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Doctoral Thesis

Root-secreted phosphomonoesterases mobilizing phosphorus from the rhizosphere A molecular physiological study in Solanum tuberosum

Author(s): Zimmermann, Philip

Publication Date: 2003

Permanent Link: https://doi.org/10.3929/ethz-a-004583500

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ETH Library Diss. ETH Nr. 15027

Root-secreted phosphomonoesterases mobilizing phosphorus from the rhizosphere

A molecular physiological study in Solanum tuberosum

A dissertation submitted to the

SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZURICH

for the degree of

DOCTOR OF SCIENCES

presented by

PHILIP ZIMMERMANN

Dipl. Ing. Agronom, ETH Zurich born January 31st, 1974 from Charmoille, JU

accepted on the recommendation of

Prof. Dr. Emmanuel Frossard, examiner Prof. Dr. Nikolaus Amrhein, co-examiner Dr. Marcel Bücher, co-examiner Dr. Markus Wyss, co-examiner

2003

Dedication

To Daman's, for her love and support.

Table of contents I

Table of contents

Table of contents /

List of abbreviations ///

Abstract V

Résumé VI

Zusammenfassung VII

11ntroduction 1

1.1 Recent developments in agricultural crop production 1

1.2 How can plant biotechnology contribute? 2

2 Literature review 3

2.1 Phosphate availability and the plant's responses to P-deficiency 3 2.1.1 Phosphorus cycle in an agro-ecosystem 3 2.1.2 Plant responses to P-deficiency: the lupin model 4 2.1.3 Root hairs and the P-deficiency response 5

2.2 Organic phosphates and 7 2.2.1 Organic phosphates in soils 7 2.2.2 Phosphatases in the rhizosphere 7 2.2.3 Phosphatases in the plant: the case of 10

2.3 Inositol phosphates and 16 2.3.1 Inositol phosphates 16 2.3.2 Phytases 23 3 Objectives of dissertation research 30

4 Materials & Methods 31

4.1 Materials and chemicals 31

4.2 Methods 35 4.2.1 Molecular biology 35 4.2.2 Physiological and biochemical measurements 37 4.2.3 Plant growth conditions and tissue harvest 41 4.2.4 Computer analyses 45

5 Purple acid phosphatases from potato 47

5.1 Introduction 47

5.2 Results 48 Secreted Phosphomonoesterase activity of potato roots 48 Cloning of StPAPI, StPAP2 and StPAP3 49 Protein sequence analysis 49 Tissue-specific expression of StPAPI, StPAP2 and StPAP3 53 Induction of expression after P deprivation 53 Effect of mycorrhizal symbiosis 53 Regulatory aspects of expression of StPAPI, 2 and 3 54

5.3 Discussion 56

6 Expression of a consensus in potato root hairs 59 Il

6.1 Introduction 59

6.2 Results 61 Potato root hair growth in P-deficient conditions 61 The root hair-specific promoter LeExtl. 1 61 Generation of transgenic potato lines expressing a consensus phytase 62 The consensus phytase properties are maintained in transgenic plants 63 The PHY protein is secreted from the roots 64 Kinetics of phytic acid degradation by root exudates 64 Phenotype of PHY plants 66

6.3 Discussion 68

7 General conclusions and outlook 71

7.1 Potato purple acid phosphatases 71

7.2 P mobilization from phytate in transgenic plants secreting phytase 76

7.3 Designing plants more effectively mobilizing P 77

7.4 Phosphatases, phytases and beyond 80

8 References 83

9 Appendix 97 Appendix 1. DNA and amino acid sequences of StPAPI 98 Appendix 2. DNA and amino acid sequences of StPAP2 99 Appendix 3. DNA and amino acid sequences of StPAP3 100 Appendix 4. DNA and amino acid sequences of the SP/PHY chimeric gene 101 Appendix 5. Analysis of the signal sequence of SP/PHY 102

10 Acknowledgements 103

11 Curriculum vitae 105 List of abbreviations III

List of abbreviations

AMF arbuscular mycorrhizal fungus / fungi Ampr ampicillin resistent ANOVA analysis of variance APase acid BLAST basic local alignment search tool bp basepair(s) BSA bovine serum albumin CaMV cauliflower mosaic virus cDNA complementary DNA CIP (CIAP) calf intestinal (alkaline) phosphatase ddH20 double distilled water dH20 deionized water DNase DTT dithiothreitol DW dry weight DMF dimethylformamide dNTP deoxynucleoside triphosphate EDTA sodium ethylenediaminetetraacetate EST expressed sequence tag EtOH ethanol GFP green fluorescent protein GFP buffer glyoxal / formamide / phosphate buffer GPI glycosylphosphatidylinositol GUS ß-glucuronidase H PLC high performance liquid chromatography ICP inductively coupled plasma Kanr kanamycin resistent kbp kilobasepair(s) M ES 2-morpholinoethanesulfonic acid monohydrate mRNA messenger RNA MS medium Murrashige and Skoog medium NaAc sodium acetate OD optical density ORF open reading frame PCR polymerase chain reaction P phosphorus PAP purple Pase phosphatase Pi orthophosphate (inorganic phosphate) PDEase PMEase Phosphomonoesterase pNPP p-nitrophenyl phosphate Po organic phosphate RACE rapid amplification of cDNA ends RNase RT-PCR reverse transcription PCR TIGR The Institute for Genomic Research X-Gluc 5-bromo-4-chloro-3-indolyl-beta-D-glucuronicacid IV Abstract V

Abstract

Phosphorus (P) is one of the most limiting plant nutrients in crop production worldwide. Owing to the strong reactivity of phosphates with soil minerals, P is largely unavailable to plants. Furthermore, P is exported from the field in the harvested products. The addition of P fertilizers to sustain crop production is thus required. However, not only are natural resources of P for the production of fertilizers limited and non-renewable, but also, the excessive use of P in high-input agricultural systems has resulted in environmental pollution. Low-input systems, in contrast, use low amounts of P fertilizers and crop production depends on the amount of available P in the soil, on the efficiency of their cropping system and on the plant genotypes used.

This thesis deals with increasing the ability of crop plants to mobilize soil P to improve crop production. The specific objective was to study, on the one hand, the plant's natural responses to P deficiency, and, on the other hand, to engineer a crop plant for improved mobilization of P from soils. Between 30 and 65% of P in soils is in organic form and largely unavailable for plant uptake. This work therefore focused specifically on the study of phosphatases secreted from plant roots and on engineering the secretion of phytase from roots by expressing a phytase gene in root hairs of potato.

Three cDNAs encoding polypeptides belonging to the family of purple acid phosphatases (PAP) were isolated from Solanum tuberosum, and the expression of the corresponding genes was characterized. StPAPI was shown to be expressed in roots, stems, leaves, flowers and stolons, and did not respond to P deprivation. Both StPAP2 and StPAP3 were induced by P starvation and expressed mainly in roots, with StPAP3 being additionally expressed in stem. Based on sequence analysis, all three PAPs are predicted to be secretory proteins. The precise function of these genes could not be elucidated within the frame of this work. However, the data obtained suggests that StPAP2 and StPAP3 contribute to the phosphatase activity in potato root exudates and therefore presumably function in the mobilization of P from organic P sources in the rhizosphere.

The expression of a synthetic phytase in root hairs of potato resulted in a more than 50-fold increased phytase activity in root exudates. The recombinant phytase showed high thermo¬ stability and was able to degrade extraradical phytic acid to lower inositol phosphates. In a soil-quartz substrate to which phytate had been added, transgenic plants secreting the synthetic phytase exhibited a 40% higher P concentration in the leaves, were on average 20% taller, but in our experimental conditions did not have a significantly higher biomass production. From the data obtained we cannot conclude whether phytate availability in soils or phytase activity in the rhizosphere is the limiting step in the mobilization of P from soil phytate for plant uptake. These issues are discussed in the light of our observations. Résumé VI

Résumé

Le phosphore (P) est un des éléments nutritifs végétaux les plus limitants pour la production agricole mondiale. En raison de la forte adsorption des phosphates aux éléments minéraux du sol, le P est largement indisponible pour le prélèvement par les plantes. Par conséquent, la fertilisation en phosphates est essentielle pour une production agricole soutenue. Non seulement les réserves en P naturel sont limitées, mais l'utilisation excessive de P dans les systèmes de production intensive cause des problèmes de pollution environnementale. Par contre, les systèmes de production extensifs n'ont souvent pas les moyens de se procurer les fertilisants P nécessaires, ce qui les rend davantage dépendants de la capacité de leurs cultures à prélever le P présent dans leurs sols.

Le concept de cette thèse est d'augmenter la capacité des plantes à mobiliser le P du sol afin d'améliorer la production végétale. L'objectif spécifique est, d'une part, d'étudier les réponses naturelles de la plante à une carence en P, et d'autre part, de créer une plante transgénique capable de mobiliser davantage de P du sol. 30-65% du P dans le sol est sous forme organique (Po), dont une partie, telle les inositols phosphates (phytates), n'est pas disponible pour le prélèvement par les plantes. Le travail présenté a donc porté surtout sur l'étude de phosphatases sécrétées par les racines et sur l'augmentation de la sécrétion de phytase par les racines en exprimant un gène de phytase dans le poil absorbant des racines de pommes de terre.

Trois gènes de pomme de terre codant pour des polypeptides appartenant à la famille des phosphatases acides pourpres (PAP) furent isolés et characterises au niveau de leur expression dans la plante. Il s'avère que StPAPI est exprimée dans les racines, feuilles, fleurs et stolons, et qu'elle ne réagit pas à l'apport de P. StPAP2 et StPAP3, par contre, sont exprimées en conditions de carence en P principalement dans les racines (StPAP2 et 3) et dans la tige (StPAPS). L'analyse des séquences d'acides aminés suggère que les trois gènes contiennent un signal de sécrétion, et que StPAP2 pourrait avoir un signal d'ancrage dans la membrane par GPI. La fonction précise des ces gènes ne fut pas possible dans le cadre du travail présenté. Néanmoins, les données obtenues permettent de supposer que StPAP2 et StPAP3 jouent un rôle dans l'activité phosphatasique des sécrétions racinaires et par conséquent dans la mobilisation de P du P organique dans la rhizosphere.

L'expression d'une phytase synthétique dans le poil absorbant des racines de pomme de terre produisit une activité de phytase plus de 50 fois plus élevée dans les exsudats racinaires. La phytase synthétique montra une grande thermo-stabilité et fut capable de dégrader l'inositol hexa/c/'sphosphate en formes réduites d'inositols phosphates. Dans un substrat composé de sol et de quartz contenant de la phytate, les plantes transgéniques sécrétant une phytase artificielle purent prélever davantage de P, étaient 20% plus grandes, mais dans nos conditions expérimentales n'accumulèrent pas davantage de biomasse. Sur la base de ces données, il n'est pas possible de déterminer si la mobilisation de P des phytates d'un sol naturel est limitée par la disponibilité des phytates ou par la quantité de l' de phytase. Néanmoins, certains aspects sont discutés sur la base des résultats obtenus. Zusammenfassung VII

Zusammenfassung

Phosphor (P) ist weltweit einer der am meisten limitierenden essentiellen Pflanzennährstoffe in der landwirtschaftlichen Pflanzenproduktion. Aufgrund des starken Bindungsvermögens von Phosphaten an Bodenelemente ist er nur in geringen Mengen für die Pflanzenaufnahme verfügbar. Demzufolge ist eine P Düngung zur Aufrechterhaltung der Pflanzenproduktion unentbehrlich. Die natürlichen Ressourcen zur Herstellung von P Dünger sind aber begrenzt. Zudem hat der übermässige Gebrauch dieser Ressourcen in den intensiven landwirtschaftlichen Systemen zu Umweltbelastungen beigetragen. In vielen anderen Gebieten werden hingegen nur sehr wenige P Dünger verwendet, und diese Gebiete sind daher stärker vom P Gehalt ihrer Böden, des Anbausystems, sowie der verwendeten Genotypen der Kulturpflanzen abhängig. Die vorgelegte Arbeit basiert auf dem Konzept einer Vergrösserung der Fähigkeit landwirtschaftlich nutzbarer Pflanzen, Boden P zu mobilisieren, und damit zur Verbesserung der Produktion und somit zur Linderung der Abhängigkeit von externen P Quellen beizutragen. Das Ziel war einerseits, die natürlichen biologischen Reaktionen von Pflanzen auf P Mangel zu studieren, und andererseits, eine Pflanze mit erhöhter P-

Mobilisierungsfähigkeit durch gentechnische Methoden zu erzeugen. Da der Boden 30-65% des P in organischer Form enthält, wovon ein wesentlicher Teil in Form von Phytat nicht pflanzenverfügbar ist, wurde diese Arbeit auf die Untersuchung der von Wurzeln ausgeschiedenen Phosphatasen sowie auf die Erzeugung transgener Pflanzen mit erhöhter Sekretion von Phytase in den Wurzeln ausgerichtet.

Drei cDNAs aus Kartoffel, die für Polypeptide kodieren, die der Familie der purpuren sauren Phosphatasen (PAP) angehören, wurden isoliert und auf Expressionsebene charakterisiert. StPAPI wird in Wurzeln, Stengeln, Blättern, Blüten sowie Stolonen exprimiert und reagierte nicht auf Änderungen der P Konzentration im Nährmedium. StPAP2 und StPAP3 wurden hingegen unter P Mangel stark induziert und waren vor allem in den Wurzeln exprimiert. StPAP3 war zusätzlich noch im Stengel stark exprimiert. Die Analyse der Aminosäuresequenzen ergab, dass alle drei PAPs eine mögliche Sekretionssignalsequenz aufweisen und dass StPAP2 eine Signalsequenz für GPI Verankerung in der Membran haben könnte. Die genaue Funktion dieser drei Gene konnte im Rahmen dieser Arbeit nicht bestimmt werden, doch die erhaltenen Resultate lassen auf eine mögliche Funktion von StPAP2 und StPAP3 in der P-Mobilisierung von organischem P in der Rhizosphäre schliessen. Die Expression einer synthetischen Phytase in Wurzelhaaren der Kartoffel ergab eine mehr als 50-fache Erhöhung der Phytaseaktivität in den Wurzelexudaten. Die rekombinante Phytase zeigte eine ausgesprochen hohe Hitzestabilität und konnte Phytinsäure degradieren. Die Phytase sezernierenden transgenen Pflanzen, die auf einem Bodensubstrat mit Phytatzusatz angezogen wurden, hatten 40% mehr P in den Blättern als Wldtyp Pflanzen. Der Spross war 20% höher als beim Wldtyp, aber die gesamte Biomasse war in diesem Experiment statistisch nicht signifikant unterschiedlich. Diesen Daten kann nicht entnommen werden, ob in einem natürlichen Boden die Verfügbarkeit des Phytats oder die Menge an Phytase für die Mobilisierung von P aus Phytat limitierend wirken. Abschliessend werden mögliche Hypothesen diskutiert. VIII 1 Introduction 1

1 I Introduction

1.1 Recent developments in agricultural crop production

The last century has experienced an unprecedented evolution in world agriculture. Up to the 1950's, increases in agricultural production were largely achieved by an expansion of the area under cultivation, by the use of chemical fertilizers, and also by the more recent use of chemicals for crop protection. Since then, significant increases have also been obtained in

many regions of the world by crop breeding in combination with the use of fertilizers and

irrigation. Nevertheless, despite significant increases in crop production, food security remains a major concern. To improve food security means to enhance both the access to and the availability of food

(Runge-Metzger, 1995). A major factor in improving food availability in many countries is the capability of increasing local production of foods. Increased local production can be achieved

by increased input and/or improved agricultural techniques and crop varieties. In the case of

phosphorus (P), which is the most limiting nutrient in crop production worldwide, increased

input is logistically and technically difficult to achieve for many regions. In addition, limited P reserves and the inefficient use of P bear the potential for a future phosphate crisis in agriculture (Abelson, 1999). In this context, the use of alternative cropping systems and the breeding of plants adapted to low nutrient soils can significantly contribute to increased local production of foods. Although the use of external fertilizer inputs remains indispensible to compensate losses by crop removal from fields, improved crop varieties may increase the efficiency of utilization of nutrients from soils and from external inputs. In order to achieve this goal, a deeper understanding of the processes involved in nutrient cycling, mobilization, and

uptake by crop plants is needed. Although much research has been done to improve agricultural systems in regions where P

is limiting, the basic processes governing the movement of P in agro-ecosystems, fixation of P in soils, mobilization by plants and uptake by the roots are still not fully understood. Recent advances in describing these systems (Oberson et al., 1999; Raghothama, 1999; Frossard et al., 2000b; Bucher et al., 2001) raise new possibilities in developing both efficient

management systems and crops more efficient in P mobilization and uptake. These improvements would result in increased productivity in those regions where P fertilizers are a rare good. Recent developments in root research, including molecular biological approaches, set the stage for new technologies that can be used to improve crops. In fact, plant biotechnology is expected to make a major contribution to the development of plants efficient in P acquisition (Hell and Hillebrand, 2001).

1.1 Recent developments in agricultural crop production 1 Introduction 2

1.2 How can plant biotechnology contribute?

The application of biotechnology as a tool for increasing food security by crop improvement has been discussed at length (Ortiz, 1998; Conway and Toenniessen, 1999; Herrera- Estrella, 1999; Kishore and Shewmaker, 1999; Serageldin, 1999). Advances in plant

breeding and in the understanding of basic biological processes have allowed new

biotechnologies to develop. Initially, products of crop biotechnology embraced mainly resistance to herbicides and to pathogens, and were commercialized primarily in industrialized countries such as the United States and Canada. New developments in

research, however, have revealed a significant potential for crop improvements for developing countries. Some examples of such achievements include the modification of seed contents for improvement of the nutritional value of crops (Ye et al., 2000; Lucca et al., 2002), resistance to pathogens (Clausen et al., 2000), and tolerance to drought, salt and freezing (Kasuga et al., 1999; Zhang and Blumwald, 2001). Recent advances have shown possibilities in improving the ability of plants to mobilize nutrients from soils, such as Fe and P (Samuelsen et al., 1998; Koyama et al., 2000; Lopez-Bucio et al., 2000; Delhaize et al., 2001; Richardson et al., 2001a; Takahashi et al., 2001a). In parallel to the introduction of

new traits in transgenic crops by expression of transgenes, advances in plant functional genomics are leading to a better understanding of the metabolic pathways governing plant

responses to stress. Ultimately, one can expect crop biotechnology to evolve from a straightforward, simple approach to more complex and comprehensive strategies in combination with plant breeding to engineer crops better adapted to their environment. It is thus important both to assess the expression of transgenes on plant productivity as well as to

understand the function of endogenous genes, to identify target genes for transgene expression, and, furthermore, to identify genetic loci of interest by marker-assisted breeding.

In addition, gene knock-out mutants and plants expressing foreign genes can serve as model plants to other sciences such as soil sciences, phytopathology and entomology, which, in turn, would yield new knowledge resulting in improved crop management.

This work embraces all three aspects, namely, the characterisation of genes involved in the

potato plant's response to P deficiency, the expression of a foreign phytase gene to improve P acquisition, and, finally, the possibility to use recombinant technology as a tool to modify the rhizosphere by targeted expression of genes in root hairs.

1.2 How can plant biotechnology contribute? 2 Literature review 3

2 ^h Literature review

2.1 Phosphate availability and the plant's responses to P-deficiency

2.1.1 Phosphorus cycle in an agro-ecosystem

Sibbesen and Runge-Metzger (1995) proposed a model of P cycling in the agro-ecosystem composed of three main compartments: soils, crops and animals (Figure 2.1).

" Milk, eggs and animals for slaughter

Surface runoff, wind erosion (Leaching)

Figure 2.1 P-cycling in agroecosystems. Modified from Sibbesen and Runge-Metzger (1995).

In intensive agricultural systems, large quantities of P are transferred from one compartment to another. Nevertheless, P is one of the most limiting essential plant nutrients in agricultural production worldwide. The availability of P in soils to plants remains limited owing to the

2.1 Phosphate availability and the plant's responses to P-deficiency 2 Literature review 4

strong reactivity of phosphates with the soil matrix (Holford, 1997). In fact, many soils contain pools of organic and inorganic P that would be large enough for sustained agricultural crop production, but only a reduced fraction of P is available to the roots (Marschner, 1995; Frossard et al., 2000a). Up to 30 million tons of non-renewable phosphate fertilizers are applied each year worldwide (Prud'homme, 2001), of which only a small fraction is actually utilized by the crops (Gallet et al., 2003). In low-input systems, where P fertilization is scarce, crops are regularly removed from the surfaces, and little or no P is returned back to the soil, resulting in soil mining and poor crop production. To cope with these problems, new management systems and the development of crops more efficient in P mobilization from soils have been and are being pursued. To better target research for crop improvement, it is a prerequisite to understand which mechanisms naturally occurring in plants allow them to cope with P deficiency stress. For this purpose, model plants such as white lupin (Lupinus albus, L.) have been used. White lupin, a non-mycorrhizal plant, is an interesting case because it has a very high capacity to mobilize P from low-P soils. Another model plant would be Arabidopsis, also non-mycorrhizal, which has the advantage of a fully sequenced genome and therefore can easily be studied via functional genomics. Lotus and Medicago are also well described plants, having the advantage of being mycorrhizal.

2.1.2 Plant responses to P-deficiency: the lupin model

Plants react to low levels of available P in the rhizosphere by activating a large number of morphological and physiological responses. The P-efficient model plant lupin (Lupinus albus, L.) has been extensively studied and has been shown to initiate various strategies to acquire P from soils (Figure 2.2).

Some of these responses appear also more generally in higher plants and include the secretion of increased amounts of organic acids and phosphatases (Ozawa et al., 1995; Johnson et al., 1996; Ascencio, 1997; Gilbert et al., 1999; Neumann et al., 2000; Gaume et al., 2001), modification of root architecture (Johnson et al., 1996; Wlliamson et al., 2001), induction of H+-ATPases and subsequent secretion of protons (Yan et al., 2002), activation of high-affinity P transporters (Smith et al., 1997; Daram et al., 1998; Liu et al., 1998), production of anthocyanins (Ticconi et al., 2001; Bloor and Abrahams, 2002), and activation of P-scavenging (Plaxton, 1998; Neumann et al., 1999). Some species possess other adaptive features like increased seed size (Milberg and Lamont, 1997) and slower growth.

The multiplicity of P-starvation responses occurring simultaneously in lupin, the intensity of these responses, and their colocalisation in root clusters (proteoid roots) suggest that the effectiveness of phosphate mobilization from the soil may depend on the joint activation of these different strategies and on the respective concentrations of secreted products involved.

However, it is known from other (non-proteoid) plant species, e.g. Arabidopsis thaliana and

Zea mays, that the development of root hairs, as well as the secretion of acid phosphatases and organic acids, is higher in P-efficient varieties than in their inefficient counterpart genotypes (Narang et al., 2000; Gaume et al., 2001). In other words, although these

2.1 Phosphate availability and the plant's responses to P-deficiency 2 Literature review 5

strategies considered alone may not be as effective as in combination, they seem to play a role in the P mobilization from soils. Of particular interest in this work are the increased growth of root hairs (see below) and the secretion of phosphatases from plant roots (see chapter 2.2.2).

Activation of P Production of anthocyanins scavenging enzymes

Secretion of organic acids Modification of redox-potential Secretion of phosphatases "^ Induction of high-affinity H+-release or uptake P transporters

Secretion of Secretion of phenolics chelating compounds

Figure 2.2 Morphological, physiological and molecular responses of white lupin to P deficiency

2.1.3 Root hairs and the P-deficiency response

Root hairs are tubular-shaped tip-growing root epidermal (rhizodermal) cells which extend into the ambient soil. Root hair length and density vary between plant species. Whereas certain species produce almost no root hairs, others such as maize, alfalfa and ryegrass produce several hundred hairs per mm2 surface area of the root cylinder (Jungk, 2001). Root hairs generate an extended area of contact between the soil and the root, resulting in the mobilization and uptake of nutrients from larger soil volumes. Comparable to the thin hyphae of symbiotic mycorrhizal fungi, root hairs are particularly efficient in mining the soil for scarcely abundant nutrients such as P. In fact, zones around roots have been shown to become increasingly depleted in P, largely due to the action of root hairs, resulting in P- depletion zones up to a few mm distance from the root surface (Fusseder and Kraus, 1986; see Figure 2.3).

2.1 Phosphate availability and the plant's responses to P-deficiency 2 Literature review 6

Since root hairs represent the major root surface area (up to 90% of the total root surface area; Itoh and Barber, 1983; Bates and Lynch, 1996), genes involved in P mobilization and uptake are predominantly expressed in this tissue (Bucher et al., 2001). In fact, several genes encoding high-affinity Pi transporters have been cloned and shown to be predominantly expressed at two root interfaces, i.e. in rhizodermal cells including root hairs, and in root zones below the rhizodermis colonized by arbuscular-mycorrhizal fungi (Daram et al., 1998; Liu et al., 1998; Muchhal and Raghothama, 1999; Rausch et al., 2001; Harrison et al., 2002; Karthikeyan et al., 2002; Paszkowski et al., 2002; Smith and Barker, 2002). In

response to P deprivation, increased root hair length was measured, for example, in Arabidopsis thaliana (Bates and Lynch, 1996), barley (Gahoonia and Nielsen, 1997), and wheat (Gahoonia et al., 1997). The role of root hairs in P uptake has been demonstrated repeatedly, for instance in six different species (Itoh and Barber, 1983), in wheat and barley cultivars (Gahoonia et al., 1997), and by using root hair mutants of Arabidopsis (Bates and Lynch, 2000). Bailey et al. (2002) reported that root hairs also promote anchorage against uprooting forces.

Figure 2.3 3-dimensional model of a P-depletion zone around roots A = root cylinder, B = root hair cylinder, C = maximal depletion zone Source Fusseder and Kraus (1986)

While, in an initial phase, plants tend to increase their root surface area under P deficiency stress (Mollier and Pellerin, 1999), they generally also secrete various compounds. The secretion of high molecular weight compounds such as phosphatases may play an important role in the mobilization of organic P sources and has been widely mentioned in the literature. This topic will be dealt with in the next chapter.

2.1 Phosphate availability and the plant's responses to P-deficiency 2 Literature review 7

2.2 Organic phosphates and phosphatases

2.2.1 Organic phosphates in soils

During soil development, organic P (Po) accumulates in the upper soil horizon (Syers and Curtin, 1989) and represents between 30% and 65% of the total P in soils (Harrison, 1987). Due to the dynamic nature of Po in soils, the composition and turnover of these fractions are difficult to assess. Soil Po occurs as monoesters ((RO)P03H2) and diesters ((RO)(R'0)P02H) and their chemical diversity resides in the organic moieties (R and R'). Although the structure of numerous forms of Po in soils remains unknown, the use of 31P-NMR techniques has given new insights into soil Po forms (Hawkes et al., 1984; Condron et al., 1990). The most

prevalent forms of Po include inositol phosphates, sugar phosphates, nucleic acids and phospholipids. Of particular relevance for our work are inositol phosphates, which represent

up to 50% of the organic P in soils (Dalai and Hallsworth, 1977; Anderson, 1980). For a more detailed analysis of inositol phosphates and phytases, see chapter 2.3 in this thesis.

