Research Collection
Doctoral Thesis
Root-secreted phosphomonoesterases mobilizing phosphorusfrom the rhizosphereA 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 phosphatases 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 purple acid phosphatases 10
2.3 Inositol phosphates and phytases 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 phytase 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 / fungiAmpr ampicillin resistent
ANOVA analysis of variance
APase acid phosphataseBLAST basic local alignment search tool
bp basepair(s)BSA bovine serum albumin
CaMV cauliflower mosaic virus
cDNA complementary DNA
CIP (CIAP) calf intestinal (alkaline) phosphataseddH20 double distilled water
dH20 deionized water
DNase deoxyribonucleaseDTT dithiothreitol
DW dry weightDMF dimethylformamidedNTP deoxynucleoside triphosphateEDTA sodium ethylenediaminetetraacetateEST expressed sequence tagEtOH ethanol
GFP green fluorescent proteinGFP buffer glyoxal / formamide / phosphate buffer
GPI glycosylphosphatidylinositolGUS ß-glucuronidaseH PLC high performance liquid chromatographyICP inductively coupled plasmaKanr kanamycin resistent
kbp kilobasepair(s)M ES 2-morpholinoethanesulfonic acid monohydratemRNA messenger RNA
MS medium Murrashige and Skoog medium
NaAc sodium acetate
OD optical densityORF open reading frame
PCR polymerase chain reaction
P phosphorusPAP purple acid phosphatasePase phosphatasePi orthophosphate (inorganic phosphate)PDEase phosphodiesterasePMEase Phosphomonoesterase
pNPP p-nitrophenyl phosphatePo organic phosphateRACE rapid amplification of cDNA ends
RNase ribonuclease
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'enzyme 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
1I 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 enzymes (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
scavenging enzymes
Modification of
redox-potential
Induction of
high-affinityP transporters
"^
Secretion of phenolics
Production of anthocyanins
Secretion of organic acids
Secretion of phosphatases
H+-release or uptake
Secretion of
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, phosphodiesterases, diadenosine tetraphosphatases, exonucleases, 5'-
nucleotidases, 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 ribonucleases (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
0 2 4 6 8 1Ü 12 14
Distance from root surface (mm)
'J Sil85
6.
2 x
1300
1200
1100
1000
900
800
—9
Q 2 4 6 8 10 12 14
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 active site. 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 alkaline phosphatase (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 binding site (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
H203P0^5
H203PO
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).
pK2 pKj pK*
1.5 1.5 1.7
pK,; pKz pK#
2.1 5.7 6.9
pK« pK|j pK,2
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
(adapted from Costello et al, 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.
pK,
1.1
pKs
2.1
pK»
7.6
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 -
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
10 20 30 40 50 60
P in final solution (ug P ml1)
70
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 complexModified 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.
3-phytase
(EC 3.1.3.8)H203PO
H203PO
H203PO
OP03H2
OP03H2
H203PO
H203PO
H203PO
OP03H2
1 OP03H2H203PO
1 -D-myo-inositol hexatosphosphate
6-phytase
(EC 3.1.3.26)
1-D-myo-mositol 1,2,4,5,6-penta/flsphosphate
H203PO
H,0,PO
H203PO
OP03H2
3 OP03H2
