Download MAKING SURE HUNGRY PLANTS GET FED: THE DUAL-TARGETED PURPLE ACID PHOSPHATASE

MAKING SURE HUNGRY PLANTS GET FED:
THE DUAL-TARGETED PURPLE ACID PHOSPHATASE
ISOZYME AtPAP26 IS ESSENTIAL FOR EFFICIENT
ACCLIMATION OF ARABIDOPSIS THALIANA TO NUTRITIONAL
PHOSPHATE DEPRIVATION
by
Brenden A. Hurley
A thesis submitted to the Department of Biology
In conformity with the requirements for
the degree of Master of Science
Queen’s University
Kingston, Ontario, Canada
(August, 2009)
Copyright ©Brenden A. Hurley, 2009
Abstract
Acid phosphatases (APases; E.C. 3.1.3.2) catalyze the hydrolysis of phosphate (Pi) from Pi
monoesters and anhydrides within the acidic pH range. Induction of intracellular and secreted
purple acid phosphatases (PAPs) is a widespread plant response to nutritional Pi-deficiency. The
probable function of intracellular APases is to recycle Pi from expendable intracellular
organophosphate pools, whereas secreted APases likely scavenge Pi from the organically bound
Pi that is prevalent in most soils. Although the catalytic function and regulation of plant PAPs
have been described, their physiological function in plants has not been fully established. Recent
biochemical and proteomic studies indicated that AtPAP26 is the predominant intracellular
(vacuolar) and a major secreted purple APase isozyme upregulated by Pi-starved (-Pi)
Arabidopsis thaliana. The in planta function of AtPAP26 was assessed by molecular,
biochemical, and phenotypic characterization of a homozygous Salk T-DNA insertion mutant.
Loss of AtPAP26 expression resulted in the elimination of AtPAP26 transcripts and 55-kDa
immunoreactive AtPAP26 polypeptides, correlated with a 9- and 5-fold decrease in extractable
shoot and root APase activity, respectively, as well as a 40% reduction in secreted APase activity
of –Pi seedlings. The results corroborate previous findings implying that AtPAP26 is: (i) the
principal contributor to Pi starvation inducible APase activity in Arabidopsis, and (ii) controlled
post-transcriptionally mainly at the level of protein accumulation. Total shoot free Pi level was
about 40% lower in –Pi atpap26 mutants relative to wild-type controls, but unaffected under Pisufficient conditions. Moreover, shoot, root, inflorescence, and silique development of the
atpap26 mutant was impaired during Pi deprivation, but unaffected under Pi-replete conditions,
or during nitrogen or potassium-limited growth, or oxidative stress. The results suggest that the
hydrolysis of Pi from organic-phosphate esters by AtPAP26 makes an important contribution to
Pi-recycling and scavenging in –Pi Arabidopsis.
ii
Co-Authorship
The work presented in this thesis was derived from collaboration with colleagues to whom I am
permanently indebted for their contributions. Contributions were made by Hue T Tran. Ms. Tran
was responsible for collection and concentration of the liquid culture media from Arabdiopsis.
She also performed the APase activity assays and immunoblotting experiments on this material.
Her work is presented in Fig 2.3.
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Acknowledgements
Those who raised and loved me,
Kevin, Maria, Peter, Laura, Jeremias, Louise, Anthony
You lit the fire, and taught me more than I can ever know.
My inspiration,
WCP, WAS, SMR
You stoked the fire to heights previously unimaginable.
My colleagues,
HT, BMO, GRU, SG, TAF, KB, LK, PL, JK, BP, AG, SKR, BV, VK, HES, AN, JD, CC,
SC
You are the girders on which my success is built.
My Brethren and Beloved,
OSH, AW, SS, AW, DM, SC, SP, KM, SMH, KH, AG, NM
You sustained my faith in Love and brought me Peace.
I owe you more than I could ever repay, you are the riches of my life.
I also owe a debt to the Natural Sciences and Engineering Research Council and the Ontario
Graduate Scholarship for financial support. Without this continued faith and support, this thesis
would have been impossible.