The microbial activity in soils significantly contributes to the transformation processes involved in P cycling (Stewart and Tiessen, 1987; Oberson et al., 1996; Rodriguez and Fraga, 1999). However, gamma-sterilized soils also actively degrade Po because of biochemical P mineralisation due to the remaining activity of phosphohydrolases (Burns,

1982). In fact, enzymes can be stabilized in soils by sorption onto soil compounds such as clay minerals while maintaining their activity (Leprince and Quiquampoix, 1996; Pant and Warman, 2000).

Although several of the mechanisms involved in the cycling and transformation processes of organic phosphates are not fully understood, there is increasing evidence that these forms of

phosphate may play a role in plant nutrition (Stewart and Tiessen, 1987; Magid et al., 1996; Rodriguez and Fraga, 1999; Oehl et al., 2001; Oehl et al., 2002). In fact, a number of authors have shown that phosphatases generally have low substrate specificity and thus are

probably able to hydrolyze P from a large array of organic P compounds from soils.

2.2.2 Phosphatases in the rhizosphere

Terminology

The name "phosphatase" has generally been attributed to any enzyme that can hydrolyze

phosphate esters and anhydrides. This group includes for example phosphoprotein phosphatases, , diadenosine tetraphosphatases, , 5'- , phytases, alkaline and acid phosphatases, respectively, and other types of phosphomonoesterases. In rhizosphere research, this term more generally refers to proteins from an unknown composition of enzymes able to cleave chromogenic substrates such as p- nitrophenylphosphate, which has been the method of choice for measuring soil phosphatase activity since 1969 (Tabatabai and Bremner, 1969). Phosphate ester hydrolysing activities would more correctly be indicated by the substrate used, for example p-nitrophenyl

2.2 Organic phosphates and phosphatases 2 Literature review 8

phosphatase activity. However, for practical reasons, the use of the general term "phosphatase" has been maintained in the scientific vocabulary. Some more recent publications refer to the more restrictive term "Phosphomonoesterase" (PMEase), "acid Phosphomonoesterase" (AcPMEase), "alkaline Phosphomonoesterase" (alkPMEase) or

"phytase" to account for the substrate or pH range, respectively, used for activity measurements.

Microbially-secreted phosphatases

Many soil microorganisms studied to date show a propensity to produce and secrete phosphatases. Some fungi, including ecto-mycorrhizal fungi, are well known for their secretion of PM Eases (e.g., Greaves and Webley, 1965; Antibus et al., 1992; Yadav and Yadav, 1996; Leake, 2002). PMEase secretion has also been described for microorganisms such as Bacillus (Kerovuo et al., 1998; Idriss et al., 2002), Pseudomonas (Rodriguez and Fraga, 1999; Home et al., 2002), Rhizobium (Rodriguez and Fraga, 1999), Pénicillium (Reyes et al., 2002), various Enterobacteriaceae, and Paecilomyces (Silva and Vidor, 2001).

Root-secreted phosphatases

Most plants respond to P deficiency by producing increased amounts of acid phosphatases in roots. As mentioned in chapter 2.1.2, the model plant lupin has been extensively studied in this respect (Adams and Pate, 1992; Ozawa et al., 1995; Li and Tadano, 1996; Li et al., 1997a; Olczak et al., 1997; Gilbert et al., 1999; Neumann et al., 1999; Miller et al., 2001), but increased acid Phosphomonoesterase activities in root protein extracts or root exudates have also been reported for a number of other species including rice (Chen et al., 1992), barley (Asmar et al., 1995), wheat (Szabo-Nagy and Erdei, 1995), maize (Gaume et al., 2001), as well as gray birch and red maple (Antibus et al., 1997), tomato (Kaya et al., 2000), and a variety of other plants (Tadano and Sakai, 1991; Dinkelaker and Marschner, 1992; Ascencio, 1997; Hayes et al., 1999). Several authors have demonstrated that plants do not only secrete

PMEases, but a more general group of P-cleaving enzymes including, additionally, phosphodiesterases (PDases; Asmar and Gissel-Nielsen, 1997; Abel et al., 2000; Chen et al., 2002) and (RNases; Nürnberger et al., 1990; Löffler et al., 1992; Bosse and Koeck, 1998; Bariola et al., 1999; Abel et al., 2000). In terms of Pi release from organic sources, PMEases are thought to have a major function compared to other phosphohydrolases due to their broad substrate specificity and to the significant proportion of monoester P forms in soils (Condron et al., 1990).

The level of PMEase activity depends on different physiological factors such as root age and morphology, the P-status, the supply in other nutrients, genotype, and various forms of stress (Grierson and Comerford, 2000; Chen et al., 2002). The secretion of PMEase from roots does not result from a higher leakiness of cell membranes due to P deficiency, but from an active export of enzymes via the cellular secretory pathway (Gaume et al., 2001; Yadav and Tarafdar, 2001). This finding is further corroborated by the discovery of several phosphatase genes which are specifically expressed in roots under phosphate starvation. Most of these

2.2 Organic phosphates and phosphatases 2 Literature review 9

phosphatases contain a signal sequence for secretion via the endoplasmatic reticulum (Deng et al., 1998; del Pozo et al., 1999; Haran et al., 2000; Wasaki et al., 2000; Baldwin et al.,

2001; Miller et al., 2001; Li et al., 2002; see also chapter 2.2.3). To sum up, many plants respond to P deficiency by secreting increased amounts of phosphatases from the roots. It is generally held that the function of these phosphatases is to mobilize P from organic sources in the rhizosphere, thereby increasing the P availability in that soil compartment.

Phosphatases and P availability in the rhizosphere

Phosphatase activity can be detected in virtually all cultivated soils. The orthophosphate present in different Po forms in soils (see chapter 2.2.1) can theoretically be released by at least one group of phosphatases present in soils. Owing to the relatively high Po concentration in soils, and to the presence of root-secreted phosphatases together with fungal phosphatases and bacterial alkaline and acid phosphatases, they are believed to be potentially important sources of P for plants. The ability of phosphatases to mobilize P from soil organic sources has been shown in a number of cases (Tarafdar and Claassen, 1988; Helal, 1990; Adams and Pate, 1992; Fox and Comerford, 1992). Others (Thompson and Black, 1970) have reported, however, that the addition of phosphatases to the soil did not decrease its organic P content. The quantitative contribution of plant-secreted phosphatases to plant nutrition and the contribution of individual substrates to the enzymatic release of P are thus not clear (Häussling and Marschner, 1989; Jungk, 1996). Tarafdar and Jungk (1987) measured the PMEase activities in soil around plant roots in relation to the depletion of different P fractions in the rhizosphere and showed that there is a strong correlation between both factors. A similar study was performed by Chen et al. (2002) in a pot experiment with ryegrass and Pinus radiata. The results obtained revealed that pine roots occasioned a larger depletion zone of Po than ryegrass (Figure 2.4). This depletion correlated with greater concentrations of water-soluble organic carbon, higher microbial biomass and increased alkaline and acid phosphatase and PDase activities.

A B

1300

1200

'J Sil 85 1100 — 9 6.

2 x 1000

900

800

0 2 4 6 8 1Ü 12 14 Q 2 4 6 8 10 12 14

Distance from root surface (mm) Distance from root surface (nun)

Figure 2.4 (A) Sodium hydroxide extractable organic P (NPo) content, and (B) acid PMEase activities in an Orthic Brown Soil (Dystrochrept) at different distances from roots of Pinus radiata D Don (T) and Lohum perenne L (o) As a control soil only (•) Figures from Chen et al (2002)

2.2 Organic phosphates and phosphatases 2 Literature review 10

Asmar et al. (1995) reported that the depletion of bicarbonate-extractable Po by barley (Hordeum vulgare, L.) roots was positively correlated with phosphatase activity in the rhizosphere. Häussling and Marschner (1989) found that readily hydrolysable Po in the

rhizosphere of 80 year-old Norway spruce (Picea abies, (L) Karst.) was accompanied by a higher phosphatase activity. Recently, Bhadraray et al. (2002) compared phosphatase activities in rhizosphere soil to plant biomass production and P uptake in rice varieties and found that among the enzymatic activities that were compared, alkaline PMEase activity was found to be most significantly correlated to plant biomass and P uptake. Whether there was a causal relationship between alkaline PMEase activity and biomass production is not known. The contribution of phosphatases from microbial origin to the hydrolysis of Po in soils is unclear. While ecto-mycorrhizal fungi are known to secrete phytase and are thought to be able to mobilize P from phytate (see chapter 2.3.1), the quantitative contribution of extracellular phosphatases from AM fungi to Po hydrolysis is thought to be insignificant (Joner et al., 2000).

Despite this wealth of evidence, other authors have suggested that increased phosphatase activities in the rhizosphere were in some cases rather a natural plant response to P- or other nutrient starvation stresses and to an increase in root density, and that there was no causal relationship between phosphatase secretion and mobilization of P from Po (Hedley et al., 1983; Furlani et al., 1984). Boreo and Thien (1979) equally found that the increased levels of phosphatase activity in the rhizosphere were not related to the mineralization of organic P. In light of these controversies, new methods must be developed to provide new insight into the effective roles of phosphatases in soils. The study of root-secreted phosphatases, and

more precisely, the identification of genes responsible for phosphatase synthesis, is one approach that may help answer some of these questions. The next chapter will deal with a particular family of plant phosphatases, some members of which have been shown to be synthesized in increased amounts in roots in response to P deficiency.

2.2.3 Phosphatases in the plant: the case of purple acid phosphatases

Biochemical characteristics

Purple acid phosphatases (PAPs) comprise a family of metal-containing glycoproteins that catalyse the hydrolysis of a wide range of phosphate esters and anhydrides. Members of this group have been identified in plants, animals, fungi and bacteria (Oddie et al., 2000; Schenk et al., 2000a). Their active sites exhibit a binuclear, redox-active Fe(lll)-M(ll)/M(lll) center, where M(ll) is either Fe, Zn or Mn, and M(lll) is Fe (Doi et al., 1988; Vincent and Averill, 1990; Barford et al., 1998; Schenk et al., 2001). The characteristic purple colour is due to a tyrosine-M(lll) charge-transfer transition (Klabunde and Krebs, 1997). PAPs are also known as tartrate-resistant acid phosphatases (TRAPs) due to their insensitivity to inhibition by tartrate.

In animals, PAPs appear to form a homogeneous group of 35 kDa monomeric proteins containing an antiferromagnetically coupled binuclear Fe(lll)-Fe(ll)/Fe(lll) center. Plant PAPs appear to be more diverse than animal forms, both in size and in the nature of the second

2.2 Organic phosphates and phosphatases 2 Literature review 11

metal involved in the . Two families of different molecular size can be distinguished. The low-molecular weight (LMW) PAPs (-35 kDa) occur both as monomers and as dimers derived from disulphide-linked monomer fragments. High-molecular weight (HMW) PAPs (-55 kDa) are unique to plants and form homodimers through disulfide bonds.

In Arabidopsis thaliana, several genes from each family have been identified based on sequence similarity (Li et al., 2002). Members of the HMW PAP family possess an extended

N terminus without catalytic function, and their C terminus has sequence similarity with the LMW family of PAPs. Five conserved motifs containing seven residues involved in metal binding are conserved throughout all known plant, animal and cyanobacterial PAPs. The binuclear catalytic center of plant PAPs has been shown to possess both Fe(lll)-Zn(ll) as well as Fe(lll)-Mn(ll) complexes (Beck et al., 1986; Schenk et al., 2001). The replacement of

Zn(ll) by Fe(ll) in a plant PAP yielded an enzyme with full activity and spectral properties similar to animal PAPs (Beck et al., 1988). As far as their substrate specificities are concerned, plant PAPs have not been well characterized. They are generally thought to be non-specific acid phosphomonoesterases (Li and Tadano, 1996).

Purple acid phosphatases in plants

Since the first purification and isolation of a human PAP protein in 1971, a number of PAP proteins have been characterized in the literature. The majority of these PAPs were discovered in the context of biomedical research, mainly in beef and rat spleen (Davis et al., 1981; Davis and Averill, 1982; Averill et al., 1983; Hara et al., 1984) and in the uterine secretion of sows (Murray et al., 1972). Owing to their iron content and their suspected role in iron transport, the latter proteins were often called uteroferrins. These proteins all belong to the family of low molecular weight PAPs. In plants, PAPs were first discovered in sweet potato (Uehara et al., 1974a; Uehara et al., 1974b) and have since been isolated from other plant species, including spinach leaves (Fujimoto et al., 1977), red kidney beans (Beck et al., 1986), sweet potato tubers (Hefler and Averill, 1987), soybean suspension cultures (Lebansky et al., 1993) and yellow lupin seeds (Olczak et al., 1997). A more detailed characterisation of a plant PAP was undertaken with a purified PAP (initially named PvPAPI ; in this work named PvPAPI; see chapter 7.1) from red kidney bean (Beck et al., 1986). This protein was later characterized with respect to its secondary structure and amino acid and metal composition (Cashikar and Rao, 1995). PvPAPI was found to be a dimeric glycoprotein with a molecular mass of 110 kDa, thus belonging to a new class of high molecular weight PAPs. Its primary structure and the structure of its oligosaccharides were also reported (Klabunde et al., 1994; Stahl et al., 1994). The first crystal structure of a plant PAP was published shortly thereafter (Strater et al., 1995) and led to a better understanding of the structure and functioning of the active site.

The characterisation of plant genes encoding PAPs has only been reported recently. Nakazato et al. (1997) reported the cloning of a cDNA encoding a putative glycosylphosphatidylinositol (GPI) anchored phosphatase from Spirodela oligorrhiza. One year later, it was reported that this phosphatase is a member of the PAP family of phosphatases (Nakazato et al., 1998) and it was recently shown by immunohistochemical staining to be preferentially distributed in the cell walls of the outermost cortical cells but not

2.2 Organic phosphates and phosphatases 2 Literature review 12

at the epidermis. Furthermore, PAP was released by digestion of the cell wall fraction with cellulases, indicating that in fact it is GPI anchored in the plasma membrane and positioned to the cell wall (Figure 2.5), thus possibly having a role in acquiring Pi from Po close to the cell surface. Since then, a moderate number of cDNA clones encoding PAPs have been described. Durmus (1999) published the sequences of cDNA fragments of two PAP isozymes of sweet potato. The encoded amino acid sequences of these two isozymes showed a similarity of 72-77% not only to each other, but also to the primary structure of the purple acid phosphatase from red kidney bean (PvPAPI). The biochemical and biophysical characterisation of three PAPs from soybean and sweet potato revealed that they had >66% sequence identity with the previously characterized PvPAPI, and all of the metal ligands were conserved (Schenk et al., 1999). Moreover, the metals involved in the active site were, in contrast to the characteristic Fe-Fe pattern found previously in mammals, Fe-Mn in the sweet potato enzyme and Fe-Zn in soybean. This report was the first to unambiguously demonstrate the involvement of Mn in the active site of an enzyme.

Figure 2.5 Simplified model of the addition of the GPI lipid to the protein by a transamidation reaction mechanism The process takes place within the lumen of the endoplasmatic reticulum (Model redrawn from diverse sources)

A more specific gene expression and functional analysis, respectively, of a plant PAP was reported for AtACP5 (AtPAPU), a gene induced by P starvation and by some other types of

P mobilizing/oxidative stresses in Arabidopsis (del Pozo et al., 1999). This gene encodes a secretory protein homologous to the mammalian LMW PAPs. Promoter-GUS analysis revealed transcription activation in older leaves and in roots upon P deprivation. Treatment with abscisic acid (ABA) and hydrogen peroxide (H202) also induced gene expression, mainly in leaves. Wasaki et al. (2000) reported the cloning and characterisation of two HMW PAPs from white lupin (LaPAPI and LaPAP2). LaPAP2 is expressed in roots under P

2.2 Organic phosphates and phosphatases 2 Literature review 13

starvation and contains a predicted secretory signal sequence. Both proteins were suspected to play a role in P mobilization from organic P sources in the rhizosphere. Miller et al. (2001) reported the cloning of another HMW PAP from white lupin which is preferentially expressed in proteoid roots under P starvation. The promoter sequence revealed a 50 bp region showing 72% identity to the promoter of AtPAPU. The cloning of a novel phytase from soybean with sequence similarity to PAPs was reported by Hegeman and Grabau (2001).

This enzyme exhibited a high affinity for phytic acid and had low pH optimum, indicating that it could be a vacuolar protein. Very recently, Li et al. (2002) published a list of 29 putative

PAPs of Arabidopsis thaliana identified in several sequence databases. Semi-quantitative

RT-PCR of fragments from 7 genes done on RNA extracted from suspension cell cultures showed differential transcriptional responses to P deprivation. Only one PAP clone was shown to be strongly inducible by P starvation (AtPAPU), a second appeared to be moderately induced (AtPAP12), while the remaining clones more or less did not respond to P nutrient stress (AtPAPl, AtPAPS, AtPAP9, AtPAPIO and AtPAP13). None of the 29 genes was characterized on a whole plant level, except for one previously reported cDNA clone (AtPAPU; del Pozo etal., 1999).

Possible functions of PAPs

The biological roles of PAPs are still unclear. Mammalian PAPs have been implicated in iron transport (Nuttleman and Roberts, 1990) and in bone resorption because of the high level of expression of such a gene in bone-resorbing osteoclasts (Hayman and Cox, 1994). Plant PAPs are thought to play a role in P mobilization or scavenging. In fact, five plant PAPs have been implicated in the P-starvation responses of Arabidopsis thaliana, Lupinus albus and Spirodela oligorrhiza (Nakazato et al., 1998; del Pozo et al., 1999; Haran et al., 2000). In microorganisms, PAPs are found only in a restricted number of organisms (myco- and cyanobacteria). Schenk et al. (2000a) suggested that PAPs may have a function in survival of eukaryotic parasites in their hosts by inhibiting the respiratory burst of their host and removing reactive oxygen species in a Fenton-type reaction (Sibille et al., 1987). The localisation of GPI-anchored PAPs at the surface of the cell may indicate that some of them could play a role in defense or in parasite recognition by signal transduction through the plasma membrane (Hiscox et al., 2002). PAPs have also been suggested to have intracellular metabolic functions. The functional analysis of plant PAPs using mutants has not yet been described.

In summary, few PAP genes have so far been isolated from plants and their functions in plant metabolism remain to be elucidated. An interesting observation is that there are a number of Arabidopsis mutants that are either defective in P starvation-induced phosphatase secretion, or which secrete phosphatase constitutively (Trull et al., 1998). In the latter case, given the high number of PAP genes identified in Arabidopsis, this finding could indicate that in this mutant either one of the phosphatase genes is overexpressed, or that several secretory phosphatase genes are regulated via a common signaling transduction pathway.

2.2 Organic phosphates and phosphatases 2 Literature review 14

Transcriptional regulation of plant phosphatases

In many respects, the molecular genetics of the yeast Saccharomyces cerevisiae and of higher plants are relatively well conserved. This may hold true for some aspects regarding the P-starvation responses in yeast. As in other microorganisms, Saccharomyces cerevisiae responds to low levels of P in its environment by activating the transcription of a number of genes involved in Pi scavenging. These genes are clustered in what is called the PHO regulon, comprising three genes coding for secretory phosphatases (PH05, PHO10 and PH011), a vacuolar (PH08), and a high-affinity Pi transporter (PH084)

(Yoshida et al., 1989). The expression of these genes is often coordinated in some sort of

"collective emergency response" to Pi deficiency. Although the induction is very strong for PH05 (more than 500-fold increase in level of transcript) and PH084 (60-fold), it is weaker in the case of the alkaline phosphatase (PH08). This differential regulation is in accordance with their putative functions as Pi scavenger, transporter and storage management proteins, respectively. In fact, the mobilization of Pi from Po is probably the most limiting factor in case of P-limitation. The PHO regulon components are transcriptionally regulated by at least five other proteins: PH081 (a cyclin-dependent kinase (CDK) inhibitor), PHO80 (a cyclin), PH085 (a CDK), PH02 and PH04 (both transcription factors; Lenburg and O'Shea, 1996; Oshimaetal., 1996).

It is hypothesized that plants may possess a regulatory network homologous to the yeast PHO regulon (Wykoff et al., 1999; Rubio et al., 2001; Tomscha, 2001). The first argument supporting this hypothesis is that there are a number of Arabidopsis mutants defective in multiple aspects of P deficiency symptoms. One example is the pho3 mutant, which exhibits a number of characteristics normally associated with low-P stress and does not seem to be able to respond to low internal P levels (Zakhleniuk et al., 2001). Characteristics of this mutant include a lack of increase in acid phosphatase activity in response to P deprivation, reduced accumulation of P in roots and shoots when supplied with sufficient amounts of P in growth media, delayed flowering, less shoot biomass when grown in soil, accumulation of starch, lower chlorophyll content when grown in P-sufficient agar media, low fertility, and anthocyanin accumulation. This multiplicity of deficiency in typical P-starvation responses indicates that the pho3 mutant may lack a regulatory component of a putative PHO regulon homolog (Zakhleniuk et al., 2001).

The second argument for the existence of a plant PHO regulon is based on evolutionary considerations. The PHO regulon is relatively well conserved throughout different organisms, from prokaryotes up to fungi, such as yeast and Neurospora crassa. The latter controls expression of phosphatases and Pi transporters with a system similar to the yeast PHO regulon, in addition to controlling the production of vacuolar and secreted ribonucleases.

Recently, mutants of the unicellular green alga Chlamydomonas reinhardtii defective in a number of specific Pi starvation responses were identified (psr, phosphorus starvation response; Shimogawara et al., 1999). psrl is a single recessive mutation that results in the inability to activate transcription of secreted phosphatases and high-affinity Pi transporters in response to P-stress. psr2 is a single dominant mutation resulting in constitutively high expression of secretory phosphatase genes in P-sufficient conditions (Shimogawara et al.,

1999). The cloning of the Psr1 gene responsible for the psrl mutation revealed that it has

2.2 Organic phosphates and phosphatases 2 Literature review 15

sequence characteristics associated with transcriptional activators. However, the Psrl

protein has no sequence homology to yeast PHO regulon proteins. A similar gene (PHR1) was found in Arabidopsis (Rubio et al., 2001). In contrast to PH04 in yeast, both Psrl and PHR1 were shown to be permanently detectable in the nucleus, indicating that

photosynthetic organsims possess a different, and probably more complex, gene regulatory system responding to P-deficiency. However, there appear to be some common pathways

between the yeast PHO regulon and a putative plant PHO regulon in that some genes

induced by low cellular P-concentrations contain a phosphate response domain within their promoter which has homology to the PH04 (Karthikeyan et al., 2002; Rausch and Bucher, 2002). In view of the large number of phosphatase genes found in the

Arabidopsis genome, and the fact that there exist both phosphatase underproducing (pup) and constitutive phosphatase secretion (cps) mutants (Trull et al., 1998), it is possible that

many, if not most, plant secreted phosphatases are transcriptionally regulated via a common signal transduction pathway.

2.2 Organic phosphates and phosphatases 2 Literature review 16

2.3 Inositol phosphates and phytases

2.3.1 Inositol phosphates

Nomenclature of inositol phosphates

Inositol phosphates are a group of phosphate esters of hexahydroxycyclohexane (inositol). A number of stereoisomers exist, including myo-, neo-, scyllo-, and D-c/7/ro-inositol phosphates

(Figure 2.6 (A)). The number of phosphate groups may vary between one and six, indicated by the prefixes mono, bis, tris, tetra/c/'s, penta/c/'s and hexa/c/'s (IUPAC, 1971). The positions of the phosphate groups are given by the position number of the carbon in the inositol ring to which they are attached. For example, lns(1,2,3,5,6)P5 is a D-myo-inositol penta/c/'sphosphate, i.e. with no phosphate moiety at carbon position 4. By convention, the abbreviation "Ins" always refers to myo-inositol with the numbering of the ID-configuration. The most common form of inositol phosphates in nature is myo-lnsP6 (Figure 2.6 (B)) and is referred to by a number of trivial names. "Phytic acid" refers to the free acid form, while

"phytate" is the salt of phytic acid. A more ancient name, "phytin", refers specifically to the Ca-Mg salt of phytic acid, which is the dominant form of inositol phosphate in cereal grains (Wheeler and Ferrel, 1971).

myo - inositol neo - inositol scyllo - inositol 1 D-chiro - inositol

B

H203PO

H203P0^5

lnsP6 (l-D-myo-inositol hexa/a'sphosphate)

Figure 2.6 (A) Some stereoisomers of inositols (B) lnsP6 (1D-myo-mositol hexa/asphosphate, or "phytic acid") in the energetically most favorable configuration

2.3 Inositol phosphates and phytases 2 Literature review 17

Chemistry of inositol phosphates

Phytic acid has 12 ionizable protons. Correspondingly, 12 pKa values will define the charge of lnsP6 at a given pH (Figure 2.7). Due to their high anionic charge, inositol phosphates act as strong ligands. Their cation complexing properties have been extensively studied in the medical and biological fields (Lutrell, 1992; Martin, 1995). The affinity of lnsP6 for polyvalent cations varies with pH. At low pH, phytic acid binds metal ions with the following affinity: Cu(ll) » Zn(ll) = Cd(ll) > Mn(ll) > Mg(ll) > Co(ll) > Ni(ll) (Turner et al., 2002). At pH 6-8, insoluble complexes are formed with ion:lnsP6 ratios of 6:1 for Co, Ni and Cu, while Zn and Ca show ion:lnsP6 binding ratios of 3.5 and 4.8, respectively (Martin and Evans, 1987). The high potential charge density of phytic acid results in its strong adsorption to different materials. For example, phytate binds strongly to iron oxides and competes with inorganic phosphate for binding sites. The addition of Ca2+ increases the adsorption of lnsP6 to these surfaces, and the chemical reduction of iron oxides results in insoluble Fe-lnsP6 (4:1) precipitates (Bowman et al., 1967; De Groot and Golterman, 1993).

pK, pK2 pKj pK*

1.1 1.5 1.5 1.7

pKs pK,; pKz pK#

2.1 2.1 5.7 6.9

pK» pK« pK|j pK,2

7.6 10.0 10.0 12.0

pH

Figure 2.7 pKa data and estimated net charge on myo-lnsP6 over a range of pH values from Costello et al (adapted , 1976)

Phytic acid

Phytic acid is thought to have been discovered as early as 1872 by Wlhelm Pfeffer, who showed that subcellular particles in wheat endosperm contained a calcium/magnesium salt of organic phosphate (cited in: Cosgrove, 1980). Two structures were proposed for phytic acid: in 1908, Neumann proposed a structure based on three cyclic pyrophosphate moieties, while Anderson proposed a structure based on esterification of six hydroxyl groups of myo¬ inositol by orthophosphate moieties (cited in: Cosgrove, 1980). The final proof for the structure of phytic acid was given by Johnson and Tate (1969b) using NMR-spectroscopy, confirming the second hypothesis.

