Elucidating the Roles of MAP Kinases in the Moss Physcomitrella Patens

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PhD thesis Sabrina Stanimirovic

Elucidating the roles of MAP kinases in the moss Physcomitrella patens

Supervisor: John Mundy Handed in: 17/05/2017

Table of contents

Preface ...... 7

Acknowledgements...... 8

Abstract ...... 9

Resume ...... 10

List of abbreviations ...... 11

Introduction ...... 13

Physcomitrella patens ...... 13

Evolution ...... 13

The life cycle and morphology ...... 14

Homologous recombination ...... 16

Plant innate immunity...... 17

2 layered defenses – PAMP and effector triggered immunity (PTI & ETI) ...... 17

MAMPs/PAMPs and LRRs ...... 18

ROS and cytoplasmic calcium ...... 19

Mitogen activated protein kinase signaling pathways ...... 20

Immunity in Physcomitrella patens...... 24

Abiotic stress ...... 26

Salinity and osmotic stress ...... 26

Phytohormones ...... 28

Abscisic acid...... 28

Auxin & cytokinin ...... 32

Strigolactone ...... 36

Light ...... 38

Results ...... 40

Identification of Physcomitrella patens homologs of MPKs in Arabidopsis thaliana ...... 40

Generation of MPK knockout mutants ...... 42

Phenotypic analysis of generated mutants ...... 44

Sporophyte induction ...... 44

Growth of protonemata ...... 53

3 weeks phenotypic analysis ...... 55

6 weeks phenotypic analysis ...... 58

Gravitropism ...... 63

Finding the needle in the haystack – what activates the MPKs? ...... 66

Biotic stress ...... 66

Abiotic stress – Drugs ...... 67

Calyculin A – protein Serine/Threonine phosphatase inhibitor ...... 67

Zeomycin – DNA damage ...... 69

Abiotic stress – Salt and osmotic stress ...... 71

Vertical growth ...... 72

Stress survival ...... 74

Abiotic stress – Hormones ...... 78

Auxin...... 78

Abscisic acid ...... 80

Cytokinin ...... 82

Strigolactone ...... 83

Abiotic stress – Light ...... 86

Dark ...... 86

Red and Blue light ...... 88

Expression of RAK1, RAK2, MPK3, MPK5 and NATH ...... 94

Discussion ...... 101

Auto-phosphorylation ...... 101

Rosettas – RAK1/RAK2 and their single-domain NATH homolog ...... 102

Characterizations of mutant KO lines...... 103

Protonemata and secondary growth ...... 104

MPK phosphorylation and activation ...... 105

MPK3 and MPK5 – involvement in ion homeostasis upon salt treatments? ...... 106

MPK regulation by phytohormones? ...... 108

Light responses ...... 110

Concluding remarks ...... 111

Material and Methods ...... 111

Plant growth conditions ...... 111

Phenotypic analysis ...... 112

Generation of Physcomitrella patens mutants ...... 113

Microscopy and statistical analysis ...... 113

Protein extraction and immunoblotting ...... 114

RNA extraction and quantitative RT-PCR ...... 114

Supplemental ...... 115

References ...... 152

Manuscript 1: Remarkable Regulatory Rosettas in the bryophyte Physcomitrella patens ..... 162

Abstract ...... 162

Introduction ...... 163

Results ...... 164

Identification of Physcomitrella patens homologs of MPKs in Arabidopsis thaliana ...... 164

Generation of rak1, nath and rak1-rak2 knockout mutants ...... 165

Phenotypic analysis of rak1, rak1-rak2 and nath ...... 165

Activation of the MPKs ...... 173

Abiotic stress – Hormones ...... 175

Blue light ...... 179

Expression of RAKs and NATH in P. patens ...... 184

Discussion ...... 189

Concluding remarks ...... 192

Material and Methods ...... 193

Supplemental ...... 196

References ...... 218

Chapter 15: Chitin and Stress Induced Protein Kinase Activation………………………...…221

Preface This thesis concludes my PhD work at the Department of Functional Genomics, Biology, University of Copenhagen. The main goal of my research was to understand the impact of evolution on plant innate immunity by using the moss, Physcomitrella patens as a model organism. I produced multiple moss mutants in homologs to Arabidopsis genes involved in signaling PAMP triggered immunity, more specifically MAP kinase signaling. I worked on the functions of these kinases on immunity, development and response to abiotic stresses. Many of the mutant lines have interesting phenotypes with and without exposure to abiotic stresses. One of these is a fascinating story of a novel Rosetta kinase such that my research provides links between protein phosphorylation and acetylation.

The thesis consists of:

I: A general introduction of plant immunity and the role of MPKs in several regulatory processes.

II: Observations and results from the PhD work (all results were conducted by Sabrina Stanimirovic. Figure 13, was adopted from PhD thesis of Simon Bressendorf).

III: A draft manuscript regarding a novel pair of Rosetta proteins in the moss P. patens, which combine a protein N-terminal acetyltransferase (NATD) and a MAP kinase (MPK). Title: “Remarkable Regulatory Rosettas in the bryophyte Physcomitrella patens”

IIII: Chapter in a methods manuscript (Kenchappa et al. 2017 Chitin & Stressed-induced Protein Kinase Activation. In Methods Mol Biol. – Plant Pattern Recognition Receptors. Vol 1578, L. Shan & P. He (Eds.), Springer Nature Series.

Acknowledgements 1.185 days later, I am now finishing my PhD, and what an interesting, scary, hard and mind-blowing ride it have been. I would like to thank my supervisor, John Mundy and co-supervisor Morten Petersen for helping me to analyze weird results and always being there when I needed to talk. A big thanks to my two Portuguese amigos, Raquel and Eleazar that always made the lab work fun and interesting. Special thanks to Raquel - my Moss-Oracle. Thanks for being there for me from the start and I could not have done this PhD without your insights and fantastic friendship. Thanks to Chandra, for teaching me laboratory methods and making it fun to teach students, and being a great friend. Thanks to my USA travelling buddy, Signe for always being there during my up’s and down’s. The remaining ones from the PMB lab, it was a pleasure to meet everyone, and I can imagine that the lab will not be the same without the crazy-Sabrina 

I cannot express how thankful I am for the support from my parents, brother, in-laws and my bestie Minela. You people have always been there for me. A special thanks to my husband who through these rollercoaster years has been supporting, understanding and loving. I love you all.

Many thanks for a great collaboration with the Department of Plant Biology, at Sveriges lantbruksuniversitet (SLU), Swedish University of Agricultural Sciences, were I learned how to dissect and characterize the moss. Furthermore, thanks to the Center for Applied Bioimaging (CAB) for the use of their stereo fluorescence microscope. Thanks to Professor Catherine Rameau from the French National Institute for Agricultural Research, Centre de Recherche de Versailles-Grignon, Paris, France for letting us use the PpCCD8 mutant as a control in experiments with the phytohormone, strigolactone.

Abstract The evolutionary transition of plants from aquatic to terrestrial environments resulted in adaptations to cope with various stresses and threats. Plant plasma membrane receptors, recognize extracellular signals and initiate immune and abiotic stress responses. MAP kinase (MPK) cascades transduce signals from such receptors by phosphorylating substrate proteins, which effectuate appropriate responses. By generating deletion lines of MPK genes in the simple, non-vascular moss Physcomitrella patens, I provide interesting evidence that MPKs may be important in the understanding of evolutionary changes of plant immunity required for the conquest of land by plants. I describe the role of MPKs (MPK3, MPK5, RAK1 & double knockout RAK1/RAK2) upon abiotic stress by characterizing the phenotypes and morphological changes there may be during stress treatments. I characterized the mutant phenotypes during treatment with phytohormones and osmotic and light stress. This thesis contains of a general introduction to plant immunity and the role of MPKs in signaling processes related to immunity, abiotic stress, and plant development in both vascular and nonvascular plants. The focus in this thesis is on abiotic stress and development changes in the MPK mutants. Results are presented in the result part of the thesis, and in a draft manuscript summarizing data on novel rosetta proteins which combine a protein N-terminal acetyltransferase (NATD) and a MPK, here we called RAKs (Rosetta-Acetyltransferase-Kinase). This thesis and work on these MPK mutants gives the laboratory a great start on several future publications, since many of the mutant lines have interesting phenotypes with and without exposure to abiotic stresses

Resume Den evolutionære overgang fra vand til jord, og de derved følgende miljøforandringer, for planter, har resulteret i udviklingen af og tilpasninger til nye forsvars strategier overfor et væld af biotiske- og abiotiske stressfaktorer. Receptorer i planters plasmamembran, genkender disse ekstracellulære stressfaktorer og iværksætter beskyttelse og adaptering imod biotisk- og abiotisk stress. Receptorerne aktiverer MAP kinase (MPK) signalerings kaskader der videre phosphorylerer forskellige substrater med det formål at frembringe et passende respons til den pågældende udfordring. Ved at generere og analysere individuelle MPK knock-out linjer i den simple, ikke-vaskulære mos Physcomitrella patens, dokumenterer jeg signifikansen af MPKerne (MPK3, MPK5, RAK1 og dobbelt knock-out RAK1/RAK2 mutanter). Fænotyper og morfologiske forandringer der opstår under stressbehandlinger, er blevet karakteriseret og derved er betydningen af MPKerne under stress blevet udforsket. Mutanterne er blevet karakteriseret under behandlinger med plante hormoner, osmotisk og lys stress. Denne afhandling indeholder en generel introduktion til planters immunitet og MPKernes roller i forskellige signalerings processor, så som beskyttelse mod biotiske- og abiotiske stressfaktorer. Hovedfokus i denne afhandling vedrører abiotisk stress og morfologiske forandringer, grundet knockout af MPKerne samt stressbehandlinger. Udover en klassisk gennemgang af resultaterne er der inkluderet et tidligt stadie udkast til et manuskrift. Manuskriftet omhandler nogle nyligt identificerede rosetta proteiner, der er fusionsproteiner bestående af en N-terminal acetyltransferase (NATD) og en MPK. Vi kalder proteinerne for RAKer (Rosetta-Acetyltransferase-Kinase). Da der er mange interessante aspekter ved de fremstillede MPK mutant linjer inklusiv kraftige fænotyper og tydelige reaktioner på abiotisk stress, kan dette arbejde forhåbentlig i fremtiden føre til en bedre forståelse and MPKernes rolle i mos og adskillige publikationer.

List of abbreviations

MAMPs Microbe-associated molecular patterns PAMPs Pathogen-associated molecular patterns MPKs Mitogen activated protein kinases PRRs Pattern recognition receptors ECD Extracellular domain TM Transmembrane PTI PAMP-triggered immunity MTI MAMP-triggered immunity ETI Effector-triggered immunity R-proteins Resistance proteins NB-LRRs Nuclear binding leucine rich repeat PCD Programmed cell death HR Hypersensitive response LRR Leucine rich repeat LRR-RLK Leucine rich repeat receptor like kinase EF-TU Elongation factor Tu EFR Elongation factor receptor SUMM1 Suppressor of mkk1/mkk2 1 MKS1 MAP kinase substrate 1 ET Ethylene BAK1 BRI1-associated receptor kinase 1 PAD3 Phytoalexin deficient 3 PAT1 Protein associated with topoisomerase II PGN Peptidoglycan LPS Lipopolysaccharide PM Plasma membrane FLS2 Flagellin Sensing 2 CEBiP Chitin elicitor-binding protein CERK1 Chitin elicitor receptor kinase 1 ROS Reactive oxygen species DAMPs Damaged-associated molecular patterns CDPKs Calcium dependent protein kinases TLR4 Toll-like receptor 4 NADPH Nicotinamide Adenine Dinucleotide Phosphate Hydrogen RbohD Respiratory burst (NADPH) Oxidase homologue D MEKKs or MAPKKKs MPK kinase kinases MKKs or MAPKKs MPK kinases NTF Nuclear transfer factor TEY Thr-Glu-Tyr TDY Thr-Asp-Tyr ERK Extracellular signal-regulated kinase SA Salicylic acid JA Jasmonic acid WT Wild type SOS1 Salinity overly sensitive 1 HKT High-affinity potassium transporter NHX1 Na+/H+ antiporter tonoplast ENA Exitus natru – exit of sodium HOG High-osmolarity glycerol 2+ PCA1 PIIB-type CA ATPase 1 EDS1 Enhanced disease susceptibility 1 protein PAD4 Phytoalexin deficient 4 CTR1 Constitutive triple response 1 ERF Ethylene response factor

EREBPs Ethylene response element-binding proteins ABA Abscisic acid NCED 9-cis-epoxycarotenoid dioxygenase ZEP Zeaxanthin epoxidase AAO3 Abscisic aldehyde oxidase 3 ARs ABA-responsive genes PYR/PYL/RCAR Pyrabactin resistance/pyrabactin resistance-like/regulatory component PP2C Protein phosphatase 2C SnRK2s Sucrose nonfermented 1 related protein kinases 2 CCD7 & CCD8 Carotenoid cleavage dioxygenases 7 and 8 IAA Indole-3-acetic acid BPA Cytokinin ARK ABA and abiotic stress-responsive Raf-like kinase SAGs Senescence-associated genes TF Transcription factor OST1 Open Stomata 1 SLAC1 Slow anion channel 1 CAT Catalase IBR5 Indole-3-butyric acid-response 5 ANP1 Arabidopsis, Nicotiana protein kinase 1 (NPK)-like Protein kinase AUX/LAX1 Auxin resistant/like AUX1 AUX/IAA Auxin/indole acetic acid ARFs Auxin response factors SCF Skp1-Cull-Fbox TPL TOPLESS TIR1 Transport inhibitor response 1 NTG N-methyl-N'-nitro-N-nitrosoguanidine NAA 1-naphthalene acetic acid NAR NAA resistant ARBs Auxin signaling F-box proteins BPA 6-benzyl aminopurine BAR BPA resistant CRE1 Cytokinin response 1 AHK4 Arabidopsis histidine kinase 4 AHP Arabidopsis histidine phosphotransfer protein ARR Arabidopsis response regulator HPT Histidine phosphotransfer AHP Arabidopsis histidine phosphotransfer CHASE Cyclases/histidine kinases associated with sensory extracellular CHK CHASE domain-containing histidine kinase ER Endoplasmatic reticulum RRB Type-B response regulator RRA Type-A response regulator BA Benzyladenine MAX More axillary growth D14 Dwarf14 KAI2 Karrikin insentistive2 CRY Crytochrome PHYB Phytochrome B PHOT Phototropin HY Elongated hypocotyl PPS Pleiotropic photo-signaling

Introduction The evolutionary transition of plants from aquatic to terrestrial environments resulted in adaptations and new strategies to cope with numerous stresses and threats. These adaptations included the expansion of a repertoire of genes encoding receptors for direct recognition of microbe-associated molecular patterns (MAMPs) or pathogen-associated molecular pattern (PAMPs). These genes provide a first line of defense against a range of pathogens and are therefore central to disease resistance in plants and crops.

This introduction focuses on the activation of downstream signaling mitogen activated protein kinase (MPK) cascades during immune responses, and stress signaling and hormonal signaling. The moss Physcomitrella patens is a reasonable model to study plant innate immunity since mosses are found largely unaltered in ~450 million year-old fossils and therefore represent early, nonvascular plant lineages. The role of MPKs and abiotic stress on the growth of the moss P. patens will also be referred in this introduction.

Physcomitrella patens Evolution Bryophytes (hornworts, mosses and liverworts) arose from an ancestor related to unicellular aquatic algae (5, 13, 14), and they diversified from aquatic environments to adapt to different terrestrial habitats. This adaptation and spread in turn modified terrestrial environments. Two of these successful bryophytes (15) are the moss P. patens and the liverwort Marchantia polymorpha (Figure 1) (3, 16, 17). Rensing et al. 2008 published the genome of the P. patens, which has enabled it to become the bryophyte model for reverse genetic analyses. Information about the P. patens genome and other resources is accessible at www.cosmoss.org, www.phytozome.jgi.doe.gov/pz/portal.html, and www.ncbi.nlm.nih.gov.

Numerous cellular processes are conserved among eukaryotes, hence functional, and comparative studies of mosses can enlighten different genetic and biological processes in other organisms (14). The evolutionary transition from aquatic to terrestrial environments led to the loss of genes associated with aquatic environments and the evolution of genes for enhanced osmoregulation, desiccation- and freezing tolerance, heat resistance, synthesis and accumulation of protective “sunscreens” against e.g. UV-light, and enhanced DNA repair mechanisms (14, 18, 19).

Figure 1: A phylogeny of plants. Times of divergence (million years ago (Mya)) are indicated at nodes in the tree. Phyla and phylum-level groupings are indicated on the right (3).

The life cycle and morphology Mosses such as P. patens have a simple morphology compared to vascular plants. In the moss, the haploid gametophyte is the predominant stage or generation. The development of the moss body plant is highly dependent on polar growth of tip cells, and the haploid phase is initiated by tip growth during spore germination (Figure 2A) which leads to protonemata and rhizoid development. Protonemata is the filamentous body morphology formed by chloronemal and caulonemal cells (Figure 2B-C)(5). As in other land plants, moss growth is regulated by light and phytohormones including cytokinin and auxin (20-22). The first cells formed after germinatiom are the chloronema (Figure 2B). These cells are quite easy to visualize since they contain 50-100 chloroplasts and transverse cell walls

14 perpendicular to the growth axis. In contrast, caulonema cells contain fewer and less-developed plastids, have oblique transverse cell walls, and grow faster than chloronema cells. These differences make it easy to distinguish chloronemal versus caulonemal cells during phenotypic analyses (5, 21, 23-25). From protonemata, and more specifically from side branches, buds form and leafy gametophytes (thallus) develop into gametophores. The gametophores are composed by nonvascular stem and rhizoids (Figure 2D), and have hydroid cells to conduct water internally as xylem vessels in vascular plants (26). After 2-6 weeks under sporophyte induction conditions (8h light, 16h dark and 15-17°C) which mimic seasonal change, adult gametophores are able to induce gametogenesis with organ bundles emerging from the top of the leafy gametophores (24, 27). The male reproductive organ (antheridia) contains motile spermatozoids that are released upon water irrigation (Figure 2E, arrow heads) (5, 22, 23). Antheridia appear as round bundles with shinny surfaces localized around the female reproductive organs (archegonia) (Figure 2E, arrows). There are numerous archegonia per apex, however normally only one develops into a mature sporophyte. Archegonia have a tube/neck-like structure, with a long canal and a basal envelope with the egg. Upon egg maturation, a small opening of the tube/neck is visible (Figure 2E, arrow, left tube) and water expands the canal making it possible for the spermatozoids to swim down the canal to the egg. After fertilization, the canal becomes dark brown to red (27, 28), and the zygote develops into the diploid sporophore (Figure 2F) (22, 24). The sporangium then undergoes different maturation steps indicated by the colorations of the spore capsule (18), and the connections between the gametophytic apex and the sporophytes are called seta (Figure 2F)(27). At the seta, a brown ring (vaginula) appears under the sporophytes, indicating the transition from gametophore to sporophyte tissue (27). Meiotic divisions in the sporophyte capsule produce up to 4000 haploid spores, and the development of the capsules is first oblong and later spheroidal. The apical part of the sporophyte capsule are the neck of the archegonia and forming the calyptra. During maturation and enlargement of the sporophyte capsule the calyptra falls off or can be removed by mechanical stress (27). At full maturation the wall of sporophyte capsules will either dry or burst and release the spores (Figure 2A) (5, 23, 24, 28). The simple life cycle and morphology of P. patens is advantageous for working with the organism in the laboratory compared to flowering plants like Arabidopsis thaliana. P. patens is easy to vegetatively propagate since any tissue of the moss will differentiate into chloronemal cells and produce new colonies. In the laboratory, the life cycle of P. patens is completed within 10-12

15 days, and the lack of roots and vascular tissue restricts their size so that they are is easily grown on petri dishes. Spore germination to development of protonemata takes approximately 5-7 days at 25°C, and it is possible to regenerate protonemata from protoplasts. This method has been used as a tool for mutagenesis, transformation etc., in P. patens (22, 24).

Figure 2: P. patens life cycle. (A) Haploid spore germinates into protonema filaments consisting of (B) chloronemal cells and (C) caulonemal cells. (D) Gametophores emerge from protonema filaments and are anchored by rhizoids that expand by tip growth from the gametophore. (E) At the apex of the gametophore organ bundles are produced with both female, archegonia (arrows), and male, antheridia (arrowheads) reproductive organs. After irrigation the egg is fertilized by motile sperm and the (F) sporophyte developes at the apex of the gametophore. (5)

Homologous recombination Genetic transformation of protoplasts of P. patens was established in 1991. The method is based on direct DNA uptake using polyethyleneglycerol (PEG) followed by heat shock and is similar to transformation method for the yeast S. cerevisiae (20, 24, 29). The transformation efficiency in P. patens is as high as for S. cerevisiae and gene knockouts and knockins can be fairly routinely made by homologous recombination with vectors containing borders of 500-1000bps of homologous sequences. This amenability makes P. patens gene replacement a powerful reverse genetic tool (24, 29). It is also possible to work with another early land plant, the haploid liverwort M. polymorpha. In this liverwort, it is possible to perform Agrobacterium-mediated transformation using immature thalli grown from asexual bud-like structures called gemmae. Many different functional genomics and transformation techniques have been developed in both M. polymorpha and P. patens, contributing to their predominant use in numerous laboratories (28, 30).

Plant innate immunity In nature, plants rely on the recognition of patterns that may be perceived as threatening or benefical to them. Such an ability to discriminate between self and non-self in all organisms is important for survival. The ensuing responses mediated by specific signaling cascades is likewise important for survival. Plants and animals rely on the recognition of patterns that may be threating or benefical to them. The ability to discriminate between self and non-self in all organisms are necessary for survival. Vertebrates have a two-fold defense system with innate and acquired immunity against pathogens. Activation of vertebrate innate immune responses is central to the activation of the adaptive immune system in which lymphocytes and other specialized cells and receptors are components. Plants do not have adaptive immune system, but are able to distinguish between self and non-self and to mount defense responses in every single plant cell by their innate immune system. (31, 32). In addition to preformed defenses, the so-called phytoanticipins such as cuticular waxesm secondary metabolites and certain anti-microbial enzymes, plants have two major layers of innate immunity (19, 31).

2 layered defenses – PAMP and effector triggered immunity (PTI & ETI) In the first layer, MAMPs (microbe-associated molecular patterns) and PAMPs (pathogen-associated molecular patterns) are recognized by plasmamembrane localized pattern recognition receptors (PRRs). PRRs recognize such microbial patterns whether they are pathogenic or not, and MAMPs/PAMPs are characteristics of classes of microbes. This recognition triggers PAMP-triggered immunity (PTI) or MAMP-triggered immunity (MTI) (33). Plants are able to recognize many different MAMPs due to the formation of complexes with different receptors. The perception of PAMPs/MAMPs leads to many downstream event, such as MAP kinase cascade pathway and the expression of defense genes. MPKs are conserved among eukaryotes in which they transduce signals into adaptive and programmed resonses via pjosphorylation of substrate proteins including transcription factors. The kinases thus transduce extracellular signals from the cell surface to the nucleus by a phosphorylation cascade in which MPK kinase kinases (MEKKs) phosphorylate MPK kinases (MKKs) that phosphorylate MPKs. MPKs regulate many different cellular processes such as cell differentiation, innate immunity, stress and hormonal responsesvia the activation of other kinases, enzymes and transcription factors (34, 35).

Some pathogens are able to evade or suppress PTI by delivering effector molecules into the host cell. Plants have evolved a surveillance system to avoid suppression of the immune system. This surveillance system recognizes effector molecules directly or indirectly by recognition of the effector molecules by their effects on host cell proteins or pathways. This recognition is mediated by resistance proteins (R-proteins) that guard targets in the host cell. R-proteins are present in the cytoplasm and encodes a class of nuclear binding, leucine rich repeat (NB-LRRs) proteins that are the main factors to trigger ETI (35-37). ETI may trigger localized programmed cell death (PCD) designated as the hypersensitive response (HR) (19). ETI is very important in preventing colonization by biotrophic pathogens; however, ETI is ineffective towards necrotrophic pathogens since these causes death of the infected and surrounding cells (35, 36, 38, 39).

MAMPs/PAMPs and LRRs Biochemical approaches in A. thaliana and other flowering plants including tobacco, rice and tomato have identified several MAMPs and their respective PRRs. One such MAMP is the 22 amino acid peptide flg22 of the bacterial flagellin protein (40, 41). Flagellin is also recognized in mammals by a Toll-like PRR receptor (42). In A. thaliana the flg22 peptide is recognized by the FLS2 PRR which contains an extracellular leucine rich repeat (LRR), a transmembrane domain and a cytoplasmic serine/threonine kinase domain (LRR-RLK) (40, 43). Another MAMP that is recognized by plant PRRs is the peptide elf18 derived from bacterial elongation factor Tu (EF-TU) (40, 43, 44). Elf18 is recognized by the LRR-RLK elongation factor receptor (EFR) (44). P. patens does not have homologs to AtFLS2 and AtEFR in its genome, and full-length flagellin, or different length flg peptides, do not trigger downstream immune signaling including the activation of the moss MAP kinases PpMPK4a&b (39, 45). A. thaliana FLS2 and EFR are able to form complexes with another LRR-RLK receptor, the BRI1-associated receptor kinase 1 (BAK1) that mediates PTI/MTI responses (39, 46-49). Other well studied MAMPs include the bacterial wall components peptidoglycan (PGN) and lipopolysaccharide (LPS) (50, 51), and fungal and oomycete cell wall components such as chitin, chitosan and β-glucans (45, 48, 52). Chitin (β-(1,4)-linked oligosaccharide of N-acetylglucosamine) elicits rapid defense responses in plants (41, 53). Kaku et al. (2006) showed that in rice (Oryza sativa) a plasma membrane (PM) glycoprotein called chitin elicitor-binding protein (OsCEBiP) binds chitin oligosaccharides at the cell surface. Knockout of OsCEBiP abolishes chitin induced defense responses (52, 54, 55). Since OsCEBiP does not contain a functional intracellular domain for signaling, Miya et al. 2007 showed that OsCEBiP needs chitin elicitor receptor kinase1 (OsCERK1)

18 for signaling via the cytoplasm. The closest A. thaliana homolog of OsCEBiP (AtLYM2) is not involved in chitin signaling. Rather Arabidopsis relies on another LRR-RLK homolog called AtCERK1 that binds chitin oligosaccharides (52, 56). The CERK1 receptor is localized at the PM and has three extracellular LysM motifs and an intracellular serine/threonine kinase domain (52, 55). Ligand binding by AtCERK1 leads to activation of a MAP kinase cascade and generation of reactive oxygen species (ROS) burst (52). The moss ortholog of CERK1 receptor has been identified and described as a first functional PRR in bryophytes (45). AtCERK1 does not only perceive chitin oligosaccharides, but also perceives PGNs by forming a hetero-oligomer with AtLYM1 and AtLYM3 (51). Besides MAMPs/PAMPs, microbes can produce lytic enzymes that damage host cells. Such damage, including cell wall fragments and peptides are known as damaged-associated molecular patterns (DAMPs). DAMPs act as danger signals for the host and generally trigger the same responses as MAMPs/PAMPs (39, 57). Plants are able to recognize many different MAMPs due to the formation of complexes with different receptors. The perception of PAMPs/MAMPs leads to many downstream events, such as ion fluxes, ROS production, activation of MAP kinase cascade pathway, calcium dependent protein kinases (CDPKs) and the expression of defense genes.

ROS and cytoplasmic calcium ROS (reactive oxygen species) are rapidly generated within seconds upon PAMP/MAMP perception. ROS accumulates apoplastically in a reaction known as the ROS burst, and is known to occur throughout the plant kingdom and in animal innate immunity under phagocytosis (32, 58, 59). In mammals such ROS act as second messengers, for example following LPS perception by Toll-like receptor 4 (TLR4) on the surface of macrophages. This results in secretion of cytokines and activation of other immune cells, as well as the activation of MAP kinase signaling cascades (32, 60). In plants, the ROS burst is generated by NADPH oxidases such as the plasma-lemma localized respiratory burst oxidase homologue D (RbohD) enzymes (39, 61, 62). ROS in plants can also act as antibiotic agents during PTI/MTI and can cause cell wall crosslinking as a defense against microbes. As in mammalian innate immunity, ROS act as a second messenger which may lead to the activation of MAP kinase cascades (39, 63). ROS production is also induced in osmotically stressed plants, and has a role in abiotic stress signaling responses (6). Besides ROS, increased concentrations of cytoplasmic calcium may act as a second messenger upon MAMP

19 perception. Perception appears to result in an influx of calcium ions from the apoplast that increases the calcium concentration in the cytoplasm. The resulting imbalance or waves in calcium concentration are then thought to activate several Ca2+ sensors, designated as calcium-dependent protein kinases (CDPKs) and the opening of membrane ion-channels (62, 64). Salt and osmotic stress increases cytosolic Ca2+ that will trigger many signaling cascades such as activation of MPKs and type IIA ATPase Ca2+ pump. Quideimat et al. 2008, showed that type IIA ATPase Ca2+ pumps are activated by salinity to provide an adaptive response in P. patens. Likewise, in A. thaliana the CaM- regulated Ca2+ ATPase pump as also been associated salinity (6, 65). Such Ca2+ signaling is important for germination of P. patens spores and the initial formation of buds (22). Many different biochemical and pharmacological approaches have been used to identify PTI/MTI signaling and thus the perception of elicitors. A robust and sensitive approach is quantitation of the ROS burst upon treatment with different elicitors (45, 61, 66). The ROS burst has thus been detected in P. patens within seconds upon treatment with the fungal elicitor chitosan and the fungal necrotrophic pathogen Botrytis cinerea (19, 45, 59, 67). Besides its production during PTI/MTI, ROS production has also been studied under abiotic stress responses and the HR (59, 68, 69).

Mitogen activated protein kinase signaling pathways

The model flowering plant A. thaliana encodes 60 MEKKs, 10 MKKs and 20 MPKs, which indicates that there may often become levels of functional redundancy in MPK signaling (35, 70, 71). MEKKs are serine or threonine kinases phosphorylating MKKs that as a result phosphorylate MPKs on threonine and tyrosine residues in the MPK activation loop (Figure 3). MPK activity and deactivation are regulated by tyrosine and serine/threonine specific phosphatases (35). MPK signaling in plants like A. thaliana show how complicated and diverse the different signaling pathway are. Hence, to understand and help elucidate MPKs in flowering plants, it may help to study MPKs in a less complicated model such as P. patens. P. patens has 22 predicted MEKKs, 7 MKKs and 8 MPKs (45). Plant MKKK proteins can be divided into two large subfamilies, with similarity to yeast STE11 and BCK1 and to mammalian MEKK1 (MEKK1-like clade) and RAF-like kinases similar to mammalian RAF1. Physcomitrella has 6 homologs in this clade, while Selaginella has 8 and 60 homologs in Arabidopsis (70, 72, 73).

MKK proteins are divided by sequence similarities into four groups (A-D). MKK1, 2 and 6 in A. thaliana are group A MKKs, and activate the downstream MPKs, MPK4 and MPK6, respectively. Group B MKKs include AtMKK3 and tobacco NPK2 that are characterized by the presence of an nuclear transfer factor (NTF) domain and these MKKs are involved in cytoplasmic to nuclear trafficking (70, 72-74). MKK3 is involved in the regulation of a cascade elicited by pathogens and is dependent on regulation of Jasmonic acid (JA) signaling. Moreover, MKK3 phosphorylates downstream the MPK1, 2, 7 and 14 and, interestingly, upon fungal elicitor flg22 phosphorylates MPK6 rather than MPK7 (74). Group C includes A. thaliana MKK4 and 5 and orthologs of tobacco NtMKK. AtMKK4 and 5 upon flg22 elicitation phosphorylates AtMPK3 and 6 (70, 75, 76). Group D includes the remaining A. thaliana MKK7-10, which are most closely related to two Physcomitrella and two Selaginella MKKs (70).

Figure 3: Simplified model of MPK signaling cascade. Extracellular stress stimulus from the cell surface to the nucleus by a phosphorylation cascade that results in phosphorylation and activation of MEKK, and further phosphorylation and activation of MKK. The activated MKK activates MPK by phosphorylation of threonine and tyrosine residues. The activation of the MPK cascade initiates targeted activation of response genes by transcription factors in nucleus. MPKs activate cytosolic targets such as proteins, kinases and TFs. PM – Plasma membrane, P – phosphorylation, TF – transcription factor (Adopted from (2))

MPKs regulate many different cellular processes such as cell differentiation, innate immunity, activation of transcription factors, stress and hormonal responses, activation of other kinases and enzymes (Figure 4) (4, 70, 71). As mentioned, MPKs are phosphorylated by MKKs at threonine and tyrosine in the Thr-Glu-Tyr (TEY) motif of the regulatory activation loop. This regulatory motif is also seen in the mammalian extracellular signal-regulated kinase (ERK) and another phosphorylation

21 motif unique for plants are the Thr-Asp-Tyr (TDY). MPKs are divided in five groups among which, proteins with a TEY motif in the activation loop are divided into groups A-C. Proteins with a TDY motif are group D that have a common C-terminal docking domain that may act as a docking site for MKKs. Group E are proteins with a TEY motif but without known docking domains for MKKs (70, 73). AtMPK3 and 6 are members of group A, while AtMPK4 is in group B (70, 73, 77). MPKs in Group D have similarities to the ERK proteins in mammals. The proteins are found in ancient lineage such as the alga Chlamydomonas reinhardtii and Physcomitrella, with 8 and 10 MPKs, respectively (70). AtMPK9 is a member of group D, which has a TDY motif and lacks the MKK binding dock site. Interestingly, AtMPK9 also has a TEY motif in its N-terminal, and can through autophosphorylation get activated (78).

Figure 4: Convergence points in abiotic and biotic stress signaling networks (Adopted from (4)).

The following sections focus on studies of these kinases in A. thaliana innate immunity, both PTI and ETI, and during abiotic stress and hormone signaling. As noted above, A. thaliana encodes 60 MEKKs, 10 MKKs and 20 MPKs which indicates that there may often be levels of functional redundancy in MPK signaling (71, 72, 79). MPK activity and deactivation are regulated by tyrosine and serine/threonine specific phosphatases (70, 71). P. patens has 22 MEKK homologs compared to the 60 homologs in A. thaliana. AtMEKK1 has been shown to be involved in PTI and the ROS burst (45, 70, 79). The first MPK signaling cascade described in A. thaliana is activated by the PRR FLS2 upon flg22 elicitor and involves MEKKα/MEKK1, MKK4/MKK5 and MPK3/MPK6) (75, 76). Gao et al. (2008) and Qiu et al.

(2008), described another MPK cascade in A. thaliana downstream of AtFLS2 consisting of AtMEKK1, AtMKK1/AtMKK2 and AtMPK4 (80). AtMPK3, 4 and 6 have also been shown to be activated upon abiotic stressors like salt, drought, cold, UV-light and wounding (72, 81-83). Other studies with the MAMPs/PAMPs flg22, efl18 and chitin have been described for AtMPK3, 4, 6 and 11, showing activation within minutes of perception (46, 77, 84-87). KO of AtMPK4 results in a dwarfed phenotype, and due to constitutive expression of defense related genes, ROS levels, high levels of salicylic acid (SA) and cell death, it was initially reported to exhibit autoimmunity and function as a negative regulator of plant immunity (88). However, newer results showed that AtMPK4 is activated in response to pathogens and PAMP treatments that is counterintuitive for a negative regulator (81, 89). KO of AtMEKK1 and double KO of AtMKK1 and 2 have similar developmental phenotype and disease resistance as Atmpk4, indicating functional connections (70, 80, 89). Zhang et al. (2012) showed that the phenotypes of Atmekk1, Atmkk1/Atmkk2 and Atmpk4 are due to activation of ETI through the R protein AtSUMM2 (suppressor of mkk1/mkk2 2). AtSUMM2 was discovered in a suppressor screen of the severe double mkk1/2 mutant, implying that AtSUMM2 induces ETI in response to disruption of the AtMEKK1-AtMKK1/AtMKK2-AtMPK4 cascade by pathogens. In addition, the triple mutant Atsumm2/Atmkk1/Atmkk2 and the double mutant Atsumm2/Atmekk1 display normal growth phenotypes, expression of defense genes and ROS levels. Interestingly, the double mutant Atsumm2/Atmpk4 did not display a normal phenotype, and had still high levels of ROS, indicating that AtMPK4 itself may be guarded by another R-protein. These results provide additional evidence that plants have a surveillance system to avoid suppression and to guard the immune system by R-proteins. However, AtSUMM2 does not directly interact with AtMEKK1, AtMKK1/AtMKK2 and AtMPK4, indicating that one or more downstream targets of the MPK cascade may be guarded by AtSUMM2 (37). Kong et al. 2012 found that mutation of another R-protein (AtSUMM1, suppressor of mkk1/mkk2 1) suppresses cell death and defense responses in Atmekk1, Atmkk1/Atmkk2 and Atmpk4. AtSUMM1 encodes the triple MPK AtMEKK2. They showed that disruption of the signaling cascade AtMEKK1, AtMKK1/AtMKK2 and AtMPK4 activates AtMEKK2 that activates AtSUMM2 mediated immune responses. However AtMEKK2 and AtSUMM2 did not show direct interaction (90), so how this works remains to be discovered. In a yeast two-hybrid cDNA library screen the MPK4 substrate AtMKS1 (MAP kinase substrate 1) was discovered. AtMPK4 interacts with and phosphorylates MKS1, which activates the transcription factors WRKY25 and WRKY33. Interestingly, WRKY33 is not phosphorylated and

23 activated by MPK4 but rather by MPK3 and 6 (91). Thus, MPK4 via MKS1 sequesters WRKY33, which is released from MPK4 upon its phosphorylation of MKS1, which may then permit WRKY33 activation by phosphorylation by MPK3 & 6. WRKY 25 and WRKY33 are involved in the expression of defense genes in response to pathogens, such as SA-, ethylene (ET)- and JA mediated responses. Moreover, WRKY33 induced the expression of the Phytoalexin Deficient 3 (PAD3) gene, which is a part of the biosynthesis of the antimicrobial compound camalexin that is required for resistance to B. cinerea (77, 92-94). The same yeast two-hybrid screen that identified MKS1 also identified the mRNA decapping enhancer PAT1 (protein associated with topoisomerase II) as an MPK4 partner or substrate. AtPAT1 is phosphorylated in response to flg22 and interacts with AtMPK4 and AtSUMM2 in planta. Roux et al. 2015, showed that AtPAT1 is regulated by AtMPK4 and, when disrupted, AtSUMM2 is activated and ETI is triggered (94). AtPAT1 may therefore be a substrate of the MPK4 cascade which is more or less directly guarded. These above studies and observations of MPK signaling in flowering plants like A. thaliana show how complicated and diverse signaling pathways are. Hence, to understand and help elucidate MPKs in flowering plants, it may help to study MPKs in a less complicated model plant like P. patens. P. patens has 22 MEKKs, 7 MKKs and 8 MPKs (45, 70, 74). Since the moss has fewer members of the MPK gene family compared to A. thaliana, it may be easier to study the functions of its MPKs. One might also expect some reduction in redundancy between the genes. Thus, it may be possible to assign a function for each MPKs in P. patens. Immunity in Physcomitrella patens

The molecular and cellular responses of P. patens under PAMP/MAMP perception are similar to those in flowering plants and includes elevated ROS, expression of defense genes, and PCD. There are a few studies on innate immunity in P. patens including studies of relevant microbial pathogenesis (19, 45, 59, 67, 95). These include studies with the necrotrophic fungus B. cinerea, the gram-negative enterobacterium Erwinia carotovora ssp. carotovora (E.c. carotovora), and the necrotrophic oomycetes Pythium irregulare and Pythium debaryanum. All of these pathogens cause browing, cell death, elevated ROS, elevated SA production, cytosolic shrinkage, chloroplast breakdown and expression of defense related genes including PpPAL, PpCHS, PpLOX and PpPR-1 (19, 45, 96, 97). Different PAMPs/MAMPs treatments of P. patens also induce defense responses, indicating a functional PTI/MTI system (45, 59, 67, 97). Treatment of the moss with Pectobacterium

24 carotovorum ssp. carotovorum (formerly named Erwinia carotovora ssp. carotovora) and the P.c. carotovorum SCC3193, which produces the elicitor HrpN, as well as a harpin (HrpN)-negative control strain, detected elevated defense responses such as ROS accumulation, expression of defense related genes, PCD and cytoplasmic shrinkage (97). Lehtonen et al. 2009 & 2012 treated P. patens with the MAMP chitosan that resulted in an increase in peroxidase activity and ROS accumulation. In a screen they determined that the peroxidase activity is due to PpPRX34. Ppprx34 mutants were not able to produce a ROS burst when treated with chitosan, making the moss more susceptible to saprophytic and pathogenic fungi isolated from another moss (59, 67). Bressendorf et al. 2016 from our laboratory recently identified an MPK cascade in P. patens. KOs and KIs generated by homologous recombination implicated a PRR, two MEKKs, three MKKs and two MPKs in the perception of the PAMP chitin. P. patens has four homologs of the A. thaliana chitin receptor CERK1 (98), and KO lines of the PRR PpCERK1 mutant (Ppcerk1) did not show cell wall modifications seen in wild type (WT) following chitin treatment. Ppcerk1 mutants were also unable to induce downstream MPK phosphorylation upon chitin and PGN treatment. This was in contrast to the response to a cell free culture filtrate from the harpin-negative strain Pectobacterium wasabiae SCC3193, which readily activated downstream kinases measured by immunoblotting with anti-phospho-p44/42 MPK antibody (α-pTEpY). Moreover, the expression of the defense related genes PpPAL4 and PpCHS were compromised in the Ppcerk1 mutant, further indicating that this PRR is involved in the perception of chitin. Two partially redundant PpMEKKs were identified, PpMEKK1a and PpMEKK1b. Ppmekk1a had reduced cell wall modifications upon chitin treatment, as also seen for Ppcerk1. While chitin induced cell wall modifications in Ppmekk1b was close to that in WT, analysis of the double KO of PpMEKK1a and PpMEKK1b showed that there is functional redundancy between the two homologs. Three P. patens MKKs, PpMKK1a, 1b and 1c which are homologs of AtMKK1 and 2, were also targeted by KOs. The three single PpMKK mutants showed reduced chitin cell wall modifications, but not to the same degree as Ppmekk1a and Ppcerk1. MPK phosphorylation upon chitin treatment in the MKK mutants was also slightly reduced compared to WT. Two P. patens MPKs, PpMPK4a and PpMPK4b, which are homologs to AtMPK4, were used for targeted KO and KI. PpMPK4 KI tagged GFP lines, MPK4a-GFP and MPK4b-GFP were activated upon chitin treatment. All these observations identify an MPK signaling pathway cascade in P. patens which responds to chitin which includes the PRR PpCERK1, PpMEKK1a/1b, PpMKK1a/1b/1c and the single MPKs, PpMPK4a and PpMPK4b (45). Bressendorf et al. 2016 did

25 not observe activation of this MPK pathway upon abiotic stress treatments. This indicates that the PpMPK4a and PpMPK4b pathway functions primarily in PTI in response to biotic stress by chitin.

Abiotic stress There are several reports showing MPK signaling pathways triggered by abiotic stressors such as, osmotic, salt, cold and drought stress and hormonal treatments in vascular plants like, A. thaliana, in yeast S. cerevisiae and in P. patens. P. patens has a high tolerance towards abiotic stress, thus making this organism a good model to understand and identify genes effecting such responses (99, 100).

Salinity and osmotic stress Salinity stress is primary due to ion toxicity in the plants. Stress occurs when plants are exposed to high soil concentration of NaCl, and can affect growth and development, and impact crop yield. A. thaliana is a salt-sensitive plant, since continued exposure to 50mM effects its. Of the cereals, O. sativa is the most sensitive, while barley (Hordeum vulgare) is the most tolerant (101). Moreover, P. patens protonemata and gametophores are tolerant to high levels of NaCl (500mM) and are able to survive and recover from the stress (99, 100). Response to high salinity involves the salinity overly sensitive 1 (SOS1) Na+/H+ antiporter that removes Na+ from the cells. The high-affinity potassium transporter (HKT) regulates intracellular K+/Na+ ratios, while Na+/H+ antiporter tonoplast (NHX1) antiporter maintains Na+ in the vacuole (71, 101, 102). A. thaliana, MEKK1, MKK2, and MPK3, 4 and 6 are activated upon elevated levels of NaCl and osmotic stress, and specifically MPK6 phosphorylates the SOS1 Na+/H+ antiporter (Figure 5) (2, 6, 71, 81, 103, 104). More specifically, during salinity and osmotic stress AtMPK6 phosphorylates the zinc finger transcription factor ZAT6 that results in seed germination during stress (105). P. patens has 3 SOS Na/H+ antiporters, SOS1-3. SOS2 and 3 interacts during salt stress and are required for sodium and potassium ion homeostasis in A. thaliana. In the presence of intracellular Ca2+ SOS3, a calcium binding protein activates the SOS2 serine-threonine protein kinase (106). In addition to the SOS transporters, fungi and bryophytes have a vacuolar Na+ efflux ATPase pump, called the ENA1 pump (107-109). P. patens has 3 ENA pumps (PpENA1-3), while S. cerevisiae has 4 (ScENA1-4) and the liwerworth, M. polymorpha has 2 (MpENA1-2) (108, 109). PpENA1 is expressed in the protonemata, mature stem, and in the central hydroids and the epidermal cell layer (109). The ENA1 pump has previously been described in S. cerevisiae, and is transcriptionally regulated in response to salt and glucose starvation (110). The regulation of ENA1 upon salt stress depends on a calcineurin pathway and the high-osmolarity glycerol (HOG) MPK pathway. Elevated sodium levels activates the

26 phosphoprotein phosphatase calcineurin, which leads to increased intracellular Ca2+ (101). Calcineurin also induces ENA1 by activation of the transcription factor Crz1/Hal8. The HOG MPK pathway is composed of Ssk2/22p-PBs2p and Hog1p MPKs. Hop1p activates the transcription factor bZIP (Sko1p) repressor that binds to the ENA1 promotor and inhibits the transcription of ENA1 by other corepressor complexes (110-112).

Figure 5: Signaling pathways involved in Na+ efflux in Arabidopsis under salt stress. Exogenous Na+ and high osmolarity are detected by unknown plasma membrane sensors that upregulates cytosolic Ca2+ concentrations. The elevated Ca2+concentration is sensed by the Ca2+-binding protein SOS3 that activates SOS2. The activated SOS2/SOS3 complex phosphorylates Na+/H+ antiporter SOS1 on the plasma membrane, resulting in increased Na+ efflux. High osmolarity initiates the accumulation of ABA, which indirectly regulates SOS1 (Modified from (6)).

Interestingly, the PpSOS1 Na+/H+ antiporter has been described to be active at low pH values, while PpENA1 Na+ ATPase is active at high pH values (113). The ability to pump Na+ out of P. patens cells by ENA1 has been utilized to increase biomass production and salt tolerance in O. sativa plants under high salinity conditions (102). As mentioned, high salinity will indirectly upregulate the intracellular Ca2+ concentration, but also triggers osmotic stress that will trigger the accumulation of ABA. Moreover, elevated levels of Ca2+ and thus upregulation of Ca2+ pumps are thought to be a way for plants to adapt to the stress. Ca2+ ATPase pumps are used by plants to restore ion homeostasis by activating signal transduction pathways like MPK signaling and maintaining low levels of 2+ 2+ cytoplasmic Ca (6, 65). For example, the moss PIIB-type Ca ATPase (PCA1) gene is activated by salt stress (65), and the regulation of cytoplasmic Ca2+ concentration is important during spore

27 germination to chloronemal filaments, protoplast division and bud formation that also is induced by cytokinin (65, 114). Phytohormones The development of plants and response to abiotic and biotic stress environment are controlled by phytohormones. SA, JA and ET are known to be involved in defense responses mediated by biotic stress. SA is involved in protection against biotrophic fungi, oomycetes and bacteria, while JA and ET signaling and accumulation are involved in defense against necrotrophic fungi (115). For example, AtMPK4 regulates SA and JA/ET mediated responses by the defense regulators enhanced disease susceptibility 1 protein (EDS1) and phytoalexin deficient 4 (PAD4) (115). Moreover, the MAP kinase kinase AtMKK3 is involved in the regulation of signaling stimulated by biotic stress that is dependent on the regulation of JA signaling (74). Furthermore, AtMPK3 and 6 phosphorylates the transcription factor WRKY33 that is involved in biotic stress by SA-, ET- and JA mediated responses (91-93). Yasumura et al. 2015, identified in P. patens the known A. thaliana regulator constitutive triple response1 (CTR1) that is involved in ET signaling. ET inactivates PpCTR1l, since the ET binding to ET receptors results in inactivation of the receptors, and thus inactivates PpCTR1L. This permits the activation of downstream signaling and thus activation of ethylene response factor (ERF) transcription factors or ethylene response element-binding proteins (EREBPs) (71, 116, 117). Interestingly, PpCTR1L also regulates signaling of abscisic acid (ABA) (116). This shows that evolution and adaptation of regulatory mechanisms in plants vary between genera making it problematic and interesting to understand hormonal regulation.

The following sections focus on studies of the phytohormones, ABA, auxin, cytokinin and strigolactone in development, abiotic stress and regulation in A. thaliana and P. patens.

Abscisic acid The phytohormone ABA is important for responses to abiotic stress. Under normal growth conditions, plants have low ABA levels but in response to environmental stress ABA levels increase (118-120). Studies of transgenic A. thaliana and O. sativa during drought stress described the induction of leaf senescence by downstream phosphorylation and upregulation of senescence-associated genes (SAGs) (121). PpMPKs are not linked to ABA synthesis, but exogenous ABA application induces morphological changes in P. patens. This includes the differentiation of cells into chains of brachycytes and brachycytes with tmema cells. Brachycytes can be reverted to germinate to protonemata when removed from ABA stress (21). The induction of brachycytes with and without tmema is thought to be due to expression of genes regulating cell wall enzymes that remodel cell

28 walls (21). ABA signaling in P. patens leads to expression of ABA-responsive genes (PpARs) upon freezing tolerance to protect the development of protonemata (99, 122). ABA induced phosphoproteins are shown to be MPK targets, and hence involved in ABA signaling. An MPK cascade in A. thaliana, MEKK17/MEKK18-MKK3-MPK1/2/7/14 is apparently activated downstream of ABA receptors. These receptors (pyrabactin resistance/pyrabactin resistance- like/regulatory component (PYR/PYL/RCARs) form signaling complexes that results in many downstream events. In the presence of ABA, the receptors inhibit Protein Phosphatases 2C (PP2Cs) activity, which results in the activation of Sucrose nonfermented 1 (SNF1)-related protein kinases (SnRKs) by auto-phosphorylation. SnRKs initiate responses by phosphorylation of transcription factors of ABA responsive genes, membrane proteins and ion channels (Figure 6) (2, 123).

Figure 6: ABA-signaling in plants. (A) The PYR/PYL/RCAR, ABA receptor binds ABA and inhibits the negative regulator PP2C. This stops dephosphorylation of SnRK2, which is activated by auto-phosphorylation resulting in ABA-response via activation of transcription factors (TF). (B) Low levels of ABA or no ABA (Adopted from (2)).

PP2Cs act as a negative regulator of ABA signaling and in P. patens, PP2C-mediated ABA signaling is involved in the formation of brachycytes formation. Moreover, PP2Cs regulate the development of archegonia, since transgenic P. patens overexpressing abi1-1 (ABI1-related type 2C protein phosphatases (PP2Cs)) generated longer gametophytes, extensive growth of archegonia with multiple sporophytes per apex. However, these morphological changes in the transgene line could be due to the water irrigation and low temperature during sporophyte induction, and thus the plants might be less tolerant to dryness (124). Furthermore, PpABI1A (PP2C) is important during the elongation of protonemata and gametophytes during normal growth conditions, although it is not involved in developmental regulation (125). In the liwerwort, M. polymorpha Tougane et al. (2010) characterized

29 an ABI1-related type 2C protein phosphatase as a negative regulator of ABA signaling as shown in P. patens and A. thaliana (126). Bressendorf et al. (2016), showed that PpSnRK2a is phosphorylated during osmotic stress and ABA, indicating that PpSnRK2a functions in ABA signaling. Moreover, in response to ABA and hyperosmotic stress PpSnRK2a activity is dependent on PpARK (ABA and abiotic stress- responsive Raf-like kinase) (127). In higher plants ABA signaling is involved in many morphological responses, such as seed and seedling development, closure of stomata and during drought and salt stress. Salt and drought stress affects the opening and closure of stomata regulated by ABA (120). Stomatal ABA responses in the A. thaliana ABA-regulatory protein kinase Open Stomata 1 (OST1)- SnRK mutant (128) were rescued by the PpOST1-1 homolog. This suggests that stomatal aperture have evolved more than 400 million years ago. The PpOST1-1 KO mutant had reduced response of stomatal aperture in sporophytes upon ABA stress (Figure 7). Stomata in P. patens are only present in the base of the sporophyte. Phosphorylated AtOST1 phosphorylates the Slow Anion Channel- associated 1 (SLAC1), that triggers membrane depolarization and is required for stomatal closure (Figure 7) (1, 2, 129).

Figure 7: ABA-signaling in stomatal aperture in A. thaliana, illustrating the role of AtOST1 (Open Stomata 1 (OST1))-SnRK2. The PY/PYL/RCAR, ABA receptor binds ABA and then inhibits the negative regulator PP2C. This will stop dephosphorylation of OST1 (SnRK2), which is activated by auto-phoshorylation resulting in ABA- induced stomatal closure by SLAC1 that triggers membrane depolarization required for stomatal closure (Modified from (1, 2)).