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Table of Contents
Abstract............................................................................................................................................ii
Co-Authorship ................................................................................................................................iii
Acknowledgements.........................................................................................................................iv
Table of Contents............................................................................................................................. v
List of Figures ................................................................................................................................vii
Abbreviations................................................................................................................................viii
Chapter 1 Introduction and Literature Review ................................................................................ 1
1.1 General Introduction .............................................................................................................. 1
1.2 Literature Review................................................................................................................... 2
1.2.1 Phosphate nutrition is determined by plant and soil interactions.................................... 2
1.2.2 Plant adaptations to Pi starvation .................................................................................... 3
1.2.2.1 Morphological and ultrastructural adaptations to Pi starvation ............................... 3
1.2.2.2 Transcriptional responses to Pi starvation................................................................ 5
1.2.2.3 Transcriptional control of PSI gene expression ....................................................... 6
1.2.2.4 Post-Transcriptional and post-translational control of PSI gene expression............ 7
1.2.2.5 Metabolic adaptations to Pi starvation ..................................................................... 9
1.2.3 Purple Acid Phosphatases ............................................................................................. 11
1.2.3.1 Purple acid phosphatases – Structure and function............................................... 12
1.2.3.2 Purple acid phosphatases of Arabidopsis thaliana................................................. 16
1.2.3.3 Phosphate starvation inducible purple acid phosphatases: Identification and control
........................................................................................................................................... 19
1.3 Thesis Objectives ................................................................................................................. 20
Chapter 2 The dual-targeted purple acid phosphatase isozyme AtPAP26 is essential for efficient
acclimation of Arabidopsis thaliana to nutritional phosphate deprivation .................................... 22
2.1 SUMMARY......................................................................................................................... 23
2.2 INTRODUCTION ............................................................................................................... 24
2.3 RESULTS AND DISCUSSION .......................................................................................... 26
2.3.1 Identification and validation of an atpap26 mutant allele ............................................ 26
2.3.2 Post-transcriptional control of AtPAP26 expression by Pi nutrition ............................ 28
2.3.3 AtPAP26 is the predominant intracellular and a major secreted APase isozyme
upregulated by -Pi Arabidopsis.............................................................................................. 29
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2.3.4 AtPAP26 loss of function compromises total shoot Pi levels under –Pi, but not +Pi
growth conditions................................................................................................................... 32
2.3.5 AtPAP26 is essential for acclimation of Arabidopsis to Pi starvation.......................... 35
2.3.6 Concluding remarks ...................................................................................................... 38
2.4 ACKNOWLEDGEMENTS ................................................................................................. 41
2.5 EXPERIMENTAL PROCEDURES .................................................................................... 42
2.5.1 Plant material and growth conditions............................................................................ 42
2.5.2 Isolation of T-DNA insertion mutants .......................................................................... 42
2.5.3 RNA isolation and semi-quantitative RT –PCR ........................................................... 43
2.5.4 Protein extraction .......................................................................................................... 43
2.5.5 APase assays and determination of protein concentrtaion ............................................ 43
2.5.6 Protein electrophoresis and immunoblotting ................................................................ 44
2.5.7 Analysis of root structure architecture .......................................................................... 44
2.5.8 Phosphate extraction and assays ................................................................................... 44
2.5.9 Statistics ........................................................................................................................ 44
Chapter 3 General Discussion........................................................................................................ 45
3.1 Overview.............................................................................................................................. 45
3.2 Future Directions ................................................................................................................. 47
Literature Cited .............................................................................................................................. 52