2.3 Inositol phosphates and phytases 2 Literature review 18

Phytic acid and inositol phosphates in plants

Phytic acid accumulates mainly in seeds and pollen, but is also found in tubers and fruits, and is a rare component in leaves (Plaami and Kumpalainen, 1997). The synthesis of phytic acid occurs through different pathways, but basically starts with the synthesis of myo-inositol from D-glucose-6-phosphate. This is the first committed step in the production of inositol- containing compounds. D-glucose-6-phosphate is initially transformed to 1L-myo-inositol-1-

phosphate (lns(1)P) by the enzyme myo-inositol-1-phosphate synthase (INPS; Nelson et al., 1998) and then dephosphorylated to myo-inositol by the myo-inositol-monophosphatase

(IMP) enzyme (Gillaspy et al., 1995). Yoshida et al. (1999) characterized a cDNA encoding an INPS homolog from rice (pRINOI), and showed that corresponding transcripts accumulated in embryos, while they were absent from other plant tissues. In situ hybridisation analysis revealed that pRINOI transcripts temporally and spatially colocalized with the accumulation of lnsP6-containing globoids in the scutellum and aleuron layer. An alternative pathway includes the formation of D-myo-inositol-3-P from D-glucose-6-P, resulting in the synthesis of a number of inositol phosphate compounds and concomitant accumulation of lnsP6 as the final product. The first complete description of the synthetic sequence of lnsP6 in the plant kingdom was suggested by Brearley and Hanke (1996). In Spirodela oligorrhiza, the synthesis pathway leading to lnsP6 proceeded through lns(3)P, lns(3,4)P2, lns(3,4,6)P3, lns(3,4,5,6)P4, lns(1,3,4,5,6)P5 to lnsP6. This pathway differed in the sequence and forms of inositol phosphates from that reported in the slime mold Dictyostelium discoideum (Van der Kaay et al., 1995).

lnsP6 has a number of functions in plant metabolism, such as providing a P and mineral storage form (Raboy, 1997), a pool in inositol phosphate pathways, as a second messenger ligand (Sasakawa et al., 1995), complexation of multivalent cations and thereby regulation of the levels of inorganic ions (Cosgrove, 1980; Loewus and Murthy, 2000), and DNA double strand break repair (Hanakahi et al., 2000). More recently, lower phosphate esters of myo¬ inositol have been shown to have several functions in cell metabolism and in cellular signalling in mammalian (Streb et al., 1983; Taylor, 1998) and plant cells, respectively (DeWald et al., 2001; Takahashi et al., 2001b; Mueller-Roeber and Pical, 2002). The inositol phosphate-mediated signal transduction pathways in plants are related to phospholipid

metabolism and Ca2+ homeostasis, and ultimately to the response of plants to hyperosmotic stress (DeWald et al., 2001; Takahashi et al., 2001b). Both in plants and animals, subtypes of inositol trisphosphate (lnsP3) receptors are involved in forming intracellular Ca2+ channels that are regulated both by lnsP3 and Ca2+. These channels release Ca2+ from intracellular stores, thus modifying the Ca2+ concentration in different compartments and leading to the activation of precise cellular functions (Lutrell, 1992; Dasgupta et al., 1996). The physiological significance of these channels is not always clear, but they could take part in rapid reaction cascades induced by external stimuli. Inositol phosphates also play an important role in membranes by the formation of inositol-containing lipids such as phosphatidyl-inositol phosphates (mainly Ptdlns(4)P and Ptdlns(4,5)P2; for a review see Mueller-Roeber and Pical, 2002). Inositol is further important by serving as a potential signal promoting Na+ uptake (Nelson et al., 1999). Worth mentioning is also that lnsP3 and

2.3 Inositol phosphates and phytases 2 Literature review 19

phospholipids have been shown to be involved in the short- and long-term responses to gravistimulation (Perera etal., 1999).

The dephosphorylation of phytic acid to lower inositol phosphate intermediates is mediated by phytase (see chapter 2.3.2). In plants, phytases are found mainly in germinating seedlings and serve to mobilize Pi from the phytate stored in the seed (Hegeman and Grabau, 2001).

Since inositol phosphates exhibit such a large array of biological functions, the question arises as to whether and how they might be affected by the presence of phytase in the cytosol. The study of the intra- and extracellular location of acid phosphatases, alkaline phosphatases and phytase in six different fungi revealed that only 25% of the total acid phosphatase and 22% of the total alkaline phosphatase were secreted, while more than 97% of the total phytase was released into the extracellular space (Tarafdar et al., 2002). Although few plant phytases have been cloned to date, those that have been characterized frequently were found to possess signal sequences for targeting to the endoplasmatic reticulum (Maugenest et al., 1997; Hegeman and Grabau, 2001). It is possible that compartmentation or secretion of phytase proteins avoids their interactions with inositol phosphates in the cytosol. Interestingly, no particular phenotypic changes have been reported for Arabidopsis plants constitutively expressing an Aspergillus niger phytase gene without secretory signal sequence (Richardson et al., 2001a). Brinch-Pedersen et al. (2000) observed that wheat transformants with constitutive heterologous expression of an Aspergillus phytase gene without secretory signal sequence exhibited a highly complex integration pattern of the transgene as compared to the lines transformed with the phytase gene including a signal sequence. In addition, phytase activity levels were eight times lower in these lines. These findings may indicate that cytosolic accumulation of phytase may have an impact on cellular metabolism affecting normal growth. In contrast, no anomalies were noted for soybean (Denbow et al., 1998), canola (Zhang et al., 2000b) or Arabidopsis (Richardson et al., 2001a) expressing a secretory Aspergillus phytase.

In summary, inositol phosphates have been described to exhibit numerous functions in plant metabolism. Quantitatively, myo-lnsP6 is the most prevalent form of inositol phosphates in the plant kingdom. Its accumulation in seeds is of particular ecological and pedological relevance, since seeds are easily transported throughout the terrestrial ecosystem by animals and natural processes like wind, resulting in a widespread occurrence of lnsP6 in the terrestrial ecosystem and ultimately in soils.

The inositol phosphate cycle in the terrestrial ecosystem

Inositol phosphates are synthesized in plants as a storage form of P and represent between

50% and 86% of total P in seeds (Lolas et al., 1976), almost exclusively as the myo- stereoisomer of lnsP6. Kasim and Edwards (1998) reported that myo-lnsP6 often comprises 100% of the total inositol phosphates detectable in most cereals, legumes and oil seeds. Phytate is generally degraded during germination (Centeno et al., 2001) or follows different pathways down to the soil either via decomposition of ungerminated seeds, or through the digestive tract of animals, where, particularly in the case of monogastric animals, it remains

2.3 Inositol phosphates and phytases 2 Literature review 20

undegraded and is excreted (Poulsen, 2000). Manure and sewage sludge are thus potentially important sources of phytate in intensive agro-ecosystems, containing large amounts of orthophosphate monoesters (Turner et al., 2002).

Although the soil inputs from plant and animal wastes are principally in form of myo-lnsP6, all possible forms of inositol-P have been detected in soils (Minear et al., 1988) including other types of stereoisomers generally not found elsewhere in nature. These are present mostly in the order myo > scyllo > D-chiro > neo, with myo-lnsP6 representing up to 90% of total lnsP6. Scy//o-lnsP6 (up to 20-50%) and D-c/7/'ro-lnsP6 (up to 10%) are the other forms present in significant amounts (McKercher and Anderson, 1968).

1000 -j

900 -

4p 800 -

- q. 700

- a 6oo

û- 500 -

£ 400 -

| 300 -

200 -

100 -

0 -

10 20 30 40 50 60 70

P in final solution (ug P ml1)

Figure 2.8 Sorption of organic phosphates on a clayey soil (Adapted from McKercher and Anderson, 1989)

A major proportion (60-90% in alkaline soil extracts) of identifiable organic P exists as monoester-P (Condron et al., 1990; CadeMenun and Preston, 1996). Since inositol phosphates often comprise more than 60% of the soil orthophosphate monoester-P fraction (Dalai and Hallsworth, 1977; Harrison, 1987), they represent a major component of the organic P fraction. However, the total amount of phytate and its contribution to the organic P vary greatly between soils (Turner et al., 2002). The composition of organic P inputs to the soil from fresh plant material and animal wastes does not reflect the proportions of different organic P forms found in soils (Magid et al., 1996). In fact, inositol-P accumulates in soils, while other organic P sources, in particular diester-P forms, are rapidly degraded. This may be explained by a differential stabilization of organic P compounds in the soil. Since adsorption of organic P compounds is principally determined by the number of phosphate groups, sugar phosphates and phosphodiesters are only weakly adsorbed and remain prone to degradation by soil enzymes, while lnsP6 strongly interacts with soil particles, resulting in preferential stabilization, preventing hydrolysis by phytases and phosphatases (Greaves et

2.3 Inositol phosphates and phytases 2 Literature review 21

al., 1963; McKercher and Anderson, 1989; Celi et al., 2001; see figure 2.8). The concentrations of inositol-phosphates in soils thus depend on the interplay between enzymatic activity and adsorption processes. The latter are controlled by a number of factors including Po content, total P content, pH, total organic carbon, and total nitrogen (Turner et al., 2002). These factors, finally, will affect the biological availability of lnsP6 in soils.

The accumulation of inositol phosphates in soils suggests that they are relatively unavailable for biological uptake. In fact, although a number of plants have been shown to secrete phytases (Li et al., 1997c; for phytases, see also chapter 2.3.2), and despite the high levels of phytase activity detected in many microorganisms (Chen, 1998; Richardson and Hayes,

2000; Tarafdar et al., 2002), few plants have been shown to possess the ability to mobilize P from phytate in P-limited environments. There is evidence for P-uptake from lnsP6 in a Carex species (Corona et al., 1996) and in the arctic tundra plant Eriophorum vaginatum (Kroehler and Linkins, 1991). Adams and Pate (1992) reported that in sand culture, phytate was at least equal to KH2P04 as a source of P for the growth of lupins, but a much poorer source in soils, where RNA and glycerophosphates were more readily taken up by lupin plants. They concluded that the difference in availability of different P sources is related to their solubility in soils rather then their susceptibility to degradation by phosphatases and phytases. Similarly, Jackman and Black (1952) showed that increasing the phytase activity of soils did not increase the level of extractable Pi. The addition of soluble phytate to the soil, however, increased the level of soil extractable Pi. These findings indicate that the availability of lnsP6 is a crucial factor influencing its degradation by phytase in soils. It appears from the work of Martin and Cartwright (1971) that the P fixing capacity of a soil strongly affects the biological availability of lnsP6. In fact, lnsP6 added to soils was more available to plants in soils with low P-adsorbing capacity than in strongly P-adsorbing soils. On the other hand, a number of authors have pointed out that in some cases, the utilization of lnsP6 is clearly limited by the quantity of phytase present in the soil solution, both of plant and microbial origin (Findenegg and Nelemans, 1993; Hayes et al., 2000b).

The contribution of microorganisms to the mobilization of P from soil phytates has been demonstrated by Richardson et al. (2001b), Greenwood and Lewis (1977) and Kim et al. (2002). However, it is not clear which mechanisms determine the availability of lnsP6 as a substrate for microbial phytase enzymes in the soil solution. In fact, plant growth on a soil inoculated with a high phytase-secreting Pseudomonas species was lower than when grown in the same soil to which a species-rich mix of soil microorganisms had been added (Richardson et al., 2001b). This finding confirms the work by Greaves and Webley (1965) who reported that, although 30-50% of soil bacterial isolates were able to secrete phytases, their ability to access lnsP6 in the presence of soil was extremely limited. Ectomycorrhizal fungi may play an important role in P-mobilization from soil phytate. Leake (2002) reported that phytase activity was detected in all 20 species of ectomycorrhizal fungi so far tested. However, the role of the ectomycorrhizal associations in improving the P-acquisition efficiency of plants from lnsP6 sources is not clear. Antibus et al. (1992) showed that the mycelium of four basidiomycete species could hydrolyse and take up 32P from radio-labelled, insoluble lnsP6. Monoester-P forms, of which phytate is the major component, were reduced in coniferous plantations grown on pasture soils as compared to the pasture control and

2.3 Inositol phosphates and phytases 2 Literature review 22

these activities were attributed to ectomycorrhizal fungi (Antibus et al., 1992). For AM fungi, no such information could be found in the literature.

myo-inositol hexa/t/sphosphate

Figure 2.9 Model configuration of the myoinositol hexa/asphosphate-Goethite complex Modified from Ognalaga et al (1994)

The current knowledge of the complex interplay of bacterial, fungal and root activities in the rhizosphere with respect to P-mobilization from lnsP6 is still very limited. Whether, and to which extent, substrate availability or enzyme activity, respectively, govern these processes and how these processes are influenced by the different soil factors still have to be clarified.

Root-induced changes in the rhizosphere, such as secretion of organic acids or protons, may significantly modify the adsorption/desorption properties of lnsP6, creating new opportunities for enzymes to access the substrate. As a matter of fact, protons and organic acids such as citric acid have been shown to play an important role in the solubilization of inorganic P minerals and phosphate rock in a number of cases (Hedley et al., 1982; Hinsinger, 2001). Hayes et al. (2000a) showed that only a small fraction of soil Po extracted by water and NaHC03 was hydrolyzable by phytase, while the addition of citric acid resulted in the solubilization of a greater amount of enzyme-labile Po. One may also ask the question as to whether phytosiderophores and other chelating molecules can induce desorption of lnsP6. Although there is no direct evidence for this hypothesis in the literature, some data suggests that they could play a role in P mobilization (Shen et al., 2001). The lowering of soil pH by active secretion of H+ from the roots, in contrast, is rather expected to decrease the solubility of lnsP6 owing to precipitation reactions with Fe and Al-oxides (Jackman and Black, 1951).

Furthermore, the binding of lnsP6 to clays is dependent on the presence and type of cations present in the soil solution and on soil pH (Celi et al., 2001). In fact, P sorption was more strongly increased by addition of Ca2+ than of K+ to the solution, especially at higher pH values. Ognalaga et al. (1994) showed that lnsP6 was adsorbed by ligand exchange to the

2.3 Inositol phosphates and phytases 2 Literature review 23

surface hydroxyls of Goethite and proposed a tentative configuration for the organophosphate-Goethite complex (Figure 2.9).

Further research is thus required to better characterize and understand the processes revolving around inositol phosphates in the soil and their availability for plant nutrition.

2.3.2 Phytases

A first reference to phytase in the literature dates from 1907 (Suzuki et al., 1907) and describes phytase as an enzyme capable of lowering phytic acid content in rice-bran. This first report opened the way to a large number of industrial applications for this enzyme, particularly in animal nutrition. In fact, phytic acid is the major storage form of P in plant seeds, as 60-90% of all organically bound P is found in this form (Plaami and Kumpalainen, 1997). In foods and feeds, phytic acid reduces the availability of inositol, phosphorus, and essential minerals by forming non-assimilable salt complexes.

OP03H2 3-phytase (EC 3.1.3.8) H203PO

H203PO OP03H2 H203PO

OP03H2 H203PO 1-D-myo-mositol 1,2,4,5,6-penta/flsphosphate

H203PO

H203PO 1 OP03H2 H203PO OP03H2 H,0,PO 1 -D-myo-inositol hexatosphosphate

H203PO 3 OP03H2 H203PO

6-phytase 1-L-myo-mositol 1,2,3,4,5-pentatosphosphate (EC 3.1.3.26) (identical to 1-D-myo-mositol 1,2,3,5,6-pentatosphosphate)

Figure 2.10 Dephosphorylation of myo-lnsP6 by 3-phytase and 6-phytase Carbon position 4 in the D-configuration corresponds to position 6 in the L-configuration Modified from Dvorakova (1998)

2.3 Inositol phosphates and phytases 2 Literature review 24

Classification and properties of phytases

Phytase (myo-inositol hexa/c/'sphosphate phosphohydrolase) belongs to a group of phosphoric monoester catalyzing the hydrolysis of myo-inositol hexa/c/'sphosphate to inorganic phosphate and lower phosphoric esters of myo-inositol. One classification distinguishes between purple acid phosphatase, alkaline phosphatase, high- and low- molecular-weight acid phosphatase and (Vincent et al., 1992). Phytases belong to a subfamily of the high-molecular-weight histidine acid phosphatases. Based on the first phosphate group attacked by the enzyme, two types of phytases are distinguished by the Enzyme Nomenclature Committee of the International Union of Biochemistry. 3- phytase and 6-phytase. 3-Phytases are mainly associated with microorganisms, while 6- phytases are found predominantly in plants (Cosgrove, 1969; Johnson and Tate, 1969a; Cosgrove, 1970b; see Figure 2.10).

However, other dephosphorylation patterns have been described in the literature. For example, Nakano et al. (2000) established by nuclear magnetic resonance spectrometry that the dephosphorylation of lnsP6 by phytases from wheat bran followed two alternative pathways (see Figure 2.11). Greiner et al. (2002) confirmed one of the two dephosphorylation pathways for phytases from legume seeds using high performance ion chromatography (HPIC) analysis and kinetic studies. The dephosphorylation of lnsP6 by a

phytase from Paramecium sp., however, appeared to occur in the sequence 6/5/4/1 (Van der Kaay and Van Haastert, 1995).

Many naturally occurring phytases are thermostable up to 70 °C (Wyss et al., 1998; Wyss et al., 1999b; Lehmann et al., 2000b; Simon and Igbasan, 2002). With respect to their pH activity profiles, fungal phytases typically exhibit enzymatic activity within the range pH 2.5 to pH 8.0 (Wyss et al., 1999a; Simon and Igbasan, 2002). The proteolytic stability of phytases varies widely and strongly depends on the type of protease. For example, Aspergillus niger phytase was found to be more resistant against pepsin than wheat phytase (Phillippy, 1999), and E.coli AppA phytase exhibited even higher stability than A. niger phytase in the presence of the same protease (Rodriguez et al., 1999; Golovan et al., 2000). A Bacillus phytase was found to be extremely resistant to proteolytic degradation by papain, pancreatin and trypsin, but appeared to be highly susceptible to pepsin (Kerovuo et al., 2000).

Plant phytases have not been characterised as extensively as microbial phytases. Of importance to this work are root-secreted and soil-borne phytases.

2.3 Inositol phosphates and phytases 2 Literature review 25

Ins Pe

I

Ins (1,2,5,6) P4 Ins (1,2,3,5,6) P5 Ins (1,2,3,6) P4

I I

/ Ins (1,2,3) P3

Figure 2.11 Proposed dephosphorylation pathway of phytic acid by phytases from wheat bran et al (Nakano , 2000)

2.3 Inositol phosphates and phytases 2 Literature review 26

Root-secreted and soil microbial phytases

Early in the 1950's, soil scientists started to investigate the role of the soil microbial community in the dephosphorylation of soil-bound lnsP6. Jackman and Black (1952) reported the presence of phytase activity in soils and correlated it to the occurrence of microorganisms. In the same year, Courtois and Manet (1952) showed that some bacilli are efficient in mineralizing inositol phosphates. Later on, Greaves et al. (1963) demonstrated that a significant proportion of soil and root-surface microorganisms secreted enzymes capable of P-hydrolysis from Na-phytate. Saxena (1964) published a first detailed study of the role of plants in the overall inositol-phosphate mineralizing process, both in the presence and absence of microorganisms. Since that time, a large number of papers have been published showing that although some plants do secrete certain amounts of phytases, the importance of these enzymes for the release of Pi from lnsP6 remains unclear.

More recently, Li et al. (1997c) measured phytase activity in root exudates of several plant species under P deficiency, while Asmar (1997) reported different levels of phytase secretion from barley roots between different genotypes. A number of authors reported on phytase activity in crude protein extracts from roots (Li et al., 1997b; Hayes et al., 1999). The relevance of phytase secretion in terms of mobilization of P from soil-bound phytate was not assessed in either of these experiments. It is generally assumed that plants have low ability to acquire P from phytate. In fact, although some authors have published that phytate could be used as a P source by plants (Islam et al., 1979; Tarafdar and Claassen, 1988), others have shown that this ability is, at most, restricted (Findenegg and Nelemans, 1993; Richardson et al., 2000). Richardson et al. (2000) proved in experiments conducted in sterile conditions that wheat plants were able to take up P from a variety of organic monoester-P compounds but were largely unable to use phytate as a source of P. At the same time, Hayes et al. (2000a) examined different soil Po substances as substrates for hydrolysis by acid phosphatases and phytases. Phytases with narrow substrate specificity and high specificity against phytate could hydrolyse only a limited proportion of Po extracted both with NaHC03 and citric acid, while phosphomonoesterases with broader substrate specificity were able to hydrolyze up to 79% of extracted Po. It thus appears that plants synthesize and secrete phytases in modest amounts under P-starvation conditions, and that these phytases are unlikely to play a significant role in the hydrolysis of soil phytate. On the other hand, soil microorganisms, which are known to secrete large amounts of phytase, may have a function in this respect. In fact, Richardson et al. (2001b) demonstrated that plant growth was significantly improved in the presence of soil microorganisms effective in phytase secretion.

However, the response to inoculation was dependent on the source of microorganisms and the growth medium. Cultured populations of soil microorganisms were more effective in releasing P from lnsP6 than inoculation with a single isolate of Pseudomonas sp. selected for its efficiency in P hydrolysis from phytate. The ability of phytases to mobilize P from phytates in soils thus seems to be additionally related to other factors that may act in changing substrate availability and affinity, as well as adsorption of released P.

2.3 Inositol phosphates and phytases 2 Literature review 27

Industrial applications of phytase

The use of phytase in animal feeding began in the 1960's, as one became aware that monogastric animals were unable to digest phytic acid, resulting in a reduced uptake of P (Nelson, 1967). The treatment of soybean meals with a mold phytase improved the uptake of P from this diet (Nelson et al., 1968). The search for microorganisms producing phytases resulted in the identification of a number of species particularly efficient in phytase synthesis. Shieh and Ware (1968) tested over 2000 microorganisms isolated from soil and could isolate only 30 secreting significant amounts of phytase. All phytase producing strains were filamentous fungi, of which the genus Aspergillus accounted for 28, the other two belonging to the genera Pénicillium and Mucor. Since then, a number of other organisms have been shown to produce phytase, including Escherichia coli (Greiner et al., 1993), Pseudomonas sp. (Cosgrove, 1970a; Richardson and Hadobas, 1997), Klebsiella terrigena (Greiner et al., 1997), Bacillus subtilis (Kerovuo et al., 1998; Kim et al., 1998), anaerobic ruminai bacteria (Yanke et al., 1998), Talaromyces thermophilus (Pasamontes et al., 1997b), and a few other fungal strains (Wyss et al., 1998; Igbasan et al., 2000; Lassen et al., 2001).

The use of phytase as a feed additive is now widely spread and has been shown to improve the availability of P in pigs (Jongbloed et al., 1992; Cromwell et al., 1993; Lei et al., 1993; Simoes Nunes and Guggenbuhl, 1998) and broilers (Simons et al., 1990; Perney et al., 1993; Broz et al., 1994; Denbow et al., 1995). As phytic acid is known as an anti-nutrient, especially in the case of Fe, Ca, Zn, Cu, Mn (Cheryan, 1980; Simpson and Wise, 1990; Lonnerdal, 2002), the addition of phytase to diets has been shown to improve uptake of these elements (Lei et al., 1993; Rimbach et al., 1995; Sebastian et al., 1996; Stahl et al., 1999). More recently, transgenic mice and pigs expressing an E. coli phytase in their salivary glands were able to use phytate as a source of P, resulting in a decreased excretion of P by up to 70% (Golovan et al., 2001a; Golovan et al., 2001b). The development of transgenic plants expressing fungal phytase genes for human and animal nutrition has also been pursued (Brinch-Pedersen et al., 2000; Lucca et al., 2001; Brinch-Pedersen et al., 2002; Lopez et al., 2002; Lucca et al., 2002; Zimmermann and Hurrell, 2002).

An alternative use of phytase has resulted from an increasing need of preparations of various

inositol phosphates and derivatives. The latter can be used as enzyme stabilizers (Siren,

1986), substrates for metabolic investigation, enzyme inhibitors, and as chiral building blocks (Laumen and Ghisalba, 1994).

To be more effective in the digestive tract of animals and humans, and to resist the high temperatures occurring during the pelleting process of feeds (60-90 °C; Simon and Igbasan, 2002), phytases need to exhibit particular properties like high thermo- and protease stability, as well as low pH optimum. For this reason, the development of new, engineered phytases has become a high priority. The next paragraph will summarize attempts to improve phytases for applications in animal feeding, resulting in the development of so-called consensus phytases, of which one has been used in the frame of this thesis.

2.3 Inositol phosphates and phytases 2 Literature review 28

Development of the consensus phytases at Roche Vitamins Ltd.

The development of the so-called consensus phytase proteins was preceded by the study of fungal phytases (Pasamontes et al., 1997a). A genomic library of Aspergillus fumigatus was screened for the phytase gene using a PCR fragment amplified with degenerate primers derived from conserved sequences of A. niger phytase and other histidine acid

phosphatases. The purified A. fumigatus phytase recovered after heat-inactivation up to 100 °C with only a loss of 10% of its activity. During the same period, the thermostability of three histidine acid phosphatases, i.e. A. fumigatus phytase, A. niger phytase, and A. niger optimum pH 2.5 acid phosphatase, were assessed. Of these, A. fumigatus phytase revealed the highest temperature stability

(refolding after exposure to >90 °C; Wyss et al., 1998).

Wyss et al. (1999b) overexpressed six different wild-type fungal phytase genes in either filamentous fungi or yeast and purified the recombinant proteins. All proteins examined were monomeric. Compared to Escherichia coli phytase, which is not glycosylated, all fungal phytases exhibited highly variable patterns of glycosylation. The different extents of glycosylation of single phytases expressed in different organisms did not affect their activity, thermostability or refolding properties. Expressed in A. niger, the phytases were weakly susceptible to proteolysis, and site-directed mutagenesis at the cleavage sites significantly increased the resistance of the proteins towards proteolysis. It was concluded that engineering of surface loops may increase the stability of the phytase protein in the digestive tract of animals.

The biochemical characterisation of wild-type phytases was extended by characterisation of the catalytic properties of the enzymes. At high enzyme concentrations, all of the considered

phytases were able to release five phosphate groups from phytic acid. The final product was generally myo-inositol 2-monophosphate. A combination of phytases was able to release all six phosphates. For some enzymes, the final products that accumulated were myo-inositol tris- and bisphosphates, respectively. The specificity of activity towards phytic acid was high in A. niger, A. terreus, and E. coli phytases, respectively, whereas A. fumigatus, Emericella nidulans, and Myceliophthora thermophila phytases were able to hydrolyse a large spectrum of phosphate compounds (Wyss et al., 1999a).

