frames and the resulting amino acid sequences were used as a database for similarity searches.

Most often, the methods described in the following sections were combined in order to completely identify and annotate genes in the potato genome.

4.2.2.1 Gene annotation by POCI EST sequences

The starting point of most analyses were POCI EST sequences representing genes of interest. The POCI ESTs, in turn, were found by key word search with the POCI online tool or by blasting sequences from related species against the POCI database, both available online (http://pgrc-35.ipk-gatersleben.de/pls/htmldb_pgrc/f?p=194:1:63062385 117638). Roughly 40% of all POCI ESTs have been assigned descriptions according to BLAST search results against the NCBI database, the rest are either ‘unknown’ or

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‘unclassified’, so that for a thorough identification of candidate ESTs, both search methods were used.

An example of how a gene of interest was identified from EST sequences is shown in Figure 23. As a first step, EST sequences were entered into Geneious and blasted against the set of 3301 DMv3 scaffolds in order to find out which scaffold they matched to. The best BLAST hit(s) - usually the one(s) with an E-value of 0.0 - were then used for a pairwise alignment with the search sequence. Annotation of introns and exons on the scaffold sequence was based on the alignment result: areas where EST and scaffold matched were marked as exons; areas between two exons which were not covered by the EST and followed the gt/ag rule were marked as introns. If the EST spanned introns larger than ~200 bp, the alignment algorithm was unable to properly align scaffold and EST sequence. In these cases, only a part of the EST sequence was properly aligned to the scaffold. Sequence section that did not fit, were ‘extracted’ by the software and fitted to the scaffold by a second alignment. Thus, multiple rounds of alignments were employed for genes containing several introns. If several EST sequences matched the same scaffold, all alignments and intron/ exon annotations were done before proceeding. After intron/ exon annotation, intron sequences were removed in order to see if an open reading frame (ORF) is formed.

Figure 23: Example of a gene annotated from two EST sequences Two ESTs, MICRO.15036.C1 and POAD094TV, were compared to DMv3 scaffolds in a BLAST-like search and found to have Scaffold PGSC0003DMS000002089 as best hit. Alignments of the EST sequences to the scaffold showed that MICRO.15036.C1 spans one intron, that POAD094TV spans two introns and that the two EST sequences share 35 bp at their respective 3’ and 5’ end. From the alignments, four exons and three introns were annotated on the scaffold sequence. Exons are marked by grey arrows, introns are marked in white. After removal of the introns, the ORF prediction tool displayed an ORF of 1152 bp, marked by an orange arrow, spanning three of the four exons. 65

For verification, predicted ORF sequences were blasted against NCBI’s non-redundant nucleotide collection using the megablast search algorithm. Additionally, the sequences were subjected to NCBI’s conserved domain search to see if functional domains of the GOI are present and complete in the protein.

If the identified ORF sequences turned out to be incomplete, the scaffold sequence up- or down-stream of the annotated area was blasted against the NCBI non-redundant EST collection or the POCI collection in order to find new ESTs covering the scaffold area. If additional ESTs were found, the procedure of aligning, annotating introns/ exons, ORF identification and verification was repeated. In cases where no new ESTs were found, methods described below were utilized.

4.2.2.2 Employing BLAST searches of known sequences from other species

Although the POCI collection contains EST sequences from many potato tissues, treatments and developmental stages, it probably does not cover the complete transcriptome of the potato plant. Therefore, for some genes of interest, EST sequences might be lacking.

In cases where no matching EST sequences were found within the POCI collection, the following two steps were carried out to identify the potato sequence of a gene of interest (GOI):

1) If a sequence of the GOI was available from a member of the Solanaceae family, this sequence was used for direct blasting against the DMv3 scaffolds. Subsequently, alignments between the scaffold and GOI sequence were done, using the same procedures as described in the previous section. If the exon/ intron and ORF identification had been successful, the newly discovered potato sequence was verified by BLAST against NCBI’s non-human, non-mouse nucleotide collection. By additionally blasting the sequence against the POCI database, ESTs representing the GOI were looked for. Any ESTs not previously identified were then incorporated into the scaffold - GOI alignments.

2) If the GOI had not been identified in a member of the Solanaceae family and only a sequence from a more distantly related plant was available, that sequence was translated into the corresponding protein sequence and employed in a similarity search against the translated scaffold sequences in order to find the matching scaffold. After a scaffold had been found, exon and intron annotations were based on multiple rounds of alignments between the translated GOI sequence and the scaffold’s translations. Any

66 Results annotations made on the scaffold’s protein sequences were afterwards transferred onto the genomic sequence using the ‘Edit > Find in Document > Find in translations’ search tool. Scaffold sequences were also blasted against NCBI’s EST collection. If any ESTs were found, they were aligned to the genomic sequence in order to find supporting evidence for annotated exons and introns. ORF identification and final verification of ORF sequences were then performed as described previously.

4.2.2.3 Gene identification via functional domains

In some cases neither the EST alignments nor the sequence search with known sequences from other species succeeded in identifying a complete ORF. If the encoded protein harboured a conserved functional domain, however, the genomic sequence could be identified via the domain it encodes, as shown for an example in Figure 24.

A

B

C

D

E

F

Figure 24: Example of a gene annotated by its encoded conserved domain(s) (A) Alignment of POCI EST bf_mxflxxxx_0059a04.t3m.scf.and Scaffold PGSC0003DMS000000650; the EST was annotated as a cytokinin oxidase (CKX), but no other ESTs were found which also fit the scaffold. (B) Result of the Conserved Domain Search using one of the translation frames of Scaffold PGSC0003DMS000000650. (C) Domain fragments (red) were annotated on the Scaffold sequence. (D) Introns and exons were afterwards annotated in the spaces between the domain fragments, with consideration of the GT/AG rule. (E) After removal of the introns, an open reading frame of 1581 bp was found (orange arrow) (F) Verification of the ORF sequence by blasting revealed that an almost identical sequence is deposited in NCBI’s nucleotide collection.

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This method was often used when only a single EST sequence was available that did not cover the complete GOI. In such a case, annotation by functional domain was performed by translating the genomic sequence into the corresponding three reading frames. Each reading frame was then entered into NCBI’s Conserved Domain Search and domain fragments as shown by the search result were annotated on the scaffold sequence. Exons and introns were subsequently annotated, taking the location of the domain fragments and likely intron/ exon borders (GT/AG rule) into account. After annotation of exons and introns, gene identification by removal of introns, ORF prediction and final verification of ORF sequences were performed as described previously.

If neither scaffold nor EST sequences for a GOI were available by previously described methods, the conserved domain of the encoded protein could be used for scaffold search. To this end, the protein sequence of the conserved domain was blasted against the collection of scaffold translations. Subsequent pairwise alignments between the resulting protein sequences and the conserved domain were used for tentative annotation of exons and introns. These preliminary annotations were then transferred onto the genomic scaffold sequence. Further gene identification was then done as described in the earlier sections.

4.2.3 Cytokinin metabolism genes

Cytokinins regulate numerous processes within the plant and CK homeostasis is finely tuned by biosynthesis, degradation and conversion of the hormone. Central to these three processes are isopentenyltransferases (IPT) in the biosynthesis pathway, cytokinin oxidases (CKX) for CK degradation and glycosyl- and xylosyltransferases which catalyse the hormone’s conjugation. For a better understanding of cytokinin pathways in potato, the genes encoding the above mentioned enzymes were identified in the potato genome.

4.2.3.1 Identification of adenylate IPT and tRNA IPT genes

There are two types of isopentenyltransferases: Adenylate IPTs transfer an isoprene moiety onto AMP, ADP or ATP, and tRNA IPTs utilize tRNA-bound adenine as an acceptor (Miyawaki et al., 2006). Within the POCI EST collection, three EST sequences were annotated as ‘isopentenyltransferase’ or ‘tRNA isopentenyltransferase’. Blasting against the DMv3 scaffolds showed that two of these ESTs, MICRO.12320.C1 and MICRO.4469.C1, match to the same scaffold. Alignments to the scaffold sequence confirmed that the two ESTs represent the same gene. The third EST, bf_mxlfxxxx_0007a09.t3m.scf, showed high sequence identity to two different scaffolds, leading to the identification of two slightly differing genomic sequences. In the order of 68 Results their identification, genes were termed StIPT1 to StIPT3. An overview of the three genes is given in table 8. Based on sequence length, StIPT1 was predicted to encode a tRNA IPT, because these enzymes carry an extension at the C-terminus which is needed for tRNA binding (Zhou and Huang, 2008). All sequences were verified on the nucleotide and amino acid level by blasting against NCBI databases.

Table 8: Overview of three StIPT genes identified by mapping of EST sequences This table lists names, scaffold numbers in the DMv3 genome, predicted enzyme type and sequence lengths for StIPT1 to StIPT3. Chromosome number was determined by BLAST search of each genomic sequence against the twelve ‘pseudomolecules’ representing the potato’s twelve chromosomes in the published version of the genome. CDS and protein sequences of StIPT1-3 can be found in the appendix.

Predicted Scaffold No. Introns / length Name POCI ESTs Chromosome Type PGSC0003DMS Exons genomic CDS protein [bp] [bp] [aa] MICRO.12320.C1 StIPT1 t-RNA IPT 000001642 12 10 / 11 6087 1353 451 MICRO.4469.C1

adenylate bf_mxlfxxxx_0007a0 StIPT2 000000767 9 0 / 1 960 960 320 IPT 9.t3m.scf

adenylate StIPT3 / 000001615 4 0 / 1 1005 1005 335 IPT

As shown in figure 25 for AtIPT9, protein sequences of known IPTs harbour conserved domains, most often a ‘P-loop NTPase’ superfamily domain and one or more multi- domains like ‘PLN02840, tRNA dimethylallyltransferase’, ‘PLN02748, tRNA dimethylallyl- transferase’ or ‘PLN02165, adenylate isopentenyltransferase’. Additional blast searches within the DMv3 genome with various IPT-encoding query sequences did not yield new potato IPT genes, although numerous fragments of ‘P-loop NTPase’, ‘PLN02840’ and ‘PLN02165’ domains could be annotated within several scaffolds. None of these fragments, however, could be joined together correctly to form a complete IPT gene.

Figure 25: Conserved domains of AtIPT9 The protein sequence of AtIPT9 was used as a query for BLAST against the Conserved Domain Database (Marchler-Bauer and Bryant, 2004; Marchler-Bauer et al., 2009; Marchler-Bauer et al., 2011). The sequence contains a P-loop_NTPase superfamily domain and tRNA dimethylallytransferase domain PLN02840.

After the publication of the DM genome version 3.4 in 2011, the analysis was repeated using the ‘PGSC_DM_v3.4_cds_representative’ collection of predicted coding 69

sequences as a search pool. StIPT1 was found to be identical to sequence PGSC003DMC400026970. StIPT2 and StIPT3 are identical to sequences PGSC003DMC400001825 and PGSC003DMC400010681, respectively.

Additionally, the BLAST search against the 39031 DMv3.4 CDS sequences yielded more than sixty possible candidates for IPT genes. These candidates were then tested by blasting against the nucleotide database of NCBI and by checking for conserved functional domains. Thirty-one of these sequences could be dismissed because they were too short and encoded only domain fragments. Another sequence (PGSC0003DMC400068199) was also dismissed as it seemed to be incompletely sequenced, containing a 2509 bp long stretch of ‘NNNNN’. After testing for conserved domains, nine putative IPT sequences remained, because most protein sequences encoded by the candidate sequences comprised multiple successive domain fragments instead of one complete domain. The newly found sequences and their provisional nomenclature are listed in table 9. A phylogenetic tree based on all twelve potato as well as nine Arabidopsis IPT protein sequences can be found in Fig.26. The tree showed that StIPT1 and StIPT5 clustered with the two tRNA IPT of Arabidopsis, AtIPT2 and AtIPT9, separately from Arabidopsis adenylate IPTs.

Figure 26: Unrooted phylogenetic tree of tRNA IPT and adenylate IPT protein sequences of potato and Arabidopsis Protein sequences of twelve potato and nine A. thaliana isopentenyltransferases (accession numbers: AtIPT1- AEE34796; AtIPT2-AEC080039; AtIPT3-AEE80436; AtIPT4-AEE84938; AtIPT5-AED92645; AtIPT6-AEE30619; AtIPT7-AEE76788; AtIPT8-AEE76200; AtIPT9-AED92784) were used for calculation of a phylogenetic tree. Arabidopsis tRNA IPT sequences are marked in blue, A. thaliana adenylate IPT sequences are labelled in red and potato sequences are noted in black. Tree calculation was based on a global alignment with free end gaps, BLOSUM62 cost matrix and Jukes-Cantor genetic distance model. The tree was built by the Geneious 5.4.6 tree builder module employing a neighbour-joining method. The scale bar at the bottom represents 0.2 substitutions per amino acid site.

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Table 9: Overview of putative IPT genes from the DMv3.4 genome This table lists sequence lengths, BLAST hit and names of putative IPT genes from the DMv3.4 ‘CDS_representative’ collection of the published potato genome.

CDS number sequence best BLAST hit at NCBI Name (PGSC0003DMC...) length [bp]

AF346892 400025657 852 StIPT4 Petunia x hybrida Sho gene, complete cds XM_002263711 400046119 1140 PREDICTED: Vitis vinifera tRNA dimethylallyltransferase 2-like StIPT5 (LOC100251208) XM_002314377 400055892 993 StIPT6 Populus trichocarpa cytokinin biosynthetic isopentenyltransferase XM_002519046 400056697 825 Ricinus communis tRNA delta(2)-isopentenylpyrophosphate transferase, StIPT7 putative GQ981408 400057939 846 StIPT8 Vitis vinifera cultivar Red Globe isopentenyltransferase (IPT1), partial cds HQ585948 400059210 819 StIPT9 Malus x domestica cultivar Fuji adenylate isopentenyltransferase (IPT3b) HQ585947 400061315 777 StIPT10 Malus x domestica cultivar Fuji adenylate isopentenyltransferase (IPT3a) HQ585947 400062081 975 StIPT11 Malus x domestica cultivar Fuji adenylate isopentenyltransferase (IPT3a) GQ981408 400065376 750 StIPT12 Vitis vinifera cultivar Red Globe isopentenyltransferase (IPT1), partial cds

As a final step in the analysis, StIPT4 to StIPT12 were used in a BLAST search against the POCI EST collection in order to identify ESTs which represent these genes on the array. This led to the re-annotation of three ESTs: bf_swstxxxx_0059g04.t3m.scf and MICRO.9536.C1 represent StIPT4 and MICRO.12726.C1 corresponds to StIPT5. Expression data for IPT genes can be found in the appendix, table A23.

4.2.3.2 Identification of cytokinin oxidase/ dehydrogenase genes

Basis for the identification of potato CKX genes were four POCI EST sequences which matched to two scaffolds in the potato genome. The first two ESTs that were mapped to a scaffold, cSTA13J24TH and bf_mxflxxxx_0059a04.t3m.scf, most likely represent a gene of 5,4 kb length spanning four introns. The two remaining ESTs mapped to scaffold PGSC0003DMS000000386 and covered a genomic sequence of 2 kb containing five exons and four introns. When the corresponding CDS sequences were blasted against the NCBI nucleotide database, it was found that five CKX sequences of potato, derived from cultivar Russet Burbank, had already been submitted there and that the query sequences were 98% identical to StCKX4 and StCKX3, respectively. The ‘Russet Burbank’ sequences were subsequently used for identification of CKX genes in the DMv3 genome. In total, six CKX genes were identified, five of which showed between 98% and 99% sequence identity with the NCBI-submitted ‘Russet Burbank’ sequences. The sixth gene, tentatively named StCKX6, shares between 76% to 85% identical sites with StCKX1 to StCKX5. Verification of StCKX6 by blasting against NCBI nucleotide and 71

conserved domain (CDD) databases confirmed the presence of domains commonly found in CKX sequences (Fig.27).

Figure 27: Conserved domains of StCKX6 The protein sequence of StCKX6 was used as a query for BLAST against the Conserved Domain Database (Marchler-Bauer and Bryant, 2004; Marchler-Bauer et al., 2009; Marchler-Bauer et al., 2011). The sequence contains ‘FAD-binding_4’ and ‘Cytokin-bind’ superfamily domains as well as cytokinin dehydrogenase multi- domain PLN02441.

Four additional scaffolds, PGSC0003DMS000000598, PGSC0003DMS000001184, PGSC0003DMS000002044 and PGSC0003DMS000002087 also emerged as candidates when blasting the NCBI-submitted StCKX sequences against the DMv3 genome. None of these scaffold sequences, however, coded for a protein with a full- length PLN02441 cytokinin dehydrogenase multi-domain. Comparison with the CDS collection of the 2011 DMv3.4 genome publication also did not reveal new CKX sequences. In a reverse search of the POCI ESTs using the identified sequences as a query, no additional ESTs representing cytokinin oxidases were found.

Figure 28: Unrooted phylogenetic tree of potato and Arabidopsis CKX protein sequences Protein sequences of six potato and seven A. thaliana CK oxidases/ dehydrogenases (accession numbers: AtCKX1-AEC09992; AtCKX2-AEC06889; AtCKX3-AED96828; AtCKX4-AEE85669; AtCKX5-AEE35721; AtCKX6- AEE80482; AtCKX7-AED92951) were used for calculation of a phylogenetic tree. Arabidopsis sequences are marked in blue and potato sequences are labeled in black. Tree calculation was based on a global alignment with free end gaps, BLOSUM62 cost matrix and Jukes-Cantor genetic distance model. The tree was built by the Geneious 5.4.6 tree builder module employing a neighbour-joining method. The scale bar at the bottom represents 0.09 substitutions per amino acid site.

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A summary of the six CKX sequences is given in table 10 and all CDS and amino acid sequences can be found in the appendix, section 8.1.2. A phylogenetic tree comparing potato and Arabidopsis thaliana CKX protein sequences is shown in Fig.28. Except StCKX1 and StCKX6, each of the six potato protein sequences clusters with a different A. thaliana protein. This might indicate distinct functions of the potato isoforms as shown, for example, for AtCKX3 and AtCKX5 (Bartrina et al., 2011).

Table 10: Overview of StCKX genes identified in the potato genome This table lists names, scaffold numbers in the DMv3 genome and sequence lengths for six potato CKX genes. Chromosome number was determined by BLAST search of each genomic sequence against the twelve ‘pseudomolecules’ which represent the potato’s twelve chromosomes in the published version of the genome. Five of the six genes were found to be already submitted to NCBI’s nucleotide database and their accession numbers are given in brackets.

Introns Scaffold No. length Name POCI ESTs Chromosome / genomic protein PGSC0003DMS CDS [bp] Exons [bp] [aa] StCKX1 / 000001089 4 4 / 5 2401 1632 544 (FJ751238)

StCKX2 / 000001425 12 4 / 5 3642 1584 528 (FJ751239)

StCKX3 SSBN002L17u.scf 000000386 1 4 / 5 2062 1602 534 (FJ888605) MICRO.12470.C1 cSTA13J24TH StCKX4 bf_mxflxxxx_0059a04.t3 000000650 4 4 / 5 5416 1581 527 (FJ888606) m.scf StCKX5 / 000000737 4 3 / 4 3146 1554 518 (FJ888607)

StCKX6 / 000000849 10 4 / 5 2424 1611 537

4.2.3.3 Identification of zeatin glycosyl- and xylosyltransferases

Genomic analysis of zeatin-conjugating transferases was initially started in order to identify full-length sequences of three ESTs annotated as ‘zeatin-O-xylosyltransferase [Phaseolus vulgaris]’. Two of these ESTs had been found to be 3.2-fold and 5.4-fold up- regulated in the transgenic line CKX1-4 (compare Fig.17) while being less strongly up- regulated in WT and IPT-6. Although the three EST sequences showed high sequence similarity, they matched different scaffolds and three genes could be identified from the EST’s alignments to their respective scaffolds. Beside these three matching scaffolds, BLAST of the EST sequences had also returned fifteen additional candidates. For nine of these candidates, open reading frames could be identified and verification by BLAST against the NCBI nucleotide collection suggested that they most likely encoded glycosyltransferases. Subsequently, the analysis was extended to include seven POCI ESTs annotated as

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‘zeatin-O-glycosyltransferase’, three Arabidopsis thaliana zeatin glycosyltransferases (ZOG1 to ZOG3) and two maize cis-zeatin glycosyltransferases (cisZOG1 and cisZOG2) as query sequences. This analysis gave a total of 46 ORF sequences which were provisionally termed StGT1 to StGT46. An overview of the candidate sequences can be found in the appendix, table A21. Despite the high number, these candidates probably amount to only a fraction of the gene family of glycosyltransferases: Using the amino acid sequence of the PSPG box, a conserved sequence motif found in plant secondary glycosyltransferases (Hughes and Hughes, 1994), a BLAST search against the peptide collection published with the DMv3.4 genome was performed. Results returned by this search suggested a gene family with more than 200 members in potato.

Figure 29: Unrooted phylogenetic tree of StGT, AtZOG, PvZOX, PlZOG and Zm cisZOG proteins Protein sequences of StGT1 to StGT46, Arabidopsis thaliana ZOG1 to ZOG3 (accession numbers: AtZOG1- Q9ZQ99; AtZOG2- Q9SK82; AtZOG3- Q9ZQ94), Zea mays cisZOG1 and cisZOG2 (accession numbers: ZmcisZOG1-Q93XP7; ZmcisZOG2-Q8RXA5), Phaseolus lunatus ZOG1 (Q9ZSK5) and Phaseolus vulgaris ZOX1 (P56725) were used for calculation of a phylogenetic tree. Sequences of known glycosyl-/ xylosyltransferase genes are marked in red, StGT sequences are noted in black. Tree calculation was based on a global alignment with free end gaps, BLOSUM62 cost matrix and Jukes-Cantor genetic distance model. The tree was built by the Geneious 5.4.6 tree builder module employing a neighbour-joining method.

There are numerous glycosyltransferases differing from each other in their substrate and, most likely, few of the 46 candidates singled out in this analysis are specific for zeatin. In order to further refine the search for potato zeatin-conjugating transferases, a phylogenetic tree based on protein sequences deduced from StGT genes and known zeatin glycosyl- and xylosyltransferases from Arabidopsis thaliana, maize and Phaseolus

74 Results sp. was calculated (Fig.29). Although the tree showed which of the potato sequences were most closely related to the known sequences of other species, it did not allow conclusions on zeatin binding capacity of the candidates. Recently, mutation studies on Phaseolus sp. glycosyl- and xylosyltransferases by Meek et al. (2008) led to the discovery of four regions with one essential amino acid each, which are required for zeatin binding. In a similar analysis of the 46 potato candidates, several potato sequences featured those motifs, but none also showed all four essential amino acids. Therefore, no final selection based on sequence information alone could be made.

4.2.4 Cytokinin signalling genes

The most prominent components of the cytokinin signalling pathway are the membrane- spanning cytokinin receptors and the A-type and B-type response regulators, transcription factors involved in CK response gene activation and feedback regulation of the signalling pathway. In the light of recent studies which showed localization of CK receptors within the ER membrane (Wulfetange et al., 2011), purine permeases (PUPs) which can transport cytokinins over the plasma membrane into the cell (Bürkle et al., 2003), might play an important role for CK signalling. Genes encoding these four protein groups were analysed in the potato genome.

4.2.4.1 Identification of cytokinin receptors

While compiling a MAPMAN mapping file for potato cytokinin-related genes, twelve ESTs from the POCI collection had been identified as putative cytokinin receptors, either because of their annotation as ‘sensor histidine kinase’, ‘Arabidopsis histidine kinase’ (AHK) or ‘cytokinin receptor’, or because of sequence similarity to known CK receptor genes from Arabidopsis. When blasting the EST sequences against the DMv3 genome, they were found to fit to five individual scaffolds. Alignments between the EST and the corresponding scaffolds showed that none of the ESTs covered a complete CK receptor gene and that, compared to CDS sequences from Arabidopsis, between 1,100 and 1,600 bp at the 5’ end, were missing. Lacking further EST sequence information, possible exons and introns were annotated by aligning different translation frames of the region upstream of EST coverage with CRE1b and AHK4 protein sequences from Arabidopsis.

The sequences resulting from this analysis were validated by nucleotide and protein blast. In all five sequences, the histidine kinase A (HisKA), histidine kinase-like ATPase (HATPase) and REC signal receiver domains were present and complete, confirming that StHK1 to StHK5 likely encode histidine kinases. The CHASE domain, however,

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which is required for cytokinin binding, could only be detected in StHK1, StHK2 and StHK5. As an example, figure 30 shows the domain structure of the StHK2 protein as determined by CDD search.

Figure 30: Verifcation of StHK2 via conserved domain search The protein sequence of StHK2 was used as a query for BLAST against the Conserved Domain Database (Marchler-Bauer and Bryant, 2004; Marchler-Bauer et al., 2009; Marchler-Bauer et al., 2011). The sequence contains the CHASE domain required for CK binding, the HisKA (histidine kinase A) domain needed for dimerization and trans-autophosphorylation, a histidine kinase-like ATPase domain and the REC signal receiver domain which is part of the phosphorelay system in the CK signalling pathway.

With release of the DMv3.4 genome in 2011, all five sequences could be compared to the list of CDS sequences (‘cds_representative’) published in addition to the genome sequence. These comparisons revealed mistakes in the hand-annotated StHK as well as the PGSC sequences:

StHK1 mapped to PGSC0003DMC400051320 and an alignment of the two sequences showed that the PGSC sequence was 36 bp longer due to a different intron/ exon border between exon 4 and intron 4. StHK2 is almost identical to PGSC0003DMC400056405, except that it was longer at the 3’ end. It seemed that the PGSC sequence ended prematurely because it retained 21 bp of an intron. This intron had been correctly annotated in StHK2 based on an alignment with POCI EST MICRO.8367.C1. StHK3 matched to PGSC0003DMC400051432, but was 188 bp shorter than the PGSC sequence because two small exons were lacking in the hand-annotated sequence. StHK4 was 273 bp shorter than, but otherwise identical to its counterpart PGSC0003DMC400006388, due to the wrong annotation of two small introns. StHK5 matched CDS sequence PGSC003DMC400010898, so that both intron/ exon borders of intron nine could be re-annotated, leading to a 198 bp longer ORF of StHK5. An overview of the five corrected sequences is given in table 11.

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Table 11: Overview of StHK genes identified in the potato genome This table lists names, scaffold numbers in the DMv3 genome and sequence lengths for five potato HISTIDINE KINASE genes. All sequences have been confirmed by and, if needed, corrected after alignment with the corresponding DMv3.4 ‘cds_representative’ sequences. Chromosome number was determined by BLAST search of each genomic sequence against the twelve DMv3.4 ‘pseudomolecules’ which represent the potato’s twelve chromosomes.

Scaffold No. Introns / length Name POCI ESTs Chromosome genomic protein PGSC0003DMS Exons CDS [bp] [bp] [aa] bf_arrayxxx_0037g12.t7 m.scf StHK1 bf_ivrootxx_0040h04.t3m 000000187 4 10 / 11 7615 2979 993 .scf MICRO.15394.C1 MICRO.8367.C1 StHK2 000000029 5 9 / 10 5176 2994 998 MICRO.9450.C1/C3 bf_suspxxxx_0014a11.t3 StHK3 m.scf 000000477 8 11 / 12 7791 3045 1015 MICRO.8924.C1 MICRO.8161.C1 StHK4 000000741 2 8 / 9 5628 3636 1221 MICRO.7190.C1

MICRO.15958.C1 StHK5 000001750 7 11 / 12 7314 2946 981 MICRO.8157.C1

For classification of the five histidine kinases, a phylogenetic tree of potato and Arabdidopsis HK protein sequences was calculated. StHK1, 2 and 5 formed pairs with the three Arabidopsis CK receptors CRE1b/AHK4, AHK3 and AHK2, respectively (Fig.31), strongly indicating that the three StHKs constitute the sub-family of CK receptors in potato.

Figure 31: Unrooted phylogenetic tree of potato HK and Arabidopsis CK receptor sequences Protein sequences of five potato histidine kinases and three A. thaliana CK receptors were used for calculation of a phylogenetic tree. Arabidopsis sequences are marked in blue and potato sequences are labeled in black. Tree calculation was based on a global alignment with free end gaps, BLOSUM62 cost matrix and Jukes-Cantor genetic distance model. The tree was built by the Geneious 5.4.6 tree builder module employing a neighbour- joining method. The scale bar at the bottom represents 0.2 substitutions per amino acid site.

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4.2.4.2 Identification of A-type and B-type response regulators

A- and B-type response regulators are transcription factors central to CK signalling output. They share the REC signal receiver domain (Fig.32), but B-type RRs are longer and contain a DNA binding domain the A-type RRs are lacking. For the identification of response regulators (RRs), seven POCI ESTs representing A-type response regulators and eleven ESTs representing B-type response regulators were available.

Figure 32: Conserved domains in A-type and B-type response regulators. The protein sequences of A-type response regulator AtARR4 (A) and B-type response regulator AtARR1 (B) were used as a query for BLAST against the Conserved Domain Database (Marchler-Bauer and Bryant, 2004; Marchler-Bauer et al., 2009; Marchler-Bauer et al., 2011). Both sequences encode the REC signal receiver domain. B-type response regulators additionally contain a SANT ('SWI3, ADA2, N-CoR and TFIIIB') DNA binding domain.

From the genomic analysis, eight A-type and five B-type response regulators were identified in potato. The genes were named StRRA1 to StRRA8 and StRRB1 to StRRB5, an overview is given in table 12. As before, all identified sequences were cross-checked by blasting against NCBI protein, nucleotide and conserved domain databases and confirmed to contain domains commonly found in these transcription factors. By comparison with the DMv3.4 CDS representative list, all eight StRRA sequences were confirmed to be identical to CDS sequences in this list. When comparing StRRB sequences with the DMv3.4 ‘CDS representative’ list, five additional CDS sequences that potentially represented B-type response regulators were identified. When these sequences were checked for conserved domains, however, only PGSC0003DMC400046203 and PGSC0003DMC400062422 contained both the REC signal receiver as well as the SANT DNA binding domain. Following the nomenclature, these two sequences were named StRRB6 and StRRB7. StRRB6 is represented on the POCI microarray chip by MICRO.3817.C1 and MICRO.3817.C3. StRRB7 is not represented on the POCI chip.

78 Results

Table 12: Overview of StRRA and StARRB genes identified in the potato genome This table lists names, scaffold numbers in the DMv3 genome and sequence lengths for eight potato A-type and seven potato B-type response regulator genes. Chromosome number was determined by BLAST search of each genomic sequence against the twelve DMv3.4 ‘pseudomolecules’ which represent the potato’s twelve chromosomes.

Scaffold No. Introns / length Name POCI ESTs Chromosome genomic protein PGSC0003DMS Exons CDS [bp] [bp] [aa]

StRRA1 MICRO.10525.C1 000000956 3 4 / 5 1548 603 201

StRRA2 MICRO.8742.C1 000000527 5 4 / 5 3161 747 249

StRRA3 MICRO.9222.C1 000000849 10 4 / 5 1903 681 227

StRRA4 MICRO.9222.C2 000000849 10 4 / 5 1853 681 227

MICRO.9362.C1 StRRA5 000000788 6 4 / 5 2266 447 148 MICRO.9362.C2

StRRA6 MICRO.9692.C1 000001055 2 1 / 2 588 492 163

StRRA7 - 000000759 4 4 / 5 3456 645 215

StRRA8 - 000001280 6 4 / 5 1853 471 156

147F09AF.esd StRRB1 MICRO.8717.C1 000000201 5 5 / 6 6986 1992 664 POAD373TV MICRO.14717.C1 StRRB2 000001518 1 5 / 6 4435 2028 676 MICRO.12644.C1

MICRO.14593.C1 StRRB3 000000187 4 5 / 6 7338 1962 654 POCCU71TP MICRO.9679.C1 BF_LBCHXXXX_0028F0 StRRB4 000002758 7 6 / 7 6350 2322 774 6_T3M.SCF STMJM10TV

StRRB5 MICRO.13665.C1 000000762 12 3 / 4 2732 1935 645

MICRO.3817.C1 StRRB6 000003002 6 10 / 11 4059 1665 555 MICRO.3817.C3

StRRB7 - 000000879 12 4 / 5 3712 1680 560

For comparison with Arabidopsis response regulators, all protein sequences of potato and Arabidopsis were used for calculation of a phylogenetic tree (Fig.33). In this tree, A- and B-type response regulators were clearly separated from each other. Except StRRB6, all potato StRRB sequences clustered with B-type response regulators of Arabidopsis. Similarly, all StRRA sequences were found in a subtree with Arabidopsis A-type response regulators.

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Figure 33: Unrooted phylogenetic tree of potato and Arabidopsis A- and B-type response regulators Protein sequences of eight A-type and seven B-type response regulators from potato and ten A-type and seven B-type RRs from A. thaliana were used for calculation of a phylogenetic tree. Arabidopsis A-type RR sequences are marked in red, Arabidopsis B-type RRs are marked in blue and potato sequences are labeled in black. Tree calculation was based on a global alignment with free end gaps, BLOSUM62 cost matrix and Jukes-Cantor genetic distance model. The tree was built by the Geneious 5.4.6 tree builder module employing a neighbour- joining method. The scale bar at the bottom represents 0.3 substitutions per amino acid site.

The StRRB6 protein, despite its clustering in a subtree with A-type response regulators, showed the B-type RR-specific domains REC and SANT. In a BLAST against NCBI’s protein database, its closest Arabidopsis ortholog was PSEUDO-RESPONSE REGULATOR 2 (PRR2), a protein similar to regular two-component response regulators, but lacking the conserved aspartate residue required for the phosphorelay (Perochon et al., 2010). As the alignment in figure 34 showed, StRRB6 also lacked this invariant residue, indicating that it might not act as a mediator of the CK signal.

Figure 34: Alignment of the REC signal receiver domain sequences of StRRB proteins The alignment was calculated by the Geneious Alignment Tool using standard settings. The conserved Asp residue required for phosphorylation is marked in bold letters and indicated by the black arrow.

80 Results

4.2.4.3 Identification of purine transporters (PUP)

Purine permeases are a family of membrane-spanning transporters some of which have been shown to preferentially transport iP and tZ into the cell (Bürkle et al., 2003). PUP transporters share a conserved EamA superfamily domain in the C-terminal part of the protein.

The POCI EST collection contained fourteen sequences representing putative PUP genes. These could be aligned to the genomic sequence to form ten StPUP genes. Three more StPUP sequences were identified by their similarity to the first identified StPUPs. An overview of the identified genes is given in table 13. Two sequences, StPUP4 and StPUP8, might be incompletely annotated, as they are shorter than the other eleven sequences. For comparison, all StPUP sequences were blasted against the ‘CDS_representative’ list of the DM3.4 genome and - if needed - corrected according to results from the ensuing pairwise alignments.

Table 13: Overview of StPUP genes identified in the potato genome This table lists names, scaffold numbers in the DMv3 genome and sequence lengths for 13 potato purine permease genes. Chromosome number was determined by BLAST search of each genomic sequence against the twelve DMv3.4 ‘pseudomolecules’ which represent the potato’s twelve chromosomes.

Scaffold No. Introns / length Name POCI ESTs Chromosome genomic protein PGSC0003DMS Exons CDS [bp] [bp] [aa] bf_mxflxxxx_0025e05.t StPUP1 000002718 12 1 / 2 1951 1107 369 3m.scf

StPUP2 MICRO.5974.C1 000002718 12 1 / 2 2092 1050 350 bf_ivrootxx_0031e08.t3 StPUP3 m.scf 000002107 6 1 / 2 2624 1026 341 MICRO.11601.C1 StPUP4 cSTB39I6TH 000002718 12 1 / 2 2193 693 231

StPUP5 MICRO.15202.C1 000000585 7 1 / 2 2680 1110 370

MICRO.13577.C1 StPUP6 000001069 1 0 / 1 1167 1116 372 MICRO.1929.C1 cSTE26M15TH StPUP7 MICRO.10221.C1 000003141 2 0 / 1 1593 1275 424 cSTE22E13TH StPUP8 - 000000379 10 0 / 1 789 789 263

bf_mxflxxxx_0065a08.t StPUP9 000000701 12 1 / 2 2549 1047 349 3m.scf

StPUP10 - 000002107 6 1 / 2 5405 1065 354

StPUP11 MICRO.14757.C1 000002166 12 1 / 2 1826 1068 355

StPUP12 - 000001642 12 1 / 2 2024 1077 359

StPUP13 MICRO.9961.C1 000001511 4 1 / 2 3467 1065 354

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Figure 35: Unrooted phylogenetic tree of potato and Arabidopsis PUP sequences Protein sequences of thirteen potato and nineteen A. thaliana purine permeases were used for calculation of a phylogenetic tree. Arabidopsis sequences are marked in blue and potato sequences are labeled in black. Tree calculation was based on a global alignment with free end gaps, BLOSUM62 cost matrix and Jukes-Cantor genetic distance model. The tree was built by the Geneious 5.4.6 tree builder module employing a neighbour- joining method. The scale bar at the bottom represents 0.4 substitutions per amino acid site.

In Arabidopsis thaliana, PUP1 and PUP2 specifically transport cytokinins whereas the other PUP proteins were shown to transport different adenine derivates (Gillissen et al., 2000; Bürkle et al., 2003). In a phylogenetic tree with Arabidopsis and potato PUP protein sequences, four of the identified potato sequences, StPUP3, StPUP9, StPUP10 and StPUP11, cluster in a subtree with the CK-transporting purine permeases of Arabidopsis (Fig.35), making them suitable candidates for analysis in the future.

82 Results

4.3 Beta-1,3-glucanases

A tuber’s transition from the dormant to the sprouting state is associated with phytohormonal, transcriptional as well as many structural changes. In many species, dormancy cycles and stages of meristem reorganisation such as the formation of overwintering buds in perennial plants (Rinne et al., 2001) or the transition from vegetative growth to flowering in the shoot apex of Arabidopsis (Gisel et al., 1999) depend on the temporary symplasmic isolation of the meristem. Most often this isolation is achieved by substantial deposition of callose at the neck regions of PD, resulting in a decrease of PD SEL and restriction of PD traffic (Rinne et al., 2001). Callose then needs to be removed, most likely mediated by β-1,3-glucanases as has been shown in poplar recently (Rinne et al., 2011), in order to re-establish symplastic connections and resume meristem activity.

Following the observation that, during tuber dormancy, bud meristems are symplasmically isolated from their surrounding tissue and that cell-to-cell connectivity is re-established prior to the onset of sprouting (Viola et al., 2007), a similar mechanism for the dormant-to-sprouting transition of tuber meristems was proposed. Beta-1,3- glucanases, as the major callose-degrading enzymes, were hypothesised to be involved in regulating dormancy release. Therefore, the gene family of beta-1,3-glucanases was thoroughly analysed in the present thesis and selected candidates were examined for their role in the regulation of bud breakage.

4.3.1 Identification of potato beta-1,3-glucanases in the potato genome

4.3.1.1 EST sequences representing beta-1,3-glucanases

As a starting point for the identification of beta-1,3-glucanase (B13G) genes in potato, the POCI EST collection was checked for ESTs annotated as B13G or showing sequence similarities to known B13G genes. The search resulted in 62 EST sequences of varying size. CDD search with a full length B13G protein sequence revealed the occurrence of two B13G-typical domains (Fig 36): The glyco_hydro_17 (GH_17) domain is the common domain of glycosyl hydrolases family 17 enzymes, to which beta-1,3- glucanases belong. The X8 domain, also known as CBM43 (carbohydrate binding module family 43), contains at least six conserved cysteine residues which presumably form three disulphide bridges. It is found at the C-terminus of several glycosyl hydrolase families and thought to be involved in carbohydrate binding (Barral et al., 2005).

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Figure 36: Conserved domains in beta-1,3-glucanases. The protein sequence of a Medicago truncatula beta-1,3-glucanase (XP_003626949) was used as a query for CDD search (Marchler-Bauer et al., 2011), revealing two B13G-typical domains.

When the EST sequences were examined for occurrence of these domains, many encoded only one or partial domains, already hinting at a degree of redundancy among the sequences.

4.3.1.2 Identification of full-length beta-1,3-glucanases

For identification of full-length B13G sequences in the potato genome, POCI ESTs were utilised in a blast search against the DMv3 scaffold sequences and subsequently aligned to their respective scaffold sequence in order to annotate introns and exons. Following the procedures described in the previous sections, the 62 EST sequences could be matched to 35 different scaffolds and from them, 34 full-length sequences were deduced. Solely for one EST, STMET40TV, no open reading frame could be found. Two other ESTs were left out of the analysis because one did not match to any scaffold in the DMv3 genome and the other encoded a domain of a different glycosyl hydrolase domain. An overview of the ESTs and scaffolds analysed and the resulting sequences, their nomenclature, lengths and predicted protein properties can be found in table A22 in the appendix.

SP Glyco_hydro_17 StB13G_03, _07, _13, _15a, _15b, _26, _28

SP Glyco_hydro_17 X8 StB13G_01, _02, _04, _05, _06, _10, _11, _12, _14, _16, _19, _21, _22, _23

SP Glyco_hydro_17 GPI StPdB13G_1

SP Glyco_hydro_17 StPdB13G_2

SP Glyco_hydro_17 X8 GPI StB13G_20

Glyco_hydro_17 StB13G_31

SP X8 StB13G_08, _09, _18, _25, _27, _29

SP X8 GPI StB13G_24, _30

Figure 37: Characterization of beta-1,3-glucanase gene family members in potato The left-hand panel shows different structures of beta-1,3-glucanase family members with the pink arrow representing the predicted signal peptide (SP), the red and blue boxes representing the glyco_hydro_17 domain and the X8 domain, respectively, as well as a grey box symbolizing a predicted GPI anchor site. The different sequence elements are not drawn to scale. On the left hand panel, beta-1,3-glucanase sequences identified in the potato genome are listed beside their corresponding structure. Two sequences with similarity to Arabidopsis PD-associated beta-1,3-glucanase AtBG_PAP are highlighted by the black box.

84 Results

For further characterisation of the members of this gene family, the StB13G sequences were sorted according to their protein architecture: All amino acid sequences were subjected to CDD search and prediction of signal peptides and GPI anchor sites. The two B13G-specific domains in combination with the predicted protein features theoretically allowed twelve different architectures. Only seven were actually found within the family, the three most prominent being the combination of signal peptide and GH_17 domain, of signal peptide, GH_17 and X8 domain and of signal peptide and X8 domain (compare Fig.37).

Figure 38: Unrooted phylogenetic tree of StB13G protein sequences and Arabidopsis and poplar beta-1,3-glucanases Protein sequences of StB13G_01 to StB13G_33, several Arabidopsis thaliana and ten poplar beta-1,3- glucanases were used for calculation of a phylogenetic tree. Arabidopsis sequences are marked in blue, poplar sequences are noted in red and potato sequences are labeled in black. The subbranch containing PD-associated and putative PD-localised proteins is highlighted by a red box. Tree calculation was based on a global alignment with free end gaps, BLOSUM62 cost matrix and Jukes-Cantor genetic distance model. The tree was built by the Geneious 5.4.6 tree builder module employing a neighbour-joining method.

Alongside the structural analysis, a phylogenetic tree based on amino acid sequences of all identified potato candidates, thirty-three Arabidopsis thaliana and ten poplar beta-1,3- glucanases was calculated (Fig.38). Strikingly, two of the potato sequences formed a separate branch, together with a PD-associated B13G from Arabidopsis (At5g42100; Levy et. al, 2007a) and two putative PD-localised B13G from poplar (GH_33 and GH_65; Rinne et al., 2011). For a better discrimination from the remaining potato sequences, these two candidates were renamed StPdB13G_1 and StPdB13G_2 for Solanum tuberosum putative Pd-associated beta-1,3-glucanase. In the structural analysis,

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StPdB13G_1 and _2 stood out by their unique protein architecture, comprising a C- terminal extension, but without an X8 domain. For StPdB13G_1 a GPI anchor was predicted, thus showing the same protein architecture as PD-associated beta-1,3- glucanase AtBG_PAP from Arabidopsis (Levy et al., 2007). Additional experiments involving these two candidates are described in section 4.3.2.

4.3.1.3 Clustering of identified beta-1,3-glucanases according to expression data

In another attempt to select candidate StB13G genes possibly involved in the regulation of dormancy release, available microarray data for the ESTs underlying the different genes was analysed for accordant expression patterns. As a first criterion, expression of the StB13G genes in an elicitor experiment was assessed. In this experiment, potato wild-type leaves had been treated with Pep13, an elicitor of plant defense responses from Phytophthora species (Brunner et al., 2002; Halim et al., 2004). Transcription data of treated and untreated plants was kindly provided by Sabine Rosahl, IPB Halle. ESTs representing six of the thirty-four B13G genes were up-regulated at least 2-fold after elicitor treatment, indicating that they may have a role in plant defense. Of the remaining 28 genes, expression of the candidate genes in a side shoot experiment (PhD thesis M. Senning, 2010) was examined. For this experiment, the tips of tissue-culture grown potato plants had been removed in order to re-activate side shoot meristems and induce growth. Side shoot samples had been taken before and two, four and twelve hours after capping of the shoot apical meristem (SAM). Transcript data was kindly provided by M. Senning and differentially expressed, statistically significant transcripts were obtained by one-way ANOVA tests of each time point compared to the non-induced ‘0h’ samples. In total, ESTs representing nine B13G genes, including StPdB13G_2, were found to be significantly up-regulated at one or more time-points after capping, suggesting a possible role in meristem re-activation. Data from a third microarray experiment was used to further divide the remaining twenty-one genes. A SRA experiment was used to near- synchronously induce sprouting in WT tuber discs and samples were taken by separating the newly formed sprouts from the parenchyma tissue directly below the sprouts. Transcription profiles of the separated tissues revealed six StB13G genes that were more strongly expressed in sprouts, including StPdB13G_1, and two genes that were mainly expressed in the parenchyma, pointing at distinct functions for these genes. An overview of the grouping is shown in figure 39.

86 Results

34 beta-1,3-glucanase genes

induced by elicitor Pep13?

YES NO

6 genes 28 genes

StB13G_03 StB13G_04 StB13G_06 StB13G_13 StB13G_22 expressed in expressed up-regulated in side not expressed/ StB13G_24 parenchyma in sprouts shoot meristem inconclusive (3d S vs P) (3d S vs P)

2 genes 6 genes 9 genes 11 genes

StB13G_10 StB13G_08 StB13G_01 StB13G_02 StB13G_33 StB13G_11 StB13G_05 StB13G_07 StB13G_16 StB13G_09 StB13G_15a, b StB13G_19 StB13G_12 StB13G_18 StB13G_21 StB13G_14 StB13G_20 StPdB13G_1 StB13G_29 StB13G_23 StB13G_30 StB13G_25 StB13G_31 StB13G_26 StPdB13G_2 StB13G_27 StB13G_28

Figure 39: Chart for categorization of potato beta-1,3-glucanases according to expression data The chart depicts the grouping of 34 beta-1,3-glucanase genes by their expression data in different microarray experiments. Genes possibly involved in plant defense were distinguished by being up-regulated after treatment with the defense elicitor Pep-13. Putative PD-associated genes (marked bold) had been identified by phylogenetic analysis and protein architecture. Genes up-regulated after induction of side shoots through SAM capping might be involved in meristem reactivation. Separate analysis of sprout and parenchyma tissue three days after induction of sprouting (‘3d S vs P’) by GA3 treatment revealed possible candidates specific for growth or developmental processes in the sprout and structural changes in the parenchyma underlying the sprout. For eleven genes adequate data was lacking (compare table A25 in the appendix). Several StB13G genes could not be grouped because expression data was lacking. One gene, StB13G_15b, was not categorized, because of the ESTs representing this gene, one was up-regulated in the parenchyma, two others were up-regulated in the sprout and one was not expressed. Expression data underlying this analysis can be found in the appendix, table A25.

4.3.2 Putative PD-associated beta-1,3-glucanases

In the preceding analyses, two potato beta-1,3-glucanases have been found to cluster with PD-localized glucanase AtBG_PAP from Arabidopsis and putative PD-associated beta-1,3-glucanases from poplar. In order to confirm that the two genes are expressed during tuber sprouting, a SRA experiment was conducted and RNA from samples taken at different time points after GA3 treatment were reverse-transcribed and subjected to real-time quantitative PCR (qPCR) with gene-specific primers. As shown in Figure 40, both genes were up-regulated during GA3-induced tuber sprouting. Noticeably, up-

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regulation already occurred before visible sprouting set in after three days and was more pronounced for StPdB13G_2. Taken together, expression data and structural analysis of these two genes point toward a possible role in tuber dormancy release and StPdB13G_1 and _2 were chosen for further analysis.

26 24 StPdB13G_1 22 20 StPdB13G_2 18 16 14 12 10 8 Relative Quantity (dR) 6 4 2 0 0d0 12h12 GA 1d24 GA 36h36 GA 2d48 GA 60h60 GA 3d72 GA 4d96 GA 5d120 GA hat

+ 50µM GA 3

Figure 40: Analysis of expression of putative Pd-associated Glucanases during GA3-induced tuber sprouting For qPCR analysis, gene specific primer pairs AH331/ AH332 (StPdB13G_1) and AH251/ AH 252 (StPdB13G_2) were used. Relative expression of StPDB13G_1 and StPDB13G_2 was normalized to ubiquitin (ubi3) expression and calibrated to expression at the ‘0d’ time-point. hat - hours after treatment

4.3.2.1 GFP-Fusions of StPdB13G_1 and StPdB13G_2

In order to study the localisation of StPdB13G_1 and StPdB13G_2, the open reading frames of the two genes minus the stop codon were amplified by PCR using primers AH98/ AH133 and AH97/ AH132, cloned into Gateway entry vector pENTR/ D-TOPO and confirmed by sequencing. Potato cDNA from a mix of potato tissues was used as a template. Via the Gateway LR reaction, the genes were transferred into expression vector pK7FWG2 carrying GFP as a reporter gene. By this, the ORF fragments were fused in frame to the GFP gene (Fig. 41A). As a reference, a similar construct with the PD-associated glucanase from Arabidopsis, AtBG_PAP, was created. Agrobacterium- mediated expression of the GFP constructs was imaged on a Confocal Laser Scanning Microscope (CLSM) two days after infiltration of Nicotiana benthamiana leaves. As seen in figure 41B, none of the three constructs showed a clear dot-like localization at the plasma membrane indicative of PD localisation. In contrast, A. thaliana line Col-16, which stably expresses a GFP-fusion construct of viral movement protein MP17, showed clear GFP fluorescence at Pd. Nonetheless, GFP fluorescence of the glucanase constructs was seen along the periphery of the cell, partly overlaying the propidium iodine-stained cell wall in a speckled manner. 88 Results

A Beta-1,3- p35S eGFP T35S attB2 attB1 Glucanase

1028 25 25 720 226 bp StPdB13G_1 : 1257 bp StPdB13G_2 : 1245 bp AtBG_PAP : 1275 bp

B

StPdB13G_1:GFP StPdB13G_2:GFP

AtBG_PAP:GFP MP17:GFP

Figure 41: C-terminal GFP-fusion constructs and their transient expression in Nicotiana benthamiana (A) Graphical representation of the GFP fusion proteins for two putative PD-associated beta-1,3-glucanases and PD-localized AtBG_PAP (Levy et al., 2007). p35S – CaMV35S promoter; T35S – terminator. (B) Agrobacterium-mediated transient expression of the beta-1,3-glucanase:GFP constructs in leaves of N. benthamiana two days after Agrobacterium infiltration and stable expression of MP17:GFP in Arabidopsis thaliana (bottom right); leaf epidermis cells were imaged under a Confocal Lase Scanning microscope (CLSM) and cell walls were counterstained with propidium iodine. The length of the white bar differed and is given within the image.