AtMPK9 and 12 are highly expressed in guard cells, and the mpk9-1/mpk12-1 double mutant exhibits enhanced water loss and an ABA insensitive stomatal response. The two MPKs act upstream of anion channels like SLAC1 in guard cells during ABA signaling. Moreover, the MPKs positively regulates ABA signaling downstream of ROS production (Figure 8) (2, 12). Expression of catalase (CAT) is induced during ABA stress and is important for stress adaptation. Furthermore, increased levels of

ABA induce cellular H2O2 controlled by catalase. AtMKK1 was found to mediate the expression of

CAT1 in response to H2O2 signaling during ABA. Interestingly, overexpression of AtMKK1 and

AtMPK6 enhanced expression of CAT1 and H2O2 production (71, 104, 123). Interestingly, AtMKK1 and AtMPK6 signaling has also been shown to influence seed germination during ABA-induced ROS production, since overexpression lines were hypersensitive to ABA and glucose during germination (104).

Figure 8: ABA-signaling in guard cells in A. thaliana. MPK9 and 12 are involved in ABA induced stomatal closure in guard cells. Activated OST1 (SnRK2) phosphorylates the NADPH oxidase RbohF resulting in ROS accumulation. This activates MPK9 and 12 that positively regulate ABA induced stomatal closure. The MPKs activate the SLAC1 anion channel, resulting in membrane depolarization required for stomatal closure. (Modified from (2, 12)).

Auxin & cytokinin In vascular plants like A. thaliana, auxin was shown to be an essential phytohormon regulating development in roots, shoots and leaves in both young and adult plants, lateral organ formation and embryogenesis (10, 130). A. thaliana MPK12 was shown to be activated in vivo by auxin, and to act as a negative regulator in auxin signaling since knock-down of MPK12 resulted in induction of auxin responsive gene expression. Moreover, the MPK phosphatase indole-3-butyric acid-response 5 (IBR5) was characterized as a positive regulator of auxin signaling by inactivating AtMPK12 by dephosphorylation (71, 130). Kovtun et al. (1999) elucidated the role of MEKKs, ANPs (Arabidopsis,

Nicotiana protein kinase 1 (NPK)-like Protein kinase) and their induction by H2O2 during oxidative stress. ANP1 further activates AtMPK3 and 6 which, somehow activate stress-responsive genes and repress expression from auxin-inducible promotors. The authors proposed that during osmotic stress the activation of AtMPK3 and 6 by ANP1 might enable plants to shift energy from auxin activities to ABA regulated stress responses (131). In-silico studies and yeast two-hybrid assays revealed that O. sativa MPK3, 4 and 6 target O. sativa Auxin resistant 1/like auxin (AUX1/LAX1) protein. The study suggested that OsMPK3/4/6 are directly involved in auxin signaling by physically interacting with OsAUX/LAX1 protein (132).

Figure 9: Model of auxin signaling. In low auxin, AUX/IAA binds to the auxin response factor (ARF) and represses transcription of auxin responses. Auxin acts as a molecular “glue” between the receptor TIR1 and the AUX/IAA repressor. When the TIR1-Auxin-AUX/IAA complex is formed, the ubiquitin ligase SCF binds to the complex and the ubiquitinated AUX/IAA is degraded. In high auxin, ARF is free to transcriptionally activate auxin responses (Modified from (10, 11)).

The synthesis, transport and catabolism of auxin are regulated at the transcriptional and post- transcriptional levels. Auxin/indole acetic acid (AUX/IAA) are repressor proteins that interact with the transcriptional co-repressor TOPLESS (TPL). They are transcriptionally activated by auxin via the auxin response factors (ARFs). AUX/IAA are ubiquitinated by the protein ligase (Skp1-Cull- Fbox) SCFTIR1 complex followed by proteolytic degradation, and thereby release the activation of ARFs from TPL repression. The activation of ARFs results in the activation of auxin responsive genes. Auxin binds to the F-box protein TIR1 (Transport inhibitor response 1) auxin receptor, and forms a complex with AUX/IAA that interacts with the ubiquitin ligase SCF (Figure 9) (10, 133, 134). During ETI in A. thaliana stimulated by the bacteria Pseudomonas syringae, the effector AvrRpt2 has been shown to stimulate auxin responses by inducing the turnover of the negative auxin- signaling regulator AUX/IAA. Hence, P. syringae promotes pathogenicity by AvrRpt2-stimulated auxin signaling, degradation and changing auxin physiology in A. thaliana (11, 135). There are as yet no studies of MPKs in P. patens that are activated and phosphorylated in response to auxin or cytokinin, although there are many studies of the influence of auxin and cytokinin on the development of the moss. In P. patens, auxin regulates the transition from chloronema to caulomena, and initiates tip growth and development of rhizoids. Application of IAA accelerates the transition from chloronema to caulonema filaments, stem elongation and development of rhizoids from leafy shoots (136-139). Cytokinin regulates the development of secondary branching from chloronemal and caulonemal filaments. Auxin and cytokinin regulate the formation of buds, which is the developmental stage before leafy gametophytes (21, 22, 140). Treatment with 1µM of the cytokinin analog 6-benzyl aminopurine (BPA) increases bud formation, although the buds do not develop into gametophytes. Moreover, high concentrations of applied cytokinin result in callus-like growth of buds. In contrast, treatment with 1µM of the auxin analog 1-naphthalene acetic acid (NAA) results in buds without gametophore axes and with abundant rhizoids (24, 140). Studies of WT spores treated with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine (NTG) on different concentrations of cytokinin and auxin were conducted in 1979 by Ashton et al. (140). Mutants were categorized by their sensitivity toward applied cytokinin and auxin during the development of gametophytes. Category 2 mutants were resistant to NAA and named NAR (NAA resistant) mutants. Interestingly, when treated with exogenous cytokinin, NAR mutants restored the gametophytic phenotype to WT morphology, suggesting that these mutants were defective in cytokinin production (137, 140). Prigge et al. (2010), provided evidence that the Arabidopsis TIR1 homologs PpAFB (auxin signaling F-box) mediate auxin signaling in chloronemal cells. Moreover, mutants resistant to BPA called BAR (BPA

33 resistant) mutants, were rescued by exogenous auxin and defined as defective in auxin biosynthesis (140). These studies concluded that P. patens required both auxin and cytokinin for normal growth morphology. Three cytokinin receptors (CRE1/AHK4, AHK2, and AHK3) were identified in Arabidopsis which transduce signals depending on the concentration of the hormone (9, 141-144). Kakimoto et al. (1996), provided evidence that overexpressing the CKI1 receptor histidine kinase gene in Arabidopsis, induced cytokinin responses. Although, the function of CKI1 as a cytokinin receptor has not been fully clarified, it is thought to be a cytokinin sensor. However, this study initiated the characterization of cytokinin receptors: CRE1 (cytokinin response 1)/AHK4 (Arabidopsis histidine kinase 4) and two homologs AHK2 and AHK3 (145). Moreover, components of the downstream signaling cascade were also identified as AHPs (Arabidopsis histidine phosphotransfer proteins) and ARRs (Arabidopsis response regulators) (9).

Figure 10: Model of signaling during cytokinin signal transduction via His-to-Asp phosphorelay. Cytokinin binding to the receptors (CRE1/AHK4, AHK2 and AHK3) induces receptor dimerization and autophosphorylation. The phosphoryl group is transferred to HPTs (AHPs) that transduce the signal from cytoplasm to type-B RRs (RRBs) in the nucleus. RRBs transcribe target genes, type-A RRs (RRAs/ARRs) resulting in downregulation of the cytokinin signaling response via a negative feedback loop and module downstream activities of cytokinin. D, aspartate residue, H, histidine residue, P, phosphoryl group, bent arrow = negative feedback loop. (Modified from (9)).

Cytokinin is bound by the ER-localized, histidine kinase receptor via the cyclases/histidine kinases associated with sensory extracellular (CHASE) domain. The binding of cytokinin results in a two- component system through a His-to-Asp phosphorelay signaling cascade. Autophosphorylation of CHK, results in phosphorylation of histidine phosphotransfer proteins (HPTs) that shuttle between the cytoplasm and nucleus. HPTs (AHPs) initiate downstream signaling by activation of Myb class transcription factors, e.g. type-B response regulators (RRBs). Furthermore, the transcription factors activate the transcription of target genes, e.g. type-A response regulators (RRAs) (ARRs) proteins with a phosphor-acceptor receiver domain, resulting in negative feedback of cytokinin signaling (Figure 10) (9, 142, 143). Recently, three P. patens CHASE domain-containing histidine kinase (CHK) cytokinin receptors have been characterized. Single, double and triple mutants of CHK1, 2 and 3 were characterized. chk1 and 2 mutants had reduced colony size and fewer peripheral filaments, and the size of the gametophores in chk2 and 3 were reduced. Moreover, the double chk1/chk2 mutant had significantly smaller gametophores than the WT and the single mutants, suggesting that CHK1 and 2 are necessary for the development of gametophores. Interestingly, the triple chk1/chk2/chk3 mutant displayed significantly fewer gametophores consisting mainly of protonemata, and treatments with the cytokinin benzyladenine (BA) resulted in cytokinin insensitivity. Taken together, the CHK1, 2 and 3 receptors are necessary for cytokinin perception in P. patens. Moreover, the receptors are necessary during bud formation, development of gametophores and of the reproductive antheridia and archegonia (142).

Strigolactone Strigolactone controls the extension of filaments in the development of P. patens gametophytes and rhizoids (146, 147). Furthermore, strigolactones controls shoot branching and signaling during symbiotic and parasitic interactions (146, 147). In vascular plants, strigolactones positively regulate secondary growth, leaf senescence, root hair elongation and primary root growth, while they negatively regulate axillary bud growth and adventitious root formation. Strigolactones negatively regulate the development of P. patens protonemta including, chloronemata branching and colony extension (Figure 11) (8).

Figure 11: Strigolactones control many developmental features in plants. In vascular plants, strigolactones positively regulate secondary growth (A), leaf senescence (B), root hair elongation (C) and primary root growth (D), while they negatively regulate axillary bud growth (E) and adventitious root formation (F). In P. patens strigolactones negatively regulate the developmemt of protonemta including, chloronemata branching (G) and colony extension (H). Red arrows indicate positive effects. T-bars in blue illustrate negative effects. SLs = strigolactones (8).

The strigolactone biosynthesis pathway is characterized in vascular plants (148). Moss carotenoid cleavage dioxygenases 7 and 8 (CCD7 and 8) cleave carotenoid substrates in plastids that result in the formation of strigolactones. PpCCD8 is involved in the regulation of branching of filaments related to the inhibition of shoot branching by strigolactones in the moss (146). In Arabidopsis, the more axillary growth (max) mutant was characterized as having an excessive shoot branching phenotype. The Max3 and 4 genes were shown to encode CCD7 and CC8, respectively, suggesting that they inhibit shoot branching. Furthermore, MAX1, 3 and 4 were shown to participate in the biosynthesis of carotenoid-derived signal and further biosynthesis of strigolactones. MAX1 is a cytochrome P450 that degrades carotenoid cleaved product to strigolactone(s). MAX2 is an F-box

36 protein, as is the TIR1 protein in auxin perception and signaling transduction, and is thus predicted to be a component of the SCF complex involved in regulated proteolysis. The strigolactone receptor protein D14 (dwarf14) is a α/β hydrolase involved in the perception of strigolactone (Figure 12) (7, 8, 147). Interestingly, algae, moss and liwerworts lack the D14 receptor protein, and recently possible strigolactone receptors in P. patens have been characterized as the D14 related PpKAI2 (karrikin insentistive2) receptor. Lopez-Obando et al. (2016) studied PpKai2 genes and their role in strigolactone perception, and showed that GR24 induces transcriptional responses of the genes, and thus PpKAI2s may be possible strigolactone receptors (149).

Figure 12: Model of the biosynthesis and signaling/perception of strigolactones in Arabidopsis. Strigolactones are carotenoid derivatives cleaved by CCD7 (AtMAX3) and CCD8 (MAX4) in plastids. In the cytosol, the carotenoid cleavage product is further processes by a cytochrome P450 (AtMAX1) to strigolactones. Signaling and perception is initiated by the receptor α/β hydrolase (D14) and further regulated by an F-box protein (AtMAX2), resulting in initiation of plant responses, such as branching inhibition (Modified from (7)).

Proust et al. (2011), discovered the strigolactone biosynthesis gene, PpCCD8. Knockouts of Ppccd8 showed early spore germination and excessive chloronemata branching. Interestingly, Ppccd8 showed continuous extension of the colony and the phenotype was reverted by application of the strigolactone analog GR24. Furthermore, strigolactones in P. patens appear to function in signaling resembling bacterial quorum-sensing. This is because, in a cross-feeding experiment the mutant colony gained a WT phenotype with decreased colony diameter when surrounded by WT plants (146). Other hormones, like auxins and cytokinins in P. patens are released into the medium to control the

37 development of protonemata, and Proust et al. (2011) provided evidence that strigolactones also are released in the medium. Furthermore, PpCCD8 and PpCCD7 were found to be expressed in differentiated buds and at the base of the gametophores (146).

Light Besides phytohormones and innate immunity to adapt to various environmental changes/threats, plants have evolved photosensory pigments to respond to different light wavelengths and intensities. These includes red/far-red reversible phytochromes and blue-light absorbing phototropins and cryptochrome. Arabidopsis has five phytochromes, two cryptochromes and two phototropins (150- 153). Phytochromes control developmental processes such as flowering time and inhibition of hypocotyl elongation (152, 154). Phototropins (PHOTA1, PHOTA2, PHOTB1 and PHOTB2) in P. patens mediate chloroplast movements during blue and red light, in contrast PHOT1 and PHOT2 in Arabidopsis which mediates chloroplast movement only by blue light (154, 155). The Arabidopsis cryptochromes CRY1 and CRY2 are involved in the inhibition of hypocotyl elongation and the regulation of flowering time (150, 152, 156). The two cryptochromes in P. patens, PpCRY1a and PpCRY1b have been implicated in many developmental processes such as induction of side branching and position, and the induction and development of gametophores (150, 157). cry1a, cry1b and cry1a-cry1b mutants had a higher ratio of caulonemal filaments, and were inhibited in the transition from chloronemal to caulonemal filaments. The transition from one filament to the other is regulated by auxin, suggesting that the deletion of CRY altered auxin responses, including the expression of auxin-inducible genes (150). In Arabidopsis, the response regulator 4 (ARR4) which, activates auxin gene responses interacts with the far-red absorbing Pfr form of phytochrome B (PhyB). Overexpression of ARR4 resulted in hypersensitivity to red light in hypocotyls, and inhibited the conversion of PhyB from active to inactive form (158). Three years later, To et al. (2004) showed that ARR3-6 are involved in red light responses (159). ARRs have been suggested to be involved in a cytokinin-signaling pathway that is interrupted during phytochrome-mediated signaling. Su & Howell (1995) studied cytokinin responses in Arabidopsis light insensitive long hypocotyl (hy) mutants. The hy3-1 (phyB-1) mutant was insensitive to cytokinin, since the hormone did not impact the elongation of hypocotyls in seedlings. The group provided evidence that cytokinin and light interact to inhibit hypocotyl elongation in seedlings in an indirect fashion, since the cytokinin response in the roots of the mutant was as the WT (160).

Beside auxin and cytokinin, strigolactones are also implicated in photomorphogenesis in Arabidopsis. As mentioned, the F-protein AtMAX2 is part of the signaling and perception pathway downstream of strigolactones. In 2007, a mutant in Arabidopsis was isolated from a genetic screen under red light as pleiotropic photo-signaling (pps). The PPS gene encodes MAX2. Under red, far- red and blue light, pps plants had longer hypocotyls, and smaller cotyledons compared to WT. Moreover, during red and far-red light induced seed germination, the pps were hyposensitive. From these observations MAX2 has been suggested to be involved in red, far-red and blue light signaling pathways, and thus to promote photomorphogenesis (161). In 2012 the same group identified MAX2 with SCF potentially target substrates in response to light in multiple hormonal signaling pathways at different developmental stages. Seeds of max2 mutants were hyposensitive to gibberellic acid (GA) and hypersensitive to ABA in seed germination in response to light. mRNA levels of ABA biosynthetic and catabolic genes were upregulated in max2 seeds, while biosynthesis genes in the GA pathway were downregulated and catabolic genes were upregulated. Moreover, treatment with an auxin transport inhibitor resulted in long hypocotyls in max2 seedlings under light. Since MAX2 regulates downstream signaling in the strigolactone pathway, the group examined the upstream max1, 3 and 4 mutants. Interestingly, max1, 3 and 4 were not inhibited in seed germination and growth of seedling in response to light. Hence, the light-signaling phenotypes seen in max2 result from defective regulation of hormone biosynthetic, signaling and transport pathways. Thus, MAX2/PPS may act as a positive regulator of photomorphogenesis in Arabidopsis (162). Moreover, Tsuchiya et al. (2010) elucidated the role of strigolactone in the absence of red, far-red and blue light photoreceptors in Arabidopsis. Exogenously applied GR24 did not inhibit the growth of seedling hypocotyls in the elongated hypocotyl 5 (hy5) and in max2 mutants. HY5 is a transcription factor that is degraded in the nucleus during dark by the negative regulator of light signaling, COP1 (constitutive photomorphogenic 1), resulting in reduced responses towards light. Interestingly, exogenous GR24 inhibited the nuclear localization of COP1, followed by a greater HY5 accumulation in the nucleus, which leads to elevated light signaling responses (163).

Results

Identification of Physcomitrella patens homologs of MPKs in Arabidopsis thaliana A reverse BLASTp search against the A. thaliana MPK genes (AtMPKs) with the highest P. patens hits (PpMPKs) was used to showed phylogenetic relationships between the 20 MPKs from A. thaliana and the 8 MPKs from P. patens (Figure 13). Two of the 8 P. patens MPKs with closest homology to AtMPK4 were initially called PpMPK4a and PpMPK4b and characterized as part of a MAP kinase cascade regulating immunity (45).

Figure 13: Phylogenetic relationship of 8 MPK homologs in Physcomitrella patens and the 20 Arabidopsis thaliana MPKs. Human ERK1 is included as an outgroup. Pp=Physcomitrella patens, At=Arabidopsis thaliana, Hs=Homo sapiens. A remaining, daunting task was the characterization of the remaining 6 MPKs in P. patens. From the phylogenetic relationship and multiple alignment of the proteins, we noted that MPK2/MPK5, RAK1/RAK2 and MPK3/MPK7, like MPK4a&b, exhibit highly sequence similarity (Table 1). These pairs of MPKs were characterized as paralogs and could be redundant. Further analysis of the multiple alignment of the proteins (Supplemental Figure 1), showed that RAK1/RAK2 and MPK2/MPK5 differ in both the N-terminal and C-terminal regions. MPK2/MPK5 paralogs have a distinctive domain in their C-terminal regions, and contain a Thr-Glu-Tyr (TEY) motif in the N-terminal region (Supplemental Figure 1, TEY motif in green). This TEY motif is not part of the regulatory loop as

40 are other TEY motifs in the other MPK proteins (Supplemental Figure 1, TEY motif in yellow). MPK2/MPK5 also have a Thr-Asp-Tyr (TDY) motif in their regulatory loop regions (Supplemental Figure 1, TDY motif in red). A reverse BLASTp search against AtMPKs, showed that AtMPK9, AtMPK15 and AtMPK16 also have a TEY motif in their N-terminal regions and a TDY motif in the activation loop (Supplemental Figure 1). Moreover, when searching in the Phosphat database (www.phosphat.uni-hohenheim.de/), no phosphorylations were found on N-terminal TEY peptides in AtMPKs. This suggests that the TEY motif in the N-terminal regions either has not been detected or does not occur.

MPKs Size (kDa) ID MPK2 60,85 Pp1s80_71V6.1 MPK5 61,04 Pp1s87_157V6.1 RAK1 78,03 Pp1s29_285V6.1 RAK2 73,75 Pp1s99_26V6.1 MPK3 42,37 Pp1s207_63V6.1 MPK7 42,49 Pp1s138_117V6.1 MPK4A 42,84 Pp1s149_39V6.1 MPK4B 43,49 Pp1s59_325V6.1 Table 1: 8 MPK homologs in Physcomitrella patens with their protein size and ID from Phytozome 12 (https://phytozome.jgi.doe.gov/pz/portal.html).

The most striking discovery was the separate N-terminal sequence of approximately 30kDa in RAK1/RAK2. Using BLASTp in www.phytozome.com, the sequence was identified as an N- terminal acetyltransferase (NAT). The NATH (N-terminal AcetylTransferase Homolog) (Table 2) was by sequence similarity identified in baker’s yeast Saccharomyces cerevisiae, bogmoss Sphagnum fallax, A. thaliana and Homo sapiens (Supplemental Figure 2).

NAT Size (kDa) ID NATH 28,95 Pp3c17_14350V3.1 Table 2: NATD homolog (NATH) in Physcomitrella patens with protein size and ID from Phytozome 12 (https://phytozome.jgi.doe.gov/pz/portal.html).

There are no sequence similarity of RAK1 and RAK2 in other embryophytes like Marchantia polymorpha and S. fallax. This indicates a fascinating identification of a novel rosetta kinase that provides links between protein phosphorylation and acetylation, hence the name RAK (Rosetta NAT Kinase). At this time there are no characterizations and publications on the identified 4 MPKs and the 2 RAKs in P. patens.

Generation of MPK knockout mutants We generated single and double knockouts (KO) mutants and knockin (KI) versions with GFP-tag by homologous recombination using targeted gene disruption to understand the function and role of these MPKs. The single KOs were generate in P. patens var. Gransden wild type by transforming the specific MPK deletion constructs, and the rak1-rak2 double KO was generated by introducing the RAK2 KO construct into the rak1 background. The deletion construct was generated by cloning 900- 1500bp of genomic flanking regions of the target gene into left side (left border, LB) and right side (right border, RB) of the selection marker in the vector. The vector was transformed into protoplast of P. patens by a PEG and heat shock method (29). The transformation vectors used have USER containing cassettes, thus USER cloning were performed under the construction of all the deletion vectors (164).

Knockout MPKs ID Plasmid name Selection MPK2 Pp1s80_71V6.1 pMBLU-MPK2 Hygromycin/G418 MPK5 Pp1s87_157V6.1 pMBLU-MPK5 G418 RAK1 Pp1s29_285V6.1 pMBLU-RAK1 G418 RAK2 Pp1s99_26V6.1 pMBLU-RAK2 Hygromycin/G418 MPK3 Pp1s207_63V6.1 pMBLU-MPK3 G418 MPK7 Pp1s138_117V6.1 pMBLU-MPK7 Hygromycin/G418 NAT ID NATH Pp3c17_14350V3.1 pMBLU-NATH Hygromycin Knockin RAK1 Pp1s29_285V6.1 pMBLU-GFP-RAK1 G418 RAK2 Pp1s99_26V6.1 pMBLU-GFP-RAK2 G418 MPK3 Pp1s207_63V6.1 pMBLU-GFP-MPK3 G418 MPK5 Pp1s87_157V6.1 pMBLU-GFP-MPK5 G418 NATH Pp3c17_14350V3.1 pMBLU-GFP-NATH G418 Table 3: Generation of plasmids with cloned flanking regions of MPKs and NATH and their ID from Phytozome 12. Plasmids that have failed to generate KOs despite several attempts are designated in red. Flanking regions of MPK2, RAK2 and MPK7 were cloned in two plasmids with to different selection markers (Hygromycin and G418).

The cloning of the flanking regions in the USER compatible vector is a four fragment cloning method such that it was possible to clone in one step the LB and RB onto the selection marker and the plasmid backbone. The KI constructs with GFP tag were cloned in the pMBLU vector containing GFP. The resulting 12 plasmids are listed in Table 3 with name, size and ID# of the corresponding target gene. After the protoplast transformation, the protoplasts underwent two antibiotic selection- and one nonselective rounds. The surviving colonies and possible KO’s were initial genotyped by PCR using specific primers in the gene (primers, p3 and p4, Figure 14) to verify whether a gene disruption had

42 occurred. After verifying gene disruption, colonies were propagated on new plates for a new DNA extraction. Primers for the external part of the LB and RB with outward oriented primers that specifically targets the selectable marker cassette (Figure 14, Supplemental Figure 3) were used to genotype the borders for a correct integration of the borders in the KOs.

Figure 14: Overview of a WT locus of a target gene and the expected KO locus after homologous recombinant integration of a single selection marker (nptII). The primers used for genotyping are indicated P1-P8. LB – Left Boarder and RB – Right Boarder.

For the initial genotyping of KIs, primers targeted in the GFP tag and in the target gene (LB) were used to verify the integration of the tag. The colonies were propagated on new plates for a new DNA extraction and further genotyped for the LB and RB (Figure 15, Supplemental Figure 3). Western Blotting verified the expression of the GFP with antibody against GFP (anti-GFP) (Supplemental Figure 4).

Figure 15: Overview of a WT locus of a target gene and the expected KI locus after homologous recombinant integration of a single selection marker (nptII). The primers used for genotyping are indicated P11-P14. LB – Left Boarder and RB – Right Boarder.

The generated lines are listed in Table 4. Unfortunately, despite numerous attempts, it was not possible to generate KO lines of MPK2, MPK7 and RAK2, the respective paralogs of MPK5, MPK3 and RAK1. We transformed two different vector constructs for gene deletion with the selection markers for G418 and Hygromycin to have a greater chance of generating the KOs of MPK2, MPK7 and RAK2 (Table 3). The struggle of generating these KOs suggests that MPK2, MPK7 and RAK2 are essential genes and that their disruption could be lethal. Simplistically, since A. thaliana has 20

MPKs while and P. patens only has 8 MPKs, it is possible that the individual MPKs in the moss have multiple functions and that a gene deletion might well be lethal.

Gene KO Selection mpk5 2 G418 rak1 5 G418 mpk3 3 G418 rak1/rak2 2 G418-Hygromycin nath 2 Hygromycin Gene KI Selection MPK5+GFP 3 G418 RAK1+GFP 4 G418 MPK3+GFP 1 G418 RAK2+GFP 4 G418 NATH+GFP 3 G418 Table 4: Generation of KO and KI lines. Generated 2 mpk5, 5 rak1, 3 mpk3, 2 rak1-rak2 and 2 nath KO lines. Generated 3 MPK5-GFP, 4 RAK1-GFP, 1 MPK3-GFP, 4 RAK2-GFP and 4 NATH-GFP reporter KI lines. All KI lines have the G418 selection marker likewise for mpk5, rak1 and mpk3, while nath and rak1-rak2 have hygromycin selection marker.

Phenotypic analysis of generated mutants

Sporophyte induction Physcomitrella patens has different morphologic characteristics during the stages in its life cycle (Supplemental Figure 5). The mutant rak1, mpk3 and mpk5 lines were phenotypically compared during the development of female (archegonia) and male (antheridia) reproductive organs (Figure 16). Under sporophyte induction conditions, WT and rak1, mpk3 and mpk5 are all able to generate reproductive organs (Figure 16I-L). mpk5 has the most striking phenotype since the organ bundle, with both reproductive organs, seems to emerge higher up at the apex of the leafy gametophore. Figure 16H and L show the gametophore apex of mpk5, which has not been dissected. Normally organ bundles are “hidden” under leaves and needs to be removed to reveal the organ bundle. The gametophore of WT, rak1 and mpk3 in Figure 16A-C have all been dissected to reveal the organ bundles (Figure 16E-I, F-J & G-K). All lines have generated archegonia (I-L, black arrows). It seems that all the lines contain tube-like archegonial structures with a long canal with either open or closed opening. Some of the archegonial canals (Figure 16I-K, black arrows) are dark brown, indicating that spermatozoids have fertilized the eggs after irrigation of the colonies.

Antheridia appear as round bundles with shinny surfaces localized around the archegonia (Figure 16, white arrows, and Supplemental Figure 5, arrowheads). The antheridia in WT, rak1 and mpk3 appear shinny and round, in contrary to mpk5, which seems to have some abnormal structured antheridia (Figure 16L, red arrows). Comparing the archegonia of mpk5 to the antheridia-like organs, it seems clear that the antheridia are not round but have a form resembling a pyramid. These observations suggest that MPK5 may be involved in the development and structure of antheridia.

Figure 16: Sporophyte induction of 6 week-old WT (A, E, I), rak1 (B, F, J), mpk3 (B, G, K) and mpk5 (D, H, L) plants. After 6 weeks, prior to the first irrigation the canal of the archegonia was dark brown, indicative of spermatozoids have fertilized the eggs (I & J, black arrows). Antheridia appear as round bundles with shinny surfaces localized around the archegonia (I & J, white arrows). Antheridia of mpk5 have abnormal structure, resembling a pyramid (L, red arrows). Bar = 2 mm.

The sporophyte induction conditions that mimic seasonal change induce altered morphology of the adult gametophores (Figure 16A-D) and colonies (Supplemental Figure 6). Gametophores of rak1 and mpk5 on the top and bottom of the colonies show early and delayed senescence, respectively (Figure 17A, Supplemental Figure 6). Central leafy gametophytes of rak1 clearly undergoes chlorosis (Figure 17B; Supplemental Figure 6B) and the bottom of mpk5 colony shows that the rhizoids are green compared to WT, rak1 and mpk3 (Figure 17A; Supplemental Figure 6H). The length of rhizoids from both central and peripheral gametophytes (Figure 17C) of mpk5 are significantly shorter than WT, rak1 and mpk3, indicating that deletion of MPK5 affects the growth of P. patens under sporophyte induction conditions. MPK5 also has a significant impact on the growth of the

45 gametophytes, since the length of mpk5 gametophytes from periphery and center of the colony are significant longer than in the other lines (Figure 17D).

Figure 17: Sporophyte induction after 6 week under sporophyte induction conditions. (A) Colonies of WT, rak1, mpk3 and mpk5. (B) Central and periphery gametophytes of 6 weeks-old colonies grown under sporophytes induction conditions. (C) Length of the rhizoids and (D) gametophytes of the central and periphery gametophytes. Analysis of variance by T-test determined statistical differences indicated by A-C (central gametophytes) & a-c (periphery gametophytes) (P <0.05), n = 16. Standard deviation as error bars.

After 6 weeks after fertilization, the zygote develops into the diploid sporophore and later sporangium. The sporangium undergoes different maturation steps indicated by the coloration of the spore capsules (Figure 18) (165). To phenotypically analyze the maturation of the spore capsules, they were grouped as followed: pre-capsule, early green stage, late green/yellow stage, yellow/orange stage, red stage (matured) and brown stage. As mentioned earlier, all the lines were able to generate both reproductive organs, but with some abnormalities of the antheridia in mpk5. WT, rak1 and mpk3 generated matured (red) sporophytes, and no matured sporophytes were found in mpk5 (Figure 18). This could be due to the abnormal pyramid form of antheridia and the growth and excess branching of the leafy gametophytes (Supplemental Figure 7). The pre-capsule stage is characterized as a cone shaped capsule with light brown coloration, and the upper part of the canal of archegonia is visible. This maturation stage is present in all lines (Figure 18, “precapsule”). WT, rak1 and mpk3 generated capsules of the early green stage when the capsules are light green and the upper part of the archegonia has fallen off. At this stage the capsules develop a pointy top and the seta (the neck of the sporangium) has a dark coloration (Figure 18, “early green stage”). The early green stage is not easy to characterize in mpk5. In Figure 18,

46 designated with white arrows in mpk5, it appears that there are three spore capsules at different maturation stages. One capsule could be in the early green stage (lower white arrow, number 1), but there is no indication of a dark colored seta. Likewise, no dark colored seta is visible for the capsule designated with number 2. However, capsule number 2 has a clear yellow coloration, showing that the spores in the spore sacks are undergoing maturation. Multiple fertilization of eggs in one organ bundle is quite normal and is highlighted in Figure 18 with yellow arrows. The yellow/orange stage was challenging to characterize due to the phenotypes of rak1 and mpk5 (Figure 18, red arrows). No clear orange coloration of the capsules was visible in rak1 and mpk5, but they appeared to have odd cone-like capsules with early green coloration and dark red coloration in the middle of the capsules. The red colorations highlight the spores in the spore sacks and their maturation stage, like in the red stage (matured capsule). rak1, mpk3 and mpk5 all generated less sporophytes then WT. rak1 generated most spore capsules in the early green stage, which could lead to maturation of more capsules than the WT (Supplemental Figure 8). This was not the case however, since senescence occurred shortly after a couple of weeks in rak1 (Supplemental Figure 6, Figure 17). mpk3 generated more matured capsules than WT, this may be due to the ability to undergo multiple fertilization that were seen in pre-capsule, late green/yellow and yellow/orange stages (Figure 18, yellow arrows, Supplemental Figure 8). mpk5 only generated capsules with maturation stages early green and late green/yellow (Supplemental Figure 8). The gene disruptions of RAK1, MPK3 and MPK5 show clear impacts on the generation of sporophytes, since less are generated compared to WT.

Figure 18: Sporophyte induction of 6 week-old WT, rak1, mpk3 and mpk5 plants. All lines are able to generate organ bundles. WT and rak1 plants were able to generate sporophyte capsules in the different maturation steps; pre-capsule, early green, late green/yellow, yellow/orange, red (matured) and brown stages. rak1 and mpk5 sporophyte capsules at the yellow/orange stage shows abnormal red coloration in the capsule, indicating the spores are matured (red arrows). mpk5 generated sporophyte capsules in maturation steps: pre-capsule, early green and yellow/orange. No matured sporophytes (red stage), were detected in mpk5. WT, mpk3 and mpk5 are able to generate multiple sporophytes in one organ bundle (yellow arrows). mpk5 were able to generate 3 sporophytes with 3 different maturation stages: early green, pre-capsule and late green/yellow stage (white arrows, 1, 2 and 3). Bar = 2mm.

Interestingly, 3 weeks after the first irrigation of the colonies rak1-rak2 and nath developed sporophyte capsules at the yellow/orange maturation stages (Figure 19B-C, black arrows). This capsule development and maturation was approximately 3 weeks earlier than the capsule development in WT. At this time point, rak1, mpk3 and mpk5 had all developed the organ bundle (data not shown), as also seen in the WT (Figure 19A1-3). The canal of the archegonia in the WT seems to be dark, indicative of egg fertilization (Figure 19A2-3, dark arrows), and the antheridia are visible underneath the archegonia (Figure 19A2-3, yellow arrows).

Figure 19: Organ bundles and sporophyte formation in WT (A), nath (B) and rak1-rak2 (C) 3 weeks after the first irrigation of the colonies. WT generated organ bundles with female (archegonia) and male (antheridia) reproductive organs (A1-3, black & yellow arrows). nath generated sporophyte capsules in the yellow/orange maturation stage (B1-2), and organ bundles with archegonia (black arrows) and antheridia (yellow arrows) (B3-8). rak1-rak2 generated sporophyte capsules in the yellow/orange and green maturation stages (C1-6). Black- and yellow arrows (C7-8) highlight archegonia and antheridia in the organ bundles for rak1-rak2.

The double rak1-rak2 mutant produced 3 sporophyte capsules in the green- and yellow/orange maturation stage (Figure 19C1-6), while nath produced 1 in the yellow/orange maturation stage (Figure 19B1-2). No sporophytes in the earlier maturation stages of sporophyte capsules were visible for nath. Interestingly, rak1 also showed evidence of early sporophyte capsule development, since this mutant generated capsules in the green maturation stage (Figure 20, black arrows). No sporophytes in the later maturation stages were visible. The archegonia (Figure 19B3-8, black arrows) in nath were clearly protruding out from organ bundles, while the antheridia were more problematic to identify (Figure 19B3-8, yellow arrows). The canal and egg sack of the archegonia in rak1-rak2 also clearly protruded from the organ bundle, although the egg sacks appeared rounder and were more visible than the sacks in nath (Figure 19C7-8, black arrows). The antheridia were also more visible in rak1-rak2 than the antheridia in nath (Figure 19C-8, yellow arrows). Interestingly, the calyptra (the womb) in rak1-rak2 and nath are clearly visible in figure 19B1-2 & C2-3, black- & red arrows).

Figure 20: Sporophyte formation in rak1 3 weeks after the first irrigation of the colonies. Black arrows highlight sporophyte capsules in the green maturation stage in rak1.

Since rak1, rak1-rak2 and nath generated sporophytes earlier than WT, the genes RAK1, RAK2 and NATH may be important factors in the regulation of sporophyte development. Spore capsules from the different maturation stages were ruptured for spore germination and phenotypic analysis. The spores from the latest maturation stage for WT, rak1 and mpk3 were able to germinate after 7 days (Figure 21). In contrast, spores from mpk5 were unable to germinate, indicating that the red coloration of the capsules did not necessarily specify matured spores. Interestingly, mpk3 spore capsules in the yellow/orange maturation step are able to germinate, which could indicate premature spores at this stage (Figure 21, mpk3 “yellow/orange stage”). Protonemata and rhizoid development are initiated by tip growth during spore germination. The first cells that are formed are chloronema (Figure 21).

Spores from early-developed sporophytes of rak1-rak2 and nath were able to germinate (Figure 22A- H). Interestingly, less than 10% of nath spores (Figure 22B-D) were able to germinate, compared to 90% of rak1-rak2 spores were able to germinate (Figure 22F-H). Since the spores of nath did not germinate in the same degree as rak1-rak2, may indicate that NATH are important in spore germination. Nonetheless, after 21 days no more nath spores germinated than the one seen in figure 22D. This may indicate that the protonemata seen in figure 22D could be sporophytic tissue that has regenerated. Moreover, there could be some redundancy in the NATs between RAK1, RAK2 and NAT, since rak1-rak2 spores fully germinated.

Figure 21: Germination of WT, rak1, mpk3 and mpk5 spores. (A) Spores from the latest maturation stages were picked and sterilized, before plated on BCDAT overlaid with cellophane (Bar = 5mm). (B) Spores prior germination (Bar = 50µm). (C) 7 days after plating the spores, where protonemata are initiated from spores (Bar = 1mm). (D) Protonemata filamentous body after 21 days of spore germination (Bar = 1mm). mpk3 spores of the yellow/orange stage (Bar = 5mm), were able to germinate, where the protonemata filamentous body are visualized after 21 days (Bar = 1mm).

The phenotypic analysis of the sporophyte induction experiments are summarized in Table 5. The gene deletions of the MPKs and NATH, clearly influence the ability to grow under seasonal changes, as the sporophyte induction conditions mimics. As the colonies of rak1 and mpk5, were strongly impacted by the seasonal changes, as the mutants had early and delayed senescence, respectively. mpk5, rak1-rak2 and nath had abnormal growth of antheridia and archegonia compared to WT, that clearly also influenced the formation of spore capsules. Interestingly, rak1, rak1-rak2 and nath generated sporophytes approximately 4 weeks earlier than WT, indicative of redundancy between RAK1, RAK2 and NATH, and the genes perhaps have a negative regulatory role during generation of sporophytes.

Figure 22: Germination of nath (A) and rak1-rak2 (B) spores. (B-C) nath spores that have not germinated (white circle) and the ruptured sporophyte capsule (white arrow). (D) After 7 days, nath protonemata from germinated spore, with chloronema cells. (F-G) rak1-rak2 spores that have not germinated (white circle) and the ruptured sporophyte capsule (white arrow). (D) After 7 days, rak1-rak2 protonemata from germinated spore, with chloronema cells.

Table 5: Summary of the phenotypic analysis of sporophyte induction experiments.

Growth of protonemata The filamentous body morphology of protonemata is formed by chloronemal and caulonemal cells. To characterize protoplasts of the mutant lines, rak1, mpk3 and mpk5 tissues were grinded and grown for 10 days on BCDAT plates overlaid with cellophane. There were no significant differences in the sizes of protoplasts between WT and the mutant lines (Supplemental Figure 9). Chloronema cells are visualized in WT by their transverse cell walls perpendicular to the growth axis (Figure 23A, white arrow). In contrast, caulonema cells contains fewer plastids and have oblique transverse cell walls (Figure 23A, black arrow). The distinctive cell walls of the two cell types can be difficult to visualize, but normally the secondary tip growth are chloronema cells and protrude after caulonema cells with oblique transverse cell walls (Figure 23B, blue- & red arrows). After 2-3 weeks under normal growth conditions, the WT forms buds from the side branches

(Figure 23A, white circle). The leafy shoots then develop from the buds (Figure 23C, black circles) where the development of the rhizoids can be visualized (Figure 23C-D, grey arrows).

Figure 23: Phenotypic analysis of WT protonemata filamentous body after 3 week on minimal media (BCD). (A) Identification of buds (white circle), chloronema (white arrow) and caulonema (black arrow) (Bar = 500µm). (B) Chloronema filaments (blue arrows) and caulonema filaments in WT (red arrows) (Bar = 500µm). (C-D) Leafy gametophytes are generated by the growth of buds (black circle), and rhizoids protruding from the buds (blue arrows) (Bar = 500µm and 1mm).

While phenotypic analysis of chloronema- and caulonema cells can be tedious, it is practicable to measure the length of the filaments from the tip to the 6th cell as the WT cells grow longer until the 4th cell. Normally, at the 4th cell the first secondary side branch will protrude (Supplemental Figure 10B). In contrast, rak1 developed the longest tip- and 2th cell, compared to WT, mpk3 and mpk5. The first secondary branching for the mutant lines were visualized at the 6th cell for rak1 and at the 5th cell for mpk3 and mpk5 (Supplemental Figure 10C). These observations suggest that RAK1, MPK3 and MPK5 may impact the tip growth of the filaments. Likewise, the most striking phenotype is the delay in secondary branching for all the mutant lines, indicating that the genes could have an important role in the secondary growth and thus in the growth of leafy gametophytes.

3 weeks phenotypic analysis WT, rak1, mpk3, mpk5, double KO rak1-rak2 and nath were grown for 3 and 6 weeks to analyze the growth of the filaments, leafy gametophytes and rhizoids (Figure 24A-F & a-r). Figure 24A-F shows characteristic colony growth for all the lines, and all the lines generated gametophytes, as indicated by white arrows (a-f). Leafy gametophytes were clearly seen for WT, rak1, mpk3 and mpk5 (A-D), but were challenging to visualize for rak1-rak2 and nath (E-F) without dissecting the colonies. The caulonema, chloronema and buds are observable at the periphery of WT, rak1, mpk3 and mpk5 colonies. In contrast, no buds were visible in rak1-rak2 and nath. Buds should have been generated in these mutants, since they also generated gametophytes as the other lines. nath has a discoloration of the filaments, that makes it difficult to characterize the protonema and distinguish between chloronemal and caulonemal cells and rhizoids (Figure 24F, f, l & r).

Figure 24: Phenotypic comparison of P. patens WT rak1, mpk3, mpk5, rak1-rak2 and nath mutants. (A-F) 3 week- old plants grown on minimal media (BCD) (Bar = 2mm). (a-f) Colonies from the center to the periphery, white arrows highlight gametophytes (bar = 1mm). (g-l) Periphery of the colonies, black circles highlight buds (Bar = 1mm). (m-r) Caulonema and chloronema filaments (Bar = 1mm). WT (A, a, g & m), rak1 (B, b, h & n), mpk3 (C, c, i & o), mpk5 (D, d, j & p), rak1-rak2 (E, e, k & q) & nath (F, f, l & r).

It seems that the protonema tissue of rak1-rak2 and nath have a ‘split identity’, since it is unclear whether the filaments are chloronema or caulonema cells (Figure 25B1-2 & C1-2). Moreover, the filaments are bent and have hook-like structures (Figure 25, black arrows). The dark coloration of nath filaments could be due to autophagy, since chlorosis under nutrient starvation is a result of autophagy in P. patens (166). Furthermore, post-translational modifications (PTMs) like acetylation have been shown to be part of the control of autophagy (167).

Figure 25: Phenotypic comparison of P. patens rak1, rak1-rak2 and nath mutants. (A-C) 3 week-old planst grown on minimal media (BCD) (bar = 1mm). (A1-2) rak1 filaments with buds and early stage of leafy gametophytes (black circles) (bar = 1mm). (B1-2) rak1-rak2 filaments with distinctive phenotype (black arrows) (bar = 1mm). (C1-2) nath filaments with hook-like structures and dark coloration (black arrows). Bar = 1mm.

Gametophytes from the central and peripheral part of the colonies were counted and measured (Figure 26 & Figure 27). All the mutant lines produced significantly different amounts of gametophytes than WT. This is seen when grown on media with (BCDAT) and without nitrogen source (BCD). There was no significant difference between mpk3 and mpk5. rak1 generated significantly the most gametophytes, while rak1-rak2 and nath generated fewer gametophytes. This observation correlates with the fact that it was problematic to visualize buds, the pre-maturation stage for leafy gametophytes, in rak1-rak2 and nath. In contrast, buds in rak1 were seen on many of the filaments, which will result in more leafy gametophytes (Figure 25A1-2, black circles). There was a significant difference in the amount of gametophytes when grown on BCDAT between rak1-rak2 and nath. However, there was no significant difference when the mutants are grown on media without nitrogen (BCD). Colonies of rak1-rak2 and nath grown on both BCD and BCDAT look similar. The colonies

56 of both mutant lines do not have many visible gametophytes compared to the other lines (Figure 26). In contrast, mpk5 seems to grow less filamentously on BCDAT with a compact growth of gametophytes (Figure 26).

Figure 26: Phenotypic comparison of WT, rak1, mpk3, mpk5, rak1-rak2 and nath mutants. 3 week-old plant grown on full media (BCDAT) and minimal media (BCD) (bar = 2mm). Gametophytes from central and peripheral part of 3 week-old colonies were dissected: Central and peripheral gametophytes for WT, rak1, mpk3, mpk5, rak1-rak2 (Bar = 1mm) & nath (Bar = 2mm).

Figure 27: Amount of gametophytes after 3 weeks on minimal media (BCD) and full media (BCDAT). Analysis of variance by T-test determined statistical differences indicated by A-E (BCD 3 weeks) & a-e (BCDAT 3 weeks) (P <0.05), n = 3. Standard deviation as error bars. Length of gametophytes (mm) from the base of the gametophore to the tip of the apex. Central and peripheral gametophytes after 3 weeks of growth on minimal media (BCD). Analysis of variance by T-test determined statistical differences indicated by A-E (central gametophytes) & a-e (periphery gametophytes) (P <0.05), n = 16. Standard deviation as error bars.

The gametophytes from all lines were measured after growing on BCD media, which will induce more leafy shoots compared to BCDAT media (Figure 26 & 27). The length was measured from the base of the gametophore to the tip of the leaf at the apex, thus the rhizoids were not measured. The central gametophytes would be more matured than the peripheral gametophytes, hence the localization of the gametophytes would have an impact on their length. Although the amount of gametophytes for rak1 and WT were significantly different, there was no difference between the length of the gametophytes from the central part of the colonies. The length of mpk3 and mpk5 central gametophytes were significantly different from WT and rak1. This was also the case for rak1-rak2 and nath. To conclude, the length of the peripheral gametophytes shows that rak1, mpk5, rak1-rak2 and nath were significantly different from WT and mpk3 (Figure 26 & 27). There were strong phenotypic differences between the rhizoids of WT and the mutant lines. The coloration of the rhizoids are were clearly different between the lines (Figure 26).

6 weeks phenotypic analysis After 6 weeks on BCD the amount of gametophytes were significant higher than 3 weeks on BCD for WT, mpk3, mpk5, rak1-rak2 and nath. There was no significant difference between 3- and 6 weeks of growth for rak1. In contrast, there was no significant difference from 3- and 6 weeks of growth for rak1-rak2 when grown on BCDAT (Supplemental Figure 12). The 6 weeks growth on BCD showed more compact rhizoids, especially for nath and rak1-rak2 for which there were visible differences between 3- and 6 weeks of growth on BCD (Figure 26 & 28. The central part of rak1-rak2 and nath colonies were also more filamentous than WT, rak1, mpk3 and mpk5. rak1 has an interesting phenotype of light green/white filaments in the central part of the colony. These types of filaments were also seen in nath, although the coloration was darker (Figure 28). When grown on BCDAT, the colonies for rak1-rak2 and nath had a white coloration of the filaments, which could be an indication of early senescence (Supplemental Figure 13). An additional 3 weeks on BCD and BCDAT did not significant change the different amounts gametophytes in WT and the mutant lines, rak1, rak1-rak2 and nath. However, the additional growth changed the differences between WT and the mpk3 and mpk5 mutants (Figure 27 & 29). There were significant differences between all lines in the length of central gametophytes when grown on BCD (Figure 29). In contrast, there was no significant difference in the length from the peripheral gametophytes for WT, mpk3 and mpk5 (Figure 29).

Figure 28: Phenotypic comparison of WT, rak1, mpk3, mpk5, rak1-rak2 and nath mutants. 6 week-old plant grown on minimal media (BCD) (Bar = 2 mm) and central part of the colonies (Bar = 2 mm). Gametophytes from central and peripheral part of 6 week-old colonies were dissected: Central and peripheral gametophytes for WT, rak1, mpk3, mpk5, rak1-rak2 & nath (Bar = 1mm).

Figure 29: Amount of gametophytes after 6 weeks on minimal media (BCD) and full media (BCDAT). Analysis of variance by T-test determined statistical differences indicated by A-E (BCD 6 weeks) & a-e (BCDAT 6 weeks) (P <0.05), n = 3. Standard deviation as error bars. Length of gametophytes (mm) from the base of the gametophore to the tip of the apex. Central and peripheral gametophytes after 6 weeks of growth on minimal media (BCD). Analysis of variance by T-test determined statistical differences indicated by A-E (central gametophytes) & a-e (periphery gametophytes) (P <0.05), n = 16. Standard deviation as error bars.

To compare the growth of the lines, the areas of the colonies were analyzed after growing 3- and 6 weeks on BCD. rak1, rak1-rak2 and nath grew significantly different from WT. rak1 had the biggest colonies after 3 and 6 weeks, while rak1-rak2 and nath had the smallest. There were no significant difference between WT, mpk3 and mpk5. Likewise, there were no differences between the double KO rak1-rak2 and nath after 3 weeks of growth (Figure 30).

Figure 30: Colony area of 3 and 6 week-old WT, rak1, mpk3, mpk5, rak1-rak2 and nath. The plants were grown on minimal media (BCD) for 3 and 6 weeks. The area of the colonies were measured by ImageJ, with the function Gaussium blur. Analysis of variance by T-test determined statistical differences indicated by A- D (BCD 3 weeks) & a-d (BCD 6 weeks) (p <0.05), n = 3. Standard deviation as error bars.

Figure 31: Total chlorophyll content of Chla and Chlb (mg pr gram FW) of 3- and 6 weeks old central and periphery gametophytes of WT, rak1, mpk3, mpk5, rak1-rak2 and nath. The plants were grown on minimal media (BCD) for 3 and 6 weeks, and the central and periphery gametophytes were dissected. Analysis of variance by T-test determined statistical differences indicated by A-B (central gametophytes) & a-d (periphery) (p <0.05), n = 16. Standard deviation as error bars.

Since there were visible differences in the coloration of rhizoids and gametophores after 3 and 6 weeks on BCD, the chlorophyll contents of the central and periphery gametophytes were analyzed (Figure 31). After 3 weeks on BCD, the central and peripheral gametophytes of rak1 and nath had the highest chlorophyll content, likewise for the peripheral gametophytes of rak1-rak2. WT, mpk3 and mpk5 had the same content of chlorophyll in both central and peripheral gametophytes. After 6 weeks on BCD, nath had less chlorophyll in the central gametophytes than WT and the other mutant lines. This was not the case for the peripheral gametophytes since there were no significant difference between nath, WT, rak1, mpk3 and mpk5. However, peripheral gametophytes of rak1-rak2 had significantly lower chlorophyll content (Figure 31.

The phenotypic analysis of protonemata and gametophytes after 3- and 6 weeks of growth on BCD and BCDAT are summarized in Table 6.

Table 6: Summary of the phenotypic analysis of protonemata and gametophytes after 3- and 6 weeks growth on BCD and BCDAT.

Gravitropism To characterize the growth of caulonema filaments in the mutant lines, they were grown vertically for 2 weeks in dark on BCD supplemented with glucose. From the propagated tissue, the filaments grew vertically, and the length of the filaments were measured. Likewise, the ability of the mutants to sense the directional change of the plates would assess their gravitropism responses.

Figure 32: (A) WT, rak1, mpk3, mpk5, rak1-rak2 and nath grown vertically in dark for 14 days on minimal media (BCD) supplemented with 50mM glucose (bar = 5mm). (B) Colonies of WT, rak1, mpk3, mpk5, rak1-rak2 and nath after growing vertically in dark for 14 days (bar = 2mm).

Figure 32A-B shows the ability WT and the mutant lines to grow vertically. The filaments of WT, rak1, mpk3 and mpk5 are clearly seen in Figure 32A, while the filaments for rak1-rak2 and nath are clearly visible. The filaments are protruding both from the top and the bottom of the colonies, however most of the filaments protrude from the top (Figure 32B).

Figure 33: (A) rak1-rak2 and (B) nath colonies after growing vertically in dark for 14 days on minimal media (BCD) (bar = 2mm and 1mm), with caulonema filaments protruding from the colonies (black arrows).