The study of these different enzymes led to the development of a synthetic, thermostable

phytase based on protein sequence comparison (Lehmann et al., 2000b; Lehmann et al., 2000a). Although A. niger phytase is being successfully used in animal feeding, where it is either added to feed pellets before or after the pelleting process, the need to develop more themostable phytases remains. In fact, feed pelleting reaches temperatures between 60 and 90 °C, partially denaturing phytases added prior to pelleting. To improve thermostability, a consensus enzyme was constructed using primary protein sequence comparisons of 13 fungal phytases. While the enzyme exhibited similar catalytic properties as each of its "parents", there was a 15-22 °C increase in the unfolding temperature. The crystal structure of the purified consensus phytase was determined and compared with known structures (Lehmann et al., 2000b). Most consensus amino acids contributed to the stability of the protein. Some consensus residues, however, predicted by structural comparisons to

2.3 Inositol phosphates and phytases 2 Literature review 29

destabilize the protein, in fact increased the unfolding temperature of the protein. On the whole, protein sequence conservation and stability of proteins were correlated, confirming the consensus concept for protein engineering.

In addition to the requirements in terms of thermostability, phytases used for animal feeding should be active in the low pH range. For this purpose, A. fumigatus and consensus phytases were engineered to exhibit high activity at low pH (Tomschy et al., 2002). In a first attempt, glycinamidylation of the surface carboxy groups lowered the pH optimum but also reduced the phytase activity by 70%. In a second strategy, active site amino acid residues considered to be correlated to a low pH activity profile in other fungal phytases were identified by sequence alignments and by three-dimensional structure analyses. Site-directed mutagenesis of a number of residues in A. fumigatus wild-type phytase gave rise to a second pH optimum at pH 2.8-3.4 (the first being at pH 6.0), while a single mutation in the consensus phytase backbone could decrease the pH optimum with phytic acid as a substrate by approximately 0.5-1 unit.

Recently, six new phytase sequences (additionally to the 13 previously used) were taken into account for designing a new, more thermostable consensus phytase sequence (Lehmann et al., 2002). Thirty eight amino-acid residues from the first consensus sequence were replaced by newly ranked conserved residues at the respective positions in either one or both of the new consensus phytases-10 and -11. It was observed that mutations could yield both stabilizing and destabilizing effects. Single mutations always resulted in changes in unfolding temperatures of smaller than 3 °C. The introduction of all stabilizing amino acid residues in a multiple mutant of consensus phytase-1 resulted in an increase of the unfolding temperature from 78 °C to 88.5 °C. The back-mutation of the four destabilizing amino acids, as well as the introduction of a stabilizing residue in consensus phytase-10 further increased the unfolding temperature from 85.4 °C to 90.4 °C. The improvements observed were the result of the exchange of a specific combination of individual mutations rather than that of dominating mutations alone, or the replacement of all amino acid residues considered with their respective conserved residues.

2.3 Inositol phosphates and phytases 3 Objectives and hypothesis 30

3 \J Objectives of dissertation research

Plants respond to P deficiency by secreting higher amounts of phosphatases from the roots.

The first objective was to identify and study the expression of phosphatase genes in potato to further the knowledge on the regulation of these genes and possible functions associated with them.

• The first hypothesis is that there are genes encoding secretory phosphatases in potato that are expressed in roots under P starvation.

• The second hypothesis is that these genes may be transcriptionally regulated via a common signal transduction pathway.

Based on the fact that plants have a low ability to take up P from phytate in the soil, another objective was to test whether the overexpression of a synthetic, secretory consensus

phytase gene in the root hairs could improve the Pi-acquisition efficiency of potato.

• The first hypothesis is that the expression of a synthetic secretory phytase in root hairs will result in an increased phytase activity in the root exudates.

• The second hypothesis is that under conditions where lnsP6 is present in the soil

solution, plants secreting a phytase protein will possess an advantage over wild-type plants in terms of P acquisition from lnsP6, both under sterile and non-sterile conditions. 4 Materials and methods 31

4 Materials & Methods

4.1 Materials and chemicals

Bacterial strains

Escherichia coli DH5a (GIBCO, Basel, Switzerland) Agrobacterium tumefaciens C58C1 pGV2260, Rif, Ampr (Deblaere et al., 1985)

Plasmid DNA

pBluescript SK (+/-) Ampr (Stratagene Europe, Amsterdam, Netherlands) pBin19 Kanr (Bevan, 1984) pCRScript Ampr (Stratagene Europe, Amsterdam, Netherlands) pUC18 Ampr (Stratagene Europe, Amsterdam, Netherlands)

Enzymes

Restriction enzymes (MBI Fermentas GmbH, Nunningen, Switzerland) T4 DNA (MBI Fermentas GmbH, Nunningen, Switzerland) Taq polymerase (MBI Fermentas GmbH, Nunningen, Switzerland) Pfu polymerase (Stratagene Europe, Amsterdam, Netherlands) SuperScript II, moloney murine leukemia virus reverse transcriptase (GIBCO, Basel, Switzerland) Mung Bean (New England Biolabs)

4.1 Materials and chemicals 4 Materials and methods 32

Oligonucleotides

Oligonucleotides were synthesized at Microsynth GmbH (Balgach, Switzerland) in the desalted grade at a scale of 40 nmol.

Kits

• QIAprep Spin Plasmid Kit (Qiagen, Basel, Switzerland)

• DNA Extraction Kit (MBI Fermentas GmbH, Nunningen, Switzerland)

• Nucléon Phytopure Plant DNA Extraction Kit (Amersham Biosciences, Dübendorf, Switzerland)

• Prime-It II Kit (Stratagene Europe, Amsterdam, Netherlands)

• Megaprime DNA Labelling System (Amersham Biosciences, Dübendorf, Switzerland)

• ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction-Kit (Perkin Elmer, Boston, USA)

• HighPure PCR Product Purification-Kit (Roche Molecular Biochemicals, Mannheim, Germany)

Radiochemicals

(a-32P) dCTP (Hartmann Analytics, Braunschweig, Germany) KH232P04 (ICN Biomedicals, Eschwege, Germany)

General chemicals and materials

Agar, granulated Difco (New Jersey, USA)

Antibiotics (kanamycin, ampicillin, gentamycin, rifampicin) Sigma (Fluka, Buchs, Switzerland) a-naphthyl phosphate Fluka (Buchs, Switzerland)

ß-naphthyl phosphate Fluka (Buchs, Switzerland)

Biomax MS film Kodak (product ordering via Integra Biosciences,

Eschenbach, Switzerland)

BSA (fraction V) Serva (Catalysis, Wallisellen, Switzerland)

Daichin agar Brunschwig Chemie (Basel, Switzerland)

Fast Black K Fluka (Buchs, Switzerland)

Fast Blue BB Fluka (Buchs, Switzerland)

Fast Red TR Serva (Heidelberg, Germany)

Glyoxal 40%; 7.1 M Ultrapure Clontech (Allschwil, Switzerland)

4.1 Materials and chemicals 4 Materials and methods 33

Hybond NX membrane Amersham Biosciences (Dübendorf, Switzerland)

Meat peptone Life Technologies Inc. (Basel, Switzerland)

MicrospinTM S-400 HR columns Amersham Biosciences (Dübendorf, Switzerland)

MS salts Sigma (Fluka, Buchs, Switzerland)

Nitro-cellulose membranes Schleicher & Schuell (Dassel, Germany)

Phytic acid (from corn and rice) Sigma (Fluka, Buchs, Switzerland)

Protease inhibitor cocktail Sigma (Fluka, Buchs, Switzerland)

Timenten Smith Kline Beecham Pharmaceuticals (Thoerishaus,

Switzerland)

Tryptone, yeast extract Difco (New Jersey, USA)

X-Gluc Biosynth AG (Staad, Switzerland)

Zeatin riboside Sigma (Fluka, Buchs, Switzerland)

All other chemicals were purchased from Fluka Chemie AG (Buchs, Switzerland)

Plants

Solanum tuberosum L. var. Désirée

Materials for plant growth

Sterile culture 2MS medium (Murrashige and Skoog, 1962), containing:

- 4.3 g/l MS salts (Sigma, without vitamins)

- 5 ml/l MS vitamins (see below)

- 20 g/l sucrose

- 8 g/l agar (Difco)

medium set to pH 5.8 with 0.1 M KOH and autoclaved

MS vitamins:

0.1 g/l nicotinic acid 0.1 g/l pyridoxine HCl 0.02 g/l thiamine HCl 0.4 g/l glycine 20 g/l myo-inositol

solution was dissolved, filtered through a 0.2 urn sterile filter and stored at -20 °C

Aeroponics An aeroponic system (see figure 4.1) was developed in collaboration with AIRWATECH (Bern, Switzerland).

4.1 Materials and chemicals 4 Materials and methods 34

Plexiglas shield

Temperature and humidity sensor

Temperature

' sensor for root chamber

Root growth chamber

Alternative Electronic outlet control unit

Atomizing disc Air-flow propeller

Nutrient solution reservoir

Control valve Cooling unit Water pump UV sterilizing lamp

Figure 4.1 Schematic representation and photography of the aeroponic system used for the controlled growth of plants and root systems.

Pot growth substrate Contained the following substrates:

• 10% loess subsoil from Frick, Switzerland, containing

550 mg total P / kg (obtained from Dr. Paul Mäder, FIBL, Frick, Switzerland)

• 85% quartz sand 0.7-1.2 mm diameter

• 5% peat (Type P; Einheitserdewerk, Sinntal-Jossa,

Germany) containing 100 mg total P per kg dry matter.

Plant-available P in this substrate was determined to be 8 mg Pi / kg dry soil by extraction with NaHC03 (Olsen et al., 1954).

4.1 Materials and chemicals 4 Materials and methods 35

4.2 Methods

4.2.1 Molecular biology

DNA recombinant methods

All standard DNA manipulation methods were done basically according to Sambrook et al. (1989). Enzymes and kits used are described above.

DNA cloning of StPAPI, StPAP2, and StPAP3

The cDNA clone of StPAPI was isolated by phage screening of a cDNA library generated from RNA originating from P-starved potato roots (Leggewie et al., 1997) using a sequence fragment from the cDNA of AtPAPU (del Pozo et al., 1999) as a probe. A protein sequence based BLAST search in the TIGR potato sequence database (http://www.tigr.org/tdb/tgi/stgi/) revealed the existence of two further PAP expressed sequence tag (EST) clones. The clones EST393242 and EST519948 were ordered at ResGen, Invitrogen Corporation (Carlsbad,

California, USA). The 5'-end sequence of EST393242 was lacking and cloned by the rapid amplification of 5' cDNA ends method (5'-RACE; see below). The full length clones corresponding to EST393242 and EST519948 were subsequently named StPAP2 and StPAP3, respectively.

5'-RACE and PCR amplification

The missing 5'-end of the StPAP2 sequence was cloned using a modified protocol of a 5'- RACE method described by Frohman et al. (1988). First, a reverse transcription-PCR (RT-PCR) was performed on RNA of P starved potato roots with primer A (5'-ttggtagctcttcctcaagcc-3'):

1 ul total RNA (2 ug/ul) 4 ul of 5x first strand buffer

1 ul of 10mM dNTP mix 1 ul of 10 uM primer A 2 ulofO.1 mM DTT H20 to 18 ul total volume

This reaction was heated for 5 minutes at 65 °C and immediately cooled on ice. After addition of 2 ul of reverse transcriptase, samples were incubated at 42 °C for two hours. The resulting cDNA-RNA duplex was subsequently purified using the StrataPrep PCR purification kit (Stratagene Europe, Amsterdam, Netherlands). Second, an adaptor was created by heating to 95 °C and slow cooling to room temperature of both complementary sequences 5'-gaccacgcgtatcgatgtcgacttttttttttttttttv-3' and 5'-

4.2 Methods 4 Materials and methods 36

gtcgacatcgatacgcgtggtc-3' (Primer B). The adaptor was ligated to the cDNA-RNA duplex strands by incubation of the following mixture overnight at 16 °C:

36 ul cDNA-RNA duplex 4 ul adaptor (20 pmol/ ul) 5 ul 10x ligation buffer (Fermentas) 5 ul T4 DNA ligase HC (Fermentas)

A first PCR was performed using primers A and B and 3 ul of the cDNA-mRNA duplex - anchor ligation solution. Two sequential nested PCR amplifications were performed with primer B in combination with primers C (5'-gtattgaggagtgtatttgccatatgc-3') and D (5'- ctcgcttgattgaataccaaagtgg-3').

DNA cloning of the consensus phytase construct

A gene expression cassette (named LeExt1.1:. SP/PHY-OCS, see figure 4.2) was constructed using the root hair-specific promoter LeExtl.1 from tomato (Bucher et al., 2002), a secretory signal peptide sequence (SP) from ß-1,3 glucanase from barley (Leah et al.,

1991) and a gene encoding a thermostable consensus phytase (PHY) from Roche Vitamins

Ltd, Basel (Lehmann et al., 2000b). The PHY gene was synthesized using the maize codon usage and was based on Consensus-Phytase-1 with four point mutations (Q24T, E32A, R265I, G378A, with reference to the start methionine of the PHY gene). This numbering is shifted by 3 amino acids as compared to the standard numbering used by Roche Vitamins Ltd. In their system, the mutations would be numbered Q27T, E35A, R268I and G381A.

pBin19-LeExt1.1-SP/PHY-OCS

[Sal l/Ncol] Kpnl

' ' Xbal I Smal Smal Xbal Neo I Smal Smal Pstl

Figure 4.2 Genetic construct used for the root hair-targeted expression of a secretory phytase in potato roots

The LeExtl.1 promoter was cloned from a pBin19 vector (Bucher et al., 2002) into pBluescript KS (-) (Stratagene, Amsterdam, The Netherlands) containing the gene terminator sequence from octopine synthase (OCS). An additional ATG site at the 3' end of LeExtl.1 was removed using Mung Bean Nuclease (New England Biolabs). Suitable restriction sites were introduced into the signal sequence and the phytase gene via PCR according to standard procedures (Sambrook et al, 1989), and both sequences were fused in frame and cloned downstream of LeExtl. 1. Between the predicted cleavage site of SP and the start methionine of PHY there are four additional amino acids (IGVS), and the first amino acid

4.2 Methods 4 Materials and methods 37

after the start methionine of PHY was mutated from S to G (see appendix 4). This construct was sequenced and inserted into the binary vector Bin19 for plant transformation.

Sequencing

DNA sequencing was performed on DNA extracted from E. coli cultures with the QIAprep Spin Plasmid Kit (Qiagen, Basel, Switzerland) and purified by NaAc-EtOH precipitation (Sambrook et al., 1989). The sequencing PCR reaction was done with the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction-Kit (Perkin Elmer, Boston, USA) and samples were sent to Microsynth (Balgach, Switzerland) for sequence analysis.

RNA isolation and gene expression analysis

RNA extraction from potato (Verwoerd et al., 1995) and RNA gel blot analysis were performed as previously described (Bûcher et al., 1997). Radioactively labelled cDNA fragments of roughly 600-900 bp were used (PHY: BamHI fragment at 3'-end; StPAPI: Ncol- Ncol fragment; StPAP2: Xhol-Ncol fragment including 3' non-translated region; StPAP3: Ncol-Smal fragment). Hybridisation was performed with 5 X SSC, 5 X Denhardt's and 0.5% SDS (w/v) at 65 °C with a final washing using 0.1 X SSC and 0.1% SDS at 55 °C.

4.2.2 Physiological and biochemical measurements

ß-Glucuronidase (GUS) assay

Plant roots from either root tip cultures, hairy root cultures, or from aeroponically grown plants were stained for ß-glucuronidase activity by vacuum-infiltration for 2x1 min in GUS- staining solution and incubation at 37 °C for 30 min to 4 h.

GUS staining solution:

0.1 g X-Gluc dissolved in DMF 1ml 10% Triton X-100 5 ml 1 M sodium phosphate buffer, pH 7.2 mixed in a final volume of 100 ml and stored in aliquots at -20 °C

Microscopy

Root hair length was measured by digital analysis of root photographs obtained from the stereo-microscope (Olympus SZX12, Olympus Optical Co., Tokyo, Japan) using SIS image analysis software (Soft Imaging System, Munster, Germany). Tissue sections were photographed at 50x to 200x magnification under a compound microscope Olympus Provis AX70 (Olympus Optical Co., Tokyo, Japan).

4.2 Methods 4 Materials and methods 38

Protein extraction from roots and determination of protein concentration

Crude protein extracts from roots were prepared as described by Aarts et al. (1991) using an extraction buffer containing 100 mM Tris-acetate at pH 7.9, 100 mM potassium-acetate, 10% (v/v) glycerol, 2 mM EDTA, 0.1 mM PMSF, 5 mM DTT and 250 mM sodium-ascorbate. Three ml extraction buffer were used for protein extraction from 1 g root fresh weight. Protein concentration in root extracts was measured basically according to Bradford (1976) relative to standard solutions of bovine serum albumin (BSA). One volume of protein extract (10-20 ul, usually around 20 ug) was diluted into 39 volumes of water and 10 volumes of Bradford reagent (BioRad Laboratories, Inc.). After 10 min incubation, optical density was measured spectrophotometrically by absorbance at 595 nm.

Enzymatic activities in crude protein extracts

Total Phosphomonoesterase (PMEase) activities were determined by incubating 20 ug crude protein extract in 25 mM MES buffer at pH 5.5 containing 10 mM p-nitrophenyl phosphate (pNPP), 5 mM cysteine and 1 mM EDTA for 20 min at 37 °C. The reaction was stopped by addition of a half sample volume of 0.5 M NaOH. The activity was calculated from the production of p-nitrophenol as determined by spectrophotometry at 410 nm relative to standard solutions. For phytase activity measurements, crude protein extracts were diluted to a final protein concentration of 0.1 ug/ul. Two ug of protein extract was mixed with 49 volumes of 25 mM MES buffer at pH 5.5 containing 2 mM lnsP6 (Sigma, Cat. No. P8810), 1 mM EDTA, and 5 mM cysteine. Phytase activity was calculated from the release of phosphate (Pi) as determined spectrophotometrically at 610 nm relative to standard solutions using the malachite green method (Ohno and Zibilske, 1991) and after subtraction of concentrations of contaminant Pi in reaction solutions and protein extract. Enzyme activities were calculated as mU per unit protein content, where 1 unit (U) releases 1 umol Pi / min under the conditions described above.

Collection of root exudates and measurement of enzymatic activities

To determine enzymatic activities in root exudates, rooted potato cuttings were grown aeroponically for two weeks, carefully removed from the aeroponic system and rinsed three times in deionized water. Roots were then incubated in a buffer (exudation solution) containing 5 mM maleate buffer, pH 5.5, 2% sucrose, 2 mM CaCI2 and 0.01% (v/v) protease inhibitor cocktail (SIGMA, Cat. No. P9599). Plants were illuminated with cool white fluorescent tubes with a mean photon flux density of 110 umol m"2 s"1 at canopy level. Exudates were collected 90 min after start of incubation and assayed for PMEase and phytase activity. For PMEase activity, exudation solution was mixed with an equal volume of 100 mM MES buffer at pH 5.5 containing 10 mM pNPP, 5 mM cysteine and 1 mM EDTA, and incubated for 60 min at 37 °C. The reaction was stopped by addition of half a volume of 0.5 M NaOH and the activity calculated as descibed above. For phytase activity measurements, exudation solution was mixed with 250 mM MES buffer (9:1, v/v), pH 5.5, containing 5 mM lnsP6 (Sigma P8810), 1 mM EDTA, 5 mM cysteine, and incubated for 60 min at 37 °C. The reaction was stopped with half a volume of 15% trichloroacetic acid (TCA). Phytase activity

4.2 Methods 4 Materials and methods 39

was calculated from the release of inorganic phosphate as described above (Ohno and

Zibilske, 1991). Enzyme activities were calculated as mU per unit root fresh weight or per root tip, where 1 unit (U) releases 1 umol Pi / min under the conditions described above.

Visual staining for Phosphomonoesterase activity

Visual staining for PMEase activity on roots was adapted from the description given by Dinkelaker and Marschner (1992). The staining solution was a 50 mM Tri-Sodium-Citrate (TSC) buffer adjusted to pH 5.5, containing 37.5 mM a-naphthyl phosphate and 2.7 mM Fast Red TR. To obtain darker colors, Blue B was added at equal amounts (w/w) to Fast Red TR to the staining solution.

Native Polyacrylamide Gel Electrophoresis (PAGE) of a thermostable phytase

Native PAGE was performed basically as described (Bucher et al., 1994). Eight ul of loading buffer was mixed to an aliquot of protein extract containing 25 ug of total protein. Because the recombinant phytase was thermotolerant, but endogenous phosphatases were not, samples were heated for 10 minutes at 70 °C prior to loading to the gel. Gel electrophoresis was performed during 2 h in the BioRad Mini-Protean II apparatus cooled on ice and set to 150 V and 80 mA (for two gels). The gel was stained for PMEase activity during 2 h in a 50 mM TSC buffer containing 0.5 mg/ml Fast Black K and 0.3 mg/ml ß-naphthyl phosphate.

HPLC analysis of the time course of accumulation of phytic acid degradation intermediates

23000

18000 lnsP3 lnsP5

13000 =; lnsP4

TO lnsP6 c •2> 8000 CO

3000

-2000 123456789

Time [min]

Figure 4.3 Example of HPLC analysis of inositol phosphates using the method described by Egh (2001)

Roots of plants grown aeroponically for two weeks were washed in deionized water and incubated in 50 ml of 5 mM maleate buffer, pH 5.5, containing 2 mM CaCI2, 0.01% protease inhibitor cocktail (Sigma, P9599) and 2 mM lnsP6 from rice (Sigma, cat. no. P3168). Every hour after beginning of incubation, 200 ul and 20 ul samples were collected for PMEase and

4.2 Methods 4 Materials and methods 40

phytase activity measurements, respectively. In addition, 1 ml was sampled every hour for HPLC analysis, set on ice, and phytase activity stopped by addition of 0.5 ml 15% TCA. Samples were centrifuged to remove cell debris and 1.45 ml of the supernatant was adjusted to pH 2-3 by addition of 1.4 ml 0.5 M KOH, purified through an anion exchange resin and used for HPLC analysis basically as described (Egli, 2001). Peaks were obtained for lnsP6, lnsP5, lnsP4 and lnsP3. lnsP2, InsPI and Ins could not be resolved from background noise (Figure 4.3). Because the sum of InsP forms was relatively constant (+/- 6%), values for the sum of lnsP2, InsPI and Ins were calculated as the difference from the total quantity of initial lnsP6 to the sum of other forms present in solution.

Concentrations of total P, Ca, Cu, Fe, Mg, Mn, Na and Zn in plant tissue

Leaf, root and tuber samples (approx. 1 g) were harvested and plant tissue dry weight was measured after drying at 80 °C for 36 h (leaves, roots) or 72 h (peeled tuber slices). Dried samples were incinerated at 550 °C for 8 h. The ash was solubilized with 2 ml of 6.0 M HCl

(20% vol.), shortly heated to 100 °C, filtered through Whatman No. 40 ashless filter papers and diluted to 50 ml with double distilled water (see figure 4.4).

Figure 4.4 Schematic representation of the extraction of total P from ashes from plant tissue

Phosphate concentration in the extracts was measured by the malachite green method (Ohno and Zibilske, 1991). Additionally, the concentrations of P and other elements (Ca, Cu, Fe, Mg, Mn, Na and Zn) were measured in these extracts using ICP emission-spectroscopy (Varian Liberty 220 equipped with an ultrasonic nebulizer CETAC U-5000 AT+, Varian Inc., Palo Alto, CA, USA).

4.2 Methods 4 Materials and methods 41

Measurement of soluble Pi in leaves

To measure soluble Pi content in plant tissue, leaf samples (approx. 1 g) were harvested and an extract was prepared as described (Hurry et al., 2000). Pi measurements in the extract were done using the malachite green method (Ohno and Zibilske, 1991).

Plant growth parameters

Total root and shoot dry weight were measured after drying in an oven for 72 h at 80 °C. Total leaf area was measured using a portable leaf area meter (LI-COR, LI-3000A combined with the LI-3050A Transparent Belt Conveyer Accessory).

4.2.3 Plant growth conditions and tissue harvest

Sterile culture

Wild-type and transgenic potato plants (Solanum tuberosum var. Désirée) were propagated in-vitro under sterile conditions in glass pots containing 100 ml of 2MS medium (Murrashige and Skoog, 1962) supplied with various sources of P and set at pH 5.8. Plants were grown at 22 °C with a 16 h/8 h light/dark cycle.

Preparing plants for culture in the greenhouse

For experiments in the greenhouse, plants were first grown in quartz or under aeroponic conditions supplied with half-strength Hoagland's solution. After excision of the primary shoot, lateral shoots were allowed to grow for 1-2 weeks. Lateral shoots (3-5 cm long) were excised and transferred to stonewool supplied with deionized water for 1 week and with % Hoagland solution for another week. Rooted potato cuttings were either transferred to an aeroponic system (Figure 4.5) or to solid substrates.

Figure 4.5 Use of lateral shoots from potato plants for rooting and growth under aeroponic conditions.

4.2 Methods 4 Materials and methods 42

Aeroponics

A spinning-disc operated aeroponic system (see Figure 4.1) was supplied with half-strength Hoagland's solution at pH 5.5 and cooled using an external cooling facility set at 5 °C. The temperature in the root growth chamber varied between 18 °C and 20 °C. Roots were sprayed for 1 minute every 4 minutes (or 1 minute every 2-3 minutes in summer). Plant shoots were exposed to daylight or to a minimum photon flux density of 100 umol m"2 s"1 at canopy level with incandescent light (Philips sodium bulbs, HPL-N 400 W) and to temperatures between 22 °C and 25 °C.

Harvest of root hairs

Roots of aeroponically-grown potato plants were removed from the aeroponic system and immediately frozen in liquid nitrogen. After gently breaking larger root pieces with a plastic pipette, the liquid nitrogen containing root pieces was mechanically stirred with a glass rod for 15-20 minutes. Every 5 minutes, the liquid nitrogen was filtered through a 200 urn mesh and new liquid nitrogen added to the roots. The filtering procedure was continued until sufficient root hairs were harvested. The quality of the harvested tissue was verified under the microscope (Figure 4.6).

Figure 4.6 (A) Harvest of root hairs from plant roots grown aeroponically using liquid nitrogen and a 200 urn mesh filter. (B) Photograph of root hairs harvested with this method; bar size is 50 urn.

Nutrient starvation experiments

For nutrient starvation analysis, ten plants were grown under aeroponic conditions with half- strength Hoagland's solution until the root system reached an average length of 20 cm. To start starvation, the aeroponic system was rinsed with deionized water and the nutrient

4.2 Methods 4 Materials and methods 43

solution replaced with half-strength Hoagland without P, where (NH4)H2P04 was replaced by equimolar amounts of NH4CI. Plants were starved for P for eight days and resupplied with P from day 8 to day 16. Approximately 1-2 g of root material was harvested at each time point for RNA extraction and Northern blot analysis.

Measurement of plant-available P in substrates

To obtain an estimate of soil P available for plant uptake, P was extracted from substrates using sodium bicarbonate (NaHC03), which decreases the ionic activity of Ca2+ by precipitation of CaC03, thus increasing P solubility (Olsen et al., 1954). P was extracted during 30 min by incubation in a 0.5 M NaHC03 solution at pH 8.5, with a soil-solution ratio of

1 g : 20 ml at ambient temperature. After filtration of the extracts (Sartorius, mesh 0.2 urn), P concentration was determined using malachite green colorimetry (Ohno and Zibilske, 1991).