Parallel to the transient expression in N. benthamiana, the GFP fusion constructs were stably transformed into potato plants. Primary transformants were screened by Northern Blot for expression of the transgenes (Fig. 42A) and for each construct, at least three strongly expressing lines were identified. When the plants were grown in the greenhouse, they showed no obvious phenotype, except for StPdB13G_1:GFP lines 18 and 24 which were smaller than the wild-type (table 14). At harvest, several lines were found to produce smaller – and in one case also fewer - tubers, leading to a reduced yield (table 14). None of the transgenic tubers differed from the wild-type concerning their sprouting behaviour (data not shown). 89

Both source leaves and hand-sectioned dormant and sprouted tubers of the transgenic lines were imaged under the CLSM, but no GFP fluorescence was detectable. In order to see whether this was due to lack of protein expression, protein extracts from leaves were prepared and analysed by Western Blot. Immunodetection of the fusion proteins was carried out by using an anti-GFP antibody. As seen in figure 42B, a strong signal was detected in three of the four StPdB13G_2:GFP lines.

Table 14: Phenotypic characteristics of transgenic potato plants expressing StPdB13G_1:GFP, StPdB13G_2:GFP or AtBG_PAP:GFP Parameters were determined from soil-grown plants at harvest. Results are shown for two independent experiments and represent means ± SE of 5 plants per line. Statistically significant differences from the WT were determined using one-tailed t tests assuming unequal variance and are indicated by asterisks (P≤ 0.05).

Plant Stem Height Tuber Yield No. of Tubers per Plant

cm g plant -1 Wild-type (Solara) 52.6 ± 1.0 113.0 ± 9.2 6.6 ± 0.8

StPdB13G_1:GFP #10 52.5 ± 0.6 96.5 ± 11.4 6.9 ± 0.7 #11 53.9 ± 1.1 89.0 ± 5.4* 6.5 ± 0.5 #12 53.4 ± 1.0 66.3 ± 7.9* 5.5 ± 0.8 #18 44.7 ± 1.0* 22.3 ± 7.0* 2.6 ± 0.7* #24 50.4 ± 0.6* 89.7 ± 5.4* 5.4 ± 0.3 #25 53.7 ± 0.8 72.2 ± 9.5* 5.1 ± 0.7 #28 54.4 ± 1.2 106.5 ± 6.1 6.6 ± 0.6

StPdB13G_2:GFP #5 53.5 ± 1.1 107.3 ± 9.9 7.2 ± 0.8 #15 54.3 ± 1.3 106.7 ± 6.2 7.2 ± 0.8 #28 53.7 ± 0.9 112.2 ± 9.7 7.3 ± 0.8 #37 53.8 ± 1.2 85.3 ± 7.5* 5.0 ± 0.5

AtBG_PAP.GFP #5 54.1 ± 1.0 108.1 ± 7.0 7.6 ± 1.1 #15 53.4 ± 0.9 113.8 ± 8.2 8.1 ± 1.2 #16 52.8 ± 0.9 110.4 ± 6.6 7.3 ± 0.5

90 Results

A StPdB13G_1:GFP StPDB13G_2:GFP AtBG_PAP:GFP WT 10 11 12 18 24 25 28 WT 5 15 28 37 WT 5 15 16 GFP

RbcS

B GFP

Figure 42: Detection of StPdB13G_1:GFP, StPdB13G_2:GFP and AtBG_PAP:GFP in stably transformed potato (A) Northern Blot screening for transgene expression in potato leaves of StPdB13G_1:GFP, StPdB13G_2:GFP and AtBG_PAP:GFP- transformed plants. 30 µg of total RNA had been loaded onto the gel and, after transfer of the RNA, the membrane was hybridized with radioactively labeled GFP-specific probe. Hybridization with the small subunit of Rubisco served as a loading control. (B) Western Blot with antiGFP antibody. 20 µl of total protein extract from source leaves was loaded onto the gel and after blotting, fusion proteins were detected by anti-GFP antibody.

In the case of the other two fusion constructs, weak signals were detected in lines 11, 12 and 28 (StPdB13G_1:GFP) as well as line 5 (AtBG_PAP:GFP), not explaining the lack of visible GFP fluorescence.

4.3.2.2 Generation of stably transformed PdB13G_RNAi and 35S:AtBG_PAP potato plants

To investigate whether the two putative PD-associated glucanases are directly involved in the regulation of potato tuber dormancy, transgenic plants with increased or decreased PD-glucanase levels were generated. A decrease in StPdB13G_1 and _2 expression was aimed at by an RNAi (RNA interference) approach. A 384 bp fragment of the StPdB13G_1 gene, of a region with high sequence identity to StB13G_2 in order to target both genes, was amplified by PCR (primers AH99/ AH100) and cloned into Gateway entry vector pENTR/ D-TOPO via the 5’-CACC-3’ sequence added to the forward primer. The integrity of the fragment sequence was confirmed by sequencing. The gateway LR reaction with destination vector pK7GWIWG2(II) generated the expression vector PdB13G_RNAi (Fig. 43A) whose expression yields a self- complementary hairpin RNA. The construct was transformed into potato and 45 primary transformants were generated. In a primary screening via Northern Blot (data not shown), these transformants were inspected for a reduced StPdB13G_1 signal and fourteen lines were selected for detailed analysis via qPCR. In the qPCR screen, only line 30 was found to be reduced in expression of both putative Pd-associated glucanases (Fig 43C). The other lines either showed a reduction in only the StPdB13G_1 gene or displayed merely a slight reduction in both genes which did not differ significantly from

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WT expression levels. Apart from line 30, lines 3, 4, 20 and 24 were chosen for a more detailed analysis of morphological parameters.

For overexpression (OE), the complete ORF of the AtBG_PAP gene from Arabidopsis (At5g42100) was amplified by PCR (primers AH309 and AH310), cloned into vector pCRblunt and confirmed by sequencing. Via the restriction sites Acc65I and BamHI, which were added to the primers, AtBG_PAP was excised from pCRblunt and inserted into the binary expression vector pBinAR (Fig. 43B). The construct was stably transformed into potato and more than forty primary transformants were generated. The lines were tested for AtBG_PAP overexpression by Northern Blot with leaf RNA, yielding thirteen strongly expressing lines, five of which were selected for further analysis (Fig.43D).

A B HindIII/SacI EcoRI XbaI/ApaI HindIII/SacI Acc65I BamHI XbaI/ApaI

StPdB13G_01/02 Intron CmR Intron StPdB13G_01/02 T35S p35S OCS p35S fragment AtBGPAP attB2 attB2 fragment attB1 attB1

1035 25 384 25 275 702 376 25 384 25 225 bp 1035 1278 710 bp

C 2 D

) 1,8 StPdB13G_1 StPdB13G_2 dR 1,6 1,4 1,2 1 0,8 0,6 0,4

Relative Quantity ( 0,2 0 WT 3 4 20 24 30

wildtype PdB13G_RNAi

StPdB13G RNAiStPdB13G #3 RNAi #4 Figure 43: Expression StPdB13G of RNAiStPdB13G PdB13G_RNAi #20 RNAiStPdB13G #24 RNAi #30 and 35S:AtBG_PAPin potato (A) Graphic representation of the PdB13G_RNAi construct containing the CaMV35S promoter (p35S) and terminator (T35S) and sense and antisense StPdB13G_01/02 fragments cloned via the att recombination sequences. (B) Graphic representation of the AtBG_PAP OE construct containing the AtBG_PAP gene, which encodes an Arabidopsis PD-localised beta-1,3-glucanase, under control of the CaMV35S promoter. (C) Screening of pre-selected PdB13G_RNAi lines by qPCR. Gene-specific primers for StPdB13G_1 (AH331/ AH332) and StPdB13G_2 (AH251/ AH252) were used to test for residual expression of these genes. Expression was normalized to ubi3 expression and calibrated to expression level in the WT (set to 1). (D) Screening of AtBG_PAP OE primary transformants. 25 µg of leaf RNA from tissue culture plants were loaded onto the gel and, after transfer to the membrane, hybridized with a radioactively marked probe. Hybridization with the small subunit of Rubisco (RbcS) served as a loading control. WT and 5 selected transgenic lines are shown.

The plants were subsequently grown in the greenhouse, harvested and tubers were analysed for alterations in sprouting behaviour.

Phenotypically, neither the PdB13G_RNAi nor the AtBG_PAP OE plants differed significantly from the WT. At harvest, tuber number and tuber yield were determined.

92 Results

Except for line 24 which produced only small tubers, PdB13G_RNAi lines showed no alterations in these attributes when compared to WT (table15). Apart from AtBG_PAP OE line 13 which displayed a significantly reduced tuber yield, AtBG_PAP- overexpressing plants were comparable to the wild-type concerning yield and tuber number.

Table 15: Phenotypic characteristics of transgenic potato plants expressing the PdB13G_RNAi or the AtBG_PAP OE construct Parameters were determined from soil-grown plants at harvest. Results are shown for one (AtBG_PAP OE) or two (PdB13G_RNAi) independent experiments and represent means ± SD of 3 to 5 plants per line.

Plant Stem Height Tuber Yield No. of Tubers per Plant

cm g plant -1 Wild-type (Solara) 58.6 ± 1.7 183.3 ± 40.2 8.6 ± 2.7

PdB13G_RNAi #3 56.8 ± 2.4 161.3 ± 23.1 6.9 ± 1.7 #4 56.8 ± 1.7 153.0 ± 28.4 7.3 ± 1.4 #20 56.3 ± 2.2 165.9 ± 31.9 7.3 ± 2.2 #24 55.9 ± 2.7 49.1 ± 23.4* 7.3 ± 1.7 #30 56.8 ± 2.7 174.8 ± 23.1 8.4 ± 1.4

Wild-type (Solara) 59.1 ± 1.1 154.0 ± 7.8 5.4 ± 0.9

AtBG_PAP OE #4 58.9 ± 0.9 131.0 ± 12.4 4.8 ± 1.3 #9 57.7 ± 1.7 146.2 ± 8.0 6.0 ± 1.6 #13 56.6 ± 2.8 78.0 ± 22.5* 3.6 ± 0.5 #27 57.8 ± 3.1 121.0 ± 6.3 4.4 ± 1.8 #36 57.0 ± 2.6 144.8 ± 7.9 6.4 ± 1.5

After harvest, tubers were stored at room temperature in the dark for several weeks and sprouting was scored regularly. Progressive sprouting of WT and transgenic lines is shown in the graphs in figure 43A and B. When sprouting of StPdB13G_RNAi tubers was observed for the first time with tubers of one plant per line, it seemed that line 30, which is strongly reduced in StPdB13G_1 and _2 expression, sprouted slightly later than the wild-type (data not shown). This would be in accordance with the hypothesized requirement of beta-1,3-glucanases for re-opening callose-blocked plasmodesmata. However, when the experiment was repeated with more tubers, such a delay in sprouting was not observed anymore (Fig. 44A). The first AtBG_PAP OE tubers of five plants per line have been harvested recently (Dec. 1st, 2011) and sprouting behaviour has been observed for fourteen weeks. During this time, tubers of selected transgenic lines seemed to start sprouting approximately one

93

week later than the wild-type (Fig.44B), in contrast to the hypothesis that increased B13G levels would lead to earlier sprouting. At 14 weeks after harvest, WT tubers have started sprouting completely whereas in the transgenic lines, between 44% and 85% of tubers showed visible sprouts.

A B 100 100 WT 90 WT 90 #3 # 4 80 80 #4 # 9 70 70 #20 # 13 60 #24 60 # 27 50 #30 50 # 36 40 40 30 30 20 20 10 10 0 0 6 8 10 12 14 6 8 10 12 14

Figure 44: Sprouting of PdB13G_RNAi and AtBG_PAP OE-expressing tubers After harvest, tubers of WT and transgenic lines were stored in the dark at room temperature and sprouting was observed regularly until all tubers had sprouted. (A) Sprouting of WTand selected PdB13G_RNAi lines, calculating sprouted tubers as percentage of total tubers per line. Data points and error bars are means ± SD of two independent experiments with 5 to 52 tubers per line. (B) Sprouting of WT and selected AtBG_PAP OE lines. Preliminary results for the first plant set harvested Dec 1, 2011; Percentages were calculated from the total number of tubers per line (n=11-32).

In potato tubers, decreasing the expression of putative PD-associated B13G seemed to have no effect on sprouting. Conversely, overexpression of a PD-localised B13G gene appeared to delay sprouting. Therefore, if beta-1,3-glucanases are involved in the regulation of tuber sprouting, it is probably in a different manner than hypothesized before.

4.3.3 Callose turnover during GA3-induced sprouting of wild-type tubers

As the previous results indicated that the reviewed putative PD-associated beta-1,3- glucanases most likely do not play the proposed role in dormancy regulation, the question arose whether callose turnover and degradation are part of the regulatory mechanisms controlling dormancy termination in potato. Experiments with carboxyfluorescein applied to dormant and non-dormant tubers had showed that the bud meristem is isolated from its surrounding tissue during the dormant phase and it was postulated that symplasmic isolation needs to be removed before sprouting can occur (Viola et al., 2007). Reversible deposition of callose at Pd has been assumed responsible, but callose deposition prior and during sprouting has never been examined. In order to address the question if callose deposition during the bud meristem’s transition

94 Results from dormant to sprouting is changed, sprouting of wild-type tubers was artificially induced by GA3 application in a SRA and callose was detected using an anti-callose antibody. Samples were taken immediately before and one, two, three and six days after the treatment and were wax-embedded using a Leica TP2010 tissue processor. Sections of 10 µm thickness were cut with a rotary microtome, the wax was removed and tissues were re-hydrated in a graded ethanol series before being equilibrated in TBS buffer. Callose within the tissues was detected by incubation with anti-callose and a Cy2 (GFP)- labelled secondary antibody which could be imaged under a CLSM. Figure 45 shows a selection of images from dormant buds (upper panel), sprouts (middle panel) and a non- sprouted water control.

Figure 45: Callose detection in potato tuber (modified from C. Prasch) Detection of callose in dormant (0d) and GA3- or water-treated tuber buds. Transmitted light images were made using a 10x dry optic lens. Fluorescence images were taken with a 20x immersion lens. The black bar represents 250µm, the white bar represents 100 µm.

Green fluorescence indicating callose presence could be detected both in the dormant and the sprouting state during the course of the experiment. An accumulation of callose around the dormant meristem’s periphery, as hypothesized for its state of symplastic isolation, could not be observed. In fact, callose was mainly localised to vascular strands well below the meristem (C. Prasch, MSc. thesis).

95

When the callose depostitions in the vasculature were examined more closely, it seemed that more callose was detectable after the onset of sprouting. This observation is likely due to an increased number of vascular strands leading to the newly formed sprout, as evidenced by different staining methods (Fig. 46). Microscopic images of stained dormant and sprouted samples suggested that extensive de novo formation of vascular tissue occurred in the course of GA3-induced tuber sprouting.

Ruthenium Red Phloroglucinol staining staining

0 d dormant tuber

5 d GA3 sprouted tuber

Figure 46: Formation of new vascular bundles at onset of sprouting Left column: Detail images of ruthenium red staining of 10 µm sections from dormant (top) and sprouted (bottom) wax-embedded tuber discs. Ruthenium red stains non-lignified pectin-containing structures such as plant cell walls. Tissue sections were stained with 0.05% (w/v) aqueous Ruthenium red solution. Right column: Detail images of phloroglucinol-stained 100 µm vibratome sections from dormant (top) and sprouted (bottom) tuber discs. Phloroglucinol stains lignin in the xylem stands. Tissue sections were stained with 0.1% phloroglucinol solution.

Together with the previous findings concerning the effects of altered B13G gene expression, these results suggest that callose deposition and callose degradation probably play a different role in dormancy release than proposed earlier. The newly formed vascular bundles are most likely needed to support the outgrowth of the young sprout which remains a strong sink tissue for metabolites and micronutrients until it has formed fully functional leaves and roots. This marked vascular development might be a process accompanying the formation of a new sprout or it could even constitute a new layer of regulation of dormancy release.

96 Discussion

5 Discussion

Dormancy in general and dormancy of potato tubers in particular have been a subject of research for a long time and considerable progress in this field has been made within the last couple of years. Especially on the molecular level, transcriptomics, phytohormones and changes of cellular structures have come to the fore. Nevertheless, information about the regulatory mechanisms of dormancy termination remained elusive. Understanding the processes underlying tuber dormancy release and how they are regulated could advance processing industries and agribusinesses by providing tubers with tailor-made dormancy length or improved storage stability.

5.1 Alteration of endogenous cytokinin content

The effect of exogenously applied cytokinins on potato tubers has already been described in the 1970ies: Apical sections of tubers treated with either zeatin or kinetin (N6-furfuryladenine, a cytokinin) sprouted after two to three days (Hemberg, 1970). Numerous later studies described an increase of endogenous cytokinins coinciding with dormancy break (Turnbull and Hanke, 1985b, 1985a; Sukhova et al., 1993; Suttle, 1998). Changes in the CK composition during the tuber’s life cycle, as well as changes in responsiveness to exogenously applied CKs have been described (Suttle, 2001). These observations indicated that cytokinins likely play a role in the regulation of potato tuber dormancy. Although transgenic potato plants with elevated CK levels were available (Ooms and Lenton, 1985; Gális et al., 1995; Sergeeva et al., 2000), they have never before been studied with regard to their dormancy and sprouting behaviour.

Several publications report that overexpression of a bacterial IPT gene led to increased levels of CKs and morphological changes like impaired root growth and reduced apical dominance in transgenic tobacco and Arabidopsis (Medford et al., 1989; Smigocki, 1991; Eklöf et al., 1996). By transformation of potato plants with Agrobacterium tumefaciens T- DNA or the ipt gene alone, transgenic plants with up to 200-fold elevated CK content could be generated (Ooms and Lenton, 1985). Phenotypically, these transformed potato plants differed greatly from the WT, forming only short shoots, but no roots so that they could only be propagated in tissue culture (Ooms and Lenton, 1985). Intact potato plants forming normal tubers were only obtained with low expression of the transgene (Gális et al., 1995). In the present thesis, 35S:IPT potato plants with a strong expression of the transgene showed a stunted, bushy growth and inhibition of root formation. One line, IPT-6, showed only low expression of the transgene and resembled the WT

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phenotypically. As these results are similar to previous findings, it could be assumed that the transgenic lines used in this study had an altered CK content. Sprouting of strong IPT expressors could not be assessed as these lines either did not produce tubers at all or, as in the case of line IPT-9, formed only few very small tubers which were not suited for analysis of sprouting behaviour. Low expression of the IPT gene in IPT-6 did not inhibit tuber formation. IPT-6 tubers stored at room temperature sprouted only little earlier than the wild-type. The sprouts formed by IPT-6 tubers were longer and thicker than those of WT tubers. As cytokinin has been shown to be crucial for shoot apical meristem size (Werner et al., 2001), an increase in meristem size due to an elevated CK level is likely for IPT-6 tubers. In an in vitro sprouting assay, IPT-6 tubers were shown to start sprouting significantly earlier than the WT, illustrating the importance of CK for dormancy release. It can be speculated that the effect of earlier sprouting due to raised endogenous CK levels would have been more evident if strong expressors had formed enough tubers for analysis or that it could have been enhanced by usage of more specific promoters. However, increased endogenous CK levels did lead to premature sprouting in transgenic plants expressing a bacterial 1-deoxy-D-xylulose 5-phosphate synthase (DXS). At harvest, these plants had already undergone bud break and hormone measurements confirmed that they mainly differed from the wild-type by increased trans-Zeatin levels (Morris et al., 2006).

Expression of cytokinin oxidase genes has been widely used to study CK deficiency in plants. Transgenic Arabidopsis plants expressing different AtCKX isoforms showed a 3 to 10-fold increase in CK activity and a residual CK content of 30-45% of the WT level (Werner et al., 2003). CKX-transgenic Arabidopsis plants were found to have greatly enhanced root growth, reduced biomass and shoot size, an increased number of lateral root branches and a diminished activity of the SAM (Werner et al., 2003; Yang et al., 2003). Similarly, expression of individual Arabidopsis thaliana CKX genes (AtCKX1 to AtCKX4) in transgenic tobacco provoked a reduced CK content and a retardation of shoot and leaf growth while simultaneously enhancing root growth and lateral root formation (Werner et al., 2001). When potato plants were transformed with a 35S:CKX1 construct, positive transformants also showed a reduction in shoot growth, strongly increased root growth and a reduction of the number of leaflets per compound leaf. The latter is in accordance with findings from tomato where compound leaf development is controlled by cytokinin (Shani et al., 2010). The reduction in leaflet number was inversely correlated with CKX1 expression, so that the stongest expressing line, CKX1-4, only formed single lanceolate leaves. CKX1-4 even showed changes in tuber morphology,

98 Discussion forming only small, drop-shaped tubers. Phenotypic alterations concerning root, shoot and leaf phenotypes are in accordance with previous descriptions of CK-deficient plants. Therefore it could be assumed that transgenic 35S:CKX1 plants examined in this thesis were indeed reduced in their CK content. When natural sprouting of 35S:CKX1 tubers was studied, the transgenic lines were delayed up to six weeks, with the length of the delay correlating to CKX1 transcript abundance. Sprouts formed by the strongest line, CKX1-4, were diminutive and did not grow out further. In an in vitro sprouting assay,

CKX1-4 tuber discs did not sprout upon GA3 treatment and the sprouting phenotype could only be restored by application of cytokinin, for example 6-benzylaminopurin (BAP). Taken together, these results confirm that cytokinin is essential for the termination of tuber dormancy and that GA requires CK in order to induce sprouting.

5.1.1 Transcriptional analysis confirmed that cytokinin is needed for bud break, but not sufficient for sprout growth

SRA experiments with wild-type tubers had shown that both GA3 and BAP, a cytokinin, are able to induce sprouting. When GA3 failed to trigger sprouting in CK-deficient tubers, this led to the conclusion that GA requires the presence of CK and that CK is essential for the inititation of bud break. This assumption was supported by two observations: When sprouting was induced with BAP, bud break occurred, but the sprouts did not grow out further. And in a ‘reverse’ SRA experiment to GA treatment of CK-deficient tubers, GA20-ox tubers which accumulate higher levels of gibberellins, were induced to sprout by BAP treatment (Hartmann et al., 2011).

CKs are known for their ability to stimulate cell division in vivo and in vitro (Werner et al., 2001). They regulate G1/ S-phase transition during the cell cycle by inducing CycD-type cyclins, for example through induction of CycD3 (Tréhin et al., 1998; Riou-Khamlichi et al., 1999). In addition, elevated CycD3 transcript levels have been found in Arabidopsis mutants with high CK content (Chaudhury et al., 1993). The onset of sprouting was also found to be accompanied by a marked increase in cell division (Campbell et al., 1996). In accordance with these findings, almost all cell cycle regulators, including CycD3, A- and

B-type cyclins and CDC20, were up-regulated in WT and IPT-6 upon GA3 treatment, but not in transgenic tubers expressing CKX1. Genes described as molecular markers for the meristem’s transition from dormant to sprouting, such as histone H4 and dUTPase (Senning et al., 2010) were activated considerably in WT and IPT-6 tubers, but were not or only slightly up-regulated in CKX1-4 tubers. Genes involved in cell wall biosynthesis and modification, fatty acid biosynthesis and transcription factors were found to show different expression patterns between the sprouting WT and IPT-6 tubers and the non- 99

sprouting CKX1-4 tubers. More genes of these two functional categories, which are involved in processes necessary to initiate cell division and growth, were activated in WT and IPT-6 tubers upon GA3 treatment than in CKX1-4 tubers, reflecting that sprout growth had been initiated in the former, but not in the latter tubers. Werner et al. (2001, 2003) suggested that CKs are not only required to uphold cell division but might also be involved in cell differentiation. Accordingly, a strongly increased expression of transcription factors like GRF3 or OVATE which control organ differentiation and (out)growth (Kim et al., 2003; Hackbusch et al., 2005), was detectable in WT and IPT-6 tuber discs, but absent in CKX1-4 discs, indicating that organ differentiation is not promoted in CK-deficient tubers.

Furthermore, differences in the response to GA3 were found with respect to auxin biosynthesis and signalling. Auxin has been reported to be essential for primordium initiation and outgrowth of lateral organs (Carraro et al., 2006; Ding and Friml, 2010). Accordingly, auxin-responsive SAUR and GH3 genes were poorly induced in CKX1-4 tubers after GA3 treatment compared to WT and IPT-6. Transcripts with homology to transcription factors ARF4 and ARF5, reported to control plant organ development and to be involved in vascular patterning (Hardtke and Berleth, 1998; Dettmer et al., 2009; Scarpella et al., 2010), accumulated in WT and IPT-6 tubers, but were less strongly induced or even down-regulated in CKX1-4 tubers. Interestingly, ESTs representing

ARF8 were 3.6 to 4-fold induced by GA3 in CKX1-expressing tubers, but accumulated not as much in WT and IPT-6. In Arabidopsis, ARF8 was described to be involved in a transcriptional feedback loop between auxin and brassinosteroid regulating growth processes in an interdependent manner (Jung et al., 2010). Distinct differences between the sprouting and non-sprouting genotypes were also seen in the accumulation of transcripts representing auxin transport proteins which occurred solely in the WT and IPT-6, such as PIN proteins which are required to generate the local auxin maxima necessary to initiate organ outgrowth (Reinhardt et al., 2000; Benková et al., 2003). Besides auxin signalling and transport genes, at least two YUCCA-like genes of the auxin biosynthesis pathway appeared to be transcriptionally activated in wild-type and IPT-6 tubers. This correlates with observations of Sorce et al. (2000) who measured increasing IAA concentration near the end of tuber dormancy. From their results of immunolocalization studies that showed accumulation of auxin in the apical meristem and the vascular tissue beneath in dormant tubers, the same authors later proposed that auxin is involved in early processes accompanying dormancy break (Sorce et al., 2009). Further support for this assumption arises from expression studies of another auxin

100 Discussion signalling component, ARF6. Faivre-Rampant et al. (2004) found a strong up-regulation of ARF6 expression at dormancy release, especially in the developing vasculature, and debated it as a marker for meristem activation. In the transcript profiling experiment at hand, ARF6 was not differentially expressed in any of the three genotypes which might be a result of the time points examined: at 0d, bud meristems were still dormant whereas after three days, sprouting had already begun. Nevertheless, the present transcript data endorse a role for auxin in the onset of tuber sprouting, possibly by inciting formation and differentiation of vascular tissues and determination of leaf primordia formation through polar auxin transport. These auxin-dependent processes seem to be mediated by GA3 and require CK biosynthesis and signalling.

ESTs homologous to component of the ethylene signalling pathway also showed different expression patterns in the three GA3-treated genotypes. Expression of transcripts representing ethylene receptors RTE1, ERS1, ETR2 and EIN4 showed a roughly similar expression level at 0d in WT and CKX1-4 tuber discs, but are lower expressed in IPT-6 tuber discs. Upon GA3 treatment, these ESTs accumulate in all three genotypes with CKX1-4 showing the highest induction. In Arabidopsis, a direct correspondence between receptor mRNA levels and ethylene binding activity has been reported (O'Malley et al., 2005). Therefore, expression of these ESTs might reflect differences in ethylene content of the three genotypes. ESTs homologous to ERF1 and

ERF5 were induced upon GA3 treatment in WT and CKX1-4, with a stronger activation in the transgenic line. IPT-6, however, showed no such induction and ERF5 transcription levels were even down-regulated after 3d. ERF1 and ERF5 are primary ethylene reponse genes which in turn activate expression of secondary response genes by binding to the GCC box in the promoters of these genes (Solano et al., 1998). This indicates a different signalling output for IPT-6, but the difference to the wild-type may be due to the time point examined, as IPT-6 started sprouting at two days after GA3 treatment whereas in the WT, visible sprouting occurred only after three days. Differences in transcript levels could also be seen for potato EBF1 and EBF2 homologues which are up-regulated in all three genotypes with the highest induction found in CKX1-tubers. EBF1 and EBF2 are F-box proteins that target the central transcription factors EIN3 and EIL1 to proteasome-dependent degradation (Stepanova and Alonso, 2009; Yoo et al., 2009). The key transcription factors EIN3 and EIL1 in turn activate expression of ERF1 and other primary response genes (Solano et al., 1998).

Interestingly, potato EIN3 transcript levels decreased in all three genotypes after GA3 treatment, most prominently in CKX1-4 tubers. This might be caused by the strong up-

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regulation of EBF1 and EBF2 homologues, which likely constitute a layer of regulation of the signalling pathway (Stepanova and Alonso, 2009), observed in these tubers. Conversely, a MKK9 (mitogen-activated protein kinase kinase 9) homologue was 5.5-fold up-regulated in CKX1-4 tubers after GA3 induction, but not WT or IPT-6 tubers. MKK9 is part of a positively acting signalling cascade and may stabilize EIN3. Although these results seem to be conflicting, it has to be taken into account that very little is known about ethylene signalling in potato and that even in Arabidopsis the pathway has not been fully elucidated yet (Stepanova and Alonso, 2009). Ethylene has been shown to play a pivotal role in the initiation and maintenance of tuber dormancy, but concerning its influence on dormancy termination, experimental results have been conflicting (Suttle, 1998; 2004b; 2009). It has been reported that transient ethylene treatment accelerated dormancy release whereas continuous treatment led to inhibition of sprout growth (Rylski et al., 1974). Suttle (2004b) reviewed reports on increasing ethylene production accompanying the onset of sprouting. Inspection of the present array data also revealed up-regulation of transcripts coding for ethylene biosynthesis genes 1-amino- cyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase after GA3 treatment, with different ESTs induced in the sprouting WT and IPT-6 tubers and the non-sprouting CKX1-4 tubers (table A24). As the expression data for ethylene receptors, this also indicates an increased ethylene formation. The overall induction of the ethylene signalling pathway seemed to be much stronger in CKX1-4 tubers than in the other two genotypes, suggesting that increased ethylene signalling negatively influences sprout outgrowth. In WT and IPT-6 tubers, a reduced ethylene response in concert with augmented auxin biosynthesis, transport and signalling might support cell differentiation and sprout outgrowth following GA3 treatment. An examination of earlier time-points after

GA3 application might further elucidate the initial steps and the crosstalk between the two hormonal signalling pathways.

Differences between the three genotypes were less prominent concerning the CK and GA metabolism and signalling pathways. Most transcripts of the GA biosynthesis pathway genes were clearly down-regulated in all three genotypes examined, likely due to a feedback mechanism as had been shown in other species (Martin et al., 1996; Dai et al., 2007). Accumulation of ESTs coding for GA-responsive genes in tubers of WT and IPT-6, in contrast to their non-induction in CKX1-4 tubers, highlights the difference between sprouting and non-sprouting tuber discs. Transcripts coding for CK biosynthesis and modification, with the exception of two ESTs with homology to zeatin xylosyltransferase genes of Phaseolus sp., were lower

102 Discussion

expressed in CKX1-4 at 0d and down-regulated after GA3 treatment. In WT and IPT-6, on the other hand, the same transcripts were mostly up-regulated at 3d, indicating that GA induces CK metabolism at least to some extend. So far, crosstalk between the GA and CK pathways has been reported for CK and GA signalling in Arabidopsis (Greenboim-Wainberg et al., 2005), but not for metabolic pathways of the two hormones. In CKX1-4 tubers, ESTs coding for A-type response regulators were very low expressed at both time points examined while they were partly up-regulated in WT and IPT-6 tubers. Recently, cross talk between GA and CK has been examined in tomato, observing inhibition of CK response genes, namely A-type response regulators, by GA and inhibition of GA responses by CK (Fleishon et al., 2011). However, reactivation of meristematic activity required for sprouting clearly depended on the availability of cytokinin, as evidenced by CKX1-expressing lines which did not sprout upon GA3 treatment and by suppressed cell proliferation and differentiation in these transgenic tubers. Ultimately, all data points to CK as the essential regulatory instance in dormancy release. CK stimulates bud break, but cannot support further sprout growth by itself. Gibberellin requires cytokinin in order to incite meristem re-activation and is adequate for sprout outgrowth after bud breakage has taken place.

5.1.2 Ectopic expression of IPT changed meristem fate

Transgenic potato plants with altered CK content have proven the essential role of CKs in tuber dormancy release. Constitutive expression of CK metabolism genes, however, led to severe growth defects and reduced tuber yield in transgenic plants. Therefore, IPT and CKX1 were also brought under control of the A. thaliana UFO promoter, which is expressed in vegetative and floral meristems (Long and Barton, 1998), for a meristem- specific expression in transgenic potato plants.

UFO:CKX1 transgenic plants showed a reduced shoot size, but were otherwise similar to the WT. In the shoot apical meristem, CKs are required for meristem maintenance by positively regulating cell division (Shani et al., 2006). Transgenic tobacco and A. thaliana plants expressing different CKX genes had been shown to have a reduced shoot size and smaller meristems (Werner et al., 2001; 2003). Thus, reduced shoot size of UFO:CKX1 potato plants is likely due to to reduced SAM size in the transgenic potato plants. The reason why UFO:CKX1 plants lack other phenotypic abnormalities probably lies in the phytohormone’s nature: Cytokinins can act as both short-distance and long- distance regulatory signals, they are synthesized in different parts of the plant and transported to their site of activity (Kudo et al., 2010). Therefore it is unlikely that

103

localized CKX1 expression would affect morphology of other tissues than its site of expression.

Surprisingly, UFO:IPT expression displayed a very distinct phenotype. In tissues containing a meristem, such as the shoot tip, axillary buds at the junction of stem and petiole, the tips of side branches and even some stolons, rudimentary inflorescences with petite flower-like structures were formed, suggesting at least partial re-programming of those meristems from the vegetative to the floral fate. Floral transition has been intensely studied in Arabidopsis and four main signalling pathways leading to flowering, including inductive photoperiods, GA status, exposure to cold periods (vernalisation) and autonomous factors, have been revealed, as reviewed by Davis (2009). These signalling pathways converge on three co-called ‘floral pathways integrators’, FT (FLOWERING LOCUS T), SOC1 (SUPPRESSOR OF CONSTANS1) and LFY (LEAFY) which elicit expression of floral meristem identity genes (Bernier and Périlleux, 2005; Davis, 2009). A role for CK in flowering has been debated since the early 1990ies when the amp1 mutant of Arabidopsis, which flowers early, was shown to have an increased CK content (Chaudhury et al., 1993). The AMP1 protein turned out to be a putative glutamate carboxypeptidase that acts as a negative regulator of CK biosynthesis (Nogué et al., 2000; Helliwell et al., 2001). Further evidence for a role of CK in flowering came from transgenic CK-deficient A. thaliana plants that showed a delay in flowering (Werner et al., 2003). Measurements of CKs in leaf tissues and leaf exudates of plants exposed to a long day (LD) flowering stimulus showed increased CK concentrations compared to non- induced plants as well as presence of CK in the phloem, indicating that it acts as a mobile signal regulating floral transition (Corbesier et al., 2003). Recent findings by D’Aloia et al. (2011) showed that CK treatment of Arabidopsis plants grown under non- inductive conditions led to flowering via a signalling pathway independent of the known floral pathways integrators. Although the exact role of CK in flowering is not yet understood, the UFO:IPT phenotype observed is in league with present knowledge about the hormone’s flower-inducing capacity. The defects observed, that the structures formed do not develop into full flowers, are probably caused by the localized IPT expression and lack of other flower-inducing signals.

5.2 Analysis of the potato genome reveals sequences of cytokinin metabolism and signalling genes

Before the release of the potato genome, only a fraction of this species genes was known. A comprehensive EST collection compiled by the Potato Oligo Chip Initiative

104 Discussion

(POCI) represented the biggest library of potato sequences available (Kloosterman et al., 2008) and, in the form of the POCI microarray, was utilized for transcriptome analysis of potato samples. Lacking genomic sequences for comparison at the time of collection, the POCI ESTs comprise a certain level of redundancy and several individual ESTs might represent the same gene, hampering analysis of transcriptome data.

In the present thesis, this problem was addressed by using sequence analysis tools and online database search to match EST sequences from the POCI collection to genomic sequences released by the Potato Genome Sequencing Consortium (PGSC), thereby developing a procedure for annotation and identification of full-length genomic sequences of genes of interest, even if no POCI ESTs for this sequence is available. With the ‘final’ genome release for the time being, new instruments for the ‘gene hunt’ became available: lists containing 39031 CDS/ protein/ transcript sequences experimentally supported by RNA sequencing (PGSC, 2011) which were used for comparison and, if needed, adjustment of identified sequences. This comparison often showed that genes identified in silico ‘by hand’ were identical to those experimentally identified, proving the validity of the employed toolset for gene identification.

5.2.1 Isopentenyltransferase genes

The first IPT genes in higher plants have been identified at the beginning of the 21st century, in the model plant Arabidopsis thaliana (Takei et al., 2001a; Golovko et al., 2002). In potato, three putative IPT genes (StIPT1 to StIPT3) were identified by hand and an additional nine putative IPT genes were found among the ‘DMv3.4_CDS representative’ list. Two Arabidopsis sequences, AtIPT2 and AtIPT9, are tRNA isopentenyltransferases, the others use ATP/ ADP/ AMP as a substrate (Miyawaki et al., 2006). Based on sequence length - tRNA IPTs carry an extension at the C-terminus which is needed for tRNA binding (Zhou and Huang, 2008) – StIPT1 and StIPT5 were predicted to encode tRNA-specific isopentenyltransferases. An alignment with Arabidopsis IPT protein sequences later showed that StIPT1 and StIPT5 cluster away from the other potato IPT sequences, in a separate branch with AtIPT2 and AtIPT9, respectively (Fig. 25). A ‘reverse’ search in the POCI database for ESTs representing the newly identified genes turned up three ESTs that match StIPT4 and StIPT5. It is likely that potato IPTs also show tissue-specific expression and regulation by various substances like auxin or cytokinin, as had been shown in Arabidopsis (Miyawaki et al., 2004), but that remains to be determined. Of the four StIPT genes represented on the

105

POCI chip, none is up-regulated during GA3-induced sprouting (compare table A23), indicating that, most likely, GA does not mediate sprouting by activating CK biosynthesis.

5.2.2. Cytokinin oxidase/ dehydrogenase genes

The enzymatic activity of the CKX enzyme had been known for more than 25 years when the first sequence of a CKX gene was identified in maize (Morris et al., 1999). Cytokinin oxidases were found to constitute small gene families whose members have different catalytic properties and are expressed in different tissues (Schmülling et al., 2003). In the potato genome, six CKX gene sequences were identified. Five of these were found to be almost identical to CKX sequences already deposited at NCBI, confirming the power of the toolset used to identify them. Although the last gene identified, StCKX6, had not been known before, its expression in potato could already be shown by PCR amplification of the gene from cDNA of mixed potato tissues (S. Bachmann, BSc thesis, 2011). In a phylogenetic tree with CKX protein sequences from Arabidopsis (Fig.27), the six members of the potato CKX family cluster with five different Arabidopsis sequences. Due to their important function in regulating CK homeostasis (Jones and Schreiber, 1997), potato cytokinin oxidases are likely to possess different biochemical properties and to be expressed in a tissue-specific manner, as had been shown for enzymes from Arabidopsis (Schmülling et al., 2003; Galuszka et al., 2007). On the POCI array, only two StCKX genes (StCKX3 and StCKX4) are represented. One EST for StCKX4 was down- regulated 4.2-fold one and two days after GA3-treatment. Although this is only a weak indication, a shift in CK homeostasis through GA treatment is conceivable and could be addressed in future experiments.

5.2.3. Cytokinin glycosyl- and xylosyltransferase genes

Glycosyltransferases form a very large family of enzymes which transfer sugar moieties onto target proteins. Glycosylation is often very important for protein function and can be found frequently as one of the last steps in the biosynthesis or modification of plant secondary products (Vogt and Jones, 2000). Glycosyltransferases specific for cytokinins have been isolated from Arabidopsis, maize and Phaseolus species (Martin et al., 1999b; 1999a; 2001; Veach et al., 2003; Hou et al., 2004). In Phaseolus, enzymes specifically transferring either UDP-glucose or UDP-xylose could be isolated (Martin et al., 1999a) and potato transcripts homologous to the corresponding Phaseolus genes were found to be differentially expressed in our in vitro sprouting experiment. In an attempt to identify the complementary potato genes, numerous potential glycosyltransferase gene sequences were identified, reflecting that glycosyltransferases form a very large family:

106 Discussion

In the CAZY database, 94 glycosyltransferase families are listed and GT family1, to which zeatin-GT and -XT belong, contains 4434 members, only 337 of them characterized (www.cazy.org/GT1.html). Although the occurrence of residues required for cytokinin binding has been reported, none of the putative GT sequences contained all sites described by Meek et al. (2008). This does not exclude that there are glycosyltransferases among the identified family members that use zeatin as substrates, because the characterized A. thaliana ZOG proteins do not contain all of these sites either (Frébort et al., 2011). Thus, for elucidation of zeatin-glycosyltransferases, the candidate enzymes identified from the potato genome sequence would have to be expressed, the proteins purified and tested for their binding and activity.

5.2.4. Cytokinin receptors

Cytokinin receptors have first been isolated in higher plants in 2001 (Inoue et al.) and the history of their discovery has recently been reviewed (Kieber and Schaller, 2010). In Arabidopsis, CK receptors belong to a small, 16-member gene family of hybrid sensory histidine kinases involved in different signalling pathways (Shi and Rashotte, 2012). The sub-family of three CK receptors (AHK2, AHK3, AHK4/CRE1b) is distinguished by the CHASE domain which is specific and sufficient for CK binding (Anantharaman and Aravind, 2001). By potato ESTs and homology search, five potato sequences potentially encoding CK receptors were identified. Three of these, StHK1, StHK2 and StHK5, were found to encode proteins containing all domains specific to Arabidopsis CK receptors. Indeed, in a phylogenetic tree of the five potato and three Arabidopsis protein sequences, StHK1, 2 and 5 were found to cluster with the three A. thaliana receptors whereas StHK3 and StHK4 formed a non-matching outgroup. Whether the three potential potato CK receptors show similar differences in properties and functions as the three Arabidopsis proteins (Heyl et al., 2011; Stolz et al., 2011) remains to be determined.

5.2.5. Response regulators type A and B

The phosphorelay systems of cytokinin signalling leads to the phosphorylation of two types of proteins: A-type response regulators are primary CK-responsive genes which act as negative regulators of CK signalling (To et al., 2004). They are short proteins with a REC signal receiver domain, form a small gene family of ten members in Arabidopsis and likely have overlapping, yet distinct functions (Ren et al., 2009). B-type response regulators are transcription factors mediating the CK response (Hwang and Sheen, 2001; Argyros et al., 2008). They are longer than A-type response regulators, contain the REC

107

domain in combination with a DNA-binding domain and are encoded by eleven genes in Arabidopsis (Ishida et al., 2008). In potato, putative sequences of eight A-type and five B-type response regulators were identified. In an alignment with Arabidopsis A- and B- type RR protein sequences, both types were clearly distinguishable from each other. One identified gene, StRRB6, probably does not constitute a part of the CK signalling pathway, as it lacks the conserved aspartate residue required for the phosphorelay (Makino et al., 2000). Nevertheless, StRRB6 might be involved in other signalling processes like its closest Arabidopsis ortholog APRR2 (Perochon et al., 2010). On the POCI array, six of the A-type and all identified B-type RR genes are represented.

Available microarray data for GA3- and BAP-induced WT tuber sprouting (compare table A23) showed strong up-regulation of ESTs representing A-type RRs at nearly all time- points investigated. In BAP-treated tubers, this could be explained by a negative feedback loop due to the treatment. In GA3-treated tuber discs, however, up-regulation of StRRA4 and StRRA5 expression might indicate GA- CK crosstalk during sprouting. In the same experiments, only few StRRB transcripts were differentially regulated, but a marked increase of StRRB4 transcript abundance was observed with onset of visible sprouting three days after GA3 incubation, possibly reflecting CK signalling required for growth processes of the newly formed tuber sprout. Given the insufficient data and complexity of the signalling pathway, a thorough inspection of the identified genes and their function is needed to determine the role of CK signaling and GA- CK crosstalk during the sprouting process.

5.2.6. PUP purine permeases

Purine permeases constitute a gene family of 17 members in Arabidopsis. Based on sequence homology, thirteen putative PUP genes were identified in the potato genome. Only two PUPs, PUP1 and PUP2, have actually been found to transport cytokinins over the plasma membrane (Gillissen et al., 2000; Bürkle et al., 2003). In a phylogenetic tree with PUP protein sequences from potato and Arabidopsis, StPUP3, 9, 10 and 11 form a subtree together with AtPUP1 and 2. In an in vitro sprouting experiment using BAP, StPUP9 and StPUP11 transcripts accumulated within a few hours of application, hinting at a rapid uptake of the hormone. During GA3-mediated sprouting, StPUP genes were not differentially expressed, but due to the time-points investigated, a possible regulation of StPUP expression by GA might have been missed. Although current knowledge and data on PUP transporters is scarce, they could be interesting candidates for further investigation.

108 Discussion

5.3 The role of beta-1,3-glucanases in tuber dormancy and sprouting

Beta-1,3-glucanases (B13G) are callose-degrading enzymes which function in stress response, pathogen defense as well as numerous developmental processes (Doxey et al., 2007). B13G are encoded by large gene families with more than ten members in tobacco, at least fourteen members in rice and even fifty members in Arabidopsis (Linthorst et al., 1990; Romero et al., 1998; Doxey et al., 2007). In the present thesis, 34 putative B13G genes were identified in the potato genome, fitting in well with gene numbers in other species. In order to get a clearer notion on possible functions and to simplify candidate gene selection, the potato sequences were grouped according to their expression patterns from microarray experiments and according to predicted protein structures, as had been done earlier in Arabidopsis and poplar (Doxey et al., 2007; Rinne et al., 2011).

5.3.1 PD gating by callose deposition and removal might not be a regulatory mechanism in tuber dormancy release

In 2007, when Viola et al. showed that the dormant bud meristem in the tuber is symplastically isolated from the underlying parenchyma and that this isolation is removed prior to sprouting, plasmodesmata and their gating by callose deposition moved into focus. The Pd gating model and evidences for it were comprehensively summarized recently (Levy and Dean, 1998; Zavaliev et al., 2011). In brief: Pd size exclusion limit (SEL) which regulates traffic through the cell-to-cell connections, is fine-tuned by a balance of callose deposition and removal at the neck of Pd. For symplastic isolation of a tissue, the balance is shifted towards callose synthesis, thus blocking Pd by callose deposition. For callose removal, the balance is shifted towards degradation via B13Gs, leading to an increase in SEL. Such mechanisms of callose deposition and removal have been observed in dormancy processes, e.g. in poplar (Rinne et al., 2001; Rinne et al., 2010), and for developmental processes in which the meristem undergoes a transition, e.g. from vegetative to flowering (Gisel et al., 1999). By monitoring the movement of symplastic tracers, symplastic isolation of dormant and symplastic connection of actively growing buds were evident (Viola et al., 2007), leading to the assumption that tuber dormancy release might be regulated in a similar manner and that B13Gs were required for its regulation. Indeed, among the 34 B13G genes identified in the genome, two candidates, tentatively termed StPdB13G_1 and _2, stood out by their similarity to a known Pd-associated B13G from Arabidopsis, AtBG_PAP, on the protein level. In order to investigate their role in potato tuber dormancy release, these genes were cloned and

109

transgenic plants carrying an AtBG_PAP overexpression construct or an RNAi construct for simultaneous knock-down of StPDB13G_1 and _2 were generated. Plants positive for either construct were cultivated in the greenhouse and after harvest, tubers were stored for examination of sprouting behaviour. While StPdB13G_RNAi transgenic tubers showed no significant differences in sprouting to the wild-type, preliminary results for AtBG_PAP-expressing tubers suggested a delay in sprouting rather than the expected acceleration. Although it cannot be ruled out that residual B13G expression in the RNAi lines was sufficient to produce adequate amounts of protein, down-regulation of putative Pd-glucanases seemed to have no impact on tuber sprouting. The observation that AtBG_PAP overexpression resulted in a delay of sprouting contradicted the hypothesis of removal of callose at PD. According to that hypothesis, increased levels of Pd- associated beta-1,3-glucanase should lead to a shift of callose turnover towards callose degradation. In turn, increased callose degradation should lead to early sprouting of potato tubers, as Pd callose deposition would be removed prematurely. That the opposite effect was seen in AtBG_PAP OE tubers could indicate that Pd callose might be less directly involved in tuber sprouting, rather regulating accompanying processes. Indeed, evidences in favour of callose-mediated gating of Pd during sprouting were lacking, as immunodetection of callose showed no changes between dormant and sprouting buds or buds just undergoing bud break. Nonetheless, analysis of callose deposition was only indirect, albeit supportive confirmation that the tested Pd-associated B13G might not play a role in tuber bud break and it cannot be ruled out that minor local changes in callose deposition were overlooked because they could not be seen by the method used. Moreover, the involvement of a different candidate B13G in dormancy release is still conceivable, since the two candidates tested had simply been selected by their sequence similarity to the Arabidopsis PD-associated glucanase.

In summary, B13G-driven callose removal at Pd is most likely not a regulatory mechanism of tuber dormancy release. Although it cannot be dismissed completely, evidences for any changes concerning callose depostition were not found. Delayed sprouting of AtBG_PAP-overexpressing tubers even suggested that beta-1,3-glucanase activity might be involved in processes accompanying sprouting rather than dormancy release itself.

110 Discussion

5.3.2 Tuber sprouting is accompanied by de novo formation of vascular tissue

Despite the failure to detect major changes in callose deposition during the transition from dormant to sprouting bud meristem, a surprising observation was made during the callose detection experiment: Numerous new vascular strands had formed between day two and day six of the experiment. Plant vascular tissues mainly serve both as conductive network for water and nutrients as well as physical support. Thus, an increasing number of vascular strands very likely demonstrates the increasing flow of nutrients from the tuber into the newly formed sprout in order to support its growth.

Vascular strands are composed of two distinct tissue types, the phloem which primarily transports organic nutrients from photosynthetic tissues to sink tissues, and the xylem which mostly serves as water and mineral conduit. Both tissues comprise specialised cell types, tracheary elements (TEs) in the xylem or sieve elements and their companion cells in the phloem, for example, and their organisation is heavily regulated on the genetic level (Zhou et al., 2011). The process of vascular development from meristematic (pro)cambial cells to differentiated vessels has been studied intensely in roots, shoots and leaves and insights into vascular patterning has progressed greatly in the last decade as evidenced by the increasing number of reviews dealing with this topic (Dengler, 2001; Fukuda, 2004; Dettmer et al., 2009; Caño-Delgado et al., 2010; Scarpella et al., 2010; Vera-Sirera et al., 2010; Zhou et al., 2011). Two phytohormones, auxin and cytokinin, play major roles in the control of vascular development: According to the auxin canalization hypothesis, first proposed by T. Sachs (1981), auxin is gradually channelled into narrow strands by polar transport which then differentiate into veins. Indeed, vascular stem cell specification was found to be dependent on expression of ARF5/ MONOPTEROS, an auxin-resposive transcription factor. ARF5, in turn, directly activates expression of AtHB8, a homeobox gene specific to procambial cells of newly forming vascular tissue (Hardtke and Berleth, 1998; reviewed by Zhou et al., 2011). The Polar auxin transporter PIN1 was also found to be expressed prior to AtHB8 expression and procambium formation, marking the routes along which the conductive tissue is formed (Scarpella et al., 2006). In addition to auxin signalling, de novo synthesis of auxin also plays a role in regulating vascular pattern formation, as Arabidopsis mutants defective in auxin biosynthesis gene YUCCA (YUC1, 2, 4, or 6) displayed abnormal vein initiation and development (Cheng et al., 2006). Although cytokinin has been implied in vascular formation for a long time (Aloni, 1993), it has been found only recently that both cytokinin signalling and cytokinin transport are 111

required for normal development of vascular tissues (De León et al., 2004; Kuroha et al., 2006; Mähönen et al., 2006; Nieminen et al., 2008; Muraro et al., 2011) and that extensive crosstalk between the two hormones auxin and cytokinin defines cell specification (Aloni et al., 2006; Dettmer et al., 2009; Bishopp et al., 2011). Downstream signalling events leading to vascular cell type specification have been unravelled for xylem formation: Transcriptional regulatory networks involving antagonistic activity of transcription factors of the HD ZIP III family (PHB - PHABULOSA, REV - REVOLUTA, PHV - PHAVOLUTA, CRN - CORONA) and the KANADI family (KAN1 to KAN3) were found to control xylem development (reviewed by Helariutta (2007), Elo et al. (2009) and Ilegems et al. (2010)). In the Zinnia elegans model system, transdifferentiation of mesophyll cells to TEs could be studied in vitro (Demura et al., 2002), leading to the discovery of the NAC-domain transcription factors VND6 and VND7 which control xylem cell identity (Kubo et al., 2005). In contrast to xylem differentiation, few is known about phloem development. Bonke et al. (2003) characterized a MYB coiled-coil type transcription factor, ALTERED PHLOEM DEVELOPMENT (APL), which initiates phloem differentiation, but further knowledge is lacking.