1-L-myo-mositol 1,2,3,4,5-pentatosphosphate
(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-configurationModified 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 hydrolases 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 protein phosphatase (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
(Nakano et al, 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 ligase (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 Nuclease (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
Antibiotics (kanamycin, ampicillin,
gentamycin, rifampicin)
a-naphthyl phosphate
ß-naphthyl phosphate
Biomax MS film
BSA (fraction V)
Daichin agar
Fast Black K
Fast Blue BB
Fast Red TR
Glyoxal 40%; 7.1 M Ultrapure
Difco (New Jersey, USA)
Sigma (Fluka, Buchs, Switzerland)
Fluka (Buchs, Switzerland)
Fluka (Buchs, Switzerland)
Kodak (product ordering via Integra Biosciences,
Eschenbach, Switzerland)
Serva (Catalysis, Wallisellen, Switzerland)
Brunschwig Chemie (Basel, Switzerland)
Fluka (Buchs, Switzerland)
Fluka (Buchs, Switzerland)
Serva (Heidelberg, Germany)
Clontech (Allschwil, Switzerland)
4.1 Materials and chemicals
4 Materials and methods 33
Hybond NX membrane
Meat peptone
MicrospinTM S-400 HR columns
MS salts
Nitro-cellulose membranes
Phytic acid (from corn and rice)
Protease inhibitor cocktail
Timenten
Tryptone, yeast extract
X-Gluc
Zeatin riboside
Amersham Biosciences (Dübendorf, Switzerland)
Life Technologies Inc. (Basel, Switzerland)
Amersham Biosciences (Dübendorf, Switzerland)
Sigma (Fluka, Buchs, Switzerland)
Schleicher & Schuell (Dassel, Germany)
Sigma (Fluka, Buchs, Switzerland)
Sigma (Fluka, Buchs, Switzerland)
Smith Kline Beecham Pharmaceuticals (Thoerishaus,
Switzerland)
Difco (New Jersey, USA)
Biosynth AG (Staad, Switzerland)
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 glycine20 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
Plexiglasshield
Temperatureand humidity
sensor
Temperaturesensor for
'
root chamber
Root growthchamber
Electronic
control unit
Air-flow
propeller
Nutrient
solution
reservoir
Water
pump
Alternative
outlet
Atomizing disc
Control valve
UV sterilizing lamp
Cooling unit
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
Neo I Smal
Smal'
Xbal '
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
=; 13000
TO
c
•2> 8000CO
3000
-2000
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
lnsP3 lnsP5
lnsP4
lnsP6
123456789
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 purpleacid 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 23 4
1
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|JVAAF6 0315 1 MAVYSGISMVLCLWVGWFGVCLASAIVELPTFHHPTKGDGSJSfIvStPAPI 1 KYMASMKILNIFISFLLLLLFPAAMAELHRLEHPVNTDGslsEjlvAAF60316 1 MGTQRSKPSCTIVAIFLAFCCFVSSSKAKLESLQHAPKADGsjsFjjVAAF60317 1 MAGLG¥WLAFIGVCFLNVSALLQRLEHPVKADGs|jsL|V
AAL34 937 1 MAVALALLAAMSALSSCTSPATAELTRHEHPVAAGApIrlIv
P1368 6 1 MDMWTALLILQALLLPSLADGATPaIreÏaNP 485726 1 MNLKRRQFLFLSSLSAVGTGLLAWKFAHKYYQSSDLAIASPPKKDLlIreÏs
AtPAP17
AAF60315
StPAPI
AAF60316
AAF60317
AAL34937
P13686
NP 485726
-RRBSFNQS LVAYŒ
-RKjDYNQS QVAFQ
-RF*TFNQS---QVAQQ|-RKjAYNQS LVAFQ
-RKjTYNQS EVSAQ
-rkIgynqt—rvaeq!
gvpn|pfhtaremanake|artvqil&t[argqy—avara|||tlyhkqnpy
GKIGEKIDL
GEIGDQLAI
GIIGEKLNI
GVIGEKLDV
GRVGAKLNI
GKVAEETEI
LFSEHDPN
LTGEHDD,
LTGVDDP,
LTGVFDPS
LSGVDDP
LAGVDDM,
vqdindk:
EIEKVNA
ÏEQS
|TES
EES
EES
ELS
HDS
jERP|fcg
AtPAP17
AAF60315
StPAPI
AAF60316
AAF60317
AAL34937
P13686
NP 485726
SYTAPSLQKQ-fi--Ytaeslqkq|ysvl|
loi tnIytapslqkn-IynvlI
106
103 SNiYTAPSLQKQjYSVLl
ytapslqkk-IynvlIytakslqkq-Iysvlytaqslhkp-1ylvl|fsdrslrkvpIyvl.