Mycorrhization of roots

To test the effect of mycorrhization on gene expression of StPAPI, StPAP2 and StPAP3, mycorrhized and non-mycorrhized roots were obtained as described by Rausch et al. (2001) and RNA was extracted from these roots.

Split-root experiment

A split-root experiment in the aeroponic system was devised to study possible gene regulatory effects of leaves and P status on PAP expression (Figure 4.7). Root systems of four plants per treatment were split into two equal parts and sprayed with half-strength Hoagland's solution without P for seven days. Subsequently, in treatments (a) and (b) one half of the root system was supplied with nutrient solution containing P, while in treatment (c), both halves remained starved as a negative control. In (a) and (c), plants were not defoliated, whereas in treatment (b) all leaves were removed except for the three youngest visible leaves at the shoot tip to test whether PAP expression in roots is regulated by a signal originating from source leaves. At times 0 (just before resupply), 4 h and 24 h after P resupply, 3 root tips (approximately 3 cm long) were harvested from each part of the root systems and the PMEase activity of secreted phosphatases was subsequently assessed. An average value was calculated for each part of the root systems, and the average for each treatment was calculated from the resulting four values. Approximately 1 g of root material was harvested in both parts of root systems for each treatment at time 0, 4 h and 24 h for

RNA extraction and gene expression analysis. In addition, one to two leaves per plant were harvested at the three time points for the measurement of soluble Pi.

4.2 Methods 4 Materials and methods 44

Figure 4.7 Aerial view of a modified setup of three aeroponic systems (1, 2 and 3) for use in a tri- chamber split-root design. The root systems of four plants per treatment (a, b, c) were split into two and each half exposed to a different growth chamber supplied from a separate aeroponic device.

Pot experiments

A substrate was established containing 85% quartz, 10% loess subsoil from Frick and 5% of a peat-derived substrate (Typ P). Rooted potato cuttings were transferred to this substrate mixture (4 kg per pot) and were irrigated with deionized water and supplemented twice a week with 300 ml of half-strength Hoagland either without P, or with 100 uM Na-lnsP6 (myo¬ inositol hexa/c/'sphosphate dodecasodium salt; SIGMA, Cat. No. P8810; Mr = 932 g/mol).

Each plant was thus supplemented with 56 mg Na-lnsP6 per week, corresponding to approximately 35 mg Pi or 11 mg P. Eight plants per treatment were cultivated in a randomized complete block design in a conventional greenhouse under daylight, supplemented by incandescent light from Philips sodium bulbs (HPL-N 400 W) with a maximum of 120 umol s"1m"2 at canopy level. Relative ambient humidities varied between 40- 80% and temperatures were between 22-28 °C during the day and 18-22 °C during the night. The experiment was carried out over a period of five weeks. Element concentrations in leaves were measured in week three, while biomass production and leaf area were measured at the end of the experiment.

4.2 Methods 4 Materials and methods 45

4.2.4 Computer analyses

Statistics

All numerical data was analysed by one-way ANOVA using SYSTAT 10.0 (SYSTAT Software Inc., CA, USA). LSD-based F tests were performed at a 5% and 1% significance level (P<0.05 and P<0.01, respectively) to identify significant differences between treatment

means.

Restriction site analysis

Restriction site analysis was performed online at:

• Webcutter (http://www.firstmarket.com/cutter/cut2.html) and at

• N EBcutter (httpJ/tools. neb. com/NEBcutter/index.php3)

BLAST

Searches for similar sequences in nucleotide and protein databases were performed using BLAST (Altschul et al., 1990):

• NCBI, National Center for Biotechnology Information (httpJ/www. ncbi. nlm. nih. gov/blast/)

• TIGR, The Institute for Genomic Research (http://www.tigr.org/tdb/tgi/)

Sequence alignments

Sequence alignments were performed at various sites:

• Multalin (http://prodes.toulouse.inra.fr/multalin/multalin.htmf)

• ClustalW (Thompson et al., 1994) at EBI, European Bioinformatics Institute (http://www. ebi.ac. uk/clustalwl)

• Other sources listed in httpJ/www. techfak. uni-bielefeld. de/bcd/Curric/MulAli/welcome. html

Phylogeny analysis

Phylogenetic relationships were inferred using web-applications of the PHYLIP Program Package using the neighbour joining algorithm (Felsenstein, 1993) at http://bioweb.pasteur.fr/

4.2 Methods 4 Materials and methods 46

Signal sequence prediction analysis

Signal sequence prediction servers are available at:

• CBS, Center for Biological Sequence Analysis (http://www. cbs. dtu. dk/services/SignalP-2.0/)

• Leeds University (http://bioinformatics.leeds.ac.uk/prot_analysis/Signal.html)

GPI-anchoring signal prediction analysis

The analysis for prediction of GPI-anchoring sites was performed at:

• http://mendel. imp. univie. ac. at/sat/gpi/gpi_server. html

• http://129.194.185.165/dgpi/index_en.html

4.2 Methods 5 Potato purple acid phosphatases 47

5 \tw Purple acid phosphatases from potato *

5.1 Introduction

The secretion of acid phosphatases from plant roots in response to P deficiency is thought to play a major role in the mobilization of phosphate (Pi) from organic P sources in the rhizosphere and in P scavenging (Duff et al., 1994). Although there are numerous reports describing increased phosphatase activities in plant root exudates under P starvation, few genes encoding secretory phosphatases which are expressed in roots have been isolated so far (Deng et al., 1998; Nakazato et al., 1998; del Pozo et al., 1999; Haran et al., 2000;

Wasaki et al., 2000; Miller et al., 2001). Those isolated belong to different gene families, of which the purple acid phosphatase (PAP) family is of particular interest due to the large number of its members and therefore the possible diversity of functions.

Purple acid phosphatases comprise a family of metal-containing glycoproteins that catalyse the hydrolysis of a wide range of phosphate esters and anhydrides. Members of this group have been identified in plants, animals and fungi (Oddie et al., 2000; Schenk et al., 2000a). In plants, two families of different molecular weight have been identified (Nakazato et al., 1998; Schenketal., 2000b).

The functions of PAPs in plants are still unclear. Five plant PAPs have been implied in the P- starvation responses of Arabidopsis thaliana, Lupinus albus and Spirodela oligorrhiza (Nakazato et al., 1998; del Pozo et al., 1999; Wasaki et al., 2000; Miller et al., 2001). AtACP5 (recently renamed AtPAPU; Li et al., 2002) was shown to be additionally involved in oxidative stress by exposure to H202. The high number of PAP genes found in plants and the structural and biochemical diversity of PAPs may reveal a multiplicity of functions originating from this gene family. At the same time, functional investigations may be hindered because of genetic and functional redundancies (Li et al., 2002).

We report the isolation and characterisation of three novel PAP genes from potato and their pattern of expression in different tissues. The primary structure of the encoded PAP proteins gives indications to their possible roles in plant metabolism. In a split-root experiment, we examine some regulatory aspects related to the expression of these three genes in potato roots.

* Major parts of this chapter were submitted for peer-reviewed publication with the title "Differential regulation of three purple acid phosphatases from potato", by Philip Zimmermann, Babette Regierer, Jens Kossmann, Emmanuel Frossard, Nikolaus Amrhein and Marcel Bucher

5.1 Introduction 5 Potato purple acid phosphatases 48

5.2 Results

Secreted Phosphomonoesterase activity of potato roots

The activity of PMEase secreted from sterile potato roots grown in P-sufficient and P- deficient conditions was visualized on agar by the intensity of precipitated red a-Naphthol- Fast Red complex (Dinkelaker and Marschner, 1992). Potato roots responded to P deficiency by an increased activity of secreted PMEase all along the root (Figure 5.1 (A)). Exudates from root tips showed higher activities, both in P-sufficient and P-deficient conditions, than the older parts of the roots. The induction of PMEases appeared to be more pronounced in the root tips than in other root zones.

B

460 aa 330 aa

-< 1 1 1

1 23 4 5 67

stPAPi mm i I 1 1

stPAP2 warn i 1 1 1 11

StPAPS WÊÊKÊ 1 I I i 1

| Metal coordinating amino acid residues

M Predicted secretion signal sequence

Predicted GPI-anchoring signal sequence

Figure 5.1 (A) Potato roots stained for activity of secreted phosphomonoesterases under P- sufficient (+P) and P-deficient (-P) conditions. (B) Primary structures of three PAPs from potato with lengths of 328 (StPAPI), >448 (StPAP2) and 477 (StPAP3) amino acids. All three sequences have a predicted secretory signal sequence. StPAP2 is predicted to possess a sequence encoding a GPI-anchoring signal. Seven metal coordinating amino acid residues characteristic for PAPs are indicated. Sequences are aligned according to the first conserved residue.

5.2 Results 5 Potato purple acid phosphatases 49

Cloning of StPAPI, StPAP2 and StPAP3

StPAPI is 984 bp long and contains an open reading frame encoding a 328-amino acid polypeptide with a molecular mass of 34.8 kDa. This polypeptide has sequence homology to the family of PAP proteins with low molecular weight (LMW; -35 kDa). Members of the LMW family are found in mammals, plants, fungi and cyanobacteria (Schenk et al., 2000a) and are relatively well conserved in sequence (Figure 5.2).

A sequence similarity search through the TIGR potato expressed sequence tag (EST) database (http://www.tigr.org/) revealed the existence of at least two more potato PAPs expressed in roots (EST393242 and EST519948), of which the cDNAs are referred to here as StPAP2 and StPAP3, respectively. StPAP2 is -1390 bp long and contains an open reading frame encoding a - 462 amino acid polypeptide, whereas StPAP3 has 1431 bp encoding 477 amino acids. The protein sequences derived from both genes show homology to the second family of PAP proteins with higher molecular weight (HMW; - 55 kDa). StPAP2 exhibits a high degree of sequence identity (58%) to StPAP3, while both have a low degree of identity to the LMW protein StPAPI The mature proteins of StPAPI, StPAP2 and StPAP3 (after cleavage of the N-terminal signal peptide sequence) have 304, 441 and 456 amino acids, respectively. (At the time of writing this thesis, the N-terminal sequence of StPAP2 was still lacking approximately 10-15 amino acids (see Figure 5.3)).

Protein sequence analysis

Protein sequence analysis revealed that the three potato PAPs show structural features typical for PAPs. In fact the analysis of the N-terminal sequences of the three potato PAP amino acid sequences using the signal IP program (Nielsen et al., 1997) indicated that all three proteins contain a predicted secretory signal sequence (Figure 5.1 (B)). This structure is common to many previously described PAPs (Hegeman and Grabau, 2001; Li et al., 2002). Five conserved motifs containing the seven described residues involved in metal binding are found throughout all compared sequences (Figures 5.2 and 5.3). GPI-prediction analysis using the algorithm described by Eisenhaber et al. (1999) indicated that the C- terminal end of StPAP2 may contain a GPI-modification signal, which was not detected in the other two PAPs.

Protein sequence alignments between StPAPI, 2 and 3 and other plant, animal and cyanobacterial PAPs show a high degree of conservation throughout all kingdoms (Figures 5.2 and 5.3). A proposed phylogenetic tree based on the neighbour-joining method (PHYLIP

Program v. 3.5) reveals two distinct families of PAPs in plants, with StPAP2 and StPAP3 belonging to the HMW PAPs (-55 kDa) and StPAPI belonging to the LMW PAPs (-35 kDa), the latter group also comprising families of PAP proteins from mammals and cyanobacteria (Figure 5.4).

5.2 Results 5 Potato purple acid phosphatases 50

AtPAP17 1 MNSGRRSLMSATASLSLLLCIFTTFVWSNGELQRFIEPAKSDGS|SE|JV AAF6 0315 1 MAVYSGISMVLCLWVGWFGVCLASAIVELPTFHHPTKGDGSJSfIv StPAPI 1 KYMASMKILNIFISFLLLLLFPAAMAELHRLEHPVNTDGslsEjlv AAF60316 1 MGTQRSKPSCTIVAIFLAFCCFVSSSKAKLESLQHAPKADGsjsFjjV AAF60317 1 MAGLG¥WLAFIGVCFLNVSALLQRLEHPVKADGs|jsL|V

AAL34 937 1 MAVALALLAAMSALSSCTSPATAELTRHEHPVAAGApIrlIv

P1368 6 1 MDMWTALLILQALLLPSLADGATPaIreÏa NP 485726 1 MNLKRRQFLFLSSLSAVGTGLLAWKFAHKYYQSSDLAIASPPKKDLlIreÏs

AtPAP17 -RRBSFNQS LVAYŒ GKIGEKIDL LFSEHDPN ÏEQS AAF60315 -RKjDYNQS QVAFQ GEIGDQLAI LTGEHDD, |TES StPAPI -RF*TFNQS---QVAQQ| GIIGEKLNI LTGVDDP, EES AAF60316 -RKjAYNQS LVAFQ GVIGEKLDV LTGVFDPS EES AAF60317 -RKjTYNQS EVSAQ GRVGAKLNI LSGVDDP ELS AAL34937 -rkIgynqt—rvaeq! GKVAEETEI LAGVDDM, HDS P13686 gvpn|pfhtaremanake|artvqil& vqdindk: NP 485726 t[argqy—avara|||tlyhkqnpy EIEKVNAfcgjERP|

AtPAP17 106 --Ytaeslqkq|ysvl|SYTAPSLQKQ-fi jLSSVLREIDSRWICLRSl WDAELVEMFF AAF60315 103 SNiYTAPSLQKQjYSVLl |lsshlrkldsrwpclrs| 1WNTETVDLFF StPAPI loi tnIytapslqkn-IynvlI |lspilkqkdnrwicmrs| 'IVNTDVAEFFF AAF60316 ytapslqkk-IynvlI jlSHVLRYRDNRWVCFRsi (TLNSENVDFFF AAF60317 ytakslqkq-Iysvl |LNTILQKIDPRWICQRs| :IVDTEIAEFFF AAL34937 ytaqslhkp-1ylvl| jlDPALRKIDSRFICMRsi :IVSAGIVDFFF P13686 fsdrslrkvpIyvl. IIA—YSKISKRWNFPSP1 [YRLHFKIPQTNl NP 485726 lkqg vkj§qacl| GDPQVRYPGFNMNGRRYI ITFRRDRVQFFA

AtPAP17 165 DTTPFVKEYYT EADGHSYDWRAVPSRNSYVK-AL lEVSjKSSKA: AAF60315 162 DTTPFVEEYFN SPE-HVYDWRGVFPQQTYTK-NV IeyaImkstt: StPAPI 160 DTTPFQDMYFT TPKDHTYDWRNVMPRKDYLS-QV Ire s sa: AAF60316 162 DTTPYVDKYFI EDKGHNYDWRGILPRKRYTS-NL |rqstat| AAF60317 154 DTTPFVDKYFL KPKDHTYDWTGVLPRDKYLS-KL jkdsta: AAL34937 157 DTTPFQLQYWT DPGEDHYDWRGVAPRDAYIA-NL ikkstati P13686 14 9 SVAIFMLDTVT LCGNSDDFLSQQPERPRLTART AAARE NP 485726 160 DTN SNADWQN QÜIekeIsssnap|

AtPAP17 224 IGHH dtkelneell CLQHMSDEDSPIC SKAWRGDI AAF60315 22 0 AGHH dtkelverll SLEHISDDESPIC SKAWRGDV StPAPI 219 AGHH sseelgvhil CLEHISSSDSPLC SKSWRGDM AAF60316 221 IGHH dtqellihfl CLEHISSLDSSVC SKAWRGDT AAF60317 213 IGHH dtqelirhll CLEHISSTSSQI SKAWKGDH AAL34937 216 VSAH dtqellelll| CLEHISSRNSPI SKAWRGIF P13686 209 IAEH pthclvkql NLQYLQDENG-Vi INFMDPSKR NP 485726 197 SGVY SNQAFIKTFTl SYERTRAIDG-TTl —AGNR

AtPAP17 284 NPVTINPKLLKFYYDGQ' MSARFTHSDAEIVFYDVFGEILHKWVTSKQLLHSSV- AAF60315 280 TMDRKGVSFFYDGQ' iMSVQLVETDIGIVFYGC StPAPI 27 9 N—WWNPKEMKFYYDGQ' iMAMQITQTQVWIQFFDIFGNILHKWSAS-KNLVSIM- AAF60316 281 K—QSEGDEMKFYYDGQ' (MSVHISQTQLRISFFDVFGNAIHKWNTC-KFDSSDM-

AAF60317 273 L — IKMGKMGQRFTMMD' ILQVWRFKKSIPKLFIMIFLAKFCKLLICPRGYVMCMP AAL34 937 27 6 Q QNEDKLQFFYDGQ' 1LSLELSENRARFAFYDVFGEALYHWSFSKANLQKVQS P13686 268 HQRKVPNGYLRFHYGTEDS LGG iAYVEISSKEMTVTYIEASGKSLFKTRLPRRARP NP 485726 252 P VGRSKWTEYSTSD— LSlATYEVYPDRIELNAIATNNRIFDRGIIRRVEVSGV-

AtPAP17

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Figure 5.2 Multiple sequence alignment of LMW PAP ammo acid sequences from potato (StPAPI), soybean (AAF60316), Arabidopsis (AtPAP17), sweet potato (AAF60315), red kidney bean (AAF60317), rice (AAL34937), human (P13686) and Nostoc sp (NP_485726) Conserved ammo acid residues involved in metal binding are indicated with asterisks Ammo acids conserved throughout all sequences aligned are shaded in black, while those conserved to a lower degree but having similar biophysical properties are shaded in grey Predicted signal peptides are underlined

5.2 Results 5 Potato purple acid phosphatases 51

StPAP2 1 SGPTSGEVTSS

IbPAP2 1 MGASRTGCYLLAWLAAVMNAAIAGITSS

GmPAPl 1 —MGWEGLLALALVLSACVMCNGGSSSP

PvPAPI 1 —MGWKGLLALALVLNWWSNGGKS SN LaPAP2 1 MGYSSFVAIALLMSWWCNGGKTSTI StPAP3 1 MLLHIFFLLSLFLTFIDNGSAGITS

StPAP2 HVE WSEKSKLKNKAN k|tt In T IbPAP2 WSENSQHKKVAR n|rt T| In T GmPAPl WSENSDKKKIAE iJIvt R1J In S PvPAPI WSEKNGRKRIAK kJIst Rjl In S LaPAP2 WSDSSLQNFTAE EjjjjFT Tl In T StPAP3 GLSEGKYDVTVE t|nn T| «Be

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StPAP2

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Figure 5.3 Multiple sequence alignment of HMW PAP ammo acid sequences from potato (StPAP2 and StPAP3), lupin (LaPAP2), sweet potato (lbPAP2), soybean (GmPAPl) and red kidney bean (PvPAPI) Conserved ammo acid residues involved in metal binding are indicated with asterisks Ammo acids conserved throughout all sequences aligned are shaded in black, while those conserved to a lower degree but having similar biophysical properties are shaded in grey Predicted signal peptides are underlined

5.2 Results 5 Potato purple acid phosphatases 52

Plants

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Figure 5.4 Phylogenese relationships between proteins identified as PAPs from mammalian, cyanobacterial and plant origin based on CLUSTALW protein alignment and the neighbour joining method in the PHYLIP program 1000 bootstrap replicates were used to generate the consensus tree Bootstrap values are indicated for major branches One can distinguish between high molecular-weight (HMW) and low molecular-weight (LMW) proteins, respectively The three potato PAPs reported in this work are shaded Plant proteins mentioned in the literature are indicated with their respective names, others are given by accession number Mammalian PAPs shown are from Bos taurus (B27035), Homo sapiens (P13686), Mus scrofa (AF292105) and Sus scrofa (P09889) Cyanobacterial PAPs are from Aphanizomenon sp (AAL16924), Aphanizomenon baltica (AAL16926), Nodulana spumigena (AAL16925) and Nostoc sp (NP_485726) PAPs from plant origin are from Arabidopsis thaliana (AtPAP3-AtPAP25, see also Li et al, 2002), Glycine max (GmPAPl (AF200824), GmPhy (AAK49438), and AAF60316), Ipomoea batatas (IbPAPI (AAF19821), lbPAP2 (AAF19822), lbPAP3 (CAA07280), and AAF60315), Lupinus albus (LaPAPI (AB023385) and LaPAP2 (AB037887)), Phaseolus vulgaris (PvPAPI (S51031) and AAF60317), Oryza sativa (AAL34937), Solanum tuberosum (StPAPI, StPAP2, StPAP3), and Spirodela oligorrhiza (SoPAPI (AB039746))

5.2 Results 5 Potato purple acid phosphatases 53

Tissue-specific expression of StPAPI, StPAP2 and StPAP3

To determine the expression patterns of the three PAP genes in different plant organs, we extracted RNA from potato plants grown in an aeroponic system either containing or lacking Pi. Root tissue was harvested from root tips (distal 2 mm), root hair elongation zone (encompassing the zone from 5 to 10 mm distance from the root tip) and root hair zone (at least 30 mm distant from the root tip), respectively. Each PAP had a specific pattern of expression distinct from those of the other two, as determined by RNA gel blot analysis. StPAPI was strongly expressed in roots and stem, but also was moderately expressed in young leaves (Figure 5.5 (A)) and at intermediate levels in stolons and flowers (data not shown). This gene was not responsive to P starvation. StPAP2, in contrast, responded strongly to P deficiency stress and was expressed highly in roots and less in leaves and stem. Similar to StPAP2, StPAP3 was P starvation-inducible, but showed highest expression in the stem, intermediate levels of expression in roots and moderate levels of expression in leaves (Figure 5.5 (A)).

Secreted phosphatases are presumed to function in P mobilization from organic P sources in the rhizosphere. Since all genes were expressed in roots, we analyzed gene expression patterns more precisely in different root fractions to get an indication of the precise sites of expression and possible functions of the encoded proteins in the roots. Root hairs and stripped roots, as well as root tip, elongation zone and root hair zone tissues, respectively, were isolated from aeroponically-grown plants in P-deficient conditions. RNA gel blot analysis showed that StPAPI was expressed both in root hairs and in whole roots, while transcripts of StPAP2 and StPAP3 were almost absent from root hairs (Figure 5.5 (B)). The latter two showed higher levels of expression in the root tips than in the elongation and root hair zone, respectively. StPAPI transcripts, on the other hand, were more abundant in the root hair zone and elongation zone, where root hair development is initiated, but were almost absent at the root tip (Figure 5.5 (B)).

Induction of expression after P deprivation

Aeroponically grown roots were harvested at different time points after initiation of P-deficient conditions and resupply of P. Expression analysis of RNA extracted from these tissues revealed that StPAP2 was the most strongly P starvation-inducible PAP gene, with intensity of induction comparable to the P starvation marker gene StPT2, which encodes a high- affinity phosphate transporter (Leggewie et al., 1997; Figure 5.5 (D)). StPAPI expression was not induced under P deficiency, while StPAP3 expression was moderately activated by low-P conditions in comparison to its homolog StPAP2.

Effect of mycorrhizal symbiosis

To test whether mycorrhizal infection and symbiosis would affect the abundance of PAP transcripts in roots. RNA was extracted from plants grown with and without mycorrhizal association under both P-sufficient and P-deficient conditions (Rausch et al., 2001). For StPAPI, StPAP2 and StPAP3, no effect of mycorrhizal colonisation was observed on the

5.2 Results 5 Potato purple acid phosphatases 54

level of gene expression in root tissues (Figure 5.5 (C)), while transcripts of the mycorrhiza- specific Pi transporter StPT3 were absent in non-mycorrhized roots but highly abundant in infected roots (Rausch et al., 2001). (Roots from the sames plants were used for the experiments presented in Rausch et al. (2001) and for the experiment described here).

A

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Figure 5.5 Transcript levels of StPAPI, StPAP2 and StPAP3. (A) Gene expression in young leaves, old leaves, stem and roots both under P-sufficient (+P) and P-deficient (-P) conditions. (B) Gene expression in different root tissues under P-deficient conditions. (C) Effect of mycorrhization on the expression of StPAPI, StPAP2 and StPAP3 in roots with (+myc) and without (-myc) mycorrhizas, both under P-sufficient (+P) and deficient (-P) conditions. (D) Analysis of PAP gene expression in potato roots at different time points after transfer to P-deficient growth conditions. At day 8, plants were resupplied with P until day 16. The high-affinity P transporter StPT2 is used as a P starvation inducible marker gene.

Regulatory aspects of expression of StPAPI, 2 and 3

A split-root experiment in an aeroponic system (see chapter 4.1) was carried out to identify conditions involved in the regulation of phosphatase expression in roots. Plants were initially starved for P for 7 days and resupplied with P for the remaining period of investigation. The addition of high levels of Pi to one half of the plant roots resulted in a decreased activity of secreted PMEase for both halves of the separated roots (Figure 5.6 (A)). This reduction started to be measurable within four hours after application of Pi to one half of the root

5.2 Results 5 Potato purple acid phosphatases 55

system and was statistically significantly different (P<0.05) 24 h after resupply of Pi. In plants from which older leaves had been removed (treatment b) just after beginning of resupply (t = 0), the decrease in PMEase activity was not statistically significant (P<0.05) but showed a trend towards lower values after 24 h (Figure 5.6 (B)). In plants continuously supplied with nutrient solution without P, in contrast, the PMEase activity remained constant in both parts of the root system (Figure 5.6 (C)). In all three treatments, there was no significant difference between both parts of the root system. Total soluble inorganic P (Pi) in leaves remained constant in all three treatments within the experimental period (Figure 5.6 (D)).

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Figure 5.6 Phosphomonoesterase (PMEase) activities in exudates of root tips, and gene expression StPAP2 in P-starved roots after P resupply in a split-root setup. Half of the root systems remained in P-deficient conditions (-P), while the other half was sprayed with a nutrient mist either with (+P) or without (-P) phosphate. (A-C) PMEase activity of exudates from root tips from both halves of the root systems of plants possessing all leaves (A and C) or stripped of the fully developed leaves (B). In (A) and (B), half of the root systems was resupplied with P after time 0 (grey columns), while the other halves remained under P starvation conditions (black columns). In C, both halves of the root system remained in P-deficient conditions as a negative control. (D) Soluble Pi content in leaves of plants from treatments A-C. (E) Gene expression of StPAP2 in roots of plants grown in the conditions as described above.

5.2 Results 5 Potato purple acid phosphatases 56

Gene expression analysis of StPAP2 in roots of both parts of the root systems in the treatments A to C (see also Figure 4.7) did not correlate with the levels of secreted PMEase measured in root tips from these same parts (Figure 5.6). It appears that there are irregularities in gene expression throughout the experiment. In fact, there is no consistent relationship between P nutrition and level of gene expression, except perhaps for treatment C, the roots of which were continuously lacking P in the nutrient solution, and from which the levels of StPAP2 transcripts were generally higher. These results could be attributed to the sampling of the roots, or to unknown factors influencing the expression of StPAP2 in roots. Since transcripts of StPAP2 are more abundant in root tips than in other root parts (Figure

5.5), the proportion of root tips harvested can be expected to influence the level of gene expression in harvested root tissue and may yield an explanation to the inconsistencies observed. The measurements of secreted PMEase activity, in contrast, were performed with root tips of similar length and age, which was not possible for RNA extraction, for which larger amounts of root tissue were required.