In potato, vascular development could, at least in part, be followed on the transcriptional level by analysis of microarray data from in vitro sprouting experiments (table A23). Although important determinators of cell fate like VND6 and VND7 are not represented on the chip, up-regulation of transcripts for ARF5 and AtHB8, indicative of the start of vascular tissue formation and in accordance with array data from in vitro transdifferentiation of Zinnia elegans cells (Demura et al., 2002), could be seen after induction of sprouting with both BAP and GA3. Up-regulation of ESTs representing YUC1 and YUC4 in tubers treated with either BAP or GA3 is consistent with an increased auxin production required for proper vascular patterning. Two days after BAP and three and five days after GA3 treatment, expression of the APL transcript is up-regulated, reflecting the difference between bud break by cytokinin and additional sprout outgrowth triggered by gibberellin. Expression of ESTs for transcription factors involved in xylem cell speciation, like PHV, REV, PHB and CRN is not consistent over the different experiments. This might be due to the manner of sample taking, where larger parts of the parenchyma tissue below the newly formed sprout was removed in order to reduce contamination with non-meristematic tissue. Microarray data from one SRA in which the newly formed sprout and the subjacent parenchyma at three days after GA3 treatment had been sampled and analysed separately, showed expression of markers for vascular tissue formation in both tissues, illustrating that vascular bundles are required for nutrient

112 Discussion supply from the tuber to the sprout as well as for nutrient distribution within the sprout. Taken together, the microarray data point toward vascular tissue formation and differentiation as a process accompanying sprouting. Whether this process also represents a layer of regulation for dormancy release, remains to be determined.

113

5.4 Future prospects

The present thesis has helped advance the knowledge of potato tuber dormancy release by determining cytokinins as a major regulator of the transition from the dormant to sprouting state. With this knowledge, directed engineering of potato tubers with customized dormancy length by expressing CK biosynthetic, modifying or CK degrading enzymes in a specific (sub)set of cells at a certain time point is conceivable. Such a directed modification of the tuber’s dormancy, aided by the use of tuber-specific promoters, could have a tremendous effect on storage stability and industrial handling as tubers might be stored for a long time without quality loss due to untimely sprouting.

With the toolset described in this thesis, the identification of CK-related genes from the potato genome and the observation of vascular tissue formation as a process occurring during sprouting, this thesis could mark a starting point, fueling intense research on molecular processes during sprouting, such as phytohoromone cross talk, signalling networks or tissue- and development-specific expression of CK-related genes. With an even deeper understanding of the sprouting process and of meristem reactivation than we have now, more regulators of tuber dormancy release could be identified, extending again the set of instruments for directed manipulation of dormancy length at our disposal.

114 References

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Internet resources http://faostat.fao.org http://pgrc.ipk-gatersleben.de/poci http://pgrc-35.ipk-gatersleben.de/pls/htmldb_pgrc/f?p=194:1:63062385 117638 http://potatogenomics.plantbiology.msu.edu/index.html http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi http://www.potatogenome.net/index.php/introduction https://www.gabipd.org/projects/MapMan/ www.cazy.org/GT1.html www.plant-hormones.info/gibberellinhistory.htm www.potato2008.org/en/potato/biodiversity.html www.potato2008.org/en/world/index.html www.umaine.edu/umext/potatoprogram/pest_control_guide.htm

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Appendix

7 Abbreviations aa amino acid ABA absisic acid Ade adenine Ado adenosine AHP Arabidopsis histidine-containing phosphotransfer protein ARE auxin-responsive element ARF auxin response factor ATP adenosine triphosphate Aux auxin β-ME β-mercapto ethanol B13G beta-1,3-glucanase BAP 6-benzylaminopurin BH-FDR Benjamini-Hochberg false discovery rate bp base pairs CaMV cauliflower mosaic virus cv. cultivar cDNA complementary DNA CHASE ‘cyclases/ histidine kinases- associated sensory extracellular’ domain CK cytokinin CKX cytokinin oxidase/ dehydrogenase CLSM Confocal Laser Scanning Microscope CRF cytokinin response factor cRNA copy RNA, in vitro amplified RNA cZ cis-zeatin cZR cis-zeatin riboside d day(s) dat days after treatment DEPC diethylpyrocarbonate DMAPP dimethylallyl pyrophosphate DMSO dimethyl sulfoxide DMv3 potato genome release version 3 DNA deoxyribonucleic acid DNase deoxyribonuclease dNTP deoxyribonucleotide DT desmotubule DZ dihydrozeatin DZR dihydrozeatin riboside E. coli Escherichia coli EDTA ethylenediaminetetraacetic acid ENT equilibrative nucleoside transporter ER endoplasmic reticulum EST expressed sequence tag FW fresh weight g gram GA gibberellic acid GFP green fluorescent protein GOI gene of interest GT glycosyltransferase h hour(s) HK histidine kinase HPLC high performance liquid chromatography IAA indole-3-acetic acid iP N6-(Δ2-isopentenyl)adenine iPR iP-riboside IPT isopentenyltransferase IPTG Isopropyl β-D-1-thiogalactopyranoside LOG ‘lonely guy’; riboside 5’ monophosphate phosphoribohydrolase mg milligram I

min minute(s) ml millilitre MMLV Moloney murine leukemia virus mRNA messenger RNA NAA 1-naphthaleneacetic acid nos nopaline synthase ocs octopine synthase oD optical density oN over night ORF open reading frame PCR polymerase chain reaction PD Plasmodesmata PGSC Potato Genome Sequencing Consortium PM plasma membrane POCI Potato Oligo Chip Initiative PUP purine permease qPCR quantitative real-time PCR RbcS Rubisco small subunit RNA ribonucleic acid RNAi RNA interference RR response regulator RT room temperature or reverse transcription SAM shoot apical meristem SD standard deviation SE standard error SEL size exclusion limit SRA sprout release assay tZ trans-zeatin tZR trans-zeatin riboside UFO unusual floral organs wah weeks after harvest WT wild-type XT xylosyltransferase ZRED zeatin reductase µg microgram µl microlitre

II Appendix

8 Appendix 8.1 DNA- and amino acid sequences 8.1.1 IPT DNA- and amino acid sequences identified in the potato DMv3 genome 8.1.1.1 StIPT1 ATGATAAGCACCGTCGGGACTGGTGGCGTTCGGATAAGCAGCTATCTCCGGCTGCCGTACACGGTGGAGACACTTCTCCGCCGCCGTTGCCGT M I S T V G T G G V R I S S Y L R L P Y T V E T L L R R R C R CTCTTCACCTCCTGCTCTGCCTCCTCTACTAAGGAGAAAGTTATTGTCATTTCCGGACCTACCGGTGCCGGTAAAAGCAAGCTAGCATTAGAG L F T S C S A S S T K E K V I V I S G P T G A G K S K L A L E CTCGCTAAGCGACTCAATGGTGAAATCATCAGCGCGGACTCTGTACAGGTGTATCGAGGGCTTGATGTCGGTTCAGCGAAGCCTTCCTTTGAT L A K R L N G E I I S A D S V Q V Y R G L D V G S A K P S F D GAACGAAAGGAAGTAGTACATCATCTGGTTGACATATTGCATCCATCCGAAGATTATTCTGTTGGACAATTTTTTGAGGATGCTAGGCAAACT E R K E V V H H L V D I L H P S E D Y S V G Q F F E D A R Q T ACCAGAGATATCCTTGATAGGGGCCGTGTTCCAATAGTCGCTGGTGGTACTGGATTATATTTGCGATGGTTTATTTATGGGAAGCCAGATGTT T R D I L D R G R V P I V A G G T G L Y L R W F I Y G K P D V CCAAAAGCCTCTCGAGAGATTGCTGCTGAAGTTGACTCAGAGCTTGCAAGTTTACAGAATGATGAAGAGTGGAATGCTGCTGTTCAGTTGTTG P K A S R E I A A E V D S E L A S L Q N D E E W N A A V Q L L GTTAAAGCTGGTGATCCTGGTGCCCAGTCTTTACCGGTAAATGATTGGTATCGCTTGCGTCGCAGGCTTGAAATTGTTAAGTCTACTGGGTCA V K A G D P G A Q S L P V N D W Y R L R R R L E I V K S T G S CCTCCATCAGCTTTTGAAGTACCATATGATTCTTTCAGAGAACAGCTCATATCAGGTCAAATAGATGCTGCTGATGTCAAGACTTCTGCTGAC P P S A F E V P Y D S F R E Q L I S G Q I D A A D V K T S A D AAATTGCAGCAGAATGAAATAAAGGATTTGGATTATGATTTTCTTTGTTATTTCCTTTCCACCAATAGGATAGATCTCTATAGATCAATTGAT K L Q Q N E I K D L D Y D F L C Y F L S T N R I D L Y R S I D TTTCGGTGTGAAGATATGCTCTTAGGAGCAGATGGAATTCTATCTGAGGCAGGGTGGCTTCTTGATGTGGGTCTTTTGCCGAACTCGAATTCT F R C E D M L L G A D G I L S E A G W L L D V G L L P N S N S GCTACTCGAGCAATTGGTTACCGACAAGCAATGGAATACCTTCTAAGATGTAGGGAAAATGGAGGATGGAGTTCTGCTGGGGACTTCTATGAA A T R A I G Y R Q A M E Y L L R C R E N G G W S S A G D F Y E TTTTTATCTGAATTTCAGAAAGGATCACGAAATTTTGCAAAAAGGCAGATGACATGGTTTCGCAATGAACAGATTTATGAGTGGATAGATGCT F L S E F Q K G S R N F A K R Q M T W F R N E Q I Y E W I D A TCTAAACCTTTGGAAAAGGTGCTTAGCTTCATATGTGATTCATATAACAGTCAGGATGGCCATCTCCAGATGCCTGAGTCACTTCGTATGCGG S K P L E K V L S F I C D S Y N S Q D G H L Q M P E S L R M R AAAGATATAAGAAATCACCGTCAAGCTGCAGAATTGAAGACCTATCGTACAATAAACAGGCATTTCATTGGGCATGAAGATTGTGTGGATGTT K D I R N H R Q A A E L K T Y R T I N R H F I G H E D C V D V CTGGACTGGATAAAGAAGACCTATGGCCAACCGACCGACTCTCTTTGTTAA L D W I K K T Y G Q P T D S L C * 8.1.1.2 StIPT2 ATGAACTCTGCACAAATGTGCAAACAAGTAAGAAGGCCAAATTTGCTACAATTGCGAAATTTTCTTCCAAAGGACAAGATTGTGGTGGTCATG M N S A Q M C K Q V R R P N L L Q L R N F L P K D K I V V V M GGAGTCACCGGTGCCGGAAAATCAAAACTCTCTATAGACTTAGCCACTCAATTCAATGGAGAAATAATTAATTCCGACAAAATACAAGTGTAC G V T G A G K S K L S I D L A T Q F N G E I I N S D K I Q V Y AAAGGTCTTGAAATAGCGACTAACAAAATAACAGAAGAAGAACGTTGCGGTGTACCACATCATTGCCTAGGGGTAATTGATCCTTACAAAGAA K G L E I A T N K I T E E E R C G V P H H C L G V I D P Y K E TTCACCACCAAAAACTTTTGTAACATGGCTTCATTTACCGTTAACTCTATAACCAACCGCGGTAAACTTCCTATCATCGTTGGAGGGTCAAAT F T T K N F C N M A S F T V N S I T N R G K L P I I V G G S N TCATTCATCGAGGCATTTGTCCACAACAATAATTCATATAATTTTAGTACAAGGTACGATTTTTGTTTCCTATGGGTCGATGCATCAATGAAC S F I E A F V H N N N S Y N F S T R Y D F C F L W V D A S M N GTGCTAAATTCGTTCTTATACGAACGAGTAGATAAAATGGTGGATCAAGGATTGGTAGATGAGGTAAGACAAATGTTCAATCCAAAAAACACA V L N S F L Y E R V D K M V D Q G L V D E V R Q M F N P K N T GATTATACCAAAGGCATTCGTAAAGCCATTGGTGTCCCGGAATTTGACAGTTACTTTCGTGCTGAACTGTCAAATTCCGTGGACAGGCAAACT D Y T K G I R K A I G V P E F D S Y F R A E L S N S V D R Q T CGTGAGAGGATGCTAAAGGAAGCAATTACTGAAGTAAAGATCAATAATTGTATACTCGCAAGTAAACAATTAGACAAAATTAACCGATTAATT R E R M L K E A I T E V K I N N C I L A S K Q L D K I N R L I AACGTTAAAGGATGGAAAATCCATCGATTAGATGCAACTGAGGTTTTCAAAAAACAGAGGATAGCAGAGGAAAAAGAGGCAGAGGAAATATGG N V K G W K I H R L D A T E V F K K Q R I A E E K E A E E I W AAGAATATCGTGATGGGACAGAGCAGAAAGATAGTGCATAAATTTTTATACGAAAATTATAGGAATTCAATGGTTTATAAAAGTGATGGGACA K N I V M G Q S R K I V H K F L Y E N Y R N S M V Y K S D G T CCCATTATAGCAGCAGCATCTCAGTATTAA P I I A A A S Q Y *

8.1.1.3 StIPT3 ATGAGAAAATTATCATTTTTATCAACAACAACGAGTACTAATCATTCTAACACGTTGTTTTTCTTCAAACCAAAAAACATTTTCTTCTTCCCC M R K L S F L S T T T S T N H S N T L F F F K P K N I F F F P AAATGGACTCGTAATGTGGTTATATCAGCAACCAGTAGTAATGTACCACGACGATCCAAAAAGATCGTCGTGATAATGGGTGCTACTGGTTGT K W T R N V V I S A T S S N V P R R S K K I V V I M G A T G C GGAAAATCAAAACTCTCTATCGACCTTGCCACTCATTTCTTCCCATCTATCGAAGTCATTAACTCCGATAAAATCCAAGTTTACAATGGTTTA G K S K L S I D L A T H F F P S I E V I N S D K I Q V Y N G L GAAATCACTACAAACAAAATTTCAATGACTGAACGCTGCGGTATCCACCATCATCTTCTCGGAGAGTTCAAACCTACTGAATCACACCCGGAG E I T T N K I S M T E R C G I H H H L L G E F K P T E S H P E

III

TTTTCCCCTTCCGATTTCCGATCAGCTGGAGATTTCAGAATCAACGAAATCGTTAATCGAGGGAAAATCCCTTTTATTGTAGGTGGGTCTAAC F S P S D F R S A G D F R I N E I V N R G K I P F I V G G S N TCATTCATCTATGCTCTGTTAGTAAAACAATTCGATTCAGGATTCGACATTTTCGAATCGTTAAATCCGGTGATTAACGAGCTTCGTTACCAG S F I Y A L L V K Q F D S G F D I F E S L N P V I N E L R Y Q TGTTGCTTCATCTGGGTTGATGCCATAGCACCTGTATTGAATCAGTATTTAGATAAACGAGTCGATGAAATGCTCGATTCGGAGATGTTCGAA C C F I W V D A I A P V L N Q Y L D K R V D E M L D S E M F E GAGCTAAAAGAGTATTTCGAGAAAGAGGGGTTTTCCGATTCCAGTTCCGACGGGATCCGGAAGGCAATAGGAGTACCGGAGTTCGAGAAATAC E L K E Y F E K E G F S D S S S D G I R K A I G V P E F E K Y TTTAAAGGGAAGATATCATACGAAGAAGCAGCCATGGAGATAAAGGAGAACACAAGGGTATTAGCAGAAATACAGGTGAAGAAGATCATGCGG F K G K I S Y E E A A M E I K E N T R V L A E I Q V K K I M R CTGAGAGAAGGTGGATGGAACATACAAAGAGTGGATGCCACAGCGACGTTAACGGCGAAAATGGGGTCGGAAAAAATAGCCGGCGGCGAGAAT L R E G G W N I Q R V D A T A T L T A K M G S E K I A G G E N CCGGCGAGGAAAATTTGGGAAGAACAGGTACTGGAACCAAGTGCCAAGATTGTGAAGCAGTTCTTGTTGGAGTAG P A R K I W E E Q V L E P S A K I V K Q F L L E *

8.1.2 CKX DNA- and amino acid sequences identified in the potato DMv3 genome 8.1.2.1 StCKX1 ‘DMv3’ This sequence was identified from Scaffold PGSC0003DMS000001089, based on an alignment with StCKX1 from S.tuberosum cv. Russet Burbank (Acc. No. FJ751238). Nucleotide and amino acid differences to FJ751238 are marked in red and blue, respectively.

ATGAAGTCATCACCAACTCATTTCTTCTTTAAACACAATAGTATGCTTCTAAGGATTCTTATATTCATACTAGGCATTTGCTCAATAAACAGA M K S S P T H F F F K H N S M L L R I L I F I L G I C S I N R AGTAACCTCTGTTGTGACCAACTTTTTGCCACCCCTTCAAGTTTCTCAGTGATTCAATCATCACTGAAACAGTTAAAGATTGAAGGGTACTTT S N L C C D Q L F A T P S S F S V I Q S S L K Q L K I E G Y F AGTTTCAAGAATTTCGATCACGTCGCCAAGGATTTCGGCAACAGATATCACTTCCTTCCATCAGCTGTTCTGTACCCGAAATCGGTTTCAGAC S F K N F D H V A K D F G N R Y H F L P S A V L Y P K S V S D ATATCATCCACTTTAAAACATATTTTTGACATGGGAACAACAACTGACCTAACTGTTGCTGCTAGAGGACATGGTCACTCTCTAGAAGGACAA I S S T L K H I F D M G T T T D L T V A A R G H G H S L E G Q GCTCAAGCTTACCGGGGAGTAGTGATCAGCATGGAATCGCTTCGAGCACCAGCTATGCGTTTCCACCATGCAGGGGAACTGCCTTTTATTGAT A Q A Y R G V V I S M E S L R A P A M R F H H A G E L P F I D GTCTCTGCCGGAGAACTTTGGATAAACATCCTGCATGAAAGTCTTAAACTTGGATTAACACCAAAATCTTGGACTGATTATCTTCACCTTACA V S A G E L W I N I L H E S L K L G L T P K S W T D Y L H L T GTTGGAGGTACTTTGTCGAATGCCGGAATCAGCGGGCAAGCATTCAAACATGGACCTCAGATAAATAATGTCTACCAACTTGAAGTTGTCACT V G G T L S N A G I S G Q A F K H G P Q I N N V Y Q L E V V T GGTAAAGGAGAGGTGATTACTTGTTCAAAGGAGCAGAATGCTGACCTGTTCTATGGTGTACTAGGAGGACTAGGCCAGTTTGGTATCATCACA G K G E V I T C S K E Q N A D L F Y G V L G G L G Q F G I I T AGGGCTAGGATTGCTCTTCAACCAGCACCTAAAAAGGTAAAGTGGATCAGAGTGCTGTATTCAGATTTCTCAACATTTTCCAATGATCAAGAA R A R I A L Q P A P K K V K W I R V L Y S D F S T F S N D Q E CAGTTGATATCATCCAAGGATTCTTTCGACTATGTAGAAGGATTTGTTATTATCAATAGAACAGGATTGCTGAACAACTGGAGGTCTACTTTC Q L I S S K D S F D Y V E G F V I I N R T G L L N N W R S T F AATCCTAAAGATCCACTTCTAGCCAGAAAGTTCAGTTCTGAAGGAAAAGTTCTCTACTGCCTAGAAGTTGCCAAATACTTCAATCCAGAAGAC N P K D P L L A R K F S S E G K V L Y C L E V A K Y F N P E D ACCCCTAATACTGATCAGAATATTGATGTCCTCTTATCTAAGTTGAATTATATCGAATCCACGCTGTTCCAATCAGAAGTCTCCTACGTGGAA T P N T D Q N I D V L L S K L N Y I E S T L F Q S E V S Y V E TTCCTTGACAGAGTCCACGTGTCCGAGATGAAACTCCAAGAGAAGGGGTTATGGGATGTCCCTCATCCATGGCTAAACCTTTTAATTCCAAAG F L D R V H V S E M K L Q E K G L W D V P H P W L N L L I P K ACCAGGATTCATGACTTTGCACAAGAAGTTTTTGGGAAGATTCTTACTGACACTAGCCATGGTCCTATACTCATCTACCCAGTCAACAAATCG T R I H D F A Q E V F G K I L T D T S H G P I L I Y P V N K S AAGTGGATAAAAGGAACATCAATGGTTACGCCTGAAGAAGATGTCATGTATTTAATAGCATTTCTATCTTCTGCCATGCCATCTTCAACAGGA K W I K G T S M V T P E E D V M Y L I A F L S S A M P S S T G AAGGATGGACTAGAACATATTCTAAATAAGAACAAGAAGATACTGAACTTTTGCAATAAAGCACATATTGGAATGAAACAGTATTTGCCACAT K D G L E H I L N K N K K I L N F C N K A H I G M K Q Y L P H TACACAACACAGGAAGACTGGAAAGTTCACTTTGGCCCTCGATGGGAAACATTTGCTAGGAGGAAATCCACTTATGACCCTTTGTCTATCCTA Y T T Q E D W K V H F G P R W E T F A R R K S T Y D P L S I L GCTCCTGGACACAAAATTTTTGAAAGAGCATCACTCCTTCAACAACAATAA A P G H K I F E R A S L L Q Q Q * 8.1.2.2 StCKX2 ‘DMv3’ This sequence was identified from Scaffold PGSC0003DMS000001425, based on an alignment with StCKX2 from S.tuberosum cv. Russet Burbank (Acc. No. FJ751239). Nucleotide and amino acid differences to FJ751239 are marked in red and blue, respectively.

ATGGCTAAGTTTTTTTTATCCTATGGTTATAATCTTATTATTTTCTTTATTATTAGTCATTTAATGTCCATTTTAGGAAAGTTAAATCCATGG M A K F F L S Y G Y N L I I F F I I S H L M S I L G K L N P W AATCCTTCAATTCCTTATGAAATTCTTTCACTTAATATTTCATCAAAACTTAGTACAAATTCTCATGATATTAAAGAATCTTCCAAAGATTTT N P S I P Y E I L S L N I S S K L S T N S H D I K E S S K D F GGAAAAATTATTCAAGAAATATTACCAGCTGCTGTTCTTTATCCTTCTTGTGTTAATGACATAATTGACCTCATTCAATTTTCTCATGACCTT G K I I Q E I L P A A V L Y P S C V N D I I D L I Q F S H D L TCTGTCCCTTTTCATGTAGCAGCCAAAGGTCATGGACATTCCATTAGGGGACAAGCCATGGCAAAAAATGGGGTAATTGTGGAAATGAGTTCT S V P F H V A A K G H G H S I R G Q A M A K N G V I V E M S S

IV Appendix

TTAAATAATAATAATAATGAGAATTGTGGTGTTAGGGTTTCTTGGGATTCGGGTTTAGGGTTTTACGCGGATGTTGGAGGTGAACAATTATGG L N N N N N E N C G V R V S W D S G L G F Y A D V G G E Q L W ATTGATGTTCTTCGTAGCACCCTAGAGTATGGCCTAGCACCTGTTTCGTGGACAGATTATTTGTACCTTACCGTTGGTGGTACTCTCTCTAAT I D V L R S T L E Y G L A P V S W T D Y L Y L T V G G T L S N GCTGGAATTAGTGGTCAAACTTTCCGATATGGTCCTCAAATAAGTAACGTTCATGAGATGGATGTTATTACAGGTAAAGGGGAATTAGTGACT A G I S G Q T F R Y G P Q I S N V H E M D V I T G K G E L V T TGCTCCAAAGATATGAATTCAGAATTGTTTTTTGGAGTTTTAGGAGGTTTAGGACAGTTTGGAATAATAACAAGAGCAAGAATTGTCTTGGAT C S K D M N S E L F F G V L G G L G Q F G I I T R A R I V L D AAAGCACCAACAAGAGTGAAATGGGTGAGAATGTTGTATGATGATTTCTCAAAATTCACAAAAGATCAAGAACATCTTATTTCAATTCATCAT K A P T R V K W V R M L Y D D F S K F T K D Q E H L I S I H H AATGGATTGGATTATGTTGAAGGCTCTCTAATGATGGAACAAAGCTCTCTAAATAATTGGAGATCTTCATTTTTTTCACCTTCCAATCAAACC N G L D Y V E G S L M M E Q S S L N N W R S S F F S P S N Q T AAAATTGCTTCATTATTATCCAAAAATAAAATTATGTATTGCTTGGAAATAGTGAAGTACTATGATGACCAAAATGCTAATACTATTGATGAG K I A S L L S K N K I M Y C L E I V K Y Y D D Q N A N T I D E GAGTTGAAGAAGTTGGTAAAAGGATTGAAGTATTTAGGTGGATTTATGTTCAAGAAAGATGTGAGTTTTGTGGAATTTTTGAATAGAGTTAGA E L K K L V K G L K Y L G G F M F K K D V S F V E F L N R V R AGTGGGGAATTAGAGTTACAATCAAAAGGAAAGTGGGATGTTCCACATCCATGGCTCAATTTGTTTGTACCAAAGTCTTCTATCATGCATTTT S G E L E L Q S K G K W D V P H P W L N L F V P K S S I M H F CATGCTGCTGTTTTTGTGGACATAATCCTCAGACAAAACAAGACAACTGGACCCATACTTGTCTACCCAACAAGTAGGAAAAGATGGGATGAT H A A V F V D I I L R Q N K T T G P I L V Y P T S R K R W D D AGGATGTCAACAATGATACCAGAAGAGGAGACATTTTATTGTGTGGGACTATTACATTCTTCAAGTGGATATAAAGAATGCAAGATTTTGGAT R M S T M I P E E E T F Y C V G L L H S S S G Y K E C K I L D AATCAAAATGAAGAAATACTAAATTATTGTGATAAAGTTGGCCTCAATATAAAGCAATATCTTCCACATTACAAGACAAAAGAGGATTGGATC N Q N E E I L N Y C D K V G L N I K Q Y L P H Y K T K E D W I AAACATTTTGGTAAAAAATGGAATATTTTTCAACAAAGAAAAGATCTATTTGATCCAAAGATGATTCTATCACCAGGACAAAGAATTTTTAAT K H F G K K W N I F Q Q R K D L F D P K M I L S P G Q R I F N TAG * 8.1.2.3 StCKX3 ‘DMv3’ This sequence was identified from Scaffold PGSC0003DMS000000386 based on an alignment with POCI EST SSBN002L17u.scf and by Conserved Domain Search of all translation frames of the genomic sequence. The final ORF sequence was found to be 98% identical to the StCKX3 ORF (FJ888605). Nucleotide and amino acid differences to FJ888605 are marked in red and blue, respectively.

ATGACATGCCAGAACTTAGTATTAACTCGAAAAACGAACATTTTGTTCGTAAGAAGCTTTACAATTTTGGTACTAATATGGATAGTTATGAAG M T C Q N L V L T R K T N I L F V R S F T I L V L I W I V M K CCAGAAGTTTGTTTCTCTAGTGTCCTGTCTTCATTGAAGGCTCTTCATCTACAAGGCTATATAACTTTTGAAAACAATGAATTTGCATCCCGA P E V C F S S V L S S L K A L H L Q G Y I T F E N N E F A S R GATTTCGGGAATCAAATTCATTCACATCCTTTGGCAGTAGTACATCCGAAATCTGTTACTGACATTTCAGAGATAGTAACACATGTCTGGCAA D F G N Q I H S H P L A V V H P K S V T D I S E I V T H V W Q ATGGGCCCGGCCTCAGAACTGACCGTGGCAGCAAGAGGCCACGGTCATTCTCTTCAAGGCCAGGCCCAAGCGCGTGGAGGAGTTATAATCAAT M G P A S E L T V A A R G H G H S L Q G Q A Q A R G G V I I N ATGGAATCATTACGGCAGGATCAAGAAATGCAAGTTTACTACAGAGGAGTACAATTCCCTTATGTTGATGTCTCAGCTGGTGAATTGTGGATA M E S L R Q D Q E M Q V Y Y R G V Q F P Y V D V S A G E L W I AACATATTGCATGAGACATTGAAATATGGATTAGCACCAAAATCTTGGACTGATTATCTACATCTTACTGTTGGAGGTACTCTGTCTAATGCT N I L H E T L K Y G L A P K S W T D Y L H L T V G G T L S N A GGAATCAGTGGACAGGCATTTCGTCACGGCCCTCAAATCAGTAATGTCCGCCAGCTGGAAGTTGTAACAGGTAAAGGAGAAGTCCTAATTTGT G I S G Q A F R H G P Q I S N V R Q L E V V T G K G E V L I C TCACAAGAACAGAATGCTGACCTCTTTCATGCTGTTCTTGGAGGACTTGGTCAGTTTGGCATAATAACTAGAGCAAGAATCTCACTCGAAAGA S Q E Q N A D L F H A V L G G L G Q F G I I T R A R I S L E R GCCCCGAAAATGGTGAAATGGATAAGAGTGTTGTACTCTGATTTCTCCACATTTGCTAGAGATCAAGAGCGTTTGATATCTGCATCGAAAACA A P K M V K W I R V L Y S D F S T F A R D Q E R L I S A S K T TTTGATTACATAGAAGGCCTTGTGATTAAGAACAAAACAGGTCTACTGAATAACTGGAGAACATCTTTTGACCCTCAAGATCCTGTTCAAGCA F D Y I E G L V I K N K T G L L N N W R T S F D P Q D P V Q A AGCCACTTTGTATCAGATGGAAGAACACTCTATTGCCTTGAACTAACCAAAAATTTATACCCCGAAAAATTCGATACAGTAAATCAGGAAATT S H F V S D G R T L Y C L E L T K N L Y P E K F D T V N Q E I GAGGACTTATTATCCCAACTAAGTTATATTCCATCAACACTTTTCATGTCAGAAGTTCCATACATAGATTTTTTGGACAGAGTTCATGCATCA E D L L S Q L S Y I P S T L F M S E V P Y I D F L D R V H A S GAGCTAATACTTCGATCGAAAGGACTTTGGGATCTTCCACATCCATGGCTAAACCTTCTAGTCCCTAAAAGCAAAATACAACACTTTGCTAAT E L I L R S K G L W D L P H P W L N L L V P K S K I Q H F A N GAAGTTTTTGGCAACATACTAAGTGATACCAACAATGGACCTGTTCTTGTCTATCCAATTCATAAATCAAAGGTGGATAACAGGACTTCATTT E V F G N I L S D T N N G P V L V Y P I H K S K V D N R T S F GTTTGTCCAGATGAAGATATTATCTATCTTGTGGCCTTTTTATCCCATGCAAATCCTTCATCTAATGGAACTGACAGTTTACAACATGTTTTA V C P D E D I I Y L V A F L S H A N P S S N G T D S L Q H V L ACTCAGAACAAAAGAATTTTAGACTTCTGTGAGGTGTCACACCTTGGAGTCAAGCAATATTTACCTCATTACACAACACAGGAACAATGGAGG T Q N K R I L D F C E V S H L G V K Q Y L P H Y T T Q E Q W R ACACACTTTGGTCCAAAATGGGAAGTTTTTGTACAGAGGAAGTCTGTTTATGACCCTTTAGCTATGCTTGCTCCTGGACAGAATATTTTCCAA T H F G P K W E V F V Q R K S V Y D P L A M L A P G Q N I F Q AAATCAGTATCAGTTTCATAA K S V S V S *

V

8.1.2.4 StCKX4 ‘DMv3’ This sequence was identified from Scaffold PGSC0003DMS000000650 based on an alignment with POCI ESTs cSTA13J24TH and bf_mxflxxxx_0059a04.t3m.scf as well as by Conserved Domain Search of all translation frames of the genomic sequence. The final ORF sequence was found to be 98% identical to the StCKX4 ORF (FJ888606). Nucleotide and amino acid differences to FJ888606 are marked in red and blue, respectively.

ATGGCTACGAAATTATTGTTAACGTTAGCGATATGTCGCTTAATTGTTACCGTGGGATTTACTTTTAACCCGAATGACCTTTTACTGCTCGGG M A T K L L L T L A I C R L I V T V G F T F N P N D L L L L G CTCAATGGGAAACTCAGCGTGGCTCCAGCCGATTTACAATCCGCTTCGGTTGATTTCGGCGGAGTGTATAAAGCCGAGCCGATGGCGGTTCTT L N G K L S V A P A D L Q S A S V D F G G V Y K A E P M A V L CACCCGGCTACATCCGAGGATGTAGCGCGGTTAGTGAAGGCCGCGTACGACTCGGCTCGTGGGTTCACCGTGTCGGCTAGAGGTCATGGACAT H P A T S E D V A R L V K A A Y D S A R G F T V S A R G H G H TCTATAAATGGTCAAGCCATGACGACAAATGGCGTTGTGGTTCAAATGAGCGGCGGTGGCGGCTCGAAAAATAAGATGTTAACTATTTCGGAG S I N G Q A M T T N G V V V Q M S G G G G S K N K M L T I S E AAATTCATGTACGCTGATGTTTGGGGTGGAGAGTTATGGATTGATGTGTTGACGTCCACATTAGAATACGGTCTCGCACCGAAATCATGGACT K F M Y A D V W G G E L W I D V L T S T L E Y G L A P K S W T GATTATTTGTACTTAACTGTGGGTGGTACTCTCTCTAATGCTGGAATTAGTGGTCAAGCTTTTAATCATGGCCCTCAAATTAGCAATGTCCAT D Y L Y L T V G G T L S N A G I S G Q A F N H G P Q I S N V H GAACTCGATGTTGTTACAGGTAAGGGTGAGCTATTGACATGTTCAGAAAAAGAAAATTCTGAGCTGTTTCAAGCCGTTCTTGGTGGATTAGGA E L D V V T G K G E L L T C S E K E N S E L F Q A V L G G L G CAATTTGGGATCATAACAAGAGCAAGAATTGCACTTGAACAAGCTCCTCAAAGGGTGAGGTGGATTCGAGTACTATATTCCAATTTTTCAACA Q F G I I T R A R I A L E Q A P Q R V R W I R V L Y S N F S T TTTACACAAGACCAAGAATATTTAATATCATTGCATGGAAAACCAGCTTCACAAAAATTTGATTATGTTGAAGGATTTGTTATAGTTGATGAA F T Q D Q E Y L I S L H G K P A S Q K F D Y V E G F V I V D E GGTTTGATAAATAATTGGAGGTCTTCTTTTTTCTCACCAAGTAACCCTGTGAAAATTTCTTCTCTCAAGGCTGAAGGAGGTGTTTTATATTGC G L I N N W R S S F F S P S N P V K I S S L K A E G G V L Y C TTGGAAATTACCAAAAATTATCACCTTTCAAATGCTGATACCATTGATCAGGAGATTGAAATATTATTGAAAAAATTAAATTATATACCAGCT L E I T K N Y H L S N A D T I D Q E I E I L L K K L N Y I P A TCAGAGTTCAGAACGGACCTTCCTTACGTGGATTTTTTGGACCGGGTTCACAAGGCCGAGTTAAAACTCCGGTCTAAAGGTTTATGGGAAGTG S E F R T D L P Y V D F L D R V H K A E L K L R S K G L W E V CCACATCCATGGCTAAACTTATTTGTACCAAAATCAAGAATTGCTGAGTTTGATAAAGGAGTATTTAAGGGCATTTTGGGAAACAAAACTAGT P H P W L N L F V P K S R I A E F D K G V F K G I L G N K T S GGTCCAATACTCATATACCCTATGAACAAAAACAAATGGGACGATAGGAGTTCGGTAGTTACACCAGAAGAAGATGTGTTTTACTTGGTCGCG G P I L I Y P M N K N K W D D R S S V V T P E E D V F Y L V A TTTTTGAGGTCTGCATTGGAGAATGGTGATGAGACACAAACGTTGGATTACCTAAGTAATCAAAATTATGAAATATTGAAGTTTTGTGAGGAT F L R S A L E N G D E T Q T L D Y L S N Q N Y E I L K F C E D GAAAAGATCAATGTGAAACAATATTTGCCTCATTATGACAATCAAAGAGAATGGAGGGACCATTTTGGAGAAAAATATTGGACAAGATTTCAA E K I N V K Q Y L P H Y D N Q R E W R D H F G E K Y W T R F Q CAAAGAAAATTAGAGTTTGATCCAAGGCATATATTAGCAACCGGCCAAAAGATTTTTATGCCTTCTTTTAATCCTAATATTGCTACTTGGTGA Q R K L E F D P R H I L A T G Q K I F M P S F N P N I A T W *

8.1.2.5 StCKX5 ‘DMv3’ This sequence was identified from Scaffold PGSC0003DMS000000737 based on an alignment with StCKX5 from S.tuberosum cv. Russet Burbank (Acc. No. FJ888607). Nucleotide and amino acid differences to FJ888607 are marked in red and blue, respectively.

ATGTTAGCATGTTTAGTTGAACGATTGGTAGCAGAGAATGACACTGATTCGATACCTGACCCTGAAAAAGATGTCGATGTTGATGGCGTTTTG M L A C L V E R L V A E N D T D S I P D P E K D V D V D G V L AAAGACTTGAATATTGAAGGAAGCATTGATTATGGAGTAACGGCAATAAGTTTAGGCAGTAGAGATTTCGGCGGCTTATATTCAGAGAAGCCG K D L N I E G S I D Y G V T A I S L G S R D F G G L Y S E K P TTAGCCGTTATACGTACAGGCGGAGCCGATGACGTTGTGCGGGTGATTAGGCGAGCGTTAGAGTCACCAACATTAACGGTAGCGGCGAGGGGT L A V I R T G G A D D V V R V I R R A L E S P T L T V A A R G AACGGTCATTCGATTAACGGCCAAGCTATGGCCCACCGTGGACTCGTAATCGATATGAAATCAATGGCTGATAATAACAGAATCGATGTGAAT N G H S I N G Q A M A H R G L V I D M K S M A D N N R I D V N GTCAATTCCATGTGCGTCGATGTAGGTGGCGGAGCATTATGGAGTGATGTATTGAAACATTGCGTTTCGGAATACGGCTTGGCTCCTAAATCA V N S M C V D V G G G A L W S D V L K H C V S E Y G L A P K S TGGACGGATTATCTTCATTTAACAGTCGGAGGTACTCTGTCTAATGCCGGCGTTAGTGGTCAAACTTTCCGTTTTGGTCCCCAAACGTCAACT W T D Y L H L T V G G T L S N A G V S G Q T F R F G P Q T S T GTAACGGAATTGGAAGTTGTTACCGGCAACGGAGAAATAATAGTCTGTTCAAATTCTCATAATTCTCAACTCTTCTTCTCCGTTCTGGGTGGA V T E L E V V T G N G E I I V C S N S H N S Q L F F S V L G G CTTGGTCAGTTTGGTATCATTACTAGAGCTCGGCTTTTGCTTCAACCCGCCCCAGATATGGTGAGGTGGATAAGAGTAGTATATAGTGAATTT L G Q F G I I T R A R L L L Q P A P D M V R W I R V V Y S E F GACGAGTTCACTCATGATGCTGAGTTACTGATAACAAGTCAAGAATCATTCGATTATGTGGAAGGGTTTGTGTTCGTGAACAGTGATGACCCG D E F T H D A E L L I T S Q E S F D Y V E G F V F V N S D D P GTAAATGGATGGTTATCGGTGCTATTAGATTCAAATCAGGCATTTGACCCGACCCATTTACCCAAGAAAACTGGTCCGGTTCTCTATTGTCTT V N G W L S V L L D S N Q A F D P T H L P K K T G P V L Y C L GAAGTGGCCTTGCATTATAACAACAACCATGACGATCCCTTCATGATGGTTGAGAAATTGCTAGGCAAATTGAGATATTTGAAGCACTTCAGA E V A L H Y N N N H D D P F M M V E K L L G K L R Y L K H F R TTTGAGATCGACTTGACTTATATGAATTTTTTATCACGAGTTGACCATGTAGAAGAAGCGGCTAGAGGTAGTGGTATATGGGCTACACCTCAT F E I D L T Y M N F L S R V D H V E E A A R G S G I W A T P H CCATGGCTTAACATGTTTGTTTCCAAGAAAGATATTGATGCATTCAATCGAATTGTATTTCAAAATATCTTAAAAAATGGTGTCAATGGCCCT P W L N M F V S K K D I D A F N R I V F Q N I L K N G V N G P

VI Appendix

ATGTTAACCTATCCTCTCCTGCGTAGCAAGTGGGATAATCGATGGTCAGTGGCGTTGCCCAAAAATGAAATGTTTTATTTAGTGGCTTTGCTA M L T Y P L L R S K W D N R W S V A L P K N E M F Y L V A L L AGGTTTACCCATGCACATCCAACAGAGTCTGAAATAAATGAGATGGTGGAACAAAATGAGGAGATTGTACAAACTTGTATCAAGAATGGATTT R F T H A H P T E S E I N E M V E Q N E E I V Q T C I K N G F GATTTTAAAATGTATCTTCCTCATTACAACTCCACAGTTGAATGGAAAAGGCACTTTGGGGAACAATGGGGAAGATTTGTCAACAGAAAGAGG D F K M Y L P H Y N S T V E W K R H F G E Q W G R F V N R K R CAGTTTGATCCAAAGTATGTCCTTGCACCTGGCCAAAAAATATTTACTAGAAATCATCAATTCTAG Q F D P K Y V L A P G Q K I F T R N H Q F * 8.1.2.6 StCKX6 This sequence was identified from Scaffold PGSC0003DMS000000849, based on an alignment with StCKX1 which showed between 76% and 85% identical sites for the 5 exons. Testing for conserved domains showed that the protein coded by this nucleotide sequence contains a complete cytokinin dehydrogenase superfamily domain.

ATGAAGTCACCAACCCAATTTTTCTTTATTCAAAAGCATATACTTCTAAAATTGCTTATATTCATACTCTTCATTTGTTCAAACAATAAAAGT M K S P T Q F F F I Q K H I L L K L L I F I L F I C S N N K S AACATATGTTGTAATCATCATTTTACTAACACTTCAAGTCTATCATTGAAACAACTAAAACTTGAAGGGTACCTTAGTTTTGAAAAACTAAAT N I C C N H H F T N T S S L S L K Q L K L E G Y L S F E K L N CATGCAGCTAAAGACTTTGGCAATAGATGTCATTTCCTTCCATTAGCAATTTTGTATCCAAAATCTGTTCTTGATATATCATCCACCTTAAAA H A A K D F G N R C H F L P L A I L Y P K S V L D I S S T L K CATGTCTTCGAAATCGGTACTAGAACAGACTTAACTGTTGCTGCTAGAGGCAATGGACACTCTTTAGAAGGCCAATCTCAAGCTTATCAAGGA H V F E I G T R T D L T V A A R G N G H S L E G Q S Q A Y Q G CTAGTTATTAACATGAAATCACTTCAAGAACTAGAAATGAAGTTCAAGATTGAGGAATTGTCTTATGTTGATGTTTCTGCTGGAGAGCTTTGG L V I N M K S L Q E L E M K F K I E E L S Y V D V S A G E L W ATAAATGTTCTGCATGAAAGTCTTAAACTTGGGCTTGCACCTAAATCTTGGACTGATTATCTTCACCTCACTGTTGGTGGTACTTTGTCTAAT I N V L H E S L K L G L A P K S W T D Y L H L T V G G T L S N GCTGGAATCAGTGGACAAGCTTTTAAGCACGGGCCGCAGATCAATAATGTCTACCAACTCGAGGTTGTCACTGGTAAAGGAGAGGTGATTACT A G I S G Q A F K H G P Q I N N V Y Q L E V V T G K G E V I T TGTTCAGAGGAGCAGAATGCAGACTTGTTCCATGGTGTACTAGGAGGACTAGGGCAGTTTGGTATTATAACTAAGGCAAGAATTGCTCTTGAA C S E E Q N A D L F H G V L G G L G Q F G I I T K A R I A L E ACAGCACCAAAGCAGGTCAAGTGGATCAGAGTGTTGTATTCAGATTTTGCTACATTTTCGAATGATCAAGAGAACTTGATATCATCTCAGAAT T A P K Q V K W I R V L Y S D F A T F S N D Q E N L I S S Q N ACATTTGACTATATCGAAGGATTCGTCATCATAAACAGCACTGGATTATTGAATAACTGGAGGTCTACTTTCAATCCTAAAGATCCACTTCTA T F D Y I E G F V I I N S T G L L N N W R S T F N P K D P L L GCTAGCAATTTTAGTTCAGAAGGCAGAGTTTTATTCTGTTTAGAAGTTGCCAAATACTTCAATCCAGAGGACACATACAGTACTGATCAGGAC A S N F S S E G R V L F C L E V A K Y F N P E D T Y S T D Q D ATTGATATACTCTTATCAAAGTTGCATTATATTCGATCAACGTTGTTCCTATCAGAAGTCTCCTACGTTGAATTCCTCGATAGAGTTCATGTC I D I L L S K L H Y I R S T L F L S E V S Y V E F L D R V H V TCCGAGATGAAGCTACAAGAAAAGGGGTTGTGGGATGTTCCTCATCCATGGCTAAATCTTCTAATACCAAAAAGCAGGATTCTTGAATTTGCA S E M K L Q E K G L W D V P H P W L N L L I P K S R I L E F A CAAGAAGTTTTCGGGAAGATTCTTACTGATACAAGCAATGGTCCTATACTCATCTACCCTGTCAACAAATCAAAGTGGAGAAAAGGAACATCA Q E V F G K I L T D T S N G P I L I Y P V N K S K W R K G T S ATGGTTACACCTGATGAAGATGTTTTCTACCTCATCGCGTTCCTGTCATCTGCTATGCCTTTTTCAACAGGAAAGGATGGACTAGAACATATT M V T P D E D V F Y L I A F L S S A M P F S T G K D G L E H I ATGAATCAGAACAAAAGGGTATTAAGTTTTTGTGAAAAAACGCGTATTGGAATGAAACAATATTTACCAAATCACAAGACTCAAGAAGAGTGG M N Q N K R V L S F C E K T R I G M K Q Y L P N H K T Q E E W AAACATCATTTTGGTTCTCAATGGGAAGCATTTGCTAGGAGGAAATCTACATATGACCCTTTGGCTATACTTGCTCCAGGACAGAGGATTTTC K H H F G S Q W E A F A R R K S T Y D P L A I L A P G Q R I F AAAAGGACAGCAGCCTGTGAACAACAATGA K R T A A C E Q Q *

8.1.3 Cytokinin receptor DNA and amino acid sequences identified in the DMv3 genome 8.1.3.1 StHK1 ATGGGTGAGAAGATGCAAAGCCACCATATGGTGAGTGTGAAGGGGAGTGAGCAATTTAACTCAAAGAGAAAGCATAGATTTGTACCTTCACAG M G E K M Q S H H M V S V K G S E Q F N S K R K H R F V P S Q GGTTATCTTCCAAAGCTTTTTGCTTTATGGATTATCTGGTGTACATTTTTCAGCATTGCTTTGTACTTTTATATGGATGCTAATCACAAGGAG G Y L P K L F A L W I I W C T F F S I A L Y F Y M D A N H K E AAAAGAAAAGAAGGGCTTGTGAGTATGTGTGATCAAAGGGCAAGGATGTTACAAGATCAATTCAGTGTTAGTGTGAACCATGTACATGCCCTT K R K E G L V S M C D Q R A R M L Q D Q F S V S V N H V H A L GCTATACTTGTATCAACTTTCCATTATGAGAAGAATCCATCAGCAATTGATCAGAATACTTTTGCTGAGTACACTGCTAGAACTGCCTTTGAG A I L V S T F H Y E K N P S A I D Q N T F A E Y T A R T A F E AGGCCACTATTAAGTGGGGTGGCATATGCAGAGAGAGTTTTAAACTCAGAAAGGGAAGAATTTGAGAGAGAACATGGATGGACTATTAAAACA R P L L S G V A Y A E R V L N S E R E E F E R E H G W T I K T ATGGAAAAAAAGCCTTCACCAATCAGAGATGAGTATTCTCCAGTTATATTTTCTCAAGAGACTGTTTCCTACATAGAATCACTTGACATGATG M E K K P S P I R D E Y S P V I F S Q E T V S Y I E S L D M M TCAGGAGAGGAGGATCGCGAAAACATCTTAAGAGCTAGGGCCAGTGGGAAGGCAGTTCTTACAAGCCCCTTCAGGCTTCTGGGTTCTCATCAC S G E E D R E N I L R A R A S G K A V L T S P F R L L G S H H CTTGGTGTTGTTTTGACATTCCCTGTTTACAGATCCAAGCTTCCGGAAAACCCGACGGAGCACGAACGGGTTGAAGCAACTGCAGGGTTTCTT L G V V L T F P V Y R S K L P E N P T E H E R V E A T A G F L GGTGGAGCCTTTGATGTTGAGTCTCTCGTTGAGTGTCTACTCGGGCAACTTGCTGCAAATCATCCAATAATTGTAAATGTCTATGATGTTACA G G A F D V E S L V E C L L G Q L A A N H P I I V N V Y D V T VII