lkqg vkj§qacl|
jLSSVLREIDSRWICLRSl|lsshlrkldsrwpclrs||lspilkqkdnrwicmrs|jlSHVLRYRDNRWVCFRsi|LNTILQKIDPRWICQRs|jlDPALRKIDSRFICMRsiIIA—YSKISKRWNFPSP1
GDPQVRYPGFNMNGRRYI
WDAELVEMFF
1WNTETVDLFF
'IVNTDVAEFFF
(TLNSENVDFFF:IVDTEIAEFFF
:IVSAGIVDFFF
[YRLHFKIPQTNlITFRRDRVQFFA
AtPAP17
AAF60315
StPAPI
AAF60316
AAF60317
AAL34937
P13686
NP 485726
165 DTTPFVKEYYT
162 DTTPFVEEYFN
160 DTTPFQDMYFT
162 DTTPYVDKYFI
154 DTTPFVDKYFL
157 DTTPFQLQYWT
14 9 SVAIFMLDTVT
160 DTN
EADGHSYDWRAVPSRNSYVK-AL
SPE-HVYDWRGVFPQQTYTK-NV
TPKDHTYDWRNVMPRKDYLS-QV
EDKGHNYDWRGILPRKRYTS-NL
KPKDHTYDWTGVLPRDKYLS-KL
DPGEDHYDWRGVAPRDAYIA-NL
LCGNSDDFLSQQPERPRLTART
SNADWQN
lEVSjKSSKA:IeyaImkstt:
Ire s sa:
|rqstat|jkdsta:ikkstati
AAARE
QÜIekeIsssnap|
AtPAP17
AAF60315
StPAPI
AAF60316
AAF60317
AAL34937
P13686
NP 485726
224 IGHH
22 0 AGHH
219 AGHH
221 IGHH
213 IGHH
216 VSAH
209 IAEH
197 SGVY
dtkelneell
dtkelverll
sseelgvhil
dtqellihfl
dtqelirhll
dtqellelll|pthclvkql
SNQAFIKTFTl
CLQHMSDEDSPIC
SLEHISDDESPIC
CLEHISSSDSPLC
CLEHISSLDSSVC
CLEHISSTSSQI
CLEHISSRNSPI
NLQYLQDENG-Vi
SYERTRAIDG-TTl
SKAWRGDI
SKAWRGDV
SKSWRGDM
SKAWRGDT
SKAWKGDH
SKAWRGIF
INFMDPSKR—AGNR
AtPAP17 284 NPVTINPKLLKFYYDGQ'
AAF60315 280 TMDRKGVSFFYDGQ'
StPAPI 27 9 N—WWNPKEMKFYYDGQ'
AAF60316 281 K—QSEGDEMKFYYDGQ'
AAF60317 273 L—IKMGKMGQRFTMMD'
AAL34 937 27 6 Q QNEDKLQFFYDGQ'
P13686 268 HQRKVPNGYLRFHYGTEDS
NP 485726 252 P VGRSKWTEYSTSD—
LGG
MSARFTHSDAEIVFYDVFGEILHKWVTSKQLLHSSV-
iMSVQLVETDIGIVFYGC
iMAMQITQTQVWIQFFDIFGNILHKWSAS-KNLVSIM-
(MSVHISQTQLRISFFDVFGNAIHKWNTC-KFDSSDM-
ILQVWRFKKSIPKLFIMIFLAKFCKLLICPRGYVMCMP
1LSLELSENRARFAFYDVFGEALYHWSFSKANLQKVQS
iAYVEISSKEMTVTYIEASGKSLFKTRLPRRARP
LSlATYEVYPDRIELNAIATNNRIFDRGIIRRVEVSGV-
AtPAP17
AAF60315
StPAPI
AAF60316
AAF60317
AAL34937
P13686
NP 485726
327 YNSLI—
32 9 SASVTEE
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
SGPTSGEVTSSStPAP2 1
IbPAP2 1 MGASRTGCYLLAWLAAVMNAAIAGITSS
GmPAPl 1 —MGWEGLLALALVLSACVMCNGGSSSP
PvPAPI 1 —MGWKGLLALALVLNWWSNGGKSSN
LaPAP2 1 MGYSSFVAIALLMSWWCNGGKTSTI
StPAP3 1 MLLHIFFLLSLFLTFIDNGSAGITS
StPAP2
IbPAP2
GmPAPl
PvPAPI
LaPAP2
StPAP3
HVE WSEKSKLKNKAN
WSENSQHKKVAR
WSENSDKKKIAE
WSEKNGRKRIAK
WSDSSLQNFTAE
GLSEGKYDVTVE
k|tt In T
n|rt T| In T
iJIvt R1J In S
kJIst Rjl In S
EjjjjFT Tl In T
t|nn T|«Be
StPAP2
IbPAP2
GmPAPl
PvPAPI
LaPAP2
StPAP3
StPAP2
IbPAP2
GmPAPl
PvPAPI
LaPAP2
StPAP3
StPAP2
IbPAP2
GmPAPl
PvPAPI
LaPAP2
StPAP3
StPAP2
IbPAP2
GmPAPl
PvPAPI
LaPAP2
StPAP3
StPAP2
IbPAP2
GmPAPl
PvPAPI
LaPAP2
StPAP3
StPAP2
IbPAP2
GmPAPl
PvPAPI
LaPAP2
StPAP3
81 LP Mil TE puÉMifl s
99 LPm TE ünSi s
97 LP
8_\
TE Sural s
7 0 LR SE liiflÉSÉl s
95 LP
95 FK
SE
EK si
Ie|YA|EA|WW
niyS ByaIea
m ^nn|tt
kiwvnIrvIhp jdestaaks—
smwvs|rfIhp Iddstttkl—slwsf|ry|hp Iddstahvsh-svwffBrhIyp IDDSTklwlf|ry|np Indstihip—sftlhBqybgsglrrrklnknhInsviserpfs
StPAP2
IbPAP2
GmPAPl
PvPAPI
LaPAP2
StPAP3 475 ARL
Figure 5.3 Multiple sequence alignment of HMW PAP ammo acid sequences from potato (StPAP2and 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
AtPAP12
lbPAP3
IbPAPI
AtPAPIO
StPAP2 |bPAP2N
PvPAPI.