5.3 Discussion

The primary structures of StPAPI, StPAP2 and StPAP3 reveal the presence of at least three distinct types of PAPs in potato (Figure 5.1). StPAPI is a LMW protein homologous to mammalian and cyanobacterial PAPs, while StPAP2 and StPAP3 are HMW types unique to plants. StPAP2 may have a predicted putative GPI anchoring signal for anchoring of the protein in the plasma membrane (Figure 5.1). Plant homologs to StPAPI were found in Ipomoea batatas (AAF60315), Glycine max (AAF60316) Arabidopsis thaliana (AtPAP8 and AtPAP17), Phaseolus vulgaris (AAF60317) and Oryza sativa (AAL34937). The characterisation of the expression of a LMW PAP at the transcript level in plants has been reported only in Arabidopsis (del Pozo et al., 1999). This gene was found to be expressed in roots and shoots under P-deficient conditions, but not in the presence of P. Furthermore, promoter-GUS fusions showed activation of the GUS gene under other types of stresses, such as oxidative stress under hydrogen peroxide (del Pozo et al., 1999). GUS staining was not found in the stem, in contrast to expression of StPAPI in potato plants. StPAPI is furthermore expressed both in P-deficient and P-sufficient conditions and appears to be expressed in most tissues analyzed. One can therefore expect it to have a more general role in plant metabolism, probably not related to the plant's P-starvation response.

HMW plant PAPs have been characterized in Spirodela oligorrhiza (Nakazato et al., 1998; Nishikoori et al., 2001), white lupin (Wasaki et al., 2000; Miller et al., 2001) and soybean (Hegeman et al., 2001). With the exception of a PAP with homology to phytases that was induced in cotyledons during germination of soybean seedlings (Hegeman et al., 2001), all other PAPs appeared to be regulated by P-starvation. In the present work, transcripts of both StPAP2 and StPAP3 started to accumulate within a few hours after transfer of the plants to medium without P (Figure 5.5 (D)). Infection by mycorrhizal fungi did not affect gene expression of StPAP2 and StPAP3 in roots under P-deficient conditions (Figure 5.5 (C)). Another type of phosphatases has been shown to be induced by pathogen attack in potato

5.2 Discussion 5 Potato purple acid phosphatases 57

leaves infected with Pseudomonas syringae pv. maculicola and Phytophthora infestans

(Petters et al., 2002), but so far no plant phosphatase gene could be associated with mycorrhizal infection.

All reported HMW plant PAPs contained a predicted signal sequence for secretion. The functions of these genes were thus assumed to be related to P nutrition and P remobilization.

The regulation of these genes has been poorly investigated. Whether gene expression is controlled locally upon environmental stimuli, or systemically is not known. The measurement of phosphatase activity of root tips from two parts of the roots of potato plants that were P- deficient revealed that resupply of P to one part of the root system affected secreted phosphatase activity in both parts (Figure 5.6 (A)). In fact, both parts of the root system of the plants reduced their excretion of phosphatase to a similar extent, while plants that were continuously starved maintained the level of phosphatase secretion in both parts of the roots

(Figure 5.6 (B)). These results would indicate that there is a systemic signal regulating gene expression of secreted phosphatases in potato. To test whether this signal is Pi, Pi concentrations in leaves were measured. In all treatments there was no significant difference, at any time point of this experiment, between plants supplied with P in part of their root systems and plants that were maintained in P starvation conditions. Stripping the shoot of all leaves except for the three youngest visible leaves resulted in a delayed reduction in phosphatase activity. From this experiment alone, it is not possible to conclude whether Pi concentrations in leaves are in fact involved in the sigaling cascade controlling gene expression of PAPs in roots. However, on can hypothesize that post-transcriptional events may control the level of PAP protein concentration and activity in the cells.

5.2 Discussion 5 Potato purple acid phosphatases 58

5.2 Discussion 6 Consensus phytase 59

\# Expression of a consensus phytase in

potato root hairs *

6.1 Introduction

Plants are sessile organisms, and their survival and productivity depend largely on their ability to cope with the local environmental conditions. To tolerate stress originating in the rhizosphere, plants must exhibit a specific subset of genetically controlled mechanisms of which many are targeted to the root-soil interface. The knowledge of such mechanisms has provided new opportunities for the breeding or genetic engineering of crop plants more tolerant to rhizosphere stress. To date, a limited number of transgenic plants have been developed that show a potential for improved tolerance to rhizosphere-related stress (Samuelsen et al., 1998; Kasuga et al., 1999; Richardson et al., 2001a; Takahashi et al., 2001a; Zhang and Blumwald, 2001). The model plants thus developed expressed the transgenes either constitutively or simultaneously in several plant parts, under the direction of the constitutive cauliflower mosaic virus (CaMV35S) or other promoters not specifically driving expression in roots, respectively.

The constitutive expression of certain transgenes may result in metabolic disorders and growth retardation, as was recently illustrated by the constitutive expression of a stress- inducible transcription factor (Kasuga et al., 1999). In contrast, the expression of this gene under the control of its own promoter had no detrimental consequences. Therefore, the analysis of tissue-specific gene expression and the availability of suitable promoters is a prerequisite for the successful application of molecular-genetic tools towards modification of root properties (Atkinson et al., 1995; Bücher, 2002). Expression of endogenous or heterologous genes can then be targeted in a precise spatial and temporal manner to alter root characteristics, avoiding undesired side-effects.

Root hairs have a large surface area which is in direct contact with the soil environment.

They make up between 70% and 90% of the total root surface area (Bates and Lynch, 1996). Root hairs are tubular extensions of root epidermal cells extending from the root surface by tip growth. This cell type has been shown to play a dominant role in a number of root

* Major parts of this chapter were submitted for peer-reviewed publication with the title "Engineenng the root-soil interface via targeted expression of a synthetic phytase gene in tnchoblasts", by Philip Zimmermann, Gerardo Zardi, Martin Lehmann, Christophe Zeder, Emmanuel Frossard, Nikolaus Amrhein and Marcel Bücher

6.1 Introduction 6 Consensus phytase 60

functions. For example, membrane proteins responsible for nutrient uptake from the soil solution have often been localized to the root epidermal layer, including root hairs. Nitrate and ammonium transporter genes were reported to be expressed in root hairs (Lauter et al., 1996; von Wren et al., 2000). Phosphate, sulfate and ammonium transporters have also been localized to the external root layers, including root hairs (Daram et al., 1998; Hartje et al., 2000; Takahashi et al., 2000; Yoshimoto et al., 2002). Two genes related to iron mobilization and uptake were predominantly expressed in the external root cell layers (Vert et al., 2002; Waters et al., 2002). Increases in root hair length and density in response to Fe and P deficiency have been reported (Bates and Lynch, 1996; Ma et al., 2001; Schmidt and Schikora, 2001), and the study of root hairless mutants revealed an important role of root hairs in nutrient uptake from the soil solution (Bates and Lynch, 2000, 2001). In addition, root hairs are instrumental in the establishment of the Rhizobium symbiosis in legumes (Kalsi and Etzler, 2000; Cullimore et al., 2001; Wubben et al., 2001) as well as in the anchorage of the plants in the soil (Bailey et al., 2002). Promoters directing root hair-specific expression in crop plants are therefore of advantage in engineering new traits in biotic and abiotic stress tolerance.

Here, we report on the use of a synthetic phytase gene and its tissue-specific expression to engineer plants able to modify the rhizosphere for improved plant nutrition. We show that the targeted secretion of the synthetic phytase exhibiting higher stability to thermal inactivation and protease degradation (Wyss et al., 1999b; Lehmann et al., 2000a) in root hairs of potato induces changes in the rhizosphere which result in higher P mobilization from substrates containing phytate.

6.1 Introduction 6 Consensus phytase 61

6.2 Results

Potato root hair growth in P-deficient conditions

The number, density and elongation of root hairs increase in response to nutrient stress in a number of species (Bates and Lynch, 1996; Gahoonia et al., 1997; Gilroy and Jones, 2000; Jungk, 2001). Root hairs are sites of phosphate transporter (Daram et al., 1998) and H+-

ATPase (Moriau, 1999) activity, respectively, both of which are necessary for P uptake from the soil solution. To investigate how potato root hairs respond to P starvation, we measured

root hair length and the expression of the high-affinity phosphate transporter gene StPT2

(Leggewie et al., 1997) in root hairs of aeroponically grown potato plants both in P-deficient and P-sufficient conditions. After 4 days of P deprivation, average root hair length of newly grown root hairs increased by approximately 40%, from an average of 390 to 570 urn, while it decreased to control levels after resupplying the roots with P for four days (Figure 6.1 (B)). Under low-P conditions, the variability of root hair length was high, varying from 400 to 850 urn for 90% of the root hairs (Figure 6.1 (A)). Roots supplied with sufficient P, however, developed root hairs with a more homogeneous hair length distribution, 90% of root hairs having lengths between 300 and 550 urn. Due to the high density of hairs, it was not possible to measure root hair density, which has been reported to increase in response to P starvation in other plant species (Jungk, 2001). It is safe to assume, however, that the root surface area increased (as a minimal estimate) in proportion to root hair length, i.e. 40%, under P deficiency.

The expression of StPT2 in root hairs was induced within 6 hours of P deprivation and reached high levels within 4 days, while little or no StPT2 transcripts were detected in the control plants supplied with P (Figure 6.1 (C)). The data confirmed the role of potato root hairs in increasing both root surface area and phosphate uptake. Root hairs are thus suitable target cells for the expression of transgenes involved in P mobilization and uptake.

The root hair-specific promoter LeExt1.1

The root hair-specific promoter LeExt1.1 of tomato (Bucher et al., 1997; Bucher et al., 2002) was used to direct ß-glucuronidase (GUS) reporter gene expression in potato. As in tomato, expression was root hair-specific and was additionally found in dry pollen, growing pollen tubes and occasionally in vascular tissues of potato tubers (Figure 6.1, (D) and (E)). GUS activity was observed in root hairs, but not in root tips, of transformed hairy roots and of

potato plants grown either in tissue culture, in an aeroponic system, or in the soil (data not shown).

6.2 Results 6 Consensus phytase 62

Root hair length (pm)

Figure 6.1 Root hair growth and gene expression analysis. (A) Distribution of root hair lengths of roots grown under high and low phosphorus (P) conditions. Insets show root segments of aeroponically grown potato roots at high and low P. (B) Average root hair length of roots grown permanently in P-sufficient conditions (black columns), and under 4 days of P starvation and after four days of resupply, respectively (grey columns). (C) Expression of the high-affinity phosphate transporter gene StPT2 in root hairs after 6 h, 24 h and 4 days of P deprivation (-P) and under P- sufficient conditions (+P) during the same period. (D and E) Expression analysis of the GUS gene under the control of the root hair-specific promoter LeExt1.1 in whole plants (D) and in longitudinal root section, dry pollen, germinating pollen, root tip, transverse section, and potato tuber slice (E, from left to right and top to bottom, respectively).

Generation of transgenic potato lines expressing a consensus phytase

To test the suitability of root hairs to achieve a modification of the rhizosphere, we engineered root hairs to secrete the enzyme phytase. To this end, a gene expression cassette was constructed (Figure 6.2 (A)) using the LeExt1.1 promoter (Bucher et al., 1997;

Bucher et al., 2002) and a consensus phytase gene (PHY; Lehmann et al., 2000b) fused to the barley ß-glucanase signal peptide (Leah et al., 1991; Figure 6.2 (A)). Transgenic potato lines obtained after leaf-disc mediated genetic transformation using Agrobacterium tumefaciens were tested for expression of the PHY gene by RNA gel blot analysis. A number of lines with high (lines 120 and 124) or intermediate (lines 127 and 129) levels of PHY transcripts were used for further analysis (Figure 6.2 (B)). The transcript levels of PHY increased moderately in aeroponically grown roots under P deficiency as compared to plants

6.2 Results 6 Consensus phytase 63

supplied with control levels of P. This may reflect the higher metabolic activity of root hairs when plants are exposed to P nutrient deprivation stress.

Figure 6.2 (A) T-DNA fragment used for plant transformation containing the root hair-specific promoter (LeExt1.1), an ER targeting signal sequence (SP), the consensus phytase gene (PHY) and the octopine synthase terminator sequence (OCS). (B) Expression of the PHY gene in different transgenic lines and in response to P deprivation (from day 0 to day 8) and after resupply of P (from day 8 to day 16), with the Pi transporter StPT2 as a P-starvation marker gene. (C) PMEase activity staining of heat-treated roots of wild-type (left) and transgenic line PHY129 (right) using a-naphthyl phosphate and Fast-Red TR / Blue B reagents. (D) Phosphomonoesterase (PMEase) activity staining of a native Polyacrylamide gel of heat-denatured crude protein extracts from different transformed lines, as well as of authentic purified phytase, using the same reagents as in (C).

The consensus phytase properties are maintained in transgenic plants

As the consensus phytase is thermotolerant (Lehmann et al., 2000b), visualisation of the activity of the recombinant PHY protein was achieved using standard staining methods for Phosphomonoesterase (PMEase) activity after heating the potato roots to 90 °C for 5 minutes to inactivate endogenous root PMEases. Roots from transgenic plants (PHY129) had high PMEase activity, while roots of untransformed plants had no PMEase activity under these conditions (Fig. 6.2 (C)). In order to verify that the PMEase activity observed was related to the phytase protein, heat-inactivated crude protein extracts were subjected to PAGE under non-denaturing conditions and stained for PMEase activity. Only a single band migrating in the gel like authentic consensus phytase was found in each of the transgenic lines, while no activity was detected in the extracts of control plants (Fig. 6.2 (D)). In non-denaturated crude protein extracts from roots, total PMEase activity was 30% higher in PHY124 as compared to wild-type (Figure 6.3 (A)). The pH activity profiles for total PMEase were similar for both PHY124 and wild-type, with an optimum at pH 5.5 (data not shown). Specific phytase activity was measured as Pi released from phytate added to the crude protein extracts. The phytase activity of crude protein extracts was more than 4 times higher in PHY124 than in wild-type plants (Fig 6.3 (B)). This activity results from both cellular phytase and secreted phytase bound to the cell walls of root hairs.

6.2 Results 6 Consensus phytase 64

B

400 200

PHY124 WT PHY124 WT

D

18 12 -i

16 -i 10 14 12 £ 8 0 10 S e- 8 4.5 5.0 5.5 6.0 6.5 7.0 6 > 4- ~r 4 PH £ 2 2 r__JL_t 0 0

PHY124 WT PHY124 WT

Figure 6.3 Specific enzymatic activities in crude protein extracts and exudates from roots. PMEase (A) and phytase (B) activities in crude protein extracts from roots of wild-type (WT) and transgenic (PHY124) lines, respectively. PMEase (C) and phytase (D) activities in root exudates of WT and PHY124, respectively. (E) pH activity profile of phytase activity in root exudates from wild-type (o) andPHY124(«).

The PHY protein is secreted from the roots

To be able to hydrolyze P from phytates in the soil, the PHY protein must be secreted from the root hairs into the rhizosphere. To verify this, plants were grown in an aeroponic system, in which root hair growth is not impaired and roots are not injured upon removal, and were subsequently incubated in a buffer for collection of exudates. Total PMEase activity in root exudates of PHY124 plants was 2 to 3 times higher than in controls (Figure 6.3 (C)). The level of specific phytase activity secreted by wild-type plants was hardly above background, whereas high levels of activity were measured in exudates collected from line PHY124 (Figure 6.3 (D)). The pH optimum of the root-secreted phytase activity was around pH 6.0 (Figure 6.3 (E)), which is similar to the pH activity profile as determined for recombinant consensus phytases expressed in yeast (Lehmann et al., 2000b).

Kinetics of phytic acid degradation by root exudates

Fungal and consensus phytases hydrolyze myo-inositol hexa/c/'sphosphate (lnsP6) to lower inositol phosphates following a particular degradation sequence (Wyss et al., 1999a). We therefore determined the kinetics and intermediates of phytic acid degradation by HPLC of potato root exudates. The roots of transgenic and wild-type plants, grown aeroponically under low P conditions, were incubated for 16 hours in a buffer containing 2 mM lnsP6 as the

6.2 Results 6 Consensus phytase 65

sole P source. Samples were collected every hour to determine myo-inositol phosphates. PMEase and phytase activities were monitored during the entire period (Figure 6.4 (A) and (B)). The PMEase activity followed a sigmoidal pattern for both PHY124 and wild-type, with an initial lag phase and a saturation of product accumulation after 6 h (Figure 6.4.(A)).

Phytase activity, measured as Pi released to the incubation solution, was very low for control plants, while Pi accumulated rapidly in exudates of PHY124 after an initial lag phase of 6 h, the rate levelling off after 16 h of incubation (Fig 6.4 (B)). HPLC analysis of inositol phosphates revealed that exudates from wild-type roots were unable to degrade lnsP6 (Figure 6.4 (C)), while PHY124 exudates rapidly degraded lnsP6 with concomitant accumulation of lnsP2, InsPI and InsP (Figure 6.4 (D)). lnsP5 and lnsP4 were observed as intermediates. The calculation of the rates of degradation of intermediates revealed that lnsP4 and lnsP5 were more rapidly degraded than lnsP6 and lnsP3 (data not shown). These findings confirm the kinetics of phytic acid degradation observed in fungal and bacterial phytases (Wyss et al., 1999a) and furthermore provide proof that the root-secreted recombinant phytase is able to degrade phytic acid present in soluble form in a liquid medium.

A B

700 300

00 A° 600 250

500 200 <^^*° O) 400 y 150 300 100 o 200 E 50 100 0 0 o-^*^

0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 18

D

100 100

80 80

60 60

S3 40 40 o

01 a. 20 20

Bg-~~§j fj-~~gi 8~~ffi gt~~~~ig'g*g g-ffi ffi~~—jp——^ 0

0 2 4 6 8 10 12 14 16 18 0 2 4 6 8 10 12 14 16 18

Time (h) Time (h)

Figure 6.4 Phosphomonoesterase activity in, and kinetics of phytic acid degradation by root exudates of wild-type (WT) and transgenic (PHY124) plants (A) Phosphomonoesterase activity in root exudates of PHY124 (o) and WT (•) plants (B) Release of Pi from phytic acid by root exudates from PHY124 (o) and WT (•) (C and D) Intermediates of phytic acid degradation by root exudates of WT and PHY124 plants, respectively Percent values indicate percent of total InsP forms present in solution, (•) lnsP6, (o) lnsP5, (T) lnsP4, (V) lnsP3, () lnsP2 + InsPI + Ins

6.2 Results 6 Consensus phytase 66

Phenotype of PHY plants

Having established that the transgenic plants excrete enzymatically active consensus phytase under aeroponic conditions, we next tested whether plants can utilize phytic acid as a P source under various growth conditions. Potato cuttings grown in sterile conditions were starved for P during two growth cycles prior to transfer to agar containing either no P (-Pi), Pi as Na2HP04 (+Pi) or as Na-lnsP6 (+lnsP6). No significant differences in growth were measured between PHY124 and wild-type plants, respectively, in the treatments -Pi and +Pi. However, in the +lnsP6 treatment, PHY124 produced a 30% higher biomass than wild-type (data not shown).

Since PHY124 plants were obviously able to utilize lnsP6 as a source of P in sterile conditions, in contrast to control plants, we tested whether this effect could be observed under non-sterile conditions in an artificial substrate containing quartz (85%), loess (10%) and peat (5%). Ten day-old rooted potato cuttings were transferred to this substrate supplied with half strength Hoagland solution containing 100 uM lnsP6 as sole source of P. After ten days of growth, first differences were visible in plant height, and these differences were observed throughout the experiment (i.e. during 4 weeks). Three week-old PHY124 plants were up to 20% taller than wild-type (Figure 6.5 (B)). Leaf shape of some leaves was altered in the transgenic plants, with a predominant growth of the terminal leaflet and reduced growth of the lateral leaflets (Figure 6.5 (C)). The ratio of leaf area to shoot dry weight was significantly higher in the wild-type as compared to PHY124 (P<0.05), concomitant with a higher root-shoot ratio in the wild-type plants (Figure 6.5 (A)). This finding may reflect the P status of the plant. In fact, PHY124 had a 40% higher total P concentration in leaves (P<0.01) as compared to wild-type. As plants of both genotypes had similar dry matter production, the total P mobilization and uptake from the soil substrate was 40% higher in line PHY124 than in wild-type. With respect to micronutrients, no statistically significant increases in Fe, Ca, Zn and Mn were measured (P<0.01; Figure 6.5 (A)). In a parallel experiment, however, PHY124 plants exhibited significantly higher concentrations not only of P, but also of Fe and Zn in the tubers when grown in substrates supplemented with Na-lnsP6 (data not shown). Both results confirm that transgenic plants secreting phytase via their root hairs are able to take up additional P from an unsterile, P-sorbing substrate supplemented with phytic acid in soluble form in the nutrient solution. Moreover, phytase-mediated P-mobilization from phytate may interfere with the acquisition of some micronutrient elements.

6.2 Results 6 Consensus phytase 67

A

1) DW shoot DWroot DW total Root/shoot Leaf area/ (g) (g) (g) Ratio (%) DW shoot (cm2 / g)

* PHY124 8 63 ±0 16 161 ±0 15 10 24 ±0 23 18 7 68 4 ± 1 9

WT 8 44 ±0 20 178 ±0 11 10 22 ±0 28 211 75 5 ± 1 6

2)a P Ca Fe Zn Mn

PHY124 4076 ±276** 8390 ± 646 103±70 32 2 ± 2 4 36 6 ± 1 6

WT 2886 ±242 8918 ±663 99 ± 4 5 27 1 ± 1 9 38 8 ± 1 5

a ** * AN values are given m mg/kg dry weight Significant at P < 0 01 Significant at P < 0 05 n=8

PHY 124 WT

Figure 6.5 Growth response of transgenic (PHY124) and wild-type (WT) plants in a soil substrate (A) Growth parameters and total nutrient concentrations in leaves of PHY124 and WT (B) Phenotype of PHY124 and WT (C) Shape of some leaves from PHY124 and WT plants

6 2 Results 6 Consensus phytase 68

6.3 Discussion

To test the approach of biochemically modifying the root-soil interface via root hair-mediated secretion of recombinant proteins, we chose to express in root hairs of potato a synthetic gene encoding a secretory phytase engineered for high heat and proteolytic stability.

Previously, the constitutive expression of an Aspergillus niger phytase gene in all organs of transgenic Arabidopsis thaliana plants and the subsequent secretion of the recombinant protein were shown to improve the ability of the plants to mobilize P from soluble phytate in sterile agar culture (Richardson et al., 2001a). In our case, root hair-specific secretion of the engineered phytase was shown to be sufficient to improve the ability of the transgenic plants to acquire P from phytate both in sterile agar and in an unsterile substrate (Figure 6.5).

The increased length of root hairs and the expression of the high-affinity phosphate transporter gene StPT2 in root hairs under low P conditions (Figure 6.1 (A) to (C)) confirmed that a root hair-specific promoter is suitable for the expression of genes involved in P mobilization and uptake. Although the total root surface area was measured to increase up to 40% in our experimental conditions for potato, other reports show that increases in root hair length in response to nutrient starvation in other species can result in an increased total root surface area by up to 340% (Gahoonia et al., 1997).

LeExt1.1 promoter-GUS analysis revealed that expression in potato roots is limited to the root hair cells and is absent at the root tip and in the root cylinder (Figure 6.1 (D) and (E);

Bûcher et al., 2002). We thus chose LeExt1.1 as a root hair-specific promoter to express the

PHY gene.

The rhizosphere represents a harsh environment for enzymes due to high microbial activities and numerous soil chemical processes. For this reason, the secretion of a highly stable phytase from roots could confer an advantage over soil microbial phytases and phosphatases. Assessment of PMEase activity of heat-treated roots and in crude protein extracts revealed that none of the endogenous PMEases matched the level of thermostability exhibited by the recombinant protein (Figure 6.2 (C) and (D)). Secretion experiments and HPLC analysis demonstrated that root hairs can produce and secrete sufficient amounts of active PHY protein within a few hours to hydrolyse lnsP6 in the medium (Figure 6.4 (D)). The phytase activities measured were comparable to those obtained by Richardson et al. (2001a) after constitutive expression of a fungal phytase gene in Arabidopsis. The improved P uptake in an unsterile, artificial soil substrate by transgenic plants as compared to wild-type (Figure

6.5) appears to indicate that the phytase activity of the recombinant protein is in excess of the activities of phytases produced by soil microorganisms at the root-soil interface. Apart from the amounts of PHY protein secreted by the transgenic lines, the higher level of P mobilization could also be related to the particular stability of the PHY protein.

Exudates from PHY124 plants were able to degrade lnsP6 at rates more than 100 times higher than exudates from wild-type plants (Figure 6.4). One would therefore expect the

PHY124 plants to be able to take up substantially more P than wild-type plants from a low-P substrate supplied with phytic acid. In the pot experiment reported here, however, biomass

6.3 Discussion 6 Consensus phytase 69

production was not higher in PHY124 than in wild-type plants, while P concentration in leaves was 40% higher. Moreover, P concentrations in the leaves of wild-type plants were in a near to normal range (3.8 g / kg DW). One can therefore conclude that, either the wild-type plants could have access to P released from phytate (by root-secreted or microbial enzymes), or there may have been other sources of P in the growth substrate or in the irrigation water allowing wild-type plants to produce similar biomass as PHY124 plants. To address this issue, the possible sources of P for plant nutrition in that experiment were calculated and are discussed.

First, in a prelimary experiment to determine phytic acid intermediates by HPLC of potato root exudates (see also chapter 4.2.2), it was observed that the phytate (P8810 from Sigma, Fluka, Buchs, Switzerland) added to the soil substrate was composed of 69% of lnsP6, 25% of lnsP5 and 6% of lnsP4 (data not shown). It is thought that extracellular enzymes other than phytase can dephosphorylate lower forms of inositol phosphates such as lnsP5, lnsP4 and lnsP3, and that therefore wild-type plants may have been able to hydrolyse P from lower forms of inositol phosphates. This hypothesis cannot be confirmed by the data. In fact, after 24 h of incubation, the proportions in the buffer containing roots of wild-type potato plants changed to 67%, 24% and 9%, with respect to lnsP6, lnsP5 and lnsP4, respectively. It therefore appears that inositol phosphates, including lnsP6, were partially degraded in solution in the presence of roots of wild-type plants, but the rates of hydrolysis were more than 100 times lower than those observed in the presence of roots from PHY plants (data not shown).

Second, the source of phytate used (P8810) contained Pi as a contaminant at 0.1% (w/w). The total Pi thus added to each pot throughout the whole experiment was approximately 0.28 mg. Based on the measurements of Pi content in leaves and on biomass production, one can estimate the total Pi uptake throughout the whole experiment to be approximately 29 (wild- type) to 41 (PHY124) mg per plant, which is two orders of magnitude higher than the contaminant Pi in the phytate added. Therefore, the level of Pi contaminant also cannot explain the relatively high amounts of Pi taken up by wild-type plants in this substrate.