AACTCCTCTGATCCTTTAATCATGTACGGACACCAGAACCCCAATGGTGATGCCTCACTTAAGCAAGTAAGCAAGCTTGATTTTGGTGATCCA N S S D P L I M Y G H Q N P N G D A S L K Q V S K L D F G D P TTTCGTAAGCATGAGATGATTTGTAGGTATCTTTACGAAGATCCTATATCTTGGGGTGCAGTGACCACTGCAGTTTTCATTTTTACCATCTTC F R K H E M I C R Y L Y E D P I S W G A V T T A V F I F T I F CTCTTAATTGGCTACACGGGTTACAAATCTGCAAGCCACATTAATAAAGTAGAGGATGATTTCCATAAAATGCAGGAGCTAAAGGTTCAAGCT L L I G Y T G Y K S A S H I N K V E D D F H K M Q E L K V Q A GAAGCAGCTGATGTTGCCAAATCCCAGTTCTTGGCTACTGTTTCACATGAAATAAGAACTCCTATGAATGGAATCCTAGGAATGCTTGCTTTG E A A D V A K S Q F L A T V S H E I R T P M N G I L G M L A L CTTCTAGATACGGATCTGAGTTCAACTCAAAGAGATTATGCTCAAACTGCTCAAGATTGTGGAAAGTCACTGATAAGATTGATAAATGAAGTG L L D T D L S S T Q R D Y A Q T A Q D C G K S L I R L I N E V CTTGATCGGGCTAAAATTGAAGCTGGCAAGTTAGAACTTGAGGCAGTTCCATTTGACCTTCGCTCTATACTGGATGATGTTCTCTCTTTATTC L D R A K I E A G K L E L E A V P F D L R S I L D D V L S L F TCTGATGAGTCAAGGCGCAAAGGTGTTGAGTTGGCCGTCTTTGTTTCTGATAAAGTGCCTGAAATTGTTATGGGGGATCCAGGAAGATTCAGA S D E S R R K G V E L A V F V S D K V P E I V M G D P G R F R CAAGTGATAACAAATCTGGTGAACAACTCGGTCAAATTCACTCTACGAGGGCATATATTTGTTCAAGTTCATCTGGCAGAACAAAAAAAAGAT Q V I T N L V N N S V K F T L R G H I F V Q V H L A E Q K K D GGTGACAAAACTGACACCTGCTTGAATGGAGGATCTGAAAGTATTATTTCTTCTAGTGCTTTTCATTTCAAGACCTTAAGTGGTTATGAAACT G D K T D T C L N G G S E S I I S S S A F H F K T L S G Y E T GCTGATAGCCAGAACACTTGGAACACATTCAAGCATATAATTGCTGACAATGGGTTGTACTATGAATCTGCAACTAAGGTGGTGAATGATGAT A D S Q N T W N T F K H I I A D N G L Y Y E S A T K V V N D D CTCTCTCGGGATGTCACTGTAATGGTTAGTGTTGAAGATACTGGGATTGGGATCCCTTTGAAAACACAAGATCGAGTTTTTACACCATTCATG L S R D V T V M V S V E D T G I G I P L K T Q D R V F T P F M CAGGCAGACAGTTCAACTTCTAGAAAGTATGGAGGAACTGGTATTGGGTTAAGCATTAGCAAGTGTCTCGTCGAGCTGATGGGCGGTCATATA Q A D S S S R K Y G G T G I G L S I S K C L V E L M G G H T S AGTTTCATTAGCCGTCCCAAGATTGGAAGCACATTTTCTTTCAGTGTTAGCTTCCTAAGATGCGAGAAACATGCTGTTGGTGATCTGAAAAAA F I S R P K I G S T F S F S V S F L R C E K H A V G D L K K K TCTCATTCTGATGATTTGCCTACTTCATTCAAAGGTCTAAATGCTATTATAGTTGATGAAAAGCCTGTAAGAGCTGCTGTAACAGGATACCAT S H S D D L P T S F K G L N A I I V D E K P V R A A V T G Y H CTGAAGAGACTTGGTATTCGGGCAGAAGTTGTCAGTAGCATCAAGAGAGCAGCAGCCACTCTTGGGAAAAATGGTTCTGTTGTTTCCAAGAAG L K R L G I R A E V V S S I K R A A A T L G K N G S V V S K K CTGGACATGATTCTGGTCGAGAAGGACTCGTGGATATCTGAAGATGTTGACCTAAATTTACACTTTCCAGACATCAATCAAAATGGACACATG L D M I L V E K D S W I S E D V D L N L H F P D I N Q N G H M TACAAGTTACCGAAGATGATCCTTCTGGCGACTAATTTTACCAACACTGCTGAACATGAAAAGGCTAAAGCAGTAGGTTTTTCAGTGATAATG Y K L P K M I L L A T N F T N T A E H E K A K A V G F S V I M AAACCTCTTAGAGCAAGTATGCTGGCTGCATGCCTTCAGCAGCTAATTGGAATTGGAAACAAGAGCCGAGGAAAAGATATGTGCAATGGATCT K P L R A S M L A A C L Q Q L I G I G N K S R G K D M C N G S CCATCCCTTCGTGGCCTACTATGTGGTATGAAAATTTTGGTGGTTGATGATAATAGAGTAAATCGCAGGGTTGCTGCTGGTGCACTCAAAAAG P S L R G L L C G M K I L V V D D N R V N R R V A A G A L K K TTTGGGGCCGAGGTAGAGTGTGCTGAGAGTGGGAAAGCTGCTCTTGCATTGCTGCAGTTACCACACAATTTTGATGCATGCTTTATGGATATT F G A E V E C A E S G K A A L A L L Q L P H N F D A C F M D I CAGATGCCAGAAATGGATGGGTTTGAGGCTACACGTCGAATTAGGGAACTGGAGAGCATTGCAAATGAACAACAGAATGGAGTGTTGAACTGG Q M P E M D G F E A T R R I R E L E S I A N E Q Q N G V L N W GATGGAGGTACAAAGAGGCACATGTGGCACATGCCGATATTGGCCATGACTGCTGATGTAATTCATGCCACATTAGAGAAATGCCTCAAGTGT D G G T K R H M W H M P I L A M T A D V I H A T L E K C L K C GGAATGGATGGTTACGTCTCAAAACCATTCGAGGAAGAGAATCTTTATGAAGCTGTATCCAAGTTCTTCGAATCCAAGCCTAACTCGGACAAA G M D G Y V S K P F E E E N L Y E A V S K F F E S K P N S D K TAG *

8.1.3.2 StHK2 ATGACTAGCAGCAAGACTTTGCTTGGTGACGGAGAGCACATAGTGAAGAAATTGTGGGAGTTGAGTGCTAAGATTTATTATTGTTACCCTCAG M T S S K T L L G D G E H I V K K L W E L S A K I Y Y C Y P Q TATGTTGGAAATAGGAAAGTGGGAAACAAGTGGTGGAGGAAGCTTTTGATAGTATGGTTATTATTTTGGATTGTTGTTTCTTTTTCAGTCTTG Y V G N R K V G N K W W R K L L I V W L L F W I V V S F S V L TGGTATATGAACTCTAAAGCTGTTGAAAAGAGAAAAGAAACACTTACAAGTATGTGTGATGAGAGAGCTAGGATGTTACAAGATCAGTTTAAT W Y M N S K A V E K R K E T L T S M C D E R A R M L Q D Q F N GTTAGTATGAACCATGTGCAGGCCATGTCCATTCTCATCTCAACTTTCCACCATGCCCGGAATCCTTCTGCTATTGATCAGTGTACTTTTGCA V S M N H V Q A M S I L I S T F H H A R N P S A I D Q C T F A AGCTATACAGAAAGAACAGCATTTGAGAGGCCTCTTACTAGTGGGGTGGCATATGCAGTAAGAGTGCTCCACTCAGAAAGAAAAGAGTTTGAG S Y T E R T A F E R P L T S G V A Y A V R V L H S E R K E F E AAGCGACACGGTTGGAGTATTAAGAGAATGGATACACGTGAACCAACTCCAGTTCACAAAGACAATGAGTATGATAGAGATGGCTTGGAGCCA K R H G W S I K R M D T R E P T P V H K D N E Y D R D G L E P TCTCCAATTCAGGCAGAATATGCCCCTGTTATTTTTGCACAGGATACGATAGCACATGTAATTTCTGTTGATATGCTCTCTGGAAAGGAAGAT S P I Q A E Y A P V I F A Q D T I A H V I S V D M L S G K E D CGCGAGAATGTTTTACGCGCGAGGGAATCAGGAAAGGGTGTCCTCACAGCTCCCTTCAGGCTACTTAAAACAAATCACCTTGGGGTGATAAAG R E N V L R A R E S G K G V L T A P F R L L K T N H L G V I K ACATTTGCAGTTTACAAAACTGATCTTCCTTCCAATGCAACTCCAAATGAAAGGATCCAAGCAACTGACGGGTACCTTGGTGGAGTACTTGAC T F A V Y K T D L P S N A T P N E R I Q A T D G Y L G G V L D ATTGAATCACTTGTAGAGAAGCTTCTTCAGCAGCTTGCAAGCAAACAAACTATCCTTGTAAACGTCTATGATATGACTAATATCTCCCACCCT I E S L V E K L L Q Q L A S K Q T I L V N V Y D M T N I S H P ATTAGCATGTATGGTTCAAATGTGTCGAGTGATGGTCTGGAGCATGTCAGTGCCCTCAACTTTGGGGATCCATTTAGAAGGCATGAGATGCGT I S M Y G S N V S S D G L E H V S A L N F G D P F R R H E M R TGCAGATTCAAACAGAAACCACCATGGCCTTGGCTAGCCATCACAACTGCAACAGGAATCCTCATAATTGCATTGCTTATAGGGCAAATATTT C R F K Q K P P W P W L A I T T A T G I L I I A L L I G Q I F CATGCAACCATCAACAGAATCGCCAAAGTTGAGGATGATTATCATGAGATGATGATGCTGAAAAAACGTGCAGAGGCTGCTGATGTTGCAAAA H A T I N R I A K V E D D Y H E M M M L K K R A E A A D V A K

VIII Appendix

TCACAGTTTCTTGCTACTGTTTCCCATGAGATCAGGACCCCCATGAACGGTGTTCTTGGCATGCTTCATATGCTTACGGACACCAATCTAGAT S Q F L A T V S H E I R T P M N G V L G M L H M L T D T N L D GTGACACAACAAGATTATGTCAGCACTGCTCAGGCCAGTGGTAAAGCTTTAGTTTCACTCATAAATGAGGTTTTGGACCAAGCAAAGATTGAA V T Q Q D Y V S T A Q A S G K A L V S L I N E V L D Q A K I E TCTGGTAAGCTTGAGCTTGACGCAGTTTGTTTTGACGTGAGAGCTACTCTGGATGAGGTTCTGTCACTCTTTTCAGGGAAATCTCAAGAAAAA S G K L E L D A V C F D V R A T L D E V L S L F S G K S Q E K GGAGTGGAGTTGGCAGGTTACATCTCTGATAAGGTTCCTGATGTGCTCATTGGTGACCCTGGACGGTTTCGTCAGATTATCACCAACTTGGTG G V E L A G Y I S D K V P D V L I G D P G R F R Q I I T N L V GGAAACTCTATCAAATTCACTGAAAAAGGCCATATATTTGTGACTGTCCATCTTGTCGAGGAAGTGACCGAGTCAGCTGAGGAGTTCAAGGTG G N S I K F T E K G H I F V T V H L V E E V T E S A E E F K V AATTCATTGTTTAAGAGCACTTTGAGTGGATTGCCTGTAGCCGATAAACGACAGAGCTGGAGAAGTTTCATGGGTTTCAATCAAGAAGGATCT N S L F K S T L S G L P V A D K R Q S W R S F M G F N Q E G S TCTTTCACATCTTCCTCATTGGACCAGATTACTTAATGGTTTCAGTAGAAGACACTGGTGTTGGAATTCCTTTAGATGCTCCAATCTCGTATA S F T S S S L D Q I T L M V S V E D T G V G I P L D A Q S R I TTCACCCCGTTCATGCAGGTTGGTCCTTCTATTGCTCGCATCCATGGAGGAACTGGTATTGGACTTAGCATAAGCAAATGTTTGGTACAGCTT F T P F M Q V G P S I A R I H G G T G I G L S I S K C L V Q L ATGAAAGGTGAAATTGGATTTGTAAGTTTGCCAAAGATTGGATCCACCTTTACTTTTACTGCTGTTTTCACCAATGGCCGTAATAATTGGAAT M K G E I G F V S L P K I G S T F T F T A V F T N G R N N W N GAGAAGAAGAGCCAGCAAATAAACAATCAATCGAACTCTATCTCTTCAGATTTCCATGGCATGAGAGCCTTAATTGTTGACCCAAGAACTGTC E K K S Q Q I N N Q S N S I S S D F H G M R A L I V D P R T V CGTGCAAGGGTCTCACAGTATCACATGAAACGACTTGGAGTACATACTGAAGTGGTCTCAGATTTGAATCATGGTTTGTCTTATGTAAGAACT R A R V S Q Y H M K R L G V H T E V V S D L N H G L S Y V R T GAAAATGGAGTTACGAATATGATCCTCATTGAACAGGAGATCTGGGATACTGATTCGGGAAAGTCAAGTCTCTTTGTCAAAATCTTAAGAAAG E N G V T N M I L I E Q E I W D T D S G K S S L F V K I L R K TTCAATACTAGTAGTTCTCCCAAACTTTTCATATTAGCCAATTCTATAAATTCTAGCCGAGTGGGTGTCTCAGTCAATGGTTTTCCTACTCCA F N T S S S P K L F I L A N S I N S S R V G V S V N G F P T P TTTATCATCATGAAGCCATTAAGGGAAAGTATGCTTGCCGCATCACTCCAACGTGCCATGGGTGTTGGTAACAAAGGAAATTGTACAAATGGA F I I M K P L R E S M L A A S L Q R A M G V G N K G N C T N G GAGCTCTCCGGTCTCTCTCTTTCCAAGCTCCTTCAAGGGAGAAAAATTTTGATTGTAGATGACAACAACGTGAACCTTAGAGTAGCTGCTGCT E L S G L S L S K L L Q G R K I L I V D D N N V N L R V A A A GCACTGAAGAAGTATGGTGCTGATGTCGTCTGCACAGACAGTGGGAAAAAAGCACTCACTTTTCTACAACCACCTCATCAATTCGATGCCTGT A L K K Y G A D V V C T D S G K K A L T F L Q P P H Q F D A C TTCATGGATATTCAGATGCCAGAAATGGATGGGTTTCAAGCTACAAAAATAATCCGCGAAATGGAATCTGATATCAATAGCCGTATCAAACTT F M D I Q M P E M D G F Q A T K I I R E M E S D I N S R I K L GGGCAACTTCCTCCTGAAGCATACGGAAACATTTCAAGCTGGAAAGTACCCATTCTGGCCATGACCGCTGACGTAATTCAAGCTACAAATGAA G Q L P P E A Y G N I S S W K V P I L A M T A D V I Q A T N E CAGTGTCAAAAATGTGGAATGGATGGTTATGTTTCGAAGCCATTTGAAGCTGAGCAGCTTTATGAAGAAGTATCACGCTTTTTCCAGATCAAG Q C Q K C G M D G Y V S K P F E A E Q L Y E E V S R F F Q I K CCAACTCAGAATACCTGA P T Q N T *

8.1.3.3 StHK5 CTGTCTCAACAACTTCATCCACTACAGCAGTTGCAGCAGCAGCAAGCTCAGATTTGCTCTCGAACTGGTGGGAAGTGGAGGAAGAAGGCTCTT M S Q Q L H P L Q Q L Q Q Q Q A Q I C S R T G G K W R K K A L GTTATCTTTGTTATTGGTGGGGTGATCCTAGCCATCTGGTTGTATTTGTACCTGAGTGCAGACATTGCATTGAGGAGGAAAGAAACACTGACA V I F V I G G V I L A I W L Y L Y L S A D I A L R R K E T L T AGCATGTGTGACGAACGAGCACGAATGTTGCAGGACCAGTTCAACGTAAGCATGAACCATGTTCATGCATTGGCTATTCTTGTTTCCACATTT S M C D E R A R M L Q D Q F N V S M N H V H A L A I L V S T F CACCATGGAAAACAACCTTCTGCAATAGACCAGAAAACTTTTGAAGAATATACTGAGAGAACAGCTTTTGAAAGGCCACTTACAAGTGGTGTT H H G K Q P S A I D Q K T F E E Y T E R T A F E R P L T S G V GCCTATGCTTTAAGGGTTCGTCACTCAGAGAGAGAAGAGTTTGAGAAGCTGCATGGGTGGACTATAAAGAAAATGGAATCAGAGGACCAAACT A Y A L R V R H S E R E E F E K L H G W T I K K M E S E D Q T TTAGCACAGGATTATATCCCTGCAAACTTGGATTCTGCTCCTGATCAAGATGAATATGCACCTGTCATATTTTCACAACAAACAGTTTCCCAT L A Q D Y I P A N L D S A P D Q D E Y A P V I F S Q Q T V S H ATAGTCTCTATTGATATGATGTCTGGAAAGGAAGATCGTGAGAACATTTTGCGAGCAAGGGCTTCTGGCAAGGGGGTTTTGACGTCACCCTTT I V S I D M M S G K E D R E N I L R A R A S G K G V L T S P F AAGCTATTGAAATCCAATCACCTGGGTGTTGTTCTTACATTTGCGGTCTATAATACTCATCTCCTTCCTTATGCTACCCCAGTGGACCGCATC K L L K S N H L G V V L T F A V Y N T H L L P Y A T P V D R I AATGCTACTGTTGGGTACATTGGTGCTTCTTACGATGTTCCATCATTAGTCGAAAAACTTCTGCATCAGCTTGCAAGCAAGCAAACTATTGTG N A T V G Y I G A S Y D V P S L V E K L L H Q L A S K Q T I V GTAAATGTTTATGATACAACTAACAAGTTTGCTCCAATTAAAATGTATGGCATGGATGAGAACGACACAGGATTACTTCGCGTTAGCAACCTT V N V Y D T T N K F A P I K M Y G M D E N D T G L L R V S N L GATTTTGGGGATCCTGCAAGGAACCACGAGATGCATTGCAGGTTCAAGCAGAAACCTTCTCCACCCTGGACTGCAATAACTCTATCTGTCGGA D F G D P A R N H E M H C R F K Q K P S P P W T A I T L S V G GTCCTTGTTATCACTCTGCTTATTGGTCATATCTTCCATGCTGCCATCAACAGAATTGCTGAAGTTGAGGGTCAGTATCAGGAAATGATGGAG V L V I T L L I G H I F H A A I N R I A E V E G Q Y Q E M M E CTCAAACATCGTGCTGAGGCTGCAGATATAGCAAAATCTCAGTTTCTTGCAACGGTTTCTCATGAAATCAGGACTCCGATGAATGGTGTTTTA L K H R A E A A D I A K S Q F L A T V S H E I R T P M N G V L GGCATGCTTCAAATGCTCATGGACACGAACCTTGACCTAACGCAACTGGATTATGCACAGACTGCTCTTGCTAGTGGGAACGATCTGATATCA G M L Q M L M D T N L D L T Q L D Y A Q T A L A S G N D L I S TTGATTAATGAGGTGCTGGATCAGGCTAAGATTGAATCAGGAAGGCTTGAGCTGGAGGCTGTTCCTTTTGATCTCCGTGCTGCACTTGATAAT L I N E V L D Q A K I E S G R L E L E A V P F D L R A A L D N GTTTCATCGCTCTTCTCAGGAAAATCTCATAAAAAAGGGATTGAGTTGGCTGTTTATGTTTCTGACCTAGTCCCAGAAGTTGTTATCGGAGAT V S S L F S G K S H K K G I E L A V Y V S D L V P E V V I G D CCAGGACGGTTCAAGCAGATAATTACGAATCTAGTTGGAAACTCAGTTAAGTTCACAAATGACAAAGGGCACATATTTGTCACAGTACATTTA P G R F K Q I I T N L V G N S V K F T N D K G H I F V T V H L

IX

GCTGATGAAGTGAGGAACCCTCATGATGTGACGGATGAAGTCTTGAAACAAAGCTTAACCTTTGTTCAAGAAAGGTCAAATGCATCTTGGAAT A D E V R N P H D V T D E V L K Q S L T F V Q E R S N A S W N ACCTTAAGTGGATTTCCTGTAGTTGACAGATGGCAAAGTTGGCAAAAGTTTGATAGGCTGAGCAGCACAGAGGAAGAAGTGGGAAAGATCAAG T L S G F P V V D R W Q S W Q K F D R L S S T E E E V G K I K TTGTTAGTGACGATAGAAGACACTGGTGTGGGAATTCCTCTTGAAGCACAGGCCCGCATTTTCACACCTTTTATGCAGGCTGACAGTTCAACA L L V T I E D T G V G I P L E A Q A R I F T P F M Q A D S S T TCTCGAACATATGGTGGAACAGGGATAGGATTGAGCATTAGCAAACGTTTGGTGGACCTCATGGGGGGAGAAATAGGCTTTTTCAGTGAACCT S R T Y G G T G I G L S I S K R L V D L M G G E I G F F S E P GGCAGAGGGAGTACATTTTCATTCACTGCAGCCTTCACAAGAGGAGAAGAAGGTTCTTTAGAACATAAATGGAAACAGTATGATCCAGCTTTT G R G S T F S F T A A F T R G E E G S L E H K W K Q Y D P A F CCAGAATTTCGTGGGTTAAGAGCATTAGTGATTGATGATAAAAGCATTCGGGCAGTGGTCACAAAATACCATTTGCAGAGACTGGGAATATGC P E F R G L R A L V I D D K S I R A V V T K Y H L Q R L G I C GTCAACATAACTTCCACAATGCATTCAGCATGTTCATATCTCTCTAATTACTCCAATACTAGCGCATTGGAACATTTAGCCGTGGTTTTTGTT V N I T S T M H S A C S Y L S N Y S N T S A L E H L A V V F V GACCAAGATAGTTGGGATAAAGAAACTTCTCTTACACTAAGTAATATGTTGAAAGAGCTCAGAACAAATGGCTCAACTACTACTTTGGGGAAG D Q D S W D K E T S L T L S N M L K E L R T N G S T T T L G K CCTCCAAAAATCTGTCTCTTATGTATGAGCTTTATGGAGAAAGATGATCTTAAATCAGCTGGAATTGTGGACCACGTGTTAACAAAACCTGTG P P K I C L L C M S F M E K D D L K S A G I V D H V L T K P V CGGTTGAGTGGGTTGATAACATGCTTTCAAGAAGCCATTGGCTACCAAAATAAGAAGCGAGTGACTCAACCTTCAACTCTTGGAAGTCTGCTG R L S G L I T C F Q E A I G Y Q N K K R V T Q P S T L G S L L ACAGGAAAGCATATATTGGTGGTAGACGATAATAATGTAAACAGGAGAGTGGCAGAAGGTGCTCTAAAGAAGTATGGGGCGATAGTGACCTGT T G K H I L V V D D N N V N R R V A E G A L K K Y G A I V T C GTAGACAGTGGAAAGGCTGCTTTGACACATCTTAATCCACCACACAATTTTGATGCTTGTTTTATGGACCTCCAAATGCCTGAAATGGATGGG V D S G K A A L T H L N P P H N F D A C F M D L Q M P E M D G TTTGAAGCGACACGACAAATACGCAATCTAGAAAACAAATACAATGAAAAGGTCGATTCTGGTGCGTTACTTCCCGGCATGTCTGCCAGAGTG F E A T R Q I R N L E N K Y N E K V D S G A L L P G M S A R V GCTCATTGGCACACGCCAATATTAGCAATGACAGCAGATGTAATTCAAGCAACAAATGAAGAGTGCATGAAGTGCGGTATGGATGATTATGTA A H W H T P I L A M T A D V I Q A T N E E C M K C G M D D Y V TCAAAACCGTTTGAAAAAGGGCAGCTTTATTCAACAGTGGCACGCTTCTTCGGGTCAGGTTGA S K P F E K G Q L Y S T V A R F F G S G *

8.1.4 Type A and B response regulator DNA and amino acid sequences identified in the DMv3 genome 8.1.4.1 StRRA1 ATGGCACAAGAGTTGCATGTTCTGGCTGTTGATGATAGTCATGTTGATCGGAAAGTTATTGAGCGATTGTTGGAGATTTCTTGCTGTAAAGTT M A Q E L H V L A V D D S H V D R K V I E R L L E I S C C K V ACGGCAGTGGAAAGTGGAACGAGGGCTTTGCAGTATTTGGGGTTGGATGGAGAGAAGAGTTCTATGGGGATCGATGGTTTGAAGGTAAATCTG T A V E S G T R A L Q Y L G L D G E K S S M G I D G L K V N L ATACTGACGGACTATTCTATGCCTGGGATGACTGGCTATGAGCTTCTCAAAAAAATCAAGGAATCATCGGTGTTGAGCAAGATACCCGTTGTT I L T D Y S M P G M T G Y E L L K K I K E S S V L S K I P V V ATTATGTCATCTGAGAAGATTTTACCTCGCATTGATAGATGTTTGGAGGAAGGTGCTGAAGAATTTCTCCTGAAGCCTGTTAAGCTGTCTGAT I M S S E K I L P R I D R C L E E G A E E F L L K P V K L S D GTTAAACGTCTCCGAGACTTCATCCTGAGAGGCGAGGGGGATGACAACAAAGAAACAGAGAATAATATCAAACAAGTATGTTCTAGAAAAAGA V K R L R D F I L R G E G D D N K E T E N N I K Q V C S R K R AAATTTCAAGACGATTCAACGACACAATCAATGCTATCTGCTGTAGTTACAGCCCATGACATTGAGTCGTCATCACCAGAATCTTCAGCATCA K F Q D D S T T Q S M L S A V V T A H D I E S S S P E S S A S TCAGAATCTCAACAGCCTTTGTCGAAGCAATCCAAGATTGGATAG S E S Q Q P L S K Q S K I G *

8.1.4.2 StRRA2 ATGGCAAGAAACGGTATGTTTTCACGGCGGCGTAGGGCGGAGAAGGTGGAGGAGTACGATGAGTTGTTGCCGTTAACAGAGGCTACTCATGAA M A R N G M F S R R R R A E K V E E Y D E L L P L T E A T H E GTTCATGTTCTTGCTGTTGATGATAGTCTTGTGGACAGAATAGTCATTGAACGTCTCCTCAAAATTACATCTTGTAAAGTGACTACTGTGGAT V H V L A V D D S L V D R I V I E R L L K I T S C K V T T V D AGTGGGATGAGAGCTTTGAAATATCTTGGATTGGATGAAGAAGAGAGTTCTGTTACTATTGATGATTTGAAGGTGGATCTGATAATTACAGAT S G M R A L K Y L G L D E E E S S V T I D D L K V D L I I T D TATTGTATGCCTGGAATGACTGGTTATGATTTGCTCAAAAAGATTAAGGGTTCATCTTTTAGGGAAGTTCCAGTTGTAATCATGTCTTCTGAA Y C M P G M T G Y D L L K K I K G S S F R E V P V V I M S S E AATGTTTTGGCTAGAATCGACAGATGTCTGGAAGAAGGCGCTGAAGACTTCCTATTGAAGCCGGTGAAATTGGCTGATGTAAAACGTTTGAAG N V L A R I D R C L E E G A E D F L L K P V K L A D V K R L K AGTTACATGTTCAACGAGGATCGATTTAGGGGTGAAGAAAAAGGAATGAACAAGAGAAAGTTGCCAGAATCATCGTCTGATGATTCATCAACA S Y M F N E D R F R G E E K G M N K R K L P E S S S D D S S T CCAACTCTCTCACCTTCACCATCATCACTTGACCTCTCATCAACACCTTCACCGCCATCCACTTCCTCTCCATCATCCCCTAAAGCATTCTCG P T L S P S P S S L D L S S T P S P P S T S S P S S P K A F S TCATCCCTCTCTACTGATTCACCGACACATTCCACTGATTCCTCCATGCCTTCTTCGCCAACAAGACGACTCAAAATGATCAGCCAAGATTTG S S L S T D S P T H S T D S S M P S S P T R R L K M I S Q D L TGA * 8.1.4.3 StRRA3 ATGGGAATGGCAGCTGCAGATCTACAGTTTCATGTTCTAGCTGTTGATGATAGTTTAATTGATAGAAAACTCATTGAAAGACTCTTTAAAATC M G M A A A D L Q F H V L A V D D S L I D R K L I E R L F K I

X Appendix

TCTTCTTGCCAAGTTACTACTGTGGATTCTGGAAGTAAAGCTTTGGAATTTCTTGGTTTACAAGAAGAACATGATGAACAGAATCACCCTTGT S S C Q V T T V D S G S K A L E F L G L Q E E H D E Q N H P C GTTTCTCCTAGCAACCAACAGGAAGTGGAAATTAATCTTATTATAACAGATTATTGTATGCCTGGGATGACAGGCTATGATTTGTTAAAGAAA V S P S N Q Q E V E I N L I I T D Y C M P G M T G Y D L L K K ATTAAGGAATCTGCATCTCTGAGGAACATACCAGTAGTCATTATGTCATCTGAAAATGTTCCTTCAAGAATCAGTAGATGCTTAGAAGAAGGA I K E S A S L R N I P V V I M S S E N V P S R I S R C L E E G GCAGAAGAATTTTTCTTGAAGCCAGTGAGATTATCAGATGTTAATAAACTTAAACCTCATATGATGAAAACCAAATGTAAAAGCCCCAAACAA A E E F F L K P V R L S D V N K L K P H M M K T K C K S P K Q CAACAACAACCTGAGATAATTGTGACACAAGACAATCAAGAATCAATAGACATAACATATGTTGTTCCACCAGAAGAAAATCAAGAATCAGAC Q Q Q P E I I V T Q D N Q E S I D I T Y V V P P E E N Q E S D AACACATTACAACAACAGAAATTACAAGACAACAGTAACAACAACAACAAGAGAAAGGCCATAGAAGAAAATCTATCACCAGACAGAACAAGA N T L Q Q Q K L Q D N S N N N N K R K A I E E N L S P D R T R CAAAGATTGAGTAGCCTCACAACAGTCTAA Q R L S S L T T V * 8.1.4.4 StRRA4 ATGGGAATGGCAGCTGTTGAGTTTCAGTTTCATGTTTTAGCTGTTGATGACAGTGTAATTGACAGGAAACTCATTGAGAGGTTGTTGAAAACC M G M A A V E F Q F H V L A V D D S V I D R K L I E R L L K T TCTGCTTGTCAAGTTACAACAGTAGATTCAGGAAGTAAAGCTTTAGAATTTCTTGGTTTACAAGAAAATGACAAGAATCAAACAAATCAGACT S A C Q V T T V D S G S K A L E F L G L Q E N D K N Q T N Q T TGTGTTTCTCTTGATAATCATCAGGAAGCTGAAATCAATCTTATTATTACTGATTACTGTATGCCTGGGATGACTGGCTATGATTTGCTCAAG C V S L D N H Q E A E I N L I I T D Y C M P G M T G Y D L L K AAACTTAAGGAATCTGCATCTCTGAGGAACATACCAGTAGTCATTATGTCATCTGAAAATGTTCCTTCAAGAATCAGTAGATGCTTAGAAGAA K L K E S A S L R N I P V V I M S S E N V P S R I S R C L E E GGGGCAGAAGAATTTTTCTTGAAGCCAGTAAGATTATCAGATGTTAATAAACTTAGACCTCATATGATGAAAACCAAATGTAAAAGCCCCAAA G A E E F F L K P V R L S D V N K L R P H M M K T K C K S P K CAACAACAACAACCTGAGATAATTGTCACACAAGACAATCAAGAATCAAGAGACATTACATATGTTGTTCCACCAGAAGAAAATCAAGAATCA Q Q Q Q P E I I V T Q D N Q E S R D I T Y V V P P E E N Q E S GACAACATAGTACAACAACAGAAATCACAAGACAACAGTAACAACAACAAGAGAAAGGCCATAGAAGAAAATTTATCACCAGACAGAATAAGA D N I V Q Q Q K S Q D N S N N N K R K A I E E N L S P D R I R CAAAGATTAAGTAGCCTTACTGCTATCTAA Q R L S S L T A I * 8.1.4.5 StRRA5 ATGGATTTTTATTCTTCATCTTCAACAATTTCTTCTCATGAAGAACCTCATGTTCTAGCTGTTGATGACAACCTTATTGATCGAAAACTTGTC M D F Y S S S S T I S S H E E P H V L A V D D N L I D R K L V GAAAAATTACTCAAGAAATCATCTTGCAAAGTGACTACTGCAGAAAATGGTCTAAGGGCCTTGGAGTACTTGGGATTAGGAGCTGATCAGGAG E K L L K K S S C K V T T A E N G L R A L E Y L G L G A D Q E CACTCTACAAATAACAATGGTTCAAAGGTTAATATGATAATAACAGATTATTGCATGCCAGGAATGACTGGTTATGAATTGCTTAAGAAAATT H S T N N N G S K V N M I I T D Y C M P G M T G Y E L L K K I AAGGAATCGTCGATTTTGAAGGATGTACCAGTTGTTATTATGTCATCTGAGAATATCCCAACTCGCATTGATCAATGCATGGAAGAAGGGGCT K E S S I L K D V P V V I M S S E N I P T R I D Q C M E E G A CAGATGTTCATGTTGAAGCCTCTCAAACACTCTGATGTCAAGAGATTACGATGTCAACTCATGCAATGCCAATGA Q M F M L K P L K H S D V K R L R C Q L M Q C Q * 8.1.4.6 StRRA6 ATGGATACTCAATTTCATGTTTTAGCTGTTGATGACAGTATTATTGATAGAAAACTCATTGAAAGACTTCTCAAGACTTCCTCTTATCAAGTT M D T Q F H V L A V D D S I I D R K L I E R L L K T S S Y Q V ACTGTTGTGGATTCTGGAAGTAAAGCTTTGGAGTTTCTTGAAGTAAGAGAAGTGGGAGTGAATTTGATAATTACAGATTATAGCATGCCTGAA T V V D S G S K A L E F L E V R E V G V N L I I T D Y S M P E ATGACTGGTTATGATTTATTGAGGAAGATTAAAGGATCATCTTATTTGAAGGATATTCCAGTTATAATTATGTCATCAGAAAATGTTCCATCA M T G Y D L L R K I K G S S Y L K D I P V I I M S S E N V P S AGAATTAATAGATGTTTGGAAGAAGGAGCTGAAGAGTTTTTTCTAAAGCCAGTTCAACAATCAGATGTTAATAGAATTAAGCCTCATTTAATG R I N R C L E E G A E E F F L K P V Q Q S D V N R I K P H L M AGGGAAAAACCAAATTCTCTCAAGAGAAAAGCTATGGTGGAATGCATTTCACCTGACAAAACTAGAAAATATAGCAACAACAATTTGTGTAGC R E K P N S L K R K A M V E C I S P D K T R K Y S N N N L C S ATCTTTTTGCAGAACATTTGTGAGTGA I F L Q N I C E * 8.1.4.7 StRRA7 ATGGGAATGTCAGAATTTCATGTTTTGGCTGTTGATGATAGCTTAATAGATAGGAAGCTCATTGAGAGGCTCTTTAAAATCTCTTCTTGTCAA M G M S E F H V L A V D D S L I D R K L I E R L F K I S S C Q GTTACTAGTGTGGATTCTGGAAGCAAAGCTTTAGAATTTTTGGGGTTACAAGAACATAACCAAAATCCCAATCCCCCTTGTGTTTCTCCTAAT V T S V D S G S K A L E F L G L Q E H N Q N P N P P C V S P N GCCAAACAGAATTTGCAGCAAGTGGAAGTAAATCTTATTATCACAGATTATTGTATGCCTGGAATGACAGGCTATGATTTGCTCAAGAAAATT A K Q N L Q Q V E V N L I I T D Y C M P G M T G Y D L L K K I AAGGAATCTCCATCTTTGAAAAATATTCCAGTAGTCATAATGTCATCTGTGAATGTTCCTTCAAGAATAAGTAGATGCTTAGAAGAAGGGGCA K E S P S L K N I P V V I M S S V N V P S R I S R C L E E G A GAAGATTTTATCTTGAAACCAGTAAGACTAGCTGATGTCAATAAGCTTAAACCCCATATGATGAAAACCAAATGCAAAAGCTTTCATAAACCT E D F I L K P V R L A D V N K L K P H M M K T K C K S F H K P CAGAAAATTGTCACAAAGGAAAATCAAGAATCACAAGAAATTGAACCAAAAACAGAACAAATTTTACAACAATCTCAAGGCAATAATAATAAT Q K I V T K E N Q E S Q E I E P K T E Q I L Q Q S Q G N N N N AATAATTATAAGAGGAAGTCTATGGATCAACAAGGTTTATCACCAGAGAGAACAAGGCCTAGATACAACTACAATATGATCTTATGA N N Y K R K S M D Q Q G L S P E R T R P R Y N Y N M I L *

XI

8.1.4.8 StRRA8 ATGGAGATGTTTGCTAATTCATCATCGATTTCAAAGGGAATGGGGGAAGTTGTTGCAAGTGATGAACCTCATGTCTTAGCTGTTGATGACAAT M E M F A N S S S I S K G M G E V V A S D E P H V L A V D D N CTTGTTGATCGTAAACTTGTTGAAATATTACTCAAGAAATCTTCTTGCAAAGTAACTACTGCAGAAGATGGTCTGAGGGCATTAGAGTATTTG L V D R K L V E I L L K K S S C K V T T A E D G L R A L E Y L GGTTTGGCAGCACATCAAGATAGCTCGGCAAATAGCAATGGCTCAAAGGTTAATATGATAATAACAGATTATTGTATGCCAGGAATGACTGGT G L A A H Q D S S A N S N G S K V N M I I T D Y C M P G M T G TATGAACTGCTCAAGAAAATAAAGGAATCGTCGATGATGAAGGATGTGCCAGTTGTGATAATGTCATCCGAGAATATCCCAACTCGAATTAAT Y E L L K K I K E S S M M K D V P V V I M S S E N I P T R I N CAATGTATGGAAGAAGGAGCTCAAATATTCATGCTGAAGCCTCTGAAACACTCTGATGTCGAAAGGCTAAGATGCCAAATAATGCAATGCCGA Q C M E E G A Q I F M L K P L K H S D V E R L R C Q I M Q C R GGATAG G * 8.1.4.9 StRRB1 ATGAATCTTGGTGTTGGGTCAGTGGTGAAAACCATGTCTGGTGCTAGTTGTAGTGTTTCTTGGAAATCCGGTGGTAGTGATAAAGTTTCCGAC M N L G V G S V V K T M S G A S C S V S W K S G G S D K V S D CAGTTTCCGGCAGGTTTAAGAGTGCTTGTTGTAGATGATGATCCTACTTGTTTAATGATCTTGGAGAAGATGCTTAGGAATTGTCACTATGAA Q F P A G L R V L V V D D D P T C L M I L E K M L R N C H Y E GTCACCAAGTCTAATCGAGCGGAGCATGCATTATCAATGCTCCGGGAAAACAGAAATGGATTTGACATTGTTATTAGTGATGTGCACATGCCA V T K S N R A E H A L S M L R E N R N G F D I V I S D V H M P GACATGGATGGTTTCAAACTTCTTGAGCATGTTGGTCTGGAAATGGACCTCCCTGTTATAATGATGTCTGCGGATGACAGTAAGGATGTTGTT D M D G F K L L E H V G L E M D L P V I M M S A D D S K D V V ATGAAAGGTGTTACTCATGGAGCATGCGATTATCTGATCAAACCGGTGCGTATTGAGGCTTTGAAGAACATTTGGCAGCATGTGATTCGTAAA M K G V T H G A C D Y L I K P V R I E A L K N I W Q H V I R K AAGAAGCACGAGTTGAAGGACAAAGATTTTGATCAATCAACAAGCGTGGAAGATGGAGATCAACAGCAGAAACCTCCAGAAGATGTTGATTAT K K H E L K D K D F D Q S T S V E D G D Q Q Q K P P E D V D Y TCATCTTCAGCTAATGAAGGGAACTGGAAAAGCTCAAAGAGAAGAAAGGAGGAGGAAGATGAAACTGAAGAAAGGGATGATTTATCTACATCA S S S A N E G N W K S S K R R K E E E D E T E E R D D L S T S AAGAAGCCACGCGTGGTTTGGTCGGTGGAGCTTCATCAACAGTTTGTACAAGCTGTCAACCAACTTGGAATTGACAAGGCTGTTCCCAAGAAA K K P R V V W S V E L H Q Q F V Q A V N Q L G I D K A V P K K ATTCTTGAGCTGATGAATGTTCCTGGGCTAACCAGAGAAAATGTTGCTAGCCACCTACAGAAATATCGGCTGTACCTAAGGAGGTTGAGTGGT I L E L M N V P G L T R E N V A S H L Q K Y R L Y L R R L S G GTATCACAGCATCAGAATGGACTGAATAACTCATTCATGGGACACCCGGAAGCAACATATGGGACGATGACTTCTTTCAATGGGCTAGAGCTT V S Q H Q N G L N N S F M G H P E A T Y G T M T S F N G L E L CAAGCTTTAGCTGCCACCGGTCAACTACCCGCACAAAGTCTTGCTACCCTCCAGGCGGCTGCACTAGGTAGGTCTGCGACAAAATCTGCAATA Q A L A A T G Q L P A Q S L A T L Q A A A L G R S A T K S A I TCTATGCCTCTAGTAGATCAAAGAAACCTTTTCAGCTTTGAAAATCCCAAGTTGAGATTTTCCGAGGGACAACAACAATTAAATAATAGCAAT S M P L V D Q R N L F S F E N P K L R F S E G Q Q Q L N N S N AAACAAATTAACTTGCTTCATGGGATCCCAACAACCATGGAACCGAAGCAGCTGGCTGATTTGCACCAGTCCTCACAGTCCTTTGTGGCTATG K Q I N L L H G I P T T M E P K Q L A D L H Q S S Q S F V A M AATATGCAAGGGAATGCTAGAATGCAACAAAACAACGCTCTACTAATGCATATGTCTCAACAACAACAACAATCATCTAGGGCTCAAATGCTA N M Q G N A R M Q Q N N A L L M H M S Q Q Q Q Q S S R A Q M L AATGAAACCAATAATGGTCAAGTTTCACGGCCGCCATTGTCCATGTCACAACCTGCTGCAGTCTTATCACGAAATAGCATTGTTGACAACGTA N E T N N G Q V S R P P L S M S Q P A A V L S R N S I V D N V CGAGGGCCAATATACAATCCAGTCTCCCAAACATCTTCAATGGTAGATTTTTCGCTGAATCAAACTACAGAGTTGCAAAACAACAGTTTTCCT R G P I Y N P V S Q T S S M V D F S L N Q T T E L Q N N S F P CTTGTGAGTAGTAATTCAGGAATGTCAACTCTGACATCTAAAAGATTGCTTCAGGAAGAAGTTAACTCTGATATTAAAGGATCTAGAGGATTC L V S S N S G M S T L T S K R L L Q E E V N S D I K G S R G F CCTCCTGGTTACGATATATTTGAAGAGTTGCATCAGCAAAAAACGCAGGATTGGGGTTTACCGAATATCGGATCAAACTTCAGTGCGTCTGAT P P G Y D I F E E L H Q Q K T Q D W G L P N I G S N F S A S D CATTCAAGTATACCAGGAACTCTAGATGTCTCACCGTCCATGTTAGTTCAACAAGGTATTTCTTCAATGAAGAAGAATGGACAAAATGGAAAT H S S I P G T L D V S P S M L V Q Q G I S S M K K N G Q N G N TATCCTATGGGTGGACCGCAACTTAACCTATTTTCTGGCGGAAATTTGCTTCCTGTTAAAGCTGAACAACTTCCTGATACGAGCTATCAAAAT Y P M G G P Q L N L F S G G N L L P V K A E Q L P D T S Y Q N ACATTTTTTCCGGAGCAATTTGGACAGGATGATCTCATGAGTGCCCTTCTGAAGCAGCAAGAAAGTGTCGGACAAGTTGAAACTGAATTTGGC T F F P E Q F G Q D D L M S A L L K Q Q E S V G Q V E T E F G TTTGATGGATATTCACCATTGGACAATCTTCCTGTGTAA F D G Y S P L D N L P V * 8.1.4.10 StRRB2 ATGAATATTGGTAGTGGGTCTGTTCTCAGTTCTACTGGTTCTTGGAAGTCCGGTGATGTGGTTTCCGATCAGTTTCCGGTGGGACTCCGGGTA M N I G S G S V L S S T G S W K S G D V V S D Q F P V G L R V CTTGTAGTTGATGATGACCCTACTTGTTTAAAGATCTTGGAGAAGATGCTCAGGAACTGTCACTATGAAGTCACCAAGTGTAATAGGGCTGAG L V V D D D P T C L K I L E K M L R N C H Y E V T K C N R A E GTTGCACTATCATATCTCCGGGAAAATAAGAATGGTTTTGACATTGTTATAAGTGATGTACACATGCCAGACATGGATGGTTTCAAACTTCTT V A L S Y L R E N K N G F D I V I S D V H M P D M D G F K L L GAGCACATTGGTCTGGAGATGGACTTGCCTGTTATAATGATGTCTGCTGATGATAGTAAGGATGTGGTTATGAAAGGAGTTACTCATGGTGCG E H I G L E M D L P V I M M S A D D S K D V V M K G V T H G A TATGATTATTTGATCAAACCGGTGCGGATTGAGGCACTTAAGAATATTTGGCAACATGTTGTTCGTAAAAGGAAACAGGAGTGGAGGGACAAT Y D Y L I K P V R I E A L K N I W Q H V V R K R K Q E W R D N AATTTTGATCAATCAGGAAGTGTGGAAGAGGGAGATCGACAGCAGAAACAATCGGAGGATGTTGATTACTCATCTTCAGCTAATGAAGGAAAC N F D Q S G S V E E G D R Q Q K Q S E D V D Y S S S A N E G N TGGAAAAACTCCAAGAAAAGAAAGGAAGAGGATGAAGAAGGTGAAGAAAGGGATGATACATCCTCACAAAAGAAGCCACGTGTCGTTTGGTCT W K N S K K R K E E D E E G E E R D D T S S Q K K P R V V W S

XII Appendix

GTGGAGCTTCATCAACAATTTGTACAAGCTGTGCATCAACTTGGAATTGACAAGGCTGTTCCGAAGAAAATCTTGGAACTGATGAATGTTCCT V E L H Q Q F V Q A V H Q L G I D K A V P K K I L N V P G L E GGACTAACCAGAGAAAATGTTGCTAGCCACCTTCAGAAATATCGGTTGTATCTTAGAAGGTTGAGTTGTGTATCGCAGCACCAGAATGGACTG L M T R E N V A S H L Q K Y R L Y L R R L S C V S Q H Q N G L AACAACTCTTTCATGGGACGCCCAGATGCAACCTTCGGAACAATATCTTCCCTCAATGGGCTTGATCTTCAAGCTATTGCTGCTGCTGGTCAA N N S F M G R P D A T F G T I S S L N G L D L Q A I A A A G Q ATCCCTGCACAAAGTCTTGCTACCCTCCAAGCAGCTGCCCTTGGTAGATCTGCTACAAAATCTGCCATATCTATTCCCCTAGTAGATCAAAGA I P A Q S L A T L Q A A A L G R S A T K S A I S I P L V D Q R AACCTTTTCAGCTTCGAAAATTCCCAGGTGAGATTTCCAGAAGGGCAACAACAACTGAATAATAGCAATAAGCAAATCGACTTGCTGCATGGA N L F S F E N S Q V R F P E G Q Q Q L N N S N K Q I D L L H G ATTCCAACCACCATGGAACCAAAGCAGCTTGCAAATTTGCACCACCCTTCCCAGACCTTTGTGGGTATGAATATGCAAGTAAACTCCATGGCA I P T T M E P K Q L A N L H H P S Q T F V G M N M Q V N S M A CAACATAACAATTCTGTGGTTATGCGAATGCCCCAATCACAACCTACGGCTCAAATGCTATGTGGAGCTAATAATGGCAGTCAAGCTTCGAGG Q H N N S V V M R M P Q S Q P T A Q M L C G A N N G S Q A S R CTTCCATTGTCCATGCAGCAATCTTTGTCATCAGAAGGGATACCTGGTGCGGTCCTAGCACGGTGTCGTATTGTTGACAATGCACGAGCGAGT L P L S M Q Q S L S S E G I P G A V L A R C R I V D N A R A S GTTTACAATCCAGTCTCCCAAGCATCTTCAATGGTAGATTTCTCAGTAAATCAAAGCAAGGAGTTGCAAAACTACAACTTTTCTCTTGGGAGT V Y N P V S Q A S S M V D F S V N Q S K E L Q N Y N F S L G S AATAGTTCAGGGATGTCCACTTTGACTAATCGAGGGATGCTTCAGGAAGAGGCGAATTCTGATATCAAAGGATCTAGAGGTTTCCCTACTAAT N S S G M S T L T N R G M L Q E E A N S D I K G S R G F P T N TATGATATTTTCAACGACCTCCATCAGCCAAAATTGCAGAACTGGGGTTTACAGAATGTTGGTTCATCCTTTGACTCATCTCATCACCCAAGT Y D I F N D L H Q P K L Q N W G L Q N V G S S F D S S H H P S ATACAAGGAACTCAGGGTCTCTCATCACAATTGTTACTGCAACAGGGGATTTCTTCAACACACAACAATGGACAAAATAGAAATGGTCCTATT I Q G T Q G L S S Q L L L Q Q G I S S T H N N G Q N R N G P I GGGAAACCAATGTACACCAATGGTGAAGAAAGTGGACATACTAATCTTATGGGTGGACAACAACTTAACTCTGTCAGTAGGAATATGCTTGCT G K P M Y T N G E E S G H T N L M G G Q Q L N S V S R N M L A GTTAAAGCTGAAAGATTTCCCGACGCAGACTATCAGAGTACCAATTTCCCGGAGCAATTTGGACAGGATGACCTCATGAGTGCCTTCCTAAAA V K A E R F P D A D Y Q S T N F P E Q F G Q D D L M S A F L K CAGCAAGGAAGTGTTGGACCAGTTGAAACTGAATTTGGCTTCGATGGATACACATTGGATAATCTTCCAGTTTAA Q Q G S V G P V E T E F G F D G Y T L D N L P V *

8.1.4.11 StRRB3 ATGAGTGGCGACGTTGCCACGTGTAAGTCGGAAGCTACGGTTGTCACTGATCACTTTCCACTAGGGTTAAGAGTCCTTGTAGTTGATGATGAC M S G D V A T C K S E A T V V T D H F P L G L R V L V V D D D GTTGTTTGCCTCCGAATTATTGAGCAAATGCTTCGCAGATGCAAGTATTCAGTTACTACTTGCACTCAAGCTATGGTGGCATTAAACCTATTA V V C L R I I E Q M L R R C K Y S V T T C T Q A M V A L N L L CGCGAAAAAAGGGGGACTTTTGATATTGTACTGAGTGATGTTCATATGCCTGATATGGATGGTTTTAAACTTCTTGAGCTTGTTGGGTTGGAA R E K R G T F D I V L S D V H M P D M D G F K L L E L V G L E ATGGACCTTCCAGTAATAATGATGTCAGGTGATGGAAGAACCAACCTCGTCATGAGGGGAGTTCAACATGGGGCTTGTGATTATTTGATTAAG M D L P V I M M S G D G R T N L V M R G V Q H G A C D Y L I K CCTATACGGGACGAGGAGCTAAAGAATATCTGGCAACATGTTGTTAGGAAAAGATACAACTCCAGTAAAGAGCTTGAATGCTCTGGTAGCTTG P I R D E E L K N I W Q H V V R K R Y N S S K E L E C S G S L GATGATAATGATCGGTATAAGCGAGGAAGTGATGATGCTGAATGTGCTTCTTCTGTAATTGAAGGCGCAGATGGAGTGCTAAAACCACAGAAG D D N D R Y K R G S D D A E C A S S V I E G A D G V L K P Q K AAGAAAAGAGAAGCCAAAGAAGAAGACGACACTGAAATGGAAAATGATGACCCAAGTACTTCAAAAAAGCCACGTGTAGTCTGGTCAGTGGAG K K R E A K E E D D T E M E N D D P S T S K K P R V V W S V E CTTCATCAGCAATTTGTTAGTGCAGTCAACCAACTTGGCATTGATAAGGCTGTACCTAAGAGAATCCTTGAACTGATGAATGTTCCTGGTTTG L H Q Q F V S A V N Q L G I D K A V P K R I L E L M N V P G L ACAAGAGAAAATGTGGCAAGCCATCTGCAGGAAAATCAGAAATTTAGGCTATACTTGAAGAGGTTAAGTGGAGTAGTTCAACAGCAGGGTGGC T R E N V A S H L Q E N Q K F R L Y L K R L S G V V Q Q Q G G CTCCCCAGTACCTTCTGTGGGCCAATAGAACAGAATTCAGAACTTGGTTCACTAGGGAGATTTGACATTCAAGCACTGGCAGCCTCTGGCCAA L P S T F C G P I E Q N S E L G S L G R F D I Q A L A A S G Q ATTCCTCCTGAAACTTTGACAGCTCTTCATGCCGAACTTTTAGGTAGATCGACAAGTAACTTGGTTTTACCAGCAGTAGAGATACAGAATCTC I P P E T L T A L H A E L L G R S T S N L V L P A V E I Q N L CTACAAGCTTCCCTGCAGCAAGCAAAGTGCATACCTGCTGACCAAGTTATGGCTTATGGTCAACCTCTGCTGAAATGCCATCCCAGCATTTCT L Q A S L Q Q A K C I P A D Q V M A Y G Q P L L K C H P S I S AATTCTAAACATCTTTCTCAATCTATCTTGTCTGCTGAAGATGTTCATTCCGGATTTGGATCCCAGCGAGCAAAAAATATATGTCTGGTCCCT N S K H L S Q S I L S A E D V H S G F G S Q R A K N I C L V P AGCAGCAATCCCATTGGGCTAGCTGCTCCGAACAGCAACATGTTAATGGCAATGATGCAACAGCAGCAATGGCAAAAGCAGCAGCAGATGGAA S S N P I G L A A P N S N M L M A M M Q Q Q Q W Q K Q Q Q M E CTACAACATAGACGGTCTGGACCTCCTGAGGTCAACCATTCAATTAATGTGCAACCTTCTTGCCTTGTATTGCCCTCTCAGTTACCAGGTAAT L Q H R R S G P P E V N H S I N V Q P S C L V L P S Q L P G N TTCCAAGTGGGAGATAGTCCTGCTTCCATCAGCCGCGCCGGCAGTCTGAGTAAATCTTCGGTGATTGATTATGGAGTTCTCTCGCCACAATCA F Q V G D S P A S I S R A G S L S K S S V I D Y G V L S P Q S AATAATTCATCAGGTGTGGTTCAAGTGTTAGACAGGGAACTCAAACCAGAATGTGGCTTAAACAGGCTCCCCAGTGGAGGTTCTCTCTCTCGG N N S S G V V Q V L D R E L K P E C G L N R L P S G G S L S R TCATGCTCGATAAATGCTGACAATAGTGTTGGTCTGCAGCTTCATAACTCGAGTTCAGCGTTTGGTTCTTCTAAACAACTGCCAGCCCTTATC S C S I N A D N S V G L Q L H N S S S A F G S S K Q L P A L I CCAAACCATTTGGGCTCGCCAGTTCCATATTATATTAACTCAAGCCAGGTTCTTGATCAGGGACATACGAGAAATCCTGGGGTTGGTAAATGT P N H L G S P V P Y Y I N S S Q V L D Q G H T R N P G V G K C GCCTCCATTCCCAGTCGTTTTGCAGTAGATGAGTCTGATTCGCCAATGTGCAACTTCAATACTGCAAAAAACTATCTTGAGGAAACTAAAGTT A S I P S R F A V D E S D S P M C N F N T A K N Y L E E T K V AAGCAGGAGCCTAACATGAATGTCATGGAAAATGCTAAAGTTGGTCCAGCGATTTTTCAAAAATTTCAACCGGGTGATCTTATGAGTGTCTTC K Q E P N M N V M E N A K V G P A I F Q K F Q P G D L M S V F AGTGACTAG S D *