GmPAPl~
LaPAP2
AtPAPU A1PAP25
I,
StPAP3
AtPAP5
1000
AtPAP20
AtPAP22
11000/
GmPhy
AtPAP15
AtPAP13
LaPAPI -
SoPAPI
974
1000/945 S60
HMW
1000
^1000728
_
StPAPI
/lOOO
1000
::;^~~~"~" AAF60316
NNs/*^^- AtPAP3
\ \\. AtPAPS
/999\ \. AAF60315
B27035 s^j4 / ,8111\ AtPAP17
pnme'V AVAA
\ AAF60317
P09889
AF292105AAL16925 / L16924 AAL34937
/ AAL16926
! |NP_485726
i I
I
Mammals Plants
LMW
Cyanobacteria
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 potatoPAPs 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
StPAPI
StPAP2
StPAP3
rRNA
rRNA
o
ooDC
OO
oo a
LU
ooa.
£
a.
E
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 mycorrhizationon 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)).
D
ct! 300 -
£
0 4 24
time (h)
*- 0.0.0-0.0.0.0.0.0.+ i
00©000'Bt'*t'Bt
(UrUJ2.QUU(?'fl!.2
stpAP2 • it 11 m • Äfc m mÛ. û. Û- Û. û. û.
£ û. Û. + +"*" ' ' -* Tt Tf ** Tl" Tt
CU fU -O -Q
StPAP2 Ü
Time (h)
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 tipsfrom 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 longitudinalroot 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
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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.
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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 presentin 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
aAN 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.
Name in
this
report:
Authors
A1 A2 A3 A4 A5 A6 A7 A8
IbPAPI IbPAPI SP-PAP1 -
lbPAP2 lbPAP2 SP-PAP2 SpPAPI
lbPAP3 lbPAP3 SP-PAP3 SpPAP2
SoPAPI Sp1
Spirodela
oligorrhizaPAP
LaPAPI La1La1,
LASAP1
LaPAP2 LASAP2
GmPAPl GmPAPl SB-PAP
GmPhy GmPhy
AtPAP17 AtACP5 StPAPI 7
PvPAPI PvPAPIKB-PAP,
Pvu
LaAPaseLupinAPase
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
SoPAPI
LaPAPI and 2, and LaAPase
GmPAPl and GmPhyAtPAP17
PvPAPI
sweet potato (Ipomoea batatas)duckweed (Spirodela oligorrhiza)white lupin (Lupinus albus)
soybean (Glycine max)
Arabidopsis thaliana
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
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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 secretingphytase
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
Proteoid roots
Enzymes• phytase• RNase
• alkaline phosphatase• acid phosphatase• 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
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9 Appendix 97
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
301 CCTGCCTTTGAGGAATCTTTTACCAATGTCTACACAGCTCCAAGCTTACAAAAAAATTGG
112 YNVLG HDYRGDALAQLSPI
361 TATAACGTTTTGGGGAACCATGACTACAGAGGTGATGCTTTAGCACAATTAAGTCCTATT
132 LKQKDNRWICMRSYIVNTDV
4 21 CTTAAGCAAAAGGATAACAGATGGATT TGTATGAGGTCT TATAT TGT TAATACAGATGTG