The filaments for rak1-rak2 and nath were clearly shorter and not visible compared to the other lines (Figure 33A-B, black arrows). The filaments for rak1-rak2 and nath were also protruding from both the top and bottom of the colonies (Figure 33). The length of the filaments from two colonies per line were measured and analyzed. The filaments of rak1, mpk3 and mpk5 were significantly longer than WT, while the filaments of rak1-rak2 and nath were significant shorter (Figure 34). The distribution of the smallest and largest measurements of the filaments are illustrated in Figure 34A which compares the variation of the smallest and largest measurements of filaments in WT, rak1, mpk3 and mpk5 versus rak1-rak2 and nath. This illustrates the ability of the lines to grow vertically and may indicate that rak1-rak2 and nath have some difficulties. There is no significant different between the length of filaments for mpk3 and mpk5. The length of the filaments for mpk3 and mpk5 are significant longer than rak1-rak2 and nath, while the filaments are shorter for mpk3 and mpk5 than rak1 (Figure 34B).

Figure 34: (A) WT, rak1, mpk3, mpk5, rak1-rak2 and nath grown vertically in dark for 14 days on minimal media (BCD). (A) Calculated the length of caulonema filaments, seen in figure 20A, by using ImageJ. Analysis of variance by blotting the measurements in boxplot graph (A) and by T-test (B) determined statistical differences indicated by A-E (P<0.05) and by asterisks (* P = 0.05-0.01, ** P = 0.01-0.001, *** P < 0.001), n = 12. Standard deviation as error bars. It seems clear from the vertical growth experiment that the deletion of MPKs and NATH have an impact on the growth of filaments. However, the deletions of RAK1, MPK3 and MPK5 do not have an impact on the vertical growth, since these mutant lines are able to grow vertically and thus no defect in gravitropism. Figures 32 and 33 show that rak1-rak2 and nath are able to generate filaments, but whether their significantly shorter filaments are a result of a delay in growth or lack of gravitropism is not clear. The vertical growth experiment suggests that RAK2 and NATH could have significant role in gravitropism and growth of filaments. To also analyze the ability to sense shifts in gravity, the plates from the vertical experiment were rotated 90 degrees (Figure 35, white arrows) and grown for 2 more weeks in the dark. WT, rak1, mpk3 and mpk5 were able to sense the 90 degrees shift, illustrated with the black arrows on directional growth change of the filaments (Figure 35A-D).

Since the filaments for rak1-rak2 and nath are significantly shorter than the other lines, it was problematic to see the directional growth change in these two mutant lines (Figure 35E-F and 36A-B and C-D). However, it seems that the filaments for rak1-rak2 and nath are still able to grow, since the filaments seem to be longer after the directional shift (Figure 36A-B and C-D, black arrows vs. red arrows).

Figure 35: (A) WT, (B) rak1, (C) mpk3, (D) mpk5, (E) rak1-rak2 and (F) nath grown vertically in dark for 14 days on minimal media (BCD), then the plates were rotated 90 degrees and further grown for 14 days in dark (bar = 5mm). Black arrows indicates the directional change of the filaments (A-D).

Figure 36: (A) rak1-rak2 and (C) nath grown vertically in dark for 14 days on minimal media (BCD), then the plates were rotated 90 degrees and further grown for 14 days in dark (bar = 5mm and 2mm). (C) rak1-rak2 colony and filaments (red arrows) when rotated 90 degrees (white thick arrow) (bar = 1mm). (D) nath colony and filaments (red arrows) when rotated 90 degrees (white thick arrow) (bar = 1mm).

Finding the needle in the haystack – what activates the MPKs?

MPKs regulate many different cellular processes such as cell differentiation, innate immunity, stress and hormonal responses, as well as the specific activation of other kinases and enzymes (45, 70, 71).

Biotic stress Our group identified an MPK signaling pathway cascade including the PRR PpCERK1, PpMEKK1a/1b, PpMKK1a/1b/1c and the single MPKs, PpMPK4a and PpMPK4b, with responds to the fungal PAMP chitin (45). The initial study was to investigate whether RAK1, MPK3, MPK5 and NATH are activated upon biotic stress, like the fungal PAMP chitin. The phosphorylation and activation of the downstream kinases were measured by immunoblotting with anti-phospho-p44/42 MPK antibody (α-pTEpY). MPK4a and MPK4b are phosphorylated and activated in response to chitin in WT, rak1, mpk3, mpk5, rak1-rak2 and nath (Figure 37A-C, black- & white asterisk). This indicates that RAK1, MPK3, MPK5, RAK2 and NATH are not part of the MPK signaling pathway cascade upon chitin.

Figure 37: Phosphorylation and presumptive activation of the downstream kinases were measured by immunoblotting with anti-phospho-p44/42 MPK antibody (α-pTEpY). (A) Phosphorylation and activation of kinases in WT, mpk4a, mpk3 and mpk5 upon treatment with fungal PAMP chitin (CHI) and no treatment (NT). Phosphorylation of MPK4a (black asterisk) and MPK4b (white asterisk). (B) Phosphorylation and activation of kinases in WT, mpk4a, rak1 and rak1-rak2 upon treatment with fungal PAMP chitin (CHI) and no treatment (NT). Phosphorylation of MPK4a (black asterisk) and MPK4b (white asterisk). (C) Phosphorylation and activation of kinases in WT, mpk4a, rak1, rak1-rak2 and nath upon treatment with fungal PAMP chitin (CHI) and no treatment (NT). Phosphorylation of MPK4a (black asterisk) and MPK4b (white asterisk).

The mentioned MPK pathway are not activated upon biotic stress treatments, indicating that the MPK4a and MPK4b pathway functions primarily in PAMP Triggered Immunity (PTI) in response to chitin. There are several reports and observations showing MPK signaling pathways triggered by abiotic stressors such as osmotic, cold, and drought stresses as well as hormonal treatments in A. thaliana. AtMPK3, 4 and 6 have been shown to be activated upon abiotic stressors like salt, drought, cold, UV-light and wounding (72, 81-83).

Abiotic stress – Drugs Calyculin A – protein Serine/Threonine phosphatase inhibitor The paralogous pairs RAK1/RAK2, MPK3/MPK7 and MPK5/MPK2 have almost the same protein sizes, thus their individual phosphorylation and activation patterns could be difficult to characterize in single mutant lines. To characterize their phosphorylation, activation and protein size of the MPKs, RAK1, MPK3 and MPK5, the mutant lines rak1, mpk3, mpk5 and rak1-rak2 were treated with the protein Serine/Threonine phosphatase inhibitor calyculin A. Such treatment should in principle, increase basal MPK activation signals. Figure 38A shows a stronger phosphorylation of the MPKs in the WT when treated with calyculin A. The phosphorylations of RAK1/RAK2, MPK3/MPK7 and MPK5/MPK2 are highlighted in Figure 38 as black asterisks, white and red arrows, respectively. Interestingly, RAK1 and RAK2 are not phosphorylated and activated in mpk3, since no bands are seen on the immunoblot like for rak1 and the double KO rak1-rak2. This could indicate that MPK3 phosphorylates RAK1 and RAK2 or indirectly affects their activation. In mpk3 there is no difference in phosphorylation of MPK5/MPK2 when treated and not treated with calyculin A (red arrows). A strong phosphorylation is seen in mpk5 at approximately 60kDa when treated with calyculin A. This could be a strong activation of the paralog of MPK5, MPK2 (Figure 38, black arrows). Interestingly, this strong phosphorylation also is seen in rak1-rak2 (Figure 38A, white asterisk). As mentioned, MPK2 and MPK5 have a TEY motif in the N-terminal and TDY motif in their regulatory loop regions (Supplemental Figure 1). AtMPK9 have the same distinct TEY and TDY motifs. Mutagenesis studies of the TDY motif resulted in strong indication that AtMPK9 are able to be activated through auto-phosphorylation. Moreover, treatments with phosphatase inhibitors and NaCl resulted in phosphorylation of AtMPK9, but interestingly on the immunoblots two bands are visualized (78). The two bands present in the immunoblot in Figure 38A (white asterisk and black arrow) could be due to phosphorylation on the threonine and tyrosine residues in both motifs, TEY and TDY. Thus, MPK2 and MPK5 could like AtMPK9 be activated through auto-phosphorylation,

67 however this needs to be further investigated. This phenomenon has not yet been characterized in the moss P. patens. The protein size of RAK1 and RAK2 are in fact almost equally large, since there is a strong phosphorylation in WT as a result of activation of RAK1 and RAK2 (Figure 38C, black asterisk). The phosphorylation is less strong in rak1 and indicative of RAK2 phosphorylation (Figure 38C, red asterisk). No phosphorylation and activation of RAK1 and RAK2 are observable in the rak1- rak2 double mutant. The activation and phosphorylation of MPKs were analyzed in the nath mutant (Figure 38B). RAK1/RAK2, MPK5/MPK2 and MPK3/MPK7 are phosphorylated in WT, but MPK3/MPK7 seems not to be phosphorylated in nath. This suggests that NATH somehow affects the activation and phosphorylation of MPK3.

Figure 38: Phosphorylation and presumptive activation of the downstream kinases were measured by immunoblotting with anti-phospho-p44/42 MPK antibody (α-pTEpY). (A) Phosphorylation and activation of kinases in WT, rak1, mpk3, mpk5 and rak1-rak2 upon treatment with the protein serine/threonine phosphatase inhibitor calyculin A (Cal.) and no treatment (NT). Phosphorylation of RAK1 and RAK2 (asterisk), MPK3/MPK7 (white arrow), MPK5 (black arrows) and white asterisk = unknown MPK but probably MPK2. (B) Phosphorylation and activation of kinases in WT and nath, treated with calyculin A (Cal.) and not treated (NT). Phosphorylation of RAK1 and RAK2 (asterisk), MPK3/MPK7 (white arrow) and MPK5 (red arrow). (C) Phosphorylation and activation of RAK1 and RAK2 in WT (asterisk) and RAK2 in rak1 mutants (red asterisk). (D) Phosphorylation and activated MPK4a (asterisk) and MPK4b (white asterisk) in response to chitin (CHI) in WT, rak1, rak1-rak2 and nath.

The relative expression of NATH in mpk3 is significantly higher than in WT, suggesting that MPK3 may have an impact on the expression of NATH (Figure 39B). The expression of NATH in rak1 is significant lower than WT, in contrast to the expression in rak1-rak2 which is significantly higher. Interestingly, this may indicate that the gene deletion of the acetyltransferase domains in RAK1 and RAK2 could result to a higher expression of NATH (Figure 39A). The relative expression of RAK2 in rak1 is significantly higher than in WT, thus there may be some redundancy between RAK1 and RAK2. In Figure 38, it is clear that RAK1 and RAK2 are not phosphorylated and activated in mpk3, although RAK2 is expressed in mpk3 at significantly lower levels than in WT. This observation

68 suggests some relationship between MPK3 and the two acetyltransferase/kinases, RAK1 and RAK2. Furthermore, the relative expression of RAK2 in nath, is also significantly lower than WT, giving another interesting link between NATH, MPK3 and RAK1/RAK2 (Figure 39B). Another interesting link between NATH and RAK1/RAK2, is that rak1-rak2, nath and rak1 were able to develop sporophytes approximately 4 weeks earlier than WT (Figure 19 & 20). Spores of yellow/orange maturation stage were able to germinate, 10 % of nath spores and 90% of rak1-rak2 spores (Figure 22). Since mRNA levels of NATH in rak1-rak2 is significantly higher than WT, could certainly indicate redundancy in the NATs of RAK1, RAK2 and NATH. Moreover, RAK1/RAK2, MPK3 and NATH could in fact be linked, since mpk3 spores capsules in the yellow/orange maturation stage likewise was able to germinate (Figure 21).

Figure 39: (A-B) Quantitative RT-PCR of NATH (A) and RAK2 (B) transcript levels in WT, rak1, rak1-rak2, mpk3 and nath fold change relative to WT. Data points represent an average of 3 quantifications of the mRNA from the same sample (technical replicates) with standard deviation as error bars. The experiment was repeated 3 times with similar results. Analysis of variance by T-test determined statistical differences indicated by asterisks (*P = 0.05- 0.01, **P = 0.01-0.001, *** P < 0.001). Standard deviation as error bars.

Zeomycin – DNA damage Homologous recombination mediated gene targeting, as insertion of reporter genes (KI) and deletion of genes (KO) in P. patens, is facilitated by the DNA double-strand break (DNA-DSB) repair system. This DNA-DSB repair system is also important during environmental stress and during DNA synthesis (168). We attempted to analyze aspects of this system by inducing DNA-DSBs in the MPK mutants by the drug zeomycin. The mutant lines were treated with zeomycin for 1, 6 and 12 hours, and the phosphorylation of MPKs was analyzed. Figure 40A-C shows the phosphorylation of MPK4a/MPK4b, RAK1/RAK2 and MPK5/MPK2. The treatment with zeomycin does not result in a stronger phosphorylation of RAK1/RAK2 and MPK5/2. The phosphorylation of MPK3/MPK7 is problematic to analyze since the protein size of these MPKs are almost the same size as MPK4a/MPK4b. However, MPK4a and MPK4b are more phosphorylated in WT when treated with chitin (Figure 40B-C, white asterisk).

These MPKs are more phosphorylated when treated with zeomycin for 6 and 12 hours in WT, mpk5 and mpk3. There are no differences in the phosphorylation of MPK4a and MPK4b between no treatment (NT) and treatment with zeomycin in rak1 and rak1-rak2. Interestingly, there appears to be an unknown MPK of 36-40kDa that is phosphorylated in rak1-rak2 (Figure 40, black arrows). However, this phosphorylation is seen in both NT and when treated with zeomycin, this indicates that the deletion of RAK1 and RAK2 activates another MPK. Furthermore, we treated the WT and mutant lines with other drugs to analyze phosphorylation of the MPKs. We treated the plants with Camptothecin (Topoisomerase inhibitor), cycloheximide (translational inhibitor), Aphidicolin (DNA synthesis inhibitor) and Propyzamide (microtubule inhibitor) for 1 and 3 hours. However, no differences in the phosphorylation of MPKs from the WT and the mutant lines could be detected (data not shown).

Figure 40: Phosphorylation and presumptive activation of the downstream kinases were measured by immunoblotting with anti-phospho-p44/42 MPK antibody (α-pTEpY). (A) Phosphorylation and activation of kinases in WT, rak1, mpk3, mpk5 and rak1-rak2 upon treatment with zeomycin for 6 hours and no treatment (NT). Phosphorylation of RAK1 and RAK2 (black asterisk), MPK5 (red asterisk), MPK4a and MPK4b (white asterisk) and black arrow = unknown MPK. (B) Phosphorylation and activation of kinases in WT and rak1-rak2, treated with zeomycin for 1, 6 and 12 hours and not treated (NT). WT treated with chitin (CHI). Phosphorylation of RAK1 and RAK2 (black asterisk), MPK5 (red asterisk), MPK4a and MPK4b (white asterisk) and black arrow = unknown MPK. (C) Phosphorylation and activation of kinases in WT, mpk3 and mpk5, treated with zeomycin for 1, 6 and 12 hours and not treated (NT). WT treated with chitin (CHI). Phosphorylation of RAK1 and RAK2 (black asterisk), MPK5 (red asterisk), MPK4a and MPK4b (white asterisk).

Abiotic stress – Salt and osmotic stress Preliminary salt stress experiments with rak1, mpk3 and mpk5 suggested that MPK3 and MPK5 could be involved in salt stress, since mpk3 and mpk5 seem to be reacting to NaCl and KCl differently than WT (Supplemental Figure 14). All mutant lines were tested for sensitivity to NaCl and KCl by growing them on BCD plates supplemented with 100mM NaCl or KCl (Figure 41).

Figure 41: Colonies of 3 and 6 week-old WT, rak1, mpk3, mpk5, rak1-rak2 and nath on minimal media (BCD) supplemented with 100mM NaCl and KCl. Bar = 2mm.

Plate growth

After growing for 3 weeks on NaCl and KCl all the mutant lines and WT had clear phenotypic changes since the colonies were less leafy, indicative of less growth of gametophytes. On BCD the colonies of WT, rak1 and mpk3 seemed to have equally sized colonies, although after NaCl stress it seemed that mpk3 was a bit smaller. This could suggest that mpk3 may be more sensitive towards NaCl. Interestingly, the coloration of mpk3 and mpk5 colonies look a bit darker, suggesting that the salt stress could have an impact on the chlorophyll content in these mutants. rak1-rak2 and nath seemed to react to salt stress as WT. After 6 weeks on KCl , WT, rak1, mpk3 and mpk5 were drying out, as

71 visualized as white coloration of the colonies. This phenotype is not visible in rak1-rak2 and nath colonies, and could be an effect of the early senescenceseen when grown without salt stress. However, the phenotype of rak1-rak2 and nath on KCl could be due to water retention in the colonies, hence the colonies would not dry out as fast as WT (Figure 41). rak1-rak2 and nath seemed to react in the same matter on KCl and NaCl, since the phenotypes of the colonies showed a light green halo around the colonies. Likewise, the size of the colonies after 6 weeks on both salt stresses seemed identical. WT and rak1 seemed to be equally sensitive toward NaCl after growing 6 weeks on this stress factor. Colonies of mpk3 after 6 weeks on NaCl suggested that mpk3 may be more sensitive to NaCl. The coloration of mpk5 colonies also seemed to be darker than WT, which may indicate higher chlorophyll content (Figure 41). From these preliminary experiments, mpk3 and mpk5 were further analyzed for salt sensitivity and osmotic stress.

Vertical growth The growth of the filaments for WT, mpk3 and mpk5 were analyzed by growing the mutants vertically. The lines were grown vertically for 2 weeks on BCD and BCD supplemented with 100mM NaCl or

KCl (Figure 42 and 43). The lines were also grown on higher NaCl and MgCl2 concentrations, however the colonies were not able to grow filaments on these conditions (Supplemental Figure 15).

Figure 42: WT, mpk3 and mpk5 were grown in dark for 14 days on minimal media (BCD) supplemented with 100mM NaCl and KCl. Calculated the length of caulonema filaments using ImageJ. Analysis of variance by T-test determined statistical differences indicated by A-C (BCD), a-c (BCD supplemented with 100mM NaCl) and A-C (BCD supplemented with 100mM KCl) (P<0.05). Standard deviation as error bars.

There were no significant differences in the length of filaments when grown on BCD, between WT and the mutant lines (Figure 42 and 43A). Salt stress had an impact on the growth of the filaments of mpk3 and mpk5, since there were significant differences between WT, mpk3 and mpk5. Furthermore, there were significant differences in the length of filaments between mpk3 and mpk5 when grown on NaCl and KCl (Figure 42).

It seems clear that mpk3 and mpk5 grew significantly shorter filaments on NaCl compared to WT (Figure 43B). Similarly, the growth of filaments was influenced by KCl, since mpk3 grew shorter filaments and mpk5 grew longer filaments (Figure 43C). These observations suggest that MPK3 and MPK5 may have a role towards salt tolerance.

Figure 43: WT, mpk3 and mpk5 were grown in dark for 14 days on minimal media (BCD) supplemented with 100mM NaCl and KCl. Calculated the length of caulonema filaments using ImageJ. Analysis of variance by blotting the measurements in boxplot graph of BCD (A), BCD supplemented with 100mM NaCl (B) and BCD supplemented with 100mM KCl (C). Variance were calculated by T-test determined statistical differences indicated by asterisks (* P = 0.05-0.01, ** P = 0.01-0.001, *** P < 0.001), n = 12.

Stress survival To confirm the observation of differences in the coloration of colonies grown on NaCl, chlorophyll contents were measured before, during stress and after recovery from stress on BCDAT (Figure 44). The lines were also grown on mannitol to analyze the colonies upon osmotic stress (Figure 45). All the colonies were grown on BCDAT and BCDAT supplemented with 500mM NaCl and 800mM mannitol plates overlaid with cellophane. All the lines were sensitive to NaCl stress, since the colonies had less chlorophyll, and the phenotype of the colonies correlated well with the content of chlorophyll since they are bleached (Figure 44A). After 5 and 15 days of recovery on BCDAT plates, all the lines recovered from the stress and there were no significant difference in the content of chlorophyll between the lines. Before stress, after growing 2 weeks on control plates, mpk3 had higher chlorophyll content than WT and mpk5 (Figure 44B & Figure 45B). This could be a result of more tissue or more chloroplasts in mpk3. In contrast, mpk5 had less chlorophyll compared to WT, indicative of either less growth of tissue or less chloroplasts.

Figure 44: (A) WT, mpk3 and mpk5 were grown for 14 days on full media (BCDAT) (control) overlaid with cellophane, and further grown for 14 days on 500mM NaCl (stress). Cellophane were moved to BCDAT and the conolies after 5- and 15 days recovery were photographed. (B) Total chlorophyll content (mg pr gram FW) for WT, mpk3 and mpk5 after grown for 14 days on BCDAT (control), 14 days NaCl (stress) and after 5-and 15 days recovery. Analysis of variance were calculated by T-test determined statistical differences indicated by asterisks A-B (control), a (stress), A (5 days recovery) and a (15 days recovery) (P<0.05), n = 3 colonies per tube (n = 3). Standard deviation as error bars.

The 2 weeks of osmotic stress on plates supplemented with mannitol did not induce colony bleaching as it did under salt stress. Colony bleaching was seen after 5 and 15 days of recovery, where the central part of the colonies were clearly bleached (Figure 45A). All the lines recovered from the osmotic stress, although after 5- and 15 days mpk5 had significantly lower chlorophyll content then WT and mpk3. This could indicate that mpk5 is slower to recover from the stress (Figure 45B).

Figure 45: (A) WT, mpk3 and mpk5 were grown for 14 days on full media (BCDAT) (control) overlaid with cellophane, and further grown for 14 days on 800mM mannitol (stress). Cellophane were moved to BCDAT and the conolies after 5- and 15 days recovery were photographed. (B) Total chlorophyll content (mg pr gram FW) for WT, mpk3 and mpk5 after grown for 14 days on BCDAT (control), 14 days NaCl (stress) and after 5-and 15 days recovery. Analysis of variance were calculated by T-test determined statistical differences indicated by A-B (control), a (stress), A (5 days recovery) and a (15 days recovery) (P<0.05), n = 3 colonies per tube (n = 3). Standard deviation as error bars. Variance between the lines were calculated by T-test and indicated by asterisk (* P = 0.05-0.01, ** P = 0.01- 0.001, *** P < 0.001).

These observations indicate that mpk3 and mpk5 are not as impacted by salt and osmotic stress as is the dehydrin mutant, PpDHNA (100). Saavedra et al. (2006) showed that DHNA plays an important role during salt and osmotic stress in P. patens. Furthermore, the role of DHNA is important in recovery and survival of the plant after stress. Higher plant MPKs have been shown to be activated and phosphorylated by abiotic stresses like salt, cold and drought (65, 99, 118). To study the expression of genes expressed upon abiotic stress, the lines were treated with NaCl for 30 minutes and 1 hour. The salt induced genes that were analyzed have been previously described to be involved in salt response in P. patens and A. thaliana. The gene COR47 is a dehydrin and known to be involved in osmotic- and salt stress (99). In addition, several cellular responses are triggered upon salt stress. One of these responses is an increase of cytosolic Ca2+ concentration, so we analyzed the expression

75 of the PCA Ca2+ ATPase pump (65). Benito and Rodriguez-Navarro (2003) identified the sodium pump, ENA Na+ ATPase in P. patens. The ENA pump was shown to be an important pump during salt stress by improving K+/Na+ homeostasis (107-109).

Figure 46: (A-C) Quantitative RT-PCR of ENA Na+ ATPase pump (A), COR47 (dehydrin) (B) and PCA Ca2+ ATPase (C) transcript levels in WT, mpk3 and mpk5 fold change relative to WT. Plants were treated with NaCl for 30min and 1hour, and not treated (NT). Data points represent an average of 3 quantifications of the mRNA from the same sample (technical replicates) with standard deviation as error bars. The experiment was repeated 3 times with similar results. Analysis of variance by T-test determined statistical differences indicated by asterisks (*P = 0.05- 0.01, **P = 0.01-0.001, *** P < 0.001). Standard deviation as error bars.

COR47 mRNA was significantly reduced in mpk3 versus WT upon 1 hour of NaCl treatment and the expression of the gene was not significantly different from 30 minutes to 1 hour of treatment. The expression was significantly different in mpk3 and mpk5 upon salt treatment. COR47 mRNA accumulation was significantly increased in mpk5 upon stress. This indicates that mpk5 might be more sensitive to salt stress than mpk3 and WT. Overall, the increased accumulation of COR47 in mpk5 may reflect redundancy between MPK5 and its paralog MPK2, thereby resulting in activation of MPK2 thus upregulation of the salt-induced COR47 gene. The downregulation of COR47 in mpk3 could indicate that MPK3 is either a direct or an indirect factor in the expression of salt-induced genes, like COR47 (Figure 46B). Levels of PCA Ca2+ ATPase pump mRNA when not treated with salt were significantly increased in the mutants compared to WT. When not treated with salt there was no significant difference between mpk3 and mpk5. However, upon salt treatment the expression was significantly

76 different in the mutants compared to the WT. Furthermore, the expression was significantly different between mpk3 and mpk5 upon salt treatment, indicating that mpk3 and mpk5 do not react to salt stress in the same degree. The expression of PCA Ca2+ ATPase was higher in mpk5 than mpk3. Upon salt stress the concentration of cytosolic Ca2+ is increased and, together with the high upregulation of the Ca2+ pump in mpk3 and mpk5 after 1 hour of stress, could suggest a high concentration of cytosolic Ca2+ in mpk3 and mpk5 (Figure 46C). The significant level of ENA mRNA in mpk3 and mpk5 without external stress may suggest that deletion of MPK3 or MPK5 stresses the plant. Salt stress induces significantly the expression of the ENA Na+ ATPase pump in both mpk3 and mpk5 (Figure 46A). When not stressed with salt the levels of ENA mRNAs were significantly higher in mpk3 and mpk5 compared to WT. This suggests that MPK3 and MPK5 may negatively regulate the ENA pump. Since the pump is required for K+/Na+ homeostasis, the mutants might be more susceptible to Na+ hence the upregulation of the pump upon salt stress. Overall, MPK3 and MPK5 might be important factors upon salt tolerance. However, whether the MPKs directly or indirectly regulate COR47, PCA Ca2+ ATPase- and ENA Na+ ATPase pumps still needs to be established. The phytohormone ABA is important for adaptation during stress. Under normal growth conditions, plants have a low level of ABA but in response to environmental stress ABA levels increase to cope with the stress (118, 119). PpMPKs are not linked to ABA synthesis hence it could be interesting to study the expression of ABA biosynthetic genes in mpk3 and mpk5 during salt stress. The expression of 9-cis-epoxycarotenoid dioxygenase (NCED) and Zeaxanthin epoxidase (ZEP) were therefore analyzed (119). The ABA biosynthesis enzyme ZEP initiates carotenoid catalysis into violaxanxin followed by synthesis and oxidative cleavage by NCED and further transformation into xanthoxin. Xanthoxin is then converted to abscisic aldehyde and oxidized into ABA (Supplemental Figure 16). ZEP1 mRNA increased significantly in mpk3 and mpk5 before salt stress and after 1 hour of stress compared to WT (Figure 47A). In contrast, the relative expression of ZEP1 after 30 min of salt stress are significantly lower, which might indicate a delay in the expression of the ZEP1 gene during stress. Similarly, NCED mRNA levels were significantly higher in mpk3 and mpk5 before stress and after 1 hour of stress (Figure 47B). After 30 min of salt stress, NCED mRNA levels were significantly lower in the mutants compared to WT. These observations indicate that there is a correlation between ZEP1 and NCED expression in the mutants. Since 30 min. of salt stress did not induce accumulation of these ABA biosynthesis gene mRNAs in mpk3 and mpk5, ABA synthesis may well be impaired the mutants. While these observations suggest that MPK3 and MPK5 might

77 function as positive regulators of an ABA biosynthesis pathway, the higher levels of the mRNAs in untreated mutant tissue suggest a more complex regulatory scenario.

Figure 47: (A-B) Quantitative RT-PCR of ZEP1 (A) and NCED (B) transcript levels in WT, mpk3 and mpk5 fold change relative to WT. Plants were treated with NaCl for 30min and 1hour, and not treated (NT). Data points represent an average of 3 quantifications of the mRNA from the same sample (technical replicates) with standard deviation as error bars. The experiment was repeated 3 times with similar results. Analysis of variance by T-test determined statistical differences indicated by asterisks (*P = 0.05-0.01, **P = 0.01-0.001, *** P < 0.001). Standard deviation as error bars.

Abiotic stress – Hormones

To determine if any of the physiological changes described for rak1, mpk3, mpk5, nath and rak1-rak2 could be due to hormonal imbalances, all lines were analyzed after application of auxin, indole-3- acetic acid (IAA), ABA, cytokinin (BPA) and an analog of strigolactone (GR24). The hormonal analysis of nath and rak1-rak2 is described in Manuscript 1. Auxin regulates the transition from chloronema to caulomena, and initiates tip growth and development of rhizoids. Cytokinin regulates the development of secondary branching from chloronemal and caulonemal filaments. Auxin and cytokinin regulate the formation of buds, which is the developmental stage before leafy gametophytes (Supplemental Figure 17). Strigolactone is known to act as a signaling factor for controlling the extension of filaments in the development of gametophytes and rhizoids.

Auxin Application of IAA accelerates the transition from chloronema to caulonema filaments, stem elongation and development of rhizoids from the leafy shoots (21, 136-138). The WT and mutant lines, rak1, mpk3 and mpk5 were grown on BCDAT plates to slow protonemata differentiation so that morphological differences could be characterized upon IAA treatment. The lines were grown for 4 weeks on plates supplemented with 0.5, 1 and 10μM IAA (Supplemental Figure 18A-D). One week growth on BCDAT supplemented with 0.5 and 1μM IAA induced similar morphological changes in all the lines (Supplemental Figure 18A-C). 10μM IAA clearly inhibited growth of the colonies, indicating its toxicity (Supplemental Figure 18D). After 3 and 4 weeks on

BCDAT with and without IAA, all the lines were able to grow. However, there were differences in colony size from the high concentration of IAA compared to the lower IAA concentration. This plate experiment shows that it is difficult to conclude whether the phenotype of the mutant lines are impacted by the exogenous application of IAA.

Figure 48: WT, rak1, mpk3 and mpk5 were grown in dark for 14 days on full media (BCDAT) (A) supplemented with 0.5µM (B) and 1µM IAA (C). (D) Calculated the length of caulonema filaments using ImageJ. Analysis of variance by T-test determined statistical differences between treatments indicated by A-C (BCDAT), a-c (BCDAT supplemented with 0.5µM IAA and a-c (BCDAT supplemented with 1µM IAA) (P<0.05). Standard deviation as error bars.

To further characterize the impact of IAA on the growth of filaments, all the lines were grown vertically in the dark for 3 weeks on BCDAT supplemented with 0- and 1μM IAA (Figure 48A-C). IAA treated colonies were greener (Figure 48B-C) compared to the colonies grown on BCDAT without IAA (Figure 47A). This is probably due to accelerated growth of chloronema compared to caulonema filaments, indicating that the exogenous IAA also affectrd all the lines when grown vertically in dark. Furthermore, the application of IAA induced accelerated growth of gametophytes (Figure 48B-C, white arrows). Filament lengths were measured and are illustrated in Figure 48D. On control plates, rak1 had significantly shorter filaments then WT, while mpk3 had significantly longer filaments. When grown on BCDAT supplemented with 0.5 and 1µM IAA there were no significant differences between WT and rak1 (Figure 48D). This is not the case for mpk3, since IAA induces longer filaments in this mutant line compared to WT, rak1 and mpk5 (Figure 48D). Low concentrations of IAA induced longer filaments in mpk5 compared to WT (Figure 48D). These observations indicate that IAA reverts the phenotypic characterization of filaments in WT and the

79 mutant lines. Furthermore, IAA resulted in shorter filaments in rak1 indicating that the plant is reacting to IAA. Interestingly, mpk3 and mpk5 have still longer filaments than WT upon exogenous IAA, which implies that these mutants are not reacting to IAA in the same manner as WT.

Abscisic acid The WT and mutant rak1, mpk3 and mpk5 lines were grown on BCDAT supplemented with 0.5, 1 and 10μM IAA (Supplemental Figure 19A-D). The colonies exhibited morphological changes within a week of this ABA treatment (Supplemental Figure 19B-D). The colonies were more filamentous compared to the control plate (Supplemental Figure 19A). Exogenous ABA application is known to induce morphological changes in P. patens, including the differentiation of cells into chains of brachycytes and brachycytes with tmema cells (Supplemental Figure 17) (21). To investigate whether the mutant lines reacted to ABA as the WT, all the lines were grown for 4 weeks in liquid BCDAT supplemented with 1µM ABA (Figure 49A-B). All lines reacted to the ABA as they all clearly generated chains of brachycytes (Figure 49B, white arrows).

Figure 49: WT, rak1, mpk3 and mpk5 were grown in liquid BCDAT (A) and BCDAT supplemented with 1µM ABA (B) for 4 weeks. All lines reacted to the ABA as they all clearly generated chains of brachycytes cells (B, white arrows).

The lines were also grown vertically in the dark on BCDAT plates without and with ABA (Figure 50 A-D). The application of 0.5 and 1µM ABA induced accelerated filament growth in all lines (Figure 50B-C, white arrows illustrating the direction of filaments). The length of filaments of rak1 were significantly different from WT on control plates and plates with ABA (Figure 50D). On ABA, rak1 generated the longest filaments, while on control plates rak1 had significantly shorter filaments compared to WT, mpk3 and mpk5. This suggests that RAK1 may somehow affect ABA responses. Additionally, mpk5 had significantly shorter filaments than WT on plates supplemented with 0.5µM

ABA, although there was no difference between WT and the mutant when grown on 1µM ABA. From a physiological aspect, ABA concentrations higher than 0.5µM could have toxic effects on the plant. This observation suggests that MPK5 affects ABA responses, like RAK1. Since ABA induced longer filaments in rak1 and shorter filaments in mpk5, RAK1 and MPK5 appear to antagonistically affect ABA responses.

Figure 50: WT, rak1, mpk3 and mpk5 were grown in dark for 14 days on full media (BCDAT) (A) supplemented with 0.5µM ABA (B) and 1µM ABA (C). (D) Calculated the length of caulonema filaments using ImageJ. Analysis of variance by T-test determined statistical differences between treatments indicated by A-C (BCDAT), a-c (BCDAT supplemented with 0.5µM ABA) and a-c (BCDAT supplemented with 1µM ABA) (P<0.05). Standard deviation as error bars.

Cytokinin The growth of WT, rak1, mpk3 and mpk5 was initially analyzed on BCDAT supplemented with 0.5, 1 and 10µM BPA (Supplemental Figure 20A-D). The colonies showed morphological changes after one week on plates supplemented with BPA (Supplemental Figure 20B-D). The colonies were smaller and less filamentous compared to BCDAT (Supplemental Figure 20A). All the lines reacted to the applied BPA, and it was clear that 10µM had a negative effect on growth of the colonies (Supplemental Figure 20D; Figure 51A-D). Exogenous cytokinin reduced amount of differentiated gametophores and promoted bud formation with callus-like structures (Figure 51B-D, black arrows). All the lines react to the applied BPA, thus the MPKs are probably not involved in cytokinin signaling.

Figure 51: WT, rak1, mpk3 and mpk5 were grown for 21 days on full media (BCDAT) (A) supplemented with 0.5µM BPA (B), 1µM BPA (C) and 10µM BPA (D). (B-D) Exogenous cytokinin (BPA) reduced amount of differentiated gametophores and promoted bud formation with callus-like structures (black arrows).

Strigolactone Strigolactones control shoot branching and signaling during symbiotic and parasitic interactions (146, 147). The strigolactone biosynthesis pathway is characterized in A. thaliana, garden pea Pisum sativum, rice Oryza sativa, petunia Petunia hybrid and P. patens (148). Moss carotenoid cleavage dioxygenases 7 and 8 (CCD7 and 8) cleave carotenoid substrates in plastids that result in the formation of strigolactones. PpCCD8 is involved in the regulation of branching of filaments related to the inhibition of shoot branching by strigolactones in the moss (146). The phenotypes of WT, rak1, mpk3 and mpk5 were analyzed on BCDAT plates supplemented with the strigolactone analog GR24 (Figure 52). GR24 clearly changed the morphology of the colonies for all lines (Figure 52C-D), compared to control plates BCDAT and BCD (Figure 52A-B). After 3 weeks on 1 and 5µM GR24 plates, the growth of filaments and shoot branching were clearly inhibited (Figure 52C-D). Interestingly, mpk3 appeared to react more strongly to GR24 compared to the other lines, since its colony and filaments seemed to be more inhibited (Figure 52D).

Figure 52: WT, rak1, mpk3 and mpk5 were grown for 21 days on full media (BCDAT) (A) supplemented with 0.5µM GR24 (B), 1µM GR24 (C) and 5µM BPA (D).

As observed, GR24 regulates the growth of filaments and branching of shoots, hence filament growth was further analyzed. The lines were grown vertically in dark on BCD plates supplemented with 1 and 5µM GR24 (Figure 53A-D). There were significant differences between WT and the mutants

83 when grown on BCD. As expected, GR24 inhibited the growth of filaments in all the lines, since filaments were shorter compared to filaments on control plate (Figure 53D). On GR24 there was no significant difference in filament lengths between WT and rak1, indicating that RAK1 does affect strigolactone signaling. Additionally, mpk3 and mpk5 had significantly shorter filaments than WT and rak1 when grown on plates supplemented with GR24. Furthermore, mpk5 seemed to react in the same way to 1µM and 5µM GR24, since there was no significant difference on filament lengths. There results may indicate that MPK3 and MPK5 affect some aspect of strigolactone responses.

Figure 53: WT, rak1, mpk3 and mpk5 were grown in dark for 14 days on full media (BCDAT) (A) supplemented with 1µM GR24 (B) and 5µM GR24 (C). (D) Calculated the length of caulonema filaments using ImageJ. Analysis of variance by T-test determined statistical differences between treatments indicated by A-C (BCDAT), a-c (BCDAT supplemented with 1µM ABA) and a-c (BCDAT supplemented with 5µM ABA) (P<0.05). Standard deviation as error bars. Variance between the lines were calculated by T-test and indicated by asterisk (* P = 0.05-0.01, ** P = 0.01-0.001, *** P < 0.001).

As mentioned previously, the formation of strigolactone is initiated by CCD7 and CCD8 cleaving carotenoid substrates in plastids. Likewise, the formation of ABA is also initiated from carotenoid substrates, but by the enzymes ZEP and NCED. As previously shown in figure 46, NCED and ZEP mRNA levels were significantly higher in untreated mpk3 and mpk5. There was no significant difference in mRNA levels for these genes between WT and rak1 (data not shown). Since the common substrate for strigolactone and ABA biosynthesis are carotenoids, the mRNA levels for CCD8 in all lines were analyzed. Figure 54A shows the mRNA levels in 14 and 25 day-old plants. At these time points we expected filamentous growth, shoot branching and development of gametophytes. CCD8

84 mRNA levels in 14 day-old rak1 and mpk5 were significantly lower than WT, while the mRNA levels in mpk3 were significantly higher (Figure 54A). It is possible that mpk3 has more plastids and thus more carotenoids than the other lines, which might result in increased ABA and strigolactone biosynthesis, initiated by ZEP, NCED and CCD8, respectively. In 25 day-old plants, the mRNA levels of CCD8 in rak1 and mpk3 were significantly higher than WT and mpk5 (Figure 54A). At 25 days, the plants generate higher amounts of gametophytes due to additional shoot branching and secondary growth. It is therefore possible that the increased filamentous growth in rak1 and mpk3 after three weeks, compared to WT and mpk5, could be due to their elevated CCD8 transcript levels. This may correlate with the idea that strigolactone production in P. patens is reminiscent of quorum sensing described in bacteria and during symbiotic and parasitic interactions. It has been suggested that P. patens uses the hormone to regulate colony extension and as a signaling molecule related to population density (146). Exogenous GR24 were sprayed on the lines, and CCD8 mRNA levels were analyzed after 24 and 48hours (Figure 54B). There were significant differences in CCD8 mRNA levels in WT, mpk3 and mpk5, but this was not the case for rak1. CCD8 mRNA levels after 24 hours GR24 treatment are significantly higher in mpk3 compared to the other lines, while after 48 hours of treatment the mRNA levels are lower in mpk3 than in WT and mpk5. As rak1 and mpk3 have higher transcript levels of CCD8 before GR24 treated, may contribute to a lower transcripts of CCD8 upon applied GR24. These observations could suggest that RAK1 and MPK3 impact the biosynthesis of strigolactones.

Figure 54: (A-B) Quantitative RT-PCR of CCD8 transcript levels in WT, mpk3 and mpk5 fold change relative to WT, in 14- and 25 days old plants (A) and upon GR24 treatment for 24- and 48 hours (B). Data points represent an average of 3 quantifications of the mRNA from the same sample (technical replicates) with standard deviation as error bars. The experiment was repeated 3 times with similar results. Analysis of variance by T-test determined statistical differences indicated by asterisks (*P = 0.05-0.01, **P = 0.01-0.001, *** P < 0.001) and by letters (A: 14 days (A- C) & 25 days (a-b), and B: No treatment (A-B), 24 hours (a-b) and 48 hours (a-b). Standard deviation as error bars.

Abiotic stress – Light Figure 20 showed that the mutant lines contain different amounts of chlorophyll after 3 and 6 weeks growth under normal light conditions. Hence, we phenotypically compared rak1, mpk3, mpk5 and the double KO rak1-rak2 with WT in dark growth conditions.

Dark 14 days old plants were moved to dark and grown for 2 more weeks on BCDAT overlaid with cellophane. After 2 weeks in dark, the plates were moved to normal light conditions, and recovery after dark analyzed (Figure 55A-D). Chlorophyll content were measured before and after growing in dark and the recovery from dark (Figure 56).

Figure 55: WT, rak1, mpk3, mpk5and rak1-rak2 were grown for 14 days on full media (BCDAT) overlaid with cellophane (A). Cellophane with colonies were moved to dark growth condition for further 14 days (B), central gametophytes and chlorosis of protonemata are highlighted with black- and white arrows, respectively. Cellophane were moved to new BCDAT plates and the recovery after 3 days (C) and 7 days (D) were photographed.

All lines, except those of the rak1-rak2double mutant, had the same growth phenotypes when grown on BCDAT overlaid with cellophane (Figure 55A). After 2 weeks under normal light conditions the total chlorophyll content (Chla+Chlb mg/g fresh weight (FW)) in mpk5 and rak1-rak2 were significantly lower than WT and the other lines (Figure 56). After 2 weeks growth in dark, there were no signicifant differences in the chlorophyll content between the lines (Figure 56). This does not, however, correlate with the visible phenotypes of the plants (Figure 55B). rak1-rak2 seem to be

86 greener compared to the other lines, although this mutant was only generating protonemata and may have looked greener due to additional growth of chloronomal filaments.

Figure 56: Total chlorophyll content (mg per gram FW) of WT, rak1, mpk3, mpk5 and rak1-rak2 colonies were calculated after 14 days growth on BCDAT (control), after growing for 14 days in dark (after dark) and after 3-and 7 days recovery. Analysis of variance were calculated by T-test determined statistical differences indicated by asterisks (* P = 0.05-0.01, ** P = 0.01-0.001, *** P < 0.001), n = 3 colonies per tube (n = 3). Standard deviation as error bars.

The other lines are clearly able to generate gametophytes and the protonemata in the perifery of the colonies are undergoing chlorosis (Figure 55B, black & white arrows). After 3 days of recovery from the dark under normal light conditions, chlorophyll contents were increased in all lines (Figure 56). Although there were no significant difference in chlorophyll contents between the lines after 3 days recovery (Figure 56), the mutant lines of mpk3, mpk5 and rak1-rak2 seemed to be greener (Figure 56C). In particular the protonemata of mpk3, mpk5 and rak1-rak2 seemed to be greener than after dark stress (Figure 55C, black arrows). One week recovery from dark stress induced significant differences in the chlorophyll contents in rak1 and rak1-rak2 compared to WT. Specifically, rak1 had significantly higher chlorophyll content than WT, while rak1-rak2 had significantly lower chlorophyll content (Figure 56). This observation suggests RAK2 impacts recovery from dark and regeneration of chloroplasts. Interestingly, since rak1-rak2 was still not generating gametophytes after one week of recovery (Figure 55D, black arrows), it could be the reason why this mutant has significantly lower chlorophyll content. rak1 seems to be greener compared to the other lines after one week of recovery, correlating well with the chlorophyll content (Figure 55D). Perhaps most

87 importantly, these observations indicate that RAK2 probably is a significant factor in the generation of chloroplasts and also in the development of gametophytes.

Red and Blue light As noted above, all the mutants had slightly different growth phenotypes in the development of protonemata and gametophytes under normal growth conditions and under stresses. Light receptors in P. patens control chloroplast movement, induction of side branching in protonemata and induction of gametophores (150, 155, 157). In particular, blue light (BL) regulates branching in protonemata as a mechanism for P. patens to avoid light stress (150, 155, 157). We preliminarily grew WT, rak1, mpk3 and mpk5 for 6 weeks on BCDAT and BCD plates under BL and red light (RL). Normally, when grown on BCD plates, rak1 had shorter gametophytes than WT, and this phenotype seemed to be reverted upon BL. No difference between the WT and the mutants when grown in RL were seen (Supplemental Figure 21). Hence, we compared the growth and amount of gametophytes and chlorophyll content of all lines when grown for 3 and 6 weeks under BL growth conditions.

Figure 57: Phenotypic comparison of WT, rak1, mpk3, mpk5, rak1-rak2 and nath mutants upon growth under blue light (BL) and white light (WL). (A) 3 weeks old plant grown on minimal media (BCD) under BL and WL (Bar = 2mm). (B) 6 weeks old plants grown on BCD under BL and WL (Bar = 2mm).

The growth of gametophytes was increased during BL compared to growth under white light (WL) (Figure 57). After 3 weeks of growth, the colony size for all the lines seemed to be the same when grown on WL and BL. Moreover, 6 weeks the growth of WT, rak1, mpk3 and mpk5 under WL seemed

88 to induce growth of the colonies compared to BL. This may imply that the plants are preferentially growing gametophytes rather than protonemata. No filaments protruded from the colonies for WT, rak1, mpk3 and mpk5 after 3 and 6 weeks of growth. This is not the case for rak1-rak2 and nath, since filamentous structures were visible at 3 and 6 weeks of growth under WL and BL (Figure 57). Interestingly, nath after 3- and 6 weeks under BL seemed to be senescent. This senescence phenotype was also seen after 6 weeks of growth under WL. This suggest that nath has early senescence or, as mentioned earlier, a result of autophagy during nutrient starvation (166). After 3 and 6 weeks under WL and BL, the growth of gametophytes for rak1-rak2 and nath seemed not to be significantly different (Figure 57). Gametophytes were counted for all lines after 3 and 6 weeks of growth under WL and BL (Figure 58A-B). rak1 had significantly more gametophytes after 3- and 6 weeks under WL compared to WT and the other mutants (Figure 58A-B). After 3 weeks under WL, mpk5, rak1-rak2 and nath all had significantly fewer gametophytes than WT and rak1 (Figure 58A). Moreover, after 6 weeks under WL mpk5 had the same amount of gametophytes as WT (Figure 58B). Interestingly, rak1-rak2 and nath still had fewer gametophytes after 6 weeks of growth under WL (Figure 58B). 3 weeks growth under BL resulted in significantly fewer gametophytes for rak1 compared to growth under WL. Furthermore, there were no significant differences between WT, rak1 and mpk3 upon BL. mpk5, rak1-rak2 and nath still had significantly fewer gametophytes than WT. Interestingly, the amount of gametophytes under BL for WT, rak1, mpk3 and mpk5 were significantly lower than growth under WL (Figure 58A). This was not the case for rak1-rak2 and nath, as these two lines had significantly more gametophytes under BL. This observation suggests that BL influences RAK2 and NATH and the generation of gametophytes. Remarkably, there was no significant difference between the amount of gametophytes when grown under WL and BL after 6 weeks for rak1-rak2 and nath (Figure 58B). Looking at the values, the amount of gametophytes after 6 weeks under WL generates more gametophytes, however this is not the case under BL, since the amount of gametophytes are the same after 3 and 6 weeks of growth. This suggests that RAK2 and NATH may have a negative regulatory effect on the generation and early development of gametophytes within the first 3 weeks of growth under BL. As mentioned, RAK2 and NATH are acetyltransferases and may participate in UV-B induced DNA damage repair and related signaling. The Arabidopsis histone acetyltransferase HAG3 has been characterized to participate in UV-B induced DNA damage repair by negatively regulating expression of DNA repair enzymes (169). Fina and Casati (2015) showed that A. thaliana

89 with decreased transcript levels of HAG3 was less sensitive towards UV-B light, since the growth of roots- and leaves were not inhibited compared to WT plants.

Figure 58: Phenotypic comparison of WT, rak1, mpk3, mpk5, rak1-rak2 and nath mutants upon growth under blue light (BL) and white light (WL). Amount of gametophytes after 3 weeks (A) and 6 weeks (B) on BCD under BL and WL growth conditions. Analysis of variance by T-test determined statistical differences indicated by A-D (WL) & a-e (BL) (P <0.05), n = 3. Standard deviation as error bars. Statistical difference between the amount of gametophytes when grown on WL and BL were analyzed by T-test indicated by asterisks (* P = 0.05-0.01, ** P = 0.01-0.001, *** P < 0.001).

Gametophore length and chlorophyll content after 3 and 6 weeks of growth under WL and BL were analyzed. After 3 weeks of growth under BL, the leafy gametophytes seem to be longer than the gametophytes grown under WL (Figure 59A-B). The rhizoids of rak1-rak2 and nath are more visible and darker, implying that BL induces growth of rhizoids in these two mutants (Figure 59B). The length of gametophytes in all the lines were significantly longer under BL growth compared to growth under WL (Figure 59C). This observation correlates with other studies demonstrating that BL induces growth of gametophytes. Growth under BL did not revert the length of peripheral gametophytes of rak1 and both central and peripheral gametophytes in rak1-rak2 and nath to WT values (Figure 59C). The length of the gametophytes for mpk3 and mpk5 plants were not significantly different from WT, indicating that the phenotypes seen under WL are reverted by BL (Figure 59C). Interestingly, there were significant differences between the length of gametophytes when grown on WL and BL for rak1, rak1-rak2 and nath, indicating that these lines reacted to BL but not in the same degree as mpk3 and mpk5.

After 3 weeks growth under BL, the total chlorophyll content of rak1-rak2 central and peripheral gametophytes were 3 times higher than WT (Supplemental Figure 22C). The Chla and Chlb content in peripheral and central gametophytes in rak1-rak2 were also 3 times higher than in the WT (Supplemental Figure 22A-B). Likewise, the total chlorophyll Chla and Chlb content under WL versus BL growth were also 3 times higher in rak1-rak2 (Supplemental Figure 22A-C). The chlorophyll content in gametophytes for nath were also higher than the WT when grown under BL, although not to the same degree as rak1-rak2 (Supplemental Figure 22A-C). The higher chlorophyll content in rak1-rak2 and nath after 3 weeks of growth under BL could be a result of chloroplast movement and photorelocation, suggesting that RAK2 and NATH may affect chloroplast movement and photorelocation at the early gametophyte developmental stage (3 weeks).

Figure 59: Phenotypic comparison of WT, rak1, mpk3, mpk5, rak1-rak2 and nath mutants upon growth under blue light (BL) and white light (WL). Central and periphery gametophytes after 3 weeks (A) on BCD under WL and BL (B) growth conditions. (C) Length of central and periphery gametophytes. Analysis of variance by T-test determined statistical differences indicated by A-C (Central WL), A-C (Central BL), a-c (Periphery WL) and a-c (Periphery BL) (P <0.05), n = 12. Standard deviation as error bars.

Furthermore, the higher contents of Chla and Chlb in the mutants suggests their ability to absorp more BL. Here, as BL is absorbed by the pigments in the chloroplast, and if rak1-rak2 and nath have more chloroplasts, the mutants would in principle be better at absorbing the light. Moreover, Chla absorbs light within violet, blue and red wavelengths, while Chlb absorbs light primarily within blue wavelengths, and the use of both pigments would in principle enhance the ability to absorb light and convert it into chemical energy. This energy could perhaps be used to cope with BL stress after 3 weeks, leading to significantly more gametophytes in rak1-rak2 and nath. After 6 weeks of growth under WL the rhizoids of rak1 (central), rak1-rak2 (periphery) and nath (central and periphery) were more visible and darker than the rhizoids for the other lines (Figure 60A). This characteristic phenotype of the rhizoids was also seen when grown under BL for central gametophytes in rak1-rak2 and nath (Figure 60B). As observed after 3 weeks of growth under BL, the leafy gametophytes for all the lines seemed to be longer than the gametophytes grown under WL (Figure 60A-B). The length of central and peripheral gametophytes for the mutant lines grown under WL were significantly shorter than the WT. This indicates that deletions of RAK1, MPK3, MPK5, RAK2 and NATH all affect the growth of gametophytes. Interestingly, at 6 weeks of growth under WL the central and peripheral gametophytes for WT are equally long. This is also observed for rak1-rak2, but not for rak1, mpk3, mpk5 and nath. The length of peripheral gametophytes for rak1, mpk3 and mpk5 are longer than the central gametophytes. This may imply that there is an energy shift in the growth of central to peripheral gametophytes (Figure 60C). As seen for 3 weeks growth, the length of gametophytes in WT, rak1, mpk3, mpk5 and rak1-rak2 were significantly longer under BL than under WL (Figure 60C). Remarkably, this was not seen for the gametophytes in nath, since there was no significant difference between the length of WL and BL grown gametophytes (Figure 60C). This may suggest that at this developmental stage (6 weeks), the light does not affect nath as it does on the other lines. Furthermore, the observation implies that nath is more sensitive to BL, thus NATH positively affect BL sensing at this developmental stage. Moreover, when grown under BL, the length of central and periphery gametophytes for rak1-rak2 and nath are similarly long (Figure 60C). As seen after 3 weeks, 6 weeks of growth under BL did not revert the length of peripheral gametophytes in rak1 and in both central and peripheral gametophytes in rak1-rak2 and nath to WT values (Figure 60C). In contrast, the length of the gametophytes for mpk3 and mpk5 plants were not significantly different from WT, indicating that mpk3 and mpk5 are as sensitive to BL as WT (Figure 60A-C). Interestingly, after 6 weeks growth under BL, the total chlorophyll contents in nath gametophytes were as high as in the WT. The total chlorophyll content of gametophytes in rak1,

92 mpk3 and mpk5, were lower than when grown under WL. The gametophytes of rak1-rak2 had almost the same chlorophyll content when grown under WL and BL (Supplemental Figure 23C). These observations indicate that the sensitivity of nath towards BL does not affect the content of chlorophyll in the gametophytes. Since rak1-rak2 and nath after 6 weeks generated the same amount and equally long gametophytes, the difference in chlorophyll content in the gametophytes may be indicative. The interesting difference between rak1-rak2 and nath are the Chla and Chlb content of the central gametophytes in which the amount of Chla in rak1-rak2 is lower than nath under BL (Supplemental Figure 23A).