XIII

8.1.4.12 StRRB4 ATGACTGTGGAAGAAATTAGAGGGAATATGGGTGGTGGTGAAAGGGGAAATAATTATGATAATTTTCCTATTGGTATGAGAGTTCTTGCTGTT M T V E E I R G N M G G G E R G N N Y D N F P I G M R V L A V GATGATGACCCTATTTGTTTGAAGCTTTTGGATGGTTTGCTCAGAAAATGCCAGTATCAGGTAACTACAACAAGTCAGGCAAGGATGGCATTG D D D P I C L K L L D G L L R K C Q Y Q V T T T S Q A R M A L AAGATGTTGAGGGAGAATAGAGATAGATTTGATTTGGTCATCAGTGATGTTCACATGCCTGATATGGATGGCTTTAAACTTCTTGAACTTGTT K M L R E N R D R F D L V I S D V H M P D M D G F K L L E L V GGTCTTGAGATGGATCTTCCAGTCATAATGTTGTCTGCAAACAGCGATACCAAACTTGTAAGGAAGGGAATTGATCATGGTGCTTGCGACTAT G L E M D L P V I M L S A N S D T K L V R K G I D H G A C D Y CTGGTGAAACCTGTTCGAATTGAGGAGTTGAGGAACATATGGCAACATGTGATCAGAAAAAAGAAGGTTGAGCCTAAGAGCCAGAGTAAGTCT L V K P V R I E E L R N I W Q H V I R K K K V E P K S Q S K S AATGATCAAGACAAATCTTATCAGGTAAGTGGAGAAAGTGGTCGAGGGCAATCACCAACAGGTAATGTAGATCAAAACGGAAAATGTAATAAG N D Q D K S Y Q V S G E S G R G Q S P T G N V D Q N G K C N K AAAAGGAAGGATGAAGAAGATGAGAATGATGAAAATGGAAATGACGATGAAGATCCAACAACTCAGAAGAGAGCTCGTGTCGTTTGGTCCATA K R K D E E D E N D E N G N D D E D P T T Q K R A R V V W S I GAACTTCACAGGAAGTTTGTTGCAGCTGTTGGTCAGTTGGGTATCGAAAAAGCTGTCCCAAAGAGGATTCTTGACCTGATGAATGTTGACGGG E L H R K F V A A V G Q L G I E K A V P K R I L D L M N V D G CTTACAAGGGAAAATGTGGCAAGCCATCTGCAGGCACTACCAGTTGGAGAAGTTGGTGAGACGAAATCGATGTATAAAGAAATAGTATTGGGA L T R E N V A S H L Q A L P V G E V G E T K S M Y K E I V L G GGAGATGTTTGGGATCTTTTGGATGAATTGATGATTTATATGGGAAATCCAATGCAATATTATGAGAAAGATGTCAAATTTGTGCGGGGTGTG G D V W D L L D E L M I Y M G N P M Q Y Y E K D V K F V R G V CTTTTGTCTGGCCCCCCTGGAACAGGTAAAACACTGTTTGCTCGGACACTTGCAAAGGAAAGTGGGATGCCTTTTGTATTTGCTTCTGGTGCA L L S G P P G T G K T L F A R T L A K E S G M P F V F A S G A GAGTTTACTGATAGTGAAAAGAGTGGTGCTGCACGAATCAATGAAATGTTTTCTGTTGCTAGGAGAAATAAGTATAGGCTTTACTTGAAAAGG E F T D S E K S G A A R I N E M F S V A R R N K Y R L Y L K R ATCAATTCAGTTCAAACCCAACAAGCAAACATGGTTGCCGCATTAGGGGGAAGGGACTATGTGCGAATGGGCTCACTGGATGGGCTTGGAGAT I N S V Q T Q Q A N M V A A L G G R D Y V R M G S L D G L G D TTTCGAACATTGGGTGGATCAGGACGGTATACTCATGCTGCCTTATCATCGTACAGTTCAGGGGGCATGCTTGGCAGACTAAATAGTGCTGCT F R T L G G S G R Y T H A A L S S Y S S G G M L G R L N S A A GGTCTGAGCGTGCGCAACCTGGCAGCATCTCAGCTACTCCAACCTAGTCATGGTCAAAATTTGAGCAATTCCGTTAATGCTTTCACAAAACTG G L S V R N L A A S Q L L Q P S H G Q N L S N S V N A F T K L AATCCAAACATTCCGCCTGCCAGCCAGAATGCTAGTTTATTTCAAGGCATTCCTGCATCATTGGAGCTTGATCAATTGCAGCAGAGTAAGTCC N P N I P P A S Q N A S L F Q G I P A S L E L D Q L Q Q S K S TCGGCGCATATTCCTTTGGACGAGTCGAGACTGCTTACAGCTGAAGTGCTCGGTTGCTCAAATAACTCCTTATCCAATGTTCCAAATATTCCG S A H I P L D E S R L L T A E V L G C S N N S L S N V P N I P ATGTTGCTTCAAGGGAATCCCCAGCAACCACTTACTGGGGGAGGATTTGGAAATCAGCACTCTCAAAACATGACTCCGTTTAGCTCAGACTCG M L L Q G N P Q Q P L T G G G F G N Q H S Q N M T P F S S D S TTCAATACTGGTGTTAATGGATCTTCCAACTTTCTGGAACATGGTAGATGTAATGAGAACTGGCAAAATTCCATTCAGTTATCGAAGTTCCAG F N T G V N G S S N F L E H G R C N E N W Q N S I Q L S K F Q TCTAATTCTTTCCCATTGACTGAATCCTTCATTAACAGCCATTTGCCACAAAATAGTGTAAGGGAAGCCACGACTCATTTACAGAACAGCCCT S N S F P L T E S F I N S H L P Q N S V R E A T T H L Q N S P CTTGATTTTACTTCCACCACCTCAGTTTCTCCTCCTTTTGAAGATTCAAGGGGAGAAATCCAATACCGTCAAAGTATGGCAGGTGCTGTTCAG L D F T S T T S V S P P F E D S R G E I Q Y R Q S M A G A V Q AGTATGAATCAAACACCATCTCAAGCATGGGCCGATAATAAACAACATTATTCCCACAACTCAAATAATACTTTTGGCAACAACTTAAGGTCT S M N Q T P S Q A W A D N K Q H Y S H N S N N T F G N N L R S CAGGTTCCCAATGATGGTAACATGGCTTCTTTAAGCCACAGTATGAATCAAAGCAATGAGAACTTTGGCAGAAGGATGGACATGTCATTGATT Q V P N D G N M A S L S H S M N Q S N E N F G R R M D M S L I GGTAGATCGAGTGGAGGTTCTTCGACACTCGTGCAGCAAACCGAGCATGAAAAGTTGACCCTAGATTCAAGGACAAGGTCAAATGAAGACTAC G R S S G G S S T L V Q Q T E H E K L T L D S R T R S N E D Y CTCTTGGAGCCAACAAAGCAACAAGTTGGTTTTAGTCCACAAGGCTATGACTCCCTGGATGATCTAATGACTGCAATGAAACGGGTATGA L L E P T K Q Q V G F S P Q G Y D S L D D L M T A M K R V *

8.1.4.13 StRRB5 ATGGTCTGTTGTTTCACAGTAACTACGACGAGTCAGGCAAGAACGGCGTTGAAGATGTTGAGGGAAAACAAAGATAGATTTGACTTGGTGATC M V C C F T V T T T S Q A R T A L K M L R E N K D R F D L V I AGTGATGTCCACATGCCTGATATGGATGGCTTTAAACTTCTGGAGCTTGTTGGTCTCGAGATGGATCTTCCTGTCATAATGTTGTCAGCAAAC S D V H M P D M D G F K L L E L V G L E M D L P V I M L S A N AGTGATAGCAAACTTGTAATGAAGGGAATAACTCACGGTGCTTGTGACTATTTGGTGAAACCTGTCCGGCTGGAGGAGCTAAAGAATATATGG S D S K L V M K G I T H G A C D Y L V K P V R L E E L K N I W CAACATGTAATAAGGAGAAAGAAAGTTGAGCCTAAGAAGCAAAACAAGTCTGATGATCAAGACAAGGCTCACCAGGGAGGCGGAGAAGGTGAA Q H V I R R K K V E P K K Q N K S D D Q D K A H Q G G G E G E AGAGGATCGCAGCTTTCAGGTAATGCAGATCAAAATGGGAAGGTTAACAAGAAAAGGAAGGATGAAGAATATGAAAGCGATGAAAATGGAAAT R G S Q L S G N A D Q N G K V N K K R K D E E Y E S D E N G N GACGATGAAGATCCTGGAACTCAGAAGAAACCTCGTGTTGTTTGGGCTATAGAGCTTCACAAGAAGTTTGTTACAGCAGTTCATCAGTTAGGC D D E D P G T Q K K P R V V W A I E L H K K F V T A V H Q L G CTTGAAAAAGCTGTGCCTAAGAGGATTCTTGATCTGATGAATGTTGAAGGACTTACAAGAGAGAATGTGGCGAGCCATCTCCAGCAGAAGTAT L E K A V P K R I L D L M N V E G L T R E N V A S H L Q Q K Y AGGCTCTTCTTGAAAAGGATCAATGCGGCAGATGCCCAGCAAGCCAACTTGGCTGCCGCTGCATTAGGGGGTAAAGATTCTGCGTACATGCGA R L F L K R I N A A D A Q Q A N L A A A A L G G K D S A Y M R ATGGGTTCACTGGACGGGCTTGGTGGTTTCCGAACGTTGGCTGGAGCAGGAAGGTTTGGTCAGGCTAGCTTATCATCATCATATGCATCAGGA M G S L D G L G G F R T L A G A G R F G Q A S L S S S Y A S G GGCATGCTTGGCAGACTAAATAGTCCTTCTGGTGTAAGCCTTCGCAATCTAGCATCTTCACCTATGCTCCAACCTAATCATGGTCAAAATTTG G M L G R L N S P S G V S L R N L A S S P M L Q P N H G Q N L AGTAACAACTCCATCAATGCCTTGATGAAATTTAATGCAAATGTTCCACCTGCAAGCCAGAATGCTAATTTGTTCCAAGGGATTCCTGCATCG S N N S I N A L M K F N A N V P P A S Q N A N L F Q G I P A S

XIV Appendix

TTGGAGCTTGATCAACTGCAGCAGAGTAAGTGTGCCACACACATACGAGAGTTGAATCCTTTGGACGAGTCAGGGTTGCATACAATGGCCAGT L E L D Q L Q Q S K C A T H I R E L N P L D E S G L H T M A S ACATTCTCAAACTCTAGGTTGGTTGGTAGCGCAAACAGCTCTATGCCCAATGTATCAAATAATCCTATTTTGCTTCAAGTGAATTCCCAGCAA T F S N S R L V G S A N S S M P N V S N N P I L L Q V N S Q Q CCACTGACGGGGACGGGATTTGGAAATCAGACATCTCTCAACATAGCCTCTTTTAGCTCTGAGGCCTTCAATACTGGTGTAAGTGGTTCTTCC P L T G T G F G N Q T S L N I A S F S S E A F N T G V S G S S AATTTTCTGGACCATGATAGATATAATGAGAATTGGCAAACTTCTCTCCAGCCTCTGAAGTTCCAGTCAACCTCCTTTCCGTTAAATGAACCT N F L D H D R Y N E N W Q T S L Q P L K F Q S T S F P L N E P TTCAGCAACAGCCATTTGCCTCAAGACAGAGTACGAGACAATGATACTTCAACTGGCCCTCTTTTGCAGAACAACCCTGTTGATTTTTCAACC F S N S H L P Q D R V R D N D T S T G P L L Q N N P V D F S T TCCGCCATAGTTTCCGTACCTTTCGAGTCTTCAAGAGGAGAAACACAATGCCGTGAAAGTTTAGGGGGTGCTGTTCAGATTATGAACCAAGCA S A I V S V P F E S S R G E T Q C R E S L G G A V Q I M N Q A ACCTGCCAACGGTGGCCAGATCAGAACCAACATTATTCCCACAATTCAAACAATATTTTTGGTAACATGAGCTCTCAAGTTTCTAGCAATGGT T C Q R W P D Q N Q H Y S H N S N N I F G N M S S Q V S S N G GGAATGGCCTCTTTAACTCATCCTATGGACCAAAACAATGATATGTTCTGCACAAGGGTGGAGACTTCTTTGACCGGCAGATCAAATGGAGGT G M A S L T H P M D Q N N D M F C T R V E T S L T G R S N G G TCTTCAATGCACATACACAATCGTGAAAGTGAAAAATTAACTCATGACTCGAGGACAAAGACAAATGAAGACTACCTCTTTCAGACAACAAAG S S M H I H N R E S E K L T H D S R T K T N E D Y L F Q T T K CAACAAGTTGGTTTTCTTCCTCAGGGTTATGGCTCCCTTGATGATCTTATGGGTGAAATGAAACGGGTAAGCTAA Q Q V G F L P Q G Y G S L D D L M G E M K R V S *

8.1.5 Selected purine permease DNA and amino acid sequences identified in the DMv3 genome 8.1.5.1 StPUP3 ATGAAGAAAGTTCTTCTAGTCATCAACTGTATAATTCTAGCTGTAGGAACCTGTGGTGGCCCTTTGGTCATGCGCCTTTACTTCATTAAAGGA M K K V L L V I N C I I L A V G T C G G P L V M R L Y F I K G GGCAAAAGAATTTGGCTATCAAGCTGGCTACAAACTGCTGCTTGGCCAATCAATTTCATCCCTTTAGTCATTTCCTATTTCTATCGTCGCAAA G K R I W L S S W L Q T A A W P I N F I P L V I S Y F Y R R K AATAATAGTACTACCAAGCTCATTCTGATGACTCCCCGAATTTTCATGGCGACTATTGGGATCGGAATCCTTCAAGGATTTACAAACTATTTC N N S T T K L I L M T P R I F M A T I G I G I L Q G F T N Y F TACGCTTATGGCATAGGAAAATTACCCGTCTCCACTAGCGGACTTCTTTTTGCAACGCAACTTGCTTTCACTGCCTTCTTTGCTTTCCTTATA Y A Y G I G K L P V S T S G L L F A T Q L A F T A F F A F L I GTGAAGCTGAAGTTCACTCCTTACTCGGTAAACTCTGTGTTTTTGTTGACGACTGGCGCAGTGGTTTTGGCTCTACGCTCCGGTGGTGATCAA V K L K F T P Y S V N S V F L L T T G A V V L A L R S G G D Q CCGGAGGGAGAGCCAAGGAAAGAGTATATATTAGGATTTGTCATGACCCTGGCATCTGCTGCATTGACTGGCCTTATTTTTCCTTTGGTGGAG P E G E P R K E Y I L G F V M T L A S A A L T G L I F P L V E TTGATATATAAAAAGACACAACAAACTATCACTTACACATTTGTATTGGAGTTTCAAACACTCTATTGCTTTGTTGCTACTGTGCTTGCAACA L I Y K K T Q Q T I T Y T F V L E F Q T L Y C F V A T V L A T ATTGGAATGATCATAAACAAAGACTTTCAGGCAATTTCAAGGGAGGCGAAAACATTTGAACTTGGAGAAGACAAATATTATATTGTGATAATA I G M I I N K D F Q A I S R E A K T F E L G E D K Y Y I V I I TGGAGTGCAATAATTCTGCAATTCTACTTCTTAGGGTCCATTGGAGTCATCTATTCTGCTTCTTCTTTGGTATGTGGCATTTTATTATCAGTT W S A I I L Q F Y F L G S I G V I Y S A S S L V C G I L L S V CTACTTCCTCTCACTGAAGTTTTAGCTGTTTTCCTTTATGGCGAAAAATTCAATGCAGAAAAAGGAGTTTCTCTTGCCCTTTCTCTATGGGGA L L P L T E V L A V F L Y G E K F N A E K G V S L A L S L W G TTTGCTTCATACTTTTATGGTGATTATAACAAGACAGAAAATAGTAATCAATCACCGGCAATAGAGATGATTGATAAAACTAATTGTACCCCA F A S Y F Y G D Y N K T E N S N Q S P A I E M I D K T N C T P TGA * 8.1.5.2 StPUP9 ATGGCGACTCAAGAAAGTTCATCAAGAAGGAAGAATTTTCTTCTAATTATAAATTGTATAATTCTAGCTATAGGAAATTGTGGTGGCCCTTTG M A T Q E S S S R R K N F L L I I N C I I L A I G N C G G P L ATCACCCGTCTTTATTTTCTCAAAGGAGGGGAAAGAATTTGGCTATCAAGTTGGTTAGAAACAGTTGGTTGGCCAATTGTTCTCATCCCTCTA I T R L Y F L K G G E R I W L S S W L E T V G W P I V L I P L TCCTTCTCCTACTTCAATCGTCGTCGTGAATTCCAAGGCAAAATGTTATCAACTATTGATAACAATAATACAAAACTCATTTTGATGACTCCT S F S Y F N R R R E F Q G K M L S T I D N N N T K L I L M T P AGGATCTTTGTAGCGAGCATCGGGATTGGAGTTCTGACTGGATTCGATAATTACCTCTACGCTTATGGCGTGGCTAAATTACCTGTCTCCACG R I F V A S I G I G V L T G F D N Y L Y A Y G V A K L P V S T TCAGCTCTTCTCATAGCTTCTCAACTTGCTTTCACCGCGGCCTTTGCTTTCCTCCTTGTGAAGCAAAAGTTCACTTCCTACTCCATCAACTCA S A L L I A S Q L A F T A A F A F L L V K Q K F T S Y S I N S ATTTTTCTACTGACATTTGGCGCGGTAGTATTGGCCCTCCACGCGAAGGGTGACCGACCGAACGGGGAGTCTAAGAAGGAGTATGTTCTAGGG I F L L T F G A V V L A L H A K G D R P N G E S K K E Y V L G TTTATCATGACACTTGGAGCTGCAGCTTTGTATGGACTTATTTTGCCTTTGTTTGAGTTGATGTACAAAAAGGCAAAACAAGGTATCACTTAC F I M T L G A A A L Y G L I L P L F E L M Y K K A K Q G I T Y ACACTTGTTATGGAGATTCAGGCTGTCTCTTGCATTTTTTCTACTGTGGTTTGCACAATTGGGATGATAATAAACAAGGACTTTCAGTTTGAA T L V M E I Q A V S C I F S T V V C T I G M I I N K D F Q F E CTTGGAGAAGCTAGATATTATATTGTTATAATATGGTCAGCAATAATTTGGCAATGTTTTTTCTTAGGTGCCATTGGAGTTATCTATTCTTCA L G E A R Y Y I V I I W S A I I W Q C F F L G A I G V I Y S S TCATCATTGGTGTCTGGCATTTTAATTACTGTTTTACTTCCTATTACTGAAATATTAGGTGTTGTTTTTTATGGTGAAAAATTTACACCAGAA S S L V S G I L I T V L L P I T E I L G V V F Y G E K F T P E AAAGGTGTTTCTCTTGTACTTTCTATATGGGGTTTTATTTCTTACCTATATGGTGATATTAAAGCTAGCAAGAAGAAGAAAGAAAATCAATCT K G V S L V L S I W G F I S Y L Y G D I K A S K K K K E N Q S CAAGAACAAGAGATGATTGATAAAATTACTTGTACTCCATGA Q E Q E M I D K I T C T P *

XV

8.1.5.3 StPUP10 ATGAAGAAAGTTCTTCTAGTCATCAACTGTATAATTCTAGCTGTAGGAACCTGTGGTGGCCCTTTGGTCATGCGCCTTTACTTCATTAAAGGA M K K V L L V I N C I I L A V G T C G G P L V M R L Y F I K G GGCAAAAGAATTTGGCTATCAAGCTGGCTACAAACTGCTGCTTGGCCAATCAATTTCATCCCTTTAGTCATTTCCTATTTCTATCGTCGCAAA G K R I W L S S W L Q T A A W P I N F I P L V I S Y F Y R R K AATAATAGTACTACCAAGCTCATTCTGATGACTCCCCGAATTTTCATGGCGACTATTGGGATCGGAATCCTTCAAGGATTTACAAACTATTTC N N S T T K L I L M T P R I F M A T I G I G I L Q G F T N Y F TACGCTTATGGCATAGGAAAATTACCCGTCTCCACTAGCGGACTTCTTTTTGCAACGCAACTTGCTTTCACTGCCTTCTTTGCTTTCCTTATA Y A Y G I G K L P V S T S G L L F A T Q L A F T A F F A F L I GTGAAGCTGAAGTTCACTCCTTACTCGGTAAACTCTGTGTTTTTGTTGACGACTGGCGCAGTGGTTTTGGCTCTACGCTCCGGTGGTGATCAA V K L K F T P Y S V N S V F L L T T G A V V L A L R S G G D Q CCGGAGGGAGAGCCAAGGAAAGAGTATATATTAGGATTTGTCATGACCCTGGCATCTGCTGCATTGACTGGCCTTATTTTTCCTTTGGTGGAG P E G E P R K E Y I L G F V M T L A S A A L T G L I F P L V E TTGATATATAAAAAGACACAACAAACTATCACTTACACATTTGTATTGGAGTTTCAAACACTCTATTGCTTTGTTGCTACTGTGCTTGCAACA L I Y K K T Q Q T I T Y T F V L E F Q T L Y C F V A T V L A T ATTGGAATGATCATAAACAAAGACTTTCAGGTCTCTCTCTTTCTCTTCCCTCAATTTGAATATTATGCGAAAACATTTGAACTTGGAGAAGAC I G M I I N K D F Q V S L F L F P Q F E Y Y A K T F E L G E D AAATATTATATTGTGATAATATGGAGTGCAATAATTCTGCAATTCTACTTCTTAGGGTCCATTGGAGTCATCTATTCTGCTTCTTCTTTGGTA K Y Y I V I I W S A I I L Q F Y F L G S I G V I Y S A S S L V TGTGGCATTTTATTATCAGTTCTACTTCCTCTCACTGAAGTTTTAGCTGTTTTCCTTTATGGCGAAAAATTCAATGCAGAAAAAGGAGTTTCT C G I L L S V L L P L T E V L A V F L Y G E K F N A E K G V S CTTGCCCTTTCTCTATGGGGATTTGCTTCATACTTTTATGGTGATTATAACAAGACAGAAAATAGTAATCAATCACCGGCAATAGAGATGATT L A L S L W G F A S Y F Y G D Y N K T E N S N Q S P A I E M I GATAAAACTAATTGTACCCCATGA D K T N C T P *

8.1.5.3 StPUP11 ATGGAAGAAGAAGCAAATTTAAGTATCAATATGAGGAGATTCCTCTTGATTATAAATTGTTTATTACTATCCGTTGGTGTTTGTGGTGGCCCT M E E E A N L S I N M R R F L L I I N C L L L S V G V C G G P TTAATGATGCGTTTATATTTTGTTGAAGGTGGTTTAAGATTATGGTTTAATAGTTGGTTACAAACCGGTGGATGGCCACTCACAATTATACCA L M M R L Y F V E G G L R L W F N S W L Q T G G W P L T I I P TTAGTCATCCTATATTTCTATCGACGAAAAACCAAGGGCTCTGATACCAAGTTTTATTATATTACACCTCGTATTTTCATTGCATCGTTCATT L V I L Y F Y R R K T K G S D T K F Y Y I T P R I F I A S F I ATTGGCGTTTTCACGGGTGTTGATGCTTACCTTTATTCATGGGGCGGGTCGAAACTCCCCGTGTCAACGTCTTCCCTTCTCATCGCAGCTCAA I G V F T G V D A Y L Y S W G G S K L P V S T S S L L I A A Q CTTGCCTTCACGGCGATAGGGTCTTACTTCATAGCAAAGATAAAATTTACATCATATTCGATTAACGCGGTGGTTTTATTGACACTTGGCGCG L A F T A I G S Y F I A K I K F T S Y S I N A V V L L T L G A GTTTTATTGGGTATGCGATCGAACGGTGATCGACCGGAGGGTGTTACAAGTAAAGAGTATGTTATTGGTTTTATTATGACACTTTTGGCTGCA V L L G M R S N G D R P E G V T S K E Y V I G F I M T L L A A GCTTTGTATGGACTTATTTTGCCTTGTATTGAGTTGATATATTTGAAGGCAAAACAAGCTATTACAGCAACATTGGTGTTGGAGATTCAAATG A L Y G L I L P C I E L I Y L K A K Q A I T A T L V L E I Q M GTCATGGCTTTTGCTGCTACTGCTTTTTGTACCATTGGAATGATTGCTAACAAAGATTTTCAGGCAATATCAAGGGAAGCAAAACATTTTAAC V M A F A A T A F C T I G M I A N K D F Q A I S R E A K H F N CTTGGAGAAGGTAGATATTACATAGTTGTAATATGGAGTGCCATAATTTGGCAATGTTTCTTTGTTGGTGCTGTTGGAGTTATTTACCTCTCT L G E G R Y Y I V V I W S A I I W Q C F F V G A V G V I Y L S TCTTCTTTAATGTCTGGAGTTATGATTGCAGTTTTACTGCCAATTACTGAAGTATTAGGAGTAATTTTCTTTGATGAAAAATTCTCAGCTGAA S S L M S G V M I A V L L P I T E V L G V I F F D E K F S A E AAGGGACTTTCACTTTTTCTTTCTCTTTGGGGTTTTGTTTCTTATTTTTATGGAGAATTTAAACAAGCAAAGAAAGTTGAGAAGAAGAAAATT K G L S L F L S L W G F V S Y F Y G E F K Q A K K V E K K K I CAAGAAAATGAGATGACAACAACAACACAAATAGAACATGTTTGA Q E N E M T T T T Q I E H V *

XVI Appendix

8.2 Tables

8.2.1 Unique and overlapping entities of WT, IPT-6 and CX1-4 Table A1: Percentages of overlapping and unique entities of the three genotypes After VENN-diagram comparison of entities found to be differentially and significantly regulated, percentages of overlapping and unique genes were calculated in relation to the total number of entities up- or downregulated in each genotype.

Genotype Entities Overlap with both Uniquely total WT CKX1-4 IPT-6 others expressed

- up-regulated - WT 4349 ----- 17.4% 30.9% 33.1% 18.6% CKX1-4 4878 15.5% ---- 6.9% 29.5% 48.1% IPT-6 4331 31.1% 7.8% ---- 33.2% 27.9% - down-regulated - WT 2860 ----- 21.8% 24.5% 39.0% 14.7% CKX1-4 4225 14.8% ---- 6.5% 26.4% 52.3% IPT-6 3389 20.7% 8.2% ---- 32.9% 38.2%

XVII

8.2.2 Allocation of functional groups to entities found to be differentially expressed in ‘3d versus 0d’ comparisons of WT, IPT-6 and CKX1-4

Table A2: Entities in each of the 18 functional groups as percentages of the total number of entities in each list After functional classification of entities found to be differentially and significantly regulated, percentages of each category were calculated in relation to the total number of entities in the up- or downregulated list of each genotype.

WT CKX1-4 IPT-6 up- down- down- down-

regulated regulated up-regulated regulated up-regulated regulated no. % no. % no. % no. % no. % no. % Cell Cycle/ Replication/ 94 2.16 36 1.26 49 1.00 37 0.88 82 1.89 29 0.86 Chromatin-associated DNA-/ RNA- 86 1.98 86 3.01 103 2.11 105 2.49 81 1.87 100 2.95 associated Transcription/ 32 0.74 76 2.66 58 1.19 67 1.59 29 0.67 87 2.57 Translation Transcription factors 141 3.24 76 2.66 192 3.94 125 2.96 164 3.79 90 2.66

Protein fate 155 3.56 155 5.42 180 3.69 229 5.42 130 3.00 187 5.52 Cell wall biosynthesis/ 116 2.67 22 0.77 59 1.21 77 1.82 114 2.63 31 0.91 modification Cytosceleton 41 0.94 3 0.10 17 0.35 22 0.52 46 1.06 5 0.15

Stress/ defense 171 3.93 61 2.13 159 3.26 95 2.25 161 3.72 98 2.89

Storage proteins 28 0.64 4 0.14 17 0.35 41 0.97 45 1.04 4 0.12

Signalling 234 5.38 101 3.53 246 5.04 187 4.43 244 5.63 99 2.92

Photosynthesis 89 2.05 7 0.24 64 1.31 9 0.21 38 0.88 11 0.32

Phytohormones 152 3.50 82 2.87 176 3.61 142 3.36 168 3.88 93 2.74

Transport 146 3.36 122 4.27 125 2.56 226 5.35 133 3.07 143 4.22

Metabolism 590 13.57 361 12.62 650 13.33 573 13.56 481 11.11 544 16.05 Development/ 3 0.07 10 0.35 9 0.18 12 0.28 6 0.14 10 0.30 senescence Electron Transport/ 112 2.58 69 2.41 118 2.42 80 1.89 118 2.72 86 2.54 Redox unknown 1621 37.27 1170 40.91 1996 40.92 1633 38.65 1774 40.96 1295 38.21 unclassified 538 12.37 419 14.65 660 13.53 565 13.37 517 11.94 477 14.07

Total 4349 100 2860 100 4878 100 4225 100 4331 100 3389 100

XVIII Appendix

Table A3: Entities in each of 16 functional groups as percentages of the number of entities in each list, without ‘Unclassified’ and ‘Unknown’ entities For better visualization of the functional categories, the numbers of ‘Unclassified’ and ‘Unknown’ entities were substracted from the total of the corresponding list and percentages of each category were re-calculated in relation to the new sum of entities.

WT CKX1-4 IPT-6 down- down- down- up-regulated regulated up-regulated regulated up-regulated regulated

no. % no. % no. % no. % no. % no. % Cell Cycle/ Replication/ 94 4.29 36 2.83 49 2.21 37 1.83 82 4.02 29 1.79 Chromatin-associated DNA-/ RNA- associated 86 3.93 86 6.77 103 4.64 105 5.18 81 3.97 100 6.18 Transcription/ 32 1.46 76 5.98 58 2.61 67 3.31 29 1.42 87 5.38 Translation Transcription factors 141 6.44 76 5.98 192 8.64 125 6.17 164 8.04 90 5.57

Protein fate 155 7.08 155 12.20 180 8.10 229 11.30 130 6.37 187 11.56 Cell wall biosynthesis/ 116 5.30 22 1.73 59 2.66 77 3.80 114 5.59 31 1.92 Modification Cytosceleton 41 1.87 3 0.24 17 0.77 22 1.09 46 2.25 5 0.31

Stress/ defense 171 7.81 61 4.80 159 7.16 95 4.69 161 7.89 98 6.06

Storage proteins 28 1.28 4 0.31 17 0.77 41 2.02 45 2.21 4 0.25

Signalling 234 10.68 101 7.95 246 11.07 187 9.23 244 11.96 99 6.12

Photosynthesis 89 4.06 7 0.55 64 2.88 9 0.44 38 1.86 11 0.68

Phytohormones 152 6.94 82 6.45 176 7.92 142 7.01 168 8.24 93 5.75

Transport 146 6.67 122 9.60 125 5.63 226 11.15 133 6.52 143 8.84

Metabolism 590 26.94 361 28.40 650 29.25 573 28.27 481 23.58 544 33.64 Development/ 3 0.14 10 0.79 9 0.41 12 0.59 6 0.29 10 0.62 Senescence Electron Transport/ 112 5.11 69 5.43 118 5.31 80 3.95 118 5.78 86 5.32 Redox

Total 2190 100 1271 100 2222 100 2027 100 2040 100 1617 100

XIX

Table A4: Sub-allocation of phytohormone-related entities into nine sub-categories For better visualization of the ‘phytohormone-associated’ category, entities were divided into functional sub-categories representing the nine phytohormone classes. Percentages of each sub-category were calculated in relation to the total number of phytohormone-associated entities in each corresponding list.

up-regulated down-regulated WT CKX1-4 IPT-6 WT CKX1-4 IPT-6 Auxin 39.07 29.14 34.73 21.95 21.99 17.39 Ethylene 14.57 25.71 17.37 9.76 14.89 20.65 Gibberellin 12.58 4.00 9.58 15.85 17.02 15.22 ABA 11.26 9.71 10.18 18.29 17.73 14.13 Jasmonate 8.61 8.57 6.59 7.32 5.67 7.61 Cytokinin 7.95 11.43 10.78 15.85 9.93 8.70 Brassinosteroids 3.97 9.71 8.38 10.98 11.35 16.30 Salicylate 1.99 1.71 2.40 0.00 1.42 0.00 Strigolactone 0.00 0.00 0.00 0.00 0.00 0.00

XX Appendix

8.2.3 VENN-diagram derived entity lists

Table A5: Overlap list of up-regulated entites from WT [3d vs 0d], IPT-6 [3d vs 0d] and CKX1-4 [3d vs 0d] comparisons Three entity lists, ‘up-regulated WT 3d vs 0d’, ‘up-regulated IPT-6 3d vs 0d’ and ‘up-regulated CKX1-4 3d vs 0d’, were combined in a VENN diagram (compare Fig.10A) and 1440 entities found in all three genotypes were saved as ‘overlap up-regulated in all three genotypes’ list. Entities in this list were sorted according to category and, within each category, ranked by fold change (FCAbsolute [3d] versus [0d]) of the WT. This table lists fold change and normalized values for 6 selected functional (sub)categories: CC - cell cycle/ replication/ chromatin associated; H - hormone-associated (sorted further by hormone class: H_AUX - auxin, H_CK - cytokinins, H_ETH - ethylene and H_GA - gibberellic acid).

FCAbsolute [3d] versus [0d] normalized values

POCI ID WT CKX1-4 IPT-6 [WT 0d] [WT 3d] [CKX1-4 0d] [CKX1-4 3d] [IPT-6 0d] [IPT-6 3d] Description category

SDBN001F10u.scf_564 315.36 7.87 413.29 -1.54 6.76 -1.59 1.39 -2.18 6.51 B-type cyclin [Nicotiana tabacum] CC

MICRO.15499.C1_637 283.30 6.30 382.97 -2.38 5.77 -1.91 0.75 -2.97 5.61 Knolle [Capsicum annuum] CC

MICRO.15342.C1_660 145.49 3.93 161.82 -0.85 6.33 -1.57 0.41 -1.15 6.19 DNA topoisomerase II [Nicotiana tabacum] CC

MICRO.3928.C1_669 30.56 2.18 10.89 -1.24 3.69 -4.44 -3.31 0.39 3.83 Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) (dUTP pyrophosphatase) CC cSTA29A10TH_321 26.96 2.84 8.11 -1.00 3.76 -2.45 -0.94 0.35 3.37 cyclin A1 [Solanum lycopersicum] CC

MICRO.2589.C1_821 14.38 2.45 5.95 -0.88 2.97 -1.24 0.05 -0.15 2.42 cyclin A2 [Solanum lycopersicum] CC bf_swstxxxx_0004g10.t3m.scf_257 14.35 2.27 6.98 -0.75 3.09 -0.69 0.50 -0.13 2.67 cyclin A2 [Solanum lycopersicum] CC

MICRO.9550.C1_1072 6.81 2.20 5.03 -1.31 1.45 -0.56 0.58 -0.53 1.80 putative replication factor A [Capsicum chinense] CC

MICRO.338.C1_1334 2.03 2.56 2.32 -0.55 0.48 -0.36 0.99 -0.73 0.49 cyclin-dependent protein kinase p34cdc2 [Solanum lycopersicum] CC

MICRO.5000.C3_344 403.31 9.26 367.04 -2.23 6.42 -2.20 1.01 -1.76 6.76 Histone H4 CC

MICRO.1026.C2_455 213.36 9.89 89.88 -1.96 5.78 -3.04 0.27 -0.30 6.19 Histones H3 and H4 (ISS) [Ostreococcus tauri] CC

MICRO.1026.C5_493 211.05 3.45 98.93 -1.13 6.59 -2.04 -0.25 0.11 6.74 PREDICTED: similar to histone 1, H2ai (predicted) [Canis familiaris] CC

MICRO.2825.C1_425 157.35 8.51 54.21 -2.04 5.25 -3.06 0.03 -0.34 5.42 histone 3 CC

BF_TUBSXXXX_0056B12_T3M.SCF_425 97.97 4.95 46.08 -1.48 5.13 -2.07 0.23 -0.32 5.21 histone 3 CC

MICRO.4761.C2_73 14.21 5.69 6.51 -2.03 1.80 -1.75 0.76 -1.00 1.70 histone H1F [Nicotiana tabacum] CC cSTD5O13TH_55 2.99 2.86 2.58 -1.12 0.46 -1.31 0.20 -0.58 0.79 high mobility group protein 2 HMG2 [Ipomoea nil] CC

MICRO.4058.C1_1748 2.21 3.04 2.34 -0.50 0.64 0.08 1.69 -1.14 0.09 histone-lysine N-methyltransferase/ zinc ion binding [Arabidopsis thaliana] CC

MICRO.18202.C1_677 15.81 30.55 21.74 -1.59 2.39 -3.81 1.12 -2.92 1.52 DFL1 (DWARF IN LIGHT 1) [Arabidopsis thaliana] H_AUX

MICRO.614.C5_1053 14.91 42.83 10.49 -2.90 1.00 -3.26 2.16 -2.16 1.23 aldo/keto reductase AKR [Manihot esculenta] H_AUX

MICRO.614.C4_1069 14.68 37.48 9.72 -2.79 1.08 -2.91 2.32 -1.93 1.35 aldo/keto reductase AKR [Manihot esculenta] H_AUX

POADO85TP_600 13.47 53.26 9.62 -2.97 0.79 -3.09 2.65 -1.84 1.43 Auxin-induced protein PCNT115 H_AUX

MICRO.614.C6_1138 9.64 13.36 8.84 -2.03 1.23 -1.27 2.47 -1.87 1.28 Auxin-induced protein PCNT115 H_AUX bf_arrayxxx_0034d11.t7m.scf_624 9.17 6.44 10.13 -0.34 2.86 -2.41 0.28 -0.87 2.47 GH3 auxin-responsive promoter [Medicago truncatula] H_AUX

XXI

STMHX62TH_635 7.71 4.06 9.65 -1.43 1.52 -2.46 -0.44 -0.27 3.00 Probable glutathione S-transferase (Auxin-induced protein PGNT1/PCNT110) H_AUX

MICRO.15954.C1_522 7.27 5.86 6.36 -0.31 2.56 -1.75 0.80 -0.65 2.02 GH3.5 [Arabidopsis thaliana] H_AUX

MICRO.13673.C1_907 6.73 2.79 5.72 -1.50 1.25 -0.69 0.79 -1.11 1.40 IAA13; transcription factor [Arabidopsis thaliana] H_AUX

MICRO.614.C1_273 3.98 2.85 2.37 -1.12 0.88 -1.12 0.40 -0.47 0.78 aldo/keto reductase AKR [Manihot esculenta] H_AUX

MICRO.6141.C1_1937 3.98 2.93 3.44 -1.08 0.91 -0.85 0.70 -0.78 1.01 auxin-induced protein [Arabidopsis thaliana] H_AUX

MICRO.15100.C1_2259 3.86 9.81 2.29 -0.98 0.97 -1.19 2.10 -0.59 0.61 Cyclin-like F-box [Medicago truncatula] H_AUX

MICRO.3540.C6_22 3.60 7.68 4.01 -0.99 0.85 -1.02 1.92 -1.16 0.84 auxin repressed/dormancy associated protein [Lycopersicon esculentum] H_AUX

MICRO.614.C2_172 3.58 4.38 2.61 -0.97 0.87 -0.59 1.54 -0.73 0.65 Auxin-induced protein PCNT115 H_AUX

MICRO.11489.C1_1222 3.08 3.14 2.89 0.03 1.65 -4.65 -3.00 -0.09 1.44 S-adenosylmethionine-dependent methyltransferase [Arabidopsis thaliana] H_AUX

POCBV18TP_741 3.06 3.83 3.13 -1.23 0.38 -1.57 0.37 -0.44 1.21 auxin and ethylene responsive GH3-like protein [Capsicum chinense] H_AUX

ACDA03609E09.T3m.scf_372 2.48 4.05 4.09 -0.96 0.35 -1.04 0.98 -1.58 0.45 auxin repressed/dormancy associated protein [Lycopersicon esculentum] H_AUX

MICRO.259.C7_585 2.33 4.00 2.24 -0.70 0.52 -1.02 0.98 -0.66 0.50 auxin response factor 4 [Cucumis sativus] H_AUX

MICRO.5658.C1_1051 2.31 5.86 2.46 -0.48 0.72 -0.73 1.82 -0.52 0.78 AUX/IAA protein [Medicago truncatula] H_AUX

MICRO.4409.C1_979 2.25 2.22 2.22 -1.08 0.09 0.47 1.62 -1.20 -0.05 auxin response factor 3 [Gossypium hirsutum] H_AUX

MICRO.6730.C2_1253 2.11 3.80 2.53 -0.66 0.41 -1.20 0.73 -1.25 0.10 L-galactose dehydrogenase [Nicotiana langsdorffii x Nicotiana sanderae] H_AUX

MICRO.5974.C1_1055 3.86 3.32 7.61 -1.54 0.40 0.23 1.96 -2.28 0.64 ATPUP3; purine transporter [Arabidopsis thaliana] H_CK

POAD617TV_655 3.66 5.68 2.82 -0.76 1.11 -0.89 1.61 -0.95 0.55 adenine phosphoribosyltransferase-like [Solanum tuberosum] H_CK

061A10AF.esd_483 2.95 3.78 2.82 0.17 1.72 -2.48 -0.56 -0.18 1.32 CLV1 receptor kinase [Arabidopsis thaliana] H_CK

MICRO.7256.C1_1091 30.21 19.98 33.66 -3.10 1.82 -2.02 2.30 -2.94 2.14 1-aminocyclopropane-1-carboxylate oxidase homolog (Protein E8) H_ETH

MICRO.2256.C1_363 21.99 32.05 4.04 -3.05 1.41 -3.76 1.24 -0.70 1.32 ethylene response factor 1 [Lycopersicon esculentum] H_ETH

MICRO.16824.C1_963 10.92 6.72 3.98 -2.06 1.39 -0.79 1.95 -1.29 0.71 ethylene receptor-like protein [Lycopersicon esculentum] H_ETH

MICRO.2939.C3_452 10.67 2.18 13.99 -2.12 1.30 -0.49 0.64 -2.41 1.40 1-aminocyclopropane-1-carboxylate oxidase [Solanum tuberosum] H_ETH

MICRO.2939.C2_541 10.43 2.47 15.22 -2.28 1.10 -0.75 0.56 -2.39 1.54 1-aminocyclopropane-1-carboxylate oxidase [Solanum tuberosum] H_ETH bf_arrayxxx_0008a01.t7m.scf_238 10.13 2.27 16.94 -2.13 1.21 -0.43 0.75 -2.64 1.44 1-aminocyclopropane-1-carboxylate oxidase [Solanum tuberosum] H_ETH

MICRO.13070.C1_66 9.05 4.70 3.07 -2.37 0.81 -1.58 0.65 -0.63 0.98 1-aminocyclopropane-1-carboxylate oxidase homolog (Protein E8) H_ETH Ethylene-responsive transcription factor 1 (Ethylene-responsive element-binding factor bf_arrayxxx_0067e02.t3m.scf_528 4.77 31.72 2.33 -1.46 0.80 -4.48 0.51 -0.44 0.79 H_ETH 1) (EREBP-1) (ERF1-like protein) (LeERF1) MICRO.1924.C1_1931 3.73 2.08 7.52 -1.30 0.60 -0.40 0.66 -2.77 0.14 EIN3-binding F-box protein 1 [Solanum lycopersicum] H_ETH

MICRO.12438.C1_600 2.89 12.94 2.89 -0.78 0.75 -0.87 2.82 -1.13 0.40 ethylene response factor 4 [Lycopersicon esculentum] H_ETH

MICRO.2158.C1_117 2.16 2.54 3.60 -0.51 0.60 -0.39 0.95 -1.50 0.35 EIL1 [Lycopersicon esculentum] H_ETH

MICRO.3821.C1_1696 2.13 3.39 3.97 -0.31 0.78 -0.04 1.72 -1.78 0.20 EIN3-binding F-box protein 1 [Solanum lycopersicum] H_ETH

MICRO.11045.C2_125 331.58 3.27 222.97 -1.32 7.06 -1.74 -0.03 -0.87 6.93 GAST1 protein precursor H_GA

XXII Appendix

Table A6: Overlap list of down-regulated entites from WT [3d vs 0d], IPT-6 [3d vs 0d] and CKX1-4 [3d vs 0d] comparisons Three entity lists, ‘down-regulated WT 3d vs 0d’, ‘down-regulated IPT-6 3d vs 0d’ and ‘down-regulated CKX1-4 3d vs 0d’, were combined in a VENN diagram (compare Fig.10B) and 1115 entities found in all three genotypes were saved as ‘overlap down-regulated in all three genotypes’ list. Entities in this list were sorted according to category and, within each category, ranked by fold change (FCAbsolute [3d] versus [0d]) of the WT. This table lists fold change and normalized values for 6 selected functional (sub)categories: CC - cell cycle/ replication/ chromatin associated; H - hormone-associated (sorted further by hormone class: H_AUX - auxin, H_CK - cytokinins, H_ETH - ethylene and H_GA - gibberellic acid).

FCAbsolute [3d] vs [0d] normalized data

POCI ID WT CKX1-4 IPT-6 [WT 0d] [WT 3d] [CKX1-4 0d] [CKX1-4 3d] [IPT-6 0d] [IPT-6 3d] Description category

MICRO.2938.C1_1209 4.22 8.01 2.40 0.79 -1.29 1.23 -1.77 0.50 -0.76 centrin [Nicotiana tabacum] CC ATP binding / ATP-dependent helicase/ helicase/ nucleic acid binding [Arabidopsis MICRO.14217.C1_885 3.53 2.66 3.80 0.75 -1.07 1.24 -0.18 0.23 -1.69 CC thaliana] MICRO.2712.C1_1379 3.33 2.66 3.55 0.53 -1.21 1.03 -0.38 0.61 -1.21 chromatin binding [Arabidopsis thaliana] CC

MICRO.1876.C2_1106 2.92 3.17 2.74 1.11 -0.44 0.46 -1.20 0.89 -0.56 DNA helicase-like [Arabidopsis thaliana] CC BF_LBCHXXXX_0053E02_T3M.SCF_35 36.28 72.63 10.70 3.04 -2.14 4.34 -1.84 1.70 -1.72 IAA type protein [Elaeis guineensis] H_AUX 8 BF_CSCHXXXX_0017E01.T3M.SCF_59 17.39 33.07 8.03 2.26 -1.86 3.39 -1.66 1.47 -1.53 IAA type protein [Elaeis guineensis] H_AUX 8 MICRO.4627.C1_781 16.82 20.16 27.96 3.73 -0.34 0.59 -3.74 3.45 -1.35 DFL2 (DWARF IN LIGHT 2) [Arabidopsis thaliana] H_AUX

MICRO.7077.C1_392 16.04 31.22 9.18 2.49 -1.51 3.67 -1.29 1.38 -1.82 IAA type protein [Elaeis guineensis] H_AUX

MICRO.6538.C2_605 10.26 11.97 9.24 1.82 -1.54 2.44 -1.14 1.17 -2.03 Auxin responsive SAUR protein [Medicago truncatula] H_AUX

MICRO.13674.C1_714 2.15 9.77 3.22 0.96 -0.15 1.81 -1.47 0.28 -1.40 auxin repressed/dormancy associated protein [Lycopersicon esculentum] H_AUX

MICRO.1641.C1_992 5.10 11.76 6.84 1.63 -0.73 0.59 -2.97 1.46 -1.31 Response regulator, RegA/PrrA/ActR type [Medicago truncatula] H_CK

MICRO.3112.C2_766 4.38 8.35 3.54 1.47 -0.67 0.39 -2.68 1.02 -0.81 Response regulator, RegA/PrrA/ActR type [Medicago truncatula] H_CK

MICRO.3817.C3_634 3.54 4.14 3.49 1.57 -0.25 0.05 -2.00 1.18 -0.62 Response regulator, RegA/PrrA/ActR type [Medicago truncatula] H_CK

MICRO.7014.C1_1198 3.29 2.96 4.65 1.37 -0.34 0.51 -1.05 1.17 -1.05 glucosyltransferase-10 [Vigna angularis] H_CK

MICRO.12441.C1_593 40.20 1433.12 7.07 3.88 -1.45 6.48 -4.00 1.09 -1.73 ethylene-responsive late embryogenesis-like protein [Lycopersicon esculentum] H_ETH

MICRO.43.C3_673 3.97 6.58 2.84 1.34 -0.65 0.63 -2.08 1.09 -0.42 S-adenosyl-L-methionine synthetase-like [Solanum tuberosum] H_ETH

MICRO.43.C2_1316 3.92 7.07 3.36 1.16 -0.81 0.46 -2.36 0.96 -0.79 S-adenosylmethionine synthase [Nicotiana tabacum] H_ETH S-adenosylmethionine synthetase 2 (Methionine adenosyltransferase 2) (AdoMet POABI58TV_468 3.81 6.69 3.88 0.83 -1.10 2.30 -0.44 0.27 -1.68 H_ETH synthetase 2) S-adenosylmethionine synthetase 2 (Methionine adenosyltransferase 2) (AdoMet MICRO.4281.C1_1256 2.97 5.24 3.34 0.62 -0.96 2.01 -0.38 0.28 -1.46 H_ETH synthetase 2) MICRO.197.C3_1563 2.55 2.45 2.69 0.78 -0.57 0.03 -1.27 1.39 -0.04 S-adenosyl methionine synthase-like [Solanum tuberosum] H_ETH cSTB42I1TH_464 36.88 27.19 82.33 2.70 -2.51 1.61 -3.16 3.54 -2.82 gibberellin 2-oxidase 1 [Nicotiana sylvestris] H_GA bf_mxlfxxxx_0006e03.t3m.scf_439 16.78 41.76 17.42 2.16 -1.91 3.62 -1.76 1.69 -2.43 3b-hydroxylase [Solanum lycopersicum] H_GA

MICRO.7232.C1_1096 11.83 27.30 18.27 1.90 -1.66 1.52 -3.25 1.89 -2.30 gibberellin 2-oxidase 3 [Nicotiana tabacum] H_GA CYP714A1; heme binding / iron ion binding / monooxygenase/ oxygen binding MICRO.1894.C1_1910 8.05 9.72 4.00 2.06 -0.95 2.33 -0.95 0.90 -1.10 H_GA [Arabidopsis thaliana]

XXIII

CYP714A1; heme binding / iron ion binding / monooxygenase/ oxygen binding BF_TUBSXXXX_0018H08_T3M.SCF_14 7.27 8.91 3.95 1.92 -0.94 2.17 -0.98 0.55 -1.43 [Arabidopsis thaliana]///CYP714A1; heme binding / iron ion binding / monooxygenase/ H_GA 6 oxygen binding [Arabidopsis thaliana] CYP714A1; heme binding / iron ion binding / monooxygenase/ oxygen binding SSBN003F20u.scf_527 6.03 8.99 5.69 1.24 -1.35 1.63 -1.54 1.01 -1.50 H_GA [Arabidopsis thaliana] MICRO.9834.C1_914 4.23 2.40 2.76 0.85 -1.23 1.24 -0.02 0.01 -1.45 CXE carboxylesterase [Actinidia deliciosa] H_GA

MICRO.2633.C1_702 2.70 7.46 3.57 0.67 -0.76 1.43 -1.47 0.54 -1.30 ent-kaurene oxidase CYP701A5 [Stevia rebaudiana] H_GA

MICRO.10720.C2_566 2.40 4.63 2.59 0.59 -0.68 1.01 -1.20 0.53 -0.84 ent-kaurene oxidase [Prunus persica] H_GA

Table A7: Overlap list of up-regulated entites from WT [3d vs 0d] and IPT-6 [3d vs 0d] Three entity lists, ‘up-regulated WT 3d vs 0d’, ‘up-regulated IPT-6 3d vs 0d’ and ‘up-regulated CKX1-4 3d vs 0d’, were combined in a VENN diagram (compare Fig.10A) and 1345 entities found to be overlapping in the WT and line IPT-6 were saved as ‘overlap up-regulated in WT and IPT-6’ list. Entities in this list were sorted according to category and, within each category, ranked by fold change (FCAbsolute [3d] versus [0d]) of the WT. This table lists fold change and normalized values for 6 selected functional (sub)categories: CC - cell cycle/ replication/ chromatin associated; H - hormone-associated (sorted further by hormone class: H_AUX - auxin, H_CK - cytokinins, H_ETH - ethylene and H_GA - gibberellic acid).