152 AEFFFVDTTPFQDMYFTTPK
4 81 GCAGAATTTTTCTTTGTAGATACAACTCCTTTTCAAGATATGTATTTCACAACTCCTAAA
172 DHTYDWRNVMPRKDYLSQVL
541 GATCATACTTATGATTGGAGAAATGTTATGCCTCGAAAAGATTATCTTTCCCAAGTTTTG
192 KDLDSALRESSAKWKIVVG
601 AAGGATT TGGACTCAGCATTAAGGGAATCAAGTGCAAAATGGAAAATAGTAGTTGGTCAC
212 HTIKSAGHHGSSEELGVHIL
661 CACACCATTAAAAGTGCTGGACACCATGGTAGCTCTGAGGAGCTTGGAGTCCACATTCTT
232 PILQANNVDFYLNG D CLE
7 21 CCCATATTACAGGCAAACAATGTTGACTTTTACCTAAATGGGCATGACCATTGCTTGGAG
252 HISSSDSPLQFLTSGGGSKS
7 81 CATATCAGCAGT TCAGATAGTCCACTACAATT T T TGACAAGTGGTGGGGGTTCAAAATCA
272 WRGDMNWWNPKEMKFYYDGQ
8 41 TGGAGGGGTGATATGAATTGGTGGAATCCAAAGGAAATGAAATTTTATTATGATGGACAA
292 GFMAMQITQTQVWIQFFDI F
9 01 GGATTTATGGCTATGCAAATTACTCAAACACAAGTTTGGATACAATTTTTTGACATTTTT
312 GNILHKWSASKNLVSIM-
961 GGAAACATTTTGCATAAATGGAGTGCATCAAAAAACCTTGTTTCCATTATGTAAACAACT
1021 CAAAATAAAAAAAAATGTTGAACAAAAAATAGCCAAAAAGAAATTATGGATT TAATT TGT
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
361 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
363 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
903 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
21 f*'*k*'rV'l'**f'i *"*'* '••'">l<Äx%^^^^B S | G H S C D
61 TTCGCTGCTGTTCCTACGAGTGTGCAGTCCATCGGCGTATCCATGGGCCACTCCTGCGAC
41 TVDGGYQCFPEISHLWGTYS
121 ACCGTGGACGGCGGCTACCAGTGCTTCCCGGAGATCTCCCACCTCTGGGGCACCTACTCC
61 PYFSLADESAISPDVPDDCR
181 CCGTACTTCTCCCTCGCCGACGAGTCCGCCATCTCCCCGGACGTGCCGGACGACTGCCGC
81 VTFVQVLSRHGARYPTSSKS
2 41 GTGACCTTCGTGCAGGTGCTCTCCCGCCACGGCGCCCGCTACCCGACCTCCTCCAAGTCC
101 KAYSALIEAIQKNATAFKGK
3 01 AAGGCCTACTCCGCCCTCATCGAGGCCATCCAGAAGAACGCCACCGCCTTCAAGGGCAAG
121 YAFLKTYNYTLGADDLTPFG
3 61 TACGCCTTCCTCAAGACCTACAACTACACCCTCGGCGCCGACGACCTCACCCCGTTCGGC
141 ENQMVNSGIKFYRRYKALAR
421 GAGAACCAGATGGTGAACTCCGGCATCAAGTTCTACCGCCGCTACAAGGCCCTCGCCCGC
161 KIVPFIRASGSDRVIASAEK
4 81 AAGATCGTGCCGTTCATCCGCGCCTCCGGCTCCGACCGCGTGATCGCCTCCGCCGAGAAG
181 FIEGFQSAKLADPGSQPHQA
541 TTCATCGAGGGCTTCCAGTCCGCCAAGCTCGCCGACCCGGGCTCCCAGCCGCACCAGGCC
201 SPVIDVIIPEGSGYNNTLDH
6 01 TCCCCGGTGATCGACGTGATCATCCCGGAGGGCTCCGGCTACAACAACACCCTCGACCAC
221 GTCTAFEDSELGDDVEANFT
6 61 GGCACCTGCACCGCCTTCGAGGACTCCGAACTCGGCGACGACGTGGAGGCCAACTTCACC
241 ALFAPAIRARLEADLPGVTL
721 GCCCTCTTCGCCCCGGCCATCCGCGCCCGCCTCGAGGCCGACCTCCCGGGCGTGACCCTC
261 TDEDVVYLMDMCPFETVART
7 81 ACCGACGAGGACGTGGTGTACCTCATGGACATGTGCCCGTTCGAGACCGTGGCCCGCACC
281 SDATELSPFCALFTHDEWIQ
8 41 TCCGACGCCACCGAACTCTCCCCGTTCTGCGCCCTCTTCACCCACGACGAGTGGATCCAG
lii:
Start methionines of
ß-1,3 glucanase signal
sequence and of the
modified sequence of
Consensus-phytase
(see chapter 4.2)
Signal peptide (SP)for secretion
sequence followingSP in ß-1,3 glucanase
Bold: mutations in AA
sequence:
• I of ß-1,3 glucanase
changed to S
• S of consensus