Third, the amount of soluble Pi supplied to each pot by the irrigation water (deionized) throughout the whole period of growth was calculated to be 0.5 mg. This source of Pi also cannot account for the relatively large amounts of P taken up by wild-type plants.

Fourth, the soil substrate may have had sufficient available P to sustain normal plant growth. In fact, extraction of Pi from the soil substrate using the sodium bicarbonate method (Olsen et al., 1954) indicated that 8 ± 0.6 mg P / kg soil were available for plant uptake (without the added phytate). Each pot culture thus contained 32 ± 2.4 mg available Pi, which is approximately equivalent to that taken up by wild-type plants. However, knowing that Pi has a very low mobility in soils (see chapter 2.2), and that the roots did not have access to the entire soil volume within the five weeks of the experiment, it is safe to assume that the total P uptake from soil available P was lower than 32 mg Pi per plant. In the best case, this value could account for the Pi taken up by wild-type plants. However, it can not be excluded that wild-type plants could take up a fraction of P released from phytate. Since HPLC analysis of phytic acid degradation by root exudates shows that exudates from wild-type roots degrade

6.3 Discussion 6 Consensus phytase 70

inositol phosphates at a very low rate (not sufficient to hydrolyse significant amounts of phytate within the five weeks of the experiment), one must conclude that a possible P release from phytate is the result of processes other than enzyme activity of plant root exudates.

In contrast, plants of PHY124 took up approximately 11 mg Pi per plant more than wild-type plants (41 mg (PHY124) - 29 mg (wild-type)) and approximately 9 mg Pi more than the estimated available P from the soil substrate alone. This difference may therefore be attributed to the P-hydrolysis from phytate by root-secreted recombinant phytase. The total amount of hydrolysable Pi in form of phytate that was added to each pot culture throughout the experiment was 175 mg. Assuming that 10 mg Pi was released from phytate and taken up by PHY124 plants, the recovery rate of P from phytate under the culture conditions described is near to 6%, which is higher than the rates of 1.5% calculated for phytate added to natural soils (Findenegg and Nelemans, 1993). In summary, the soil substrate used had relatively high levels of available P, thus not allowing a strong effect of phytase secretion from roots on the total P mobilization and uptake from the substrate containing phytate, and thereby on improving plant growth of transgenic plants, as compared to wild-type plants. Nevertheless, the above estimates allow concluding that approximately 25% of the total P taken up by the PHY124 plants resulted from P hydrolysis from phytate. This remains to be proven, for example by using radioactively labelled phytic acid as a source of P.

6.3 Discussion 7 Conclusions and outlook 71

ff General conclusions and outlook

The results on the identification, cloning, and analysis of three purple acid phosphatase

(PAP) genes from potato will be discussed first. Particular attention will be given to link these findings to the current body of knowledge about plant PAPs. The second part of the discussion will relate the results obtained with the expression of a synthetic phytase in potato root hairs to other work in this field. Finally, a comprehensive and conceptual overview of the role of phosphatases and phytases in soils and on how the current knowledge and the presently available tools may change our perspectives in rhizosphere research will be given.

7.1 Potato purple acid phosphatases

Analysis of StPAPI, StPAP2 and StPAP3

In recent years, several names have been proposed for individual plant PAP genes and proteins in different publications. To clarify this unsatisfactory state, a comparison of amino acid sequences of known plant PAPs was performed to identify unique sequences and a systematic list of names was created (see table 7.1). In the left column are the names which are used in this report.

Early reports on plant PAPs were based on the proteins purified from red kidney bean and from tubers of sweet potato, and on the characterisation of their biochemical and biophysical properties (see table 7.1). The elucidation of the crystal structure of red kidney bean PAP (PvPAPI) confirmed previous proposals on the structure of the protein and the mechanism of phosphate ester hydrolysis. In more recent times, cDNA clones have been isolated and gene expression data, together with protein characterisation and localisation, have yielded important information on the biological functions of these proteins (see table 7.2), but in no case could the respective biological function be fully demonstrated.

However, some authors have deduced possible biological functions from their results. Using histoenzymological methods, Cashikar et al. (1997) showed that PvPAPI was localized exclusively in the cell walls of the peripheral two to three rows of cells in the cotyledons. Additionally, in vitro experiments showed that pectin, a major component of the cell wall, altered the kinetic properties of PvPAPI. Based on these two findings, they suggested that

PvPAPI may have a role in mobilizing organic phosphates during seed germination. In Arabidopsis, transcripts of AtPAPU (=AtACP5) were shown to be localized both in leaves and in roots, and gene expression to be regulated by a large number of environmental

7.1 Purple acid phosphatases 7 Conclusions and outlook 72

factors. It was therefore thought to be controlled via several signal transduction pathways (del Pozo et al., 1999). Individual functions for this protein were not suggested. However, a general role in recycling of phosphate from the plant's phosphate ester pool was proposed. The first account of a PAP-like protein to have a relatively clear metabolic function was recently reported for a soybean PAP exhibiting phytase activity (Hegeman and Grabau,

2001). In contrast to PvPAPI, which does not have any phytase activity, this protein, named GmPhy, had a high affinity for phytic acid, while it showed a 340-fold and 540-fold lower affinity for ATP and pNPP, respectively, than PvPAPI Since the GmPhy protein was highly concentrated in cotyledons of germinating soybean, its role in the mobilisation of P from seed phytate was suggested.

Authors Name in this report: A1 A2 A3 A4 A5 A6 A7 A8

IbPAPI IbPAPI SP-PAP1 -

lbPAP2 lbPAP2 SP-PAP2 SpPAPI

lbPAP3 lbPAP3 SP-PAP3 SpPAP2 Spirodela SoPAPI Sp1 oligorrhiza PAP La1, LaPAPI La1 LASAP1

LaPAP2 LASAP2

GmPAPl GmPAPl SB-PAP

GmPhy GmPhy

AtPAP17 AtACP5 StPAPI 7

KB-PAP, PvPAPI PvPAPI Pvu Lupin LaAPase APase

Authors

A1 Hegeman et al (2001) A2 Schenketal (1999,2000b) A3 Durmusetal (1999) A4 Nakazato et al (1997, 1998) and Nishikoon et al (2001) A5 Wasaki et al (2000) A6 del Pozo et al (1999) A7 Li et al (2002) A8 Miller et al (2001)

Corresponding plants

IbPAPI, 2 and 3 sweet potato (Ipomoea batatas) SoPAPI duckweed (Spirodela oligorrhiza) LaPAPI and 2, and LaAPase white lupin (Lupinus albus) GmPAPl and GmPhy soybean (Glycine max) AtPAP17 Arabidopsis thaliana PvPAPI red kidney bean (Phaseolus vulgaris)

Table 7.1 Names given to recently published plant PAPs The first occurrence in the literature is given in bold The left column indicates names that are used in this report Authors and plants corresponding to the genes mentioned are given below the table

7.1 Purple acid phosphatases 7 Conclusions and outlook 73

In the current work, three cDNA clones encoding phosphatases belonging to the PAP family have been cloned and characterized. StPAPI is homologous to the LMW PAPs also found in mammals and cyanobacteria. StPAP2 and StPAP3 are homologous to the HMW PAPs, a family of proteins exclusively found in plants to date, of which some have been extensively characterized on a biochemical and biophysical level (Beck et al., 1986; Strater et al., 1995;

Klabunde et al., 1996). Based on amino acid sequence analysis, StPAPI, as well as StPAP2

and StPAP3, have a predicted signal sequence for secretion. As typically for signal peptides,

their sequences are not conserved, in contrast to the sequences encoding the mature proteins, which are highly conserved.

Biological analysis Protein characterisation

cu CO 4—' 3 o to to 3 55 o >s o to C >s CO to Q. O fU o o C LL to to £- fU CO cü CD o > O to o M— o >s CO JU fU o fU o cu O .c o CO ÇU CO o (f) "5 M— to 1 s E 4—' Q. CD 10 o cu cu to to CO to CO CO to X o cü o > < to >s >s cu o 1 JO o cD o o Q. o cu CO 10 Q. TJ CO CO a3 CO _ < cu cu o cs cu to o .c E CO to OT < o E E o CO o o o o cu "cu Name Q cu Q cu X cu cD 5 Authors o CD C75 or CL CL CL H CL CL H a S Ö O E

IbPAP? / 1

PvPAPI / Fe-Zn 2

PvPAPI / Fe-"? 3

PvPAPI / • / • Fe-Zn 4

PvPAPI / • / / • • / Fe-Zn 5

PvPAPI / • / / • • / Fe-Zn 6

PvPAPI / • / • • 7

SoPAPI / / / / / Fe-Mn 8-10

IbPAPI / / • / Fe-Mn 11

/ / • / lbPAP2 Fe-Mn 11, 12 / / • / lbPAP3 Fe-Mn 11, 12 AtPAP17 / / / / • / / / / 13

LaPAPI / / / 14

LaPAP2 / / / / 15

GmPAPl / / / / Fe-Zn 11

GmPhy / / / • / / • / / 16

LaAPase / 17

LePAPI / / / 18

NtPAP? / / 19

StPAPI / / / This work

StPAP2 / / / This work

StPAP3 / / / This work

Table 7.2 Selected publications on plant PAPs since 1974 (1) (Uehara et al ) (1974 a, b), (2) Beck et al (1986), (3) Hefler and Averill (1987), (4) Cashikar et al (1995), (5) Strater et al (1995), (6) Klabunde et al (1996), (7) Cashikar et al (1997), (8) Nakazato et al (1997), (9) Nakazato et al (1998), (10) Nishikoon et al (2001), (11) LeBansky et al (1991), (12) Schenk et al (1999), (13) Durmus et al (1999), (14) del Pozo et al (1999), (15) Wasaki et al (2000), (16) Hegeman et al (2001), (17) Miller et al (2002), (18) Varadarajan et al (2002), (19) Kaida and Kaneko (2002)

7.1 Purple acid phosphatases 7 Conclusions and outlook 74

The search for particular sequence motifs revealed, in addition, that StPAP2 may contain a

GPI anchoring signal (Figure 5.1 (B)). Since GPI anchors do not have a very strict structure allowing high probability predictions, this information must be taken with caution. In fact, the algorithm described by Eisenhaber et al. (1999) could not unanimously detect a GPI anchor in the C-terminal sequence of the Spirodela oligorrhiza PAP, in contrast to experimental data that confirm this hypothesis. Reciprocally, a positive prediction may not necessarily prove the presence of a GPI anchor.

Although all three potato PAPs are preferentially expressed in roots and stems based on RNA gel blot analysis, some level of expression is also found in leaves, and for StPAPI additionally in stolons, growing sprouts and flowers, but not in tubers (see Figure 5.4). StPAPI was not induced by P starvation, in contrast to StPAP2 and StPAP3. Due to the fact that StPAPI is expressed in most tissues tested and, furthermore, that it is not responsive to

P starvation stress, it may have a rather constitutive function, probably in the cell walls, e.g. by interacting with ß-glucans or pectins. In fact, Kaida and Kaneko (2002) found that overexpression of a PAP in yeast enhanced the deposition of ß-glucan at the surface of protoplasts. StPAP2 and StPAP3, being responsive to P starvation and localized mainly in roots, can be expected to play a role in the remobilization of organic P, such as ATP, which is present in the extracellular matrix of multicellular organisms and in the extracellular fluid of unicellular organisms (Thomas et al., 1999), from the apoplast or at the soil-root interface. StPAP2 may additionally have other functions in conjunction with its putative localisation at the external surface of the cell membranes, possibly via interactions with cell wall polymers, or by taking part in the activation or inactivation of other GPI-anchored proteins clustered in membrane microdomains ("rafts"). Such cell surface raft microdomains composed of GPI-anchored proteins and lipids have been shown to be enriched in a number of signal transduction components in animal cells (Horejsi et al., 1999).

The results presented here do not include any precise localisation studies, such as by in situ RNA hybridisation or immunolocalisation. Furthermore, the cloning of RNA interference constructs to trace protein function via knock-out could not be terminated. Therefore, the precise function of the three potato PAPs analysed in this work cannot be presented. Further research is thus needed to give a conclusive answer to the question of the biological roles of these genes.

Potato PAPs: outlook

Table 7.2 reveals that the biochemical and biophysical characterisation of PAPs is well advanced, while biological data are scarce. Few authors have reported gene expression data, protein localisation, protein purification and substrate hydrolysis kinetics, effect of inhibitors, and promoter-reporter gene studies. Only recently, the use of knock-out mutants has been taken into consideration for Arabidopsis (Li et al., 2002). Functional analysis of these genes must be pursued with priority.

Probably the most promising approach for potato, for which single gene knock-out mutants are not yet descibed, is the establishment of transgenic lines in which gene expression of

7.1 Purple acid phosphatases 7 Conclusions and outlook 75

individual or several PAPs, respectively, is down-regulated by RNA interference (Smith et al.,

2000). The study of PAPs may also be accelerated by the concomitant analysis of

Arabidopsis PAPs, for which the sequences of 29 PAP homologs are available (Li et al.,

2002) and where gene knock-out mutants are available as well. A search of the Syngenta

Arabidopsis Insertion Library (SAIL) using an Arabidopsis sequence homolog to StPAPI revealed the existence of at least three putative knock-outs. The analysis of repressed transgenic lines or knock-out mutants is a complex task due to the redundancy of PAPs in plants and the likelihood that they may substitute for each other. Furthermore, the use of antibodies for immunolocalisation and Western blot analyses could result in cross-reactions, as illustrated by the cross-reaction of an anti-/\raö/'cfops/s PAP antibody with the S. oligorrhiza PAP (Nishikoori et al., 2001).

A complementary approach that would assist in interpreting genetic results could be the study of protein substrate hydrolysis specificity and protein-protein binding using protein microarray technology (Templin et al., 2002). This technique would allow screening of hundreds or thousands of substrates and known proteins in a relatively short time, leading to more targeted biological experiments which may allow defining precise functions to the PAP proteins tested.

Another interesting aspect of PAPs is their regulation. In fact, despite the large number of

PAPs, there are Arabidopsis mutants that have lost their P-starvation response for phosphatase secretion (see chapter 2.2.3). As many PAPs contain signal sequences for secretion, this could indicate that there is a common signal transduction pathway controlling several secretory phosphatase genes. The elucidation of these control mechanisms would be a big step towards understanding the plants' responses to P deficiency stress.

Comparative PAP promoter sequence analysis would allow the identification of conserved response elements. Gel-shift and deoxyribonuclease-l footprinting assays would then allow to identify putative transcription factors and their corresponding DNA binding sites. One such response domain has already been shown to bind homeodomain leucine zipper proteins in soybean, suggesting a role for these transcription factors in P-modulated gene expression (Tangetal., 2001).

Not to be ignored are possible functions of PAPs associated with plant-pathogen interactions. In fact, a potato phosphatase cDNA clone has recently been identified. The corresponding transcript was upregulated in leaf tissues infected with the fungal pathogen

Phytophthora infestans and the bacterium Pseudomonas syringae pv. Maculicola (Petters et al., 2002). Similarly, the transcript levels of a phosphatase gene in bean was correlated to disease resistence, but not to wounding (Jakobek and Lindgren, 2002). In our case, arbuscular mycorrhizal infection of roots did not affect the transcript levels of StPAP2 and

StPAP3. However, it has been reported that mycorrhizal infection may affect phosphatase secretion (McArthur and Knowles, 1993). Since P is thought to be transported through the mycelium of certain species mainly as polyphosphates, but also as Pi and Po (Boddington and Dodd, 1999; Ezawa et al., 2002), polyphosphates and Po must be hydrolysed for release through the fungal membrane and uptake by the plant cell. Whether organic forms of P are also released by the fungus and require the activity of a plant cell membrane-bound phosphatase for hydrolysis and subsequent uptake into the plant is not known. The GPI-

7.1 Purple acid phosphatases 7 Conclusions and outlook 76

anchoring machinery would provide the means for polar or targeted accumulation of surface proteins (Chatterjee and Mayor, 2001) within the peri-arbuscular membrane. By analogy to

PAP activation in response to pathogen attack, one can thus suggest a role of PAPs in the events leading to mycorrhizal symbiosis.

7.2 P mobilization from phytate in transgenic plants secreting phytase

It has long been debated whether the availability of phytate in soils is limited by its solubility or by the quantity of phytase present in the soil solution. There are still conflicting views in the literature about this topic. For example, in the 1950's, Jackman and Black (1952) found that the hydrolysis of soil phytate was strongly controlled by its solubility. Flaig et al. (1960), in contrast, stated that the rate of phytate hydrolysis was limiting the rate of P uptake from phytate by the plants. Since that time, depending on the conditions chosen, it was either reported that the activity of root-secreted or microbial phytase was sufficient for mobilizing P from phytate (Tarafdar and Claassen, 1988; Adams and Pate, 1992; Findenegg and Nelemans, 1993; Hayes et al., 2000b) or that the availability of phytate for enzymatic degradation was a function of its solubility in the soil (Martin and Cartwright, 1971; McKercher and Anderson, 1989; see also figure 2.8). Some experiments were done in sterile conditions (Hayes et al., 2000b; Richardson et al., 2000), while most others were performed

in non-sterile greenhouse pot experiments. Results obtained with plants grown in sand culture appear to support the second hypothesis, while experiments in pot cultures containing bulk soil emphasized the availability of phytate as the bottleneck for the release of P from phytate. One can assume that all authors were right about their conclusions, as far as their own experimental conditions were concerned, but with the limitation that the conclusions are valid only to a restricted number of other conditions. Moreover, considering the complexity of the rhizosphere, it is not surprising to be confronted with divergent results. The role of soil microorganisms, including ecto-mycorrhizal fungi, in mobilizing phytate from soils has also been demonstrated (Antibus et al., 1992, 1997; Richardson et al., 2001b; Tarafdar et al., 2001).

In the present work, transgenic plants secreting a synthetic phytase from their roots were able to degrade phytic acid in a buffer solution. One would therefore expect transgenic plants grown in a medium containing soluble phytate to have a higher P mobilization capacity and thus higher P uptake than wild-type plants. In fact, in the experiments described here, transgenic plants grown in sterile culture or in a quartz sand mixture, to which soluble phytate

had been added, grew better than wild-type plants, in terms of biomass production (in sterile culture; data not shown) and of total P content in the leaves (Figure 6.5). Up to this point, the results do not contradict those of Richardson et al. (2001a), obtained by constitutively overexpressing a fungal phytase in Arabidopsis, or other data from experiments in which

plant growth was assessed after phytase addition to sandy soil substrates or agar medium (Findenegg and Nelemans, 1993; Richardson and Hayes, 2000). In a preliminary experiment using a soil from the Federal Research Station Reckenholz (Zurich, Switzerland), however, no significant difference in total P concentrations in leaves was measured between

7.2 Plants secreting phytase 7 Conclusions and outlook 77

transgenic and wild-type plants (data not shown). Given the high rates of phytate hydrolysis measured when incubating plants in a solution containing phytate (see figure 6.4), one should assume that the phytase activity in exudates is not limiting. Knowing that soils generally contain phytate concentrations in the range of 50 - 400 mg / kg soil, representing 10-35% of the total P in soils, the potential P that could be released from phytate is in the same order of magnitude. The calculated total P uptake by the plants in this pot experiment

* (~ [20 g plant DW] [2000 mg P / kg DW]) is around 40 mg, which is far below the amounts which could theoretically be taken up by the plants if all the phytate was degraded and the P was available for plant uptake. The results of this experiment using a natural soil thus suggest that the availability of phytate for enzymatic hydrolysis is limiting the P mobilization from phytate by the enzyme in the conditions used, or that, alternatively, the enzymatic cleavage takes place to a certain extent, but the Pi released is rapidly fixed to the soil and only part of it remains available for plant uptake. Findenegg and Nelemans (1993) showed that commercial phytase added to the growth substrates of plants had an effect on plant growth only in those substrates to which soluble phytate had been added prior to the experiment. Added phytase did not stimulate P-uptake and growth when no phytate was added to the soil. The addition of very high amounts of phytase (10'000 U / kg soil, which is three orders of magnitude higher than soil phytase activity) did not result in increased P- uptake from three soils tested, but in one soil it resulted in a more than two-fold higher uptake of P by the plants. However, in the latter case, it was not known whether the increased uptake resulted from the activity of the phytase or from the P contamination (153 umol P per pot) in the solution of phytase added. Since the recovery rate of P in this soil was much higher than that which is usually measured for P (69% as compared to 10%), the authors concluded that the increased P uptake resulted from an increase in the hydrolysis rate of soil phytin.

The results from the present work are not sufficient to confirm one or the other of the two hypotheses, but in any case do not seem to contradict the general contention that the soil solubility of phytate determines its availability for plant uptake. Should this be the case, the effectiveness of a recombinant phytase secreted from plant roots would certainly be increased if the plant had the additional capacity to increase the availability of Po and phytate in the soil. The next chapter will discuss possibilities to genetically engineer plants towards that end.

7.3 Designing plants more effectively mobilizing P

Based on our current knowledge and experience, and on our understanding of plant P metabolism, it is time to think about developing a "super-mobilizing-transporting-recycling" crop plant, or more simply, a "lupinized" plant. This idea is not new, but first positive experiences in this field are encouraging to the development of such a crop. Figure 7.1 shows a model of such a plant, as well as possible requirements to achieve increased P mobilization efficiency, uptake and recycling.

7.2 Plants secreting phytase 7 Conclusions and outlook 78

P retranslocation

P sensing & signaling

phytosiderophores

Enzymes • phytase

• RNase

• alkaline phosphatase • acid phosphatase Proteoid roots • apyrase

Figure 7.1 Model of a "lupmized" plant, with addition of mycorrhizal association and elements in P sensing and signalling, P retranslocation within the plant and P metabolism

The expression of a synthetic phytase in root hairs of potato resulted in an increased

mobilization of P from phytate added to the growth substrate. It appears, however, from the data obtained, that the effect may be lower in a natural soil, where phytic acid is not soluble, but bound to the soil matrix. The particularly high adsorption of phytic acid to different soils has been demonstrated previously (McKercher and Anderson, 1989). Some soils, especially those with high organic matter content, exhibited lower adsorption profiles, suggesting that organic matter, and by extension the generally increased biological activity, may affect the adsorption of phytic acid to soil minerals (McKercher and Anderson, 1989).

It is known that plants can significantly modify the organic content and biological activity of soils by secreting organic compounds and therefore encouraging the development of the rhizosphere microflora (Zhang et al., 2000a; Nardi et al., 2002). Furthermore, lupin plants are particularly effective in secreting organic acids in root clusters, thereby concentrating exudates in a relatively small volume (Neumann and Martinoia, 2002). The concomitant secretion of phosphatases, organic acids and protons in root clusters results in the establishment of miniature "P mining factories". It is believed that organic acids such as

7.3 Designing plants more effectively mobilizing P 7 Conclusions and outlook 79

citrate play a role in making sources of organic P more effectively available to enzymatic degradation. To be effective, this process is expected to require a minimum concentration of organic acids, together with sufficiently high phosphatase and phytase activities. This could explain why lupin is more effective in P mobilization than other plants equally secreting both phosphatases and organic acids under P deprivation. By analogy, one would expect transgenic plants secreting a fungal phytase in high amounts to be more effective in P uptake if they were additionally engineered for increased organic acid secretion. The latter has been shown not to be simply done by the expression of a bacterial citrate synthase in the cytosol

(Delhaize et al., 2001). The expression of a mitochondrial citrate synthase gene, however, was shown to improve the P acquisition efficiency of Arabidopsis plants (Koyama et al., 2000). One can speculate whether in this case the effect was also a function of the chosen growth condition. In fact, it is known that aluminum ions induce the secretion of citric and malic acids by activating a citrate-permeable anion channel (Kollmeier et al., 2001; Zhang et al., 2001). The soil used in the experiment reported by Koyama et al. (2000) was rich in Al- phosphates, which could explain that in this soil an effect of increased citrate production and subsequent secretion could be measured. A more robust and reproducible strategy may be to additionally express genes encoding di- and tri-carboxylate carriers directed to the mitochondrial and cell membranes. Such carriers have been identified in Arabidopsis mitochondria (Picault et al., 2002). Even artificial membrane channels are currently being designed, including switches (Bayley, 1999), which could be used for increased secretion of citric and malic acids.

Other chelating agents like phytosiderophores could play a role in rendering organic P available for enzymatic hydrolysis. The study of the effects of exudates from roots of elephantgrass (Pennise clundestinum L., cv. Nayier 62), which is very efficient in utilizing P in acid soils, revealed that rhizosphere acidification and phosphatase activity could not account for this efficiency. Exudates contained high amounts of pentanedioic acid, a phytosiderophore, in response to P deficiency, and the presence of pentanedioic acid correlated with improved P uptake (Shen et al., 2001). Transgenic plants secreting higher amounts of phytosiderophores have been obtained and had an increased capacity to take up Fe (Takahashi et al., 2001a). It is not known whether these plants exhibited higher capacities to mobilize and take up P.

In lupin, increased proton secretion was observed in root clusters under P-deficient conditions (Yan et al., 2002). It was suggested that the activation of H+-ATPase is instrumental in the acidification of the rhizosphere by active proteoid roots. Whether acidification is essential for P mobilization is not clear. The overexpression of a H+-ATPase in plant roots could thus mimic what is observed in cluster roots.

Introducing mycorrhization capability to plants like lupin or Arabidopsis may sound like a fancy idea. However, since the control mechanisms allowing mycorrhization by different fungi are not yet well understood, the assumption can be made that non-mycorrhizal plants may have lost the ability to be mycorrhizal during evolution, and that this loss-of-function could be related to a limited number of genes, possibly even a single gene. Reactivation or reintroduction of such a gene in non-mycorrhizal plants may provide the means to allow fungi to enter mutualistic association with its host.

7.3 Designing plants more effectively mobilizing P 7 Conclusions and outlook 80

The modification of retranslocation of P throughout the plant, altering of P sensing patterns, and changing metabolic pathways to less expensive ones in terms of P are all theoretically possible ways to improve the P utilisation efficiency of crops. In the last resort, the dream plant of a plant P nutritionist would embrace increased efficiency of P mobilization from several P sources in the soil, more efficient transport and allocation, and higher P use efficiency within plant metabolism.

7.4 Phosphatases, phytases and beyond

Taking into account the large number of publications dealing with phosphatase secretion from plant roots and phosphatase activities in the soil, one would expect the current knowledge to be large enough to yield a clear concept in terms of the contribution of phosphatases to plant nutrition. Unfortunately, throughout the years, new data have either confirmed or questioned previous findings in new experimental setups, but have not necessarily shed new light in understanding the processes involved (Figure 7.2). This lack of significant progress is certainly not due to uncreative research strategies, but much more to the lack of adequate tools for rhizosphere investigation, and undoubtedly also to the complexity and variability of the rhizosphere. In fact, results obtained in a particular soil condition may not hold true for another closely related soil, not to mention other, unrelated soil types. The reductionist approach, where individual processes were studied in tightly controlled and simplified systems, resulted in interesting discoveries, but often failed at the integration step into the natural, complex system. Obviously, there is a conceptual black hole in rhizosphere research.