FCAbsolute [3d] vs [0d] normalized data

POCI ID WT IPT [WT 0d] [WT 3d] [IPT-6 0d] [IPT-6 3d] Description category cSTB42A7TH_458 134.61 53.93 -1.71 5.36 -0.89 4.86 cyclin - common tobacco CC

MICRO.105.C1_948 126.83 43.20 -1.60 5.38 -0.43 5.00 CDC20.1; signal transducer [Arabidopsis thaliana]///"CDC20.1; signal transducer [Arabidopsis thaliana]" CC

MICRO.15340.C1_604 86.11 69.36 -0.34 6.09 -0.27 5.85 cyclin A1 [Solanum lycopersicum] CC

STMDE81TV_744 59.96 75.17 -0.75 5.16 -1.43 4.80 B-type cyclin [Nicotiana tabacum] CC

MICRO.1632.C1_1 56.43 94.24 0.13 5.95 -0.53 6.03 Histone H4 CC STMCG29TH_467 47.23 17.88 -1.75 3.82 -0.17 3.99 PREDICTED: similar to histone 1, H2ai (predicted) [Canis familiaris] CC

MICRO.3256.C1_799 44.87 47.95 -0.50 4.99 -1.07 4.51 CYCB2;4; cyclin-dependent protein kinase regulator [Arabidopsis thaliana] CC

MICRO.5000.C1_302 38.18 35.76 -0.37 4.88 -0.24 4.92 Histone H4 CC

PPCAY95TH_404 37.24 28.45 -0.34 4.88 -0.16 4.67 cyclin B2 [Solanum lycopersicum] CC

MICRO.1498.C1_1152 31.45 22.19 -0.33 4.65 0.04 4.52 B2-type cyclin dependent kinase [Solanum lycopersicum] CC

MICRO.3793.C1_798 30.31 32.05 -0.07 4.85 -0.51 4.49 CDC20.1; signal transducer [Arabidopsis thaliana] CC

MICRO.1026.C1_670 28.18 36.34 -0.54 4.27 -0.53 4.65 PREDICTED: similar to histone protein Hist2h3c1 [Monodelphis domestica] CC

MICRO.15330.C1_684 24.34 28.62 -0.97 3.63 -1.40 3.44 kinesin-like protein NACK1 [Nicotiana tabacum] CC

MICRO.3203.C1_557 18.21 11.19 -0.57 3.62 -0.25 3.24 B-type cyclin [Nicotiana tabacum] CC bf_arrayxxx_0040c06.t7m.scf_563 15.51 10.33 -0.90 3.06 -0.75 2.61 kinesin-like protein NACK2 [Nicotiana tabacum] CC

MICRO.630.C1_1186 14.99 17.84 -0.42 3.48 -0.60 3.56 B1-type cyclin dependent kinase [Solanum lycopersicum] CC

MICRO.1026.C4_287 13.00 15.27 -0.16 3.54 -0.44 3.49 histone H3 (H3-1.1) CC MICRO.5000.C2_444 12.83 11.48 -0.66 3.02 -0.33 3.19 PREDICTED: similar to germinal histone H4 gene [Canis familiaris] CC

XXIV Appendix

MICRO.17911.C1_764 12.44 6.35 -0.62 3.02 0.40 3.06 SMC2-like condensin [Arabidopsis thaliana] CC PAKRP1 (PHRAGMOPLAST-ASSOCIATED KINESIN-RELATED PROTEIN 1); ATP binding / microtubule motor bf_arrayxxx_0039e05.t7m.scf_586 9.45 5.23 -0.47 2.78 0.31 2.70 [Arabidopsis thaliana]///"PAKRP1 (PHRAGMOPLAST-ASSOCIATED KINESIN-RELATED PROTEIN 1); ATP CC binding / microtubule motor [Arabidopsis thaliana]" cSTB29A8TH_341 8.82 4.95 -0.57 2.57 0.19 2.50 mini-chromosome maintenance protein MCM3 [Pisum sativum] CC

MICRO.2583.C1_1311 8.77 3.06 -1.19 1.95 0.27 1.89 MCM; Nucleic acid-binding, OB-fold [Medicago truncatula] CC

BPLI15D8TH_308 7.88 17.05 -0.32 2.65 -1.06 3.03 Histone H2A.1 (LeH2A-1) CC bf_mxflxxxx_0013h02.t3m.scf_565 7.79 3.81 -1.00 1.96 0.23 2.16 putative replication factor A [Capsicum chinense] CC

STMGV96TV_577 6.92 4.38 -0.44 2.35 -0.05 2.08 cyclin [Medicago sativa subsp. x varia] CC

MICRO.12318.C1_677 6.77 8.38 -0.74 2.02 -1.37 1.70 G2/mitotic-specific cyclin-1 (B-like cyclin) (CycMs1) CC

MICRO.9440.C2_783 6.48 5.72 -0.23 2.47 -0.17 2.34 high mobility group protein [Canavalia gladiata] CC cSTA39H13TH_212 6.47 3.45 -0.99 1.70 0.12 1.91 putative replication factor A [Capsicum chinense] CC

MICRO.14288.C1_662 5.76 3.72 -0.71 1.82 0.07 1.96 high mobility group protein [Canavalia gladiata] CC MICRO.15148.C1_840 5.64 6.72 -0.53 1.97 -0.63 2.12 high mobility group protein [Canavalia gladiata] CC

MICRO.3083.C1_707 4.60 3.78 -0.26 1.95 -0.11 1.81 Abnormal spindle-like microcephaly-associated protein homolog CC

MICRO.3107.C1_684 4.11 3.02 -0.61 1.43 -0.36 1.24 cell cycle switch protein [Arabidopsis thaliana] CC

MICRO.2468.C7_410 3.97 3.46 -0.32 1.67 -0.09 1.70 Histone H4 CC

MICRO.2269.C1_1577 3.89 4.06 -0.16 1.80 0.15 2.17 DNA primase [Arabidopsis thaliana] CC

MICRO.6216.C1_553 3.69 3.26 -0.34 1.54 0.01 1.71 Histone H2B.1 (LeH2B-1) CC

MICRO.3504.C1_499 3.69 2.87 0.02 1.91 -0.09 1.44 cyclin D3.1 [Solanum lycopersicum] CC

MICRO.12427.C1_255 3.56 3.85 -0.04 1.80 0.09 2.03 mini-chromosome maintenance protein MCM6 [Pisum sativum] CC

POCDA63TV_480 3.33 3.67 -0.10 1.64 0.06 1.93 DNA primase [Arabidopsis thaliana] CC

MICRO.16190.C1_367 3.16 2.84 0.03 1.69 -0.25 1.26 cyclin D3.1 [Solanum lycopersicum] CC

MICRO.9879.C1_475 3.01 2.17 -0.27 1.32 0.00 1.11 FAS1 (FASCIATA 1) [Arabidopsis thaliana] CC

MICRO.11151.C1_611 2.80 2.21 -0.32 1.16 0.23 1.37 mini-chromosome maintenance 7 [Pisum sativum] CC

MICRO.9000.C1_8 2.62 2.04 -0.88 0.51 -0.40 0.64 histone H2B-like [Solanum tuberosum] CC

MICRO.4616.C1_818 2.52 2.60 -0.17 1.17 0.14 1.52 CycD3;3 [Solanum lycopersicum] CC

MICRO.5137.C1_613 2.50 2.60 -0.22 1.10 -0.03 1.35 histone H2B [Cicer arietinum] CC

MICRO.1606.C1_532 2.44 2.06 -0.60 0.69 -0.46 0.59 cyclin-dependent protein kinase p34cdc2 [Solanum lycopersicum] CC FAS2 (FASCIATA 2); nucleotide binding [Arabidopsis thaliana]///"FAS2 (FASCIATA 2); nucleotide binding MICRO.12088.C1_722 2.40 2.18 -0.19 1.07 -0.11 1.02 CC [Arabidopsis thaliana]" MICRO.16598.C1_470 2.39 2.00 -0.47 0.79 -0.12 0.88 cyclin dependent kinase A [Camellia sinensis] CC

MICRO.2720.C1_551 2.21 2.05 -0.56 0.58 -0.51 0.53 cyclin D1 [Helianthus tuberosus] CC

SDBN004G07u.scf_725 2.12 4.15 -0.36 0.72 -1.38 0.68 CDC45 (CELL DIVISION CYCLE 45) [Arabidopsis thaliana] CC

MICRO.2720.C2_1166 2.08 2.78 -0.52 0.54 -0.30 1.18 cyclin D1 [Helianthus tuberosus] CC

XXV

MICRO.14118.C1_1638 45.36 21.69 -4.72 0.79 -3.86 0.58 putative AUX1-like permease [Populus tremula x Populus tremuloides] H_AUX

MICRO.8761.C1_643 11.22 9.35 -0.29 3.20 -0.81 2.42 putative auxin response factor 23 [Arabidopsis thaliana] H_AUX

MICRO.155.C3_717 9.40 6.51 -0.41 2.83 -0.28 2.42 auxin/indole-3-acetic acid [Solanum tuberosum] H_AUX

MICRO.13709.C1_754 7.97 14.35 0.05 3.05 -0.07 3.77 auxin:hydrogen symporter [Arabidopsis thaliana] H_AUX

STMGZ78TV_542 7.90 12.10 -0.21 2.77 -0.01 3.58 Auxin Efflux Carrier [Medicago truncatula] H_AUX

MICRO.14512.C1_1400 7.33 8.16 -0.27 2.60 -0.23 2.79 GH3.1 [Arabidopsis thaliana] H_AUX

MICRO.8690.C1_447 6.54 3.88 -0.01 2.70 0.00 1.95 Auxin responsive SAUR protein [Medicago truncatula] H_AUX

MICRO.14242.C1_2372 6.53 3.74 -0.40 2.31 0.39 2.30 putative auxin efflux carrier protein 10 [Medicago truncatula] H_AUX

MICRO.14165.C1_858 6.42 6.60 -0.30 2.38 -0.27 2.45 auxin transport protein [Arabidopsis thaliana] H_AUX bf_mxflxxxx_0067b09.t3m.scf_177 4.24 5.82 -0.66 1.42 -1.01 1.53 GH3.1 [Arabidopsis thaliana] H_AUX

MICRO.1210.C1_761 4.03 2.89 -0.65 1.37 0.02 1.55 flavin monooxygenase-like protein [Solanum lycopersicum] H_AUX

MICRO.4929.C1_652 3.69 2.55 -0.40 1.49 -0.02 1.33 auxin response factor 4 [Lycopersicon esculentum] H_AUX

MICRO.15975.C1_481 3.61 6.03 0.34 2.19 -0.48 2.11 PIN1-like auxin transport protein [Populus tremula x Populus tremuloides] H_AUX

MICRO.743.C1_716 3.55 2.75 -0.51 1.32 -0.27 1.19 auxin response factor 4 [Lycopersicon esculentum] H_AUX

MICRO.11792.C1_442 3.21 2.30 -0.11 1.57 0.38 1.58 flavin monooxygenase-like protein [Solanum lycopersicum] H_AUX cSTB24K20TH_232 3.12 2.75 -0.44 1.20 0.00 1.46 auxin response factor-like protein [Arabidopsis thaliana] H_AUX

STMGP45TV_727 3.09 2.47 -0.48 1.15 0.01 1.31 auxin response factor 4 [Lycopersicon esculentum] H_AUX LeAux=Arabidopsis auxin-regulated protein homolog [Lycopersicon esculentum=tomatoes, VFN8, Peptide Partial, MICRO.445.C1_981 3.01 3.40 -1.52 0.07 -1.40 0.36 H_AUX 150 aa] MICRO.15157.C1_1121 2.94 3.07 -0.47 1.08 -0.01 1.61 dopamine beta-monooxygenase [Arabidopsis thaliana] H_AUX

MICRO.7665.C1_676 2.60 2.47 -0.49 0.88 -0.50 0.80 TIR1 (TRANSPORT INHIBITOR RESPONSE 1); ubiquitin-protein ligase [Arabidopsis thaliana] H_AUX

MICRO.9052.C1_1128 2.57 3.36 -0.29 1.07 -0.48 1.27 transport inhibitor response 1 [Gossypium hirsutum] H_AUX

MICRO.2496.C1_801 2.51 2.24 -0.69 0.64 -1.29 -0.12 TIR1 (TRANSPORT INHIBITOR RESPONSE 1); ubiquitin-protein ligase [Arabidopsis thaliana] H_AUX

STMJG87TV_671 2.26 2.18 -0.78 0.39 -0.81 0.32 ubiquitin-conjugating enzyme [Capsicum annuum] H_AUX

MICRO.13137.C1_982 2.16 2.52 -0.64 0.47 -1.41 -0.08 transport inhibitor response 1 [Gossypium hirsutum] H_AUX

MICRO.5667.C1_1232 12.73 3.80 -0.92 2.75 0.62 2.54 24-sterol C-methyltransferase [Gossypium hirsutum] H_BR

MICRO.4642.C6_1607 2.88 3.24 -0.83 0.70 0.38 2.08 Cytochrome P450 76A2 (CYPLXXVIA2) (P-450EG7) H_BR bf_mxlfxxxx_0012c10.t3m.scf_522 4.68 2.06 -0.70 1.53 0.10 1.14 Conserved hypothetical protein 730 [Medicago truncatula] H_CK

MICRO.9362.C1_603 3.61 4.62 0.10 1.95 -0.34 1.87 ARR17; transcription regulator/ two-component response regulator [Arabidopsis thaliana] H_CK

MICRO.12517.C1_560 3.04 2.89 -0.23 1.37 -0.05 1.48 histidine-containing phosphotransfer protein [Catharanthus roseus] H_CK

SDBN002N24u.scf_719 2.04 3.03 0.00 1.03 -0.08 1.52 Zeatin O-glucosyltransferase (Trans-zeatin O-beta-D-glucosyltransferase) H_CK

MICRO.11534.C1_772 20.58 9.55 -0.83 3.53 -0.50 2.76 1-aminocyclopropane-1-carboxylate synthase [Solanum lycopersicum] H_ETH

STMHA07TV_808 19.88 20.84 0.42 4.73 -0.36 4.02 1-aminocyclopropane-1-carboxylate synthase [Solanum lycopersicum] H_ETH

MICRO.1924.C2_115 4.89 6.89 -1.06 1.23 -2.92 -0.14 EIN3-binding F-box protein 1 [Solanum lycopersicum] H_ETH

XXVI Appendix

bf_mxlfxxxx_0050c04.t3m.scf_58 4.28 14.88 -0.38 1.72 -1.05 2.85 EIL3 [Lycopersicon esculentum] H_ETH

MICRO.3410.C1_429 4.19 4.72 -0.20 1.87 -0.76 1.48 1-aminocyclopropane-1-carboxylate oxidase [Solanum lycopersicum] H_ETH

MICRO.3410.C3_1150 3.56 5.29 0.09 1.92 -0.29 2.11 1-aminocyclopropane-1-carboxylate oxidase [Solanum lycopersicum] H_ETH 1-aminocyclopropane-1-carboxylate oxidase 4 (ACC oxidase 4) (Ethylene-forming enzyme) (EFE) (Protein MICRO.5523.C6_733 3.11 2.60 -0.28 1.36 -0.29 1.09 H_ETH pHTOM5) POACS09TV_420 2.30 3.79 0.24 1.44 -0.50 1.42 1-aminocyclopropane-1-carboxylate oxidase [Solanum lycopersicum] H_ETH

MICRO.8954.C1_392 2.14 6.10 -0.88 0.22 -2.01 0.60 1-aminocyclopropane-1-carboxylate oxidase homolog (Protein E8) H_ETH

MICRO.63.C4_522 71.99 55.53 -0.17 6.00 -0.07 5.73 Gip1-like protein [Petunia x hybrida] H_GA bf_mxflxxxx_0037f09.t3m.scf_286 59.49 44.86 -0.36 5.54 -0.27 5.22 Gip1-like protein [Petunia x hybrida] H_GA

MICRO.12055.C1_573 20.84 17.36 -2.13 2.26 -3.23 0.88 Gibberellin regulated protein [Medicago truncatula] H_GA bf_ivrootxx_0022g11.t3m.scf_438 19.44 17.92 -3.09 1.19 -1.28 2.88 GRAS16 [Solanum lycopersicum] H_GA

MICRO.9855.C1_515 7.06 10.63 -0.75 2.07 -0.55 2.86 Gibberellin regulated protein [Medicago truncatula] H_GA

MICRO.10809.C1_780 7.04 9.89 0.05 2.87 -1.08 2.23 ent-kaurenoic acid oxidase [Pisum sativum] H_GA

MICRO.63.C3_3 5.24 4.39 -0.43 1.96 -0.95 1.19 Gip1-like protein [Petunia x hybrida] H_GA

MICRO.14676.C1_934 3.80 2.81 -0.42 1.51 0.24 1.73 cytochrome P450 monooxygenase CYP716A12 [Medicago truncatula] H_GA

MICRO.2028.C3_1229 2.57 3.28 -0.92 0.44 -0.82 0.90 geranylgeranyl diphosphate synthase [Adonis palaestina] H_GA

MICRO.13238.C1_543 2.41 4.55 -0.12 1.15 -0.27 1.92 RSI-1 protein precursor (TR132) H_GA

MICRO.14034.C1_2231 2.33 2.56 -0.12 1.10 -0.15 1.21 DELLA protein GAI (Gibberellic acid-insensitive mutant protein) H_GA

MICRO.15301.C1_587 2.20 2.98 -0.37 0.77 -0.95 0.63 Gonadotropin, beta chain; Gibberellin regulated protein [Medicago truncatula] H_GA

MICRO.15301.C2_101 2.07 2.46 -0.19 0.86 -0.83 0.46 Gonadotropin, beta chain; Gibberellin regulated protein [Medicago truncatula] H_GA

XXVII

Table A8: Overlap list of up-regulated entites from WT [3d vs 0d] and CKX1-4 [3d vs 0d] Three entity lists, ‘up-regulated WT 3d vs 0d’, ‘up-regulated IPT-6 3d vs 0d’ and ‘up-regulated CKX1-4 3d vs 0d’, were combined in a VENN diagram (compare Fig.10A) and 755 entities found to be overlapping in the WT and line CKX1-4 were saved as ‘overlap up-regulated in WT and CKX1-4’ list. Entities in this list were sorted according to category and, within each category, ranked by fold change (FCAbsolute [3d] versus [0d]) of the WT. This table lists fold change and normalized values for 6 selected functional (sub)categories: CC - cell cycle/ replication/ chromatin associated; H - hormone-associated (sorted further by hormone class: H_AUX - auxin, H_CK - cytokinins, H_ETH - ethylene and H_GA - gibberellic acid).

FCAbsolute [3d] vs [0d] normalized data

POCI ID WT CKX1-4 [WT 0d] [WT 3d] [CKX1-4 0d] [CKX1-4 3d] Description category

MICRO.6640.C1_802 3.75 6.96 -1.60 0.31 -1.91 0.89 H1 histone-like protein [Solanum lycopersicum] CC

MICRO.8023.C1_708 2.29 8.72 -0.28 0.91 -1.28 1.84 AAA ATPase, central region; DEAD/DEAH box helicase, N-terminal [Medicago truncatula] CC bf_suspxxxx_0053a09.t3m.scf_659 2.15 3.55 -0.67 0.43 -0.55 1.28 Eukaryotic-type DNA primase, large subunit [Medicago truncatula] CC

MICRO.7722.C1_792 2.14 2.34 -0.83 0.27 0.01 1.23 regulator of chromosome condensation family protein [Beta vulgaris] CC

MICRO.17626.C1_308 2.09 2.12 -0.82 0.25 -0.79 0.30 Histone H3.3a (Somatic-like histone H3-2) (Histone soH3-2) CC

MICRO.338.C2_1290 2.01 2.34 -0.54 0.47 -0.45 0.78 cyclin-dependent protein kinase p34cdc2 [Solanum lycopersicum] CC bf_acdaxxxx_0061e08.t3m.scf_643 2.02 2.39 -0.58 0.43 -1.20 0.06 3-beta-hydroxy-delta5-steroid dehydrogenase [Arabidopsis thaliana] H

MICRO.12783.C1_554 10.01 4.11 -2.77 0.55 -0.55 1.48 Flavin-containing monooxygenase FMO [Medicago truncatula] H_AUX

STMDH06TV_284 2.54 2.39 -0.97 0.38 -0.08 1.18 aldo/keto reductase AKR [Manihot esculenta] H_AUX

MICRO.259.C5_773 2.25 3.71 -0.34 0.83 -0.25 1.64 auxin responsive transcription factor [Arabidopsis thaliana] H_AUX

MICRO.9222.C2_700 3.06 4.55 -0.55 1.07 -7.30 -5.11 SRR217 [Striga asiatica] H_CK bf_mxflxxxx_0025e05.t3m.scf_364 2.57 2.98 -0.73 0.63 -0.43 1.14 ATPUP2 [Arabidopsis thaliana] H_CK

MICRO.3788.C4_980 2.10 5.38 -0.74 0.33 -1.74 0.69 Zeatin O-xylosyltransferase (Zeatin O-beta-D-xylosyltransferase) H_CK

MICRO.2856.C2_675 3.05 2.69 -1.00 0.61 -0.42 1.01 gibberellin 7-oxidase [Cucurbita maxima] H_GA cSTA34L9TH_122 2.21 2.52 -0.87 0.28 0.05 1.38 gibberellin 7-oxidase [Cucurbita maxima] H_GA

XXVIII Appendix

Table A9: Overlap list of up-regulated entites from IPT-6 [3d vs 0d] and CKX1-4 [3d vs 0d] Three entity lists, ‘up-regulated WT 3d vs 0d’, ‘up-regulated IPT-6 3d vs 0d’ and ‘up-regulated CKX1-4 3d vs 0d’, were combined in a VENN diagram (compare Fig.10A) and 337 entities found to be overlapping in the transgenic lines IPT-6 and CKX1-4 were saved as ‘overlap up-regulated in IPT-6 and CKX1-4’ list. Entities in this list were sorted according to category and, within each category, ranked by fold change (FCAbsolute [3d] versus [0d]) of line IPT-6. This table lists fold change and normalized values for 6 selected functional (sub)categories: CC - cell cycle/ replication/ chromatin associated; H - hormone-associated (sorted further by hormone class: H_AUX - auxin, H_CK - cytokinins, H_ETH - ethylene and H_GA - gibberellic acid).

FCAbsolute [3d] vs [0d] normalized data

POCI ID IPT-6 CKX1-4 [IPT-6 0d] [IPT-6 3d] [CKX1-4 0d] [CKX1-4 3d] Description category

MICRO.11350.C1_929 2.32 2.03 -1.01 0.20 0.11 1.13 ATP-dependent DNA ligase; BRCT [Medicago truncatula]///ATP-dependent DNA ligase; BRCT [Medicago truncatula] CC

MICRO.4424.C2_1345 2.28 2.18 -0.71 0.48 -0.34 0.78 SCD1 (STOMATAL CYTOKINESIS-DEFECTIVE 1) [Arabidopsis thaliana] CC cPRO3C16TH_643 10.02 3.70 -0.20 3.13 -2.13 -0.24 Probable glutathione S-transferase (Auxin-induced protein PGNT35/PCNT111) H_AUX

MICRO.8985.C1_777 2.72 2.32 -0.07 1.37 -2.46 -1.24 auxin transporter protein 1 [Populus tomentosa] H_AUX

MICRO.2063.C1_1434 2.50 2.11 -0.47 0.85 -2.48 -1.40 putative auxin influx carrier protein [Prunus avium] H_AUX

MICRO.2063.C3_1416 2.42 2.45 -0.52 0.76 -2.85 -1.56 auxin influx transport protein [Casuarina glauca] H_AUX

MICRO.8985.C2_66 2.36 2.29 -0.46 0.78 -2.27 -1.08 auxin influx transport protein [Casuarina glauca] H_AUX

MICRO.14039.C2_373 3.05 3.52 -0.77 0.83 -1.09 0.73 putative protein [Arabidopsis thaliana] H_CK

MICRO.3821.C2_948 3.91 3.56 -1.94 0.03 -0.31 1.52 EIN3-binding F-box protein 1 [Solanum lycopersicum] H_ETH

MICRO.12677.C2_1395 2.98 4.87 -0.31 1.26 -1.28 1.00 Ethylene-responsive proteinase inhibitor 1 precursor (Ethylene-responsive proteinase inhibitor I) H_ETH

MICRO.298.C1_1282 2.52 2.34 -1.35 -0.02 -0.11 1.11 ethylene receptor homolog [Solanum tuberosum] H_ETH

MICRO.6797.C1_652 2.51 2.14 -1.16 0.16 -0.01 1.08 putative ethylene receptor [Solanum tuberosum] H_ETH bf_suspxxxx_0024D12.t3m.scf_610 2.16 2.25 -1.04 0.07 -0.26 0.91 ethylene receptor homolog [Lycopersicon esculentum] H_ETH cSTA39M4TH_350 2.02 2.46 -1.10 -0.08 -0.26 1.04 ethylene receptor homolog [Lycopersicon esculentum] H_ETH

XXIX

Table A10: Overlap list of down-regulated entites from WT [3d vs 0d] and IPT-6 [3d vs 0d] Three entity lists, ‘down-regulated WT 3d vs 0d’, ‘down-regulated IPT-6 3d vs 0d’ and ‘down-regulated CKX1-4 3d vs 0d’, were combined in a VENN diagram (compare Fig.10B) and 702 entities found to be overlapping in the WT and line IPT-6 were saved as ‘overlap down-regulated in WT and IPT-6’ list. Entities in this list were sorted according to category and, within each category, ranked by fold change (FCAbsolute [3d] versus [0d]) of the WT. This table lists fold change and normalized values for 6 selected functional (sub)categories: CC - cell cycle/ replication/ chromatin associated; H - hormone-associated (sorted further by hormone class: H_AUX - auxin, H_CK - cytokinins, H_ETH - ethylene and H_GA - gibberellic acid).

FCAbsolute [3d] vs [0d] normalized data

POCI ID WT IPT-6 [WT 0d] [WT 3d] [IPT-6 0d] [IPT-6 3d] Description category

MICRO.18244.C1_813 3.13 3.88 0.85 -0.80 1.02 -0.94 DNA topoisomerase like-protein [Arabidopsis thaliana] CC

MICRO.4640.C1_1328 2.93 2.48 0.33 -1.22 -0.03 -1.34 Helicase, C-terminal [Medicago truncatula] CC

MICRO.14999.C1_1174 2.88 5.31 1.25 -0.28 1.92 -0.48 PDE316 (PIGMENT DEFECTIVE EMBRYO); ATP binding / ligase [Arabidopsis thaliana] CC

MICRO.3709.C1_1507 2.87 3.13 0.68 -0.84 0.73 -0.92 DNA binding / DNA topoisomerase/ DNA topoisomerase type I/ nucleic acid binding [Arabidopsis thaliana] CC

MICRO.2892.C1_1209 2.52 2.43 0.73 -0.60 0.18 -1.10 DEAD/DEAH box helicase, N-terminal [Medicago truncatula] CC

MICRO.15275.C1_1219 2.44 2.56 1.03 -0.25 1.19 -0.16 retinoblastoma-binding protein-like [Arabidopsis thaliana] CC

MICRO.2892.C2_1020 2.35 2.28 0.18 -1.05 0.06 -1.13 DEAD/DEAH box helicase, N-terminal [Medicago truncatula] CC

MICRO.4095.C2_711 2.27 2.02 0.30 -0.88 0.14 -0.87 ATP binding / ATP-dependent helicase/ helicase/ nucleic acid binding [Arabidopsis thaliana] CC

MICRO.4095.C1_758 2.17 2.15 0.47 -0.64 0.06 -1.05 ATP binding / ATP-dependent helicase/ helicase/ nucleic acid binding [Arabidopsis thaliana] CC

MICRO.3726.C1_919 2.04 2.13 0.89 -0.14 0.69 -0.40 RCY1; cyclin-dependent protein kinase regulator [Arabidopsis thaliana] CC

MICRO.17246.C1_735 2.01 2.07 0.37 -0.64 0.22 -0.83 mitochondrial SBP40 [Solanum tuberosum] CC cSTA20L4TH_382 2.01 2.16 0.30 -0.71 0.11 -1.00 ATP binding / ATP-dependent helicase/ helicase/ nucleic acid binding [Arabidopsis thaliana] CC

MICRO.6356.C2_571 2.01 2.31 0.25 -0.76 0.05 -1.16 ATP binding / ATP-dependent helicase/ helicase/ nucleic acid binding [Arabidopsis thaliana] CC

MICRO.6515.C1_1369 3.47 3.38 1.63 -0.17 1.70 -0.06 cytochrome P450 [Pyrus communis] H_AUX

MICRO.17060.C2_21 2.06 4.00 0.39 -0.64 1.61 -0.39 Nt-iaa4.5 deduced protein [Nicotiana tabacum] H_AUX

MICRO.1042.C1_368 2.05 2.36 0.67 -0.37 0.93 -0.31 PIN1-like auxin transport protein [Populus tremula x Populus tremuloides] H_AUX

MICRO.3788.C5_92 3.75 8.36 1.22 -0.69 2.04 -1.02 Zeatin O-glucosyltransferase (Trans-zeatin O-beta-D-glucosyltransferase) H_CK

MICRO.14039.C1_1513 2.04 2.24 0.97 -0.06 1.10 -0.07 putative protein [Arabidopsis thaliana] H_CK

MICRO.2830.C1_1022 11.25 27.22 -0.29 -3.79 0.51 -4.25 ACC oxidase [Solanum tuberosum] H_ETH

MICRO.16392.C1_448 5.50 6.53 0.68 -1.78 -0.30 -3.01 ethylene-responsive transcriptional coactivator [Lycopersicon esculentum] H_ETH

MICRO.7271.C1_651 5.73 34.75 0.34 -2.17 0.67 -4.45 ent-kaurenoic acid 13-hydroxylase [Stevia rebaudiana] H_GA

XXX Appendix

Table A11: Overlap list of down-regulated entites from WT [3d vs 0d] and CKX1-4 [3d vs 0d] Three entity lists, ‘down-regulated WT 3d vs 0d’, ‘down-regulated IPT-6 3d vs 0d’ and ‘down-regulated CKX1-4 3d vs 0d’, were combined in a VENN diagram (compare Fig.10B) and 624 entities found to be overlapping in the WT and line CKX1-4 were saved as ‘overlap down-regulated in WT and CKX1-4’ list. Entities in this list were sorted according to category and, within each category, ranked by fold change (FCAbsolute [3d] versus [0d]) of the WT. This table lists fold change and normalized values for 6 selected functional (sub)categories: CC - cell cycle/ replication/ chromatin associated; H - hormone-associated (sorted further by hormone class: H_AUX - auxin, H_CK - cytokinins, H_ETH - ethylene and H_GA - gibberellic acid).

FCAbsolute [3d] Vs [0d] normalized data

POCI ID WT CKX1-4 [WT 0d] [WT 3d] [CKX1-4 0d] [CKX1-4 3d] Description category cSTD17I2TH_671 4.74 4.55 2.02 -0.23 1.13 -1.06 Trp repressor/replication initiator [Medicago truncatula] CC

MICRO.10863.C1_885 3.86 3.88 1.28 -0.67 0.87 -1.09 Trp repressor/replication initiator [Medicago truncatula] CC

MICRO.10062.C2_1253 2.76 11.60 0.85 -0.61 1.42 -2.11 phragmoplastin [Nicotiana tabacum] CC

MICRO.14067.C1_848 2.72 11.11 1.03 -0.42 1.75 -1.72 phragmoplastin [Nicotiana tabacum] CC

MICRO.2429.C1_627 2.45 2.69 0.83 -0.47 1.10 -0.33 putative cell division cycle protein 23 homolog [Arabidopsis thaliana] CC

MICRO.10062.C1_825 2.24 2.18 0.38 -0.78 0.49 -0.63 phragmoplastin [Nicotiana tabacum] CC

MICRO.339.C6_422 3.38 2.78 0.67 -1.08 1.39 -0.08 MAR-binding protein [Nicotiana tabacum] CC

MICRO.14378.C1_1397 3.24 2.09 -0.22 -1.92 2.24 1.17 nucleosome assembly protein 1-like protein 4 [Nicotiana tabacum] CC

MICRO.7614.C1_756 2.15 2.08 1.00 -0.10 0.03 -1.02 HMG-I and HMG-Y, DNA-binding [Medicago truncatula] CC

MICRO.14827.C1_675 4.58 3.82 1.17 -1.03 2.09 0.16 putative auxin-regulated protein [Nicotiana glutinosa] H_AUX

MICRO.17422.C1_814 3.22 3.47 0.86 -0.83 0.01 -1.78 auxin:hydrogen symporter [Arabidopsis thaliana] H_AUX

MICRO.13353.C1_1039 2.43 2.79 0.44 -0.84 -0.24 -1.72 CYP81B2v2 [Nicotiana tabacum] H_AUX

MICRO.1042.C3_1621 2.21 2.07 0.62 -0.52 -0.05 -1.10 PIN1-like auxin transport protein [Populus tremula x Populus tremuloides] H_AUX

MICRO.14381.C1_48 2.18 3.57 0.27 -0.85 1.82 -0.02 cytochrome P450 [Helianthus tuberosus] H_AUX

MICRO.7814.C1_795 2.39 5.01 0.96 -0.29 1.69 -0.63 Conserved hypothetical protein 730 [Medicago truncatula] H_CK

MICRO.1090.C4_255 2.27 3.84 0.55 -0.63 0.27 -1.67 adenosine kinase isoform 2S [Nicotiana tabacum] H_CK

MICRO.9961.C1_518 2.21 5.60 0.86 -0.29 0.78 -1.71 PUP1 (PURINE PERMEASE 1); purine transporter [Arabidopsis thaliana] H_CK

MICRO.1090.C2_170 2.14 3.46 0.53 -0.57 0.17 -1.62 adenosine kinase isoform 2S [Nicotiana tabacum] H_CK

MICRO.1312.C1_665 3.42 2.78 0.76 -1.02 0.50 -0.98 gibberellic acid receptor [Gossypium hirsutum] H_GA

MICRO.7264.C1_686 3.28 3.10 1.02 -0.69 0.83 -0.80 gibberellic acid receptor [Gossypium hirsutum] H_GA

MICRO.9779.C1_866 2.86 2.51 0.82 -0.69 1.26 -0.06 catalytic [Arabidopsis thaliana] H_GA

XXXI

Table A12: Overlap list of down-regulated entites from IPT-6 [3d vs 0d] and CKX1-4 [3d vs 0d] Three entity lists, ‘down-regulated WT 3d vs 0d’, ‘down-regulated IPT-6 3d vs 0d’ and ‘down-regulated CKX1-4 3d vs 0d’, were combined in a VENN diagram (compare Fig.10B) and 276 entities found to be overlapping in the transgenic lines IPT-6 and CKX1-4 were saved as ‘overlap down-regulated in IPT-6 and CKX1-4’ list. Entities in this list were sorted according to category and, within each category, ranked by fold change (FCAbsolute [3d] versus [0d]) of line IPT-6. In this list, only one entity remains after removal of entities not belonging to one of the 6 functional (sub)categories selected for detailed analysis.

FCAbsolute [3d] vs [0d] normalized data

POCI ID IPT-6 CKX1-4 [IPT-6 0d] [IPT-6 3d] [CKX1-4 0d] [CKX1-4 3d] Description category

MICRO.14215.C2_502 2.27 5.74 0.21 -0.97 1.77 -0.75 Histone deacetylase superfamily [Medicago truncatula] CC

Table A13: List of entites uniquely up- or downregulated in the WT Two lists of entities containing 809 uniquely up- and 419 uniquely in the WT down-regulated entities were derived from VENN diagram comparisons of differentially expressed, at least 2-fold regulated, statistically significant entities (compare Fig.10). The two lists were combined and entities were sorted according to category and, within each category, ranked by fold change (FCAbsolute [3d] versus [0d]). This table lists fold change, regulation and normalized values for 6 selected functional (sub)categories: CC - cell cycle/ replication/ chromatin associated; H - hormone-associated (sorted further by hormone class: H_AUX - auxin, H_CK - cytokinins, H_ETH - ethylene and H_GA - gibberellic acid).

FCAbsolute [WT 3d] vs [WT 0d] normalized data

POCI ID FC value regulation [WT 0d] [WT 3d] Description category

STMDH63TV_374 26.22 up -0.78 3.93 DNA topoisomerase II [Nicotiana tabacum] CC bf_arrayxxx_0041e02.t7m.scf_58 23.37 up -1.31 3.23 DNA topoisomerase II [Nicotiana tabacum] CC

POAD478TP_924 14.77 up -0.62 3.26 cyclin A-like protein [Nicotiana tabacum] CC

MICRO.10069.C1_792 5.57 up -0.30 2.18 Histone H2A; Histone-fold [Medicago truncatula]///Histone H2A; Histone-fold [Medicago truncatula] CC cSTB48C5TH_616 5.03 up -0.18 2.15 cyclin-dependent protein kinase [Arabidopsis thaliana] CC

MICRO.1712.C3_1062 4.27 up -1.83 0.26 Histone H1 CC

MICRO.5330.C2_540 3.48 up -1.01 0.79 Zinc finger, CCHC-type; Replication fork protection component Swi3 [Medicago truncatula] CC

MICRO.5312.C1_1077 3.01 up -0.29 1.30 mini-chromosome maintenance 7 [Pisum sativum] CC

MICRO.5040.C1_716 2.84 up -0.69 0.81 HMG-CoA synthase 2 [Hevea brasiliensis] CC cPRO7A1TH_292 2.79 up -0.47 1.01 Histone H4 CC

MICRO.9836.C1_412 2.75 up -0.75 0.71 histone H4 CC

MICRO.8026.C1_661 2.72 up -0.44 1.01 cyclin-dependent protein kinase [Arabidopsis thaliana] CC

MICRO.11812.C1_884 2.48 down 0.50 -0.81 Helicase, C-terminal [Medicago truncatula] CC

MICRO.150.C2_1113 2.42 down 0.58 -0.69 Histone deacetylase 2a (HD2a) (ScHD2a) CC

XXXII Appendix

bf_arrayxxx_0043h04.t7m.scf_528 2.39 up -0.29 0.96 HMG-CoA synthase 2 [Hevea brasiliensis] CC

MICRO.10167.C1_1095 2.31 down 0.44 -0.76 ATP binding / ATP-dependent helicase/ helicase/ nucleic acid binding [Arabidopsis thaliana] CC cSTA25L20TH_539 2.29 up -0.51 0.69 Rad9 [Medicago truncatula] CC

MICRO.12918.C2_327 2.29 down 0.42 -0.77 single-stranded DNA binding [Arabidopsis thaliana] CC

SSBN003A10u.scf_707 2.27 down 0.24 -0.94 ATP-dependent DNA ligase [Medicago truncatula] CC

MICRO.5807.C1_1388 2.25 up -0.62 0.55 regulator of chromosome condensation (cell cycle regulatory protein) like [Arabidopsis thaliana] CC

MICRO.11720.C1_677 2.24 down 0.29 -0.87 putative Ruv DNA-helicase [Cicer arietinum] CC bf_mxflxxxx_0061c11.t3m.scf_69 2.17 up -0.52 0.60 histone H2A-like protein [Solanum melongena] CC

MICRO.6982.C1_997 2.12 down 0.57 -0.52 Helicase, C-terminal [Medicago truncatula] CC

MICRO.2468.C12_399 2.08 up -0.37 0.69 HIST2H4B protein [Homo sapiens] CC

MICRO.12355.C1_193 2.08 down 0.61 -0.45 Helicase, C-terminal [Medicago truncatula] CC

MICRO.11349.C1_775 2.08 up -1.00 0.06 DNA ligase IV [Populus nigra] CC

MICRO.1640.C1_1190 2.04 up -0.58 0.44 CycD3;3 [Solanum lycopersicum] CC

MICRO.10240.C1_1586 2.04 down 0.81 -0.22 HMG-I and HMG-Y, DNA-binding [Medicago truncatula] CC

MICRO.5165.C1_709 2.04 up -0.19 0.84 kinesin-like protein NACK1 [Nicotiana tabacum] CC

MICRO.13713.C2_471 2.04 down 0.28 -0.74 ATP binding / ATP-dependent helicase/ helicase/ nucleic acid binding [Arabidopsis thaliana] CC

MICRO.6295.C1_877 18.64 up -1.34 2.88 amino acid permease/ transporter [Arabidopsis thaliana] H_AUX

POCCF46TV_497 5.22 up 0.04 2.42 amino acid permease/ transporter [Arabidopsis thaliana] H_AUX

MICRO.14420.C1_1398 4.88 up -2.17 0.12 auxin-repressed protein [Solanum virginianum] H_AUX

MICRO.13184.C1_243 4.48 up -1.89 0.28 auxin resistance protein [Plantago major] H_AUX

MICRO.11239.C1_1440 4.35 up -2.59 -0.47 cytochrome P450 [Sesamum radiatum] H_AUX

MICRO.14165.C2_649 3.83 up -0.27 1.67 auxin transport protein [Arabidopsis thaliana] H_AUX

MICRO.16081.C1_687 3.28 up -0.11 1.60 PIN1-like auxin transport protein [Populus tremula x Populus tremuloides] H_AUX

PPCAF59TH_232 2.77 down 1.13 -0.34 Auxin-induced protein PCNT115, putative, expressed [Oryza sativa (japonica cultivar-group)] H_AUX

MICRO.3540.C4_427 2.64 up -1.07 0.33 auxin-repressed protein ARP1 [Capsicum annuum] H_AUX

MICRO.6405.C2_654 2.16 down 0.61 -0.51 perakine reductase [Rauvolfia serpentina] H_AUX bf_suspxxxx_0039c05.t3m.scf_615 2.16 down 0.53 -0.58 putative auxin response factor 10 [Gossypium raimondii] H_AUX

MICRO.199.C1_1634 2.15 up -0.11 0.99 Aux/IAA protein [Solanum tuberosum] H_AUX

XXXIII

POAC466TV_1 2.06 up -0.17 0.87 AUX/IAA protein [Lycopersicon esculentum] H_AUX bf_ivrootxx_0021f05.t3m.scf_117 2.04 up -0.16 0.87 Aux/IAA protein-like [Solanum tuberosum] H_AUX

MICRO.13334.C2_659 2.02 down 0.57 -0.44 auxin-repressed protein-like protein [Nicotiana tabacum] H_AUX cSTB43F4TH_440 14.93 up -2.57 1.33 Zeatin O-glucosyltransferase (Trans-zeatin O-beta-D-glucosyltransferase) H_CK

MICRO.9222.C1_706 3.27 up -0.39 1.31 SRR217 [Striga asiatica] H_CK

MICRO.7401.C1_378 3.15 down 0.88 -0.78 purine transporter [Arabidopsis thaliana] H_CK

MICRO.3817.C1_671 2.47 down 1.37 0.06 Response regulator, RegA/PrrA/ActR type [Medicago truncatula] H_CK

MICRO.8077.C1_1367 2.14 down 0.52 -0.58 pseudo-response regulator protein [Oryza sativa (indica cultivar-group)] H_CK

MICRO.2939.C1_658 5.38 up -1.05 1.38 NA H_ETH

MICRO.63.C1_455 7.80 up -0.81 2.15 Gip1-like protein [Petunia x hybrida] H_GA

BPLI13C5TH_322 4.88 up -0.95 1.33 geranylgeranyl pyrophosphate synthase 1 [Lycopersicon esculentum] H_GA

MICRO.3641.C2_896 4.32 up -0.64 1.46 ent-kaurenoic acid oxidase [Pisum sativum] H_GA

XXXIV Appendix

Table A14: List of entites uniquely up- or downregulated in line IPT-6 Two lists containing 1209 uniquely up- and 1296 uniquely in IPT-6 down-regulated entities were derived from VENN diagram comparisons of differentially expressed, at least 2-fold regulated, statistically significant entities (compare Fig.10). The two lists were combined and entities were sorted according to category and, within each category, ranked by fold change (FCAbsolute [3d] versus [0d]). This table lists fold change and normalized values for 6 selected functional categories: CC - cell cycle/ replication/ chromatin associated; CW - cell wall biosynthesis/ modification; DNA/ RNA - DNA-/ RNA-associated; H - hormone-associated (sorted further by hormone class: H_ABA - abscisic acid, H_AUX - auxin, H_BR - brassinosteroids, H_CK - cytokinins, H_ETH - ethylene, H_GA - gibberellic acid, H_JA - jasmonic acid, H_SA - salicylic acid); TF - transcription factors; TK/ TL - transcription/ translation.