Figure 60: Phenotypic comparison of WT, rak1, mpk3, mpk5, rak1-rak2 and nath mutants upon growth under blue light (BL) and white light (WL). Central and periphery gametophytes after 6 weeks (A) on BCD under WL and BL (B) growth conditions. (C) Length of central and periphery gametophytes. Analysis of variance by T-test determined statistical differences indicated by A-C (Central WL), A-C (Central BL), a-c (Periphery WL) and a-c (Periphery BL) (P <0.05), n = 12. Standard deviation as error bars.

Expression of RAK1, RAK2, MPK3, MPK5 and NATH

We generated knockin (KI) versions with GFP-tags by homologous recombination using targeted gene disruption to analyze the expression of RAK1, RAK2, MPK3, MPK5 and NATH. All the KI versions were phenotypically analyzed by confocal fluorescence microscopy. To analyze the expression of the genes in different tissues, 2 weeks old plants were analyzed. At 2 weeks, the plants have all the morphological characteristics such as protonemata with chloronemal- and caulonemal filaments, and leafy gametophytes with rhizoids protruding from the gametophytes. Likewise, bud formation and thallus in the gametophytes were analyzed (Figures 61-66). Figure 61A- C shows that there is background signal, but no GFP signal in the thallus (A), filaments (B), rhizoids (C) and buds (C, black arrow).

Figure 61: Confocal fluorescent pictures of 14 days old WT visualizing the background signal, but no GFP signal in the thallus (A), filaments (B), rhizoids (C) and buds (C, black arrow).

RAK1-GFP was expressed throughout the different tissues. Specifically, RAK1-GFP was expressed at the initiation of secondary growth of protonemata, the chloronemal filaments (Figure 62D-E, red arrows) and the buds (Figure 62C, white arrow). Furthermore, RAK1-GFP was also expressed cytosolic in the chloronema cells near their transverse walls perpendicular to the growth axis (Figure 62A, white arrows; D-E, red arrows), and at the cell walls in thallus (Figure 62A-B and F-J, white arrows). There was no GFP signal near the oblique transverse cell walls of caulonema, but there was signal in the cytosol of caulonema cells (Figure 62D-E, yellow arrows). Moreover, there was strong

94 signal in the thallus, spanning from the tip through the midrib and to the surrounding cells. The GFP signal seemed to be present in either the cytosol or chloroplast in the cells (Figure 62A-B and F-J, white arrows). RAK1 appears to be expressed during secondary growth, initiating from secondary growth of filaments, bud formation and generation of thallus.

Figure 62: Confocal fluorescent pictures of 14 days old RAK1-GFP. GFP signal in thallus (A-B, F-J, white arrows). Signal in buds (C, white arrow) and in chloronema cells (D-E, red arrows) and in caulonema cells (D-E, yellow arrows). Bar = 100µm. RAK2-GFP was not expressed to the same degree as RAK1-GFP, as RAK2-GFP was predominately expressed during secondary growth of protonemata. There was a RAK2-GFP signal in caulonema cells (Figure 63A, D-E, white arrows, where the oblique transverse cell walls are highlighted with yellow arrows). Chloronema cell walls are highlighted in figure 63D-E with red arrows, where RAK2-GFP was expressed in the cells. In general, there was no signal near the cell walls of caulonema and chloronema cells, but rather in the cytosol or chloroplasts of the cells. Furthermore, RAK2-GFP was expressed in the tip cells (Figure 63C-D, blue arrows). RAK2-GFP was also expressed in the thallus, but not to the same degree as RAK1-GFP. RAK2-GFP was expressed in the tip cells and in the peripheral cells of the thallus (Figure 63B-C, white arrows), and not in the midrib of the thallus as was RAK1-GFP. The strongest RAK2-GFP signal was seen at the initiation of secondary branching and initiation of buds (Figure 63F-I, white arrows), however RAK2 was not in

95 expressed in the later generation of buds, as seen in RAK1 (Figure 63J, black arrow merge picture). These observations indicate that RAK2 is expressed predominantely in chloronema cells, and initiation of buds and to some degree in the thallus while RAK1 is expressed dominantly throughout the different tissues and the later generation of buds. Comparing these observations with the phenotype of the rak1rak2 double mutant, their loss-of-functions would be expected to strongly impact secondary growth in protonemata and the formation of buds. Likewise, the lower chlorophyll content under different light conditions correlates well with the observed expression of RAK1 and RAK2 GFP fusions in the cytosol or chloroplast of cells in protonemata and thallus.

Figure 63: Confocal fluorescent pictures of 14 days old RAK2-GFP. GFP signal in thallus (B-C, white arrows). Signal in the initial generation of buds (F-I, white arrow), but not in the fully generated buds (J, black arrow in merge). RAK2 is expressed in tip cells (C-D, blue arrows) and in chloronema cells (A, D-E, red arrows) and in caulonema cells (D- E, yellow arrows). Bar = 100µm.

Initially, we generated KO line of the single domain, Nα-terminal acetyltransferase homolog (NATH), since RAK1 and RAK2 provided links between protein phosphorylation and acetylation. Remarkably, NATH-GFP was strongly expressed at thallus cell walls (Figure 64A-C, white arrows), chloronema cells in their transverse cell walls (Figure 64D-E, red arrows), and in caulonema cells oblique cell walls (Figure 64A-E, yellow arrows). NATH was predominantly, expressed in the tip cells of chloronema (Figure 64B-E, white arrows). As mentioned, NATH phenotypes, including less generation of gametophytes and morphological changes protonemata growth, correlate well with

96 these observations that NATH-GFP was expressed in initial steps of secondary growth and in the thallus. Since RAK1 and 2, and NATH are all expressed during the initial steps of secondary growth, their disruptions may be expected to result in the strong phenotypes seen in the rak1-rak2 and nath mutants (Figure 24).

Figure 64: Confocal fluorescent pictures of 14 days old NATH-GFP. GFP signal in cell membrane of thallus (A-C, white arrows). NATH is expressed in tip cells (B-E, white arrows) and in chloronema cells (B-E, red arrows) and in caulonema cells (B-E, yellow arrows). Bar = 100µm.

MPK3-GFP was expressed in both caulonema and chloronema cells (Figure 65B, D-E). No signal was visible in the perpendicular cell walls of chloronema or in the oblique transverse cell walls of caulonema (Figure 65B & D, red arrows & yellow arrows). Furthermore, MPK3-GFP was also expressed in the buds, the initial step of secondary growth (Figure 65A, white arrow; C, blue arrow). Like NATH, MPK3 was detected at the walls of the cells in thallus (Figure 65C, white arrows) and in the tip cells of chloronema (Figure 65E, white arrow).

Figure 65: Confocal fluorescent pictures of 14 days old MPK3-GFP. GFP signal in cell membrane in thallus (C, white arrows). Signal in the buds (A, white arrow; C, blue arrow). MPK3 is expressed in tip cells (E, white arrows) and in chloronema cells (B, D-E, red arrows) and in caulonema cells (B, D-E, yellow arrows). Bar = 100µm.

As mentioned, MPK3 appeared not to be phosphorylated in nath (Figure 38B), suggesting that NATH somehow affects the activation of MPK3. Furthermore, mRNA levels of NATH in mpk3 were also significantly higher than in WT (Figure 39A) indicating MPK3 may affect the expression of NATH. This would correlate with the expression of both MPK3 and NATH in the same tissues. We showed in figure 39A that mRNA levels of NATH in rak1 rak2 were significantly higher, perhaps suggesting

98 that loss of the presumptive acetyltransferase activities of the RAKs could result to a higher expression of NATH. Furthermore, we showed that there could be redundancy between RAK1 and RAK2, since the mRNA levels of RAK2 in rak1 were significantly higher than in WT. We showed that RAK1 and RAK2 were not phosphorylated and activated in mpk3 (Figure 38A). Over all the phosphorylation and mRNA levels studies, parred with the expression of the proteins, suggest an interesting link between NATH, MPK3 and RAK1/RAK2.

Figure 66: Confocal fluorescent pictures of 14 days old MPK5-GFP. MPK5 is expressed in chloronema cells (A-E, red arrows), in caulonema cells (A-E, yellow arrows) and in rhizoids (C and E, blue arrows). Bar = 100µm.

MPK5 is the only MPKs in this study that appeared to be expressed in rhizoids (Figure 66C and E, blue arrows). As MPK3 and RAK2, MPK5-GFP was expressed in both caulonema- and chloronema cells, visualized by the transverse cell walls (Figure 66B-E, yellow & white arrows and Figure 66A- E, red & white arrows). The GFP signal in the filaments are not seen in the transverse cell walls, compared to NATH and RAK1. MPK5-GFP was predominantly expressed in filaments surrounding

99 the transverse cell walls, before the secondary branching (Figure 66A-D, white arrows). The GFP signal seen in figure 66E (white arrows) may indicate that MPK5 was also expressed in the chloroplasts. No signal was seen in the buds and thallus (data not shown). We showed under sporophyte induction conditions that mpk5 rhizoids underwent delayed senescence and were significantly shorter than WT (Figure 17A and C). The observations correlate with the fact that MPK5-GFP was expressed in the rhizoids (Figure 66C & E, blue arrows), and would probably be expressed in rhizoids under sporophyte induction conditions.

Figure 67: Confocal fluorescent pictures of protoplasts from 14 day-old WT and reporter lines RAK1-, RAK2-, NATH-, MPK3- and MPK5-GFP. RAK1, RAK2 and NATH are expressed in protoplast compared to WT, while MPK3 and MPK5 seem not to be expressed in protoplasts. Bar = 20µM.

Furthermore, the expression of the reporter genes was also analyzed in protoplasts (Figure 67). Compared to WT protoplasts it seemed that RAK1, RAK2 and NATH were expressed in protoplasts, although it can be problematic to analyze GFP signals in protoplasts due to their plastids and relatively high chlorophyll signal. Comparing the merge pictures of the WT to RAK1, RAK2 and NATH protoplasts indicates these fusion proteins are expressed while MPK3 and MPK5 fusions were not detectable.

100

Discussion This study aimed to characterize functions of MPKs in the moss P. patens. A reverse BLASTp search against Arabidopsis MPK genes (AtMPKs) with the highest P. patens hits (PpMPKs) was used to assess phylogenetic relationships between the 20 Arabidopsis MPKs and 8 moss MPKs. These alignments indicated four parologous pairs of moss MPKs: MPK2/MPK5, RAK1/RAK2, MPK3/MPK7, and MPK4a & b (45). Using gene targeting I generated single KOs of MPK3, MPK5, RAK1 and double KO of RAK1/RAK2. Auto-phosphorylation The moss MPK alignments (Supplemental Figure 1) also showed that RAK1/RAK2 and MPK2/MPK5 differ from the other four in both their N- and C-terminal regions. MPK2/MPK5 have extended C-terminal regions, and contain a Thr-Glu-Tyr (TEY) motif in the N-terminal region (Supplemental Figure 1, TEY motif in green). This TEY motif is not part of the regulatory loop as are TEY motifs in the other MPKs (Supplemental Figure 1, TEY motif in yellow). MPK2/MPK5 also have a Thr-Asp-Tyr (TDY) motif in their regulatory loop regions (Supplemental Figure 1, TDY motif in red). A reverse BLASTp search against AtMPKs, showed that AtMPK9, AtMPK15 and AtMPK16 also have a TEY motif in their N-terminal regions and a TDY motif in the activation loop (Supplemental Figure 1). No phosphorylations have been detected on these N-terminal TEY peptides in AtMPKs (PhosphAT database). I detected strong phosphorylation of a presumptive MPK of ~60kDa in the mpk5 KO when treated with the PP2 phosphatase inhibitor calyculin A. I suspect that this is a strong activation of the paralog of MPK5, MPK2 (Figure 38 black arrows). Interestingly, this strong phosphorylation was also seen in rak1-rak2 (Figure 38A, white asterisk). As mentioned, MPK2 and MPK5 have a TEY motif in the N-terminal and TDY motif in their regulatory loop regions (Supplemental Figure 1). Nagy et al. 2015 showed that AtMPK9 has similar TEY and TDY motifs. Mutagenesis of the TDY motif indicated that AtMPK9 may be activated by auto-phosphorylation. Moreover, treatments with phosphatase inhibitors and NaCl resulted in phosphorylation of AtMPK9 but, interestingly, immunoblotting detected two bands (78). Since two bands are seen on the moss immunoblot (Figure 38A, white asterisk and black arrow), it is conceivable that phosphorylation occurs on the threonine and tyrosine residues in both the TEY and TDY motifs. Thus, MPK2 and MPK5 could, similarly to AtMPK9, be activated through auto-phosphorylation.

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Rosettas – RAK1/RAK2 and their single-domain NATH homolog The moss MPK alignment (Supplemental Figure 1) showed that RAK1/RAK2 have an N-terminal region of ~30kDa in addition to their C-terminal MPK region. This N-terminal region is highly similar to protein N-terminal acetyltransferases D (NATD). Moss also has a single-domain NATD homolog, what we call NATH (Table 2) which is similar to homologs in baker’s yeast S. cerevisiae, the moss Sphagnum fallax, Arabidopsis and humans (Supplemental Figure 2). No sequences with overall similarity to both regions of RAK1 and RAK2 were identified in other embryophytes including Marchantia polymorpha and S. fallax. Thus, RAK1 & 2 appear to be novel rosetta proteins which may provide links between protein N-terminal acetylation and phosphorylation. We have therefore called them RAKs for Rosetta NAT Kinases. The RAK1/RAK2 MPK sequences lack the 4-5 introns found in the other 6 moss MPKs. The rosetta therefore probably arose by retroposition of an MPK C-terminally to a NATD paralog, and this RAK was later duplicated on chromosome 9 or 15. If this was part of a large-scale genome duplication that occurred 30-45MYA (17), then RAK1/RAK2 are at least that old. Such age bespeaks functional conservation. Using gene targeting I generated single KOs of NATH. MPKs generally regulate adaptive and developmental responses by phosphorylation of substrate proteins, while NATDs N- acetylate histones H2A and H4 (170-172). Although the interplay between protein acetylation and phosphorylation is well documented in the modification of histone tails (173), a major mechanism in chromatin remodeling, direct links between them are few, particularly for MPKs (174). The moss rosettas, RAK1 and RAK2 may therefore represent a new tool for understanding protein modifications affecting chromatin, gene expression and cellular signaling. N-terminal acetylation by N-acetyltransferases have been studied in mammals, yeast and Arabidopsis. Acetylation at the N- terminus in the proteins of mammals, yeast and A. thaliana occurs frequently (70-90%, 50-70% and 70-75%, respectively (170-172). NATs change the chemical properties of their substrate proteins by acetylating the N-terminal -amino group by transfer of an acetyl moiety (Ac) from Ac-CoA, resulting in the removal of a positive charge of the substrate protein (170-172). Most NATs are composed of enzyme complexes with substrate specificity. In mammals, 6 NATs have been identified (170), while 5 NATs have been identified in yeast (175). NATs are divided into groups, NATA-F due to their composition and substrate specificity. NATF is only present in higher eukaryotes (176). The RAK1 & 2 NAT domains are similar throughout their lengths to NATD. In yeast and mammals, NATD has a catalytic unit Naa40p (Nat4) that differs from the other NATs, since this NAT is only

102 known to acetylate histones H2A and H4 (175). The other NATs (NATA, B, C, E) have 2 or 3 catalytic units and apparently acetylate many different substrates (amino acids) after methionine is removed by methionine aminopeptidases (MAPs). Substrate specificity of the catalytic units for NATA, B, C, E and F are within the first 2-5 residues, while for Naa40p (Nat4) the substrate specificity is within the first 30-50 residues of the substrate sequence Ser-Gly-Arg-Gly (170, 171, 175, 176). NATD has been associated with ribosomes and cytoplasm, indicating that NATD could be involved in post-translational protein acetylation (176). There are currently no publications on NATD activites in plants

Characterizations of mutant KO lines Sporophyte development in rak1-rak2 and nath KOs (Figure 19B-C) appeared to occur earlier than in WT, rak1, mpk3 and mpk5 (Figure 19A1-3). Interestingly, rak1 also showed evidence of early sporophyte capsule development (Figure 20, black arrows). While it is difficult to speculate on the underlying molecular mechanisms, two lines of evidence related to histone acetylation are worthy of speculation. First, it has been shown in the yeast S. cerevisiae that NATD N-acetylation of H4 cooperates with 3 lysine acetylation marks in the H4 tail (H4K5, 8 & 12) to inhibit arginine 3 dimethylation (H4R3me2), an inhibitory mark which silences ribosomal DNA (rDNA) to reduce rRNA and protein synthesis during starvation and other stresses (177). Second, expression of the Arabidopsis MADS-box floral repressor Flowering Locus C (FLC) is inhibited by asymmetric dimethylation of H4R3 by the methyltransferase PHRMT10 (178). Given the differences in moss sporophyte and Arabidopsis floral development, it is still intriguing to propose that the loss of N- acetylation of H4, and possibly of H2A, would inhibit the expression of developmental regulators and result in earlier sporophyte development in the moss. mpk5 antheridia are not round but resemble pyramids (Figure 16L) and mpk5 was unable to develop mature sporophytes. Interestingly, BLASTs of the MPK5 and MPK2 C-terminal regions showed similarity to seven PpNEKs (NIMA related-kinases) (Supplemental Figure 24). The first NIMA (Never In Mitosis A) family member, described in the fungus Aspergillus nidulans, is a serine/threonine kinase linked to cell-cycle regulation (179). In A. nidulens, NIMA is involved in the chromosome transition to daughter cells. Osmani et al. 1991 showed that without NIMA the fungus arrests at the G2/M phase transition, inducing defective mitosis. The first mammalian NIMA related- kinase 1, NEK1, was identified as a cell cycle regulator which was also highly expressed in male and female germ cells in mouse (180, 181). A. nidulans has one NIMA, while mammals have 11 NEKs, A. thaliana has 7, and O. sativa has 6. Vigneault et al. (2007) showed that NEKs are expressed in all

103 organs of A. thaliana and O. sativa, and suggested that the proteins are involved in developmental processes (182). Multiple alignment of three NEKs from P. patens, one from A. thaliana and the C- terminal of four MPKs (RAK1, MPK3, MPK2 and MPK5) show some similarities between PpNEK5, AtNIMA and the paralogs MPK2 and MPK5 (Supplemental Figure 24). These observations suggest that MPK5 may be involved in the development and structure of antheridia.

Protonemata and secondary growth The moss MPKs appear to influence the development of protonemata and secondary growth, since KOs of MPK3, MPK5, RAK1 and RAK2 showed morphological changes in 3 and 6 week-old plants. Furthermore, the KO of NATH showed strong morphological changes, resembling the double KO rak1-rak2. Caulonema and chloronema filaments of nath and rak1-rak2 had a kind of “split identity”, since the transverse cell walls were oblique and perpendicular to the growth axis (Figure 25). I generated MPK3, MPK5, RAK1, RAK2 and NATH reporter lines tagged with GFP. MPK3-GFP was detected near the cell walls of the thallus and in caulonema and chloronema cells, but not near the perpendicular and oblique transverse cell walls. MPK3-GFP was also detected in the buds and in the tip of cells of chloronema, the initial steps of secondary growth (Figure 65). MPK5-GFP was the only fusion detected in the rhizoids. Furthermore, MPK5 was predominately, expressed in filaments surrounding the transverse cell walls, before secondary branching. During sporophyte induction mpk5 rhizoids underwent delayed senescence and were significantly shorter than WT (Figure 17A and C), providing evidence that MPK5 may be involved in the development of rhizoids. RAK1, RAK2 and NATH GFP fusions were highly expressed in thallus, protoplasts, protonemata and during secondary branching. RAK1-GFP fusion was notably expressed during the maturation of buds (Figure 62), while RAK2-GFP was specifically expressed in the initial steps of bud formation (figure 63). These observations link RAK1 and RAK2 during bud formation and thus the initial growth of leafy gametophytes. This correlates with the phenotype of rak1-rak2 which had significantly lower numbers of gametophytes and thus fewer buds (Figure 27 & 29). To get a more complete functional picture of the proteins, it is necessary to investigate the MPK3, MPK5, RAK1, RAK2 and NATH reporter lines during formation of organ bundles and development of sporophytes. The filaments of nath were darker than WT and all the other mutants, indicative of chlorosis. Such darkening of the filaments might be due to hyper-activation of autophagy since chlorosis under nutrient starvation (minimal media BCD) (Figure 24 & 26) and not on full BCDAT media (Figure 26) is a result of autophagy. Mukae et al. (2015) showed that KO lines of the autophagy-related gene 5 (ATG5) turned yellow on nutrient starvation. Autophagy is a recycling

104 pathway that selectively collects cellular components for degradation in vacuoles or lysosomes. Furthermore, the atg5 mutant showed early, dark-induced senescence compared to the WT (166). Colonies of rak1, rak1-rak2 and nath during gravitopism experiments, showed that they were a much darker yellow than WT, mpk3 and mpk5 (data not shown). Moreover, rak1 and rak1-rak2 exhibited early senescence (Figure 17A, Supplemental Figure 6) and tissue whitening (Figure 28), respectively. These early senescent characteristics in the RAK and NATH KOs may conceivably be linked to reduced cytoplasmic recycling by autophagy. For example, the S. cerevisiae histone acetyltransferase Esa1 and the deacetylase Rpd3 regulate acetylation of K19 and K48 in yeast ATG3 (167, 183). If so, then the presumptive NAT activities of the RAKs and NATH may have a regulatory influence on autophagy and thereby senescence. Although I was repeatedly unable to obtain RAK2 single KO lines, other results provide evidence that RAK2 is important for the secondary growth of buds, gametophytes and filaments. For example, rak1 single KOs had many buds, correlating well with the significantly higher numbers of gametophytes in rak1. As RAK2-GFP was expressed in the initial steps of bud formation, the higher numbers of buds in rak1 could be effected via redundancy between RAK1 and RAK2. Importantly, the numbers of gametophytes after 3- and 6 weeks in rak1-rak2 was significantly lower than in rak1 (Figure 27 & 29). Likewise, the expansion of the rak1 and rak1-rak2 colonies (Figure 30) indicates that RAK2 is involved in the generation of secondary growth of bud formation and colony expansion by protonemata. Interestingly, while the extension of gametophytes was not impacted in rak1, the length of central gametophytes for mpk3 and mpk5 after 3 weeks was significantly longer than WT. This could suggest that these two MPKs negatively regulate the elongation of gametophytes. In contrast, gametophytes of rak1-rak2 and nath were significantly shorter, indicating that RAK2 and NATH have a positive regulatory effect on elongation of gametophytes. Another interesting trait is the apparent lack of gravitopism in rak1-rak2 and nath (Figure 35 & 36). However, the combination of growth on minimal media (BCD), darkness and directional change could be stressful to a degree that pleiotropic effects could occur. The phenotypic characterization of all the KO lines are summarized in Table 6.

MPK phosphorylation and activation Multiple experiments described here confirm that while MPK4a/MPK4b are activated upon biotic stress, MPK3, MPK5, RAK1, and RAK2 are not (Figure 37). Certain experiments indicate a possible link between RAK1, RAK2, and MPK3, since RAK1 and RAK2 were not phosphorylated and activated in mpk3 (Figure 38). Interestingly, MPK3 was not phosphorylated and activated in nath,

105 suggesting that NATH somehow affects the activation of MPK3. In addition, NATH mRNA accumulation is significantly higher in mpk3 than in WT, suggesting that MPK3 influences NATH expression (Figure 39A). Moreover, RAK1 and RAK2 KOs resulted in a higher expression of NATH (Figure 39A). Furthermore, RAK1 and RAK2 are probably partially redundant and the expression of RAK2 in rak1 is significantly higher than in WT (Figure 39B). Although RAK1 and RAK2 were not phosphorylated in mpk3, RAK2 was expressed in mpk3 but at significantly lower levels than in WT. This observation suggests some relationship between MPK3 and the two RAKs. Furthermore, the relative expression of RAK2 in nath was also significantly lower than WT, giving another interesting link between NATH, MPK3 and RAK1/RAK2 (Figure 39B). Interestingly, a strong phosphorylation signal was seen in mpk5 treated with calyculin A at approximately 60kDa. This is most likely a strong activation of the paralog of MPK5, MPK2 (Figure 38, black arrows), that also was seen in rak1-rak2 (Figure 38A, white asterisk). As mentioned, MPK2 and MPK5 have a TEY motif in their N-terminal regions, as well as a TDY motif in their regulatory loop regions (Supplemental Figure 1). Mutagenesis studies of the TDY motif in their close homolog AtMPK9 indicated that AtMPK9 is activated by auto-phosphorylation. Moreover, treatments with phosphatase inhibitors and NaCl resulted in such phosphorylation of AtMPK9, but interestingly on these immunoblots two bands were visualized by Nagy et al. (2015) (78). Intriguingly, the two ~60kDa bands in the immunoblot of mpk5 and rak1-rak2 (Figure 38A, white asterisk and black arrow), could conceivably be due to phosphorylation on the threonine and tyrosine residues in both the TEY and TDY motifs. Thus, I speculate that MPK2 and MPK5 like AtMPK9 may be auto-phosphoryled, although this needs to be further investigated.

MPK3 and MPK5 – involvement in ion homeostasis upon salt treatments? MPK3 and MPK5 may have a role in salt tolerance, since vertical growth experiment showed significantly shorter caulonemal filaments on plates with NaCl, while mpk5 generated longer filaments on plates supplemented with KCl (Figure 42). I also examined stress survival of WT, mpk3 and mpk5 after 5 and 15 days recovery. Saavedre et al. (2006) showed that PpDHNA is important in recovery and survival after salt and osmotic stress. mpk3 and mpk5 were sensitive to NaCl stress as the WT, since the colonies had the equal chlorophyll content as the WT after stress recovery. All the lines recovered from the osmotic stress, although after 5- and 15 days mpk5 had significantly lower chlorophyll content then WT and mpk3. This may suggest that mpk5 is slower to recover from the stress (Figure 45B). However, the chlorophyll content between WT and mpk5 after recovery were not as extreme as Saavedra et al. 2006 showed for PpDHNA mutant.

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I also studied the expression of genes following abiotic and osmotic stress (COR47, PCA Ca2+ ATPase pump and ENA Na+ ATPase pump). COR47 mRNA accumulation was significantly increased in mpk5 upon stress. This indicates that mpk5 might be more sensitive to salt stress than mpk3 and WT. Overall, the increased accumulation of COR47 in mpk5 may reflect redundancy between MPK5 and its paralog MPK2, thereby resulting in activation of MPK2 thus upregulation of the salt-induced COR47 gene. The downregulation of COR47 in mpk3 could indicate that MPK3 directly or indirectly affects expression of salt-induced genes like COR47 (Figure 46B). Levels of mRNA of the PCA Ca2+ ATPase when not treated with salt were significantly increased in the mutants compared to WT. When not treated with salt there was no significant difference between mpk3 and mpk5. However, upon salt treatment PCA expression was significantly different in the mutants compared to the WT. Upon salt stress the concentration of cytosolic Ca2+ is increased. This, together with the high upregulation of the Ca2+ pump in mpk3 and mpk5 after 1 hour of stress, could suggest increased concentrations of cytosolic Ca2+ in mpk3 and mpk5 (Figure 46C). The ENA Na+ ATPase pump was identified in fungus and bryophytes and not in higher plants. During ion homeostasis, higher plants, fungi and mammalian cells use the H+ ATPase and Na+/K+ ATPase pumps, respectively. The ENA (Exitus natru – exit of sodium) Na+ pump extrudes sodium from the plasma membrane and into the vacuole (107-109). NaCl-stress significantly induced ENA expression in both mpk3 and mpk5. When unstressed the levels of ENA mRNA were significantly higher in mpk3 and mpk5 compared to WT (Figure 46A). This suggests that MPK3 and MPK5 may negatively regulate the ENA pump. Since the pump is required for K+/Na+ homeostasis, the mutants might be more susceptible to Na+, hence the upregulation of the pump upon salt stress. P. patens has 3 PpENA pumps, and PpENA1 has been shown to complement a salt-sensitive yeast strain deficient in NA+/K+ homeostasis (107-109). In S. cerevisiae it has been shown that the regulation of ENA1 upon salt stress depends on the high-osmolarity glycerol (HOG) MPK pathway. Hog1p activates the Sko1p repressor that binds to the ENA1 promotor and inhibits the transcription of ENA1 by other corepressor complexes (110-112). Moreover, in A. thaliana, MPK3, MPK4 and MPK6 are activated upon elevated levels of NaCl and osmotic stress, and MPK6 specifically phosphorylates the SOS1 (Salt overly sensitive) Na+/H+ antiporter (6, 71, 81, 103). It is therefore possible that MPK3 and MPK5, like HOG1, since the deletion of MPK3 and MPK5 had a significantly higher mRNA level of ENA pump. However, whether the MPKs directly or indirectly regulate transcription of PCA Ca2+ ATPase- and ENA Na+ ATPase pumps still needs to be established.

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I also checked the expression levels of ABA biosynthetic genes, since ABA levels increase during abiotic stress (118, 119). This showed that during NaCl stress the mRNA levels of ZEP1 and NCED were significantly lower in mpk3 and mpk5 compared to WT. This could be directly due to the MPK3 and 5 KOs, since high salinity will change the osmolarity in the cells and trigger accumulation of ABA, and indirectly regulate the expression of AtSOS1 Na+/H+ antiporter (6, 106). Interestingly, the PpSOS1 Na+/H+ antiporter has been described to be active at low pH values, while the PpENA1 Na+ ATPase is active at high pH values (113). These preliminary results suggest interesting links between MPK3, MPK5 and the regulation of ion homeostasis which may be further investigated.

MPK regulation by phytohormones? I examined morphological changes of WT, rak1, mpk3 and mpk5 upon application of IAA, ABA, BPA and GR24. The hormonal analysis of rak1-rak2 and nath is described in Manuscript 1. Application of IAA accelerates the transition from chloronema to caulonema filaments, stem elongation and development of rhizoids from leafy shoots (5, 21, 114, 136-138). The application of IAA induced accelerated growth of gametophytes (Figure 48B-C, white arrows). Furthermore, IAA resulted in shorter filaments in rak1, indicating that rak1 reacts to IAA. Interestingly, mpk3 and mpk5 had longer filaments than WT upon IAA treatment, which implies that these mutants do not react to IAA in the same manner as WT. Exogenous ABA application is known to induce morphological changes in P. patens, including the differentiation of cells into chains of brachycytes and brachycytes with tmema cells (Supplemental Figure 17) (21). All lines reacted to the ABA as they all clearly generated chains of brachycytes (Figure 49B, white arrows). I showed that vertical growth induced longer filaments in rak1 (Figure 50). Additionally, mpk5 had significantly shorter filaments than WT on plates supplemented with 0.5µM ABA, although there was no difference between WT and the mutant when grown on 1µM ABA. From a physiological aspect, ABA concentrations higher than 0.5µM could have toxic effects on the plant. This observation suggests that MPK5 and RAK1 somehow affect ABA responses, and since ABA induced longer filaments in rak1 and shorter filaments in mpk5, RAK1 and MPK5 appear to antagonistically affect ABA responses. Exogenous cytokinin reduced the numbers of differentiated gametophores and promoted bud formation with callus-like structures (Figure 51B-D, black arrows) (21, 114). All the lines react to the applied BPA, thus the MPKs are probably not involved in cytokinin signaling.

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GR24 (strigolactone) treatment clearly changed the morphology of the colonies for all lines, such that filament branching was inhibited (Figure 52C-D) and the filaments were shorter (Figure 53D), as previously described (146, 147). On plates supplemented with GR24, mpk3 and mpk5 had significantly shorter filaments than WT and rak1. Furthermore, mpk5 seemed to react in the same way to 1µM and 5µM GR24, since there was no significant difference in its filament lengths. This suggests that MPK3 and MPK5 affect some aspect of strigolactone responses. Moss carotenoid cleavage dioxygenases 7 and 8 (CCD7 and 8) cleave carotenoid substrates in plastids that result in the formation of strigolactones. PpCCD8 is involved in the regulation of branching of filaments related to the inhibition of shoot branching by strigolactones in the moss (146). Likewise, the formation of ABA is also initiated from carotenoid substrates, but by the enzymes ZEP and NCED (Supplemental Figure 16) (119). I evaluated the expression of CCD8 in the 14 and 25 day-old mutant and WT plants, since at this timepoint we expected filamentous growth, shoot branching and development of gametophytes. CCD8 mRNA levels in 14 day-old rak1 and mpk5 were significantly lower than WT, while the mRNA levels in mpk3 were significantly higher (Figure 54A). The high CCD8 expression in mpk3 could be a result of more plastids and thus more carotenoids, which might result in increased ABA and strigolactone biosynthesis. In 25 day-old plants, the mRNA levels of CCD8 in rak1 and mpk3 were significantly higher than WT and mpk5 (Figure 54A). At 25 days the plants generate higher numbers of gametophytes due to additional shoot branching and secondary growth. It is therefore possible that the increased filamentous growth in rak1 and mpk3 after three weeks is due to their elevated CCD8 transcript levels. This appears to correlate with the idea that strigolactone production in P. patens is reminiscent of quorum sensing described in bacteria and during symbiotic and parasitic interactions. Thus, P. patens is thought to use the hormone to regulate colony extension, and as a signaling molecule related to population density (146). I also examined CCD8 transcript levels after GR24 application for 24 and 28 hours. This showed that rak1 and mpk3 had higher transcript levels of CCD8 before GR24 treatment (Figure 54B), that may contribute to a lower transcripts of CCD8 upon applied GR24. These observations suggest that RAK1 and MPK3 influence the biosynthesis of strigolactones. I note that such simple molecular phenotypes are complicated by overlapping signaling between hormones. Nonethless, I think I have provided preliminary results suggesting possible roles of moss MPKs in hormone signaling and responses.

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Light responses I examined growth of the mutants under blue light (BL), since BL has been shown to induce branch formation and secondary growth (157). Interestingly, Imaizumi et al. (2002) showed that the BL receptors, cryptochromes PpCRY1a and PpCRY1b, control the development of secondary growth by suppressing auxin sensitivity in P. patens. As nath was apparently insensitive to exogenous IAA (data not shown, Manuscript 1), I compared my results to those of Imaizumi et al. (2002). nath and rak1- rak2 plants were more filamentous than WT and the other mutants when grown under BL (Figure 57), correlating with the cry1a, cry1b and cry1a-cry1b mutants, that under BL had a higher ratio of caulonemal filaments. This indicates that the inhibition of auxin responses, and thus the transition from chloronemal to caulonemal filaments, are specific to cryptochrome signaling. Other analyses described here suggest that RAK2 and NATH may have a negative regulatory effect on the generation of secondary growth and early development of gametophytes. Interestingly, if NATH has a negative regulatory effect on BL signaling via cryptochrome, it would perhaps result in the inhibition of auxin signaling (data not shown, Manuscript 1). Furthermore, BL induces, via cryptochrome, branch induction and branch formation (157), implying that if NATH and RAK2 have negative regulatory effects on generation of branching, their KOs would result in a branched filamentous plant as seen in Figure 57. Cryptochromes have also shown to initiate the differentiation of buds and thus the development of gametophytes regulated by side branch initials (150). This leads to another link to NATH and RAK2, since I showed that nath and rak1-rak2 developed significantly less gametophytes than WT. mRNA levels of NATH in rak1-rak2 were significantly higher compared to WT and rak1 (Figure 39), indicative of possible redundancy between RAK1/RAK2 and NATH due to their shared, presumptive acetyltransferase activities. I provided evidence for this redundancy, since the mutants grown under WL and BL had the same amount of gametophytes and the phenotypes of these mutants were almost identical (Figure 57 & 58). Interestingly, the A. thaliana histone acetyltransferase HAG3 participates in UV-B induced DNA damage repair by negatively regulating expression of DNA repair enzymes (169). Fina and Casati (2015) showed that A. thaliana with decreased transcript levels of HAG3 was less sensitive to UV-B light, since the growth of roots- and leaves were not inhibited compared to WT plants. Taken together, my moss BL study may provide a fascinating link between PpMPK, PpNATH and auxin signaling as previously described in A. thaliana and P. patens. Further studies need to be elucidated to get a more complete picture.

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Concluding remarks Further studies are needed to elucidate the role of MPK3, MPK5, RAK1/RAK2 and NATH. If MPK3 and MPK5 have a role in the regulation of ion homestasis, it would be necessary to measure the intracellular concentration of ions in mpk3 and mpk5 upon salt treatments and osmotic stress. I provided evidence of an interesting link between MPKs and NATs, thus a next step would be to study the activity of the acetyltransferase in the RAKs. It could be interesting to do complementation studies with human- and yeast with PpNATH and the NATs in the RAKs. Furthermore, to get a more complete functional picture of the proteins, it is necessary to induce sporophytes in the MPK3, MPK5, RAK1, RAK2 and NATH reporter lines to elucidate whether the proteins are expressed in the organ bundle and sporophytes. I note that I have shown that the archegonia and antheridia of mpk5, rak1- rak2 and nath had interesting phenotypes, which may be born out in sporophytic analyses. Moreover, the link between RAK1, RAK2, NATH and MPK3 could be investigated by protein-protein interaction studies. During the phenotypic analysis, I found that KOs of a triple MEKK (mekk1a) and of a double MKK (mkk1a) had early senescence similar to that of the rak1 KO. It is therefore possible that a signaling cascade of the MPKs, MEKK1a-MKK1a and RAK1 could affect the regulation of senescence in P. patens. I also proposed that MPK2 and MPK5 could, like AtMPK9, be activated through auto-phosphorylation, however this needs to be further investigated. This phenomenon has not yet been characterized in the moss P. patens.

Material and Methods

Plant growth conditions Physcomitrella patens (Gransden strain) was grown on full media BCDAT (250 mg/l MgSO4·7H2O, 250 mg/l KH2PO4, 1010 mg/l KNO3, 920 mg/l Ammonium tartrate, 12.5 mg/l FeSO4·7H2O, 147 mg/l CaCl2·2H2O, trace elements (614 μg/l H3BO3, 389 μg/l MnCl2·4H20, 110 μg/l AlK(S04)2·12H2O, 55 μg/l CoCl2·6H20, 55 μg/l CuSO4·5H20, 55 μg/l ZnS04·7H20, 28 μg/l KBr, 28 μg/l KI, 28 μg/l LiCl, 28 μg/l SnCl2·2H20, 25 μg/l Na2MoO4·2H2O, 59 μg/μl NiCl2·6H20), pH 6.5, 8 g/l agar). Minimal media (BCD) without Ammonium Tartrate. BCDAT plates were overlaid with cellophane discs (AA Packaging Ltd.) when needed. Colonies were grown at 21°C in 55 µE m- 2 s-1 in a 16h light/8h dark cycle. For routine protonema propagation, plants were grown on BCDAT; protonema tissue was blended with a homogenizer (PowerGen 500, Fisher Scientific). Protonema cultures were subcultured every 5-7 days.

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Phenotypic analysis The genotypes were grown on BCD or BCDAT with addition of different amounts of hormones during several weeks so growth patterns of the different tissues could be assessed. Unless otherwise stated, all treatments were applied by dipping with 1 ml of solution to colonies grown for 2 weeks.

Hormone plates for phenotypic analysis were used at a final concentration of 0.5 μM auxin, indole- 3-acetic acid (IAA) (Sigma Aldrich), 0.5 μM GR24 (Chiralix), 0.5µM cytokinin (BPA). For the salt and osmotic stress treatments, colonies grown on BCDAT overlaid with cellophane, and transferred to medium supplemented with the indicated concentrations of NaCl or mannitol. Cellophane with the colonies were moved to BCDAT medium for stress recovery assay. The colonies were harvested at the indicated time points and the fresh weight (FW) and chlorophyll content were determined. During dark experiment, the genotypes were grown as indicated under dark at 21°C, and the colonies were harvested at the indicated time points and the FW and chlorophyll content were determined. During blue light experiment, the genotypes were grown for 3- and 6 weeks in a box were only blue light (10 µE m-2 s-1) were present. Chlorophyll content were measured by extracting chlorophyll from colonies (NaCl, mannitol and dark stress) or gametophytes (BL) by homogenize the tissue in 80% acetone. The samples were led stand for 1hr at RT and centrifuged at 16400xg for 10 min. The absorbance in the supernatant were measured at values 663nm and 645 nm with spectrometer, and calculated as following: Chla = ((12,7*663nm)-(2,6*645nm))*(1/FW) & Chlb = ((22,9*645nm)-(4,68*663nm))*(1/FW). For vertical growth experiment, plants were grown on either BCD or BCDAT for 2 weeks under dark. BCD or BCDAT plates were supplemented with hormones, salt or 50mM glucose as stated in the figures. The plated were wrapped in tin foil and placed vertically in a card box, that also were wrapped in tin foil. The length of the caulonema filaments were calculated by ImageJ. For sporophyte induction, plants were grown on BCDAT for 6 weeks under normal light and temperature conditions. Plants were moved to winter conditions (17ºC and 10-20 µE m-2 s-1) and irrigated twice after 4 and 5 weeks in consecutive weeks. Organ bundles were developed after the first irrigation and sporophyte capsules in WT developed after 2 weeks from the first irrigation. The sporophytes were harvested by separating then from gametophytic material. The spore capsules were released by pinching the base of the seta, which can be identified by a zone of pigmentation. One or more sporophytes were placed in a sterile 1.5mL microcentrfuge tube, and sterilized by adding 1mL 70% ethanol and incubated for 4 min at room temperature. The sporophytes were twice gently rinsed with 1mL of sterile H2O at room temperature. To increase spore germination, 1mL of H2O were

112 added and the tube placed for 7 days in dark at 4°C. Sporophyte capsules were crushed with fine forceps and mixed to produce a spore suspension.

Generation of Physcomitrella patens mutants All mutants were generated by cloning 900-1500bp of genomic flanking regions of the target gene into left side (left border, LB) and right side (right border, RB) (Supplemental Figure 5) of the selection marker in the previously modified pMBL6 vector with USER sites in both sides of the selection cassette (164). The left and right side of the selection gene NptII in the vector were modified. At the left side a USER cassette with a PacI restriction site and Nt.BbvCI nicking site was inserted in pMBL6 cut with SacI and KpnI. At the right side, a USER cassette containing AsiSI and Nt.BbvCI sites was inserted in pMBL6 cut with SalI and EcoRI. The modified vector was called pMBLU (45). The genomic flanking regions of PpRAK1, PpRAK2, PpNATH, PpMPK3 (unpublished), PpMPK5 (unpublished) and PpMPK4a & b (45), were cloned by PCR amplification with USER compatible primers. The PCR fragments were by four fragment USER reactions cloned in pMBLU, which was cut with PacI and AsiSI and nicked with Nt.BbvCI. GFP and a nos terminator were cloned in the pMBLU Kpn1 site to make the USER cloning vector pMBLU-GFP for tagging proteins with a C- terminal GFP. The double KO line rak1-rak2 were generated by modifying pMBLU to contain an HtpII selection cassette (hygromycin resistance) by cutting pMBLU with Sal1 and Kpn1 and inserting HtpII amplified from pUNI33 (45). PpRAK2 flanking regions was cloned into the modified vector pMBLU-hyg, and transformed into the single KO line rak1-1 to generate rak1-rak2. By PEG and heat shock method (29), 10-30µg of linearized vectors were transformed into protoplasts of P. patens by homologous recombination. Stable transformants were selected by transferring the protoplasts on cellophane to media containing 50 µg/ml G418 or 30 µg/ml hygromycin B for 2 weeks, followed by transfer to media without selection for 2 weeks, and finally by transfer onto BCDAT with selection for a further 2 weeks (29). Primers used to confirm gene deletions and for cloning are listed in Supplemental Figure 3.

Microscopy and statistical analysis Micrographs of protonemata, gametophytes, sporophyte capsules and organ bundles were taken with a Leica DFC310 FX camera mounted on Stereo Fluorescence Microscpoe Leica MZ FLII using the Leica Application Suite imaging software at the Center for Applied Bioimaging (CAB). Confocal images were acquired with a Zeiss LSM 700 and analyzed with ZEN imaging software (version 2011) with GFP settings (excitation 488 nm and detection 300 to 610 nm). The length and amount of gametophytes, sporophyte capsules, organ bundles and protonemata were measured, and further

113 analysis of the gametophytes were measured by using ImageJ. Microsoft Office was used to analyze statistical variance and were calculated by T-test.

Protein extraction and immunoblotting Unless otherwise stated, three 2 week-old colonies were frozen at the time given, and proteins were extracted by grinding in Lacus buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 15 mM EGTA, 100 mM NaCl, 2 mM DTT, 30 mM β-glycero-phosphate, 0.1% NP-40) supplemented with phosphatase inhibitor (PhosSTOP, Roche) and protease inhibitor cocktails (Complete, Roche). Samples were centrifuged at 16400xg for 30minutes, boiled for 5 min in SDS-loading buffer with DTT, and subjected to 10-15% SDS-PAGE gels and electroblotting. Immunoblots were blocked for 30 min. in TBS-Tween (0.1% v/v) and 5% milk (Sigma Aldrich). Activation and phosphorylation of MPKs were detected by probing with primary antibody anti-p42/p44-erk (1:2000, Cell Signaling Technology), followed by incubation with secondary antibody anti-rabbit-IgG-AP (1:5000, Promega). The alkaline phosphatase-conjugated antibodies were visualized by NBT/BCIP substrate (Roche).

RNA extraction and quantitative RT-PCR RNA was isolated from frozen plant tissue using TRI-reagent (Sigma) according to the manufacturer’s instructions. RNA concentrations were measured in a Nano Drop 1000 (Thermo Scientific) and 1ug of total RNA used for cDNA synthesis using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). Quantitative PCR was performed in 1/10 dilution of the cDNA using the High ROX SYBR green kit (Thermo Scientific) with 10 pmol of each primer and 12.5 ng total RNA in 10 µl. Reactions were run on a CFX 96 Thermocycler (BioRad) with cycle conditions as follows: 10 minutes incubation at 95 °C followed by 40 cycles of 15 seconds incubation at 95 °C and 20 seconds at 54°C. A dissociation stage was performed at the end of the run to confirm the amplification of specific amplicons. Relative expression levels were calculated by the ΔΔCt method using β-TUBULIN as internal normalization control. Primers used for qPCR are in Supplemental Table 7.

114

Supplemental Figure 1: Multiple alignments for MPKs Thr-Glu-Tyr (TEY) and Thr-Asp-Tyr (TDY) motifs in the activation loop are highlighted in yellow and red, respectively. Thr-Asp-Tyr (TEY) motif in MPK2 and MPK5 are highlighted in green.