Figure 7.2 Current concept of the role of phosphatases in the rhizosphere phosphatases are secreted into the rhizosphere and interact with scarcely available sources of organic P (P-org ) The soil matrix and colloids exert electrostatic interactions with enzymes, resulting in immobilisation on mineral surfaces (not shown) Organic P is located either in soil solution or in complex soil-humic complexes The uptake of Pi occurs via P transporters in conjunction with the activity of proton- secreting pumps (H+-ATPases) The rhizoplane contains scores of living organisms that also release phosphatases, phytases and other hydrolases

7.4 Phosphatases, phytases and beyond 7 Conclusions and outlook 81

Recently developed technologies in enzymology, soil sciences and root biology may help to fill the gap between basic process understanding in plants and in soil, and in the integration of both disciplines in a more complex (or natural) system. For example, the use of the APIZYME technology (BioMérieux, Lyon, France), which allows to screen for at least 20 enzymatic reactions, would rapidly give a profile of enzymatic activities in a given system. Using this method, one could then modify single parameters in the system and check the

profile for modifications. This would be a kind of soil enzyme microarray allowing the rapid detection of changes in individual enzymatic activities. A complex system cannot be understood only by reduction to its single elements, but also requires the collection of large databases of information that give some sort of fingerprint profiles for each studied system. Such fingerprint profiles would cover the fields of enzymology, microbial species diversity, mineralogy, perhaps soil organic P by using P-NMR studies in rhizo, and of course plant gene expression profiling. The computing of these different profiles into a single database, and the comparison of databsets from strictly controlled experiments by statistical analysis can be expected to yield valuable information on processes in the rhizosphere. Some of these profiles can already be achieved. As mentioned, enzyme-substrate arrays are already available on the market (Templin et al., 2002). Species diversity can be assessed for example by molecular identification of the ITS2 region of rDNA in microorganisms (Jansa et al., 2002) or by using other standard tests for bacterial identification, such as API strips from

BioMérieux. A rapid mineralogical profile may be more difficult to achieve, but is certainly

possible. The profiling approach could be extended to other parameters, but this would go

beyond the scope of this analysis.

Figure 7.3 An imaginable concept of rhizosphere research

7.4 Phosphatases, phytases and beyond 7 Conclusions and outlook 82

The combination of such data with molecular biological tools to modifiy root properties, as well as the study of genes having a function in the root-soil interface can be expected to open new fields in rhizosphere research. For example, the expression of a synthetic phytase in root hairs of potato allows the setup of novel experimental systems for the study of lnsP6 availability and mobilization in soils. The wild-type and transgenic plants differ in a single but important trait, allowing the study of mechanisms involved in P mobilization and uptake in better controlled systems. This is but one example. One can further imagine the development of transgenic plants secreting a large number of substances that would affect biological activity, biochemistry, and chemistry of soils, including toxins, enzyme inhibitors, allelopathic substances, or maybe even sugars. After engineering plants with improved nutrient uptake capabilities, better resistance to pathogens, and higher quality, one could think of engineering the rhizosphere to increase the quality of soils, not only by phytoremediation, but much more for increasing the biological activity, by improving the quality of life of those little beings which we do not see, but who do more for us than we generally assume.

7.4 Phosphatases, phytases and beyond 8 References 83

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g %J Appendix

Appendix 1. DNA and amino acid sequences of StPAPI

Appendix 2. DNA and amino acid sequences of StPAP2

Appendix 3. DNA and amino acid sequences of StPAP3

Appendix 4. DNA and amino acid sequences of the SP/PHY chimeric gene

Appendix 5. Analysis of the signal sequence of SP/PHY 9 Appendix 98

Appendix 1. DNA and amino acid sequences of StPAPI

1 MASMKILNIFI

1 TCGGCACGAGTTCTTNTCTAAAAATATATGGCTTCCATGAAAATACTCAACATTTTCATT

12 SFLLLLLFPAAMAELHRLEH

61 AGTTTCTTGTTGTTGTTACTATTTCCGGCAGCCATGGCTGAGCTCCACCGGTTAGAACAT

32 PVNTDGSISFLVVG WGRRG

121 CCGGTGAACACCGACGGCTCGATTAGTTTTTTGGTCGTCGGAGATTGGGGAAGAAGAGGA

52 TFNQSQVAQQMGIIGEKLNI 181 ACCTTTAACCAATCTCAAGTTGCTCAACAAATGGGAATAATTGGAGAGAAATTAAACATA

72 DFVVSTG NF DDGLTGVDD

2 41 GATTTTGTTGTATCAACTGGAGACAATTTCTATGATGATGGATTGACTGGTGTGGATGAT

92 PAFEESFTNVYTAPSLQKNW 3 01 CCTGCCTTTGAGGAATCTTTTACCAATGTCTACACAGCTCCAAGCTTACAAAAAAATTGG

112 YNVLG HDYRGDALAQLSPI 3 61 TATAACGTTTTGGGGAACCATGACTACAGAGGTGATGCTTTAGCACAATTAAGTCCTATT

132 LKQKDNRWICMRSYIVNTDV 4 21 CT TAAGCAAAAGGATAACAGAT GGAT T T GTAT GAGGT CT TATAT T GT TAATACAGAT GT G

152 AEFFFVDTTPFQDMYFTTPK 4 81 GCAGAATTTTTCTTTGTAGATACAACTCCTTTTCAAGATATGTATTTCACAACTCCTAAA

172 DHTYDWRNVMPRKDYLSQVL 541 GATCATACTTATGATTGGAGAAATGTTATGCCTCGAAAAGATTATCTTTCCCAAGTTTTG

192 KDLDSALRESSAKWKIVVG

6 01 AAGGAT T T GGACT CAGCAT TAAGGGAATCAAGT GCAAAAT GGAAAATAGTAGT T GGT CAC

212 HTIKSAGHHGSSEELGVHIL

6 61 CACACCATTAAAAGTGCTGGACACCATGGTAGCTCTGAGGAGCTTGGAGTCCACATTCTT

232 PILQANNVDFYLNG D CLE 7 21 CCCATATTACAGGCAAACAATGTTGACTTTTACCTAAATGGGCATGACCATTGCTTGGAG

252 HISSSDSPLQFLTSGGGSKS 7 81 CATAT CAGCAGT T CAGATAGTCCACTACAAT T T T T GACAAGT GGTGGGGGTT CAAAAT CA

272 WRGDMNWWNPKEMKFYYDGQ 8 41 TGGAGGGGTGATATGAATTGGTGGAATCCAAAGGAAATGAAATTTTATTATGATGGACAA

292 GFMAMQITQTQVWIQFFDI F 9 01 GGATTTATGGCTATGCAAATTACTCAAACACAAGTTTGGATACAATTTTTTGACATTTTT

312 GNILHKWSASKNLVSIM-

9 61 GGAAACATTTTGCATAAATGGAGTGCATCAAAAAACCTTGTTTCCATTATGTAAACAACT

1021 CAAAATAAAAAAAAAT GTT GAACAAAAAATAGCCAAAAAGAAAT TAT GGAT T TAAT T T GT

1081 TTCTGCTAATTAGCAGTTAAATATTATCCTAATCATTGATGTAATTGTATCCAATATGTT

1141 CTATTGAAATATTTATTTATGTTAAATCTGAGTTTATTTGCAGTAAAAAAAAAAAAAAAA

12 01 AAAAAACTCGAGACTAGTTCTCTCCTNCGTGCCGAATTGCGGCCGCGAATTCCTGCAGCC 9 Appendix 99

Appendix 2. DNA and amino acid sequences of StPAP2

1 SGPTSGEVTSSFVRKIEKTIDMPLDSDVFR

1 TCCGGCCCAACTTCCGGAGAAGTCACCAGTAGTTTTGTTAGGAAAATTGAGAAGACAATTGATATGCCTCTGGATAGTGATGTCTTCCGT

31 VPPGYNAPQQVHITQGDHVGKAVIVSWVTM 91 GTTCCTCCTGGATATAATGCGCCTCAACAGGTTCATATAACACAAGGAGATCATGTGGGAAAGGCGGTAATTGTTTCATGGGTGACTATG

61 DEPGSSTVVYWSEKSKLKNKANGKVTTYKF

181 GATGAACCTGGTTCAAGTACAGTAGTATACTGGAGTGAGAAAAGCAAGCTAAAGAATAAGGCAAATGGAAAAGTTACTACCTATAAGTTT

91 YNYTSGYIHHCNIKNLKFDTKYYYKIGIGH

271 TATAACTATACATCTGGTTACATCCACCACTGCAATATCAAAAATTTGAAGTTCGATACCAAATACTACTATAAGATTGGGATTGGACAC

121 VARTFWFTTPPEAGPDVPYTFGLIGDLGQS 3 61 GTGGCACGAACCTTCTGGTTCACAACCCCTCCAGAAGCCGGCCCTGATGTACCCTATACATTTGGTCTTATAGGGGATCTTGGTCAGAGT

151 FDSNKTLTHYELNPIKGQAVSFVGDISYAD 4 51 TTCGATTCAAACAAGACACTCACACATTATGAATTAAATCCAATTAAGGGGCAAGCAGTGTCGTTCGTAGGCGACATATCTTACGCAGAT

181 NYPNHDKKRWDTWGRFAERSTAYQPWIWTA 541 AACTACCCAAATCATGACAAAAAAAGATGGGACACATGGGGAAGGTTTGCAGAGAGAAGTACTGCTTATCAACCTTGGATTTGGACAGCA

211 GNHEIDFAPEIGETKPFKPYTHRYHVPFRA

631 GGAAACCATGAGATAGATTTTGCTCCTGAAATTGGGGAAACAAAACCATTCAAGCCCTACACTCATCGGTATCATGTCCCATTCAGAGCA

241 SDSTSPLWYSIKRASAYIIVLSSYSAYGKY

7 21 TCAGACAGCACATCTCCACTTTGGTATTCAATCAAGCGAGCTTCAGCGTATATCATAGTTTTATCCTCATATTCAGCATATGGCAAATAC

271 TPQYKWLEEELPKVNRTETPWLIVLVHSPW 811 ACTCCTCAATACAAGTGGCTTGAGGAAGAGCTACCAAAGGTTAACAGGACTGAGACTCCGTGGCTGATTGTTCTAGTACATTCGCCATGG

301 YNSYNYHYMEGETMRVMYE PWFVQYKVNMV 9 01 TATAACAGCTACAACTATCACTATATGGAAGGGGAAACCATGAGAGTAATGTATGAACCATGGTTTGTACAGTACAAAGTGAATATGGTT

331 FAGHVHAYERTERI SNVAYNVVNGECS PI K

9 91 TTTGCAGGTCATGTTCATGCTTATGAACGAACGGAACGGATTTCTAATGTGGCCTACAACGTTGTCAATGGAGAATGCAGTCCTATTAAA

361 DQSAPIYVTIGDGGNLEGLATNMTEPQPAY 1081 GATCAGTCTGCTCCAATTTATGTAACAATCGGTGATGGAGGAAATCTTGAAGGCCTAGCCACCAACATGACAGAGCCACAACCAGCTTAC

391 SAFREASFGHATLAIKNRTHAYYSWHRNQD 1171 TCTGCATTCCGCGAGGCTAGTTTTGGACATGCCACTCTTGCCATCAAGAATAGAACTCATGCTTATTATAGTTGGCATCGTAATCAAGAT

421 GYAVEADKIWVNNRVWHPVDESTAAKS-

12 61 GGATATGCTGTGGAAGCTGATAAAATATGGGTTAACAACCGAGTTTGGCATCCAGTTGATGAGTCCACAGCAGCCAAATCATGATGATAT

1351 ACACGAAATTTCATCTATCTTTTCTTTCCTTTTCCTCAGTAACATTGTGCACTTGTTGATGAATAAACGTTTCATTATTTCAAGCTCTTG

14 41 CTGCCTCATAATTTGTTAAACGTCCATTTGGGACATGGCAGAAGAGTCATTGTGTGGTAAACGATAAAAACGTCGTAAAAGAAAATCGAA

1531 GGACATACATTTGTTCATATTACTTATTTATCCAAATTATAATTCTAATCATTAAAAAAAAAAAAAAAAAACTCGAGGGGGGGCCCGGTA

1621 CCCA 9 Appendix 100

Appendix 3. DNA and amino acid sequences of StPAP3

1 MLLHIFFLLSLFLTFIDNGSAGIT

3 GTTAGTGGAGAGGAGACAATGTTGCTTCATATCTTCTTTTTGTTATCTCTCTTTTTGACATTTATAGACAATGGGAGTGCTGGTATAACA

31 SAFIRTQFPSVDIPLENEVLSVPNGYNAPQ 93 AGTGCATTCATTCGAACTCAGTTTCCGTCTGTTGATATTCCCCTTGAAAATGAAGTACTTTCAGTTCCAAATGGTTATAACGCTCCACAG

61 QVHITQGDYDGEAVIISWVTADEPGSSEVR 183 CAAGTGCATATTACACAAGGTGACTATGATGGGGAAGCTGTCATTATCTCATGGGTAACTGCTGATGAACCAGGGTCTAGCGAAGTGCGA

91 YGLSEGKYDVTVEGTLNNYTFYKYESGYIH

27 3 TATGGCTTATCTGAAGGGAAATATGATGTTACTGTTGAAGGGACTCTAAATAACTACACATTCTACAAGTACGAGTCTGGTTACATACAT

121 QCLVTGLQYDTKYYYEIGKGDSARKFWFET 3 63 CAGTGCCTTGTAACTGGCCTTCAGTATGACACAAAGTACTACTATGAAATTGGAAAAGGAGATTCTGCACGGAAGTTTTGGTTTGAAACT

151 PPKVDPDASYKFGIIGDLGQTYNSLSTLQH 4 53 CCTCCAAAAGTTGATCCAGATGCTTCTTACAAATTTGGCATCATAGGTGACCTTGGTCAAACATATAATTCTCTTTCAACTCTTCAGCAT

181 YMASGAKSVLFVGDLSYADRYQYNDVGVRW 54 3 TATATGGCTAGTGGAGCAAAGAGTGTCTTGTTTGTTGGAGACCTCTCTTATGCTGACAGATATCAGTATAATGATGTTGGAGTCCGTTGG

211 DTFGRLVEQSTAYQPWIWSAGNHEIEYFPS 633 GATACATTTGGCCGCCTAGTTGAACAAAGTACAGCATACCAGCCATGGATTTGGTCTGCTGGGAATCATGAGATAGAGTACTTTCCATCT

241 MGEEVPFRSFLSRYPTPYRASKSSNPLWYA

7 23 ATGGGGGAAGAAGTTCCATTCAGATCGTTTCTATCTAGATACCCCACACCTTATCGAGCTTCAAAAAGCAGTAATCCCCTTTGGTATGCC

271 IRRASAHIIVLSSYSPFVKYTPQWHWLKQE 813 ATCAGAAGGGCATCTGCTCACATAATTGTCCTATCAAGCTATTCCCCTTTTGTAAAATATACACCTCAATGGCATTGGCTGAAACAGGAA

301 FKKVNREKTPWLIVLMHVPIYNSNEAHFME

9 03 TTTAAAAAGGTGAACAGAGAGAAAACTCCTTGGCTTATAGTCCTTATGCATGTTCCTATCTACAACAGTAATGAAGCCCATTTCATGGAA

331 GESMRSAYERWFVKYKVDVI FAGHVHAYER

9 93 GGGGAAAGCATGAGATCCGCCTACGAAAGATGGTTTGTCAAATACAAAGTCGATGTGATCTTTGCTGGCCACGTCCATGCTTATGAAAGA

361 SYRISNIHYNVSGGDAYPVPDKAAPIYITV

1083 TCATATCGCATATCTAATATACACTACAATGTCTCGGGTGGTGATGCTTATCCCGTACCAGATAAGGCAGCTCCTATTTACATAACTGTT

391 GDGGNSEGLASRFRDPQPEYSAFREASYGH 117 3 GGTGATGGAGGAAATTCAGAAGGTCTTGCTTCAAGATTTAGAGATCCCCAGCCAGAATATTCTGCTTTCCGTGAAGCCAGCTATGGTCAT

421 STLDIKNRTHAIYHWNRNDDGNNITTDSFT

12 63 TCCACTCTAGATATCAAGAATAGAACACATGCTATCTACCACTGGAATCGAAATGATGATGGAAATAACATTACAACTGACTCATTTACA

451 LHNQYWGSGLRRRKLNKNHLNSVISERPFS 1353 TTGCACAACCAGTATTGGGGAAGTGGTCTTCGCAGGAGAAAGTTGAACAAGAATCATCTAAACTCTGTCATTTCCGAAAGGCCCTTCTCT

481 A R L -

14 4 3 GCGCGACTCTGAGACACAGTTTCTCAAACATGTTAGTCAAACTATGGGTAATTTTATGCCATGATCCTAGTATGTAGTTATATTATAAAA

1533 TCTATCTACTTTTGTTGGAGAGAGTGGATCAAGCTATTTTCCCAGTGTATTGGTTCATGTAAAATAAGGATTTGTGTTGTTTATATGACA

162 3 GCATTATGGAAAGTATAGCTCTTGTAAATTTGAAATAGCTACTTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

1713 AAAAAAAAAAAAAAAAAAAAA 9 Appendix 101

Appendix 4. DNA and amino acid sequences of the SP/PHY chimeric gene

GGATCCATGGCTAGAAAAGATGTTGCCTCCATGTTTGCAGTTGCTCTCTTCATTGGAGCA Start methionines of 21 f*'*k*'rV'l'**f'i *"*'* '••'">l<Äx%^^^^B S | G H S C D 61 TTCGCTGCTGTTCCTACGAGTGTGCAGTCCATCGGCGTATCCATGGGCCACTCCTGCGAC ß-1,3 glucanase signal sequence and of the 41 TVDGGYQCFPEISHLWGTYS 121 ACCGTGGACGGCGGCTACCAGTGCTTCCCGGAGATCTCCCACCTCTGGGGCACCTACTCC modified sequence of

61 PYFSLADESAISPDVPDDCR Consensus-phytase 181 CCGTACTTCTCCCTCGCCGACGAGTCCGCCATCTCCCCGGACGTGCCGGACGACTGCCGC (see chapter 4.2)

81 VTFVQVLSRHGARYPTSSKS 2 41 GTGACCTTCGTGCAGGTGCTCTCCCGCCACGGCGCCCGCTACCCGACCTCCTCCAAGTCC lii: Signal peptide (SP)

101 KAYSALIEAIQKNATAFKGK for secretion 3 01 AAGGCCTACTCCGCCCTCATCGAGGCCATCCAGAAGAACGCCACCGCCTTCAAGGGCAAG

121 YAFLKTYNYTLGADDLTPFG sequence following 3 61 TACGCCTTCCTCAAGACCTACAACTACACCCTCGGCGCCGACGACCTCACCCCGTTCGGC SP in ß-1,3 glucanase 141 ENQMVNSGIKFYRRYKALAR 421 GAGAACCAGATGGTGAACTCCGGCATCAAGTTCTACCGCCGCTACAAGGCCCTCGCCCGC

161 KIVPFIRASGSDRVIASAEK Bold: mutations in AA 4 81 AAGATCGTGCCGTTCATCCGCGCCTCCGGCTCCGACCGCGTGATCGCCTCCGCCGAGAAG sequence: 181 FIEGFQSAKLADPGSQPHQA 541 TTCATCGAGGGCTTCCAGTCCGCCAAGCTCGCCGACCCGGGCTCCCAGCCGCACCAGGCC

• I of 201 SPVIDVIIPEGSGYNNTLDH ß-1,3 glucanase 6 01 TCCCCGGTGATCGACGTGATCATCCCGGAGGGCTCCGGCTACAACAACACCCTCGACCAC changed to S

221 GTCTAFEDSELGDDVEANFT

6 61 GGCACCTGCACCGCCTTCGAGGACTCCGAACTCGGCGACGACGTGGAGGCCAACTTCACC • S of consensus

241 ALFAPAIRARLEADLPGVTL phytase changed to G 721 GCCCTCTTCGCCCCGGCCATCCGCGCCCGCCTCGAGGCCGACCTCCCGGGCGTGACCCTC

261 TDEDVVYLMDMCPFETVART Arrow: cleavage site of 7 81 ACCGACGAGGACGTGGTGTACCTCATGGACATGTGCCCGTTCGAGACCGTGGCCCGCACC signal peptide 281 SDATELSPFCALFTHDEWIQ 8 41 TCCGACGCCACCGAACTCTCCCCGTTCTGCGCCCTCTTCACCCACGACGAGTGGATCCAG

301 YDYLQSLGKYYGYGAGNPLG 901 TACGACTACCTCCAGTCCCTCGGCAAGTACTACGGCTACGGCGCCGGCAACCCGCTCGGC

321 PAQGVGFANELIARLTRS PV 961 CCGGCCCAGGGCGTGGGCTTCGCCAACGAACTCATCGCCCGCCTCACCCGCTCCCCGGTG

341 QDHTSTNHTLDSNPATFPLN 1021 CAGGACCACACCTCCACCAACCACACCCTCGACTCCAACCCGGCCACCTTCCCGCTCAAC

361 ATLYADFSHDNSMISIFFAL

1081 GCCACCCTCTACGCCGACTTCTCCCACGACAACTCCATGATCTCCATCTTCTTCGCCCTC

381 GLYNGTAPLSTTSVESIEET

1141 GGCCTCTACAACGGCACCGCCCCGCTCTCCACCACCTCCGTGGAGTCCATCGAGGAGACC

401 DGYSASWTVPFAARAYVEMM

1201 GACGGCTACTCCGCCTCCTGGACCGTGCCGTTCGCCGCCCGCGCCTACGTGGAGATGATG

421 QCQAEKEPLVRVLVNDRVVP 1261 CAGTGCCAGGCCGAGAAGGAGCCGCTCGTGCGCGTGCTCGTGAACGACCGCGTGGTGCCG

441 LHGCAVDKLGRCKRDDFVEG

1321 CTCCACGGCTGCGCCGTGGACAAGCTCGGCCGCTGCAAGCGCGACGACTTCGTGGAGGGC

461 LSFARSGGNWAECFA-LIKR

1381 CTCTCCTTCGCCCGCTCCGGCGGCAACTGGGCCGAGTGCTTCGCCTGATTAATTAAGAGA 9 Appendix 102

Appendix 5. Analysis of the signal sequence of SP/PHY

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l l l i i l i 20 30 40 50 60 Positon

Change from polar to apolar

Polarity apppaapaaaaaaaaaaaaaaappappaaaapaa

Charge 0++-000000000000000000000000000000

AA sequence MARKDVASMFAVALFIGAFAAWTSJvja^ IGVCYGV

, cleavage h site

The general model describes three domains: a positively charged, polar domain (n), a neutral and hydrophobic core (h) and a neutral, polar domain close to the cleavage site (c). The algorithm for signal sequence prediction has the following requirements:

1. apolar (hydrophobic), uncharged core surrounded by two polar (hydrophilic) sequences.

2. Clear change from polar (signal sequence) to apolar (mature protein) at cleavage site

3. Last amino acid usually S, third last usually V.

The SP/PHY construct fulfils all the theoretical requirements and was tested positive in all four tests performed by the SignallP prediction server

(http://www. cbs. dtu. dk/services/SignalP-2.0/). 10 Acknowledgements 103

1 \ß Acknowledgements

I wish to thank:

Dr. Marcel Bücher for giving me a good start in molecular biology and his continuous support throughout the project. The creative discussions, the freedom given me to try also personal ideas, and the help during dissertation writing are particularly appreciated.

Prof. Dr. Emmanuel Frossard for providing me the opportunity to pursue a PhD in his group and giving me a good and agréable place to work. Thanks also for the advices, suggestions, and conceptual ideas during thesis writing.

Prof. Dr. Nikolaus Amrhein for his support and for the good questions raised during seminars, resulting in improved experiments. Thank you for the great contribution in language style and scientific precision of content to this thesis.

Dr. Markus Wyss for providing us with the consensus phytase gene and protein samples, the good scientific discussions during the early processes of patenting, and finally for his detailed corrections and critical review of the phytase manuscript and of the whole thesis. The good collaboration with Roche Vitamins Ltd is greatly appreciated. Thanks also to Martin Lehmann for investing time and energy to synthesize a consensus phytase with maize codon usage.

My colleagues from the Bucher lab, Gerardo Zardi, Christine Rausch, Réka Nagy, Pierre Daram, Silvia Brunner, Volodya Karandashov, Sarah Wegmüller, and Cyril Steiner for the good atmosphere in the lab and their help in learning new methods in plant molecular biology.

My colleagues from the "Frossard Group" for the good times we spent together, in the office and elsewhere. Special thanks to Theres Rösch and Thomas Flura for their help in ICP analysis, and to Christiane Gujan for readily organizing the administrative parts of the project. Special thanks also to Jan Jansa, who, next to being a good scientific collaborator, always remained a dynamic and interesting friend.

Christophe Zeder and Prof. Dr. Richard Hurrell from the group of Human Nutrition, ETH Zurich, for their great help in HPLC analysis.

My wife Damaris, who always stood by my side, ever trying to help and understand; her constant support and encouragements, throughout the whole period of the thesis and despite my frequent absences from home, were greatly appreciated.

Both our families Zimmermann and Oertle, who always provided me with optimism and happiness by their cheerful receiving us at their homes.

The financial support of the Swiss Federal Institute of Technology (ETH) Zurich, and of the Hochstrasser Stiftung for a three months prolongation. 104 11 Curriculum vitae 105

11 Curriculum vitae

Name: Philip Zimmermann

Date of birth 31st January 1974

Citizenship Swiss, from Charmoille, JU

07.1999-today PhD thesis at the ETH Zurich. Project as a collaboration between the groups of Plant Nutrition (Prof. Dr. E. Frossard) and Plant Biochemistry and Physiology (Prof. Dr. N. Amrhein)

12.1998-06.1999 Training in molecular biology methods in the lab of Dr. Marcel Bucher, ETH Zurich, Group of Prof. Dr. N. Amrhein.

10.1998- 12.1998 Training course at the SRVA (Service Romand de Vulgarisation Agricole) in Lausanne

04.1998-09.1998 Diploma thesis at the Texas A&M University in Lubbock, TX, in the group of Soil Physics (Prof. Dr. R.J. Lascano). Awarded with the ETH medal.

10.1992-04.1998 Studies as agronomist in the Department of Food Science and Agriculture, ETH Zurich

07.1996- 11.1996 Training course in the Ramat Negev Agro-Research Station, Israel.

1989- 1992 Lycée Cantonal, Porrentruy, Type C, June 1992

1986- 1989 Ecole secondaire, Porrentruy, Switzerland

1984- 1986 Schweizer Sekundärschule, Papua New Guinea

1980- 1984 Primary School, Papua New Guinea

31st January 1974 Born in Kassam, Eastern Highlands Province, Papua New Guinea