FCAbsolute [IPT-6 3d] vs [IPT-6 0d] normalized data

POCI ID FC value regulation [IPT 0d] [IPT 3d] Description category

MICRO.16932.C1_159 16.74 up -1.05 3.01 dUTP diphosphatase/ hydrolase [Arabidopsis thaliana] CC

MICRO.13525.C1_803 4.56 down 0.14 -2.05 DEAD/DEAH box helicase, N-terminal [Medicago truncatula] CC

MICRO.840.C2_1114 3.84 down 1.51 -0.43 single-stranded DNA binding protein precursor [Solanum tuberosum] CC

MICRO.13531.C1_702 3.55 up -1.61 0.22 cyclin A2 [Solanum lycopersicum] CC bf_mxflxxxx_0016h06.t3m.scf_552 2.98 down 1.30 -0.27 cyclin-dependent protein kinase [Arabidopsis thaliana] CC bf_arrayxxx_0061d07.t7m.scf_709 2.97 up -0.43 1.14 RNase H family protein [Solanum demissum] CC

MICRO.6239.C1_910 2.83 up -1.38 0.13 telomere binding protein [Solanum lycopersicum] CC

MICRO.6107.C6_1338 2.79 up -1.41 0.07 Meiotic recombination protein DMC1 homolog CC bf_arrayxxx_0068h01.t3m.scf_723 2.64 down -0.12 -1.52 DNA polymerase [Nicotiana tabacum] CC

MICRO.6239.C2_585 2.57 up -1.23 0.13 telomere binding protein [Solanum lycopersicum] CC

MICRO.5385.C1_1140 2.46 down 0.89 -0.41 Glucose inhibited division protein [Medicago truncatula] CC

SSBN001L22u.scf_620 2.32 up -1.90 -0.69 RNase H family protein [Solanum demissum] CC

MICRO.1629.C1_835 2.32 down -0.09 -1.31 Helicase, C-terminal [Medicago truncatula] CC

MICRO.4669.C1_494 2.29 down 0.04 -1.15 Helicase, C-terminal [Medicago truncatula] CC

MICRO.2576.C1_573 2.29 down 0.10 -1.09 Helicase, C-terminal [Medicago truncatula] CC

MICRO.15912.C1_1212 2.21 up -1.31 -0.16 telomere binding protein [Solanum lycopersicum] CC

MICRO.6082.C1_45 2.21 up -0.09 1.05 Histone H2B.2 (LeH2B-2) CC

MICRO.17793.C1_473 2.16 up -0.18 0.94 MCM protein-like protein [Nicotiana tabacum] CC

MICRO.14533.C1_828 2.12 up -0.07 1.01 chromosome associate protein subunit H [Arabidopsis thaliana] CC

STMDB52TV_517 2.11 up 0.07 1.15 CycD3;3 [Solanum lycopersicum] CC

XXXV

MICRO.16828.C1_447 2.04 down 0.63 -0.40 Single-stranded DNA binding [Medicago truncatula] CC cSTB41C21TH_361 2.04 down 0.60 -0.43 Single-stranded DNA binding [Medicago truncatula] CC

MICRO.17918.C1_281 2.02 down 0.15 -0.86 ATP binding / ATP-dependent helicase/ helicase/ nucleic acid binding [Arabidopsis thaliana] CC

MICRO.17060.C1_21 5.94 down 1.97 -0.60 Nt-iaa4.5 deduced protein [Nicotiana tabacum] H_AUX

MICRO.9824.C1_656 5.40 up -2.86 -0.43 Nt-iaa4.5 deduced protein [Nicotiana tabacum] H_AUX

MICRO.9932.C1_2097 4.04 up -0.58 1.44 DFL1 (DWARF IN LIGHT 1) [Arabidopsis thaliana] H_AUX

MICRO.16631.C1_511 3.34 up 0.18 1.92 auxin-induced SAUR-like protein [Capsicum annuum] H_AUX bf_mxflxxxx_0060a02.t3m.scf_592 3.21 up -1.02 0.66 auxin-induced SAUR-like protein [Capsicum annuum] H_AUX

MICRO.9052.C2_9 3.18 up 0.09 1.76 transport inhibitor response 1 [Gossypium hirsutum] H_AUX

PIN4 (PIN-FORMED 4); auxin:hydrogen symporter [Arabidopsis thaliana]///PIN4 (PIN-FORMED 4); auxin:hydrogen symporter MICRO.11119.C1_605 2.76 down 0.98 -0.49 H_AUX [Arabidopsis thaliana]

MICRO.14456.C2_833 2.55 down 1.29 -0.06 auxin response factor 3 [Lycopersicon esculentum] H_AUX cSTE12I8TH_595 2.54 up -0.32 1.02 P-glycoprotein [Solanum tuberosum] H_AUX

MICRO.10332.C2_542 2.35 up -1.52 -0.29 putative auxin-induced SAUR-like protein [Capsicum annuum] H_AUX

MICRO.6405.C3_1227 2.26 up -0.96 0.22 perakine reductase [Rauvolfia serpentina] H_AUX

MICRO.6702.C1_1536 2.17 down 0.14 -0.98 auxin:hydrogen symporter [Arabidopsis thaliana] H_AUX

POACV24TP_702 2.12 down 0.85 -0.24 IAA-alanine resistance protein 1 H_AUX

MICRO.1480.C1_1175 2.10 down 1.09 0.02 putative auxin-amidohydrolase precursor [Populus alba x Populus tremula] H_AUX cPRO5I6TH_651 2.03 down 1.02 0.00 auxin response factor 3 [Gossypium hirsutum] H_AUX

MICRO.17011.C2_298 4.88 up -1.90 0.39 purine transporter [Arabidopsis thaliana] H_CK cSTB39I6TH_334 4.56 up -1.09 1.10 hypothetical protein OsJ_009351 [Oryza sativa (japonica cultivar-group)] H_CK

MICRO.7333.C1_686 3.97 down 1.13 -0.86 zeatin O-glucosyltransferase [Glycine max] H_CK

MICRO.17011.C1_418 3.82 up -1.45 0.49 purine transporter [Arabidopsis thaliana] H_CK

STMEV96TV_161 3.26 up 0.03 1.74 putative protein [Arabidopsis thaliana] H_CK

MICRO.13437.C1_575 3.18 up 0.08 1.75 putative protein [Arabidopsis thaliana] H_CK

MICRO.12396.C1_404 2.95 up -1.17 0.39 Conserved hypothetical protein 730 [Medicago truncatula] H_CK

SDBN002L20u.scf_241 2.76 down 1.00 -0.47 pseudo-response regulator protein [Oryza sativa (indica cultivar-group)] H_CK

MICRO.9080.C1_691 2.24 up -0.34 0.82 unknown protein [Arabidopsis thaliana] H_CK bf_swstxxxx_0059g04.t3m.scf_68 2.15 up -1.10 0.00 unknown [Petunia x hybrida] H_CK

XXXVI Appendix

POCCQ54TV_436 2.08 up -0.86 0.20 AHP4 (HPT PHOSPHOTRANSMITTER 4); transferase, transferring phosphorus-containing groups [Arabidopsis thaliana] H_CK

MICRO.1929.C1_659 2.05 up -0.23 0.80 ATPUP11 [Arabidopsis thaliana] H_CK cSTA34H12TH_201 16.32 down 4.24 0.21 ACC oxidase [Solanum tuberosum] H_ETH bf_suspxxxx_0025D01.t3m.scf_599 13.42 down 2.02 -1.73 ethylene-binding protein [Lycopersicon esculentum] H_ETH

MICRO.14241.C1_566 9.34 down 0.88 -2.34 ethylene response factor 3 [Solanum tuberosum] H_ETH

MICRO.3330.C1_1158 7.52 down 1.94 -0.97 ethylene responsive element binding protein C2 [Capsicum annuum] H_ETH bf_acdcxxxx_0005e04.t3m.scf_7 5.46 up -2.11 0.34 1-aminocyclopropane-1-carboxylate oxidase homolog (Protein E8) H_ETH

MICRO.2158.C3_2411 3.61 up -1.44 0.41 EIL1 [Lycopersicon esculentum] H_ETH

MICRO.773.C9_1174 3.55 down 1.31 -0.52 ethylene response factor 5 [Lycopersicon esculentum] H_ETH

MICRO.773.C2_646 3.31 down 1.34 -0.39 ethylene response factor 5 [Lycopersicon esculentum] H_ETH bf_ivrootxx_0006e10.t3m.scf_122 3.12 down 0.83 -0.82 ethylene response factor 5 [Lycopersicon esculentum] H_ETH

MICRO.773.C3_1031 2.87 down 0.89 -0.63 ethylene response factor 5 [Lycopersicon esculentum] H_ETH cPRO6I20TH_575 2.27 down 0.96 -0.22 ACC synthase [Solanum tuberosum] H_ETH

MICRO.14881.C1_1320 2.11 down 0.38 -0.69 EIL1 [Petunia x hybrida] H_ETH

MICRO.650.C1_1374 2.10 down 0.75 -0.33 1-aminocyclopropane-1-carboxylate synthase/ transaminase/ transferase, transferring nitrogenous groups [Arabidopsis thaliana] H_ETH

MICRO.11045.C1_194 9.15 up -0.56 2.63 GAST1 protein precursor H_GA

MICRO.1345.C1_1453 5.01 down 2.30 -0.02 ent-kaurenoic acid hydroxylase [Arabidopsis thaliana] H_GA

MICRO.1948.C1_1391 2.96 down 1.30 -0.26 geranylgeranyl reductase [Nicotiana tabacum] H_GA

MICRO.8269.C1_1248 2.74 up -0.39 1.07 gibberellin 2-oxidase 5 [Nicotiana tabacum] H_GA

MICRO.2020.C2_542 2.41 down 0.76 -0.51 GRAS2 [Solanum lycopersicum] H_GA

MICRO.2020.C3_1798 2.16 down 0.48 -0.63 GRAS2 [Solanum lycopersicum] H_GA

XXXVII

Table A15: List of entites uniquely up- or downregulated in line CKX1-4 Two lists containing 2346 uniquely up- and 2210 uniquely in CKX1-4 down-regulated entities were derived from VENN diagram comparisons of differentially expressed, at least 2- fold regulated, statistically significant entities (compare Fig.10). The two lists were combined and entities were sorted according to category and, within each category, ranked by fold change (FCAbsolute [3d] versus [0d]). This table lists fold change and normalized values for 6 selected functional categories: CC - cell cycle/ replication/ chromatin associated; CW - cell wall biosynthesis/ modification; DNA/ RNA - DNA-/ RNA-associated; H - hormone-associated (sorted further by hormone class: H_ABA - abscisic acid, H_AUX - auxin, H_BR - brassinosteroids, H_CK - cytokinins, H_ETH - ethylene, H_GA - gibberellic acid, H_JA - jasmonic acid, H_SA - salicylic acid); TF - transcription factors; TK/ TL - transcription/ translation.

FCAbsolute [CKX1-4 3d] vs [CKX1-4 0d] normalized data

POCI ID FC value regulation [CKX1-4 0d] [CKX1-4 3d] Description category

MICRO.16932.C1_159 17.50 down 3.56 -0.56 dUTP diphosphatase/ hydrolase [Arabidopsis thaliana] CC bf_mxflxxxx_0016h06.t3m.scf_552 10.40 up -3.30 0.08 cyclin-dependent protein kinase [Arabidopsis thaliana] CC

MICRO.13531.C1_702 8.71 down 0.52 -2.60 cyclin A2 [Solanum lycopersicum] CC

MICRO.8178.C1_673 6.21 down -0.56 -3.19 histone 3 CC

ACDA02123A11.T3m.scf_370 5.36 up -2.11 0.32 H1 histone-like protein [Solanum lycopersicum] CC

MICRO.12318.C1_677 4.99 down 0.91 -1.41 G2/mitotic-specific cyclin-1 (B-like cyclin) (CycMs1) CC

SDBN004G07u.scf_725 4.59 down 0.13 -2.06 CDC45 (CELL DIVISION CYCLE 45) [Arabidopsis thaliana] CC

ATP binding / ATP-dependent helicase/ DNA binding / helicase/ nucleic acid binding / ubiquitin-protein ligase/ zinc ion binding bf_mxlfxxxx_0009h01.t3m.scf_634 4.55 up -0.59 1.60 CC [Arabidopsis thaliana]

MICRO.8942.C1_886 4.18 down -0.11 -2.18 proliferating cell nuclear antigen [Solanum lycopersicum] CC

MICRO.4709.C1_47 3.17 down 0.23 -1.43 Micronuclear linker histone polyprotein, putative [Solanum bulbocastanum] CC

MICRO.3236.C2_757 3.08 up -0.44 1.19 DNA topoisomerase I [Daucus carota] CC

SSBN001F07u.scf_578 3.00 up -1.47 0.12 MAR-binding filament-like protein 1 CC

MICRO.4710.C1_440 2.94 down 0.61 -0.95 Micronuclear linker histone polyprotein, putative [Solanum bulbocastanum] CC

MICRO.144.C1_1610 2.89 up -0.14 1.39 Helicase, C-terminal [Medicago truncatula] CC

MICRO.4058.C2_697 2.81 up -0.02 1.47 histone-lysine N-methyltransferase/ zinc ion binding [Arabidopsis thaliana] CC

Putative SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 3-like 1 (SMARCA3- MICRO.8913.C1_659 2.79 up -0.15 1.33 CC like protein 1)

MICRO.61.C10_881 2.63 up -1.19 0.21 AN3 (ANGUSITFOLIA3) [Arabidopsis thaliana] CC

SDBN004C01u.scf_477 2.63 down -0.16 -1.56 protein kinase WEE1 [Solanum lycopersicum var. cerasiforme] CC

MICRO.168.C1_40 2.60 down 0.18 -1.20 Histone H2A; Histone-fold [Medicago truncatula] CC

MICRO.1936.C1_753 2.49 up 0.05 1.37 ATP binding / ATP-dependent helicase/ helicase/ nucleic acid binding [Arabidopsis thaliana] CC

MICRO.4732.C2_283 2.47 up -1.62 -0.31 histone H1 [Solanum lycopersicum] CC

XXXVIII Appendix

MICRO.9967.C1_1620 2.46 down 0.10 -1.20 DNA-directed DNA polymerase B [Medicago truncatula] CC

MICRO.3017.C1_1110 2.42 up -1.45 -0.17 HMG-I and HMG-Y, DNA-binding [Medicago truncatula] CC

MICRO.3273.C2_1654 2.41 down 0.17 -1.10 HMG-I and HMG-Y, DNA-binding [Medicago truncatula] CC

ATP binding / ATP-dependent helicase/ DNA binding / DNA-dependent ATPase/ helicase/ nucleic acid binding [Arabidopsis MICRO.8801.C2_485 2.39 up -0.61 0.65 CC thaliana]

MICRO.7363.C3_1 2.37 up -0.05 1.20 HMG-I and HMG-Y, DNA-binding [Medicago truncatula] CC

POABT61TP_901 2.33 down 1.13 -0.09 Helicase, C-terminal; Calcium-binding EF-hand; DNA-directed DNA polymerase B; UBA-like [Medicago truncatula] CC

STMDB52TV_517 2.27 down -1.71 -2.89 CycD3;3 [Solanum lycopersicum] CC

ATP binding / ATP-dependent helicase/ DNA binding / helicase/ nucleic acid binding / protein binding / ubiquitin-protein ligase/ MICRO.16879.C1_1137 2.26 down 0.69 -0.49 CC zinc ion binding [Arabidopsis thaliana]

MICRO.3828.C1_2610 2.22 up -0.26 0.89 Helicase, C-terminal; Zinc finger, CCHC-type; GUCT [Medicago truncatula] CC

MICRO.2307.C2_910 2.20 down 0.84 -0.29 phragmoplastin [Camellia sinensis] CC

MICRO.14533.C1_828 2.19 down -1.02 -2.15 chromosome associate protein subunit H [Arabidopsis thaliana] CC

Putative SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 3-like 1 (SMARCA3- MICRO.10886.C1_812 2.16 up -0.22 0.89 CC like protein 1)

MICRO.5702.C2_644 2.15 up -0.15 0.95 histone deacetylase [Arabidopsis thaliana] CC

MICRO.2307.C1_762 2.15 down 0.68 -0.43 phragmoplastin [Camellia sinensis] CC

AAA ATPase; DEAD/DEAH box helicase, N-terminal [Medicago truncatula]///AAA ATPase; DEAD/DEAH box helicase, N- MICRO.11316.C1_1212 2.13 down 0.83 -0.26 CC terminal [Medicago truncatula]

MICRO.15417.C1_618 2.13 up -0.69 0.40 ATP binding / ATP-dependent DNA helicase/ DNA binding [Arabidopsis thaliana] CC

ATP binding / ATP-dependent helicase/ DNA binding / DNA-dependent ATPase/ helicase/ nucleic acid binding [Arabidopsis MICRO.8801.C1_351 2.11 up -0.82 0.26 CC thaliana]

POCB894TP_157 2.11 down 1.07 -0.01 Histone deacetylase superfamily [Medicago truncatula] CC

ATP binding / ATP-dependent helicase/ DNA binding / helicase/ nucleic acid binding / protein binding / ubiquitin-protein ligase/ MICRO.2453.C6_1930 2.10 up -0.09 0.98 CC zinc ion binding [Arabidopsis thaliana] Pleckstrin-like; Regulator of chromosome condensation/beta-lactamase-inhibitor protein II; Zinc finger, FYVE/PHD-type MICRO.8783.C2_1132 2.09 down 0.47 -0.59 CC [Medicago truncatula]

MICRO.11151.C1_611 2.08 down -0.95 -2.00 mini-chromosome maintenance 7 [Pisum sativum] CC

MICRO.5819.C1_1103 2.08 up -0.62 0.44 Putative SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily C member (AtSwi3C) CC bf_ivrootxx_0045f05.t3m.scf_417 2.04 up -0.33 0.70 Putative chromatin remodelling complex ATPase chain (ISW2-like) (Sucrose nonfermenting protein 2 homolog) CC

MICRO.12776.C1_719 2.03 up -0.14 0.88 replication factor C 110 kDa subunit [Oryza sativa (japonica cultivar-group)] CC bf_arrayxxx_0038d04.t7m.scf_1 2.00 up -0.80 0.20 ATP binding / ATP-dependent DNA helicase/ DNA binding [Arabidopsis thaliana] CC

MICRO.10353.C3_1961 2.00 down 0.96 -0.05 retinoblastoma binding protein 6 , related [Medicago truncatula] CC

MICRO.5307.C1_683 20.34 down 0.98 -3.36 AUX/IAA protein [Medicago truncatula] H_AUX

MICRO.16631.C1_511 14.00 down 1.45 -2.36 auxin-induced SAUR-like protein [Capsicum annuum] H_AUX

XXXIX

MICRO.15153.C1_1582 13.38 up -3.50 0.25 auxin-regulated dual specificity cytosolic kinase [Lycopersicon esculentum] H_AUX

POADW11TP_585 11.99 up -3.17 0.41 auxin-regulated dual specificity cytosolic kinase [Lycopersicon esculentum] H_AUX

MICRO.13654.C1_700 10.60 down -0.95 -4.36 auxin:hydrogen symporter [Arabidopsis thaliana] H_AUX

MICRO.864.C1_988 6.17 down -0.36 -2.99 Nt-iaa2.3 deduced protein [Nicotiana tabacum] H_AUX

STMGR29TV_725 5.38 down -0.33 -2.76 ARF6 (AUXIN RESPONSE FACTOR 6) [Arabidopsis thaliana] H_AUX

MICRO.1935.C1_2210 5.26 down 1.99 -0.40 putative auxin growth promotor protein [Lycopersicon esculentum] H_AUX

TIR1 (TRANSPORT INHIBITOR RESPONSE 1); ubiquitin-protein ligase [Arabidopsis thaliana]///"TIR1 (TRANSPORT bf_mxlfxxxx_0055g09.t3m.scf_624 4.96 down 1.42 -0.89 H_AUX INHIBITOR RESPONSE 1); ubiquitin-protein ligase [Arabidopsis thaliana]"

MICRO.12878.C1_1607 4.69 up -1.12 1.11 auxin response factor 2 [Gossypium arboreum] H_AUX

MICRO.13019.C1_678 4.41 down 0.05 -2.09 Nt-gh3 deduced protein [Nicotiana tabacum] H_AUX

MICRO.2689.C2_803 4.31 up -1.25 0.86 F-box containing protein TIR1 [Populus tremula x Populus tremuloides] H_AUX

MICRO.2689.C1_759 4.12 up -1.07 0.97 F-box containing protein TIR1 [Populus tremula x Populus tremuloides] H_AUX

MICRO.2689.C3_719 4.08 up -1.24 0.79 F-box containing protein TIR1 [Populus tremula x Populus tremuloides] H_AUX

MICRO.15157.C1_1121 4.07 down -0.61 -2.64 dopamine beta-monooxygenase [Arabidopsis thaliana] H_AUX

MICRO.10332.C2_542 4.01 down 1.13 -0.87 putative auxin-induced SAUR-like protein [Capsicum annuum] H_AUX

MICRO.15975.C1_481 3.22 down -1.39 -3.08 PIN1-like auxin transport protein [Populus tremula x Populus tremuloides] H_AUX

MICRO.1332.C1_2122 3.21 up -0.36 1.33 transport inhibitor response 1 [Gossypium hirsutum] H_AUX

STMHS63TV_640 3.14 down 2.56 0.91 GH3 auxin-responsive promoter [Medicago truncatula] H_AUX

MICRO.859.C1_1186 3.13 up -1.77 -0.12 putative auxin-amidohydrolase precursor [Populus alba x Populus tremula] H_AUX

MICRO.15029.C1_1231 3.12 up -0.91 0.73 auxin response factor 2 [Cucumis sativus] H_AUX

MICRO.6455.C1_1086 2.92 down -0.61 -2.16 Peptidase M20 [Medicago truncatula] H_AUX

MICRO.5658.C2_173 2.92 up -0.61 0.94 AUX/IAA protein [Medicago truncatula] H_AUX

MICRO.9389.C1_676 2.82 down 0.99 -0.50 Probable glutathione S-transferase (Auxin-induced protein PCNT107) H_AUX

MICRO.13962.C2_243 2.81 up -1.38 0.11 Probable glutathione S-transferase (Auxin-induced protein PGNT1/PCNT110) H_AUX

POCCK15TP_729 2.72 up -0.01 1.44 Os03g0718000 [Oryza sativa (japonica cultivar-group)] H_AUX

MICRO.12040.C1_21 2.69 down -0.20 -1.63 auxin response factor 4 [Cucumis sativus] H_AUX

MICRO.10523.C1_451 2.67 up -0.60 0.82 putative auxin-regulated protein [Nicotiana glutinosa] H_AUX

STMCM67TV_421 2.67 up -1.51 -0.10 putative auxin-amidohydrolase precursor [Populus alba x Populus tremula] H_AUX

MICRO.5879.C1_908 2.59 up -0.28 1.09 Aldo/keto reductase [Medicago truncatula] H_AUX

XL Appendix

MICRO.6732.C1_947 2.56 down 0.55 -0.80 ER auxin binding protein 1 [Solanum lycopersicum] H_AUX cSTE9F3TH_486 2.44 down 1.27 -0.02 putative auxin growth promotor protein [Lycopersicon esculentum] H_AUX

MICRO.13758.C1_711 2.30 up -0.40 0.80 Auxin responsive SAUR protein [Medicago truncatula] H_AUX

MICRO.12039.C1_725 2.30 down -0.34 -1.54 auxin response factor 4 [Cucumis sativus] H_AUX

MICRO.4441.C1_1371 2.29 up -0.90 0.30 indole-3-glycerol-phosphate synthase [Arabidopsis thaliana] H_AUX

MICRO.5709.C1_743 2.26 up -0.23 0.95 putative auxin-resistance protein [Populus trichocarpa] H_AUX

POADO52TP_930 2.25 down -0.43 -1.60 CYP81B2v2 [Nicotiana tabacum] H_AUX

MICRO.11015.C1_683 2.21 up 0.03 1.17 catalytic/ hydrolase [Arabidopsis thaliana] H_AUX

MICRO.16131.C1_317 2.19 up -1.04 0.09 AXR1 (AUXIN RESISTANT 1); ubiquitin-like activating enzyme [Arabidopsis thaliana] H_AUX

MICRO.5508.C1_1098 2.18 down -0.03 -1.15 dopamine beta-monooxygenase [Arabidopsis thaliana] H_AUX

AXR1 (AUXIN RESISTANT 1); ubiquitin-like activating enzyme [Arabidopsis thaliana]///AXR1 (AUXIN RESISTANT 1); ubiquitin- MICRO.10849.C1_413 2.11 up -0.23 0.85 H_AUX like activating enzyme [Arabidopsis thaliana]

MICRO.6405.C4_293 2.07 up -0.97 0.08 perakine reductase [Rauvolfia serpentina] H_AUX

MICRO.9692.C1_943 28.74 down -2.38 -7.23 Response regulator receiver [Medicago truncatula] H_CK

MICRO.7190.C1_1043 7.40 up -1.76 1.13 cold inducible histidine kinase 1 [Catharanthus roseus] H_CK

MICRO.8580.C1_1653 5.81 up -3.18 -0.64 CYP735A1; heme binding / iron ion binding / monooxygenase/ oxygen binding [Arabidopsis thaliana] H_CK

MICRO.165.C20_58 3.77 up -2.90 -0.99 Zeatin O-glucosyltransferase (Trans-zeatin O-beta-D-glucosyltransferase) H_CK

MICRO.10221.C1_533 3.68 up 0.41 2.29 ATPUP4; purine transporter [Arabidopsis thaliana]///ATPUP4; purine transporter [Arabidopsis thaliana] H_CK

MICRO.1090.C1_31 3.43 down 0.43 -1.35 adenosine kinase isoform 2T [Nicotiana tabacum] H_CK cSTE26M15TH_695 3.17 up 0.59 2.26 Os01g0680200 [Oryza sativa (japonica cultivar-group)] H_CK

MICRO.11289.C1_201 3.16 up 0.15 1.81 Zeatin O-xylosyltransferase (Zeatin O-beta-D-xylosyltransferase) H_CK

MICRO.8742.C1_707 3.13 down -4.33 -5.97 ARR4 (RESPONSE REGULATOR 4); transcription regulator/ two-component response regulator [Arabidopsis thaliana] H_CK

MICRO.6056.C1_887 2.79 up -1.52 -0.05 glucosyltransferase [Nicotiana tabacum] H_CK

SDBN002N24u.scf_719 2.67 down 0.03 -1.39 Zeatin O-glucosyltransferase (Trans-zeatin O-beta-D-glucosyltransferase) H_CK bf_cswcxxxx_0010b04.t3m.scf_460 2.52 up -0.89 0.45 SRR391 [Striga asiatica] H_CK

MICRO.10581.C2_114 2.48 up -0.27 1.04 putative His-Asp phosphotransfer protein [Pisum sativum] H_CK

MICRO.10525.C1_758 2.48 down -0.40 -1.71 type-A response regulator [Catharanthus roseus] H_CK

POCCU71TP_810 2.30 up -0.09 1.11 response regulator 8 [Zea mays] H_CK

MICRO.15202.C1_1176 2.16 down -0.45 -1.56 ATPUP5; purine transporter [Arabidopsis thaliana] H_CK

XLI

PUP1 (PURINE PERMEASE 1); purine transporter [Arabidopsis thaliana]///PUP1 (PURINE PERMEASE 1); purine transporter bf_mxflxxxx_0065a08.t3m.scf_488 2.07 up -1.16 -0.11 H_CK [Arabidopsis thaliana] AHP4 (HPT PHOSPHOTRANSMITTER 4); transferase, transferring phosphorus-containing groups [Arabidopsis bf_cswbxxxx_0056e08.t3m.scf_535 2.04 up -0.30 0.73 thaliana]///AHP4 (HPT PHOSPHOTRANSMITTER 4); transferase, transferring phosphorus-containing groups [Arabidopsis H_CK thaliana] MICRO.773.C3_1031 119.26 up -5.52 1.38 ethylene response factor 5 [Lycopersicon esculentum] H_ETH

MICRO.773.C8_590 64.19 up -4.77 1.24 ethylene response factor 5 [Lycopersicon esculentum] H_ETH

MICRO.773.C9_1174 60.33 up -4.74 1.17 ethylene response factor 5 [Lycopersicon esculentum] H_ETH

MICRO.773.C2_646 48.70 up -5.09 0.51 ethylene response factor 5 [Lycopersicon esculentum] H_ETH bf_ivrootxx_0006e10.t3m.scf_122 36.48 up -3.76 1.43 ethylene response factor 5 [Lycopersicon esculentum] H_ETH cSTA34H12TH_201 35.38 up -0.92 4.23 ACC oxidase [Solanum tuberosum] H_ETH

MICRO.773.C4_126 28.99 up -4.77 0.09 ethylene response factor 5 [Lycopersicon esculentum] H_ETH

MICRO.4096.C3_460 19.12 down 1.71 -2.54 ethylene-responsive small GTP-binding protein [Lycopersicon esculentum] H_ETH

Ethylene-responsive transcription factor 1 (Ethylene-responsive element-binding factor 1) (EREBP-1) (ERF1-like protein) bf_mxlfxxxx_0044h01.t3m.scf_388 11.22 up -3.16 0.33 H_ETH (LeERF1)

MICRO.3330.C1_1158 9.98 up -1.46 1.86 ethylene responsive element binding protein C2 [Capsicum annuum] H_ETH cPRO6I20TH_575 8.42 up -2.60 0.48 ACC synthase [Solanum tuberosum] H_ETH bf_suspxxxx_0044c09.t3m.scf_562 6.27 down 3.34 0.69 Ethylene insensitive 3 [Medicago truncatula] H_ETH

POAD333TP_870 5.96 down 1.65 -0.93 ethylene response factor 3 [Solanum tuberosum] H_ETH bf_stolxxxx_0017h02.t7m.scf_136 5.75 down 0.50 -2.03 1-aminocyclopropane-1-carboxylate oxidase 3 (ACC oxidase 3) (Ethylene-forming enzyme) (EFE) H_ETH

MICRO.14004.C1_971 5.61 up -1.30 1.19 ACS10 (ACC SYNTHASE 10); 1-aminocyclopropane-1-carboxylate synthase [Arabidopsis thaliana] H_ETH

MICRO.17604.C1_330 5.53 up -1.29 1.17 ethylene responsive element binding factor 1 [imported] - Arabidopsis thaliana H_ETH bf_arrayxxx_0052g04.t7m.scf_719 5.06 up -1.34 1.00 1-aminocyclopropane-1-carboxylate synthase [Rosa hybrid cultivar 'Kardinal'] H_ETH

MICRO.5497.C1_673 4.85 up -2.60 -0.32 ethylene-responsive nuclear protein [Lycopersicon esculentum] H_ETH

MICRO.5523.C2_556 4.65 down 0.83 -1.39 1-aminocyclopropane-1-carboxylate oxidase [Solanum tuberosum] H_ETH

MICRO.14004.C2_1385 4.44 up -1.56 0.59 1-aminocyclopropane-1-carboxylate synthase [Rosa hybrid cultivar 'Kardinal'] H_ETH

MICRO.12848.C1_1294 4.40 up -1.59 0.55 1-aminocyclopropane-1-carboxylate oxidase homolog (Protein E8) H_ETH

POCA171TV_416 3.83 down -0.17 -2.10 ethylene-responsive element binding factor [Capsicum annuum] H_ETH

MICRO.7350.C1_931 3.82 down 1.11 -0.82 Ethylene-responsive transcription factor 4 (Ethylene-responsive element-binding factor 4 homolog) (EREBP-3) (NsERF3) H_ETH

MICRO.9868.C3_1278 3.66 down 1.32 -0.55 Ethylene-responsive transcription factor 3 (Ethylene-responsive element-binding factor 3 homolog) (EREBP-5) (NtERF5) H_ETH bf_suspxxxx_0005B12.t3m.scf_243 3.61 down 0.56 -1.29 Ethylene-responsive transcription factor 4 (Ethylene-responsive element-binding factor 4 homolog) (EREBP-3) (NsERF3) H_ETH

MICRO.12256.C1_769 3.48 up -0.55 1.25 Pti4 [Lycopersicon esculentum] H_ETH

XLII Appendix

POCD291TV_296 3.41 down 0.55 -1.22 Ethylene-responsive transcription factor 4 (Ethylene-responsive element-binding factor 4 homolog) (EREBP-3) (NtERF3) H_ETH bf_suspxxxx_0025D01.t3m.scf_599 3.35 up 0.14 1.88 ethylene-binding protein [Lycopersicon esculentum] H_ETH

POCC735TV_540 3.26 down 1.36 -0.34 Ethylene-responsive transcription factor 3 (Ethylene-responsive element-binding factor 3 homolog) (EREBP-5) (NtERF5) H_ETH

MICRO.12256.C2_785 3.25 up -0.56 1.14 Pti4 [Lycopersicon esculentum] H_ETH cSTA24G2TH_376 3.21 up -0.52 1.16 putative ethylene receptor [Nicotiana tabacum] H_ETH

MICRO.15363.C1_670 3.15 up -0.93 0.73 ERF transcription factor 5 [Nicotiana tabacum] H_ETH

MICRO.14057.C2_1848 3.06 up -1.25 0.37 hypothetical protein MtrDRAFT_AC139526g23v1 [Medicago truncatula] H_ETH

MICRO.16820.C1_617 2.87 up -0.47 1.05 putative ethylene receptor [Nicotiana tabacum] H_ETH

MICRO.4096.C4_1011 2.86 down 0.78 -0.74 ethylene-responsive small GTP-binding protein [Lycopersicon esculentum] H_ETH

MICRO.6336.C2_132 2.52 up 0.26 1.59 putative ethylene response protein [Capsicum chinense] H_ETH

MICRO.1296.C3_1216 2.46 up -0.81 0.49 CTR1-like protein kinase [Lycopersicon esculentum] H_ETH

BPLI7H9TH_411 2.20 down 0.73 -0.40 ethylene-responsive small GTP-binding protein [Lycopersicon esculentum] H_ETH

POCAK33TV_556 2.14 down -0.46 -1.56 Ethylene-responsive transcription factor 4 (Ethylene-responsive element-binding factor 4 homolog) (EREBP-3) (NsERF3) H_ETH

EGY1 (ETHYLENE-DEPENDENT GRAVITROPISM-DEFICIENT AND YELLOW-GREEN 1); sterol regulatory element-binding MICRO.9990.C1_417 2.13 up -0.99 0.10 H_ETH protein site 2 protease [Arabidopsis thaliana]

MICRO.8945.C1_994 2.07 down -0.44 -1.49 ACS-like protein [Gossypium hirsutum] H_ETH

MICRO.7039.C2_1180 2.02 up -0.43 0.59 EIN3-binding F-box protein 2 [Lycopersicon esculentum] H_ETH bf_suspxxxx_0056c12.t3m.scf_364 38.74 up -3.65 1.63 putative giberellin beta-hydroxylase [Arabidopsis thaliana] H_GA

MICRO.13776.C2_969 18.40 down 0.01 -4.19 gibberellin 2-oxidase [Solanum lycopersicum] H_GA

MICRO.13776.C1_387 7.93 down 0.19 -2.79 gibberellin 2-oxidase [Solanum lycopersicum] H_GA

MICRO.2924.C1_569 7.36 down 1.59 -1.29 GRAS2 [Solanum lycopersicum] H_GA

MICRO.1223.C2_2109 7.34 up -1.73 1.15 GRAS4 [Solanum lycopersicum] H_GA

MICRO.9346.C1_943 6.98 down 1.35 -1.45 GRAS2 [Solanum lycopersicum] H_GA

MICRO.1345.C1_1453 6.92 up -6.25 -3.46 ent-kaurenoic acid hydroxylase [Arabidopsis thaliana] H_GA

MICRO.4165.C15_196 5.90 down 0.87 -1.70 geranylgeranyl pyrophosphate synthase 2 [Lycopersicon esculentum] H_GA

MICRO.9738.C2_788 5.42 down -0.60 -3.04 Gibberellin regulated protein [Medicago truncatula] H_GA

MICRO.4165.C14_923 4.98 down 1.12 -1.19 geranylgeranyl pyrophosphate synthase 2 [Lycopersicon esculentum] H_GA

MICRO.6620.C1_570 4.84 down 1.27 -1.01 Ga20 oxidase [Solanum tuberosum subsp. andigena] H_GA

POAB769TP_776 3.53 down 1.26 -0.56 GIA/RGA-like gibberellin response modulator [Argyroxiphium sandwicense subsp. macrocephalum] H_GA

XLIII

CYP714A1; heme binding / iron ion binding / monooxygenase/ oxygen binding [Arabidopsis thaliana]///CYP714A1; heme bf_mxlfxxxx_0002f03.t3m.scf_136 3.14 down 0.65 -1.00 H_GA binding / iron ion binding / monooxygenase/ oxygen binding [Arabidopsis thaliana]

MICRO.10720.C1_668 2.88 down 1.96 0.43 ent-kaurene oxidase [Malus x domestica] H_GA bf_arrayxxx_0034b09.t7m.scf_559 2.22 up -1.51 -0.36 gibberellin 20-oxidase [Nicotiana tabacum] H_GA

MICRO.2820.C1_1005 2.00 down 0.70 -0.30 geranylgeranyl reductase [Nicotiana tabacum] H_GA

8.2.4 POCI Identifiers shown in MAPMAN visualizations Table A16: Entities representing cell cycle/ replication-related genes shown in Figure13 Entities are listed in the order of their appearance on the panel in Figure13 and fold change (if at least 2fold regulated) and normalized values are given because some of these entities might not be listed in any of the previous tables due to their not being statistically significant, at least 2-fold changed, differentially expressed in all of the three genotypes.

FCAbsolute [3d] vs [0d] normalized values

WT CKX1-4 IPT-6 WT [0d] WT [3d] CKX1-4 [0d] CKX1-4 [3d] IPT-6 [0d] IPT-6 [3d] CDKA MICRO.16598.C1_470 2.39 2.00 -0.47 0.79 -0.63 0.11 -0.12 0.88 MICRO.630.C1_1186 14.99 17.84 -0.42 3.48 -0.70 0.02 -0.60 3.56 CKDB MICRO.1498.C1_1152 31.45 22.19 -0.33 4.65 -1.11 -0.39 0.04 4.52 bf_swstxxxx_0004g10.t3m.scf_257 14.35 2.27 6.98 -0.75 3.09 -0.69 0.50 -0.13 2.67 cSTA29A10TH_321 26.96 2.84 8.11 -1.00 3.76 -2.45 -0.94 0.35 3.37 MICRO.13531.C1_702 -8.71 3.55 -0.21 0.62 0.52 -2.60 -1.61 0.22 Cyclin A MICRO.15340.C1_604 86.11 69.36 -0.34 6.09 -1.26 0.25 -0.27 5.85 MICRO.2589.C1_821 14.38 2.45 5.95 -0.88 2.97 -1.24 0.05 -0.15 2.42 POAD478TP_924 14.77 -0.62 3.26 -0.78 -0.49 0.36 3.38 STMGV96TV_577 6.92 4.38 -0.44 2.35 -0.59 -1.17 -0.05 2.08 MICRO.3203.C1_557 18.21 11.19 -0.57 3.62 -0.96 -0.24 -0.25 3.24 SDBN001F10u.scf_564 315.36 7.87 413.29 -1.54 6.76 -1.59 1.39 -2.18 6.51 STMDE81TV_744 59.96 75.17 -0.75 5.16 -0.84 -0.32 -1.43 4.80 Cyclin B PPCAY95TH_404 37.24 28.45 -0.34 4.88 -0.51 -0.19 -0.16 4.67 MICRO.12318.C1_677 6.77 -4.99 8.38 -0.74 2.02 0.91 -1.41 -1.37 1.70 cSTB42A7TH_458 134.61 53.93 -1.71 5.36 -1.14 -0.06 -0.89 4.86 MICRO.3256.C1_799 44.87 47.95 -0.50 4.99 -0.67 -0.08 -1.07 4.51

XLIV Appendix

MICRO.1640.C1_1190 2.04 -0.58 0.44 -2.23 -1.73 0.34 1.08 MICRO.4616.C1_818 2.52 2.60 -0.17 1.17 -1.71 -2.93 0.14 1.52 STMDB52TV_517 -2.27 2.11 -0.49 0.92 -1.71 -2.89 0.07 1.15 Cyclin D MICRO.2720.C1_551 2.21 2.05 -0.56 0.58 -0.31 0.29 -0.51 0.53 MICRO.2720.C2_1166 2.08 2.78 -0.52 0.54 -0.29 0.42 -0.30 1.18 MICRO.16190.C1_367 3.16 2.84 0.03 1.69 -0.47 -0.23 -0.25 1.26 MICRO.3504.C1_499 3.69 2.87 0.02 1.91 -0.46 -0.28 -0.09 1.44 MICRO.105.C1_948 126.83 43.20 -1.60 5.38 -0.29 0.16 -0.43 5.00 CDC20 MICRO.3793.C1_798 30.31 32.05 -0.07 4.85 -0.19 -1.19 -0.51 4.49 CCS52B MICRO.3107.C1_684 4.11 3.02 -0.61 1.43 -0.47 -0.44 -0.36 1.24 WEE1 SDBN004C01u.scf_477 -2.63 2.02 -0.10 -0.16 -1.56 0.15 0.05 E2F MICRO.17033.C1_536 2.30 -0.51 0.35 -0.19 1.01 -0.69 0.25

Table A17: Entities representing auxin transport and signalling genes shown in Figure14 Entities are listed in the order of their appearance on the panel in Figure14 and fold change (if at least 2fold regulated) and normalized values are given for all three genotypes.

FCAbsolute [3d] vs [0d] normalized values WT CKX1-4 IPT-6 WT [0d] WT [3d] CKX1-4 [0d] CKX1-4 [3d] IPT-6 [0d] IPT-6 [3d] MICRO.2063.C3_1416 2.29 2.42 0.31 0.77 -2.85 -1.56 -0.52 0.76 MICRO.8985.C2_66 2.36 0.25 0.90 -2.27 -1.08 -0.46 0.78 POCCF46TV_497 5.22 2.11 0.04 2.42 -1.15 -0.57 -0.53 2.21 Auxin influx carrier MICRO.2063.C1_1434 2.50 0.24 0.68 -2.48 -1.40 -0.47 0.85 MICRO.14118.C1_1638 45.36 21.69 -4.72 0.79 -0.34 0.39 -3.86 0.58 MICRO.6295.C1_877 18.64 2.32 -1.34 2.88 -1.36 -1.32 -0.44 2.55 MICRO.8985.C1_777 2.72 0.20 0.77 -2.46 -1.24 -0.07 1.37 MICRO.1042.C1_368 -2.05 -2.07 -2.36 0.67 -0.37 0.17 -0.71 0.93 -0.31 MICRO.1042.C3_1621 -2.21 0.62 -0.52 -0.05 -1.10 0.92 -0.01 MICRO.14242.C1_2372 6.53 -3.22 3.74 -0.40 2.31 -3.00 -2.30 0.39 2.30 PIN proteins MICRO.15975.C1_481 3.61 6.03 0.34 2.19 -1.39 -3.08 -0.48 2.11 MICRO.14165.C1_858 6.42 6.60 -0.30 2.38 -1.98 -1.33 -0.27 2.45 MICRO.14165.C2_649 3.83 -0.27 1.67 -0.01 -0.45 -0.25 1.66 MICRO.16081.C1_687 3.28 -0.11 1.60 -2.41 -2.48 0.14 1.57

XLV

MICRO.11119.C1_605 -2.76 0.65 -0.25 -0.08 -0.88 0.98 -0.49 PGP1/PGP19 cSTE12I8TH_595 -6.17 2.54 -0.03 0.85 -0.93 -0.66 -0.32 1.02 MICRO.864.C1_988 0.03 0.05 -0.36 -2.99 0.20 0.46 MICRO.17060.C1_21 -5.94 0.42 -0.90 0.65 -0.39 1.97 -0.60 Aux/IAA proteins MICRO.17060.C2_21 -2.06 -4.00 0.39 -0.64 0.47 0.16 1.61 -0.39 MICRO.9824.C1_656 5.40 -0.47 0.12 0.82 0.35 -2.86 -0.43 ARF3 MICRO.14456.C2_833 -2.55 0.10 -0.03 -0.04 -0.20 1.29 -0.06 MICRO.4929.C1_652 3.69 2.55 -0.40 1.49 -0.61 -0.15 -0.02 1.33 ARF4 STMGP45TV_727 3.09 2.47 -0.48 1.15 -0.50 -0.02 0.01 1.31 MICRO.743.C1_716 3.55 -2.53 2.75 -0.51 1.32 -0.57 0.12 -0.27 1.19 MICRO.4453.C1_685 2.88 -0.30 0.64 0.08 -1.26 -0.60 0.92 MICRO.12756.C1_343 -2.15 2.51 -0.75 0.12 -0.23 0.61 -1.16 0.17 ARF5 cSTA39D1TH_337 2.00 -4.01 2.47 -0.26 0.74 0.19 -0.92 -0.33 0.98 STMCG44TV_524 2.15 -5.38 -0.44 0.66 -0.33 -2.33 -0.09 1.19 STMGR29TV_725 -2.30 0.52 -0.01 -0.33 -2.76 -0.14 0.23 ARF6 MICRO.12039.C1_725 -2.69 0.20 -0.17 -0.34 -1.54 0.19 0.16 MICRO.12040.C1_21 3.71 0.50 0.24 -0.20 -1.63 -0.18 -0.10 MICRO.259.C5_773 2.25 3.60 -0.34 0.83 -0.25 1.64 -0.32 0.29 ARF8 MICRO.259.C7_585 2.33 4.00 2.24 -0.70 0.52 -1.02 0.98 -0.66 0.50 ARF9 cSTB24K20TH_232 3.12 2.22 2.75 -0.44 1.20 -2.01 -0.49 0.00 1.46 ARF10 MICRO.4409.C1_979 2.25 2.22 2.22 -1.08 0.09 0.47 1.62 -1.20 -0.05 bf_suspxxxx_0039c05.t3m.scf_615 -2.16 0.53 -0.58 -0.07 -0.37 0.68 -0.45 ARF16 cPRO5I6TH_651 4.69 -2.03 0.50 -0.40 0.12 -0.39 1.02 0.00 MICRO.12878.C1_1607 3.12 -0.30 -0.03 -1.12 1.11 -0.05 0.36 ARF19 MICRO.15029.C1_1231 2.30 -0.38 -0.10 -0.91 0.73 -0.20 -0.05 MICRO.13758.C1_711 -11.97 0.13 0.12 -0.40 0.80 -0.01 -0.12 MICRO.6538.C2_605 -10.26 -9.24 1.82 -1.54 2.44 -1.14 1.17 -2.03 MICRO.8690.C1_447 6.54 3.88 -0.01 2.70 -1.62 -1.31 0.00 1.95 SAUR/ SAUR-like proteins bf_mxflxxxx_0060a02.t3m.scf_592 -14.00 3.21 -0.41 0.48 -1.29 0.31 -1.02 0.66 MICRO.16631.C1_511 -4.01 3.34 -1.23 -0.58 1.45 -2.36 0.18 1.92 MICRO.10332.C2_542 2.35 0.36 0.23 1.13 -0.87 -1.52 -0.29 GH3.1 bf_mxflxxxx_0067b09.t3m.scf_177 4.24 0.00 5.82 -0.66 1.42 -0.59 1.15 -1.01 1.53

XLVI Appendix

MICRO.14512.C1_1400 7.33 3.83 8.16 -0.27 2.60 -0.11 0.10 -0.23 2.79 GH3.3 POCBV18TP_741 3.06 3.13 -1.23 0.38 -1.57 0.37 -0.44 1.21 MICRO.9932.C1_2097 6.44 4.04 0.34 0.41 -1.23 -0.13 -0.58 1.44 GH3.4 bf_arrayxxx_0034d11.t7m.scf_624 9.17 5.86 10.13 -0.34 2.86 -2.41 0.28 -0.87 2.47 MICRO.15954.C1_522 7.27 -3.14 6.36 -0.31 2.56 -1.75 0.80 -0.65 2.02 GH3.5 STMHS63TV_640 30.55 -0.24 -0.17 2.56 0.91 -1.84 -1.60 GH3.6 MICRO.18202.C1_677 15.81 30.55 21.74 -1.59 2.39 -3.81 1.12 -2.92 1.52

Table A18: Entities representing ethylene signalling signalling genes shown in Figure15 Entities are listed in the order of their appearance on the panel in Figure15 and fold change (if at least 2fold regulated) and normalized values are given for all three genotypes.

FCAbsolute [3d] vs [0d] normalized values

WT CKX1-4 IPT-6 WT [0d] WT [3d] CKX1-4 [0d] CKX1-4 [3d] IPT-6 [0d] IPT-6 [3d] MICRO.8614.C1_876 4.28 -0.64 0.31 -0.09 0.71 -1.65 0.45 RTE1 MICRO.8614.C2_648 3.35 3.66 -1.01 0.47 -1.20 0.54 -1.51 0.36 MICRO.298.C1_1282 2.34 2.52 -0.23 0.74 -0.11 1.11 -1.35 -0.02 ERS1 MICRO.6797.C1_652 2.14 2.51 -0.37 0.45 -0.01 1.08 -1.16 0.16 ETR2 MICRO.16824.C1_963 10.92 6.72 3.98 -2.06 1.39 -0.79 1.95 -1.29 0.71 bf_suspxxxx_0024D12.t3m.scf_610 2.25 2.16 -0.29 0.39 -0.26 0.91 -1.04 0.07 cSTA24G2TH_376 3.21 -0.15 0.48 -0.52 1.16 -0.02 -0.27 EIN4 cSTA39M4TH_350 2.46 2.02 -0.16 0.53 -0.26 1.04 -1.10 -0.08 MICRO.16820.C1_617 2.87 -0.12 0.28 -0.47 1.05 -0.01 -0.09 CTR1 MICRO.1296.C3_1216 2.46 -0.14 0.30 -0.81 0.49 -0.25 0.38 EIN3 bf_suspxxxx_0044c09.t3m.scf_562 -6.27 0.13 -2.34 3.34 0.69 -0.85 -1.52 MICRO.2158.C1_117 2.16 2.54 3.60 -0.51 0.60 -0.39 0.95 -1.50 0.35 EIL1 MICRO.2158.C3_2411 3.61 -0.69 0.17 -0.05 0.74 -1.44 0.41 MKK9 MICRO.12818.C1_1190 5.54 -0.41 0.28 -1.52 0.95 -0.38 0.44 MICRO.633.C3_265 2.44 -1.55 0.39 -1.40 -0.11 0.26 0.23 MPK3/6 MICRO.633.C4_356 4.60 2.29 -1.58 0.62 -1.27 -0.07 0.42 0.41 bf_arrayxxx_0067e02.t3m.scf_528 4.77 31.72 2.33 -1.46 0.80 -4.48 0.51 -0.44 0.79 ERF1 bf_mxlfxxxx_0044h01.t3m.scf_388 11.22 -1.65 0.32 -3.16 0.33 -0.10 -0.01 MICRO.12256.C1_769 3.48 -0.66 0.52 -0.55 1.25 -0.06 0.19

XLVII

MICRO.12256.C2_785 3.25 -0.73 0.41 -0.56 1.14 -0.07 0.24 MICRO.773.C2_646 48.70 -3.31 -1.86 -0.17 -5.09 0.51 1.34 -0.39 MICRO.773.C3_1031 119.26 -2.87 -3.33 0.17 -5.52 1.38 0.89 -0.63 MICRO.773.C4_126 28.99 -2.72 0.26 -4.77 0.09 0.37 -0.23 ERF5 MICRO.773.C8_590 64.19 -3.53 -0.05 -4.77 1.24 0.33 -0.52 MICRO.773.C9_1174 60.33 -3.55 -1.82 0.14 -4.74 1.17 1.31 -0.52 bf_ivrootxx_0006e10.t3m.scf_122 36.48 -3.12 -2.32 0.35 -3.76 1.43 0.83 -0.82 MICRO.1924.C1_1931 3.73 2.08 7.52 -1.30 0.60 -0.40 0.66 -2.77 0.14 MICRO.1924.C2_115 4.89 6.89 -1.06 1.23 -0.02 1.24 -2.92 -0.14 EBF1 MICRO.3821.C1_1696 2.13 3.39 3.97 -0.31 0.78 -0.04 1.72 -1.78 0.20 MICRO.3821.C2_948 3.56 3.91 -0.42 0.37 -0.31 1.52 -1.94 0.03 EBF2 MICRO.7039.C2_1180 2.02 -0.16 0.11 -0.43 0.59 -0.06 0.12 EIN5 cSTA30D8TH_422 2.11 -0.22 0.09 -0.07 1.01 0.09 -0.12

Table A19: Entities representing cytokinin metabolism and signalling genes shown in Figure16

Entities are listed in the order of their appearance on the panel in Figure16 and fold change (if at least 2fold regulated) and normalized values are given for all three genotypes.

FC [3d] vs [0d] normalized values

WT CKX1-4 IPT-6 WT [0d] WT [3d] CKX1-4 [0d] CKX1-4 [3d] IPT-6 [0d] IPT-6 [3d] CYP735A MICRO.8580.C1_1653 5.81 0.54 0.82 -3.18 -0.64 -0.43 0.54 bf_mxlfxxxx_0012c10.t3m.scf_522 4.68 2.06 -0.70 1.53 -1.43 -1.50 0.10 1.14 LOG MICRO.12396.C1_404 2.95 -0.44 0.07 0.51 0.07 -1.17 0.39 MICRO.7814.C1_795 -2.39 -5.01 0.96 -0.29 1.69 -0.63 -0.03 0.05 cSTB43F4TH_440 14.93 -2.57 1.33 -6.73 -5.88 0.76 0.38 MICRO.165.C20_58 3.77 0.21 0.41 -2.90 -0.99 0.99 -0.11 Zeatin-GT MICRO.3788.C5_92 -3.75 -8.36 1.22 -0.69 0.38 -0.36 2.04 -1.02 MICRO.7333.C1_686 -3.97 0.65 0.32 -2.92 -3.33 1.13 -0.86 SDBN002N24u.scf_719 2.04 -2.67 3.03 0.00 1.03 0.03 -1.39 -0.08 1.52 MICRO.11289.C1_201 3.16 -0.78 0.17 0.15 1.81 -0.70 0.13 Zeatin-XT MICRO.3788.C4_980 2.10 5.38 -0.74 0.33 -1.74 0.69 -0.24 0.70

XLVIII Appendix

CK receptors MICRO.7190.C1_1043 2.77 7.40 -0.87 0.60 -1.76 1.13 -0.27 0.30 bf_mxflxxxx_0025e05.t3m.scf_364 2.57 2.98 -0.73 0.63 -0.43 1.14 -1.04 0.95 bf_mxflxxxx_0065a08.t3m.scf_488 2.07 -0.34 0.07 -1.16 -0.11 0.24 0.36 MICRO.10221.C1_533 3.68 -0.59 -0.36 0.41 2.29 0.45 -0.32 PUP CK transporters MICRO.15202.C1_1176 -2.16 0.71 0.05 -0.45 -1.56 0.81 0.02 MICRO.1929.C1_659 2.05 -0.08 0.42 -0.16 -0.14 -0.23 0.80 MICRO.5974.C1_1055 3.86 3.32 7.61 -1.54 0.40 0.23 1.96 -2.28 0.64 MICRO.9961.C1_518 -2.21 -5.60 0.86 -0.29 0.78 -1.71 0.05 -0.17 bf_cswbxxxx_0056e08.t3m.scf_535 2.04 -0.50 0.35 -0.30 0.73 -0.54 0.43 MICRO.10581.C2_114 2.48 -0.30 0.22 -0.27 1.04 -0.33 0.30 AHPs MICRO.12517.C1_560 3.04 2.89 -0.23 1.37 -1.04 -0.86 -0.05 1.48 POCCQ54TV_436 2.08 -0.32 0.31 -0.28 0.50 -0.86 0.20 MICRO.10525.C1_758 -2.48 0.11 0.26 -0.40 -1.71 1.08 0.18 MICRO.8742.C1_707 -3.13 0.01 0.43 -4.33 -5.97 0.46 0.04 A-type RRs MICRO.9222.C1_706 3.27 -0.39 1.31 -4.36 -4.02 0.37 0.75 MICRO.9222.C2_700 3.06 4.55 -0.55 1.07 -7.30 -5.11 0.21 1.08 MICRO.9362.C1_603 3.61 4.62 0.10 1.95 -3.19 -2.32 -0.34 1.87 B-type RRs POCCU71TP_810 2.30 -0.15 0.09 -0.09 1.11 -0.06 -0.29

Table A20: Entities representing ethylene signalling signalling shown in Figure17 Entities are listed in the order of their appearance on the panel in Figure17 and fold change (if at least 2fold regulated) and normalized values are given for all three genotypes.