MPK4a Pp1s149_39V6.1 METSSGTPELKVISTPTYGGHYVKYVVAGTDFEVTARYKPPLRPIGRGAYGIVCSLFDTVTGEEVAVKKIGNAFDNRIDAKRTLRE IKLLRHMDHENVVAITDIIRPPTRENFNDVYIVYELMDTDLHQIIRSNQALTEDHCQYFLYQILRGLKYIHSANVLHRDLKPTNLLVNANCDLKIADFGL ARTLSETDFMTEYVVTRWYRAPELLLNCSAYTAAIDIWSVGCIFMELLNRSALFPGRDYVHQLRLITELIGTPEDRDLGFLRSDNARRYIKHLPRQSPIP LTQKFRGINRSALDLVEKMLVFDPAKRITVEAALAHPYLASLHDINDEPASVSPFEFDFEEPSISEEHIKDLIWREALDCSLGPDDMVQ*

MPK4b Pp1s59_325V6.1 MDVAGAGGGGAADGNIQGVPTHNGEYTQYNIFGNLFEVSRKYVPPIRPIGRGAYGIVCSAVNSETGEEVAIKKIGNAFDNRIDAK RTLREIKLLRHMDHENIVAIRDIIRPPTRENFNDVYIVYELMDTDLHQIIRSNQPLTEDHCQYFLYQLLRGLKYIHSAKVLHRDLKPSNLLLNANCDLKI CDFGLARTTSETDFMTEYVVTRWYRAPELLLNCSEYTAAIDVWSVGCIFMELLNREPLFPGRDYVQQLRLITELIGSPEDHDLGFLRSDNARRYIRQLPR FARQPLDRKFPNMGPAAIDLVEHMLRFDPARRITVEEALAHPYLATLHDINDEPICHSPFEFDFEQPSFTEEHIKELIMMEAIAFNPGNVGDMMS*

RAK1 Pp1s29_285V6.1 MDRHKHYDSGEGSTREKKLLGARDEFPEAEKAVRAAAAKPDPIAEFPSFLIYNRNGLKLNLEAGSGAALSATTKESMHKLLMMNM EVLFGPHEWPAEENMKRWEMVSPEARFIFVRKSTPTIEAGSSDEGHPMVGFVHFRFGLEHEVPVLYIYETQLEKTVQGKGLGKFLMQLLELVARKNNMKA VLLAVHKRNTRALTFYNERLGYKLAIRSASSQQSTQTVTEMKYEILCKTFDVEYTAVVEERQGDMDCESREESAGEASCQTVDAEDQVLDDSRPDTECES RIESVPNTLQGMKYTQYNVRGDKFEVYDKYVMIGPIGHGAYGDVCAFTNRETGEKVAIKKIGNAFQNNTTARRTLREILLLRHTEHDNIIPIRDIIVPAN IEDFHDAYIANELMDTDLHQIVRSTKLDEYHCQFLLYQLLRGLKYIHSANILHRDLKPSNLLINCNDCLLKICDFGLARTSAEDDFLTEYVVTRPYRAPE LLLGSRMYTAAVDMWSVGCIFMEMLTGQPLFPIRSRQEHPVNHLKLITELLGTPDASDLSFLQNPDARQRIQMALLGQERKPLFSRFPQTSAIACDLAEK MLRFNPSNRITAEEALAHPYLAALHDLSDEPTCHLMFDFDAYLPSLTVEHVKTLIWREATLINVQ*

RAK2 Pp1s99_26V6.1 MDRGKHYQYGEGSERQKRLLSRRHEFAEAENAVRAAAAKPNLIEEFPSFRTYNRNGLILMLEAGTGSAQSASTKERMHALLMMN MQVLFGPHEWPAEEKTKQEEMVSHEARFIFVEQNSTSEASSLDEGDSMVGFVHFKFGLEHDVPVLYVYEMQLKRTVQGVGLGKFLMQLLELVARKNNMKA ILVAVHKRNSRALAFYNGSLGYKVATRSSSTQKNTQTSTEMNYEILCKTFDLEDTAVVERQGDQNCESREESGGEATCQPVDAEDQVLEDSCPDMECESS IENVPNLLQGMRYTQYYVRDDKFEVYDKYVMIGPIGHGAYGDVCAFTNKETGEKVAIKKIGNAFQNHTTARRTLREILLLRHTEHDNIIPIRDIIVPANI EDFEDAYIANELMDTDLHQIVRSTKLDEYHCQFLLYQLLRGLKYIHSANILHRDLKPSNLLINCNDCLLKICDFGLARTSAEDDFLTEYVVTRPYRAPEL LLGSRMYTAAVDMWSVGCIFMEMLTGQPLFPIRSRQEHPVNHLKLITELLGTPDASDLSFLQNPDARQRIQMALIGQERKPLFSRFPQTSAAACDLAEKM LRFNPSNRITAEDALAHPYLSALHDVSDEPTCHLLFDFDAYLPNLSVDHVKTLIWREATLINAQ*

MPK3 Pp1s207_63V6.1 MATKVEVPEGTCPPGKHHYMLWRSVFEIDTKYAPIKPIGKGAYGVVCSAKNNETGDRVAIKKITNAFENTTDARRTLREIRLLRHL FHENIIAVKDIMKPVGRQTFNDVYIVYELMDTDLHQIIRSSQTLTDDHCQYFIYQLLRGLKYVHSANVLHRDLKPSNLLLNASCDLKICDFGLARTGSDK GQFMTEYVVTRWYRAPELLLSCDEYTSAIDMWSVGCIFAELLGRKPLFPGKDYIHQLKLIISIIGSPDETDLHFIQSQKARSYIRSLPFTPRVSLARLYP RANPLAIQLIDKMLVFDPRKRITVHEALEHPYLSMLHDATVEPSAPAPFEFDFEDEDLKEDALRERVWNEMLFYHPEAAAET*

MPK7 Pp1s138_117V6.1 MATKVEVPEGTCPPGKHHYMLWRSVFEIDTKYVPMKPIGKGAYGVVCSAKNNEAGDRVAIKKITNAFENTTDARRTLREIRLLR HLFHENIIAVKDIMKPVGRRTFNDVYIVYELMDTDLHQIIRSSQTLTDDHCQYFIYQLLRGLKYIHSANVLHRDLKPSNLLLNASCDLKICDFGLARTGS DKGQFMTEYVVTRWYRAPELLLSCDEYTSAIDMWSVGCIFAELLGRKPLFPGKDYIHQLKLIISIIGSPDETDLHFIQSHKARSYIQSLPFTPRVSLARL YPRANPLAIQLIDRMLVFDPRKRVTVHEALEHPYLSMLYDASQELSAPAPFDFDFEDEDLKEDALRERVWNEMLMYHPEAASEM*

MPK5 Pp1s87_157V6.1 MADAEFFTEYGEANRYRILEVIGKGSYGVVCSAVDTLTGEKVAIKKINDIFEHVSDATRILREIKLLRLLRHPDIVEVKHIMLPP SRRDFKDIYVVFELMESDLHQVIKANDDLTPEHYQFFLYQLLRALKYIHTANVFHRDLKPKNVLANADCKLKICDFGLARVAFSDAPTAIFWTDYVATRW YRAPELCGSFFSKYTPAIDIWSIGCIFAEVLTGKPLFPGKNVVHQLDLMTDMLGSPSSETVQRVRNEKARRYLSTMRKKLPMPFGQKFPNADPLAIRLLE RMLAFDPRDRPTAEEALADPYFKGLAKVDREPSAQPITKMEFEFERRRINKEDVRELIYREILEYHPQMLKEYLNGSDNATFLYPSAVDQFKRQFAHLEE HYGKGTSLGVP*

MPK2 Pp1s80_71V6.1 MADAEFFTEYGEANRYRILEVIGKGSYGVVCSAVDTLTGEKVAIKKINDIFEHVSDATRILREIKLLRLLRHPDIVEVKHIMLP PSRRDFKDIYVVFELMESDLHQVIKANDDLTPEHYQFFLYQLLRALKYIHTANVFHRDLKPKNVLANADCKLKICDFGLARVAFSDAPTAIFWTDYVATR WYRAPELCGSFFSKYTPAIDIWSIGCIFAEVLTGKPLFPGKNVVHQLDLMTDMLGSPSPETVQRVRNEKARRYLSTMRKKPPMPFVQKFPNADPLAIRLL ERMLAFDPRDRPTAEEALADPYFKGLAKVDREPSAQPITKMEFEFERRRINKEDVRELIYREILEYHPQMLKEYLNGSDNATFLYPSAVDQFKRQFAHLE EHYGKGGNNPPLERQHASLPRERVLEFREEASKYQREDSKLHDKHASSGQRNVYSQNSSKSQDGSVGRAGNPAAYVGAKEYTDPRRVSKTASMSTTNSYS GSINPYGRRHSSNKTDRDDRELNAVQSKTESMGLGGSRKVPAAQSVGQ*

SfMPK Sphfalx0021s0079.1 MLETEVSSSCMIGSSHSQQADRQAHRELLLRGNAAGCNLMRYNVRGDVFEVYSKYIPIGPVGRGAYGDVCAFTNSETGEKVA IKKIRNAFQNSTLARRTLREILLLRHTDHDNIIPIKDIVVPASMENFHDAYIANELMDTDLHEVVRTTKLDEYHCQFLLYQLLRGLKYIHSANILHRDLK PSNILINCSDCLLKICDFGLARTSCEDDFLTEYVVTRPYRAPELLLGSRSYSASVDMWSVGCIFVEMLTGQPLFPTQSRQEHPVNHLKLITELLGTPDQS

115

DLSFLQSPEARLRIQSALLGQERKPLASRFPQTSAIACDLAEKMLLFNPSKRITAEEALAHPYLSALHDPSDEPNCHLTFDFDSYLPNLKVEHVKHLIWR EAVAMNSS*

SfMPK Sphfalx0072s0095.1 MLETKVSSCTIDSQQTDTHTHKELLQGTTTSCNLTRYHVREDVFEVYSKYILIGPVGRGAYGDVCAFTNSKTGEKVAIKKIV SAFQNNTLARRTLREILLLRHTNHDNIIPIKDIVVPASIEDFHDAYIANELMDTDLHDVLRTTKLDEYQCQFLLYQLLRGLKYIHSANILHRDLKPSNIL INYSDCLLKICDFGLARTSCEDDFLTEYVVTRPYRAPELLLGSHSYSAAVDMWSVGCIFMEMLTGEPLFPTRSQQEHPVNHLKLITELLGTPDESDLLFL QSPEARVRIKSALVGQERKPLASRFPQTTTVACDLAEKMLQFNPTRRITAEEALAHPYLSALHDPSDEPNCYLTFDFDTYLPNLTVEHVKHLIWQEAVAI NSS*

CLUSTAL W (1.83) multiple sequence alignment

MPK2 MADAEFF------MPK3 MATKVEV------MPK4a METSSGT------MPK4b MDVAGAG------MPK5 MADAEFF------MPK7 MATKVEV------RAK1 MDRHKHYDSGEGSTREKKLLGARDEFPEAEKAVRAAAAKPDPIAEFPSFL RAK2 MDRGKHYQYGEGSERQKRLLSRRHEFAEAENAVRAAAAKPNLIEEFPSFR SfMPK MLETEVS------SfMPK_1 MLETKVS------*

MPK2 ------MPK3 ------P------MPK4a ------P------MPK4b ------GGGAA------MPK5 ------MPK7 ------P------RAK1 IYNRNGLKLNLEAGSGAALSATTKESMHKLLMMNMEVLFGPHEWPAEENM RAK2 TYNRNGLILMLEAGTGSAQSASTKERMHALLMMNMQVLFGPHEWPAEEKT SfMPK ------SSCMI------SfMPK_1 ------SCT-I------

MPK2 ------MPK3 ------MPK4a ------MPK4b ------MPK5 ------MPK7 ------RAK1 KRWEMVSPEARFIFVRKSTPTIEAGSSDEGHPMVGFVHFRFGLEHEVPVL RAK2 KQEEMVSHEARFIFVEQNS-TSEASSLDEGDSMVGFVHFKFGLEHDVPVL SfMPK ------SfMPK_1 ------

MPK2 ------MPK3 ------MPK4a ------MPK4b ------MPK5 ------MPK7 ------RAK1 YIYETQLEKTVQGKGLGKFLMQLLELVARKNNMKAVLLAVHKRNTRALTF RAK2 YVYEMQLKRTVQGVGLGKFLMQLLELVARKNNMKAILVAVHKRNSRALAF SfMPK ------SfMPK_1 ------

MPK2 ------MPK3 ------MPK4a ------MPK4b ------MPK5 ------MPK7 ------RAK1 YNERLGYKLAIRSASSQQSTQTVTEMKYEILCKTFDVEYTAVVEERQGDM RAK2 YNGSLGYKVATRSSSTQKNTQTSTEMNYEILCKTFDLEDTAVV-ERQGDQ SfMPK ------GSSHSQQADRQAHREL------SfMPK_1 ------DSQQTDTHTHKEL------

MPK2 ------

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MPK3 ------EG------TCPPG-KH MPK4a ------EL------KV--ISTPTYGGHYV MPK4b ------DG------NI--QGVPTHNGEYT MPK5 ------MPK7 ------EG------TCPPG-KH RAK1 DCESREESAGEASCQTVDAEDQVLDDSRPDTECESRIESVPNTLQGMKYT RAK2 NCESREESGGEATCQPVDAEDQVLEDSCPDMECESSIENVPNLLQGMRYT SfMPK ------LLRGNAAGCNLM SfMPK_1 ------LQGTTTSCNLT

MPK2 ------TEYGEANRYR-ILEVIGKGSYGVVCSAVDTLTGEKVAIKKINDI MPK3 HYMLWRSVFEIDTKYA-PIKPIGKGAYGVVCSAKNNETGDRVAIKKITNA MPK4a KYVVAGTDFEVTARYKPPLRPIGRGAYGIVCSLFDTVTGEEVAVKKIGNA MPK4b QYNIFGNLFEVSRKYVPPIRPIGRGAYGIVCSAVNSETGEEVAIKKIGNA MPK5 ------TEYGEANRYR-ILEVIGKGSYGVVCSAVDTLTGEKVAIKKINDI MPK7 HYMLWRSVFEIDTKYV-PMKPIGKGAYGVVCSAKNNEAGDRVAIKKITNA RAK1 QYNVRGDKFEVYDKYV-MIGPIGHGAYGDVCAFTNRETGEKVAIKKIGNA RAK2 QYYVRDDKFEVYDKYV-MIGPIGHGAYGDVCAFTNKETGEKVAIKKIGNA SfMPK RYNVRGDVFEVYSKYI-PIGPVGRGAYGDVCAFTNSETGEKVAIKKIRNA SfMPK_1 RYHVREDVFEVYSKYI-LIGPVGRGAYGDVCAFTNSKTGEKVAIKKIVSA : :* : :*:*:** **: : :*:.**:*** .

MPK2 FEHVSDATRILREIKLLRLLRHPDIVEVKHIMLPPSRRDFKDIYVVFELM MPK3 FENTTDARRTLREIRLLRHLFHENIIAVKDIMKPVGRQTFNDVYIVYELM MPK4a FDNRIDAKRTLREIKLLRHMDHENVVAITDIIRPPTRENFNDVYIVYELM MPK4b FDNRIDAKRTLREIKLLRHMDHENIVAIRDIIRPPTRENFNDVYIVYELM MPK5 FEHVSDATRILREIKLLRLLRHPDIVEVKHIMLPPSRRDFKDIYVVFELM MPK7 FENTTDARRTLREIRLLRHLFHENIIAVKDIMKPVGRRTFNDVYIVYELM RAK1 FQNNTTARRTLREILLLRHTEHDNIIPIRDIIVPANIEDFHDAYIANELM RAK2 FQNHTTARRTLREILLLRHTEHDNIIPIRDIIVPANIEDFEDAYIANELM SfMPK FQNSTLARRTLREILLLRHTDHDNIIPIKDIVVPASMENFHDAYIANELM SfMPK_1 FQNNTLARRTLREILLLRHTNHDNIIPIKDIVVPASIEDFHDAYIANELM *:: * * **** *** * ::: : .*: * . *.* *:. ***

MPK2 ESDLHQVIKANDDLTPEHYQFFLYQLLRALKYIHTANVFHRDLKPKNVLA MPK3 DTDLHQIIRSSQTLTDDHCQYFIYQLLRGLKYVHSANVLHRDLKPSNLLL MPK4a DTDLHQIIRSNQALTEDHCQYFLYQILRGLKYIHSANVLHRDLKPTNLLV MPK4b DTDLHQIIRSNQPLTEDHCQYFLYQLLRGLKYIHSAKVLHRDLKPSNLLL MPK5 ESDLHQVIKANDDLTPEHYQFFLYQLLRALKYIHTANVFHRDLKPKNVLA MPK7 DTDLHQIIRSSQTLTDDHCQYFIYQLLRGLKYIHSANVLHRDLKPSNLLL RAK1 DTDLHQIVRST-KLDEYHCQFLLYQLLRGLKYIHSANILHRDLKPSNLLI RAK2 DTDLHQIVRST-KLDEYHCQFLLYQLLRGLKYIHSANILHRDLKPSNLLI SfMPK DTDLHEVVRTT-KLDEYHCQFLLYQLLRGLKYIHSANILHRDLKPSNILI SfMPK_1 DTDLHDVLRTT-KLDEYQCQFLLYQLLRGLKYIHSANILHRDLKPSNILI ::***:::::. * : *:::**:**.***:*:*:::******.*:*

MPK2 NA-DCKLKICDFGLARVAFSDAPTAIFWTDYVATRWYRAPELCGSFFSKY MPK3 NA-SCDLKICDFGLARTGSDK---GQFMTEYVVTRWYRAPELLLS-CDEY MPK4a NA-NCDLKIADFGLARTLSE----TDFMTEYVVTRWYRAPELLLN-CSAY MPK4b NA-NCDLKICDFGLARTTSE----TDFMTEYVVTRWYRAPELLLN-CSEY MPK5 NA-DCKLKICDFGLARVAFSDAPTAIFWTDYVATRWYRAPELCGSFFSKY MPK7 NA-SCDLKICDFGLARTGSDK---GQFMTEYVVTRWYRAPELLLS-CDEY RAK1 NCNDCLLKICDFGLARTSAE----DDFLTEYVVTRPYRAPELLLG-SRMY RAK2 NCNDCLLKICDFGLARTSAE----DDFLTEYVVTRPYRAPELLLG-SRMY SfMPK NCSDCLLKICDFGLARTSCE----DDFLTEYVVTRPYRAPELLLG-SRSY SfMPK_1 NYSDCLLKICDFGLARTSCE----DDFLTEYVVTRPYRAPELLLG-SHSY * .* ***.******. . * *:**.** ****** . *

MPK2 TPAIDIWSIGCIFAEVLTGKPLFPGKN----VVHQLDLMTDMLGSPSPET MPK3 TSAIDMWSVGCIFAELLGRKPLFPGKD----YIHQLKLIISIIGSPDETD MPK4a TAAIDIWSVGCIFMELLNRSALFPGRD----YVHQLRLITELIGTPEDRD MPK4b TAAIDVWSVGCIFMELLNREPLFPGRD----YVQQLRLITELIGSPEDHD MPK5 TPAIDIWSIGCIFAEVLTGKPLFPGKN----VVHQLDLMTDMLGSPSSET MPK7 TSAIDMWSVGCIFAELLGRKPLFPGKD----YIHQLKLIISIIGSPDETD RAK1 TAAVDMWSVGCIFMEMLTGQPLFPIRSRQEHPVNHLKLITELLGTPDASD RAK2 TAAVDMWSVGCIFMEMLTGQPLFPIRSRQEHPVNHLKLITELLGTPDASD SfMPK SASVDMWSVGCIFVEMLTGQPLFPTQSRQEHPVNHLKLITELLGTPDQSD SfMPK_1 SAAVDMWSVGCIFMEMLTGEPLFPTRSQQEHPVNHLKLITELLGTPDESD :.::*:**:**** *:* ..*** :. :::* *: .::*:*.

MPK2 VQRVRNEKARRYLS-TMRKKPPMPFVQKFPNADPLAIRLLERMLAFDPRD MPK3 LHFIQSQKARSYIR-SLPFTPRVSLARLYPRANPLAIQLIDKMLVFDPRK MPK4a LGFLRSDNARRYIK-HLPRQSPIPLTQKFRGINRSALDLVEKMLVFDPAK

117

MPK4b LGFLRSDNARRYIR-QLPRFARQPLDRKFPNMGPAAIDLVEHMLRFDPAR MPK5 VQRVRNEKARRYLS-TMRKKLPMPFGQKFPNADPLAIRLLERMLAFDPRD MPK7 LHFIQSHKARSYIQ-SLPFTPRVSLARLYPRANPLAIQLIDRMLVFDPRK RAK1 LSFLQNPDARQRIQMALLGQERKPLFSRFPQTSAIACDLAEKMLRFNPSN RAK2 LSFLQNPDARQRIQMALIGQERKPLFSRFPQTSAAACDLAEKMLRFNPSN SfMPK LSFLQSPEARLRIQSALLGQERKPLASRFPQTSAIACDLAEKMLLFNPSK SfMPK_1 LLFLQSPEARVRIKSALVGQERKPLASRFPQTTTVACDLAEKMLQFNPTR : ::. .** : : .: : * * ::** *:*

MPK2 RPTAEEALADPYFKGLAKVDREPSAQPITKMEFEFERRRINKEDVRELIY MPK3 RITVHEALEHPYLSMLHDATVEPSAP--APFEFDFEDEDLKEDALRERVW MPK4a RITVEAALAHPYLASLHDINDEPASV--SPFEFDFEEPSISEEHIKDLIW MPK4b RITVEEALAHPYLATLHDINDEPICH--SPFEFDFEQPSFTEEHIKELIM MPK5 RPTAEEALADPYFKGLAKVDREPSAQPITKMEFEFERRRINKEDVRELIY MPK7 RVTVHEALEHPYLSMLYDASQELSAP--APFDFDFEDEDLKEDALRERVW RAK1 RITAEEALAHPYLAALHDLSDEPTCH--LMFDFDAYLPSLTVEHVKTLIW RAK2 RITAEDALAHPYLSALHDVSDEPTCH--LLFDFDAYLPNLSVDHVKTLIW SfMPK RITAEEALAHPYLSALHDPSDEPNCH--LTFDFDSYLPNLKVEHVKHLIW SfMPK_1 RITAEEALAHPYLSALHDPSDEPNCY--LTFDFDTYLPNLTVEHVKHLIW * *.. ** .**: * . * . ::*: :. : :: :

MPK2 REILEYHPQMLKEYLNGSDNATFLYPSAVDQFKRQFAHLEEHYGKGGNNP MPK3 NEMLFYHPEAA------MPK4a REALDCSLGP------MPK4b MEAIAFNPGNV------MPK5 REILEYHPQMLKEYLNGSDNATFLYPSAVDQFKRQFAHLEEHYGK----- MPK7 NEMLMYHPEAA------RAK1 REATLIN------RAK2 REATLIN------SfMPK REAVAMN------SfMPK_1 QEAVAIN------*

MPK2 PLERQHASLPRERVLEFREEASKYQREDSKLHDKHASSGQRNVYSQNSSK MPK3 ------MPK4a ------MPK4b ------MPK5 ------MPK7 ------RAK1 ------RAK2 ------SfMPK ------SfMPK_1 ------

MPK2 SQDGSVGRAGNPAAYVGAKEYTDPRRVSKTASMSTTNSYSGSINPYGRRH MPK3 ------MPK4a ------MPK4b ------MPK5 ------MPK7 ------RAK1 ------RAK2 ------SfMPK ------SfMPK_1 ------

MPK2 SSNKTDRDDRELNAVQSKTESMGLGGSRKVPAAQSVGQ MPK3 ------AET MPK4a ------DDMVQ MPK4b ------GDMMS MPK5 ------GTSLGVP MPK7 ------SEM RAK1 ------VQ RAK2 ------AQ SfMPK ------SS

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Peptide sequences of MPK2/5 and reverse BLASTp search for Arabidopsis thaliana MPKs Thr-Asp-Tyr (TDY) motifs in the activation loop are highlighted in red. Thr-Asp-Tyr (TEY) motif in the N-terminal are highlighted in green.

>Pp1s87_157V6.1 PpMPK5 MADAEFFTEYGEANRYRILEVIGKGSYGVVCSAVDTLTGEKVAIKKINDIFEHVSDATRILREIKLLRLLRHPDIVEVKHIMLPP SRRDFKDIYVVFELMESDLHQVIKANDDLTPEHYQFFLYQLLRALKYIHTANVFHRDLKPKNVLANADCKLKICDFGLARVAFSDAPTAIFWTDYVATRW YRAPELCGSFFSKYTPAIDIWSIGCIFAEVLTGKPLFPGKNVVHQLDLMTDMLGSPSSETVQRVRNEKARRYLSTMRKKLPMPFGQKFPNADPLAIRLLE RMLAFDPRDRPTAEEALADPYFKGLAKVDREPSAQPITKMEFEFERRRINKEDVRELIYREILEYHPQMLKEYLNGSDNATFLYPSAVDQFKRQFAHLEE HYGKGTSLGVP*

>Pp1s80_71V6.1 PpMPK2 MADAEFFTEYGEANRYRILEVIGKGSYGVVCSAVDTLTGEKVAIKKINDIFEHVSDATRILREIKLLRLLRHPDIVEVKHIMLP PSRRDFKDIYVVFELMESDLHQVIKANDDLTPEHYQFFLYQLLRALKYIHTANVFHRDLKPKNVLANADCKLKICDFGLARVAFSDAPTAIFWTDYVATR WYRAPELCGSFFSKYTPAIDIWSIGCIFAEVLTGKPLFPGKNVVHQLDLMTDMLGSPSPETVQRVRNEKARRYLSTMRKKPPMPFVQKFPNADPLAIRLL ERMLAFDPRDRPTAEEALADPYFKGLAKVDREPSAQPITKMEFEFERRRINKEDVRELIYREILEYHPQMLKEYLNGSDNATFLYPSAVDQFKRQFAHLE EHYGKGGNNPPLERQHASLPRERVLEFREEASKYQREDSKLHDKHASSGQRNVYSQNSSKSQDGSVGRAGNPAAYVGAKEYTDPRRVSKTASMSTTNSYS GSINPYGRRHSSNKTDRDDRELNAVQSKTESMGLGGSRKVPAAQSVGQ*

>AT5G19010 MPK16 MQPDHRKKSSVEVDFFTEYGEGSRYRIEEVIGKGSYGVVCSAYDTHTGEKVAIKKINDIFEHVSDATRILREIKLLRLLRHPDIVEIKHILLPPSRREFR DIYVVFELMESDLHQVIKANDDLTPEHYQFFLYQLLRGLKYIHTANVFHRDLKPKNILANADCKLKICDFGLARVAFNDTPTAIFWTDYVATRWYRAPEL CGSFFSKYTPAIDIWSIGCIFAELLTGKPLFPGKNVVHQLDLMTDMLGTPSAEAIGRVRNEKARRYLSSMRKKKPIPFSHKFPHTDPLALRLLEKMLSFE PKDRPTAEEALADVYFKGLAKVEREPSAQPVTKLEFEFERRRITKEDVRELIYRESLEYHPKMLKEYLDGSEPTNFMYPSAVEHFKKQFAYLEEHYKNGT SHNPPERQQHASLPRACVLYSDNNHPVAQQSSAEVTDGLSKCSIRDERPRGADRNAQMPMSRIPINVPQTIQGAAVARPGKVVGSVLRYNNCGAATGVEA LEQQQRRMVRNPAAASQYPKRTQPCKSNRGDEDCATAAEGPSRLKPNTQYIPQKVSAAQDTAMSRWY

>AT3G18040 MPK9 MDPHKKVALETEFFTEYGEASRYQIQEVIGKGSYGVVASAIDTHSGEKVAIKKINDVFEHVSDATRILREIKLLRLLRHPDIVEIKHVMLPPSRREFRDI YVVFELMESDLHQVIKANDDLTPEHYQFFLYQLLRGLKFIHTANVFHRDLKPKNILANSDCKLKICDFGLARVSFNDAPSAIFWTDYVATRWYRAPELCG SFFSKYTPAIDIWSIGCIFAEMLTGKPLFPGKNVVHQLDIMTDLLGTPPPEAIARIRNEKARRYLGNMRRKPPVPFTHKFPHVDPLALRLLHRLLAFDPK DRPSAEEALADPYFYGLANVDREPSTQPIPKLEFEFERRKITKEDVRELIYREILEYHPQMLQEYLRGGEQTSFMYPSGVDRFKRQFAHLEENYGKGEKG SPLQRQHASLPRERVPAPKKENGSHNHDIENRSIASLVTTLESPPTSQHEGSDYRNGTSQTGYSARSLLKSASISASKCIGMKPRNKSEYGESNNDTVDA LSQKVAALHT

>AT1G73670 MPK15 MGGGGNLVDGVRRWLFFQRRPSSSSSSNNHDQIQNPPTVSNPNDDEDLKKLTDPSKLRQIKVQQRNHLPMEKKGIPNAEFFTEYGEANRYQIQEVVGKGS YGVVGSAIDTHTGERVAIKKINDVFDHISDATRILREIKLLRLLLHPDVVEIKHIMLPPSRREFRDVYVVFELMESDLHQVIKANDDLTPEHHQFFLYQL LRGLKYVHAANVFHRDLKPKNILANADCKLKICDFGLARVSFNDAPTAIFWTDYVATRWYRAPELCGSFFSKYTPAIDIWSVGCIFAEMLLGKPLFPGKN VVHQLDIMTDFLGTPPPEAISKIRNDKARRYLGNMRKKQPVPFSKKFPKADPSALRLLERLIAFDPKDRPSAEEALADPYFNGLSSKVREPSTQPISKLE FEFERKKLTKDDIRELIYREILEYHPQMLEEYLRGGNQLSFMYPSGVDRFRRQFAHLEENQGPGGRSNALQRQHASLPRERVPASKNETVEERSNDIERR TTAAVASTLDSPKASQQAEGTENGGGGGYSARNLMKSSSISGSKCIGVQSKTNIEDSIVEEQDETVAVKVASLHNS

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Figure 2: Multiple alignments for NAT

PpNAT.H Pp3c17_14350V3.1 MDRKKQSSKEKKLKRKEELAKKQSIDEFVRVANAKAAPIEEFPSFLKYERNGLNLIMEAGRGDSLSPPVKQYVQTLLKVNMEEP YGPEEWPAEEKNKRREMVSPDARYIFVKQPCSNSTEILPTDRSNNLLWKGEGDPIVAFVHYRFVVEHEVPALYVYEIQVEQAVQGKGLGKFLMQFLELIA RKNGMKAMLLTLQKRNVRALAFYTGKLRFKIAAISPSRWANTLIGAAKSYEILCKTFDPDAKSILEDGN*

SfNAT Sphfalx0292s0009.1 MDRKKQSVSKDKKLKRKEELAKKKAIDETVRTAAAKAAPIEEFPSFLKYERNGLNLQLEAELGEKLSPPLKQYIQDLLKLNM EGPFGPDEWPAEEKKKRRDMVSPDARYIIVREKNLSQDSKFSSDSNLGRSDHHHCAGLWGANGGDPVVAFVHYRFVVEHDVPALYVFEIQLEQSIQGKGF GKFLMQFLELIARKNNMKAVLLTVQKRNTRAMAFYTQKLRFVVASISPSRWQDTLTGAEMTYEILCKTFDVEAKAILEEGTHLGAET*

AtNAT AT1G18335.1 MDPSPTESLQTWRTNETEGRESSVWRAMDLKKRRKILEKKKTIHDLIKRASSIDDPLSPFDSFRRYRRNDLSLYLESGRGDRLSSSVKH HIQKLLKTNMEGFYGSDWPIQAKVKRKEMSSADAHYIFVRELRFGKAYETSTQRTCMEGCNQIAGFVHYRFILEEEIPVLYVYEIQLESRVQGKGLGEFL MQLIELIASKNRMSAIVLTVLTSNALAMTFYMSKLGYRISSISPSKANLPTLSVKYEILCKTFDSEAKSVLENDEEPTKD*

ScNAT4 NP_013785.1 MRSSVYSENTYNCIRTSKEHLTERRRVAMAPMFQHFLNLCVEKFPESIEHKDTDGNGNFTTAILEREIIY IPEDDTDSIDSVDSLKCINYKLHKSRGDQVLDACVQLIDKHLGAKYRRASRIMYGNRKPWKANKLAEMKS AGLVYVCYWDNGVLGAFTSFMLTEETGLVEGDALHEVSVPVIYLYEVHVASAHRGHGIGRRLLEHALCDG VARHTRRMCDNFFGVALTVFSDNTRARRLYEALGFYRAPGSPAPASPTIRHTRHGGGRVVVPCDPLYYVY CLHMP

HsNAT EAW49729.1 MTLCALIRGVHQKNKSTSGFDIINMLMGFDKAELCMKNLMESLDSLLCAEGSESLKSLCLKLLLCLVTVT DNISQNTILEYVMINSIFEAILQILSHPPSRREHGYDAVVLLALLVNYRKYESVNPYIVKLSIVDDEATL NGMGLVIAQALSEYNRQYKDKEEEHQSGFFSALTNMVGSMFIADAHEKISVQTNEAILLALYEAVHLNRN FITVLAQVGHIKNPSVFFSLNFIPINSIEKPIWLYLFKPCFALSHPEMGLVTTPVSPAPTTPPMRHRKKA ADKNLPCRPLVCAVLDLMVEFIVTHMMKEFPMDLYIRCIQVVHKLLCYQKKCRVRLHYTWRELWSALINL LKFLMSNETVLLAKHNIFTLALMIVNLFNMFITYGDTFLPTPSSYDELYYEIIRMHQSFDNLYSMVLRLS TNAGQWKEAASKVTHALVNIRAIINHFNPKIESYAAVNHISQLSEEQVLEVVRANYDTLTLKLQDGLDQY ERYSEQHKEAAFFKELVRSISTNVRRNLAFHTLSQEVLLKEFSTIS

CLUSTAL W (1.83) multiple sequence alignment

AtNAT MDPSPTESLQTWRTNETEGRESSVW-RAMDLKKRRKILEK------HsNAT MTLCALIRGVH-----QKN--KSTS-GFDIINMLMGFDKAELCMKNLMES PpNAT.H MDRKKQS------SKEKKLK-RKEELAKKQSI------RAK1 MDRHKHYD------SGEGSTR-EKKLLGARDEFPEA------RAK2 MDRGKHYQ------YGEGSER-QKRLLSRRHEFAEA------ScNAT4 MRSSVYSE------NTYNCIRTSKEHLTERRRVAM------SfNAT MDRKKQSV------SKDKKLK-RKEELAKKKAI------* : .

AtNAT ------KKTIHDLIKRAS---SID HsNAT LDSLLCAEGSESLKSLCLKLLLCLVTVTDNISQNTILEYVMINSIFEAIL PpNAT.H ------DEFVRVAN---AKA RAK1 ------EKAVRAAA---AKP RAK2 ------ENAVRAAA---AKP ScNAT4 ------APMFQH---FLN SfNAT ------DETVRTAA---AKA

AtNAT DPLSPFDSFRRYRR------NDL HsNAT QILSHPPSRREHGY------DAV PpNAT.H APIEEFPSFLKYER------NGL RAK1 DPIAEFPSFLIYNR------NGL RAK2 NLIEEFPSFRTYNR------NGL ScNAT4 LCVEKFPESIEHKDTDGNGNFTTAILEREIIYIPEDDTDSIDSVDSLKCI SfNAT APIEEFPSFLKYER------NGL : . : . :

AtNAT SLYLESGRGDRLSSSVKHHIQKLLKTN------MEGFYGS-DWPIQA HsNAT V-LLALLVNYRKYESVNPYIVKLSIVDDEATLNGMGLVIAQALSEYNRQY PpNAT.H NLIMEAGRGDSLSPPVKQYVQTLLKVN------MEEPYGPEEWPAEE RAK1 KLNLEAGSGAALSATTKESMHKLLMMN------MEVLFGPHEWPAEE RAK2 ILMLEAGTGSAQSASTKERMHALLMMN------MQVLFGPHEWPAEE ScNAT4 NYKLHKSRGDQVLDACVQLIDKHLGAKYR-----RASRIMYGNR-KPWKA SfNAT NLQLEAELGEKLSPPLKQYIQDLLKLN------MEGPFGPDEWPAEE

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: . . : . . :

AtNAT KVKRKEMSSADAHYIFVRELRFGKAYETSTQRTC------MEG HsNAT KDKEEEHQSG------PpNAT.H KNKRREMVSPDARYIFVKQPCSNSTEILPTDRSN------NLL-WKGE RAK1 NMKRWEMVSPEARFIFVRKSTPTIEA-GS------SDE RAK2 KTKQEEMVSHEARFIFVEQNST-SEA-SS------LDE ScNAT4 -NKLAEMKSAGLVYVCYWDNGVLGA--FT------SFM SfNAT KKKRRDMVSPDARYIIVREKNLSQDSKFSSDSNLGRSDHHHCAGLWGANG * : *

AtNAT CNQIAGFVHYRFILEEEIPVLYVYEIQLESRVQGKGLGEFLMQ--LIELI HsNAT ------F---FSALTNMVGSMFIADAHEKISVQTN--EAILLA--LYEAV PpNAT.H GDPIVAFVHYRFVVEHEVPALYVYEIQVEQAVQGKGLGKFLMQ--FLELI RAK1 GHPMVGFVHFRFGLEHEVPVLYIYETQLEKTVQGKGLGKFLMQ--LLELV RAK2 GDSMVGFVHFKFGLEHDVPVLYVYEMQLKRTVQGVGLGKFLMQ--LLELV ScNAT4 LTEETGLVEGDALHEVSVPVIYLYEVHVASAHRGHGIGRRLLEHALCDGV SfNAT GDPVVAFVHYRFVVEHDVPALYVFEIQLEQSIQGKGFGKFLMQ--FLELI : : ::: : : : *: : : :

AtNAT ASK-----NRMSAIVLTVLT-SNAL------AMTFY----- HsNAT HLN-----RNFITVLAQVGHIKNPSVFFSLNFIPINSIEKPIWLYLFKPC PpNAT.H ARK-----NGMKAMLLTLQK-RNVR------ALAFY----- RAK1 ARK-----NNMKAVLLAVHK-RNTR------ALTFY----- RAK2 ARK-----NNMKAILVAVHK-RNSR------ALAFY----- ScNAT4 ARHTRRMCDNFFGVALTVFS-DNTR------ARRLY----- SfNAT ARK-----NNMKAVLLTVQK-RNTR------AMAFY----- : : : : * . :*

AtNAT ----MSKLGYRISSISPSKA--NLPTLSVKYEILCKTFDSEAKSVLENDE HsNAT FALSHPEMGLVTTPVSPAPTTPP------PpNAT.H ----TGKLRFKIAAISPSRWANTLIGAAKSYEILCKTFDPDAKSILED-- RAK1 ----NERLGYKLAIRSASSQQSTQTVTEMKYEILCKTFDVEYTAVVEERQ RAK2 ----NGSLGYKVATRSSSTQKNTQTSTEMNYEILCKTFDLEDTAVVERQG ScNAT4 ----E-ALGFYRAPGSPAPASPTIRHTRHGGG------SfNAT ----TQKLRFVVASISPSRWQDTLTGAEMTYEILCKTFDVEAKAILEEGT : : *.:

AtNAT ------HsNAT ------PpNAT.H ------RAK1 GDMDCESREESAGEASCQTVDAEDQVLDDSRPDTECESRIESVPNTLQGM RAK2 -DQNCESREESGGEATCQPVDAEDQVLEDSCPDMECESSIENVPNLLQGM ScNAT4 ------SfNAT ------

AtNAT ------HsNAT ------PpNAT.H ------RAK1 KYTQYNVRGDKFEVYDKYVMIGPIGHGAYGDVCAFTNRETGEKVAIKKIG RAK2 RYTQYYVRDDKFEVYDKYVMIGPIGHGAYGDVCAFTNKETGEKVAIKKIG ScNAT4 ------SfNAT ------

AtNAT ------HsNAT ------MRHRKKAADKNLPCRPLVCA--VLDLMVEFIV PpNAT.H ------RAK1 NAFQNNTTARRTLREILLLRHTEH--DNIIPIRDIIVPANIEDFHDAYIA RAK2 NAFQNHTTARRTLREILLLRHTEH--DNIIPIRDIIVPANIEDFEDAYIA ScNAT4 ------RVVVPCDP------SfNAT ------

AtNAT ------HsNAT THMMKEFPMDLYIRCIQVVHKLLCYQKKCRVRLHYTWRELWSALINLLKF PpNAT.H ------RAK1 NELMDT-----DLH--QIVRSTKLDEYHCQFLL------YQLLRGLKY RAK2 NELMDT-----DLH--QIVRSTKLDEYHCQFLL------YQLLRGLKY ScNAT4 ------SfNAT ------

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AtNAT ------HsNAT LMSNETVLLAKHNIFTLALMIVNLFNMFITYGDTFLPTPSSYDELYYEII PpNAT.H ------RAK1 IHSANI----LHRDLKPSNLLINCNDCLLKICDFGLARTSAEDDFLTEYV RAK2 IHSANI----LHRDLKPSNLLINCNDCLLKICDFGLARTSAEDDFLTEYV ScNAT4 ------SfNAT ------

AtNAT ------HsNAT --RMHQSFDNLYSMVLRLSTNA-GQWKEAAS----KVTHALVNIRA---- PpNAT.H ------RAK1 VTRPYRAPELLLG--SRMYTAAVDMWSVGCIFMEMLTGQPLFPIRSRQEH RAK2 VTRPYRAPELLLG--SRMYTAAVDMWSVGCIFMEMLTGQPLFPIRSRQEH ScNAT4 ------SfNAT ------

AtNAT ------HsNAT ------PpNAT.H ------RAK1 PVNHLKLITELLGTPDASDLSFLQNPDARQRIQMALLGQERKPLFSRFPQ RAK2 PVNHLKLITELLGTPDASDLSFLQNPDARQRIQMALIGQERKPLFSRFPQ ScNAT4 ------SfNAT ------

AtNAT ------HsNAT ------IINHFNPK--IESYAAVNHISQLSEEQVLEVVRANYDTLT PpNAT.H ------RAK1 TSAIACDLAEKMLRFNPSNRITAEEALAH------PYLAALHDLSDEPT RAK2 TSAAACDLAEKMLRFNPSNRITAEDALAH------PYLSALHDVSDEPT ScNAT4 ------SfNAT ------

AtNAT ------HsNAT LKLQDGLDQYE-RYSEQHKEAAFFKELVRSISTNVRRNLAFHTLSQEVLL PpNAT.H ------RAK1 CHLMFDFDAYLPSLTVEHVKTLIWREA------RAK2 CHLLFDFDAYLPNLSVDHVKTLIWREA------ScNAT4 ------LYYVYC SfNAT ------

AtNAT --EPTKD HsNAT KEFSTIS PpNAT.H -----GN RAK1 -TLINVQ RAK2 -TLINAQ ScNAT4 ---LHMP SfNAT -HLGAET

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Figure 3: Genotyping MPKs, NAT KO’s & primers

Vectors: Deletion constructs were generated by cloning 900-1500bp of genomic flanking regions of the target gene into left side (left border, LB) and right side (right border, RB) of the selection marker in the vector.

Primers for pMBLU-G418 Sb27R ggcaatggaatccgaggaggt Sb24F GGTATCAGAGCCATGAATAGGTC Sb59R tgtgagcggataacaatttcac Sb410F GGTTATTGTCTCATGAGCGGA Primers pMBLU-HYG Sb468F cgccaactttgaaaacaactt Sb410F GGTTATTGTCTCATGAGCGGA Sb417R TAGCTTGGCGTAATCATGGTC Sb467R TGCTCCACCATGTTGGCAAGC

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Primers with overhangs for cloning in the LB and RB:

Primers Direction Primers with overhangs Product size Borders MPK2 (Pp1s80_71V6.1) ssB1 F GGCTTAAUAGCACCTGTGGAATTAGTGAGG 901bp LB ssB2 R GGTTTAAUTCCGAATCATATTAGCGGAGGC ss57F GGCGCGAUCGATGAGTGCAGGTGAGGTA 1136bp RB ss58R GGTGCGAUCCATCGTCTTGGTTTCCTGT MPK5 (Pp1s87_157V6.1) ssD1 F GGCTTAAUAAGAAGCGTTCACAAACCATCG 1171bp LB ssD2 R GGTTTAAUACCCGTCTCACTTCTTGGAATC ssD3 F GGCGCGAUAGTGACCCCGACAAGGTATCTA 1319bp RB ssD4 R GGTGCGAUTCCTCACTCAGACATCCATCCT RAK1 (Pp1s29_285V6.1) ssA1.1 F GGCTTAAUTGTTCCCAACCATTGTCCTT LB ssA2 R GGTTTAAUCCTGAGCGGTCTGTCTAGTTTT ssA3 F GGCGCGAUAACTGCCGGAAAATGTCATGTG 1197bp RB ssA4 R GGTGCGAUGGTCACAAGAGCTCAGCTTACT RAK2 (Pp1s99_26V6.1) ssE1 F GGCTTAAUGACAAGGGAGGTGCCATTTCTA 1111bp LB ssE2 R GGTTTAAUTGCAAGGAGAAGTCACAGATCC ssE3 F GGCGCGAUTACCATTACATCGGCAACCCAA 923bp RB ssE4 R GGTGCGAUATCTAGCCATGCAACCTTCGAA MPK3 (Pp1s207_63V6.1) ssC1 F GGCTTAAUTGGCCAGGGCTTCAGATTTTAT 1019bp LB ssC2 R GGTTTAAUTACAACAACCCTCCACCACAAA ssC3 F GGCGCGAUCTTCGTGGGTGTTTTAAGGCTG 1038bp RB ssC4 R GGTGCGAUTGTTCATGAACACACGAAACGG MPK7 (Pp1s138_117V6.1) ssF1.1 F GGCTTAAUACTACCACCACGACGTCTCC 1041BP LB ssF2.1 R GGTTTAAUCGCTGCAGATTCAGGTATGA ssF3 F GGCGCGAUGTCCAGCGTTTGATCGAATCTG 1015bp RB ssF4 R GGTGCGAUACAATCGATACCTTTGGGCCTT NATH (Pp3c17_14350) ss184 F GGCTTAAUGCCCACATCTTCCAGTTCGA 1136bp LB ss185 R GGTTTAAUATAACCCTTTGTGGAGCCCG ss186 F GGCGCGAUCCCTCCGGACTGCTTCAAAT 926bp RB ss187 R GGTGCGAUTGGAAGCCAATAGCCATGCA

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Primers for genotyping KO lines:

Name Code Primer Description rak1 ss182F p1 TGCAAATATAAGCTAGTCTTTGA Genotyping LB RAK1 KO ss183R p2 CCCAATAGTCATCTCCTCTC ss11F p3 TGGTTGCTAAGCACGATGAG Genotype RAK1 KO - part of the gene ss12R p4 CGGAAATTTCTCACCGAAAA ss13F p5 GAGCATCCCGTGAATCATTT Genotype RB RAK1 KO ss14R p6 TGGAACATGCCTACCAAAAA ss144F p1 GAAAGTGTATTAAAATCAATAC Genotyping LB RAK1 KO with sb27R (in the cassette) ss145R p6 GTTGACACCTTTGACTCTCAATC Genotyping RB RAK1 KO with sb24F (in the cassette) mpk3 ss17F p1 AAGTAACGGAGCCCTTGGAC Genotype LB MPK3 KO ss18R p2 CGGTCCTATCATGGTCGAGT ss19F p3 CTCCTGTCGTGCGATGAGTA Genotype MPK3 KO - part of the gene ss20R p4 CCACAGTGGCGTCATGTAAC ss21F p5 GGCGAGATTTGGTTGATGTT Genotype RB MPK3 KO ss22R p6 CACGAAACGGAATACTGTGG p1 Genotype LB MPK3 KO with sb27R (in the cassette) p6 Genotype RB MPK3 KO with sb24F (in the cassette) mpk5 ss25F p1 AACAATGGCCTCTGATCCTG Genotype LB MPK5 KO ss26R p2 AATGTGACGCCCACCTGTAT ss27F p3 ATTAATGCCCTCTGCACACC Genotype MPK5 - part of the gene ss28R p4 AAGGCCCATGAAACTTGTTG ss29F p5 AAGCAAAAGTGATCGGGATG Genotype RB MPK5 KO ss30R p6 TCTGTCACGTCTGGCACTTC p1 Genotype LB MPK5 KO with sb27R (in the cassette) p6 Genotype RB MPK5 KO with sb24F (in the cassette) rak1-rak2 ss9F p1 AATTAGATGGTTGACCTCAATTTTT Genotype LB RAK2-RAK1 dKO with ss195R ss182F p1 TGCAAATATAAGCTAGTCTTTGA Genotype LB RAK2-RAK1 dKO with ss195R ss144F p1 GAAAGTGTATTAAAATCAATAC Genotype LB RAK2-RAK1 dKO with ss195R ss195R p2 GTATCGTCTGCGTCCTCACC ss35F p3 ATGGCCTGCTGAAGAGAAAA Genotype RAK2-RAK1 dKO - part of the gene ss36R p4 TCAGAAACTTGCCGAGTCCT ss196F p5 ACCACGTCGCAGTACTTCAG Genotype RB RAK2-RAK1 dKO ss14R p6 TGGAACATGCCTACCAAAAA p1 Genotype LB RAK2 KO in rak1 background with sb410F p6 Genotype RB RAK2 KO in rak1 background with sb417R nath ss198F p1 CTTTGTAACCCACAATGGTG Genotype LB NATH KO ss199R p2 CTCTTTGCTGGACTGCTTC ss200F p3 GCTTGAATTTGATCATGGAGGCA Genotype NATH KO - part of the gene ss201R p4 GCTACAATAGGATCACCTTCACCT ss202F p5 GAGCTGCCAAGTCCTATGA Genotype RB NATH KO ss203R p6 CACCATTGTGGGTTACAAAG ss204R p1 AGAGCAGACTTCGAGGAGGT Genotype LB NATH KO with sb410F ss205F p6 CCTCTAAACATACAAGCGAATACAT Genotype RB NATH KO with sb417R

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Figure 4: Genotyping MPK KI’s & primers

Initially, verification of the GFP tag in the KI lines tested by PCR. After numerous of attempt to verify the GFP tag by immunoblots, it was only possible to verify MPK3+GFP in 30 day- old plants (A, asterix). Proteins were extracted from 10, 14, 25 and 30 day-old KI lines, indicating that either the proteins are very low expressed or the expression of the proteins were very specific to a developmental stage and/or tissue. (B) Insertion of the GFP tag in the KI candidates of RAK1, RAK2, MPK3 and NATH were verified by PCR, with primers located in the GFP tag and the promotor. Out of these candidates 4 RAK1+GFP, 4 RAK2+GFP, 3 MPK5+GFP and 3 NATH+GFP had a signaling in the confocal fluorescence microscope.

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Primers used for verifying GFP tags:

Primers for pMBLU-GFP-G418 Sb410F GGTTATTGTCTCATGAGCGGA Sb24F GGTATCAGAGCCATGAATAGGTC Sb417R TAGCTTGGCGTAATCATGGTC ss206R CTCCTCGCCCTTGCTCACCAT In GFP tag ss207R GCGCGATCACATGGTCCTGC In GFP tag. With sb27R from tag to 35s promotor. Primers used for constructing LB in KI lines:

Name Primers Size (bp) Description RAK1+GFP ss220F GGCTTAAUTACAATGTGAGGGGCGACAAG 1047 LB forward for RAK1 knockin (KI) in pMBLU-GFP ss219R GGTTTAAUCTGGACGTTGATAAGTGTAGC LB reverse for RAK1 knockin (KI) in pMBLU-GFP RAK2+GFP ss222F GGCTTAAUGAGAAGCAACCTGCCAGCCAGTGG 1172 LB forward for RAK2 knockin (KI) in pMBLU-GFP ss221R GGTTTAAUCTGAGCGTTTATAAGAGTAGCCTC LB reverse for RAK2 knockin (KI) in pMBLU-GFP MPK3+GFP ss126F GGCTTAAUCCCGCCTGGTAAACATCACT 1215 LB forward for MPK3 knockin (KI) in pMBLU-GFP ss188R GGTTTAAUCGTCTCGGCGGCCGCTTCAG LB reverse for MPK3 knockin (KI) in pMBLU-GFP MPK5+GFP ss224F GGCTTAAUTCGCTCATCTCGAGGAACACT 934 LB forward for mpk5 knockin (KI) in pMBLU-GFP ss223R GGTTTAAUATGTCCAACACTCTGAGCTGC LB reverse for mpk5 knockin (KI) in pMBLU-GFP NAT.H+GFP ss226F GGCTTAAUGCAGTTTCTTGAGTTGATTGC 1077 LB forward for natH knockin (KI) in pMBLU-GFP ss225R GGTTTAAUATTGCCATCCTGCACAAGAAC LB reverse for nath knockin (KI) in pMBLU-GFP

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Figure 5: Physcomitrella patens life cycle

P. patens life cycle. (A) Haploid spore germinates into protonema filaments consisting of (B) chloronemal cells and (C) caulonemal cells. (D) Gametophores emerge from protonema filaments and are anchored by rhizoids that expand by tip growth from the gametophore. (E) At the apex of the gametophore organ bundles are produced with both female, archegonia (arrows), and male, antheridia (arrowheads) reproductive organs. After irrigation the egg is fertilized by motile sperm and the (F) sporophyte developes at the apex of the gametophore. (5)

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Figure 6: Colonies of mutants upon 12 weeks under sporophyte induction

Colonies of WT, rak1, mpk3 and mpk5 under sporohyte induction conditions after 12 weeks. Central part of the colonies of WT (A), rak1 (B), mpk3 (C) and mpk5 (D). Underneath the colonies were the rhizoids are visible of WT (E), rak1 (F), mpk3 (G) and mpk5 (H) plants.

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Figure 7: Colonies and central- and periphery gametophytes of mutants after 6 weeks under sporophyte induction conditions.

Colonies of WT, rak1, mpk3 and mpk5 under sporohyte induction conditions after 6 weeks. Central gametophytes of WT, rak1, mpk3 and mpk5. Periphery gametophytes of WT, rak1, mpk3 and mpk5. Bar = 1mm.

130

Figure 8: Amount of sporophytes at the different maturation stages.

Amount of sporophytes at the different maturation stages in WT, rak1, mpk3 and mpk5 induced under sporophyte induction conditions. The total amount of sporophytes (blue bar), early green sporophytes (green bar), late green/yellow sporophytes (yellow bar), yellow/orange sporophytes (orange bar), orange/red sporophytes (red bar) and brown sporophytes (brown bar).

131

Figure 9: Protoplast of WT, rak1, mpk3 and mpk5

Protoplast of 14 days-old WT, rak1, mpk3 and mpk5 plants. Bar = 50µm. Area of protoplasts of 14 days-old of WT, rak1, mpk3 and mpk5 plants. Area of protoplasts were measured by ImageJ. Analysis of variance by T-test determined statistical differences indicated (P <0.05), n = 12. Standard deviation as error bars.

132

Figure 10: Filaments of WT, rak1, mpk3 and mpk5

(A) Filaments of 14 days-old WT, rak1, mpk3 and mpk5 plants. Bar = 50µm. (B) Length of cells from the tip cell, 2th cell, 3rd cell, 4th cell, 5th cell and 6th cell of WT, rak1, mpk3 and mpk5 plants. The length were measured by ImageJ (µm). (C) Length of cell for the first secondary branching. The first secondary branching was visible for WT at 4th cell, rak1 at 6th cell, mpk3 and mpk5 at 5th cell. The length were measured by ImageJ (µm)

133

Figure 12: The difference between amount of gametophytes after 3- & 6 weeks growth on BCD and BCDAT

Difference between the amount of gametophytes after 3 and 6 weeks of growth on minimal media (BCD) and full media (BCDAT). Analysis of variance by T-test determined statistical differences indicated by asterisks (* P = 0.05-0.01, ** P = 0.01-0.001, *** P < 0.001), n = 3 colonies.

134

Figure 13: Colonies of all lines after growing for 3- and 6 weeks on BCD and BCDAT

Colonies of 3 and 6 week-old WT, rak1, mpk3, mpk5, rak1-rak2 and nath. The plants were grown on minimal media (BCD) and full media (BCDAT) for 3 and 6 weeks. Bar = 2 mm.

135

Figure 14: Preliminary salt experiments.

Colonies of 6 week-old WT, rak1, mpk3 and mpk5. (A) The plants were grown on full media (BCDAT) and supplemented with 100mM NaCl (B) and 100mM KCl (B). WT, mpk3 and mpk5 plants are marked with white circles, while rak1 is marked with white arrows.

136

Figure 15: Vertical growth experiment with 200mM MgCl2 and NaCl

Vertical growth experiment with 200mM MgCl2 and NaCl with WT, mpk3 and mpk5. The plangts were grown for 2 weeks in dark on minimal media (BCD) supplemented with (D) MgCl2 and (E) NaCl. The concentration of MgCl2 and NaCl influenced the growth of filaments and no filaments were visible.

137

Figure 16: Regulation of ABA synthesis

Regulation of ABA synthesis. Abiotic stresses such as high salinity activates the biosynthesis genes, through a Ca2+ dependent phosphorylation cascade. NCED (9- cis-epoxycarotenoid dioxygenase) is strongly upregulated by stress (indicated with a thick arrow). The enzymes involved in ABA biosynthesis are shown in small ovals: ZEP (zeaxanthin epoxidase), NCED and AAO (abscisic aldehyde oxidase). ZEP initiates carotenoid catalysis into violaxanxin followed by synthesis and oxidative cleavage by NCED transforming inot xanthoxin. Xanthoxin is then converted to abscisic aldehyde and oxidized into ABA. (Adopted from (119)).

138

Figure 17: Hormone regulation on development of protonemata

Hormone regulation under development of protonemata in P. patens. Chloronema cells can by apical division form either a new chloronema cell or caulonema cell induced by auxin, light and glucose, or a bud initial induced by cytokinin. Under influence of ABA cells differentiate into round brachycytes or tmema cells. The prevalent phase od cell cycle for the transition from chloronema to caulonema cells are indicated (G2 and G1). (Adopted from (21)).

139

Figure 18: Auxin – BCDAT plates supplemented with 0.5, 1 and 10μM IAA

Colonies of 1-, 2-, 3-, and 4- weeks old WT, rak1, mpk3 and mpk5 plants grown on (A) full media (BCDAT) supplemented with IAA: 0.5 µM (B), 1µM (C) and 10µM (D).