phytase changed to G
Arrow: cleavage site of
signal peptide
301
901
YDYLQSLGKYYGYGAGNPLG
TACGACTACCTCCAGTCCCTCGGCAAGTACTACGGCTACGGCGCCGGCAACCCGCTCGGC
321
961
PAQGVGFANELIARLTRS PV
CCGGCCCAGGGCGTGGGCTTCGCCAACGAACTCATCGCCCGCCTCACCCGCTCCCCGGTG
341
1021
QDHTSTNHTLDSNPATFPLN
CAGGACCACACCTCCACCAACCACACCCTCGACTCCAACCCGGCCACCTTCCCGCTCAAC
361
1081
ATLYADFSHDNSMISIFFAL
GCCACCCTCTACGCCGACTTCTCCCACGACAACTCCATGATCTCCATCTTCTTCGCCCTC
381
1141
GLYNGTAPLSTTSVESIEET
GGCCTCTACAACGGCACCGCCCCGCTCTCCACCACCTCCGTGGAGTCCATCGAGGAGACC
401
1201
DGYSASWTVPFAARAYVEMM
GACGGCTACTCCGCCTCCTGGACCGTGCCGTTCGCCGCCCGCGCCTACGTGGAGATGATG
421
1261
QCQAEKEPLVRVLVNDRVVP
CAGTGCCAGGCCGAGAAGGAGCCGCTCGTGCGCGTGCTCGTGAACGACCGCGTGGTGCCG
441
1321
LHGCAVDKLGRCKRDDFVEG
CTCCACGGCTGCGCCGTGGACAAGCTCGGCCGCTGCAAGCGCGACGACTTCGTGGAGGGC
461
1381
LSFARSGGNWAECFA-LIKR
CTCTCCTTCGCCCGCTCCGGCGGCAACTGGGCCGAGTGCTTCGCCTGATTAATTAAGAGA
9 Appendix 102
Appendix 5. Analysis of the signal sequence of SP/PHY
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l ! i *r "--"'^tv-.-t--^x-;"' ,/•....
G3*ttR<r»aVFAVALF1 &6FMVFTS/3S19/3XH903TCB3MCFPE 1SHWGTYSPVFSLAEESA
l l l i i l i
20 30 40 50 60Positon
Change from polar to apolar
Polarity
Charge
AA sequence
apppaapaaaaaaaaaaaaaaappappaaaapaa
0++-000000000000000000000000000000
IGVCYGV
, cleavagesite
MARKDVASMFAVALFIGAFAAWTSJvja^
h
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
\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 personalideas, 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 duringseminars, 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 goodcollaboration 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, alwaysremained 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.
1
104
11 Curriculum vitae 105
11 Curriculum vitae
Name:
Date of birth
Citizenship
Philip Zimmermann
31st January 1974
Swiss, from Charmoille, JU
07.1999-today
12.1998-06.1999
10.1998- 12.1998
04.1998-09.1998
10.1992-04.1998
07.1996- 11.1996
1989- 1992
1986- 1989
1984- 1986
1980- 1984
31st January 1974
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)
Training in molecular biology methods in the lab of Dr. Marcel
Bucher, ETH Zurich, Group of Prof. Dr. N. Amrhein.
Training course at the SRVA (Service Romand de Vulgarisation
Agricole) in Lausanne
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.
Studies as agronomist in the Department of Food Science and
Agriculture, ETH Zurich
Training course in the Ramat Negev Agro-Research Station,Israel.
Lycée Cantonal, Porrentruy, Type C, June 1992
Ecole secondaire, Porrentruy, Switzerland
Schweizer Sekundärschule, Papua New Guinea
Primary School, Papua New Guinea
Born in Kassam, Eastern Highlands Province,
Papua New Guinea