FCAbsolute [3d] vs [0d] normalized values WT CKX1-4 IPT-6 WT [0d] WT [3d] CKX1-4 [0d] CKX1-4 [3d] IPT-6 [0d] IPT-6 [3d] MICRO.10720.C1_668 -2.88 0.25 -0.04 1.96 0.43 -0.35 -0.49 ent-kaurene oxidase (KO) MICRO.10720.C2_566 -2.40 -4.63 -2.59 0.59 -0.68 1.01 -1.20 0.53 -0.84 MICRO.2633.C1_702 -2.70 -7.46 -3.57 0.67 -0.76 1.43 -1.47 0.54 -1.30 MICRO.10809.C1_780 7.04 9.89 0.05 2.87 -3.97 -3.84 -1.08 2.23 ent-kaurenoic acid MICRO.3641.C2_896 4.32 -0.64 1.46 -4.59 -4.53 0.65 0.48 oxidase (KAO) MICRO.1345.C1_1453 6.92 -5.01 0.51 0.42 -6.25 -3.46 2.30 -0.02 cSTA6G24TH_420 -4.50 0.58 -0.15 0.49 -1.67 0.41 -0.44 MICRO.6620.C1_570 -4.84 0.03 -0.70 1.27 -1.01 0.72 -0.63 GA20-ox bf_arrayxxx_0034b09.t7m.scf_559 2.22 -0.14 0.46 -1.51 -0.36 0.59 0.47

XLIX

GA3-ox bf_mxlfxxxx_0006e03.t3m.scf_439 -16.78 -41.76 -17.42 2.16 -1.91 3.62 -1.76 1.69 -2.43 MICRO.13776.C1_387 -7.93 0.04 0.15 0.19 -2.79 -0.05 0.11 MICRO.13776.C2_969 -18.40 -0.16 0.13 0.01 -4.19 -0.19 0.38 GA2-ox cSTB42I1TH_464 -36.88 -27.19 -82.33 2.70 -2.51 1.61 -3.16 3.54 -2.82 MICRO.7232.C1_1096 -11.83 -27.30 -18.27 1.90 -1.66 1.52 -3.25 1.89 -2.30 MICRO.8269.C1_1248 2.74 0.15 0.93 -1.08 -1.50 -0.39 1.07 MICRO.9834.C1_914 -4.23 -2.40 -2.76 0.85 -1.23 1.24 -0.02 0.01 -1.45 MICRO.9779.C1_866 -2.86 -2.51 0.82 -0.69 1.26 -0.06 0.08 -0.72 GID1 MICRO.7264.C1_686 -3.28 -3.10 1.02 -0.69 0.83 -0.80 0.36 -0.42 MICRO.1312.C1_665 -3.42 -2.78 0.76 -1.02 0.50 -0.98 0.25 -0.39 MICRO.14034.C1_2231 2.33 2.56 -0.12 1.10 -0.64 0.02 -0.15 1.21 POAB769TP_776 -3.53 -0.86 -0.60 1.26 -0.56 0.82 0.01 MICRO.6247.C1_920 2.17 -0.13 0.17 0.02 0.61 -0.85 0.27 bf_ivrootxx_0022g11.t3m.scf_438 19.44 17.92 -3.09 1.19 -0.08 0.20 -1.28 2.88 DELLA proteins MICRO.2020.C2_542 -2.41 0.17 -0.19 0.37 -0.01 0.76 -0.51 MICRO.2020.C3_1798 -2.16 0.13 -0.34 0.44 -0.24 0.48 -0.63 MICRO.2924.C1_569 -7.36 0.64 0.61 1.59 -1.29 -0.06 -0.41 MICRO.9346.C1_943 -6.98 0.41 0.38 1.35 -1.45 -0.01 -0.14 MICRO.1223.C2_2109 7.34 -1.55 1.16 -1.73 1.15 -0.19 0.50 PIF3 / PIF4 bf_mxlfxxxx_0069d07.t3m.scf_483 2.99 -0.72 0.20 -0.05 1.53 -0.71 0.01 transcription factors MICRO.3059.C1_1613 2.76 -0.76 0.08 -0.08 1.39 -0.81 0.04 MICRO.11045.C1_194 9.15 0.49 2.14 -0.37 -0.42 -0.56 2.63 MICRO.11045.C2_125 331.58 3.27 222.97 -1.32 7.06 -1.74 -0.03 -0.87 6.93 MICRO.12055.C1_573 20.84 17.36 -2.13 2.26 -0.52 0.37 -3.23 0.88 MICRO.9738.C2_788 -5.42 0.58 0.37 -0.60 -3.04 -0.26 0.07 MICRO.9855.C1_515 7.06 10.63 -0.75 2.07 -0.87 -1.67 -0.55 2.86 GA-responsive bf_mxflxxxx_0037f09.t3m.scf_286 59.49 44.86 -0.36 5.54 -1.14 -0.23 -0.27 5.22 genes MICRO.63.C1_455 7.80 -0.81 2.15 -0.20 -1.25 -0.53 1.67 MICRO.63.C3_3 5.24 4.39 -0.43 1.96 -0.35 -0.50 -0.95 1.19 MICRO.63.C4_522 71.99 55.53 -0.17 6.00 -3.50 -1.55 -0.07 5.73 MICRO.15301.C1_587 2.20 2.98 -0.37 0.77 -0.01 0.05 -0.95 0.63 MICRO.15301.C2_101 2.07 2.46 -0.19 0.86 -0.11 -0.22 -0.83 0.46 MICRO.13238.C1_543 2.41 4.55 -0.12 1.15 -2.49 -1.42 -0.27 1.92

L Appendix

8.2.5 Putative zeatin-O-glycosyl- and xylosyltransferases in potato Table A21: Overview of 46 candidate genes for zeatin-O-glycosyltransferases and zeatin-O-xyloslytransferases StGT1 to StGT46, their corresponding POCI EST sequences and scaffold number as well as sequence length of the genomic, CDS and protein sequences as determined by the genome analysis are given in this table. Descriptions for each candidate were determined by blasting the CDS sequences against NCBI’s non-redundant nucleotide collection and selecting the top result, ignoring hits without functional specification (e.g. ‘Solanum lycopersicum chromosome 5 clone C05HBa0065K15, complete sequence’).

sequence length Name POCI ESTs Scaffold No. genomic CDS protein Description (BLAST hit) [bp] [bp] [aa] StGT1 cSTB43F4TH PGSC0003DMS000000154 1409 1326 442 XM_002284345; PREDICTED: Vitis vinifera zeatin O-glucosyltransferase-like AB555737; Gardenia jasminoides GjUGT7 mRNA for UDP-glucose StGT2 MICRO.11289.C1 PGSC0003DMS000001089 1801 1521 507 glucosyltransferase MICRO.165.C20 AB555737; Gardenia jasminoides GjUGT7 mRNA for UDP-glucose StGT3 PGSC0003DMS000000762 1358 1221 407 MICRO.165.C8 glucosyltransferase StGT4 MICRO.3788.C2 PGSC0003DMS000000638 735 1167 389 XM_003519026; PREDICTED: Glycine max zeatin O-glucosyltransferase-like StGT5 MICRO.3788.C4 PGSC0003DMS000001089 801 801 267 XM_003553351; PREDICTED: Glycine max zeatin O-glucosyltransferase-like StGT6 MICRO.3788.C5 PGSC0003DMS000000187 1365 1365 455 XM_003519026; PREDICTED: Glycine max zeatin O-glucosyltransferase-like StGT7 MICRO.4570.C1 PGSC0003DMS000000762 1383 1383 461 XM_002284345; PREDICTED: Vitis vinifera zeatin O-glucosyltransferase-like StGT8 MICRO.7333.C1 PGSC0003DMS000001615 1380 1380 460 XM_002532089; Ricinus communis UDP-glucosyltransferase, putative StGT9 SDBN002N24u.scf PGSC0003DMS000000520 1419 1419 473 AF489877; Phaseolus lunatus clone PlGT3 putative glucosyltransferase StGT10 MICRO.4821.C1 PGSC0003DMS000000013 1398 1398 466 XM_002517308; Ricinus communis UDP-glucosyltransferase, putative StGT11 PGSC0003DMS000000013 1131 1131 377 EU561019; Hieracium pilosella glycosyltransferase UGT90A7 AB443871; Catharanthus roseus CaUGT4 mRNA for UDP-glucose:flavonoid StGT12 PGSC0003DMS000000026 1365 1365 455 glucoside 1,6-glucosyltransferase AB371297; Antirrhinum majus AmUGT36 mRNA for flavonoid glucoyltransferase StGT13 PGSC0003DMS000000068 1476 1476 492 UGT73E2 bf_suspxxxx_0025F08.t3m.scf StGT14 PGSC0003DMS000000187 1485 1485 495 XM_003553351; PREDICTED: Glycine max zeatin O-glucosyltransferase-like 172H01AF.esd StGT15 PGSC0003DMS000000370 1506 1506 502 AY082661; Lycopersicon esculentum putative glucosyltransferase gene XM_002264735; PREDICTED: Vitis vinifera cyanidin-3-O-glucoside 2-O- StGT16 PGSC0003DMS000000540 1233 1233 411 glucuronosyltransferase-like StGT17 PGSC0003DMS000000541 1416 1416 472 XM_002284345; PREDICTED: Vitis vinifera zeatin O-glucosyltransferase-like StGT18 PGSC0003DMS000000638 1431 1431 477 XM_002284345; PREDICTED: Vitis vinifera zeatin O-glucosyltransferase-like StGT19 PGSC0003DMS000000638 1332 1332 444 XM_003626736; Medicago truncatula UDP-glycosyltransferase StGT20 MICRO.1477.C2/3 PGSC0003DMS000000729 1464 1464 488 AY082661; Lycopersicon esculentum putative glucosyltransferase gene StGT21 PGSC0003DMS000000944 1341 1341 447 AY082661; Lycopersicon esculentum putative glucosyltransferase gene

LI

StGT22 MICRO.14976.C1 PGSC0003DMS000000956 1440 1440 480 AY345983; Stevia rebaudiana UDP-glycosyltransferase 89B2 XM_002265356; PREDICTED: Vitis vinifera cyanidin-3-O-glucoside 2-O- StGT23 PGSC0003DMS000001127 1341 1341 447 glucuronosyltransferase-like StGT24 PGSC0003DMS000001250 1524 1524 508 AB360626; Lycium barbarum UGT73A12 mRNA for UDP-glucose:glucosyltransferase StGT25 MICRO.10308.C3 PGSC0003DMS000001250 1425 1425 475 X85138; L.esculentum twi1 mRNA StGT26 MICRO.5442.C1 PGSC0003DMS000001353 1431 1431 477 XM_003519199; PREDICTED: Glycine max UDP-glycosyltransferase 71C3-like StGT27 bf_suspxxxx_0016d04.t3m.scf PGSC0003DMS000001353 1455 1455 485 XM_003519199; PREDICTED: Glycine max UDP-glycosyltransferase 71C3-like XM_002271551; PREDICTED: Vitis vinifera flavanone 7-O-glucoside 2''-O-beta-L- StGT28 PGSC0003DMS000001367 1374 1374 458 rhamnosyltransferase-like StGT29 MICRO.6539.C2 PGSC0003DMS000001428 1467 1467 489 AB182387; Solanum aculeatissimum SaGT6 mRNA for putative glycosyltransferase StGT30 MICRO.6539.C1 PGSC0003DMS000001428 1398 1398 466 AF006081; Solanum berthaultii UDPG glucosyltransferase (PLGT) StGT31 MICRO.13277.C1 PGSC0003DMS000001615 1416 1416 472 XM_002532089; Ricinus communis UDP-glucosyltransferase, putative StGT32 PGSC0003DMS000001698 1008 1008 336 XM_002284345; PREDICTED: Vitis vinifera zeatin O-glucosyltransferase-like XM_003614135; Medicago truncatula Cytokinin-O-glucosyltransferase StGT33 PGSC0003DMS000001908 1467 1467 489 (MTR_5g045960) MICRO.5076.C1 XM_003617024; Medicago truncatula UDP-glucuronosyltransferase 1-7C StGT34 PGSC0003DMS000002034 1497 1497 499 MICRO.9901.C1 (MTR_5g087620) StGT35 STMIA73TH PGSC0003DMS000002461 1431 1431 477 XM_002284345; PREDICTED: Vitis vinifera zeatin O-glucosyltransferase-like StGT36 PGSC0003DMS000002594 1350 1350 450 HM543573; Prunus persica UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) StGT37 PGSC0003DMS000002691 1014 1014 338 XM_002284345; PREDICTED: Vitis vinifera zeatin O-glucosyltransferase-like StGT38 PGSC0003DMS000002731 1452 1452 484 AB072918; Nicotiana tabacum NtGT3 mRNA for glucosyltransferase bf_arrayxxxx_0061g06.t7m.scf MICRO.16513.C1 XM_003633312; PREDICTED: Vitis vinifera UDP-glucose flavonoid 3-O- StGT39 PGSC0003DMS000002731 1440 1440 480 MICRO.15574.C1 glucosyltransferase 6-like bf_suspxxxx_0041f01.t3m.scf AB524720; Forsythia x intermedia RengUGT12 mRNA for UDP- StGT40 PGSC0003DMS000002818 1458 1458 486 sugar:glycosyltransferase MICRO.12411.C1 StGT41 STMEX37TV PGSC0003DMS000002818 1461 1461 487 AB072918; Nicotiana tabacum NtGT3 mRNA for glucosyltransferase BPLI8H7TH AB371297; Antirrhinum majus AmUGT36 mRNA for flavonoid glucoyltransferase StGT42 PGSC0003DMS000002919 1281 1281 427 UGT73E2 XM_002268947; PREDICTED: Vitis vinifera anthocyanidin 3-O-glucosyltransferase 5- StGT43 PGSC0003DMS000002951 1482 1482 494 like MICRO.5226.C1/C2/C3 AB371297; Antirrhinum majus AmUGT36 mRNA for flavonoid glucoyltransferase StGT44 PGSC0003DMS000003352 1473 1473 491 POACR96TV UGT73E2 StGT45 PGSC0003DMS000003447 1440 1440 480 AY082661; Lycopersicon esculentum putative glucosyltransferase gene StGT46 PGSC0003DMS000003821 1386 1386 462 XM_002284345; PREDICTED: Vitis vinifera zeatin O-glucosyltransferase-like

LII Appendix

8.2.6 Overview of potato beta-1,3-glucanases

Table A22: Overview of StB13G and StPdB13G genes identified in the potato genome

DMv3 scaffold genomic ORF protein protein Name POCI ESTs EST description Chromosome pI PGSC0003DMS [bp] [bp] [aa] [kDa]

MICRO.7538.C2 beta-1,3-glucanase [Camellia sinensis] StB13G_01 STMHC29TV beta-1,3-glucanase [Camellia sinensis] 000002259 11 3886 1497 498 53.84 5.31 STMHM64TV beta-1,3-glucanase [Camellia sinensis]

StB13G_02 MICRO.6397.C1 beta-1,3-glucanase [Medicago sativa] 000001425 12 4613 1491 496 53.95 6.62 POAD094TV Os04g0412300 [Oryza sativa (japonica cultivar-group)] StB13G_03 000002089 11 5298 1152 383 42.75 7.87 MICRO.15036.C1 hydrolase, hydrolyzing O-glycosyl compounds [Arabidopsis thaliana] MICRO.7219.C1 hydrolase, hydrolyzing O-glycosyl compounds [Arabidopsis thaliana] StB13G_04 000000477 8 1997 1470 489 52.91 5.87 STMHT66TV Glycoside hydrolase, family 17; X8 [Medicago truncatula] MICRO.1592.C1 hydrolase, hydrolyzing O-glycosyl compounds [Arabidopsis thaliana] StB13G_05 000001329 7 2046 1434 477 52.47 7.87 MICRO.1592.C2 putative beta-1,3-glucanase [Arabidopsis thaliana] MICRO.17289.C1 beta-1,3-glucanase [Camellia sinensis] StB13G_06 SSBN003C06u.s 000000187 4 5376 1359 452 49.38 5.15 beta-1,3-glucanase [Camellia sinensis] cf bf_ivrootxx_0018 beta-1,3-glucanase 1 [Ziziphus jujuba] e03.t3m.scf StB13G_07 000002017 7 2022 1101 366 40.31 8.81 bf_suspxxxx_005 beta-1,3-glucanase 1 [Ziziphus jujuba] 5c06.t3m.scf 008F09AF.esd putative glycosyl hydrolase family 17 protein [Medicago truncatula] StB13G_08 MICRO.1763.C1 putative glycosyl hydrolase family 17 protein [Medicago truncatula] 000000944 5 2619 618 205 20.45 6.20 MICRO.1763.C2 putative glycosyl hydrolase family 17 protein [Medicago truncatula]

StB13G_09 MICRO.18101.C1 X8 [Medicago truncatula] 000000721 4 2658 537 178 19.70 6.48 MICRO.15629.C1 putative beta-1,3-glucanase precursor [Arabidopsis thaliana] StB13G_10 000000695 12 2326 1371 456 50.26 6.31 MICRO.16572.C1 Glycoside hydrolase, family 17; X8 [Medicago truncatula] MICRO.2991.C1 beta-1,3-glucanase, acidic [Coffea arabica]

StB13G_11 MICRO.18084.C1 beta-1,3-glucanase, acidic [Coffea arabica] 000000913 12 2973 1410 469 51.63 4.68 SSBN003D19u.s beta-1,3-glucanase, acidic [Coffea arabica] cf StB13G_12 cSTA15O17TH putative protein (fragment) [Arabidopsis thaliana] 000003712 1 6000 1413 470 49.89 4.75

LIII

MICRO.921.C1 beta-1,3-glucanase-like protein [Olea europaea] POABU11TV glucan endo-1,3-beta-D-glucosidase [Solanum lycopersicum] StB13G_13 MICRO.6187.C1 glucan endo-1,3-beta-D-glucosidase [Solanum lycopersicum] 000000849 10 1142 1035 344 37.55 9.31 MICRO.6187.C2 glucan endo-1,3-beta-D-glucosidase [Solanum lycopersicum]

StB13G_14 MICRO.312.C2 beta-1,3 glucanase [Pisum sativum] 000002758 7 2928 1389 463 51.29 9.33

Glucan endo-1,3-beta-glucosidase precursor ((1->3)-beta-glucan StB13G_15a MICRO.18187.C1 000001128 1 1677 1065 354 39.81 6.04 endohydrolase) ((1->3)-beta-glucanase) (Beta-1,3-endoglucanase) 1,3-beta-D-glucan glucanohydrolase precursor; glucan endo-1,3-beta- MICRO.2286.C15 glucosidase A precursor [Solanum tuberosum] MICRO.2286.C17 1,3-beta-glucan glucanohydrolase [Solanum tuberosum] StB13G_15b 000001128 1 1564 1044 336 38.63 7.14 MICRO.2286.C28 acidic class II 1,3-beta-glucanase precursor [Solanum tuberosum]

MICRO.2286.C42 acidic class II 1,3-beta-glucanase precursor [Solanum tuberosum] MICRO.18200.C1 AT5g58090/k21l19_70 [Arabidopsis thaliana] StB13G_16 MICRO.256.C1 AT5g58090/k21l19_70 [Arabidopsis thaliana] 000000223 5 10688 1449 482 52.89 6.85 MICRO.256.C2 hydrolase, hydrolyzing O-glycosyl compounds [Arabidopsis thaliana]

StB13G_17 STMET40TV AT5g58090/k21l19_70 [Arabidopsis thaliana] 000001353 7 sequence information incomplete

StB13G_18 MICRO.5936.C2 unknown protein [Arabidopsis thaliana] 000000956 3 3721 558 186 18.63 6.87 SDBN006N14u.s AT5g55180/MCO15_13, putative [Medicago truncatula] StB13G_19 cf 000000134 3 1645 1182 394 42.52 6.93 MICRO.5479.C1 AT5g55180/MCO15_13, putative [Medicago truncatula]

StB13G_20 MICRO.6608.C1 hydrolase, hydrolyzing O-glycosyl compounds [Arabidopsis thaliana] 000001284 4 3483 1473 491 53.49 5.96

StB13G_21 MICRO.946.C1 Glycoside hydrolase, family 17; X8 [Medicago truncatula] 000000832 1 2883 1332 444 48.34 4.64

StB13G_22 MICRO.2526.C3 1,3-beta-glucan glucanohydrolase [Solanum tuberosum] 000000750 1 1490 1092 364 39.91 7.23

StB13G_23 MICRO.1984.C1 elicitor inducible beta-1,3-glucanase NtEIG-E76 [Nicotiana tabacum] 000001927 12 1404 1404 468 50.22 5.30

StB13G_24 MICRO.2259.C1 putative glycosyl hydrolase family 17 protein [Medicago truncatula] 000002568 7 2187 684 228 22.84 7.21

StB13G_25 MICRO.15113.C1 unknown protein [Arabidopsis thaliana] 000000261 7 1969 735 244 25.39 7.35

LIV Appendix

StB13G_26 MICRO.14688.C1 glucan endo-1,3-beta-D-glucosidase [Solanum lycopersicum] 000001089 4 1088 1005 329 35.97 5.15

StB13G_27 MICRO.13229.C1 unknown protein [Arabidopsis thaliana] 000001349 5 2097 1104 368 37.42 5.98

Glucan endo-1,3-beta-glucosidase, acidic isoform GI9 precursor ((1->3)- StB13G_28 MICRO.12659.C1 beta-glucan endohydrolase) ((1->3)-beta-glucanase) (Beta-1,3- 000001674 2 1587 1038 346 38.76 6.42 endoglucanase) (PR-2B) (PR-36)

MICRO.3866.C1 putative glycosyl hydrolase family 17 protein [Medicago truncatula] StB13G_29 000001851 1 3419 576 192 19.33 6.47 MICRO.3866.C2 putative glycosyl hydrolase family 17 protein [Medicago truncatula] MICRO.6607.C1 unknown [Arabidopsis thaliana] StB13G_30 MICRO.6607.C2 unknown [Arabidopsis thaliana] 000001958 7 809 663 221 21.76 4.69 MICRO.6607.C3 unknown [Arabidopsis thaliana] bf_arrayxxx_0067 StB13G_31 exo-1,3-beta-glucanase [Lilium longiflorum] 000001174 1 1334 756 252 27.00 5.03 f11.t3m.scf not further analysed; StB13G_32 cSTA44E6TH exo-1,3-beta-glucanase [Lilium longiflorum] 000000638 4 does not contain beta-1,3-glucanase-specific domains StB13G_33 MICRO.1005.C1 putative protein (fragment) [Arabidopsis thaliana] 000003712 4 2783 1326 441 47.23 7.60

not further analysed - no matching scaffold StB13G_34 MICRO.17601.C1 beta-1,3 exoglucanase [Hypocrea lixii] - - sequence was found MICRO.1430.C1 hypothetical protein - potato StPDB13G_1 000001626 12 1366 1260 419 42.82 6.62 MICRO.1430.C2 hypothetical protein - potato

StPDB13G_2 MICRO.2181.C1 hypothetical protein - potato 000001525 8 1378 1248 415 44.89 7.83

LV

8.3.7 Microarray data for selected genes Table A23: Microarray data for identified CK metabolism and signalling genes and genes involved in vascular development The table lists ESTs corresponding to genes of CK metabolism, CK signalling and vascular development genes as well as their fold change values in three independent SRA experiments in which sprouting had been induced by BAP (a CK) or GA3 treatment. Values given are fold changes compared to the ‘0d’ sample of each experiment. Empty cells indicate that the corresponding EST did not give a signal after hybridisation or was not differentially expressed. Abbreviations: AHP – ARABIDOPSIS HISTIDIN-CONTAINING PHOSPHOTRANSFER PROTEIN; APL – ALTERED PHLOEM DEVELOPMENT; ARF – AUXIN RESPONSE FACTOR; CKX – CYTOKININ OXIDASE; CRN – CORONA; FC – fold change; HB – homeobox; HK – HISTIDINE KINASE; IPT – ISOPENTENYLTRANSFERASE; KAN – kanadi; PHB – PHABULOSA; PHV – PHAVOLUTA; PUP – PURINE PERMEASE; RRA – A-type RESPONSE REGULATOR; RRB – B-type RESPONSE REGULATOR; YUC - YUCCA

FC BAP treatment short timecourse FC BAP treatment long timecourse FC GA3 treatment POCI EST Gene [4h] / [0d] [12h] / [0d] [1d] / [0d] [1d] / [0d] [2d] / [0d] [3d] / [0d] [5d] / [0d] [1d] / [0d] [2d] / [0d] [3d] / [0d] [5d] / [0d] MICRO.12320.C1_335 2.72 2.90 2.93 2.88 StIPT1 MICRO.4469.C1_656 -1.38 -1.78 -1.53 -1.25 cSTA13J24TH_232 3.48 3.21 2.83 3.89 StCKX4 bf_mxflxxxx_0059a04.t3m.scf_657 2.01 2.50 1.63 3.62 3.57 3.10 4.60 -4.24 -4.17 0.28 1.32 bf_arrayxxx_0037g12.t7m.scf_640 5.18 5.41 5.24 3.13 4.32 4.65 2.08 1.83 2.25 2.36 2.28 bf_ivrootxx_0040h04.t3m.scf_613 StHK1 4.89 4.22 4.36 2.81 3.87 4.61 1.64 MICRO.15394.C1_650 6.33 5.30 4.23 2.54 3.71 4.38 1.69 2.05 2.26 2.33 2.06 MICRO.8367.C1_726 0.49 0.36 0.32 -3.14 -2.29 -2.59 -1.34 MICRO.9450.C1_777 StHK2 0.49 0.42 0.53 MICRO.9450.C3_340 8.40 12.33 11.44 14.11 0.75 0.48 7.64 7.38 MICRO.15958.C1_589 2.53 1.69 1.27 -2.17 -1.86 -2.04 -1.19 1.88 2.36 1.84 2.17 StHK5 MICRO.8157.C1_777 2.15 1.39 2.60 MICRO.10525.C1_758 StRRA1 10.88 19.11 11.80 7.36 6.36 7.12 1.56 MICRO.8742.C1_707 StRRA2 5.12 5.27 6.97 7.92 5.71 5.12 3.05 MICRO.9222.C1_706 StRRA3 8.15 8.71 25.59 MICRO.9222.C2_700 StRRA4 4.88 5.19 6.20 9.71 8.22 8.51 2.86 2.75 2.42 2.81 2.90 MICRO.9362.C1_603 3.37 8.72 19.19 3.76 4.45 4.73 2.37 4.32 5.65 6.23 9.49 StRRA5 MICRO.9362.C2_666 6.66 17.46 35.03 7.35 7.67 10.22 3.28 4.70 4.94 6.26 11.31 MICRO.9692.C1_943 StRRA6 4.78 2.49 3.18 20.59 17.23 15.44 8.63 147F09AF.esd_122 -2.20 -2.42 -2.76 1.21 MICRO.8717.C1_877 StRRB1 1.13 -1.03 -1.21 3.29 POAD373TV_349 1.41 1.26 6.75 9.47

LVI Appendix

MICRO.14717.C1_951 0.76 0.45 0.55 1.72 0.94 12.60 16.98 StRRB2 MICRO.12644.C1_1077 -1.52 -1.29 -1.15 1.47 MICRO.9679.C1_703 0.72 0.70 4.96 5.76 BF_LBCHXXXX_0028F06_T3M.SCF_459 StRRB4 2.07 1.21 1.19 8.25 8.44 9.22 11.51 1.79 1.32 7.13 8.06 STMJM10TV_381 bf_mxflxxxx_0065a08.t3m.scf_488 StPUP9 3.48 3.73 5.17 -1.97 -1.78 -2.03 -2.15 MICRO.14757.C1_570 StPUP11 2.90 3.11 3.68 1.06 1.90 1.61 1.17 MICRO.16033.C1_546 APL 2.54 2.13 3.17 2.78 1.30 1.25 3.51 3.44 cSTD6F3TH_131 3.19 3.08 4.05 2.08 2.58 2.22 2.19 HB8 MICRO.369.C4_42 2.21 3.45 2.58 2.88 MICRO.2149.C1_308 AHP6 0.61 0.47 0.44 MICRO.369.C1_1514 CRN 1.95 2.86 3.62 1.55 1.86 2.14 1.35 2.01 2.35 2.74 3.31 STMDL70TH_557 1.03 1.66 2.23 1.86 cSTB42F6TH_232 1.12 2.03 2.65 2.06 1.04 1.69 1.88 2.20 PHV MICRO.5970.C1_1648 1.22 1.48 1.89 1.39 MICRO.16031.C1_275 -1.43 -1.23 -1.33 -1.05 MICRO.2368.C1_490 PHB 1.58 1.82 2.19 2.24 cSTA39D1TH_337 ARF5 3.14 6.17 6.93 2.02 1.85 1.78 -1.11 2.22 2.55 2.08 2.02 MICRO.11792.C1_442 YUC1 3.72 8.69 20.50 5.91 9.02 12.91 12.35 3.13 4.45 8.72 9.57 MICRO.1210.C1_761 YUC4 3.46 6.88 16.84 13.34 20.07 29.42 25.15 3.24 4.09 8.52 9.12 MICRO.12381.C1_679 YUC6 MICRO.12382.C1_474 0.98 2.02 4.39 MICRO.13141.C1_474 KAN2 4.20 4.30 4.42 3.45 1.80 1.31 3.22 3.03 MICRO.5902.C1_1057 KAN3 -1.86 -1.82 -1.56 -2.32 0.55 0.66 0.57 -2.55

LVII

Table A24: Microarray data for transcripts representing genes mentioned in the text. This table lists names, BLAST search-derived descriptions, functional category and FC values for ESTs representing genes mentioned in the results and discussion section. Data is given for differentially expressed, at least 2-fold regulated, statistically significant entities identified from [3d] versus [0d] comparisons of WT, CKX1-4 and IPT-6 . Abbreviations: CW – cell wall biosynthesis/ modification; CS – cytoskeleton; H_ETH – phytohormone, ethylene-associated; TF – transcription factors

FC [3d] / [0d] POCI ID Description according to BLAST hit category WT CKX1-4 IPT-6

MICRO.5924.C1_1140 cellulose synthase 3 [Eucalyptus grandis] CW 33.56 114.48 MICRO.4590.C1_956 xyloglucan endotransglucosylase-hydrolase XTH7 [Lycopersicon esculentum] CW 31.41 42.63 MICRO.7256.C1_1091 1-aminocyclopropane-1-carboxylate oxidase homolog (Protein E8) H_ETH 30.21 19.98 33.66 MICRO.11534.C1_772 1-aminocyclopropane-1-carboxylate synthase [Solanum lycopersicum] H_ETH 20.58 9.55 STMHA07TV_808 1-aminocyclopropane-1-carboxylate synthase [Solanum lycopersicum] H_ETH 19.88 20.84 bf_mxflxxxx_0063b10.t3m.scf_599 xyloglucan endotransglycosylase/hydrolase precursor XTH-1 [Populus tremula x Populus tremuloides] CW 18.37 STMIL73TV_673 cellulose synthase 1 [Eucalyptus grandis] CW 15.27 96.93 MICRO.4019.C1_1395 AtGRF3 (GROWTH-REGULATING FACTOR 3) [Arabidopsis thaliana] TF 14.78 10.04 STMHE29TV_558 ovate protein [Lycopersicon esculentum] TF 12.77 14.18 MICRO.14995.C1_919 xyloglucan endotransglycosylase/hydrolase 16 protein [Lycopersicon esculentum] CW 11.34 10.71 92.78 MICRO.2830.C1_1022 ACC oxidase [Solanum tuberosum] H_ETH 11.25 -27.22 MICRO.4636.C1_516 squamosa promoter binding-like protein [Lycopersicon esculentum] TF 11.00 20.68

MICRO.2939.C3_452 1-aminocyclopropane-1-carboxylate oxidase [Solanum tuberosum] H_ETH 10.67 2.18 13.99

MICRO.2939.C2_541 1-aminocyclopropane-1-carboxylate oxidase [Solanum tuberosum] H_ETH 10.43 2.47 15.22 bf_arrayxxx_0008a01.t7m.scf_238 1-aminocyclopropane-1-carboxylate oxidase [Solanum tuberosum] H_ETH 10.13 2.27 16.94

SDBN002K12u.scf_427 AtGRF3 (GROWTH-REGULATING FACTOR 3) [Arabidopsis thaliana] TF 9.34 5.93

MICRO.13070.C1_66 1-aminocyclopropane-1-carboxylate oxidase homolog (Protein E8) H_ETH 9.05 4.70 3.07 bf_stolxxxx_0057b09.t3m.scf_723 Homeobox protein knotted-1-like LET6 TF 8.98 5.12 6.28

MICRO.8217.C1_656 Homeobox protein knotted-1-like LET6 TF 8.54 5.37 5.88 MICRO.15441.C1_1058 xyloglucan endotransglycosylase hydrolase [Apium graveolens] CW 8.54 2.18 MICRO.8676.C1_629 xyloglucan endotransglycosylase/hydrolase precursor XTH-6 [Populus tremula] CW 6.62 52.16 3.79 bf_stolxxxx_0063g03.t3m.scf_457 squamosa promoter binding-like protein [Lycopersicon esculentum] TF 6.55 12.41 4.96

LVIII Appendix

MICRO.11215.C1_864 cellulose synthase 3 [Eucalyptus grandis] CW 5.93 2.68 19.89 MICRO.11808.C1_535 Beta tubulin; Remorin, C-terminal region [Medicago truncatula] CS 5.84 2.21 MICRO.1229.C1_1235 phantastica [Lycopersicon esculentum] TF 5.79 2.01 2.31 MICRO.15212.C1_855 cellulose synthase-like protein CslG [Nicotiana tabacum] CW 5.31 8.10 MICRO.9056.C1_548 cellulose synthase CesA-1 [Zinnia elegans] CW 5.09 10.99 MICRO.3047.C2_110 Homeotic protein knotted-1 (TKN1) TF 4.94 6.75 4.00 MICRO.67.C1_1055 Beta tubulin; Remorin, C-terminal region [Medicago truncatula] CS 4.80 cSTB17M13TH_381 xyloglucan endotransglucosylase-hydrolase XTH7 [Lycopersicon esculentum] CW 4.53 bf_mxflxxxx_0021d02.t3m.scf_651 cellulose synthase [Populus tremula x Populus tremuloides] CW 4.20 2.74 20.96 MICRO.3410.C1_429 1-aminocyclopropane-1-carboxylate oxidase [Solanum lycopersicum] H_ETH 4.19 4.72 STMDL42TV_258 Beta tubulin; Remorin, C-terminal region [Medicago truncatula] CS 3.74 MICRO.3410.C3_1150 1-aminocyclopropane-1-carboxylate oxidase [Solanum lycopersicum] H_ETH 3.56 5.29 1-aminocyclopropane-1-carboxylate oxidase 4 (ACC oxidase 4) (Ethylene-forming enzyme) (EFE) MICRO.5523.C6_733 H_ETH 3.11 2.60 (Protein pHTOM5) POACS09TV_420 1-aminocyclopropane-1-carboxylate oxidase [Solanum lycopersicum] H_ETH 2.30 3.79 MICRO.8954.C1_392 1-aminocyclopropane-1-carboxylate oxidase homolog (Protein E8) H_ETH 2.14 6.10 MICRO.3047.C1_537 Homeotic protein knotted-1 (TKN1) TF 2.13 3.06 2.44

MICRO.270.C2_1563 beta tubulin [Setaria viridis] CS 2.11 3.40 bf_acdcxxxx_0005e04.t3m.scf_7 1-aminocyclopropane-1-carboxylate oxidase homolog (Protein E8) H_ETH 5.46 STMJM75TV_237 cellulose synthase-like protein CslG [Nicotiana tabacum] CW 2.14 cSTB46E15TH_215 cellulose synthase CW 2.12 1-aminocyclopropane-1-carboxylate synthase/ transaminase/ transferase, transferring nitrogenous MICRO.650.C1_1374 H_ETH -2.10 groups [Arabidopsis thaliana] MICRO.18221.C1_505 beta tubulin [Arabidopsis thaliana] CS -2.23 cPRO6I20TH_575 ACC synthase [Solanum tuberosum] H_ETH 8.42 -2.27 cSTA34H12TH_201 ACC oxidase [Solanum tuberosum] H_ETH 35.38 -16.32 bf_arrayxxx_0052g04.t7m.scf_719 1-aminocyclopropane-1-carboxylate synthase [Rosa hybrid cultivar 'Kardinal'] H_ETH 5.06 bf_stolxxxx_0017h02.t7m.scf_136 1-aminocyclopropane-1-carboxylate oxidase 3 (ACC oxidase 3) (Ethylene-forming enzyme) (EFE) H_ETH -5.75 MICRO.12848.C1_1294 1-aminocyclopropane-1-carboxylate oxidase homolog (Protein E8) H_ETH 4.40 MICRO.14004.C1_971 ACS10 (ACC SYNTHASE 10); 1-aminocyclopropane-1-carboxylate synthase [Arabidopsis thaliana] H_ETH 5.61

LIX

MICRO.14004.C2_1385 1-aminocyclopropane-1-carboxylate synthase [Rosa hybrid cultivar 'Kardinal'] H_ETH 4.44 MICRO.5523.C2_556 1-aminocyclopropane-1-carboxylate oxidase [Solanum tuberosum] H_ETH -4.65 MICRO.8945.C1_994 ACS-like protein [Gossypium hirsutum] H_ETH -2.07 STMGM17TV_59 cellulose synthase isolog [Arabidopsis thaliana] CW 9.94 MICRO.12704.C1_684 Beta tubulin; Remorin, C-terminal region [Medicago truncatula] CS 2.66 MICRO.8706.C2_834 cellulose synthase catalytic subunit [Nicotiana alata] CW -2.69 MICRO.4016.C1_518 cellulose synthase [Solanum tuberosum] CW -3.10 MICRO.4016.C2_43 cellulose synthase [Solanum tuberosum] CW -3.45

Table A25: Microarray data for categorization of potato beta-1,3-glucanase genes The table lists ESTs corresponding to B13G genes identified in the potato genome and their expression data in the experiments used for the categorization shown in figure 36. Data on Pep13 elicitor (Brunner et al., 2002; Halim et al., 2004) treatment of potato leaves was kindly provided by Sabine Rosahl, IPB Halle. In the side shoot experiment, axillary meristems of tissue-cultures potato plants were re-activated by capping of the SAM and samples were taken at the indicated time points. Transcript data from his experiment was kindly provided by Melanie Senning. For the ‘sprout vs parenchyma tissue separation’ experiment, a SRA with GA3 to elicit synchronous sprouting of WT tuber discs was performed and at 3d after treatment, the newly formed sprouts and subjacent parenchyma tissue were sampled separately.The ratio of expression in sprouts and parenchyma indicates in which tissue expression is higher: a positive value corresponds to higher expression in the sprouts whereas a negative value stands for higher expression in the parenchyma.

PEP13 treatment side shoot experiment sprout [S] vs parenchyma [P] tissue separation POCI ID Gene FC [Pep13] / [control] FC [2h] / [0h] FC [4h] / [0h] FC [12h] / [0h] P [3d GA] / [3d H2O] S [3d GA] / P [3d GA] S [3d GA] / [3d H2O] STMHM64TV_476 -4.16 1.18 2.23 4.79 -1.31 3.67 -1.05 STMHC29TV_616 StB13G_01 -5.86 1.09 1.95 5.13 -1.28 3.98 1.08 MICRO.7538.C2_668 -4.28 1.01 1.98 4.32 -1.06 6.75 2.18 MICRO.6397.C1_717 StB13G_02 -6.53 MICRO.15036.C1_846 2.88 1.48 -4.43 -1.01 POAD094TV_353 StB13G_03 26.13 STMHT66TV_172 11.74 1.45 -4.84 -1.09 MICRO.7219.C1_623 2.02 1.58 -4.57 1.22 StB13G_04 MICRO.1592.C2_1826 1.45 1.45 2.54 -2.08 2.38 1.09 MICRO.1592.C1_749 StB13G_05 -5.81 1.82 2.15 4.19 -1.82 1.90 -1.29 SSBN003C06u.scf_499 1.67 StB13G_06 MICRO.17289.C1_667 2.59 1.50 1.65 3.45 -1.57 3.88 1.08 bf_suspxxxx_0055c06.t3m.scf_646 StB13G_07 3.32

LX Appendix

bf_ivrootxx_0018e03.t3m.scf_636 -1.17 MICRO.1763.C1_5 -1.93 MICRO.1763.C2_1171 StB13G_08 1.61 -1.43 2.17 -1.19 008F09AF.esd_477 1.09 MICRO.18101.C1_663 StB13G_09 1.33 2.00 6.12 MICRO.16572.C1_587 -2.23 -2.50 -1.07 StB13G_10 MICRO.15629.C1_696 SSBN003D19u.scf_451 MICRO.2991.C1_757 StB13G_11 -1.03 MICRO.18084.C1_783 1.68 -1.05 2.81 -1.03 MICRO.921.C1_1665 1.94 3.22 1.97 -1.75 StB13G_12 cSTA15O17TH_161 MICRO.6187.C1_994 15.39 POABU11TV_383 StB13G_13 5.03 MICRO.6187.C2_1171 11.61 3.32 1.65 1.40 MICRO.312.C2_1336 StB13G_14 -1.06 4.18 2.96 -1.25 MICRO.18187.C1_681 StB13G_15a MICRO.2286.C15_125 3.18 4.64 1.10 1.47 MICRO.2286.C17_1144 2.22 5.62 -3.66 2.72 StB13G_15b MICRO.2286.C28_194 -4.50 MICRO.2286.C42_636 9.89 6.32 1.19 1.53 MICRO.18200.C1_839 -1.34 -1.45 1.90 -1.03 MICRO.256.C1_1 StB13G_16 MICRO.256.C2_950 -1.80 STMET40TV_304 StB13G_17 -2.46 MICRO.5936.C2_659 StB13G_18 -6.18 MICRO.5479.C1_696 -1.97 3.92 1.13 StB13G_19 SDBN006N14u.scf_635 -1.74 MICRO.6608.C1_678 StB13G_20 -3.80 MICRO.946.C1_855 StB13G_21 -2.38 -1.10 2.75 1.79 MICRO.2526.C3_401 StB13G_22 3.07 MICRO.1984.C1_1464 StB13G_23 -3.32 MICRO.2259.C1_627 StB13G_24 4.19

LXI

MICRO.15113.C1_911 StB13G_25 -4.81 MICRO.14688.C1_1230 StB13G_26 MICRO.13229.C1_388 StB13G_27 -2.08 MICRO.12659.C1_1006 StB13G_28 1.70 MICRO.3866.C1_891 -3.30 1.47 1.51 2.98 -1.38 2.78 -1.01 StB13G_29 MICRO.3866.C2_1 MICRO.6607.C1_2 MICRO.6607.C2_658 StB13G_30 MICRO.6607.C3_679 -1.80 3.28 1.76 1.44 bf_arrayxxx_0067f11.t3m.scf_577 StB13G_31 -13.36 -1.26 1.83 3.16 -1.21 2.97 1.07 MICRO.1005.C1_1573 StB13G_33 -1.50 1.08 -2.58 -1.21 MICRO.1430.C2_873 -11.73 StPDB13G_1 MICRO.1430.C1_91 -8.27 2.75 3.71 6.70 MICRO.2181.C1_1314 StPDB13G_2 -7.67 1.39 2.01 1.79

LXII Publications and conference contributions

List of publications

Hartmann, A., Senning, M., Hedden, P., Sonnewald, U., and Sonnewald, S. (2011). Reactivation of meristem activity and sprout growth in potato tubers require both cytokinin and gibberellin. Plant Physiology 155:776-796.

Senning, M., Steuernagel, B., Hartmann, A., Sonnewald, U., Scholz, U., and Sonnewald, S. (2007). Regulation der Keimruhe von Kartoffelknollen. Genomxpress 3.07, 7–10.

Conference contributions

Talks

Hartmann, A., Senning, M., Sonnewald, U., Biemelt, S. Gibberellin regulates potato tuber sprouting via cytokinin. 23. Tagung Molekularbiologie der Pflanzen, Dabringhausen; 23. – 26. Februar 2010.

Senning, M., Hartmann, A., Ferreira, S.J., Sonnewald, U., and Sonnewald, S. Comparative analysis of gene expression profiles from meristem-enriched potato tissues. 4th EU-SOL meeting on Tomato Genomics, Toledo, Spain; 5. - 6. Oktober 2009

Senning, M., Hartmann, A., Ferreira, S.J., Sonnewald, U., and Sonnewald, S. Developmental changes from meristem reactivation to bud outgrowth in potato tubers revealed by microarray data. 1st International PhD School Plant Development, Retzbach; 17. - 19. September 2008

Poster

Hartmann, A., Prasch, C., Stadler, R., Palmisano, R., Sonnewald, U., and Sonnewald, S. Multi-lane supply highway: Potato sprout growth is boosted by formation of new vascular tissue rather than restored symplastic connectivity. Botanikertagung, Berlin; 18. – 23.September 2011

Hartmann, A., Senning, M., Ferreira, S.J., Sonnewald, U., and Sonnewald, S. Expression of the Agrobacterium tumefaciens ipt gene under control of UFO Promotor alters meristem identity in transgenic potato (Solanum tuberosum L.) Botanikertagung, Leipzig; 6. – 10. September 2009

Senning, M., Hartmann, A., Ferreira, S.J., Sonnewald, U., and Sonnewald, S. Comparative analysis of gene expression profiles from meristem-enriched potato tissues. Botanikertagung, Leipzig; 6. – 10. September 2009

LXIII

Acknowledgement

First and foremost I would like to thank Professor Dr Uwe Sonnewald for assigning me this fascinating research topic, for generous sharing of his vast knowledge and for constructive discussions of my work. I benefited greatly from the opportunity to attend international meetings and the ample range of methods and equipment he provided at the Lehrstuhl für Biochemie.

My heartfelt gratitude I offer to Dr Sophia Sonnewald for continuous support and encouragement, for demanding a high scientific standard and for her effort to keep me focused while leaving me room to work in my own way. Throughout my thesis she has been patient and perceptive, reminding me of my passion for research when I felt nothing worked in the lab or prompting me to work harder whenever she felt it was needed. She fostered my inclination towards computer work and sequence analysis which became a substantial part of my thesis. Without her mentoring and care this thesis would not have been completed or written.

I also would like to thank Dr Mark Taylor from the The James Hutton Institute (formerly Scottish Crop Research Institute, SCRI) for taking on the task of reviewer for my thesis. Thanks to him I had the opportunity of doing research in the labs of SCRI’s Cell Biology and Imaging Group in 2008 and 2009.

My special thanks goes to Melanie Senning who supervised me during my diploma thesis and who, when I continued to work on potato, never ceased to be a source for helpful information, constructive scientific discussions and invitations to ‘Sing Star’ parties.

As results of both have found their way into this thesis, I thank Christian Prasch for outstanding work during his master thesis on selected beta-1,3-glucanases and Sabrina Bachmann for excellent work cloning several StCKX genes during her bachelor thesis. Stephen Reid is also worthy of a major thank you, not only for expert technical assistance with microarray hybridisation, but also for guidance with methods I did not regularly do. And, not to forget, for being all ears whenever I needed to talk about scientific or non-scientific problems and for frequently entertaining our taste buds with his delicious cakes.

In addition to all the people already mentioned, in my daily work I was blessed with a great group of lab mates, first in lab 01.184 (formerly known as ‘Mädels Lab’) and later in lab 01.172 (a.k.a. ‘melting pot of the Lehrstuhl’). A thousand times ‘thank you’ to Nurcan ‘Sing Star’ Kocal, Julia ‘Die Perle’ Schuster, Jasmin Drobietz, Lien Quinh Le, Isabel Jungkunz, Katrin Link, Hannes Priller, Kathrin Volkert, Waldemar Röhrig, Stephanus Ferreira, Matthias Wimmelbacher, Dr Katharina Müller, Dr Urte Schlüter and Suayib Üstün.

LXIV Acknowledgement

Furthermore I would like to thank all members of the Lehrstuhl für Biochemie at large, for creating a stimulating work environment, and in particular the tissue culture team Anja Saalbach, Christiane Börnke and Eva Düll for plant transformation, Florian Vogel and Ralf Palmisano for confocal imaging, Dr Jörg Hofmann for cytokinin measurements, Alfred for help with any computer problem, Sabine Albert for always providing clean lab equipment and our ‘trio organizzativo’ in the secretariat, Gabriele Wabel, Iris Hammer and Ursula Lebherz.

Beyond the Biochemistry Department, there’s a whole bunch of other people who contributed significantly to my well-being and sanity all these years – my friends Eileen, Sandra, Susanne and Clara. Thanks for always being there when I needed you, for keeping me level-headed and at times teasing out my ‘crazy’ side.

I could not wish for a greater love than that which I have found in my boyfriend, Dirk Becker, who entered my life only 7 months ago. We met when the end of my thesis was drawing nearer, and whenever writing was an arduous task, the certainty of his love and support kept me going. Dirk, you’re the love of my life!

To my big sister Simone and my brother-in-law, Kai, I owe a hearty thank you, as they were always ready to offer open arms, open hearts, open ears, a filled fridge, a comfortable armchair and their luxurious bathroom whenever I came over. I love you two!

Finally, the two most important people in my life deserve the biggest thanks: I am deeply indebted to my parents Doris and Werner Hartmann whose generous funding throughout my studies at University and – most importantly – whose unwavering trust in and unconditional love for me brought me where I am today. You gave me exactly what a child needs: Roots to grow and wings to fly. I love you!

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Anja Hartmann current address:

Born August 16, 1982 Würzburger Ring 37 in Merseburg a. d. Saale 91056 Erlangen

Education

1989-1993 Primary School Geusa, Germany

1993-1997 Secondary School Domgymnasium Merseburg Merseburg a. d. Saale, Germany

1997-1999 Secondary School Landesschule Pforta Schulpforte, Germany

1999-2000 Elm Creek Collegiate - Canadian High School Diploma - Elm Creek, Manitoba, Canada

2000-2002 Secondary School Landesschule Pforta - Cambridge Certificate of Advanced English - - Abitur (German university entrance qualification) - Schulpforte, Germany

Academic Studies

2002-2007 Friedrich-Alexander-Universität Erlangen-Nürnberg Degree Program: Biology Diplom (comparable to MSc) Major: Biochemistry

2007-2012 PhD thesis at the chair of biochemistry of the Friedrich-Alexander-Universität Erlangen-Nürnberg

Research Visits

October 2008 Scottish Crop Research Institute (SCRI) Invergowrie, Scotland

July/Aug. 2009 Scottish Crop Research Institute (SCRI) Invergowrie, Scotland

Spoken Languages

German mother tongue English fluent French CEFR level B2 Italian basics Spanish basics

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