140

Figure 19: ABA – BCDAT plates supplemented with 0.5, 1 and 10μM IAA

Colonies of 1-, 2-, 3-, and 4- weeks old WT, rak1, mpk3 and mpk5 plants grown on (A) full media (BCDAT) supplemented with ABA: 0.5 µM (B), 1µM (C) and 10µM (D).

141

Figure 20: Cytokinin – BCDAT plates supplemented with 0.5, 1 and 10μM BPA

Colonies of 1-, 2-, 3-, and 4- weeks old WT, rak1, mpk3 and mpk5 plants grown on (A) full media (BCDAT) supplemented with BPA: 0.5 µM (B), 1µM (C) and 10µM (D).

142

Figure 21: Preliminary blue- and red light experiment

Preliminary blue – and red light experiment with WT, mpk1 (rak1), mpk3 and mpk5 plants. The plants were grown on full media (BCDAT) and minimal media (BCD) for 6 weeks under BL and RL.

143

Figure 22: Chlorophyll content of central and periphery gametophytes after 3 weeks growth under WL and BL.

Total chlorophyll content (mg per gram FW) of WT, rak1, mpk3, mpk5, rak1-rak2 and nath colonies were calculated after 3 weeks growth under white – and blue light (WL & BL). (A) ChlA and ChlB in central gametophytes. (B) ChlA and ChlB in periphery gametophytes. (C) Total chlorophyll content of central and periphery gametophytes. Standard deviation as error bars, n = 3 colonies and 3 gametophytes per triplicate.

144

Figure 23: Chlorophyll content of central and periphery gametophytes after 6 weeks growth under WL and BL.

Total chlorophyll content (mg per gram FW) of WT, rak1, mpk3, mpk5, rak1-rak2 and nath colonies were calculated after 6 weeks growth under white – and blue light (WL & BL). (A) ChlA and ChlB in central gametophytes. (B) ChlA and ChlB in periphery gametophytes. (C) Total chlorophyll content of central and periphery gametophytes. Standard deviation as error bars, n = 3 colonies and 3 gametophytes per triplicate.

145

Figure 24: Multiple alignment of MPK5, NEK and NIMA

C-terminal of PpMPKs

>Pp1s29_285V6.1 RAK1 MDRHKHYDSGEGSTREKKLLGARDEFPEAEKAVRAAAAKPDPIAEFPSFLIYNRNGLKLNLEAGSGAALSATTKESMHKLLMMNMEVLFGPHEWPAEENM KRWEMVSPEARFIFVRKSTPTIEAGSSDEGHPMVGFVHFRFGLEHEVPVLYIYETQLEKTVQGKGLGKFLMQLLELVARKNNMKAVLLAVHKRNTRALTF YNERLGYKLAIRSASSQQSTQPSFMFENQALFFAVDRLICRMELILKFWFALLQTVTEMKYEILCKTFDVEYTAVVEERQGDMDCESREESAGEASCQTV DAEDQVLDDSRPDTECESRIESVPNTLQGMKYTQYNVRGDKFEVYDKYVMIGPIGHGAYGDVCAFTNRETGEKVAIKKIGNAFQNNTTARRTLREILLLR HTEHDNIIPIRDIIVPANIEDFHDAYIANELMDTDLHQIVRSTKLDEYHCQFLLYQLLRGLKYIHSANILHRDLKPSNLLINCNDCLLKICDFGLARTSA EDDFLTEYVVTRPYRAPELLLGSRMYTAAVDMWSVGCIFMEMLTGQPLFPIRSRQEHPVNHLKLITELLGTPDASDLSFLQNPDARQRIQMALLGQERKP LFSRFPQTSAIACDLAEKMLRFNPSNRITAEEALAHPYLAALHDLSDEPTCHLMFDFDAYLPSLTVEHVKTLIWREATLINVQ

>Pp1s80_71V6.1 MPK2 MADAEFFTEYGEANRYRILEVIGKGSYGVVCSAVDTLTGEKVAIKKINDIFEHVSDATRILREIKLLRLLRHPDIVEVKHIMLPPSRRDFKDIYVVFELM ESDLHQVIKANDDLTPEHYQFFLYQLLRALKYIHTANVFHRDLKPKNVLANADCKLKICDFGLARVAFSDAPTAIFWTDYVATRWYRAPELCGSFFSKYT PAIDIWSIGCIFAEVLTGKPLFPGKNVVHQLDLMTDMLGSPSPETVQRVRNEKARRYLSTMRKKPPMPFVQKFPNADPLAIRLLERMLAFDPRDRPTAEE ALADPYFKGLAKVDREPSAQPITKMEFEFERRRINKEDVRELIYREILEYHPQMLKEYLNGSDNATFLYPSAVDQFKRQFAHLEEHYGKGGNNPPLERQH ASLPRERVLEFREEASKYQREDSKLHDKHASSGQRNVYSQNSSKSQDGSVGRAGNPAAYVGAKEYTDPRRVSKTASMSTTNSYSGSINPYGRRHSSNKTD RDDRELNAVQSKTESMGLGGSRKVPAAQSVGQ

>Pp1s207_63V6.1 MPK3 MATKVEVPEGTCPPGKHHYMLWRSVFEIDTKYAPIKPIGKGAYGVVCSAKNNETGDRVAIKKITNAFENTTDARRTLREIRLLRHLFHENIIAVKDIMKP VGRQTFNDVYIVYELMDTDLHQIIRSSQTLTDDHCQYFIYQLLRGLKYVHSANVLHRDLKPSNLLLNASCDLKICDFGLARTGSDKGQFMTEYVVTRWYR APELLLSCDEYTSAIDMWSVGCIFAELLGRKPLFPGKDYIHQLKLIISIIGSPDETDLHFIQSQKARSYIRSLPFTPRVSLARLYPRANPLAIQLIDKML VFDPRKRITVHEALEHPYLSMLHDATVEPSAPAPFEFDFEDEDLKEDALRERVWNEMLFYHPEAAAET

>Pp1s87_157V6.1 MPK5 MADAEFFTEYGEANRYRILEVIGKGSYGVVCSAVDTLTGEKVAIKKINDIFEHVSDATRILREIKLLRLLRHPDIVEVKHIMLPPSRRDFKDIYVVFELM ESDLHQVIKANDDLTPEHYQFFLYQLLRALKYIHTANVFHRDLKPKNVLANADCKLKICDFGLARVAFSDAPTAIFWTDYVATRWYRAPELCGSFFSKYT PAIDIWSIGCIFAEVLTGKPLFPGKNVVHQLDLMTDMLGSPSSETVQRVRNEKARRYLSTMRKKLPMPFGQKFPNADPLAIRLLERMLAFDPRDRPTAEE ALADPYFKGLAKVDREPSAQPITKMEFEFERRRINKEDVRELIYREILEYHPQMLKEYLNGSDNATFLYPSAVDQFKRQFAHLEEHYGKVGNNPPLERQH ASLPRERVLEFREEAAKYQREESKLQDKHGASGQRNTYSQNSKSLDGPVGRAGNPTSFMGLSNKEYTDPRRVTKTAAMSATNSYSAPLNPYGRRHSTSKS DRDDRELNAVQSKTESMGLGSSRKVPAAQSVGH

PpNEKs:

>Pp3c17_9390V3.1 NEK MLSTRACSSIMGSKSAETSGGSSGGRQPSPLDLYSKGTQIIELGSAVWPENPIRTGRSEVYIWGSEHAVKLCMGPTFALHEYTMS RAARNYAVRTVAMFTIHGKPNGIVMERGKSVNPVTCDLKQIAFEMVRAVQGLYSIGIIHGDIKLSSFLVCRDGCVRLCDFGTSEYKCDSVSHSEMSIPWS RPSLLRNPDRPRVKADDLYSLGLTIWELYTGKVPFVPPTSEGWESLDINEVAEEAILAGEQVDLNDILDLEIRCGCFTCERGGRAPTLPGVVTWGFRKVA KY*

>Pp3c2_17750V3.1 NEK2 MSDFQAIKDAVMHEIQDGLIEVLLHMKRQIFDELAEYFSGTGKTPQGITAPKIAPSDCKCEATLATGPNKGSKCPNKAKDNTPFC GRHKRTIVVDKDGVVQKPKRCKAIIHKYTLQKKQCEHLAKPGSLFCGVHRNYCPNGLNAVPEAFDTLHDRDESISEAQPYPNRLKEKGVKIPIPDDCVAV PSTHPDRFEQGRENLSRGIKQDVAELRVAVKMKEEEEKETEAILDQPPTEEQIKEAEESGMIPYQYKELWNVQCRMVNEKMYPILPSGCVEEAWDNIRAM TTQEEYDSLKEFQVKHPHIHQWIRIMSIGCYCEQRRDYRATPDHKYDERFELTINGEVPLTNKQKLDVIGIKRRVFEEEMKELGLPYVPSTLAAKLPNER LEESMRKNRRKPEDFFDPSDHPYYETNTITKTIYVHKKWDSRDDKILERLQDGQRVRKSTFSNNLVIKNKDNSYECDNEEYDSDCSKGSVAGDYFPRFIE CLTPRSKKFEMWTSKFRDDNQASTSIVIDFAYNSNLKDLLNSAGDILSEELKRRLVYDICCSVKCLHDRSIAHEDLKPKNILLDFGLRAKICDFGHSKHI NDYYEANSGTNIYKSPEKRETTTSSPIRKYDRIKSDIFSLGMIIEEIYRTSNETEVSSIWKLCKSSDITERPSCNELLEKFEYTRVDYTSFRGIEKYSLS EVERINIAYYWSDYSEGEDELLSRCRSLILRERYSEAYDCLRMFQNHIRDMLLGILLFHGLGCRKDNIESKQHLDNCIKSNIWMGIEPDFLLLQFYITVL DMEMNNNCSVEKLLLLSERGNSFAQIELARLYSNQGFLREHRVDSITRERFVMASAKSGHSHAQALAAIHGFSPVDGVEGKFTKDTVLFAKRSAEGGNVQ G*

>Pp3c2_28480V3.1 NEK5 MGESKMDNYEIMEQVGRGAFGSAILVNHKLEKKKYVLKKIRLARQTDRCRRSAHQEMSLVSRVQHPYVVEYKESWVEKGCYVCIV TGYCEGGDMADVIRKAHGQYFSEERLLKWFAQLLLSVDYLHSNHVLHRDLKCSNIFLTKDQDIRLGDFGLAKMLNQDDLASSVVGTPNYMCPELLADIPY GFKSDIWSLGCCMYEMAAHRPAFKAFDMQGLISKINKSTIGPLPSIYSSPLKSMIRSMLRKNPEHRPTAAELIRHPHMQPYIMQCRIQAALQCSSPEPLI RTDPPVAADSPLRKSRSVQEKAPPPIVNPVTTNVDTSTDRESFTTDAKSSPDRDTEYGDLTDGPPADEGVDHPWNYDSVGGTENSRLSRETSERENVRSS NRDWVKEECKVAAQYAINVLRPKADGTRERKEDRLVRLPEKTAPRVPRSDRNPPVAEGPPTSSPAAKIVNALKAKSDPTKRRPKQMDNPPVPKVKEANMI AAESPRVKWRADKLPPAPPKVQNSSEEEATVKKQRVPVTPAVTSAPRRSSLPLPQKVSRKTSPPTRRTSPPLASLRSSGAPSPKLRAQAGPPAYLSKLIK NDENGQSGRRSEIGHSGRLSDIGLNDRRSEIGHSDRRSDIGSGRRSDIGHSGRRSDIGHSGRRSDVGLSGRRSDIRKTPSLERGLDHDSAETVKATLQRL SSLTSRSDKDGSSMRSNRSSPSESGNIIQELGLNNHSPNVSVNAPRLDLIPEFKLTADPEPYSSQLASHEIRMPMERPKFSSSVMPTTTRSHIAVVSPRY SQQVMPKQTTEPRVSASPNRPDSSLALPASETNKPLYSMHMETSIFDKTQEKGTIQINEKSPAPPAPARPAFNDVIHVIRHSTFRLGATSDHSHTDADYA TMGEIDFRAGRTDLEPFHGKMDIGSLLDLPQRGSDVEVVSVSPGSSVTSRHPQVDMHQRHANGLDVKSYRQRAEALEGLLELSAQLLSQHRLEELAIVLK PFGRGKVSPRETAIWLTKSLKGMLGDEQPHDSPTVVI*

AtNIMA:

>NP_175853.1 NIMA-related serine/threonine kinase 1 [Arabidopsis thaliana] MEQYEFLEQIGKGSFGSALLVRHKHEKKKYVLKKIRLARQTQRTRRSAHQEMELISKMRHPFIVEYKDSW VEKACYVCIVIGYCEGGDMAQAIKKSNGVHFQEEKLCKWLVQLLMGLEYLHSNHILHRDVKCSNIFLTKE

146

QDIRLGDFGLAKILTSDDLTSSVVGTPSYMCPELLADIPYGSKSDIWSLGCCIYEMAYLKPAFKAFDMQA LINKINKTIVSPLPAKYSGPFRGLVKSMLRKNPEVRPSASDLLRHPHLQPYVLDVKLRLNNLRRKTLPPE LPSSKRIMKKAHFSEPAVTCPAFGERQHRSLWNDRALNPEAEEDTASSIKCISRRISDLSIESSSKGTLI CKQVSSSACKVSKYPLAKSSVTSRRIMETGRRSDHLHPVSGGGTTSKIIPSARRTSLPLTKRATNQEVAA YNPIVGILQNVKSPEYSINEPQVDKIAIFPLAPYEQDIFFTPMQRKTSSKSSSVSDRSITKDKCTVQTHT TWQGIQLNMVDNISDGSSSSDQNATAGASSHTTSSSSRRCRFDPSSYRQRADALEGLLEFSARLLQEGRY DELNVLLKPFGPGKVSPRETAIWIAKSLKENRDKTKMVDLNVSREIPHVGLL

CLUSTAL O(1.2.4) multiple sequence alignment

Pp3c17_9390V3.1 NEK ------Pp3c2_17750V3.1 NEK2 MSDFQAIKDAVMHEIQDGLIEVLLHMKRQIFDELAEYFSGTGKTPQGITA--PKIAPSDC Pp1s80_71V6.1 MPK2 ------Pp1s87_157V6.1 MPK5 ------Pp1s29_285V6.1 RAK1 ------MDRHKHYDSGEGSTREKKLLGARDEFPEAE Pp1s207_63V6.1 MPK3 ------Pp3c2_28480V3.1 NEK5 ------NP_175853.1 AtNIMA ------

Pp3c17_9390V3.1 ------Pp3c2_17750V3.1 KCEATLATGPNKGSKCPNKAKDNTPFCGRHKRTIVVDKDGVVQKP------KRCKAI Pp1s80_71V6.1 ------Pp1s87_157V6.1 ------Pp1s29_285V6.1 KAVRAAAAKPDPIAEFP------SFLIYNRNGLKLNLEAGSGAALSATTKES Pp1s207_63V6.1 ------Pp3c2_28480V3.1 ------NP_175853.1 ------

Pp3c17_9390V3.1 ------Pp3c2_17750V3.1 IHKYTLQKKQCEHLAKPGSLFCGVHRNYCPNGLNAVPEAFDTLHDRDESISEAQPYPNRL Pp1s80_71V6.1 ------Pp1s87_157V6.1 ------Pp1s29_285V6.1 MHKLLM------MNMEVLFG------PHEWPAEE Pp1s207_63V6.1 ------Pp3c2_28480V3.1 ------NP_175853.1 ------

Pp3c17_9390V3.1 ------Pp3c2_17750V3.1 KEKGVKIPIPDDCVAVPSTHPDRFEQ------GRENLSRGIKQDVAELRVAVKMKE Pp1s80_71V6.1 ------Pp1s87_157V6.1 ------Pp1s29_285V6.1 NMKRWEMVSPEARFIFVRKSTPTIEAGSSDEGHPMVGFVHFRFGLEHEVPVLYIYETQLE Pp1s207_63V6.1 ------Pp3c2_28480V3.1 ------NP_175853.1 ------

Pp3c17_9390V3.1 ------Pp3c2_17750V3.1 E------EEKETEAIL-DQP--PT------EEQIKEA--EE Pp1s80_71V6.1 ------Pp1s87_157V6.1 ------Pp1s29_285V6.1 KTVQGKGLGKFLMQLLELVARKNNMKAVLLAVHKRNTRALTFYNERLGYKLAIRSASSQQ Pp1s207_63V6.1 ------Pp3c2_28480V3.1 ------NP_175853.1 ------

Pp3c17_9390V3.1 ------Pp3c2_17750V3.1 SG--MIPYQYKEL----WNVQCRMVNEKMYPILPSGCVEEAWDNIRAMTTQEEYDSLK-E Pp1s80_71V6.1 ------Pp1s87_157V6.1 ------Pp1s29_285V6.1 STQPSFMFENQALFFAVDRLICRME------LILKFWFALLQTVTEMKYEILCKT Pp1s207_63V6.1 ------Pp3c2_28480V3.1 ------NP_175853.1 ------

Pp3c17_9390V3.1 ------MLST------Pp3c2_17750V3.1 FQVKHPHIHQWIRIMSIGCYCEQRRDYRATPDHKYDERFELTINGEVPLTNKQKLDVIGI Pp1s80_71V6.1 ------Pp1s87_157V6.1 ------Pp1s29_285V6.1 FDVEYTAV---VEERQGDMDCESREESA------GEAS------CQTVDA Pp1s207_63V6.1 ------Pp3c2_28480V3.1 ------NP_175853.1 ------

147

Pp3c17_9390V3.1 ------RACSSIMGS------Pp3c2_17750V3.1 KRRVFEEEMKELGLPYVPSTLAAKLPNERLEESMRKNRRKPEDFFDPSDHPYYETNTITK Pp1s80_71V6.1 ------MAD---AEFFTEYG Pp1s87_157V6.1 ------MAD---AEFFTEYG Pp1s29_285V6.1 EDQVLDD------SRPD---TECESRIESVPNTLQGMKYTQYNVRGDKFE Pp1s207_63V6.1 ------MATKVEVPEGTCPPGKHHYMLWRSVFE Pp3c2_28480V3.1 ------MGES NP_175853.1 ------

Pp3c17_9390V3.1 ------KSAETSGGSSGGRQPS-PLDLYSKGTQIIELGSAVWPENPIR-TGRS Pp3c2_17750V3.1 TIYVHKKWDSRDDKILERLQDGQRVRKS-----TFSNNLVIKNKDNSYECDNEEYDSDCS Pp1s80_71V6.1 ------EANRYRILEVIGKGSYGVVCSAVDTLTGEKVAIKKINDIFEHVSDATRILRE Pp1s87_157V6.1 ------EANRYRILEVIGKGSYGVVCSAVDTLTGEKVAIKKINDIFEHVSDATRILRE Pp1s29_285V6.1 ------VYDKYVMIGPIGHGAYGDVCAFTNRETGEKVAIKKIGNAFQNNTTARRTLRE Pp1s207_63V6.1 ------IDTKYAPIKPIGKGAYGVVCSAKNNETGDRVAIKKITNAFENTTDARRTLRE Pp3c2_28480V3.1 ------KMDNYEIMEQVGRGAFGSAILVNHKLEKKKYVLKKIRLARQTDRCRRSAHQE NP_175853.1 ------MEQYEFLEQIGKGSFGSALLVRHKHEKKKYVLKKIRLARQTQRTRRSAHQE . . : : . Pp3c17_9390V3.1 EVYIWGSEH-AVKLCMGPTFALHEYTMSRAARNYAV--RTVAMFTIHGKPNGIVME-RGK Pp3c2_17750V3.1 KGSVAGDYFPRFIECLTPRSKKFEMWTSKFRDDNQASTSIVIDFAYNSNLKDLLNS-AGD Pp1s80_71V6.1 IKLLRLLRHPDIVE------VKHIMLPPSRRDF-KDIYVVF-ELMESDLHQVIKAND-- Pp1s87_157V6.1 IKLLRLLRHPDIVE------VKHIMLPPSRRDF-KDIYVVF-ELMESDLHQVIKAND-- Pp1s29_285V6.1 ILLLRHTEHDNIIP------IRDIIVPANIEDF-HDAYIAN-ELMDTDLHQIVRST--- Pp1s207_63V6.1 IRLLRHLFHENIIA------VKDIMKPVGRQTF-NDVYIVY-ELMDTDLHQIIRSSQ-- Pp3c2_28480V3.1 MSLVSRVQHPYVVE------YKESWVEKGCY-----VCIVTGYCEGGDMADVIRKAHGQ NP_175853.1 MELISKMRHPFIVE------YKDSWVEKACY-----VCIVIGYCEGGDMAQAIKKSNGV : . . . . . : Pp3c17_9390V3.1 SVNPVTCDLKQIAFEMVRAVQGLYSIGIIHGDIKLSSFLVCR-DGCVRLCDFGTSEYKCD Pp3c2_17750V3.1 ILSE--ELKRRLVYDICCSVKCLHDRSIAHEDLKPKNILLDF-GLRAKICDFGHSKHIN- Pp1s80_71V6.1 DLTP--EHYQFFLYQLLRALKYIHTANVFHRDLKPKNVLANA-DCKLKICDFGLARVAFS Pp1s87_157V6.1 DLTP--EHYQFFLYQLLRALKYIHTANVFHRDLKPKNVLANA-DCKLKICDFGLARVAFS Pp1s29_285V6.1 KLDE--YHCQFLLYQLLRGLKYIHSANILHRDLKPSNLLINCNDCLLKICDFGLARTSA- Pp1s207_63V6.1 TLTD--DHCQYFIYQLLRGLKYVHSANVLHRDLKPSNLLLNA-SCDLKICDFGLARTGS- Pp3c2_28480V3.1 YFSE--ERLLKWFAQLLLSVDYLHSNHVLHRDLKCSNIFLTK-DQDIRLGDFGLAKMLN- NP_175853.1 HFQE--EKLCKWLVQLLMGLEYLHSNHILHRDVKCSNIFLTK-EQDIRLGDFGLAKILT- . :: .:. :: : * *:* ...: :: *** :. Pp3c17_9390V3.1 S-----VSHSEMSIPW-SRPSLLRNP-----DRPRVKADDLYSLGLTIWELYTGKVPFVP Pp3c2_17750V3.1 -----DYYEANSGTNIYKSPEKRETTTSSPIRKYDRIKSDIFSLGMIIEEIYRTSNETEV Pp1s80_71V6.1 DAPTAIFWTDYVATRWYRAPELCGSF-----FSKYTPAIDIWSIGCIFAEVLTGKPLFPG Pp1s87_157V6.1 DAPTAIFWTDYVATRWYRAPELCGSF-----FSKYTPAIDIWSIGCIFAEVLTGKPLFPG Pp1s29_285V6.1 ---EDDFLTEYVVTRPYRAPELLLG------SRMYTAAVDMWSVGCIFMEMLTGQPLFPI Pp1s207_63V6.1 --DKGQFMTEYVVTRWYRAPELLLS------CDEYTSAIDMWSVGCIFAELLGRKPLFPG Pp3c2_28480V3.1 ---QDDLASSVVGTPNYMCPELLADI------PYGFKSDIWSLGCCMYEMAAHRPAFKA NP_175853.1 ---SDDLTSSVVGTPSYMCPELLADI------PYGSKSDIWSLGCCIYEMAYLKPAFKA *. *::*:* : *: Pp3c17_9390V3.1 PTSEGWESLDINEVAEEAILAGEQVDLNDILDLEIRCGCFTCERGGRAPTLPGVVTWGFR Pp3c2_17750V3.1 SS------IWKLCKSSDITE-RPSCNELLEK------F------EYTRVDYTSFR Pp1s80_71V6.1 KN----VV-HQLDLM--TDMLG-SPSPETVQ------RVRNE Pp1s87_157V6.1 KN----VV-HQLDLM--TDMLG-SPSSETVQ------RVRNE Pp1s29_285V6.1 RSRQEHPV-NHLKLI--TELLG-TPDASDLS------FLQNP Pp1s207_63V6.1 KD----YI-HQLKLI--ISIIG-SPDETDLH------FIQSQ Pp3c2_28480V3.1 FDMQGLIS-KINKST--I------NP_175853.1 FDMQALIN-KINKTI--V------. Pp3c17_9390V3.1 KVAKY*------Pp3c2_17750V3.1 GIEKYSL---SEVERINIAYYWSDYSEGEDELLSRCRSL------ILRERYSE Pp1s80_71V6.1 KARRYLSTMRK-KPPMP---FVQKFPNADPLAIRLLERMLAFDPRDRPTAEEALADPYFK Pp1s87_157V6.1 KARRYLSTMRK-KLPMP---FGQKFPNADPLAIRLLERMLAFDPRDRPTAEEALADPYFK Pp1s29_285V6.1 DARQRIQMALLGQERKP---LFSRFPQTSAIACDLAEKMLRFNPSNRITAEEALAHPYLA Pp1s207_63V6.1 KARSYIRSLPF-TPRVS---LARLYPRANPLAIQLIDKMLVFDPRKRITVHEALEHPYLS Pp3c2_28480V3.1 ------GP---LPSIYS---SPLKSMIRSMLRKNPEHRPTAAELIRHPHMQ NP_175853.1 ------SP---LPAKYS---GPFRGLVKSMLRKNPEVRPSASDLLRHPHLQ

Pp3c17_9390V3.1 ------Pp3c2_17750V3.1 AYDCLRMFQNHIRDMLLGILLFHGLGCRKDN------IESKQHLDNCIKSNIWMGI Pp1s80_71V6.1 GLAKV------DREP---SAQ---PITKME--FEFERRRINKEDVRELIYREI Pp1s87_157V6.1 GLAKV------DREP---SAQ---PITKME--FEFERRRINKEDVRELIYREI Pp1s29_285V6.1 ALHDL------SDEP---TCH---LM--FD--FDAYLPSLTVEHVKTLIWREA Pp1s207_63V6.1 MLHDA------TVEP---SAP---AP--FE--FDFEDEDLKEDALRERVWNEM Pp3c2_28480V3.1 PYIMQ------CRIQAALQCSSPEPLIRTDPPVAADSPLRKSRSVQEKAPPPI NP_175853.1 PYVLD------VKLRLNNLRRKT------LPPEL

Pp3c17_9390V3.1 ------Pp3c2_17750V3.1 EPDFLLLQFYITVLDMEMNNNCSVEKLLLLSERGNSFAQIELARLYSN--QGFLREHRVD

148

Pp1s80_71V6.1 LE--YHPQMLKEYL-NGSDNATFLYP-SAVDQFKRQFAHLE--EHYGKGGNNPPLERQHA Pp1s87_157V6.1 LE--YHPQMLKEYL-NGSDNATFLYP-SAVDQFKRQFAHLE--EHYGKVGNNPPLERQHA Pp1s29_285V6.1 TL--INVQ------Pp1s207_63V6.1 LF--YHPEAAAET------Pp3c2_28480V3.1 V----NP--VTTNVDTSTDRESFTTD------AKSSPDRD--TEYGDLTDGPPADEGVD NP_175853.1 P----S---SK----RIMKKAHFSEP------AVTCP------AFGE------RQHR

Pp3c17_9390V3.1 ------Pp3c2_17750V3.1 SITRERFVMASAKSGH------SHAQALAAIHGFSP-VDGVEGKF Pp1s80_71V6.1 SLPRERVLEFRE------EASKYQREDSKLHDKHA------SSGQRNVY Pp1s87_157V6.1 SLPRERVLEFRE------EAAKYQREESKLQDKHG------ASGQRNTY Pp1s29_285V6.1 ------Pp1s207_63V6.1 ------Pp3c2_28480V3.1 HPWNYDSVGGTENSRLSRETSERENVRSSNRDWVKEECKVAAQYAINVLRPKADGTRERK NP_175853.1 SLWNDRALN------PEAEEDT---

Pp3c17_9390V3.1 ------Pp3c2_17750V3.1 TKDTVLFAK------RSAEGGNVQ-G*------Pp1s80_71V6.1 S------QNSSKSQDGSVGRAGNPAA------YVG--AKEYTDPRRVS------Pp1s87_157V6.1 S------QNS-KSLDGPVGRAGNPTS------FMGLSNKEYTDPRRVT------Pp1s29_285V6.1 ------Pp1s207_63V6.1 ------Pp3c2_28480V3.1 EDRLVRLPEKTAPRVPRSDRNPPVAEGPPTSSPAAKIVNALKAKSDPTKRRPKQMDNPPV NP_175853.1 ------A------SSIKCIS------RR---ISDLSI

Pp3c17_9390V3.1 ------Pp3c2_17750V3.1 ------Pp1s80_71V6.1 ---KTASMSTTN----SYS------G Pp1s87_157V6.1 ---KTAAMSATN----SYS------A Pp1s29_285V6.1 ------Pp1s207_63V6.1 ------Pp3c2_28480V3.1 PKVKEANMIAAESPRVKWRADKLPPAPPKVQNSSEEEATVKKQRVPVTPAVTSAPRRSSL NP_175853.1 ESSSKGTLICKQVS------SSACKVSKY

Pp3c17_9390V3.1 ------Pp3c2_17750V3.1 ------Pp1s80_71V6.1 SINPYGRRHSSNKTDRDDRELNAVQ------Pp1s87_157V6.1 PLNPYGRRHSTSKSDRDDRELNAVQ------Pp1s29_285V6.1 ------Pp1s207_63V6.1 ------Pp3c2_28480V3.1 PLPQKVSRKTSPPTRRTSPPLASLRSSGAPSPKLRAQAGPPAYLSKLIKNDENGQSGRRS NP_175853.1 PLAKSS------VT------

Pp3c17_9390V3.1 ------Pp3c2_17750V3.1 ------Pp1s80_71V6.1 -----SKTESMGL------GGSRKVPAAQ------SVGQ------Pp1s87_157V6.1 -----SKTESMGL------GSSRKVPAAQ------SVGH------Pp1s29_285V6.1 ------Pp1s207_63V6.1 ------Pp3c2_28480V3.1 EIGHSGRLSDIGLNDRRSEIGHSDRRSDIGSGRRSDIGH----SGRRSDIGHSGRRSDVG NP_175853.1 ------SRRIMETGRRSDHLHPVSGGGTTSKIIPSARRTSLP

Pp3c17_9390V3.1 ------Pp3c2_17750V3.1 ------Pp1s80_71V6.1 ------Pp1s87_157V6.1 ------Pp1s29_285V6.1 ------Pp1s207_63V6.1 ------Pp3c2_28480V3.1 LSGRRSDIRKTPSLERGLDHDSAETVKATLQRLSSLTSRSDKDGSSMRSNRSSPSESGNI NP_175853.1 LTKRAT------N------QE------VAAYNP

Pp3c17_9390V3.1 ------Pp3c2_17750V3.1 ------Pp1s80_71V6.1 ------Pp1s87_157V6.1 ------Pp1s29_285V6.1 ------Pp1s207_63V6.1 ------Pp3c2_28480V3.1 IQELGLNNHSPNVSVNAPRLDLIPEFKLTADPEPYSSQLASHEIRMPMERPKFSSSVMPT NP_175853.1 IVGILQNVKSPEYSINEPQVDKIAIFPLAP----YEQDIF----FTPMQRKTSSK----

Pp3c17_9390V3.1 ------Pp3c2_17750V3.1 ------Pp1s80_71V6.1 ------Pp1s87_157V6.1 ------

149

Pp1s29_285V6.1 ------Pp1s207_63V6.1 ------Pp3c2_28480V3.1 TTRSHIAVVSPRYSQQVMPKQTTEPRVSASPNRPDSSLALPASETNKPLYSMHMETSIFD NP_175853.1 ------SSSVSDRS------

Pp3c17_9390V3.1 ------Pp3c2_17750V3.1 ------Pp1s80_71V6.1 ------Pp1s87_157V6.1 ------Pp1s29_285V6.1 ------Pp1s207_63V6.1 ------Pp3c2_28480V3.1 KTQEKGTIQINEKSPAPPAPARPAFNDVIHVIRHSTFRLGATSDHSHTDADYATMGEIDF NP_175853.1 ------ITKDKCTVQTHTTWQ-G------IQ-

Pp3c17_9390V3.1 ------Pp3c2_17750V3.1 ------Pp1s80_71V6.1 ------Pp1s87_157V6.1 ------Pp1s29_285V6.1 ------Pp1s207_63V6.1 ------Pp3c2_28480V3.1 RAGRTDLEPFHGKMDIGSLLDLPQRGSDVEVVSVSPGSSVTSRHPQVDMHQRHANGLDVK NP_175853.1 ------LNM-VDNISDGSSSSDQNATAGAS-----SHTTSSSSRRCRFDPS

Pp3c17_9390V3.1 ------Pp3c2_17750V3.1 ------Pp1s80_71V6.1 ------Pp1s87_157V6.1 ------Pp1s29_285V6.1 ------Pp1s207_63V6.1 ------Pp3c2_28480V3.1 SYRQRAEALEGLLELSAQLLSQHRLEELAIVLKPFGRGKVSPRETAIWLTKSLKGMLGDE NP_175853.1 SYRQRADALEGLLEFSARLLQEGRYDELNVLLKPFGPGKVSPRETAIWIAKSLKENRDKT

Pp3c17_9390V3.1 ------Pp3c2_17750V3.1 ------Pp1s80_71V6.1 ------Pp1s87_157V6.1 ------Pp1s29_285V6.1 ------Pp1s207_63V6.1 ------Pp3c2_28480V3.1 QPHD------SPTVVI* NP_175853.1 KMVDLNVSREIPHVGLL

150

Table 7: Primers used for qPCR

PpRAK2 (Pp1s80_71V6.1) ss222F CTATTGGCCATGGAGCATAT ss223R AGGAATGATGTTGTCATGCT PpNATH (Pp3c17_14350V3.1) ss224F CGCTACATTTTCGTCAAACA ss225R CGTATAATGCTGGGACTTCA PpCHK1 (Pp3c25_8540) ss226F GAAAATATCATACGAGCGCG ss227R CAGGTCTGTGGTGTATACAG PpSHI (Pp3c21_16440) ss230F ACACATTTTCAAGGGACTGT ss231R GGACCATACATTCCAGAAGG PpCCD8 ss76 GTCGCGCAGAAGAAGTAACC ss77 GCCACTCATCTTGCTTCACA PpENA 1 ss102F GTAGTTAGATCAGCGGCGCT ss103R TATGCCAGACTGAGTCGGGA PpCOR47 (Pp1s442_22V6.2) JB03f GCTCCCACATCTGGAGGATA JB04r CGTAGGTTCGGACGTTTGTT PpPCA1 (Pp1s8_288V6.1) JB11f GGATATCGTTGTTGGGGATG JB12r CTGGTTCGCTTTCTCCAGTC PpNCED3 ss61 CCGTGAAGTTCAAGCACTGA ss62 AACAGCGTGGTGGAGGATAC B-tubulin (Pp1s93_158V6.1) sb041F gagttcacggaagcggagag sb042R atatctttcaggctccaccg

151

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Manuscript 1: Remarkable Regulatory Rosettas in the bryophyte Physcomitrella patens

Sabrina Stanimirovic1, Morten Petersen1 & John Mundy1* 1 Department of Biology, University of Copenhagen, Copenhagen, Denmark. *Corresponding author [email protected]

Abstract Bryophytes (hornworts, mosses and liverworts) arose from an ancestor related to unicellular aquatic algae, and they diversified from aquatic environments to adapt to terrestrial habitats. We have discovered a pair of novel rosetta proteins in the moss P. patens which combine a protein N-terminal acetyltransferase (NATD) and a MAP kinase (MPK). Rosettas are rare fusions between proteins of different functions. They occur by near random re-arrangements and may be maintained in a lineage because they optimize the co-expression/interactions of the original proteins. NATDs N-acetylate histones H2A and H4, while MPKs generally regulate adaptive and developmental responses by phosphorylation of substrate proteins. The moss rosettas are remarkable in combining post- translational modification enzymes which may regulate multiple pathways. The rosettas are highly expressed in leafy gametophytes, protoplasts, protonemata and during secondary branching. Double knockout lines of both rosettas, and knockout lines of their single domain NAT homolog (NATH), have strong developmental phenotypes including fewer and shorter gametophytes and early induction of sporophytes. Thus, the rosettas are required for a primary developmental switch in the moss life cycle. This report aims to determine their functions to provide mechanistic links between protein acetylation and phosphorylation by phenotypically characterizing knockout mutants of the rosettas and NATH.

Keywords: Mitogen-activated protein kinase (MAPK); N-terminal acetyltransferase (NAT); Physcomitrella patens; Cytokinin; Auxin; Strigolactone.

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Introduction The transition of plants from aquatic to terrestrial environments resulted in adaptations and strategies to cope with new stresses and threats. This led to the loss of genes associated with aquatic environments and the evolution of genes for enhanced osmoregulation, desiccation, cold and heat tolerance, synthesis and accumulation of protective “sunscreens” against e.g. UV-light, and enhanced DNA repair mechanisms (2-5). These adaptations included the expansion of repertoires of genes encoding receptors for direct recognition of microbe-associated molecular patterns (MAMPs) or pathogen-associated molecular pattern (PAMPs). These genes provide a first line of defense against pathogens and are therefore central to disease resistance in plants. Bryophytes (mosses, hornworts and liverworts) arose from an ancestor related to unicellular aquatic algae (1, 5, 6), and they diversified to adapt to terrestrial habitats. Examples of model bryophytes are the moss Physcomitrella patens and the liverwort Marchantia polymorpha (7-9). Plants and animals rely on the recognition of patterns that may be threating or beneficial to them. Vertebrates have a two-fold defense system with innate and adaptive immunity against pathogens. Activation of vertebrate innate immune responses is central to the activation of the adaptive immune system in which lymphocytes and other specialized cells and receptors are key components. Plants do not have adaptive immune systems, but are able to distinguish between self and non-self and to mount defense responses in their cells by their innate immune system (10, 11). In addition to preformed defenses, the so-called phytoanticipins such as cuticular waxes, secondary metabolites and anti-microbial enzymes (4), plants have two major layers of innate immunity. In the first layer, MAMPs or PAMPs are recognized by plasma-membrane localized pattern recognition receptors (PRRs). This recognition triggers PAMP-triggered immunity (PTI)(12). Plants are able to recognize many different MAMPs due to the formation of complexes with different receptors. The perception of PAMPs/MAMPs leads to many downstream events, including activation of MAP kinase (MPK) cascades and the expression of defense genes. Eukaryotic MPKs transduce signals into adaptive and programmed responses via phosphorylation of substrate proteins including transcription factors. The kinases thus transduce extracellular signals from the cell surface to the nucleus by a phosphorylation cascade in which MPK kinase kinases (MEKKs) phosphorylate MPK kinases (MKKs) that phosphorylate MPKs. MPKs regulate many different cellular processes such as cell differentiation, innate immunity, stress and hormonal responses via the activation of other kinases, enzymes and transcription factors (13, 14).

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The model flowering plant Arabidopsis thaliana encodes 60 MEKKs, 10 MKKs and 20 MPKs which indicates that there are levels of functional redundancy in MPK signaling (14-16). MEKKs are serine or threonine kinases phosphorylating MKKs that then phosphorylate MPKs on threonine and tyrosine residues in the MPK activation loop. MPK activity and deactivation are regulated by tyrosine and serine/threonine specific phosphatases (14). MPK signaling in plants like Arabidopsis is complex. To elucidate basic aspects of such complexity, it may help to study MPKs in potentially simpler bryophyte models. The moss P. patens has 22 predicted MEKKs, 7 MKKs and 8 MPKs (17). While some redundancy is expected among them, it may be possible to assign a function for each MPKs in P. patens. We describe here a pair of novel rosetta proteins in P. patens which combine a MAP kinase (MPK) and a protein N-terminal acetyltransferase (NATD). The relative rarity and duality of rosetta stone proteins distinguish them from common chimeric or multi-domain proteins (18). Rosetta genes arise by near-random recombination events, and may thus be specific to limited phylogenies (19). However, novel functional insights from rosettas have been limited as they are often fusions of enzymes or subunits of known biosynthetic pathways or complexes. The moss rosettas appear to be remarkable in combining two regulatory enzymes, a NATD and an MPK. While the interplay between protein acetylation and phosphorylation is well documented in the modification of histone tails (20), a major mechanism in chromatin remodeling, direct links between them are few, particularly for MPKs (21, 22). The moss rosettas, which we call RAK1 and RAK2 (Rosetta NATD-MPK), may therefore represent new tool for understanding protein modifications affecting chromatin, gene expression and cellular signaling.

Results Identification of Physcomitrella patens homologs of MPKs in Arabidopsis thaliana A reverse BLASTp search against the Arabidopsis thaliana MPK genes (AtMPKs) with the highest P. patens hits (PpMPKs) was used to showed phylogenetic relationships between the 20 MPKs from Arabidopsis and the 8 MPKs from P. patens. From the phylogenetic relationship and multiple alignment of the proteins, we noted that MPK2/MPK5, RAK1/RAK2 and MPK3/MPK7 and MPK4a/MPK4b (17), exhibit highly sequence similarity as apparent pairs (Supplemental Figure 1). Further sequence alignments (Supplemental Figure 2) showed that RAK1/RAK2 differ from the other 6 PpMPKs in their N-terminal region. This region of RAK1/RAK2 of approximately 30kDa is similar to N-terminal acetyltransferases of the NATD type including one moss protein containing just a NATD-like sequence (PpNATH) (Supplemental Figures 1 and 3). There are no apparent

164 rosettta-like sequences similar to RAK1 and RAK2 in other embryophytes like Marchantia polymorpha and S. fallax (data not shown). The RAKs are interesting as they may provide links between protein phosphorylation and acetylation, hence the name RAK (Rosetta NATD Kinase). RAK1 (locus Pp09c_11360) and RAK2 (Pp15c_11610) are encoded on syntenic chromosome 9 and 15 regions. Their NAT sequences share 6 introns with NATH (Pp17c_14350) encoded on a non-syntenic chromosome 17 region. In contrast, the RAK1 & 2 MPK sequences lack the 4-5 introns found in the other 6 moss MPKs encoded on other chromosomes. Alignments with sequences from diverse eukaryotes show that the RAKs contain both full-length enzymes separated by a short, repeated region unique to them (Supplemental Figure 4). Thus, the rosetta probably arose by retroposition of a MPK C-terminally to a NAT paralog, and this RAK was later duplicated on chromosome 9 or 15.

Generation of rak1, nath and rak1-rak2 knockout mutants We generated RAK single and double knockouts (KO) mutants and knockin (KI) versions with GFP- tags by homologous recombination using targeted gene disruption to understand their functions. The single KOs were generate in var. Gransden wild type by transforming the specific deletion constructs, and the rak1-rak2 double KO was generated by introducing the RAK2 KO construct into the rak1 background. The KI constructs with GFP tag were cloned in vector pMBLU containing GFP. After transformation, protoplasts underwent two antibiotic selection- and one nonselective rounds, and targeted disruption events were detected by PCR using gene specific primers. Primers for the external part of the targeting constructs with outward oriented primers that specifically target the selectable marker cassette (Supplemental Figure 5) were used to genotype the correct integration of the targeted disruption in the KOs. We generated 4 lines of rak1, and 2 lines of each rak1-rak2 double KO and nath single KO (Supplemental Figure 5). We were also able to generate KI lines of RAK1, RAK2 and NATH.

Phenotypic analysis of rak1, rak1-rak2 and nath Sporophyte induction As several genotypes were phenotypically analysed for sporophytic induction, the results of these experiments is summarized for clarity in Supplemental Table 1. P. patens has different morphologic characteristics during the stages in its life cycle (Supplemental Figure 6). The mutant rak1, rak1-rak2 and nath lines were phenotypically compared during the development of female (archegonia) and male (antheridia) reproductive organs (Figure 1A-C, Supplemental Figure 7A). After irrigation of colonies the canals of archegonia (Supplemental Figure 7A, black arrows) are dark brown, indicating

165 that spermatozoids have fertilized the eggs. Antheridia appear as round bundles with shinny surfaces localized around the archegonia (Supplemental Figure 7A, white arrows). Six weeks after fertilization the zygote develops into the diploid sporophore and later sporangium. The sporangium undergoes different maturation steps indicated by the coloration of the spore capsule (Supplemental Figure 7B). To phenotypically analyze the maturation of spore capsules, we staged them as followed: pre-capsule, early green, late green/yellow, yellow/orange, red (matured) and brown (2). Interestingly, 3 weeks after the first irrigation, rak1-rak2 and nath developed sporophyte capsules at the yellow/orange maturation stage (Figure 1B-C, black arrows), while rak1 generated sporophytes in the green stage (Figure 1D, black arrows). This capsule development and maturation was approximately 3 weeks earlier than development in WT. WT had at this time point developed the organ bundle but no sporophyte capsules (Figure 1A). The canal of the archegonia in the WT seemed to be dark, indicative of egg fertilization (Figure 1A, dark arrows), and the antheridia were visible underneath the archegonia (Figure 1A, yellow arrows). The double rak1-rak2 mutant produced 3 sporophyte capsules in the green- and yellow/orange stage (Figure 1C), while nath produced 1 in the yellow/orange (Figure 1B). No sporophytes in the earlier maturation stages of sporophyte capsules were visible for nath. rak1 produced 3 sporophyte capsules in the green maturation stage, and no sporophytes in later maturation stages were visible (Figure 1D). The archegonia (Figure 1B (3-8), black arrows) in nath were clearly protruding out from organ bundles, while the antheridia were more problematic to identify (Figure 1B (3-8), yellow arrows). The canal and egg sack of the archegonia in rak1-rak2 also clearly protruded from the organ bundle, although the egg sacks appeared rounder and were more visible than the sacks in nath and rak1 (Figure 1C (7- 8), black arrows, Supplemental Figure 7A (J)). The antheridia were also more visible in rak1-rak2 than the antheridia in nath and rak1 (Figure 1C (8), yellow arrows, Supplemental Figure 7A (J)). Interestingly, the calyptra in rak1-rak2 and nath were clearly visible (Figure 1B (1-2) & C (2-3), black- & red arrows). Since rak1, rak1-rak2 and nath generated sporophytes earlier than WT, RAK1, RAK2 and NATH may be important factors in the regulation of sporophyte development.

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Figure 1: Organ bundles and sporophyte formation in WT (A), nath (B), rak1-rak2 (C) and rak1 (D) 3 weeks after the first irrigation of colonies. WT generated organ bundles with female (archegonia) and male (antheridia) reproductive organs (A1-3, black & yellow arrows). nath generated sporophyte capsules in the yellow/orange maturation stage (B1-2), and organ bundles with archegonia (black arrows) and antheridia (yellow arrows) (B3-8). rak1-rak2 generated sporophyte capsules in the yellow/orange and green maturation stages (C1-6). Black- and yellow arrows (C7-8) highlight archegonia and antheridia in the organ bundles for rak1-rak2. Black arrows (D) highlight sporophyte capsules in the green maturation stage in rak1.

Spore capsules were ruptured for germination and phenotypic analysis. Spores from early-developed sporophytes of rak1-rak2 and nath were able to germinate (Figure 2A-H). Interestingly, less than 10% of nath spores (Figure 2B-D) germinated, compared to 90% of rak1-rak2 spores (Figure 2F-H). Protonemata and rhizoid development were initiated by tip growth during spore germination. The first cells that were formed are chloronema (Figure 2D-H). Since the spores of nath did not germinate to the same degree as rak1-rak2, NATH may be important in spore germination. Moreover, there

167 could be some redundancy in the NATs between RAK1, RAK2 and NAT, since rak1-rak2 spores germinated.

Figure 2: Germination of nath (A) and rak1-rak2 (B) spores. (B-C) nath spores that have not germinated (white circle) and the ruptured sporophyte capsule (white arrow). (D) After 7 days, nath protonemata from germinated spore, with chloronema cells. (F-G) rak1-rak2 spores that have not germinated (white circle) and the ruptured sporophyte capsule (white arrow). (D) After 7 days, rak1-rak2 protonemata from germinated spore, with chloronema cells.

Protonemata and secondary branching As several genotypes were analysed for protonematal and gametophyte phenotypes after 3- and 6 weeks of growth on BCD and BCDAT, the results of these experiments is summarized in Supplemental Table 2. The filamentous morphology of protonemata is formed by chloronemal and caulonemal cells (Supplemental Figure 6B-C). Chloronema contain short, broad cells with 50-100 chloroplasts and transverse cell walls perpendicular to the growth axis. In contrast, caulonema contain fewer and less-developed plastids, and have oblique transverse cell walls (1, 23, 24). WT, rak1, nath and rak1-rak2 were grown for 3 and 6 weeks on full (BCDAT) and on minimal media (BCD) to analyze the growth of filaments, leafy gametophytes and rhizoids (Figure 3). The colony of rak1 was significantly larger than the others (Supplemental Figure 8). In contrast, rak1-rak2 and nath plants were significantly smaller than WT and rak1 (Supplemental Figure 8),

168 indicating that RAK2 and NATH regulate growth of the filamentous body. All the lines had characteristic growth after 3 weeks on BCD, and they all generated gametophytes (Figure 3A-D & a- d, white arrows). Leafy gametophytes were clearly seen for WT and rak1 (Figure 3A-B), but were challenging to visualize for rak1-rak2 and nath (Figure 3C-D) without dissecting the colonies. The caulonema, chloronema and buds were observable at the periphery of WT and rak1 colonies. In contrast, no buds were visible in rak1-rak2 and nath (Figure 3e-h, black circle). Bud formation is a result of secondary branching and the initial step before leafy gametophytes. nath had discolored filaments, making it difficult to characterize the protonema and to distinguish between chloronemal and caulonemal cells and rhizoids (Figure 3D, d, h & l).

Figure 3: Phenotypic comparison of P. patens WT and rak1, rak1-rak2 and nath mutants. (A-D) 3 week-old plants grown on minimal media (BCD) (bar = 2mm). (a-d) Colonies from the center to the periphery, white arrows highlight gametophytes (bar = 1mm). (e-h) Periphery of the colonies, black circles highlight buds (bar = 1mm). (i-l) Caulonema and chloronema filaments (bar = 1mm). WT (A, a,e & i), rak1 (B, b, f & j), rak1-rak2 (C, c, g & k) & nath (D, d, h & l).

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It seems that the protonemata tissue of rak1-rak2 and nath have a ‘split identity’, since it is unclear whether the filaments are chloronema or caulonema cells (Figure 4B-C). The filaments are bent and have hook-like structures (Figure 4B-C, black arrows). The dark coloration of nath filaments could conceivably be due to increased autophagy, since chlorosis under nutrient starvation is a result of autophagy in P. patens (25). Post-translational modifications (PTMs), like N-terminal acetylation, have been shown to be important in the control of autophagy (26, 27).

Figure 4: Phenotypic comparison of P. patens rak1, rak1-rak2 and nath mutants. (A-C) 3 week-old plants grown on minimal media (BCD) (bar = 1mm). (A1-2) rak1 filaments with buds and early stage of leafy gametophytes (black circles) (bar = 1mm). (B1-2) rak1-rak2 filaments with distinctive phenotype (black arrows) (bar = 1mm). (C1-2) nath filaments with hook-like structures and dark coloration (black arrows) (bar = 1mm).

Gametophytes from the central and peripheral parts of colonies were measured after growth on BCD which induces more leafy gametophytes than BCDAT media (Figure 5). Their length was measured from the base of the gametophore to the tip of the leaf at the apex, thus the rhizoids were not measured. Central gametophytes would be more mature than the peripheral gametophytes, hence the localization of the gametophytes would have an impact on their length. 3- and 6 week-old rak1-rak2 and nath generated significantly shorter central and peripheral gametophytes compared to WT, while 3 week- old rak1 peripheral gametophytes and 6 week-old central and peripheral gametophytes were significantly shorter (Supplemental Figure 9A-B). rak1 generated significantly more gametophytes than WT, rak1-rak2 and nath (Supplemental Figure 9C). The double mutant rak1-rak2 and nath both generated significantly fewer gametophytes than WT (Supplemental Figure 9C). This observation correlates with the difficulty in visualizing buds, the pre-maturation stage for leafy gametophytes, in

170 rak1-rak2 and nath. In contrast, buds in rak1 were seen on many of the filaments, which would result in more leafy gametophytes (Figure 4, black circles). After 6 weeks on BCD the numbers of gametophytes were significant higher than at 3 weeks on BCD for WT, rak1-rak2 and nath. There was no significant difference between 3 and 6 weeks of growth for rak1 (Supplemental Figure 9C). Gametophytes of rak1-rak2 and nath were problematic to visualize compared to WT and rak1 (Figure 5A). Filaments were wrapped around gametophytes, and the rhizoids and thalli were more fragile compared those of WT and rak1. The rhizoids of rak1-rak2 and nath were greener than those of WT and rak1(Figure 5A). Gametophytes on 6 week-old rak1-rak2 and nath colonies were more visible and the rhizoids were more compact and darker compared to 3 week-old gametophytes (Figure 5B). Interestingly, rak1-rak2 and nath still had filaments wrapped around the gametophytes, which had a light green/white coloration in rak1-rak2 and dark coloration in nath (Figure 5C). This may indicate that rak1-rak2 and nath have early senescence. Furthermore, these phenotypic observations of rak1-rak2 and nath indicate that RAK2 and NATH affect the generation of gametophytes and growth of the filamentous body.

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Figure 5: Phenotypic comparison of WT, rak1 and rak1-rak2 and nath mutants. (A) 3 week-old plant grown on minimal media (BCD) (bar = 2mm). (B) 6 week-old plants grown on minimal media (BCD) (bar = 2mm). Gametophytes from central and peripheral parts of 3 and 6 week-old colonies were dissected: (A) central and peripheral gametophytes for WT, rak1, rak1-rak2 (bar = 1mm) & nath (bar = 2mm). (B) Central and peripheral gametophytes for all lines (bar = 2mm). (C) Central part of the colonies after 6 weeks on full media BCDAT (bar = 2mm).

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Activation of the MPKs MPKs regulate processes such as cell differentiation, immunity, and stress and hormonal responses by regulating the activities of other kinases, enzymes and transcription factors (13, 14, 17). Bressendorf et al. identified a moss MPK signaling pathway cascade mediating responses to the fungal PAMP chitin including the PRR PpCERK1, PpMEKK1a/1b, PpMKK1a/1b/1c and the MPKs PpMPK4a and PpMPK4b,. I investigated whether RAK1, RAK2 are activated upon biotic stress such as exposure to chitin. The phosphorylation and activation of the kinases were measured by immunoblotting with anti-phospho-p44/42 MPK antibody (α-pTEpY). MPK4a and MPK4b are phosphorylated and activated in response to chitin in WT, rak1, rak1-rak2 and nath (Figure 6D, black and red star), indicating that RAK1, RAK2 and NATH are probably not involved in signaling in response to chitin. RAK1/RAK2 have almost the same protein size, thus their individual phosphorylation and activation patterns could be difficult to characterize in single mutant lines. To attempt to characterize their phosphorylation, activation and size, the mutant lines rak1, mpk3, mpk5 and rak1- rak2 were treated with the protein Serine/Threonine phosphatase inhibitor calyculin A. Such treatment should in principle increase detectable basal MPK activation signals. mpk3 and mpk5 were included in the immunoblot to give an overall picture of the phosphorylation of MPKs upon treatment with calyculin A. Like RAK1 and RAK2, MPK2 and MPK3 are paralogous to MPK5 and MPK7, respectively (Figure 6A-C). Treatment with calyculin A increased the phosphorylation signals for RAK1/RAK1, MPK2/MPK5 and MPK3/7 in WT (Figure 6A-C, black star, black arrow and white arrow, respectively). Interestingly, RAK1 and RAK2 were not phosphorylated and activated in mpk3 (Figure 6A). This could indicate that MPK3 phosphorylates RAK1 and RAK2 or indirectly affects their activation. A strong phosphorylation was seen in mpk5 at approximately 60kDa when treated with calyculin A. This is most likely MPK2 which, although presumably paralogous to MPK5 of 45.8kDa, has a predicted size of 60.8kDa (Figure 6A, red star). The sizes of RAK1 (73.87kDa) and RAK2 (74.73kDa) are very similar, and there is more or less a single band detected in WT as a result of activation of RAK1 and RAK2 (Figure 6A & C, black and red star).

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Figure 6: Phosphorylation and presumptive activation of the MPKs was measured by immunoblotting with anti- phospho-p44/42 MPK antibody (α-pTEpY). (A) Phosphorylation and activation of kinases in WT, rak1, mpk3, mpk5 and rak1-rak2 upon treatment with the protein serine/threonine phosphatase inhibitor calyculin A (Cal.) versus no treatment (NT). Phosphorylation of RAK1 and RAK2 (asterisk), MPK3/MPK7 (white arrow), MPK5 (black arrows) and red asterisk = probably MPK2. (B) Phosphorylation and activation of kinases in WT and nath treated with calyculin A (Cal.) versus untreated (NT). Phosphorylation of RAK1 and RAK2 (asterisk), MPK3/MPK7 (white arrow) and MPK5 (red arrow). (C) Phosphorylation and activation of RAK1 and RAK2 in WT (asterisk) and RAK2 in rak1 mutants (red asterisk). (D) Phosphorylation and activation MPK4a (asterisk) and MPK4b (white asterisk) in response to chitin (CHI) in WT, rak1, rak1-rak2 and nath.

The phosphorylation is less strong in rak1 and indicative of RAK2 phosphorylation (Figure 6C, red star). No phosphorylation and activation of RAK1 and RAK2 was detectable in the rak1-rak2 double mutant, showing that rak1-rak2 is a double KO of RAK1 and RAK2. Activation and phosphorylation of MPKs were analyzed in the nath mutant (Figure 6C). RAK1/RAK2, MPK5 and MPK3/MPK7 appeared to be phosphorylated in WT, but MPK3/MPK7 seemed not to be phosphorylated in nath (Figure 6B, black star, red arrow and white arrow, respectively). This suggests that NATH somehow affects the activation and phosphorylation of MPK3/MPK7. The relative expression of NATH in mpk3 was significantly higher than in WT, suggesting that MPK3 impacts the expression of NATH (Supplemental Figure 10B). The expression of NATH in rak1 was significant lower than WT, in contrast to the expression in rak1-rak2 which was significantly higher. Interestingly, this may indicate that the gene deletion of the acetyltransferase domains in RAK1 and RAK2 results in a higher

174 expression of NATH (Supplemental Figure 10A). Since the level of RAK2 mRNA in rak1 was significantly higher than in WT, there may be some compensatory redundancy between RAK1 and RAK2. In Figure 5A it is clear that RAK1 and RAK2 are not phosphorylated in mpk3, although RAK2 is expressed in mpk3 at significantly lower levels than in WT. This observation suggests some relationship between MPK3 and RAK1 and RAK2. Furthermore, the relative expression of RAK2 in nath was also significantly lower than WT, giving another interesting link between NATH, MPK3 and RAK1/RAK2 (Supplemental Figure 10B).

Abiotic stress – Hormones To determine if any of the physiological changes described for rak1, nath and rak1-rak2 could be due to hormonal imbalances, KO lines were analyzed after application of the auxin indole-3-acetic acid (IAA), cytokinin (BPA) and an analog of strigolactone (GR24). Auxin regulates the transition from chloronema to caulomena, and initiates tip growth and development of rhizoids. Cytokinin regulates the development of secondary branching from chloronemal and caulonemal filaments. Auxin and cytokinin regulate the formation of buds, which is the developmental stage before leafy gametophytes. Strigolactone is known to act as a signaling factor for controlling the extension of filaments in the development of gametophytes and rhizoids (28-30).

Auxin Application of IAA accelerates the transition from chloronema to caulonema filaments, stem elongation and development of rhizoids from the leafy shoots (31-33). WT, rak1, rak1-rak2 and nath were grown on BCDAT plates to slow protonemata differentiation so that morphological differences could be characterized upon IAA treatment. The lines were grown for 6 weeks on plates supplemented with 500mM IAA (Figure 7A-B). As illustrated in Figure 4C, the gametophytes of rak1-rak2 and nath were problematic to visualize compared to WT and rak1. This characteristic phenotype is also seen in Figure 7A, where the application of IAA did not change the morphology of the colonies. Growth on IAA supplemented plates induced accelerated growth of gametophytes in WT, rak1 and rak1-rak2, but not in nath (Figure 7B). This suggests some involvement of NATH in auxin signaling. Moreover, IAA induces growth of rak1 colonies since colony size was significantly bigger than WT, rak1-rak2 and nath (Supplemental Figure 12B). This suggests that RAK1 may affect some aspect of auxin responses.

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Figure 7: Phenotypic comparison of WT, rak1 and rak1-rak2 and nath mutants on BCDAT and BCDAT supplemented 500nM auxin. (A) 6 week-old colonies on BCDAT with 500nM auxin (IAA). Bar = 1mm. (B) Amount of gametophytes after 6 weeks on BCDAT versus BCDAT with 500nM IAA. Analysis of variance by T-test determined statistical differences indicated by A-D (BCDAT) and a-d (BCDAT+500nM IAA) (P <0.05), n = 3. Statistical difference between BCDAT and BCDAT+500nM IAA indicated by *P = 0.05-0.01, **P = 0.01-0.001, *** P < 0.001. Standard deviation as error bars.

Cytokinin The growth of WT, rak1, mpk3, mpk5 and rak1-rak2 was initially analyzed on BCDAT supplemented with 500mM BPA (Supplemental Figure 11). Interestingly, rak1-rak2 seemed not to respond to cytokinin as the other lines, and a concentration of 2.5µM was clearly not toxic to this double mutant. Exogenous cytokinin reduced the amount of differentiated gametophores and promoted bud formation with callus-like structures (Supplemented Figure 11). We further analyzed the morphological changes of all the lines on BCDAT plates with 500mM BPA (Figure 8). Interestingly, nath reacted in the same manner as rak1-rak2. Furthermore, the colony area of rak1-rak2 and nath on BPA was significantly bigger than WT and rak1 (Supplemental Figure 12B). This indicates that the exogenous BPA induces growth of the mutants. As rak1 was not as affected by BPA as the rak1- rak2 and nath mutants, it may be that the acetyltransferase activities of RAK1/RAK2 and NATH are important in the regulation and/or signaling of cytokinin.

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Figure 8: Phenotypic comparison of WT, rak1 and rak1-rak2 and nath mutants. (A) 3 week-old plant grown on full media (BCDAT) and BCDAT supplemented with 500nM BPA (bar = 2mm). (B) 6 weeks old plants grown on BCDAT and BCDAT with 500nM (bar = 2mm).

Strigolactone Strigolactones control shoot branching and signaling during symbiotic and parasitic interactions (28, 29). Strigolactone biosynthesis is characterized in Arabidopsis and P. patens (30). Moss carotenoid cleavage dioxygenases 7 and 8 (CCD7 and 8) in plastids catalyze the formation of strigolactones. PpCCD8 is involved in the regulation of filament branching related to the inhibition of shoot branching by strigolactones in the moss (28). The phenotypes of WT, rak1, rak1-rak2 and nath were analyzed on BCDAT plates supplemented with the strigolactone analog GR24 (Figure 9A-B). GR24 inhibited the growth of gametophytes in rak1, rak1-rak2 and nath, while growth of gametophytes in WT and the Ppccd8 mutant were not inhibited (Figure 9B). These observations imply that rak1, rak1-rak2 and nath do not respond as the WT and ccd8 to strigolactone. Thus RAK1, RAK2 and NATH may be involved in strigolactone signaling, but not in its biosynthesis like ccd8. The colony areas of rak1-rak2 and nath were significantly smaller than WT and rak1 when grown on plates with GR24 (Supplemental Figure 12B), indicating rak1-rak2 and nath are more sensitive to GR24 and that RAK2 and NATH may impact strigolactone responses.

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Figure 9: Phenotypic comparison of WT, rak1 and rak1-rak2, nath and ccd8 mutants on BCDAT and BCDAT with GR24. (A) Colonies of 6 week-old WT, rak1, rak1-rak2 and nath on BCDAT with 500nM GR24. Bar = 1mm. (B) Amount of gametophytes after 6 weeks on BCDAT and BCDAT with 500nM GR24. Analysis of variance by T-test determined statistical differences indicated by A-D (BCDAT) and a-d (BCDAT+500nM BPA) (P <0.05), n = 3. Statistical difference between BCDAT and BCDAT+500nM IAA indicated by *P = 0.05-0.01, **P = 0.01-0.001, *** P < 0.001. Standard deviation as error bars.

When treated with IAA, BPA and GR24, RAK1 and RAK2 are not more activated and phosphorylated in WT than no treatment, indicating that the exogenous hormonal treatments do not directly interact with the RAK MPK activities (Supplemental Figure 13). The relative expression of the cytokinin receptor, the CHASE domain-containing histidine kinase (CHK1) (34), and of SHI, which induces the activity of auxin biosynthetic genes (31) were analyzed in WT, rak1 and rak1-rak2 upon treatment with BPA and IAA (Supplemental Figure 14A-B). mRNA levels of CHK1 were significantly higher in untreated rak1 than in WT. In contrast, there was no significant difference between WT and rak1-rak2 (Supplemental Figure 14A). Interestingly, mRNA levels of CHK1 after 30 min. of BPA treatment were significantly lower in rak1, indicating that RAK1 could affect responses to cytokinin. Moreover, longer treatment with BPA negatively affected the mRNA levels in rak1, since there was no significant difference between 30min. and 1 hour of treatment. In contrast, CHK1 was highly expressed after 1 hour of BPA

178 treatment in rak1-rak2, suggesting there could be a delayed response in rak1-rak2 (Supplemental Figure 14A). The expression on CHK1 in the rak mutants could indicate that the RAKs affect the expression of the cytokinin receptor. The relative expression of SHI untreated and IAA-treated WT and rak1showed the same tendency (Supplemental Figure 14B). Interestingly, when treated with IAA, the mRNA levels of SHI in rak1-rak2 were significantly lower than in WT and rak1, indicating that RAK2 may impact the expression of auxin biosynthesis genes. We further investigated the relative expression of the abscisic acid (ABA) biosynthetic enzyme ZEP which initiates carotenoid catalysis into violaxanxin followed by synthesis and oxidative cleavage by NCED and further transformation into xanthoxin. Xanthoxin is then converted to abscisic aldehyde and oxidized into ABA (35). ABA is important for adaptation during stress (35, 36). Since ABA synthesis is initiated from carotenoid substrates, we also studied the mRNA levels of CCD8 that cleaves with CCD7 carotenoid substrates in plastids to synthesize strigolactone (Supplemental Figure 15A-D). mRNA levels of ZEP1 after 1 hour of IAA treatment were downregulated in rak1 and rak1-rak2 (Supplemental Figure 15A), while after 30 min of BPA treatment mRNA levels of ZEP1 were lower in rak1 and rak1-rak2 (Supplemental Figure 15B). The tendency of mRNA levels of ZEP1 in rak1 and rak1-rak2 are the same, indicating that both RAKs may affect hormonal signaling. Interestingly, mRNA levels of CCD8 in rak1-rak2 after 30min and 1 hour of IAA treatment were high (Supplemental Figure 15C), likewise after 30min of BPA treatment (Supplemental Figure 15D). rak1-rak2 were significantly bigger on BPA plates, which may correlate with the idea that strigolactone production may be used by P. patens to regulate colony extension and as a signaling molecule related to population density. Thus, the high mRNA levels of CCD8 could be a result of signaling in the colony extension.

Blue light rak1-rak2 and nath have very distinct phenotypes in the development of protonemata and gametophytes in control and hormonal treatments. Light receptors in P. patens control chloroplast movement, induction of side branching in protonemata, and induction of gametophytes (37-40). In particular, blue light (BL) regulates branching in protonemata as a mechanism for P. patens to avoid light stress (37, 40). We compared the growth and numbers of gametophytes in WT, rak1, rak1-rak2 and nath grown for 3 and 6 weeks under BL and white light (WL). After 3 weeks of growth, the colony size for all lines appeared the same when grown on WL and BL. Moreover, at 6 weeks the growth of WT and rak1 under WL seemed to induce growth of the colonies compared to BL (Figure 10B). This suggests that the plants were preferentially

179 growing gametophytes rather than protonemata. No filaments protruded from the colonies for WT and rak1 after 6 weeks of growth (Figure 10B). This was not the case for rak1-rak2 and nath, since filamentous structures were visible at 3 and 6 weeks of growth under WL and BL (Figure 10A-B). Interestingly, nath after 3 and 6 weeks under BL seemed to be senescent. This senescence phenotype was also seen after 6 weeks of growth under WL (Figure 10A-B). This suggest that nath has early senescence perhaps, as mentioned earlier, a result of autophagy during nutrient starvation (25). After 3 and 6 weeks under WL and BL the growth of gametophytes of rak1-rak2 and nath seemed not to be significantly different (Figure 10A-B).

Figure 10: Phenotypic comparison of WT, rak1 and rak1-rak2 and nath under blue (BL) and white light (WL). (A) 3 week-old plant grown on minimal media (BCD) under BL and WL (Bar = 2mm). (B) 6 week-old plants grown on BCD under BL and WL (Bar = 2mm).

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Gametophytes were counted for all lines after 3 and 6 weeks of growth under WL and BL (Figure 11A-B). rak1 had significantly more gametophytes under WL compared to WT and the other mutants (Figure 11A-B). After 3 weeks under WL, rak1-rak2 and nath had significantly fewer gametophytes than WT and rak1 (Figure 11A). Interestingly, rak1-rak2 and nath still had fewer gametophytes after 6 weeks of growth under WL (Figure 11B). 3 weeks growth under BL resulted in significantly fewer gametophytes for rak1 compared to growth under WL. Furthermore, there were no significant differences between WT and rak1 upon BL. rak1-rak2 and nath still had significantly fewer gametophytes than WT. Interestingly, the amount of gametophytes under BL for WT and rak1 were significantly lower than growth under WL (Figure 11A). This was not the case for rak1-rak2 and nath, as these lines had significantly more gametophytes under BL. This observation suggests that BL influences RAK2 and NATH and the generation of gametophytes. Remarkably, there was no significant difference between the amount of gametophytes when grown under WL and BL after 6 weeks for rak1-rak2 and nath (Figure 11B).

Figure 11: Amount of gametophytes after 3 weeks (A) and 6 weeks (B) on BCD under BL and WL growth conditions. Analysis of variance by T-test determined statistical differences indicated by A-D (WL) & a-e (BL) (P <0.05), n = 3. Standard deviation as error bars. Statistical difference between the amount of gametophytes when grown on WL and BL were analyzed by T-test indicated by asterisks (* P = 0.05-0.01, ** P = 0.01-0.001, *** P < 0.001).

Looking at the values, 6 weeks under WL generated more gametophytes, however this was not the case under BL since the amount of gametophytes were the same after 3 and 6 weeks of growth. This suggests that RAK2 and NATH have a negative regulatory effect on the generation and early development of gametophytes during the first 3 weeks of growth under BL. As mentioned, RAK2

181 and NATH are predicted acetyltransferases and may participate in UV-B induced DNA damage repair and related signaling. While tenuous, this is because Arabidopsis histone acetyltransferase HAG3 participates in UV-B induced DNA damage repair by negatively regulating expression of DNA repair enzymes (41). Fina and Casati (2015) showed that Arabidopsis with decreased transcript levels of HAG3 was less sensitive to UV-B light.

Figure 12: Phenotypic comparison of WT, rak1, rak1-rak2 and nath under blue (BL) and white light (WL). Central and peripheral gametophytes after 3 weeks (A) on BCD under WL and BL (B). (C) Length of central and peripheral gametophytes. Analysis of variance by T-test determined statistical differences indicated by A-C (Central WL), A-C (Central BL), a-c (Periphery WL) and a-c (Periphery BL) (P <0.05), n = 12. Standard deviation as error bars.

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Central and peripheral gametophore lengths after 3 and 6 weeks of growth under WL and BL were analyzed (Figure 12 & 13). After 3 weeks under BL, the leafy gametophytes seemed to be longer than gametophytes under WL (Figure 12A-B). Rhizoids of rak1-rak2 and nath were more visible and darker, implying that BL induces growth of rhizoids in these two mutants (Figure 12B). The length of gametophytes in all lines was significantly longer under BL than under WL (Figure 12C). This observation correlates with other studies demonstrating that BL induces growth of gametophytes. Growth under BL did not revert the length of peripheral gametophytes of rak1 and both central and peripheral gametophytes in rak1-rak2 and nath to WT values (Figure 12C). Interestingly, there were significant differences between the length of gametophytes when grown on WL and BL for rak1, rak1-rak2 and nath, indicating that these lines reacted to BL. After 6 weeks of growth under WL the rhizoids of rak1 (central), rak1-rak2 (periphery) and nath (central and periphery) were more visible and darker than the rhizoids of the other lines (Figure 13A). This characteristic phenotype was also seen when grown under BL for central gametophytes in rak1-rak2 and nath (Figure 13B). As observed after 3 weeks of growth under BL, the leafy gametophytes for all lines seemed to be longer than the gametophytes grown under WL (Figure 12A-B). The length of central and peripheral gametophytes for the mutant lines grown under WL were significantly shorter than the WT. This indicates that deletions of RAK1, RAK2 and NATH all affect the growth of gametophytes. Interestingly, at 6 weeks of growth under WL the central and peripheral gametophytes for WT are equally long. This was also observed for rak1-rak2, but not for rak1 and nath. The length of peripheral gametophytes for rak1 was longer than the central gametophytes. This may imply that there is an energy shift in the growth of central to peripheral gametophytes (Figure 13C). As seen for 3 weeks growth, the length of gametophytes in WT, rak1 and rak1-rak2 were significantly longer under BL than under WL (Figure 13C). Remarkably, this was not seen for the gametophytes in nath, since there was no significant difference between the length of WL and BL grown gametophytes (Figure 13C). This suggests that at this developmental stage the light does not affect nath as it does on the other lines. Furthermore, the observation implies that nath is more sensitive to BL, thus NATH may positively affect BL sensing at this developmental stage. Moreover, when grown under BL, the length of central and peripheral gametophytes for rak1-rak2 and nath were similarly long (Figure 13C). As seen after 3 weeks, 6 weeks of growth under BL did not revert the length of peripheral gametophytes in rak1 and in both central and peripheral gametophytes in rak1-rak2 and nath to WT values (Figure 13C).

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Figure 13: Phenotypic comparison of WT, rak1, rak1-rak2 and nath under blue (BL) and white light (WL). Central and peripheral gametophytes after 6 weeks (A) on BCD under WL and BL (B). (C) Length of central and periphery gametophytes. Analysis of variance by T-test determined statistical differences indicated by A-C (Central WL), A-C (Central BL), a-c (Periphery WL) and a-c (Periphery BL) (P <0.05), n = 12. Standard deviation as error bars.

Expression of RAKs and NATH in P. patens We generated knockin (KI) versions with GFP-tag by homologous recombination using targeted gene disruption to analyze the expression of RAK1, RAK2 and NATH. All the KI versions were phenotypically analyzed by confocal fluorescence microscopy. We compared the expression of RAK1, RAK2 and NATH in protoplasts prepared from 2 weeks-old plants (Supplemental Figure 16). Compared to the WT, it appeared that RAK1, RAK2 and NATH are expressed in protoplasts although this can be problematic due to high chlorophyll autofluorescence. Comparing the merged pictures of the WT protoplasts to the reporter lines, RAK1, RAK2 and NATH GFP fusions could be expressed

184 in the chloroplasts and cytosol in the protoplasts (Supplemental Figure 16). To analyze the expression of the genes in different tissues, 2 week-old plants were analyzed. At 2 weeks, the plants have all the morphological characteristics such as protonemata with chloronemal- and caulonemal filaments, and leafy gametophytes with rhizoids protruding from the gametophytes. Likewise, bud formation and thallus in the gametophytes were analyzed (Figures 14-19). Figure 14A-C shows that there is background signal, but no GFP signal in the thallus (A), filaments (B), rhizoids (C) and buds (C, black arrow).

Figure 14: Confocal fluorescent pictures of 14 day-old WT visualizing the background signal, but no GFP signal in the thallus (A), filaments (B), rhizoids (C) and buds (C, black arrow).

RAK1-GFP was expressed throughout the different tissues including at the initiation of secondary growth of protonemata, the chloronemal filaments (Figure 15D-E, red arrows) and the buds (Figure 15C, white arrow). RAK1-GFP was also localized cytoplasmic in chloronema cells near their transverse walls perpendicular to the growth axis (Figure 15A, white arrows; D-E, red arrows), and near at the cell walls in thallus (Figure 15A-B and F-J, white arrows). There was no GFP signal near the oblique transverse cell walls of caulonema, but there was signal in the caulonema cells (Figure 15D-E, yellow arrows). Moreover, there was strong signal in the thallus, spanning from the tip through the midrib and to the surrounding cells. The GFP signal seemed to be present in either the cytosol or chloroplasts (Figure 15A-B and F-J, white arrows).

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RAK1 appears to be expressed during secondary growth, initiating from secondary growth of filaments, bud formation and generation of thallus.

Figure 15: Confocal fluorescent pictures of 14 day-old RAK1-GFP. GFP signal in thallus (A-B, F-J, white arrows). Signal in buds (C, white arrow), and in chloronema (D-E, red arrows) and caulonema cells (D-E, yellow arrows). Bar = 100µm. RAK2-GFP was not expressed to the same degree as RAK1-GFP, as RAK2-GFP was predominately detected during secondary growth of protonemata. There was a RAK2-GFP signal in caulonema cells (Figure 16A, D-E, white arrows, where the oblique transverse cell walls are highlighted with yellow arrows). Chloronema cell walls are highlighted in figure 16D-E with red arrows, where RAK2-GFP was detected in the cells. In general, there is signal in the cytosol or chloroplasts of the cells. Furthermore, RAK2-GFP was expressed in the tip cells (Figure 16C-D, blue arrows). RAK2-GFP was also expressed in the thallus, but not to the same degree as RAK1-GFP. RAK2-GFP was expressed in the tip cells and in the peripheral cells of the thallus (Figure 16B-C, white arrows), and not in the midrib of the thallus as was RAK1-GFP. The strongest RAK2-GFP signal was seen at the initiation of secondary branching and initiation of buds (Figure 16F-I, white arrows). However, RAK2 was not in expressed in the later generation of buds, as seen in RAK1 (Figure 16J, black arrow merge picture). These observations indicate that RAK2 is expressed predominantly in chloronema cells, and initiation of buds and to some degree in the thallus while RAK1 is expressed dominantly

186 throughout the different tissues and the later generation of buds. Comparing these observations with the phenotype of the rak1rak2 double mutant, their loss-of-function might be expected to strongly impact secondary growth in protonemata and the formation of buds. Likewise, the lower chlorophyll content under different light conditions correlates well with the observed expression of RAK1 and RAK2 GFP fusions in the cytosol or chloroplast of cells in protonemata and thallus.

Figure 16: Confocal images of 14 day-old RAK2-GFP. GFP signal in thallus (B-C, white arrows). Signal in the initial generation of buds (F-I, white arrow), but not in the fully generated buds (J, black arrow in merge). RAK2 is expressed in tip (C-D, blue arrows),chloronema (A, D-E, red arrows) and caulonema cells (D-E, yellow arrows). Bar = 100µm.

We initially generated a KO line of the single domain, Nα-terminal acetyltransferase homolog (NATH), since RAK1 and RAK2 provided links between protein phosphorylation and acetylation. Remarkably, NATH-GFP was strongly expressed at thallus cell walls (Figure 17A-C, white arrows), and in chloronema their transverse cell walls (Figure 17D-E, red arrows), and in caulonema cells near their oblique cell walls (Figure 17A-E, yellow arrows). NATH was predominantly expressed in the tip cells of chloronema (Figure 17B-E, white arrows). As mentioned, NATH phenotypes, including reduced generation of gametophytes and morphological changes to protonemata growth, correlate well with these observations that NATH-GFP was expressed in initial steps of secondary growth and in the thallus.

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Since RAK1 and 2 and NATH are all expressed during the initial steps of secondary growth, their disruptions may be expected to result in the strong phenotypes seen in the rak1-rak2 and nath mutants (Figure 5).

Figure 17: Confocal images of 14 day-old NATH-GFP. GFP near cell membrane of thallus (A-C, white arrows). NATH is expressed in tip cells (B-E, white arrows) and in chloronema (B-E, red arrows) and caulonema cells (B-E, yellow arrows). Bar = 100µm.

MPK3-GFP was also expressed in the buds, the initial step of secondary growth (Supplemental Figure 17A, white arrow; C, blue arrow). Like NATH, MPK3 was detected near cell walls in thallus (Supplemental Figure 17C, white arrows) and in the tip cells of chloronema (Supplemental Figure 17E, white arrow). As mentioned, MPK3 appeared not to be phosphorylated in nath (Figure 6B), suggesting that NATH somehow affects the activation of MPK3. Furthermore, mRNA levels of NATH in mpk3 were also significantly higher than in WT (Supplemental Figure 10A), indicating

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MPK3 may affect the expression of NATH. This would correlate with the expression of both MPK3 and NATH in the same tissues. Figure 10A showed that mRNA levels of NATH in rak1 rak2 were significantly higher, perhaps suggesting that loss of the presumptive acetyltransferase activities of the RAKs could result to a higher expression of NATH. Furthermore, there is likely redundancy between RAK1 and RAK2 given their similarity and since the mRNA levels of RAK2 in rak1 were significantly higher than in WT. We also showed that RAK1 and RAK2 were not phosphorylated and activated in mpk3 (Figure 6A). Over all then, the phosphorylation and mRNA levels studies with the expression of the proteins, suggest interesting links between NATH, MPK3 and RAK1/RAK2.

Discussion This study aimed to characterize functions of RAK1 and RAK2, which combine a type of protein N- terminal acetyltransferase (NATD) and a MAP kinase (MPK). NATDs N-acetylate histones H2A and H4, while MPKs generally regulate adaptive and developmental responses by phosphorylation of substrate proteins (42-44). Although the interplay between protein acetylation and phosphorylation is well documented in the modification of histone tails (20), a major mechanism in chromatin remodeling, direct links between them are few, particularly for MPKs (21). The moss rosettas, RAK1 and RAK2 may therefore represent a new tool for understanding protein modifications affecting chromatin, gene expression and cellular signaling. N-terminal acetylation by N-acetyltransferases have been studied in mammals, yeast and Arabidopsis. Acetylation at the N-terminus in the proteins of mammals, yeast and Arabidopsis occurs frequently (70-90%, 50-70% and 70-75%, respectively) (42-44). NATs change the chemical properties of their substrate proteins by acetylating the N- terminal -amino group by transfer of an acetyl moiety (Ac) from Ac-CoA, resulting in the removal of a positive charge of the substrate protein (42-44). NATs are composed of enzyme complexes with substrate specificity. In mammals, 6 NATs have been identified (42), while 5 NATs have been identified in yeast (45). NATs are divided into groups, NATA-F due to their composition and substrate specificity. NATF is only present in higher eukaryotes (46). The RAK1 & 2 NAT domains are similar throughout their lengths to NATD. In yeast and mammals, NATD has a catalytic unit Naa40p (Nat4) that differs from the other NATs, since this NAT is only known to acetylate histones H2A and H4 (45). The other NATs (NATA, B, C, E) have 2 or 3 subunits and apparently acetylate many different substrates (amino acids) after methionine is removed by methionine aminopeptidases (MAPs). Substrate specificity of the catalytic units for NATA, B, C, E and F are within the first 2-5 residues, while for Naa40p (Nat4) the substrate specificity is within the first 30-50 residues of the

189 substrate sequence Ser-Gly-Arg-Gly (42-46). NATD has been associated with ribosomes and cytoplasm, indicating that NATD could be involved in post-translational protein acetylation (46). The RAK1/RAK2 MPK sequences lack the 4-5 introns found in the other 6 moss MPKs. The rosetta therefore probably arose by retroposition of an MPK C-terminally to a NATD paralog, and this RAK was later duplicated on chromosome 9 or 15. If this was part of a large-scale genome duplication that occurred 30-45MYA (9), then RAK1/RAK2 are at least that old. Such age bespeaks functional conservation. There are currently no publications on NATD activities in plants. Using gene targeting we generated single KOs of NATH, RAK1, and double KO of RAK1/RAK2. We also generated RAK1, RAK2 and NATH reporter lines tagged with GFP. We characterized several morphological changes in the mutants compared to WT upon treatments with exogenous hormones or blue light (BL). RAK1 and RAK2 GFP fusions were highly expressed in leafy gametophytes, protoplasts, protonemata, during secondary branching and especially during bud formation. Furthermore, double KO lines of RAK1 and RAK2 and KO lines of their single NATD domain homolog NATH had strong developmental phenotypes including fewer and shorter gametophytes and early induction of sporophytes. Remarkably, 90% of rak1-rak2 spores were able to germinate, while less than 10% of nath spores germinated. Interestingly, the expression of the Arabidopsis MADS-box floral repressor Flowering Locus C (FLC) is inhibited by asymmetric dimethylation of H4R3 (histone 4, arginine 3) by the methyltransferase PHRMT10 (47). Given the differences in moss sporophyte and Arabidopsis floral development, it is still intriguing to propose that the loss of N-acetylation of H4, and possibly of H2A, would inhibit the expression of developmental regulators and result in earlier sporophyte development in the moss. This indicates that NATH are important during sporophyte development and spore germination, suggesting that there is redundancy between RAK1/RAK2 and NATH. Our results provide some link between RAK1, RAK2, NATH and MPK3, since RAK1 and RAK2 were not phosphorylated in mpk3. Interestingly, MPK3 was not phosphorylated in nath, indicating that NATH somehow affects the activation of MPK3. Protonemata tissue of rak1-rak2 and nath had a ‘split identity’, since it was unclear whether the filaments were composed of chloronema or caulonema cells. The expression of the GFP fusions in both caulonema and chloronema cells indicates they may be important in secondary growth of protonemata. This correlates well with the strong phenotypes of the corresponding KO lines. For example, many buds were seen on rak1 plants,

190 resulting in a filamentous body with many leafy gametophytes, correlating with the higher amount of gametophytes compared to WT, rak1-rak2 and nath. We looked at the growth of the mutants grown under BL, since BL has been shown to induce branch formation and secondary growth (40). Interestingly, Imaizumi et al 2002, showed that the BL receptors, cryptochromes PpCRY1a and PpCRY1b, control the development of secondary growth by suppressing auxin sensitivity in P. patens. As nath was apparently in sensitive to exogenous IAA (Figure 6B), we compared our results to those of Imaizumi et al. 2002. nath plants were more filamentous than WT and the RAK mutants when grown under BL, correlating with the cry1a, cry1b and cry1a-cry1b mutants which had a higher ratio of caulonemal filaments under BL. This indicates that the inhibition of auxin responses, and thus the transition from chloronemal to caulonemal filaments, are specific to crytochrome signaling. Our study suggests that RAK2 and NATH may have a negative regulatory effect on the generation of secondary growth and early development of gametophytes within the first 3 weeks of growth under BL. If NATH has a negative regulatory effect on BL signaling via cryptochrome, it would perhaps result in the inhibition of auxin signaling, as seen in Figure 6B. Furthermore, BL induces via cryptochrome branch induction and branch formation (40), implying that if NATH has a negative regulatory effect on generation of branching, a NATH disruption would result in a branched filamentous plant as seen in Figure 4A-B. Moreover, the differentiation of buds and thus the development of gametophytes regulated by side branch initials are controlled by cryptochromes (37). This leads to another link to NATH, since nath develops significantly less gametophytes than WT. mRNA levels of NATH in rak1-rak2 were significantly higher compared to WT and rak1, indicative of possible redundancy between RAK1/RAK2 and NATH due to their shared, presumptive acetyltransferase activities. This redundancy is seen when these mutants are grown under WL and BL, since the amount of gametophytes are the same and the phenotypes in these mutants are almost identical (Figure 3, 4, 5 and 10). However, the difference between the mutants in auxin responses (Figure 7) could be due to MAP kinase activities of the RAKs and not to their acetyltransferase activities. Gametophytes of rak1-rak2 and nath were problematic to visualize when grown on plates supplemented with auxin. The growth of gametophytes for WT, rak1 and rak1-rak2 were accelerated on exogenous auxin, but not in nath. This indicates some involvement of NATH in auxin signaling as noted above. Moreover, RAK1 may affect some aspect of auxin response, since auxin significantly induced growth of rak1 colonies. We also exminedcytokinin responses in rak1, rak1- rak2 and nath, and showed that rak1-rak2 and nath did not respond to exogenous cytokinin as WT.

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More specifically, the colony areas of rak1-rak2 and nath on cytokinin were significantly larger than WT and rak1. We may therefore suggest that the acetyltransferase activities of RAK1/RAK2 and NATH are important in cytokinin responses. Moreover, the lack of detectable buds in rak1-rak2 and nath versus WT and rak1 correlates with findings that cytokinin induces and regulates bud formation. It could be interesting to quantify the intracellular cytokinin level in these mutants. Furthermore, it would be insightful to count the buds during treatment with cytokinin to see if cytokinin induces bud formation in the mutants. Since rak1-rak2 and nath had significantly fewer gametophytes than WT and rak1, we studied plant growth on plates with strigolactone as this hormone regulates growth of filament branching by inhibiting shoot branching. Strigolactone inhibited the growth of gametophytes in rak1, rak1-rak2 and nath, and the colony areas of rak1-rak2 and nath were significantly smaller than WT and rak1. We suggests that RAK2 and NATH influence strigolactone responses. mRNA levels of CHK1, SHI, CCD8 and ZEP1 upon treatment with cytokinin and auxin (Supplemental 13 & 14) showed that RAK1/RAK2 could affect the expression of the cytokinin receptor, and that RAK2 may influence the expression of auxin biosynthesis genes. These results correlate with the ‘split identity’ phenotype of rak1-rak2 filaments, since the transition from chloronema to caulonema is known to be regulated by auxin. The tendency of mRNA levels of ZEP1 in rak1 and rak1-rak2 are the same, indicating that both RAKs may affect hormonal signaling. Interestingly, mRNA levels of CCD8 in rak1-rak2 after auxin and cytokinin treatment were significantly heightened. rak1-rak2 plants were significantly bigger on cytokinin plates, which may correlate with findings that strigolactone production is used by P. patens to regulate colony extension and as a signaling molecule related to population density. Thus, the high mRNA levels of CCD8 could be a result of signaling in the colony extension.

Concluding remarks Further studies are needed to elucidate the activity of the acetyltransferase in the RAKs. It could be interesting to do complementation studies with human- and yeast with PpNATH and the NATs in the RAKs. Furthermore, to get a more complete functional picture of the proteins, it is necessary to induce sporophytes in the RAK1, RAK2 and NATH reporter lines to elucidate whether the RAKs and NATH are expressed in the organ bundle and sporophytes. Moreover, the link between RAK1, RAK2, NATH and MPK3 could be investigated by protein-protein interaction studies. During the phenotypic analysis we found that KOs of a triple MEKK (mekk1a) and of a double MKK (mkk1a) had early senescence similar to that of the rak1 KO (data not shown). The central gametophyte of these three

192 mutants underwent chlorosis approximately 6 weeks prior to propagation, while for the WT chlorosis was visible around 8-9 weeks (data not shown). These observations imply that a signaling cascade of the MPKs, MEKK1a-MKK1a and RAK1 could occur in the regulation of senescence in P. patens.

Material and Methods Plant growth conditions Physcomitrella patens (Gransden strain) was grown on full media BCDAT (250 mg/l MgSO4·7H2O, 250 mg/l KH2PO4, 1010 mg/l KNO3, 920 mg/l Ammonium tartrate, 12.5 mg/l FeSO4·7H2O, 147 mg/l CaCl2·2H2O, trace elements (614 μg/l H3BO3, 389 μg/l MnCl2·4H20, 110 μg/l AlK(S04)2·12H2O, 55 μg/l CoCl2·6H20, 55 μg/l CuSO4·5H20, 55 μg/l ZnS04·7H20, 28 μg/l KBr, 28 μg/l KI, 28 μg/l LiCl, 28 μg/l SnCl2·2H20, 25 μg/l Na2MoO4·2H2O, 59 μg/μl NiCl2·6H20), pH 6.5, 8 g/l agar). Minimal media (BCD) without Ammonium Tartrate. BCDAT plates were overlaid with cellophane discs (AA Packaging Ltd.) when needed. Colonies were grown at 21°C in 55 µE m- 2 s-1 in a 16h light/8h dark cycle. For routine protonema propagation, plants were grown on BCDAT; protonema tissue was blended with a homogenizer (PowerGen 500, Fisher Scientific). Protonema cultures were subcultured every 5-7 days.

Phenotypic analysis The genotypes were grown on BCD or BCDAT with addition of hormones during several weeks so growth patterns could be assessed. Unless otherwise stated, all treatments were applied by dipping with 1 ml of solution to colonies grown for 2 weeks. Hormone plates for phenotypic analysis were final concentrations of 0.5 μM auxin, indole-3-acetic acid (IAA) (Sigma Aldrich), 0.5 μM GR24 (Chiralix), or 0.5µM cytokinin (BPA). During blue light experiment, the genotypes were grown for 3- and 6 weeks in a box were only blue light (10 µE m-2 s-1) were present. Chlorophyll content were measured by extracting chlorophyll from central and peripheral gametophytes by homogenize the tissue in 80% acetone. The samples were led stand for 1hr at RT and centrifuged at 16400xg for 10 min. The absorbance in the supernatant were measured at values 663nm and 645 nm with spectrometer, and calculated as following: Chla = ((12,7*663nm)-(2,6*645nm))*(1/FW) & Chlb = ((22,9*645nm)-(4,68*663nm))*(1/FW). For sporophyte induction, plants were grown on BCDAT for 6 weeks under normal light and temperature conditions. Plants were moved to winter conditions (17ºC and 10-20 µE m-2 s-1) and irrigated twice after 4 and 5 weeks in consecutive weeks. Organ bundles were developed after the first irrigation and sporophyte capsules in WT developed after 2 weeks from the first irrigation.

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Sporophytes were harvested by separating then from gametophytic material. The spore capsules were released by pinching the base of the seta, which can be identified by a zone of pigmentation. One or more sporophytes were placed in a sterile 1.5mL microcentrfuge tube, sterilized by adding 1mL 70% ethanol, and incubated for 4 min at room temperature. The sporophytes were twice gently rinsed with

1mL of sterile H2O at room temperature. To increase spore germination, 1mL of H2O was added and the tube placed for 7 days in dark at 4°C. Sporophyte capsules were crushed with fine forceps and mixed to produce a spore suspension. Generation of Physcomitrella patens mutants All mutants were generated by cloning 900-1500bp of genomic flanking regions of the target gene into left side (left border, LB) and right side (right border, RB) (Supplemental Figure 5) of the selection marker in the previously modified pMBL6 vector with USER sites in both sides of the selection cassette (48). The left and right side of the selection gene NptII in the vector were modified. At the left side a USER cassette with a PacI restriction site and Nt.BbvCI nicking site was inserted in pMBL6 cut with SacI and KpnI. At the right side, a USER cassette containing AsiSI and Nt.BbvCI sites was inserted in pMBL6 cut with SalI and EcoRI. The modified vector was called pMBLU (17). The genomic flanking regions of PpRAK1, PpRAK2, PpNATH, PpMPK3 (unpublished), PpMPK5 (unpublished) and PpMPK4a/b (17), were cloned by PCR amplification with USER compatible primers. The PCR fragments were by four fragment USER reactions cloned in pMBLU, which was cut with PacI and AsiSI and nicked with Nt.BbvCI. GFP and a nos terminator were cloned in the pMBLU Kpn1 site to make the USER cloning vector pMBLU-GFP for tagging proteins with a C- terminal GFP. Double KO lines of rak1-rak2 were generated by modifying pMBLU to contain an HtpII selection cassette (hygromycin resistance) by cutting pMBLU with Sal1 and Kpn1 and inserting HtpII amplified from pUNI33 (17). PpRAK2 flanking regions was cloned into the modified vector pMBLU-hyg, and transformed into the single KO line rak1-1 to generate rak1-rak2. By PEG and heat shock method (49), 10-30µg of linearized vectors were transformed into protoplasts of P. patens by homologous recombination. Stable transformants were selected by transferring the protoplasts on cellophane to media containing 50 µg/ml G418 or 30 µg/ml hygromycin B for 2 weeks, followed by transfer to media without selection for 2 weeks, and finally by transfer onto BCDAT with selection for a further 2 weeks (49). Primers used to confirm gene deletions and for cloning are listed in Supplemental Table 3.

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Microscopy and statistical analysis Micrographs were taken with a Leica DFC310 FX camera mounted on Stereo Fluorescence Microscpoe Leica MZ FLII using the Leica Application Suite imaging software at the Center for Applied Bioimaging (CAB). Confocal images were acquired with a Zeiss LSM 700 and analyzed with ZEN imaging software (version 2011) with GFP settings (excitation 488 nm and detection 300 to 610 nm). The length and amount of gametophytes, sporophyte capsules, organ bundles and protonemata were measured, and the gametophyte data were analyzed using ImageJ. Microsoft Office was used to analyze statistical variance and T-test.

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Supplemental Figure 1: Identified MPKs in Physcomitrella patens

NAT Size (kDa) ID NATH 28,95 Pp3c17_14350V3.1 Figure 2: Multiple alignments of RAK1 & 2 with NATs and MPKs from diverse eukaryotes. AmNAT , Alligator mississippiensis (944429706): HsNAT, Human NATD (189571650); XlNat, Xenopus laevis (XX); DmNAT, Drosophila melanogaster (24651109); PiNAT, Phytophthora infestans (566019326); SmNAT, Selaginella moldorffi (302765549); AtNAT, Arabidopsis thaliana (42570083); DmROL, D. melanogaster Rol (62861950); PiMPK, P. infestans (675174255); AtMPK5, Arabidopsis (297809387); HsMPK1, human (20986531); AmMPK1, A. mississippiensis (564260955); XiMPK, X. laevis (148228736); SmMPK1, S. moldorffi (302754982). , residues implicated in NAT activity (Magin et al., 2015); TEY dual phosphorylation site in MPK activation loop.

  consensus NAT s a k krl r av v aA l dpl fp fkky rnglnl ie k s l trdwi L ktNm AmNAT 1 MSVRRKSSKAKEKKQKRLEERAAMDAVCAKVEAANKLGDPLEAFPVFKKYDRNGLNVSIECKKVSGLDQATIDWAFELTKTNMQ HsNATD 1 MGRKSSKAKEKKQKRLEERAAMDAVCAKVDAANRLGDPLEAFPVFKKYDRNGLNVSIECKRVSGLEPATVDWAFDLTKTNMQ XlNAT 1 MGRKSVRAKEKKQKRLEERAAMAAVCAKVQAANQLGDPLGAFPVFKKFDRNGLNLSIECCKVSDLDQKTIDWAFELTKTNMQ DmNAT 1 MRNQDDLSQGAKQRFVETAARAKNPLESLSYQSYKAPSGEEFKLICRAKSDADSKLLKWAFKLAETNVG PiNAT 1 MPKTSKAKKTKQSVATDAPTHPTLVAANAVSDVMVDFQAFSHYARNGANVTIRGAKSKDLSKSTRDHIVELFENNMK SmNAT 1 LDRKKAIDEVIRMAYAKEDHLSEFPAFLTYQRSGLNLIMQPQSGETLPAPLKRYIQALLKENME AtNAT 1 MDPSPTESLQTWRTNETEGRESSVWRAMDLKKRRKILEKKKTIHDLIKRASSIDDPLSPFDSFRRYRRNDLSLYLESGRGDRLSSSVKHHIQKLLKTNME PpRAK1 1 MDRHKHYDSGEGSTREKKLLGARDEFPEAEKAVRAAAAKPDPIAEFPSFLIYNRNGLKLNLEAGSGAALSATTKESMHKLLMMNME PpRAK2 1 MDRGKHYQYGEGSERQKRLLSRRHEFAEAENAVRAAAAKPNLIEEFPSFRTYNRNGLILMLEAGTGSAQSASTKERMHALLMMNMQ          consensus NAT ly sewgw dr Kr Em eAryliv e s lvafvhfrF ve g VLYlYEvQle vqrkGlGkFlmqllelmArk AmNAT 85 TLYEQSEWGWKDREKREELMDDRAWYLIAWEKS------SIPYAFSHFRFDVECGDEVLYCYEVQLESKVRRKGLGKFLIQILQLMANS HsNATD 83 TMYEQSEWGWKDREKREEMTDDRAWYLIAWENS------SVPVAFSHFRFDVECGDEVLYCYEVQLESKVRRKGLGKFLIQILQLMANS XlNAT 83 LLYEQSEWGWKEREKREELTDERAWYLIARDEL------AALVAFVHFRFDVECGDEVLYCYEVQLETRVRRKGVGKFLVQILQLMANS DmNAT 70 PYYKQLKMGWQPKIKAAELNKNWARYLVAQNEK------KENVAYAMFRFDMDHGDCVLYCYEMQVAAEYRRKGLGKFIMSTLEDCARL PiNAT 78 SLYQASEWGYDAAAKRTELFEAEARYLLVSDES------ETLVGFAHFRFVDDDGALVLYLYEVQLAAMAQRQGIGKFLMQLLLLVARK SmNAT 65 GPYG-SEWPAEEKVKKREMVASEARYIIVRQLVEDPGKHDGLWR---DGGDPVVSFVQFRFLIDEEIPVLYVYELQLEKCVQKKGLGKFLMQLLELVARK AtNAT 101 GFYG-SDWPIQAKVKRKEMSSADAHYIFVRELRFGKAYETSTQRTCMEGCNQIAGFVHYRFILEEEIPVLYVYEIQLESRVQGKGLGEFLMQLIELIASK PpRAK1 88 VLFGPHEWPAEENMKRWEMVSPEARFIFVRKSTPTIEAGS------SDEGHPMVGFVHFRFGLEHEVPVLYIYETQLEKTVQGKGLGKFLMQLLELVARK PpRAK2 88 VLFGPHEWPAEEKTKQEEMVSHEARFIFVEQNS-TSEASS------LDEGDSMVGFVHFKFGLEHDVPVLYVYEMQLKRTVQGVGLGKFLMQLLELVARK       consensus NAT qmk vmltV khN gai Fyre l y iddtSps d YeIlsks s c AmNAT 168 TQMKKVMLTVFKHNHGAYQFFREALQFDIDDTSPSMSGCCG--DDCSYEILSRRTKFGESQHPHAGGHCGGCCH HsNATD 166 TQMKKVMLTVFKHNHGAYQFFREALQFEIDDSSPSMSGCCG--EDCSYEILSRRTKFGDSHHSHAGGHCGGCCH XlNAT 166 TQMKKVVLTVFKHNHGAYQFFRDALQFEIDETSPSVSGCCS--DDCTYEILSKRTKFGDTPHSHTG-HCAGCCH DmNAT 153 WHLEKVMLTVLNNNEASISFFKQLG-YVKDEISPDVL------EQADYQIFSKSMLS PiNAT 161 HGMELMVLTVFKNNTGAMRFYRERLGFEIDETSPSACG-—D--NSQDYEILSKSVVQIK SmNAT 181 NNMKAVLLTVQKRNLAAMAFYS-KLKYVVSSISPS--RVDPLVVLKNYEILCKTFDPEAKAKLEVRKIKLDRSIESAAL AtNAT 181 NRMSAIVLTVLTSNALAMTFYMSKLGYRISSISPS--KANLPTLSVKYEILCKTFDSEAKSVLENDEEPTKD DmRol 1 MEE

PiMPK 1 MASYTPTGPASSPLKSVGSPLHSSSRNLSAKGLSGGDKLGGPSENPAPIGDAASS

PpRAK1 181 NNMKAVLLAVHKRNTRALTFYNERLGYKLAIRSASSQQSTQTVTEMKYEILCKTFDVEYTAVVEERQGDMDCESREESAGEASCQTVDAEDQVLDDSRPD PpRAK2 180 NNMKAILVAVHKRNSRALAFYNGSLGYKVATRSSSTQKNTQTSTEMNYEILCKTFDLEDTAVVE-RQGDQNCESREESGGEATCQPVDAEDQVLEDSCPD AtMPK5 1 MDKEIES

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HsMPK1 1 MAAAAAAGAGPEMVRGQVFDVGPRYTNLS-YIGEGAYGMVCSAYDNVNKVRVAIKKIS-PFEHQTYCQRTLREIKILLRFRHENIIGIND AmMPK1 1 MAAVSGAAAAGGAANAGGPEMVRGQAFDVGPRYTNLS-YIGEGAYGMVCSAYDNVNKVRVAIKKIS-PFEHQTYCQRTLREIKILLRFRHENIIGIND XlMPK1 1 MAAAGAASNPGGGPEMVRGQAFDVGPRYINLA-YIGEGAYGMVCSAHDNVNKVRVAIKKIS-PFEHQTYCQRTLREIKILLRFKHENIIGIND DmROL 4 FNSSGSVVNGTGSTEVPQSNAEVIRGQIFEVGPRYIKLA-YIGEGAYGMVVSADDTLTNQRVAIKKIS-PFEHQTYCQRTLREITILTRFKHENIIDIRD PiMPK 56 TRKASTTTAVAAPPARPGTYSFVVAGTNFQIDEKYKFVK-VIGRGAYGVVISADNIETNEKVAVKKISRAFEDLVDAKRILREIKLLQHFDHENVITIVD PpRAK1 281 TECESRIESVPNTLQGMKYTQYNVRGDKFEVYDKYVMIG-PIGHGAYGDVCAFTNRETGEKVAIKKIGNAFQNNTTARRTLREILLLRHTEHDNIIPIRD PpRAK2 280 MECESSIENVPNLLQGMRYTQYYVRDDKFEVYDKYVMIG-PIGHGAYGDVCAFTNKETGEKVAIKKIGNAFQNHTTARRTLREILLLRHTEHDNIIPIRD AtMPK5 8 AADPGDTNIKGVLVHGGRYFQYNVYGNLFEVSNKYVPPIRPIGRGAYGFVCAAVDSETHEEIAIKKIGKAFDNKVDAKRTLREIKLLRHLEHENVVVIKD SmMPK1 1 MAQKVDPPGGIAHPGKHYYMLWRSLFEIDQRYAPIK-PIGKGAYGVVCSATNSESGEKVAIKKITNAFENTTDARRTLREIRLLRHLYHENIIGIKD consensus MPK a a g fmvrgq Fev rYi l IGrGAYGmVcsa d et ervAiKKIs aFe qt akRtLREIklL hf HeNii IrD

HsMPK1 92 IIRAPTIEQMKDVYIVQDLMETDLYKLLKT-QHLSNDHICYFLYQILR-GLKYIHSANVLHRDLKPSNLLLNT-TCDLKICDFGLARVADPDHDHTGFLT AmMPK1 89 IIRAPTIEQMKDVYIVQDLMETDLYKLLKT-QHLSNDHICYFLYQILR-GLKYIHSANVLHRDLKPSNLLLNT-TCDLKICDFGLARVADPDHDHTGFLT XlMPK1 97 IIRAPTIEQMKDVYIVQDLMETDLYKLLKT-QHLSNDHICYFLYQILR-GLKYIHSANVLHRDLKPSNLLLNT-TCDLKICDFGLARVADPDHDHTGFLT DmROL 102 ILRVDSIDQMRDVYIVQCLMETDLYKLLKT-QRLSNDHICYFLYQILR-GLKYIHSANVLHRDLKPSNLLLNK-TCDLKICDFGLARIADPEHDHTGFLT PiMPK 155 LLPPPSLAQFEDVYIIADLMETDLHRIIYSRQPLTDDHVQYFLYQILR-ALKYIHSANVLHRDLKPSNLLLNS-NCDLKVCDFGLSRGVTPEEDN-MELT PpRAK1 379 IIVPANIEDFHDAYIANELMDTDLHQIVRS-TKLDEYHCQFLLYQLLR-GLKYIHSANILHRDLKPSNLLINCNDCLLKICDFGLARTS---AED-DFLT PpRAK2 378 IIVPANIEDFEDAYIANELMDTDLHQIVRS-TKLDEYHCQFLLYQLLR-GLKYIHSANILHRDLKPSNLLINCNDCLLKICDFGLARTS---AED-DFLT AtMPK5 112 IIRPPKKEDFVDVYIVFELMDTDLHQIIRSDQPLNDDHCQYFLYQILR-GLKYIHSANVLHRDLKPSNLLLNS-NCDLKITDFGLARTT---SET-EFMT SmMPK1 97 IMKPVGRSSFNDVYLVYELMDTDLHQIIRSSQALTDDHCQYFIYQQLLRGLKYVHSANVLHRDLKPSNLLLNA-SCDLKICDFGLARTG---SDKGQFMT consensus MPK iirpptieqfkDvYivqdLMeTDLhkilks qhLsddHiqyflYQiLr gLKYiHSANvLHRDLKPSNLLlNt tCdLKicDFGLaR a pd dh flT

HsMPK1 186 EYVATRWYRAPEIMLNSKGYTKSIDIWSVGCILAEMLSNRPIFPGKH----YLDQLNHILGILGSPSQEDLNCIINLKARNYLLSLPHKN-KVPWNRLFP AmMPK1 194 EYVATRWYRAPEIMLNSKGYTKSIDIWSVGCILAEMLSNRPIFPGKH----YLDQLNHILGILGSPSQEDLNCIINLKARNYLLSLPHKN-KVPWNRLFP XlMPK1 188 EYVATRWYRAPEIMLNSKGYTKSIDIWSVGCILAEMLSNRPIFPGKH----YLDQLNHILGILGSPSQEDLNCIINLKARNYLLSLPHKN-KVPWNRLFP DmROL 199 EYVATRWYRAPEIMLNSKGYTKSIDIWSVGCILAEMLSNRPIFPGKH----YLDQLNHILGVLGSPSRDDLECIINEKARNYLESLPFKP-NVPWAKLFP PiMPK 262 EYVVTRWYRAPEIMLSSREYTKAIDMWSTGCIFAELLGRTPLFPGDD----YIHQLQIICDKIGTPCEEDLHFVVSERAKRFMKNQPMRP-GVPFAKLFP PpRAK1 474 EYVVTRPYRAPELLLGSRMYTAAVDMWSVGCIFMEMLTGQPLFPIRSRQEHPVNHLKLITELLGTPDASDLSFLQNPDARQRIQMALLGQERKPLFSRFP PpRAK2 472 EYVVTRPYRAPELLLGSRMYTAAVDMWSVGCIFMEMLTGQPLFPIRSRQEHPVNHLKLITELLGTPDASDLSFLQNPDARQRIQMALIGQERKPLFSRFP AtMPK5 202 EYVVTRWYRAPELLLNSSEYTSAIDVWSVGCIFAEIMTREPLFPGKD----YVHQLKLITELIGSPDGASLEFLRSENARKYVKELPKFP-RQKFSSRFP SmMPK1 193 EYVVTRWYRAPELLLSCEEYTSAIDMWSVGCIFAELLGRKPIFPGKD----YIHQLKLIINTIGSPEEDDLQFILSNKARSYIRSLPFAP-KIPLERLYP consensus MPK EYVvTRwYRAPEimLnsk YTkaiDiWSvGCIfaEmls rPiFPgk yl qLniIl ilGsP edL fiin kArnyl slp kn kvpw rlfP

HsMPK1 280 NADSKALDLLDKMLTFNPHKRIEVEQALAHPYLEQYYDPSDEPIAEAPFKFDMELDDLPKEKLKELIFEETARFQPGYRS AmMPK1 289 NADPKALDLLDRMLTFNPHKRIEVEQALAHPYLEQYYDPSDEPVAEAPFKFDMELDDLPKEKLKELIFEETARFQPGYRS XlMPK1 284 NADPKALDLLDKMLTFNPHKRIEVEAALAHPYLEQYYDPSDEPVAEAPFKFEMELDDLPKETLKELIFEETARFQPGY DmROL 294 NADALALDLLGKMLTFNPHKRIPVEEALAHPYLEQYYDPGDEPVAEVPFRINMENDDISRDALKSLIFEETLKFKERQPDNAP PiMPK 357 KATPEAIDLLQRMLVFDPVKRVSVEEALEHPYLASLHNLEDEPVADSCFSFDFEKEDLTESRLKELIFEEILRIHPDAPRSPLKASHAGINSPPTEQVL… PpRAK1 574 QTSAIACDLAEKMLRFNPSNRITAEEALAHPYLAALHDLSDEPTCHLMFDFDAYLPSLTVEHVKTLIWREATLINVQ PpRAK2 572 QTSAAACDLAEKMLRFNPSNRITAEDALAHPYLSALHDVSDEPTCHLLFDFDAYLPNLSVDHVKTLIWREATLINAQ AtMPK5 297 SMNSTAIDLLEKMLVFDPAKRITVEEALCHPYLSALHDLNDEPVCSKHFSFDFEDPSSTEEEIKELVWLESVKFNPHPTI SmMPK1 288 RANPLALNLIDQMLVFDPKKRLTVTDALEHPYLSMLHDAALEPSASAAFEFDFEDEELREDALREKVWNEMCYYHPEATNEYS consensus MPK na AldLldkML FnPhkRisveeALaHPYL lhd sdEPvae F fd elddls eklkelifeEt rfqp

197

Figure 3: Multiple alignments of NATH with NATDs. PpNAT.H; Physcomitrella patens, ScNAT4; Saccharomyces cerevisiae, SfNAT; Sphagnum fallax (bogmoss), AtNAT; Arabidopsis thaliana and HsNAT; Homo sapiens.

>PpNAT.H Pp3c17_14350V3.1 MDRKKQSSKEKKLKRKEELAKKQSIDEFVRVANAKAAPIEEFPSFLKYERNGLNLIMEAGRGDSLSPPVKQYVQTLLKVNMEEPYGPEEWPAEEKNKRRE MVSPDARYIFVKQPCSNSTEILPTDRSNNLLWKGEGDPIVAFVHYRFVVEHEVPALYVYEIQVEQAVQGKGLGKFLMQFLELIARKNGMKAMLLTLQKRN VRALAFYTGKLRFKIAAISPSRWANTLIGAAKSYEILCKTFDPDAKSILEDGN*

>SfNAT Sphfalx0292s0009.1 MDRKKQSVSKDKKLKRKEELAKKKAIDETVRTAAAKAAPIEEFPSFLKYERNGLNLQLEAELGEKLSPPLKQYIQDLLKLNMEGPFGPDEWPAEEKKKRR DMVSPDARYIIVREKNLSQDSKFSSDSNLGRSDHHHCAGLWGANGGDPVVAFVHYRFVVEHDVPALYVFEIQLEQSIQGKGFGKFLMQFLELIARKNNMK AVLLTVQKRNTRAMAFYTQKLRFVVASISPSRWQDTLTGAEMTYEILCKTFDVEAKAILEEGTHLGAET*

>AtNAT AT1G18335.1 MDPSPTESLQTWRTNETEGRESSVWRAMDLKKRRKILEKKKTIHDLIKRASSIDDPLSPFDSFRRYRRNDLSLYLESGRGDRLSSSVKHHIQKLLKTNME GFYGSDWPIQAKVKRKEMSSADAHYIFVRELRFGKAYETSTQRTCMEGCNQIAGFVHYRFILEEEIPVLYVYEIQLESRVQGKGLGEFLMQLIELIASKN RMSAIVLTVLTSNALAMTFYMSKLGYRISSISPSKANLPTLSVKYEILCKTFDSEAKSVLENDEEPTKD*

>ScNAT4 NP_013785.1 MRSSVYSENTYNCIRTSKEHLTERRRVAMAPMFQHFLNLCVEKFPESIEHKDTDGNGNFTTAILEREIIYIPEDDTDSIDSVDSLKCINYKLHKSRGDQV LDACVQLIDKHLGAKYRRASRIMYGNRKPWKANKLAEMKSAGLVYVCYWDNGVLGAFTSFMLTEETGLVEGDALHEVSVPVIYLYEVHVASAHRGHGIGR RLLEHALCDGVARHTRRMCDNFFGVALTVFSDNTRARRLYEALGFYRAPGSPAPASPTIRHTRHGGGRVVVPCDPLYYVYCLHMP

>PpRAK1 MDRHKHYDSGEGSTREKKLLGARDEFPEAEKAVRAAAAKPDPIAEFPSFLIYNRNGLKLNLEAGSGAALSATTKESMHKLLMMNMEVLFGPHEWPAEENM KRWEMVSPEARFIFVRKSTPTIEAGSSDEGHPMVGFVHFRFGLEHEVPVLYIYETQLEKTVQGKGLGKFLMQLLELVARKNNMKAVLLAVHKRNTRALTF YNERLGYKLAIRSASSQQSTQTVTEMKYEILCKTFDVEYTAVVEERQGD

>PpRAK2 MDRGKHYQYGEGSERQKRLLSRRHEFAEAENAVRAAAAKPNLIEEFPSFRTYNRNGLILMLEAGTGSAQSASTKERMHALLMMNMQVLFGPHEWPAEEKT KQEEMVSHEARFIFVEQNSTSEASSLDEGDSMVGFVHFKFGLEHDVPVLYVYEMQLKRTVQGVGLGKFLMQLLELVARKNNMKAILVAVHKRNSRALAFY NGSLGYKVATRSSSTQKNTQTSTEMNYEILCKTFDLEDTAVVERQGD

>HsNATD MGRKSSKAKEKKQKRLEERAAMDAVCAKVDAANRLGDPLEAFPVFKKYDRNGLNVSIECKRVSGLEPATVDWAFDLTKTNMQTMYEQSEWGWKDREKR EEMTDDRAWYLIAWENSSVPVAFSHFRFDVECGDEVLYCYEVQLESKVRRKGLGKFLIQILQLMANSTQMKKVMLTVFKHNHGAYQFFREALQFEIDD SSPSMSGCCGEDCSYEILSRRTKFGDSHHSHAGGHCGGCCH

PpNAT.H 1 ------MDRKKQSS-KEKKLKRKEELAK------KQSIDEFVRVANAKAAPIEEFPSFLKYERNGLNLIMEAGRGDSLSPPVKQYVQTLLKVNME SfNAT 1 ------MDRKKQSVSKDKKLKRKEELAK------KKAIDETVRTAAAKAAPIEEFPSFLKYERNGLNLQLEAELGEKLSPPLKQYIQDLLKLNME PpRAK1 1 ------MDRHKHYDSGEGSTREKKLLGAR----DEFPEAEKAVRAAAAKPDPIAEFPSFLIYNRNGLKLNLEAGSGAALSATTKESMHKLLMMNME PpRAK2 1 ------MDRGKHYQYGEGSERQKRLLSRR----HEFAEAENAVRAAAAKPNLIEEFPSFRTYNRNGLILMLEAGTGSAQSASTKERMHALLMMNMQ AtNAT 1 MDPSPTESLQTWRTNETEGRESSVWRAMDLKKRRKILEKKKTIHDLIKRASSIDDPLSPFDSFRRYRRNDLSLYLESGRGDRLSSSVKHHIQKLLKTNME HsNATD 1 ------MGRKSSKAKEKKQKRLEERAAMD------AVCAKVDAANRLGDPLEAFPVFKKYDRNGLNVSIECKRVSGLEPATVDWAFDLTKTNMQ ScNAT4 1 --MRSSVYSENTYNCIRTSKEHLTERRRVAMAPMFQHFLNLCVEKFPESIEHKDTDGNGNFTTAILEREIIYIPEDDTDSIDSVDSLKCINYKLHKSRGD consensus 1 mdrkk s kek krk elak id vr a ak pieefpsf kyeRngl l leag g ls tvk v Llkvnme

PpNAT.H 83 EPYGPEEWPAEEKNKRREMVSPDARYIFVKQPCSNSTEILPTDRSNN------LLWKGEGDPIVAFVHYRFVVEHEVPALYVYEIQVEQAVQGKGL SfNAT 84 GPFGPDEWPAEEKKKRRDMVSPDARYIIVREKNLSQDSKFSSDSNLGRSDHHH-CAGLWGANGGDPVVAFVHYRFVVEHDVPALYVFEIQLEQSIQGKGF PpRAK1 87 VLFGPHEWPAEENMKRWEMVSPEARFIFVRKSTPTIEAGSS------DEGHPMVGFVHFRFGLEHEVPVLYIYETQLEKTVQGKGL PpRAK2 87 VLFGPHEWPAEEKTKQEEMVSHEARFIFVEQNS-TSEASSL------DEGDSMVGFVHFKFGLEHDVPVLYVYEMQLKRTVQGVGL AtNAT 101 GFYG-SDWPIQAKVKRKEMSSADAHYIFVRELRFGKAYETSTQR------TCMEGCNQIAGFVHYRFILEEEIPVLYVYEIQLESRVQGKGL HsNATD 83 TMYEQSEWGWKDREKREEMTDDRAWYLIAWENS------SVPVAFSHFRFDVECGDEVLYCYEVQLESKVRRKGL ScNAT4 99 QVLDACVQLIDKHLGAKYRRASRIMYGNRKPWKANKLAEMKSAGLVYVCYWDNGVLGAFTSFMLTEETGLVEGDALHEVSVPVIYLYEVHVASAHRGHGI consensus 101 lygp ewpaeek krremvs daryifvk t g ivgfvhyrfvvEhevpvlYvyEiqle vqgkGl

PpNAT.H 173 GKFLMQFLELIARK------NGMKAMLLTLQKRNVRALAFYTGKLRFKIAAISPSRWANTLIGAAKSYEILCKTFDPDAKSILEDGN------SfNAT 183 GKFLMQFLELIARK------NNMKAVLLTVQKRNTRAMAFYTQKLRFVVASISPSRWQDTLTGAEMTYEILCKTFDVEAKAILEEGTHLGAET- PpRAK1 167 GKFLMQLLELVARK------NNMKAVLLAVHKRNTRALTFYNERLGYKLAIRSASSQQSTQTVTEMKYEILCKTFDVEYTAVVEERQGD----- PpRAK2 166 GKFLMQLLELVARK------NNMKAILVAVHKRNSRALAFYNGSLGYKVATRSSSTQKNTQTSTEMNYEILCKTFDLEDTAVVERQGD------AtNAT 186 GEFLMQLIELIASK------NRMSAIVLTVLTSNALAMTFYMSKLGYRISSISPS--KANLPTLSVKYEILCKTFDSEAKSVLENDEEPTKD-- HsNATD 152 GKFLIQILQLMANS------TQMKKVMLTVFKHNHGAYQFFREALQFEIDDSSPS--MSGCCGEDCSYEILSRRTKFGDSHHSHAGGHCGGCCH ScNAT4 199 GRRLLEHALCDGVARHTRRMCDNFFGVALTVFSDNTRARRLY-EALGFYRAPGSPAPASPTIRHTRHGGGRVVVPCDPLYYVYCLHMP------consensus 201 GkfLmqlleliark nnmkavlltv krNtrAl fy kLgfkia Sps tl em yeilcktfd e ile

198

Figure 4: Underlined nucleotides indicate intron positions, brackets and yellow highlight indicate short repeated regions between N-terminal NAT and C-terminal MPK.

PpRAK1 cDNA ATGGATAGGCACAAGCATTATGACTCTGGCGAGGGGAGTACCAGGGAGAAGAAGCTCCTGGGTGCGCGAGATGAGTTTCCAGAGGCTGAAAAGGCTGTTA M D R H K H Y D S G E G S T R E K K L L G A R D E F P E A E K A V R GAGCTGCCGCTGCCAAGCCAGACCCTATAGCGGAGTTCCCTTCGTTTCTCATTTACAATCGAAATGGTTTAAAACTGAACTTGGAAGCTGGGTCTGGCGC A A A A K P D P I A E F P S F L I Y N R N G L K L N L E A G S G A AGCTCTGTCTGCTACTACGAAAGAAAGTATGCATAAACTTTTAATGATGAATATGGAAGTGTTATTTGGTCCCCATGAATGGCCTGCTGAAGAGAACATG A L S A T T K E S M H K L L M M N M E V L F G P H E W P A E E N M AAGCGGTGGGAAATGGTATCCCCTGAAGCGCGCTTCATTTTTGTGAGGAAAAGCACTCCTACCATTGAAGCGGGCAGTTCTGATGAAGGTCACCCAATGG K R W E M V S P E A R F I F V R K S T P T I E A G S S D E G H P M V TGGGCTTCGTCCATTTTAGATTCGGTTTGGAACACGAGGTTCCAGTACTTTATATATATGAGACACAGCTGGAGAAAACAGTGCAAGGGAAAGGTCTTGG G F V H F R F G L E H E V P V L Y I Y E T Q L E K T V Q G K G L G AAAGTTTTTAATGCAGTTACTTGAGTTGGTTGCACGAAAGAACAACATGAAAGCAGTACTTTTAGCTGTGCATAAAAGAAACACAAGGGCGCTAACCTTT K F L M Q L L E L V A R K N N M K A V L L A V H K R N T R A L T F TACAATGAACGTTTAGGGTATAAGTTGGCAATTAGATCAGCATCAAGTCAACAAAGCACACAAACTGTCACAGAGATGAAATACGAGATTCTTTGTAAAA Y N E R L G Y K L A I R S A S S Q Q S T Q T V T E M K Y E I L [C K T CTTTCGATGTGGAGTACACAGCCGTTGTAGAGGAACGGCAAGGGGACATGGATTGTGAATCACGTGAAGAGAGCGCTGGAGAAGCAAGCTGCCAGACAGT F D V E Y T A V V E E R Q G D M D C E S R E E S A G E A S][C Q T V TGACGCAGAGGATCAGGTTTTGGATGACTCACGTCCTGATACAGAATGTGAGTCACGGATCGAGAGCGTGCCAAACACCCTACAAGGAATGAAGTACACA D A E D Q V L D D S R P D T E C E S R I E S V P N T L] Q G M K Y T CAGTACAATGTGAGGGGCGACAAGTTTGAAGTCTACGACAAGTATGTAATGATTGGTCCCATTGGTCATGGAGCTTATGGCGATGTGTGTGCTTTCACGA Q Y N V R G D K F E V Y D K Y V M I G P I G H G A Y G D V C A F T N ACAGGGAGACAGGGGAGAAAGTGGCCATAAAGAAGATTGGAAACGCATTTCAGAACAATACTACAGCGAGGCGCACACTTAGAGAGATTTTGTTGCTCCG R E T G E K V A I K K I G N A F Q N N T T A R R T L R E I L L L R CCATACTGAACACGACAACATCATTCCCATCAGAGATATCATTGTGCCTGCTAACATTGAGGACTTTCATGATGCCTATATCGCAAATGAGCTCATGGAT H T E H D N I I P I R D I I V P A N I E D F H D A Y I A N E L M D ACAGACCTTCACCAGATAGTGAGGTCAACAAAACTTGACGAATACCATTGCCAGTTCCTGCTTTACCAGCTGTTGAGGGGTCTCAAATACATCCACTCTG T D L H Q I V R S T K L D E Y H C Q F L L Y Q L L R G L K Y I H S A CCAATATATTGCACCGTGACCTGAAGCCCAGTAATCTCCTCATCAATTGCAACGACTGTCTACTCAAGATTTGTGATTTTGGCTTGGCTCGAACATCTGC N I L H R D L K P S N L L I N C N D C L L K I C D F G L A R T S A AGAGGATGACTTCCTTACGGAGTATGTTGTTACTCGACCATATCGAGCTCCAGAGCTCTTGCTTGGGAGCCGAATGTACACAGCGGCTGTTGATATGTGG E D D F L T E Y V V T R P Y R A P E L L L G S R M Y T A A V D M W TCAGTGGGCTGCATCTTCATGGAGATGCTTACAGGACAACCTTTGTTTCCAATCCGGTCAAGGCAAGAGCATCCCGTGAATCATTTGAAACTCATCACGG S V G C I F M E M L T G Q P L F P I R S R Q E H P V N H L K L I T E AGCTTCTAGGAACACCCGATGCTTCGGACCTGTCGTTTCTGCAGAATCCAGATGCTCGGCAAAGAATCCAAATGGCTTTGTTAGGTCAGGAAAGGAAGCC L L G T P D A S D L S F L Q N P D A R Q R I Q M A L L G Q E R K P TTTGTTTTCGAGGTTTCCTCAAACGTCTGCAATAGCTTGTGACTTAGCGGAGAAGATGCTGAGGTTTAACCCATCCAACAGAATAACTGCGGAAGAGGCC L F S R F P Q T S A I A C D L A E K M L R F N P S N R I T A E E A TTGGCCCATCCTTACTTGGCAGCGCTTCACGACCTAAGTGATGAGCCAACGTGTCATCTTATGTTCGACTTCGATGCTTACCTTCCCAGCCTAACAGTTG L A H P Y L A A L H D L S D E P T C H L M F D F D A Y L P S L T V E AGCATGTGAAAACTCTTATCTGGAGGGAAGCTACACTTATCAACGTCCAGTAA H V K T L I W R E A T L I N V Q

PpRAK2 cDNA ATGGATCGCGGCAAGCATTATCAGTATGGCGAGGGGAGTGAGAGGCAGAAGAGGCTCCTGAGCAGGCGACATGAGTTTGCAGAGGCTGAAAATGCAGTTA M D R G K H Y Q Y G E G S E R Q K R L L S R R H E F A E A E N A V R GAGCTGCGGCTGCCAAGCCCAACCTGATAGAAGAGTTCCCTTCATTTCGTACCTACAATAGAAATGGTTTAATCTTAATGTTGGAAGCTGGGACCGGCTC A A A A K P N L I E E F P S F R T Y N R N G L I L M L E A G T G S AGCTCAGTCTGCTTCTACGAAAGAACGTATGCACGCGCTTTTAATGATGAACATGCAAGTGTTATTTGGTCCCCATGAATGGCCTGCTGAAGAGAAAACG A Q S A S T K E R M H A L L M M N M Q V L F G P H E W P A E E K T AAGCAGGAGGAAATGGTATCCCATGAGGCACGCTTCATTTTCGTTGAGCAAAACTCTACCTCTGAAGCGAGCAGTTTGGATGAAGGTGACTCAATGGTGG K Q E E M V S H E A R F I F V E Q N S T S E A S S L D E G D S M V G GCTTTGTTCATTTCAAATTCGGTTTGGAACACGATGTTCCAGTCCTATATGTATATGAAATGCAGCTGAAGAGGACAGTGCAAGGGGTAGGACTCGGCAA F V H F K F G L E H D V P V L Y V Y E M Q L K R T V Q G V G L G K GTTTCTGATGCAGTTGCTTGAGTTGGTTGCTCGAAAGAATAACATGAAAGCAATACTTGTAGCTGTGCACAAAAGAAACAGCAGGGCGTTGGCGTTTTAC F L M Q L L E L V A R K N N M K A I L V A V H K R N S R A L A F Y AATGGAAGTTTAGGGTATAAAGTTGCAACTAGATCATCATCAACGCAGAAAAACACACAAACTTCTACAGAGATGAATTACGAGATTCTTTGCAAAACCT N G S L G Y K V A T R S S S T Q K N T Q T S T E M N Y E I L [C K T F TTGATTTGGAGGATACCGCCGTTGTAGAGCGTCAAGGAGACCAGAATTGCGAGTCACGCGAGGAGAGCGGTGGAGAAGCAACCTGCCAGCCAGTGGATGC D L E D T A V V E R Q G D Q N C E S R E E S G G E A T][C Q P V D A AGAGGATCAAGTTTTGGAGGACTCATGTCCGGATATGGAATGTGAGTCATCTATTGAAAATGTGCCAAACCTTTTACAAGGAATGAGGTACACACAGTAC E D Q V L E D S C P D M E C E S S I E N V P N L L] Q G M R Y T Q Y TATGTGAGGGACGATAAGTTTGAAGTATATGATAAGTACGTCATGATTGGTCCTATTGGCCATGGAGCATATGGCGATGTGTGTGCTTTCACAAACAAGG Y V R D D K F E V Y D K Y V M I G P I G H G A Y G D V C A F T N K E AGACTGGGGAGAAGGTAGCCATAAAGAAGATTGGTAATGCCTTTCAGAACCACACAACAGCGAGGCGCACTCTTAGAGAGATTTTGTTGCTCCGGCACAC T G E K V A I K K I G N A F Q N H T T A R R T L R E I L L L R H T TGAGCATGACAACATCATTCCTATCAGAGATATTATTGTGCCTGCAAACATTGAAGATTTTGAGGATGCTTATATTGCAAATGAGCTCATGGATACAGAC E H D N I I P I R D I I V P A N I E D F E D A Y I A N E L M D T D CTTCATCAGATAGTGAGATCAACTAAGCTCGACGAATACCACTGCCAGTTCCTGCTTTATCAGCTTCTGAGGGGCCTCAAGTACATTCATTCAGCTAATA L H Q I V R S T K L D E Y H C Q F L L Y Q L L R G L K Y I H S A N I TATTACACCGCGATCTGAAGCCTAGTAATCTTCTCATCAACTGCAACGATTGTCTACTCAAGATCTGTGATTTTGGCTTGGCTCGAACCTCTGCAGAGGA L H R D L K P S N L L I N C N D C L L K I C D F G L A R T S A E D TGATTTTCTTACGGAGTACGTTGTTACTCGACCTTATCGAGCTCCGGAGCTTTTGCTTGGGAGCCGAATGTACACAGCTGCTGTTGATATGTGGTCAGTG D F L T E Y V V T R P Y R A P E L L L G S R M Y T A A V D M W S V GGCTGTATCTTTATGGAGATGCTCACAGGTCAACCTTTGTTCCCAATCCGGTCAAGACAAGAGCATCCCGTGAATCACTTGAAACTTATCACCGAGCTAC G C I F M E M L T G Q P L F P I R S R Q E H P V N H L K L I T E L L TAGGGACTCCTGATGCTTCGGACTTGTCGTTCCTGCAGAATCCGGATGCTCGGCAAAGGATACAAATGGCCTTGATAGGTCAGGAAAGAAAGCCCTTGTT

199

G T P D A S D L S F L Q N P D A R Q R I Q M A L I G Q E R K P L F TTCGAGGTTCCCACAAACTTCTGCTGCAGCTTGCGACTTAGCCGAGAAGATGCTGAGATTCAACCCCTCCAACAGGATAACTGCGGAAGATGCTTTGGCT S R F P Q T S A A A C D L A E K M L R F N P S N R I T A E D A L A CATCCTTATCTGTCAGCACTTCACGACGTCAGTGATGAGCCAACCTGTCATCTTCTGTTCGACTTCGACGCCTACCTGCCCAATCTCTCTGTCGATCATG H P Y L S A L H D V S D E P T C H L L F D F D A Y L P N L S V D H V TTAAAACTCTCATTTGGCGAGAGGCTACTCTTATAAACGCTCAGTAA K T L I W R E A T L I N A Q

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Figure 5: Genotyping rak1, nath and rak1-rak2 mutants

Knockout MPKs ID Plasmid name Selection RAK1 Pp1s29_285V6.1 pMBLU-RAK1 G418 RAK2 Pp1s99_26V6.1 pMBLU-RAK2 Hygromycin/G418 NAT ID NATH Pp3c17_14350V3.1 pMBLU-NATH Hygromycin

201

Figure 6: Physcomitrella patens life cycle

P. patens life cycle. (A) Haploid spore germinates into protonema filaments consisting of (B) chloronemal and (C) caulonemal cells. (D) Gametophores emerge from protonema filaments and are anchored by rhizoids that expand by tip growth from the gametophore. (E) At the apex of the gametophore organ bundles are produced with both female, archegonia (arrows), and male, antheridia (arrowheads) reproductive organs. After irrigation the egg is fertilized by motile sperm and the (F) sporophyte develops at the apex of the gametophore. (1)

202

Figure 7: Sporophyte induction

Sporophyte induction of 6 week-old WT (A, E, I) and rak1 (B, F, J) plants. After 6 weeks, prior to the first irrigation, the canal of the archegonia was dark brown, indicating that spermatozoids have fertilized the eggs (I & J, black arrows). Antheridia appeared as round bundles with shinny surfaces localized around the archegonia (I & J, white arrows). WT and rak1 plants generated sporophyte capsules in different maturation steps; precapsule, early green, late green/yellow, yellow/orange, red (matured) and brown stages (B). rak1 sporophyte capsule at the yellow/orange stage showed abnormal red coloration in the capsule, indicating the spores are matured (red arrows). Bar = 2 mm.

203

Figure 8: Colony area of WT, rak1, nath and rak1-rak2 after 3 and 6 weeks growth on minimal media (BCD).

Colony area of 3 and 6 week-old WT, rak1, rak1-rak2 and nath. Plants were grown on minimal media (BCD) for 3 and 6 weeks (Bar = 2 mm). The areas of colonies were measured by ImageJ, with the function Gaussium blur. Analysis of variance by T-test determined statistical differences indicated by A-D (BCD 3 weeks) & a-d (BCD 6 weeks) (p <0.05), n = 3. Standard deviation as error bars.

204

Figure 9. Phenotypic comparison of the amount and length of gametophytes for WT, rak1, rak1-rak2 and nath.

(A) Length of gametophytes (mm) from the base of the gametophore to the tip of the apex. Central and peripheral gametophytes after 3 weeks of growth on minimal media (BCD). Analysis of variance by T-test determined statistical differences indicated by A-E (BCD 3 weeks) & a-e (BCD 6 weeks) (P <0.05), n = 16. Standard deviation as error bars. (B) Length of gametophytes (mm) from the base of the gametophore to the tip of the apex. Central and peripheral gametophytes after 6 weeks of growth on minimal media (BCD). Analysis of variance by T-test determined statistical differences indicated by A-E (BCD 3 weeks) & a-e (BCD 6 weeks) (P <0.05), n = 16. Standard deviation as error bars. (C) Amount of gametophytes after 3 and 6 weeks on BCD. Analysis of variance by T-test determined statistical differences indicated by A-E (BCD 3 weeks) & a-e (BCD 6 weeks) (P <0.05), n = 3 colonies. Standard deviation as error bars. (D) Difference between the amount of gametophytes after 3 and 6 weeks of growth in minimal media (BCD). The length of the gametophytes were measured by ImageJ and the analysis of variance by T-test determined statistical differences indicated by asterisks (* P = 0.05-0.01, ** P = 0.01-0.001, *** P < 0.001).

205

Figure 10: mRNA levels of NATH and RAK2 in WT, rak1, rak1-rak2, mpk3 and nath.

(A-B) Quantitative RT-PCR of NATH (A) and RAK2 (B) transcript levels in WT, rak1, rak1- rak2, mpk3 and nath fold change relative to WT. Data points represent an average of 3 quantifications of the mRNA from the same sample (technical replicates) with standard deviation as error bars. The experiment was repeated 3 times with similar results. Analysis of variance by T-test determined statistical differences indicated by asterisks (*P = 0.05-0.01, **P = 0.01-0.001, *** P < 0.001). Standard deviation as error bars.

206

Figure 11: Phenotypic analysis of WT, rak1, mpk3, mpk5 and rak1-rak2 on BCDAT supplemented with cytokinin (BPA).

Colonies of 3 week-old WT, rak1, mpk3, mpk5 and rak1-rak2 on full media (BCDAT) supplemented with 500nM or 2,5µM cytokinin (BPA). Bar = 1mm.

207

Figure 12: Colony area of WT, rak1, nath and rak1-rak2 after 6 weeks of growth on full media (BCDAT) supplemented with BPA, IAA or GR24.

Colony area of 6 week-old WT, rak1, rak1-rak2 and nath on BCDAT plates supplemented with cytokinin (BPA), auxin (IAA) or strigolactone (GR24). (A) The areas of colonies measured by ImageJ, with the function Gaussium blur. Bar = 1mm. (B) Analysis of variance by T-test determined statistical differences indicated by A-D (BCDAT), a-d (BCDAT+500nM BPA), a-d (BCDAT+500nM IAA) & a-d (BCDAT+500nM GR24) (p <0.05), n = 3. Standard deviation as error bars.

208

Figure 13: Immunoblots of the activation and phosphorylation of RAK1 and RAK2 upon hormonal treatment.

Phosphorylation and presumptive activation of the downstream kinases measured by immunoblotting with anti-phospho-p44/42 MPK antibody (α-pTEpY). (A) Phosphorylation and activation of kinases in WT, rak1 and rak1-rak2 upon treatment with 0.5 µM BPA for 15 and 30 min. and no treatment (NT). Phosphorylation of RAK1 and RAK2 (asterisk). (B) Phosphorylation and activation of kinases in WT, rak1 and rak1-rak2, treated with 0.5µM IAA for 15 and 30 min. and not treated (NT). (C) Phosphorylation of kinases in WT, rak1, rak1-rak2 and the strigolactone deficient mutant ccd8, treated with 0.5µM GR24 versus untreated (NT). Phosphorylation of RAK1 and RAK2 (asterisk).

209

Figure 14: mRNA levels of CHK1 and SHI in WT, rak1 and rak1-rak2.

(A-B) Quantitative RT-PCR of CHK1 (A) and SHI (B) transcript levels in WT, rak1 and rak1- rak2 fold change relative to WT when treated with 0.5µM cytokinin (BPA) or auxin (IAA) for 30 min. and 1 hr. Data points represent an average of 3 quantifications of the mRNA from the same sample (technical replicates) with standard deviation as error bars. The experiment was repeated 3 times with similar results. Analysis of variance by T-test determined statistical differences indicated by asterisks (*P = 0.05-0.01, **P = 0.01-0.001, *** P < 0.001). Standard deviation as error bars.

210

Figure 15: mRNA levels of ZEP1 and CCD8 in WT, rak1 and rak1-rak2.

(A-B) Quantitative RT-PCR of ZEP1 (A-B) and CCD8 (C-D) transcript levels in WT, rak1 and rak1-rak2 fold change relative to WT when treated with cytokinin (BPA) or auxin (IAA) for 30 min. and 1 hr. Data points represent an average of 3 quantifications of the mRNA from the same sample (technical replicates) with standard deviation as error bars. The experiment was repeated 3 times with similar results. Standard deviation as error bars.

211

Figure 16: Expression of RAK1, RAK2 and NATH-GFP reporter lines in protoplasts

Confocal images of protoplasts from 14 day-old WT and reporter lines RAK1-, RAK2- and NATH-GFP. RAK1, RAK2 and NATH GFP fusions are expressed in protoplasts. Bar = 20µM.

212

Figure 17: Expression of MPK3-GFP reporter line

Confocal images of 14 day-old MPK3-GFP. GFP signal near cell membrane in thallus (C, white arrows). Signal in the buds (A, white arrow; C, blue arrow). MPK3-GFP in tip cells (E, white arrows) and in chloronema (B, D-E, red arrows) and caulonema cells (B, D-E, yellow arrows). Bar = 100µm.

213

Table 1. Summary of the phenotypic analysis of sporophyte induction experiments.

214

Table 2. Summary of the phenotypic analysis of protonemata and gametophytes after 3 and 6 weeks on BCD and BCDAT.

215

Table 3: Primers Primers targeted to the selectable marker cassettes:

216

Primers for qPCR:

217

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Chapter 15

Chitin and Stress Induced Protein Kinase Activation

Chandra Kenchappa*, Raquel Azevedo da Silva*, Simon Bressendorff, Sabrina Stanimirovic, Jakob Olsen, Morten Petersen, and John Mundy

Abstract

The assays described here are pertinent to protein kinase studies in any plant. They include an immunoblot phosphorylation/activation assay and an in-gel activity assay for MAP kinases (MPKs) using the general protein kinase substrate myelin basic protein. They also include a novel in-gel peptide substrate assay for Snf1-related kinase family 2 members (SnRK2s). This kinase family-specific assay overcomes some limitations of in-gel assays and permits the identification of different types of kinase activities in total protein extracts.

Key words Acryloylated peptide substrates, In-gel assays, Myelin basic protein

1 Introduction

MAP kinases (MPKs) regulate responses to developmental and stress signals including pathogen infection [1–3]. They are activated by phosphorylation on conserved Thr and Tyr residues in a regulatory site (TEY) by MAP kinases kinases which are activated by MAP kinase kinase kinases. MPK phosphorylation is assayed by immunoblot with α-pTEpY antibodies. MPK cascades amplify stimuli to responses by phosphorylating their substrates, including transcription factors, on Ser/Thr followed by Pro (S/TP sites). MPK activities in total or purified protein extracts are routinely assayed in gels following separation by SDS-PAGE using Myelin Basic Protein (MBP) as substrate [4]. MBP contains four S/TP sites phosphorylated in vivo [5]. SnRK2s kinases are required for responses to abiotic stress and the osmoregulatory hormone abscisic acid (ABA) [6, 7]. In the ABA pathway, hormone binding to PYL receptors represses the activity of protein phosphatases 2C which otherwise dephosphorylate and

*These authors contributed equally to this work.

Libo Shan and Ping He (eds.), Plant Pattern Recognition Receptors: Methods and Protocols, Methods in Molecular Biology, vol. 1578, DOI 10.1007/978-1-4939-6859-6_15, © Springer Science+Business Media LLC 2017 185 186 Chandra Kenchappa et al.

inactivate SnRK2s. SnRK2 autophosphorylation on Ser residues [8, 9] then permits their phosphorylation of substrates including ABA-responsive transcription factors. Like other SnF1 related enzymes, SnRK2s preferentially phosphorylate substrates on Ser in the consensus ΦXRXXSXXXΦ (Φ = hydrophobic) [10, 11]. Peptides containing such sequences have been used to assay purified SnF1-related and SnRK2 activities in vitro [12, 13]. MBP is phosphorylated by SnRK2s [9] and may contain three SnRK2 phosphorylation sites. Although in-gel kinase assays are useful, they have limitations. First, during gel casting substrates are copolymerized via their Lys residues to acryl gel groups to prevent substrates from being eluted during electrophoresis and subsequent processing steps. Second, proximal Lys-acryl linkages may mask substrate phosphorylation sites. Thus, protein substrates like MBP, with multiple phosphorylation sites and/or relatively distal Lys residues, are commonly used. Third, MBP is phosphorylated by different kinases which may confound their identification. For example, immunoblotting with α-pTEpY antibodies identifies at least two MPKs of 40–42 kD that are phosphorylated in the moss Physcomitrella patens in response to treatment with the fungal elicitor chitin (Fig. 1a, b, lane 3). This immunoblot assay also shows that moss MPKs are not phosphorylated in response to osmotic stress (NaCl, lanes 4–8). Similarly, an in-gel kinase activity assay with MBP as substrate shows that one or more kinases are activated in response to chitin (Fig. 1c, lane 3). However, this activity assay also detects one or more kinases of ~39 kD that are activated in response to osmotic stress (500 mM NaCl, lanes 4–8). While these two assays indicate that the 40–42 kD bands are MPKs, the nature of the ~39 kD activities are unclear. These assays illustrate the limitations of in-gel activity assays using MBP as substrate. These limitations have also precluded the use of shorter peptide substrates lacking Lys residues for gel linking, or peptides necessarily containing Lys proximal to phosphorylation sites. We have recently shown that this limitation may be circumvented by synthesizing substrate peptides lacking Lys residues but containing an acryloylated N-terminus for acryl gel linking (Fig. 2) [14, 15]. This is shown in Fig. 1d with an N-acryloylated peptide with the consensus SnRK2 phosphorylation site ALARAASAAALARRR [13]. It identifies SnRK2s activated in response to osmotic stress (NaCl or mannitol, lanes 3–8) which are not responsive to chitin (lane 3). This indicates that peptides containing phosphorylation sites for specific kinase types can be used to study their activation and to identify them. For example, multiple substrate proteins are phosphorylated on lysine-less peptides by different kinase types in response to various cues in Arabidopsis [16, 17]. The specificity of in-gel peptide substrate assays could confirm and extend their Protein Kinase Activation 187

Fig. 1 Protein kinase assays. (a) Immunoblot with α-pTEpY antibodies; (b) Ponceau- stained gel as loading control; (c) In-gel kinase activity assay with MBP as substrate. a–c are same samples of wild type moss treated as follows in lanes: 1, 5 min H2O control; 2, 10 min H2O control; 3, 5 min 100 μg/mL chitin; lanes 4–8, 2, 5, 10, 20, 60 min 500 mM NaCl. (d) In-gel kinase activity assay with acryloylated peptide ALARAASAAALARRR containing the SnRK2 consensus phosphorylation site ΦXRXXSXXXΦ. Lanes are wild type moss treated with: 1, 5 min H2O; 2, 5 min 100μg/mL chitin; 3–5, 5, 10, and 20 min 500 mM NaCl; 6–8, 5, 10, and 20 min 800 mM mannitol. Protein molecular weight markers in kDa at left

Fig. 2 Peptide synthesized on standard resin with N-terminal 6-aminohexanoic acid (Ahx) is acryloylated with acryloyl chloride. Acryloylated peptide released from resin by HCl hydrolysis can be copolymerized as usual by ammonium persulfate (ApS) and tetramethylethylenediamine (TEMED) with acrylamide (and/or bisacrylamide, not shown) 188 Chandra Kenchappa et al.

analysis in such post-genomic models and would aid kinase studies in other models as well [18]. In addition, the cost of such synthetic, kinase-specific peptides (30 mer = $31 for 0.75 mm thick or $39 for 1 mm thick gels; Schaefer N, Copenhagen) is less than that of commercial MBP ($39 for 0.75 mm thick or $49 for 1 mm thick gels; Sigma-Aldrich).

2 Materials

2.1 MPK 1. Protein extraction buffer, 10 mL. Phosphorylation 50 mM Tris–HCl pH 7.5. by Immunoblotting 10 mM MgCl2. 15 mM EGTA. 100 mM NaCl. 2 mM DTT. 30 mM β-glycerophosphate. 0.1% NP-40.

H2O. Phosphatase (PhosSTOP, Roche) and protease inhibitors (Complete, Roche), 1 tablet each for 10 mL buffer. 2. Separation gel.

15% acrylamide, vary H2O and 30% acryl mix volumes to alter percentage. 4.5 mL for 1 gel with 0.75 mm spacer with 10 wells, for example for Bio-Rad Mini Protean Tetra system (Cat. # 165-8000 system), gel size 10 × 8 cm.

H2O 0.90 mL (1.15 mL without substrate for immunoblot). 30% acrylamide–bis mix 2.25 mL (30/0.8 w/w acrylamide–bis, Sigma Cat. # A3699). 1.5 M Tris pH 8.8 1.00 mL

Substrate* 250 μL 10% SDS 45 μL 10% APS 45 μL TEMED 5 μL *For in-gel kinase assays only: MBP (5mg/mL), SnRK peptide (1mg/mL) stocks. OR 13% acrylamide, 5 mL for 1 gel with 1 mm spacer with 10 wells. Mini protean tetra system, 1 mm thickness cat no. 165-8001 system used for immuno blotting.

H2O 1.41 mL (1.72 mL without substrate for immunoblot) 30% acrylamide–bis mix 2.17 mL Protein Kinase Activation 189

1.5 M Tris pH 8.8 1.0 mL

Substrate * 312 μL 10% SDS 50 μL 10% APS 50 μL TEMED 8 μL. *MBP (5 mg/mL), SnRK peptide (1 mg/mL) stocks. –– After pouring the acrylamide separation solution between the plates, CAREFULLY overlay with water-saturated isopropanol. –– Rinse with water after the gel has polymerized (20 min). –– Pour the stacking gel (below) and insert the comb without air bubbles! Allow the gel to polymerize (minimum10 min). 3. Stacking gel (5% acrylamide) 6 mL for 3 gels 2 mL/gel.

H2O 3.5 mL 30% acrylamide–bis mix 1.0 mL 1.0 M Tris pH 6.8 1.5 mL

10% SDS 60 μL 10% APS 60 μL TEMED 10 μL 4. 6× SDS loading buffer. 60% glycerol. 12% SDS. 375 mM Tris pH 6.8. 60 mM DTT. 0.5% bromophenol blue. 5. Broad range marker—Spectra multicolour broad range ladder, Cat. # 26634, Thermofisher, SE). 6. Electrophoresis buffer. 25 mM Tris. 250 mM glycine. 0.1% SDS. 7. Transfer buffer. 48 mM Tris. 39 mM glycine. 0.037% SDS. 20% methanol. 8. Immunoblot 2ashing buffer (TBST) 1× Tris-buffered saline (TBS, 50 mM Tris, 150 mM NaCl). 0.1% Tween. 9. Immunoblot blocking buffer. 190 Chandra Kenchappa et al.

1× TBST. 5% dry milk. 0.1% Tween. 10. Immunoblot alkaline phosphatase buffer (AP) 100 mM NaCl. 5 mM MgCl2. 100 mM Tris–HCl (pH 9.5, important).

2.2 In-Gel Kinase 1. PAGE and separation gel—Essentially the same as for immu- Activity Assay noblotting (Subheading 2.1 above) EXCEPT for the addition with MBP or of kinase substrate to the separation gel. Acryloylated Peptide 2. Kinase activity assay exchange buffer. Substrates 25 mM Tris–HCl pH 7.5. 0.5 mM DTT. 0.1 mM Na3VO4. 5 mM NaF. 0.5 mg/mL BSA. 0.1% Triton X−100. pH to 7.5 3. Kinase activity assay denaturing buffer. 50 mM Tris–HCl pH-7.5. 6 M guanidine hydrochloride (Gua-HCl). 20 mM DTT. 4. Kinase activity assay renaturing buffer. 25 mM Tris–HCl pH 7.5. 1 mM DTT. 0.1 mM Na3VO4. 5 mM NaF. 5. Kinase activity assay reaction buffer. 25 mM Tris–HCl pH 7.5. 2 mM EGTA. 12 mM MgCl2. 1 mM DTT. 0.1 mM Na3VO4. After washing with this buffer, add to 20 mL volume for kinase reaction add cold ATP to 100 nM and 3.7 MBq (100 μCi) γ-ATP32. 6. Kinase activity assay termination solution. 5% (w/v) trichloroacetic acid (TCA). 1% (w/v) sodium pyrophosphate (Na4O7P2). 7. PhosphorImaging—we use the following but other systems also work: MultiSensitive Phosphor Screens, Medium, Size, 12.5 × 25.2 cm Product No. 7001723, PerkinElmer Cyclone Phosphor Imager: http://macro.lsu.edu/psrgroup/ PersonalPages/Derek%20Dorman/SAXS%20Webpage/ TurboCad%20Drawings/PSI/Phosphorimager%20manual.pdf. Protein Kinase Activation 191

3 Methods

3.1 MPK Phosphorylated MPKs are detected by immunoblot using α-pTEpY Phosphorylation antibody following SDS-PAGE. by Immunoblotting

3.1.1 Sample Treatment Samples—For moss, three 14-day-old colonies on BCDAT medium and Extraction of a given genotype/sample. For Arabidopsis, 3–6 seedlings grown for 10–14 days in 6-well plates with 4 mL liquid MS. 1. Label Eppendorf tubes according to samples. 2. For moss: open the petri dish with three colonies and spray them evenly with 100 μg/mL chitin in H2O. For Arabidopsis: Add chitin to a final concentration of 100 μg/mL. 3. For moss: After 5 min., carefully and quickly pick the three colonies, gently blot them on tissue paper to remove excess liquid, place them in a labelled Eppendorff tube and freeze in liquid nitrogen. For Arabidopsis: After 10 min, carefully and quickly pick the 4–5 seedlings, gently blot them on tissue paper to remove excess liquid, place them in a labelled Eppendorff tube and freeze in liquid nitrogen. 4. Add 250 μL of Extraction Buffer to each sample and grind with a pestle while the sample thaws. 5. Centrifuge at 13,500 × g for 10 min at 4–8 °C. 6. Transfer 200 μL of the supernatant to a new tube without dis- turbing the pellet (see Note 1). 7. Add 40 μL of 6× SDS loading buffer. Mix well and incubate for 5 min in heating block at 95 °C.

3.1.2 SDS-PAGE The samples prepared above generally contain 2–3 μg/μL protein. and Blotting ~30 μg of protein per lane may give good results in gels of 1 mm (for western blotting) and 0.75 mm (for in-gel kinase assay) thickness. 1. Carefully and rapidly load equal amounts of protein in max. 20 μL of each sample on the polyacrylamide gel. Include a well with 10 μL of protein marker. Freeze store the remaining sample at −80 °C. 2. Run samples at 90 V for 30 min to allow proteins and dye to enter the stacking gel. 3. Run the gel at 130 V. As MPKs are 40–50 kDa, stop electrophoresis when ~32 kDa marker runs out. This takes approx. 4 h at RT. 192 Chandra Kenchappa et al.

4. Carefully dismount the gel, then incubate it in Transfer Buffer for 2 min. 5. Immobilize the proteins on a nitrocellulose membrane (Amersham Hybond Nitrocellulose blotting membrane, GE Healthcare, Cat # RPN303D) by wet transfer overnight or 2 h at 70 V (see http://www.bio-rad.com/webroot/web/pdf/ lsr/literature/4006190b.pdf).

3.1.3 Blocking 1. Stain membrane for total protein loading by adding Ponceau and Incubation in 1° stain (Ponceau S: 0.1% (x/v) Ponceau S in 1% (v/v) acetic Antibody acid) for 2 min. 2. Remove the Ponceau stain by washing 3× in TBST (approximately 5 min each wash with gentle shaking) 3. Incubate the membrane in 5% milk TBST for 30 min. 4. Wash 3× in TBST (approximately 5 min each wash with gentle shaking). 5. Add 10 mL 1° antibody (α-pTEpY—anti-p42/p44-erk) 1/2000 in 5% BSA-TBST). Incubate for 2 h at RT or at 4 °C overnight.

3.1.4 2° Antibody 1. Collect 1° antibody and wash blot 3× in TBST (approximately and Developing 5 min per wash with gentle shaking). 2. Add 10 mL 2° antibody (anti-rabbit AP 1/5000 in 5% BSA- TBST). Incubate for 1 h at RT. 3. Collect 2° antibody and wash blot 3× (approximately 5 min per wash with gentle shaking). 4. Wash once with AP buffer for 5 min, then develop blot by adding 10 mL BCIP/NBT substrate (Cat. # 11697471001,

Roche: dissolve 1 tablet in 10 mL H20). Dark bands should appear after 2–10 min. Stop reaction by washing membrane 3× in water.

3.2 In-Gel Kinase The protocol is slightly modified from an earlier one [19]. Sample Activity Assay treatments and protein extractions are the same as for ASSAY 1— with MBP or MPK phosphorylation by immunoblotting. The polyacrylamide Acryloylated Peptide gels are cast with the substrate in 0.75 mm gels and run as in Substrates Subheading 3.1. When SDS-PAGE is finished: 1. Exchange the SDS buffer by rinsing gel 4–6× in 50 mL Washing Buffer, 30 min each rinse at RT with gentle shaking. 2. Fully denature proteins by rinsing the gel 2× in 50 mL chilled Denaturing Buffer, 30 min each with shaking at RT. 3. Renature the proteins by rinsing the gel 4× in 50 mL at 4 °C to remove Gua-HCl in chilled Renaturing Buffer, with shaking at RT, followed by 1 longer rinse in Renaturing Buffer for 3–12 h at 4 °C without shaking. Protein Kinase Activation 193

4. Wash gel 2× at RT in 50 mL Reaction Buffer, 30 min each with shaking. 5. Incubate gel for 1–2 h at RT in 20 mL Reaction Buffer containing 3.7 MBq (100 μCi) γ-ATP32 with shaking. 6. Carefully discard the radioactive waste. 7. Quickly stop the reaction by adding 50 mL Termination Solution with shaking. 8. Rinse 6× in Termination Solution for a total of 3–6 h to eliminate unincorporated γ-ATP32. 9. To avoid shrinking/brittleness, treat gels with 10% glycerol/5% ethanol/7% methanol for 30 min prior to drying at 80 °C under vacuum for 1 h. 10. Expose gels to Phosphor image screen overnight.

4 Notes

1. *Optional determination of sample protein concentration for SDS-PAGE: (a) Add 795 μL water to an eppendorff tube. (b) Add 5 μL protein extract. Mix. Remember a control with 5 μL Extraction Buffer. (c) Add 200 μL commercial Bradford Reagent. Mix. (d) Incubate for 10 min and measure optical density at 595 nm (OD595). (e) Calculate [protein] using: A595 = 0.0556 c + 0.0245 where c = μg protein in 1 mL Bradford reaction.

Acknowledgments

This work was supported by grants from the Danish Basic Research Foundation (Center for Comparative Genomics) and the Danish Research Council for Nature and the Universe (Comparative & functional genomics of plant innate immunity, 1323-00267A) to JM.

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