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List of Figures
Figure 1.1 - Current model for role played by APases during Pi starvation in plant cells............. 15
Figure 1.2 – Classification scheme for Arabdiopsis PAPs.. .......................................................... 17
Figure 2.1 - Confirmation of T-DNA insert location and number in an atpap26 T-DNA
insertional mutant................................................................................................................... 27
Figure 2.2 – AtPAP26 is the predominant intracellular APase isozyme upregulated by –Pi
Arabidopsis and whose expression is nullified in atpap26 mutant seedlings. ....................... 30
Figure 2.3 - Immunological AtPAP detection and corresponding APase activities of secreted
media proteins of +Pi and –Pi Col-0 and atpap26 mutant seedlings ..................................... 33
Figure 2.4 - Influence of Pi-deprivation on shoot and root Pi levels of atpap26 mutant and Col-0
seedlings................................................................................................................................. 34
Figure 2.5 - Influence of nutrient deprivation or oxidative stress on shoot growth of atpap26
mutant and Col-0 seedlings.................................................................................................... 36
Figure 2.6 - Effect of Pi deprivation on root growth and architecture of atpap26 mutant and Col-0
seedlings................................................................................................................................. 39
Figure 2.7 - Inflorescence development and reproductive parameters of atpap26 mutant and Col-0
seedlings................................................................................................................................. 40
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Abbreviations
APase
acid phosphatase
bHLH
basic Helix-Loop-Helix transcription factor
Col-0
Arabidopsis thaliana ecotype Columbia, accession 0
EDTA
ethylenediaminetetraacetic acid
DTT
dithiothreitol
HMW
high molecular weight
IgG
immunoglobulin G
LMW
low molecular weight
miR
micro RNA
MS
Mass Spectrometry
NADH
nicotinamide adenine dinucleotide
NMR
nuclear magnetic resonance
PAGE
polyacrylamide gel electrophoresis
PAP
purple acid phosphatase
PEP
phosphoenolpyruvate
PEPC
phosphoenolpyruvate carboxylase
PFK
phosphofructokinase
Pi
phosphate
+Pi and –Pi
Pi sufficient and Pi deficient
PL
phospholipase
pNPP
para-nitrophenyl phosphate
PPi
pyrophosphate
PQ
paraquot
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PS
photosystem
PSI
phosphate starvation inducible
PSR
phosphate starvation response
PVDF
poly(vinylidene difluoride)
SNrK
sucrose non-fermenting related kinase
RNase
ribonuclease
RSA
root structure architecture
T-DNA
transferred DNA
TF
transcription factor
VSP
vegetative storage protein
ZAT
Zinc finger of Arabidopsis thaliana
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Chapter 1
Introduction and Literature Review
1.1 General Introduction
Modern agricultural practice is faced with a paradoxical reality. Crop yields are sustained
by massive application of fertilizers and other agrochemicals, yet many plants flourish in nature
without any of these exogenous inputs. A central focus of modern biotechnology is characterizing
the adaptations of plants to biotic and abiotic stresses which allow growth and reproduction in
conditions inclement for crop growth. The ultimate goal of this inquiry is the identification of
strategies for engineering crop cultivars that would alleviate the need for the unsustainable
application of agrochemicals.
Orthophosphate (Pi) plays a central role in virtually all major metabolic processes in
plants, including photosynthesis and respiration. The massive use of soluble Pi fertilizers in
agriculture demonstrates how the free Pi levels of most soils are suboptimal for plant growth. The
world’s reserves of rock-Pi, our major source of Pi fertilizers, are projected to be depleted by the
end of this century (Vance et al., 2003). Furthermore, Pi-runoff from fertilized fields into nearby
surface waters results in environmentally destructive processes such as aquatic eutrophication and
blooms of toxic cyanobacteria. In order to ensure agricultural sustainability and a reduction in Pi
fertilizer overuse, plant and soil science research must address the need to bioengineer Pi-efficient
transgenic crops. The design of effective biotechnological strategies to enhance crop Pi
acquisition necessitates our detailed understanding of Pi starvation inducible (PSI) gene
expression and the complex biochemical adaptations of Pi-deficient (-Pi) plants. The goal of this
thesis is to characterize a key biochemical adaptation utilized by -Pi plants that allow
maintenance of growth and reproduction despite being limited by a major macronutrient.
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1.2 Literature Review
1.2.1 Phosphate nutrition is determined by plant and soil interactions
Phosphate is an essential macronutrient for plant growth and is preferentially assimilated
by plants in its fully oxidized anionic state, H2PO4- and HPO42- (Vance et al., 2003). Pi is integral
to many components of cellular processes including structure of macromolecules, such as nucleic
acids and phospholipids, bioenergetics, signal transduction, as well as metabolic control and the
assimilation of other nutrients (Plaxton and Podesta, 2006; Vance et al., 2003). Despite its central
role in plant growth and metabolism, Pi is the least accessible macronutrient to plants in many
natural soils (Abel et al., 2002). This is a result of Pi’s low solubility in most soils, as it easily
forms calcium salts or complexes with iron and aluminium oxides rendering it inaccessible to
plant uptake (Abel et al., 2002; Ward et al., 2008). As a result, most natural soils have soluble Pi
concentrations that are often two or three orders of magnitudes lower than cytoplasmic Pi
concentrations required for optimal plant growth (Vance et al., 2003).
Although soluble free Pi concentrations are limiting for plant growth, vast Pi reserves
exist in soils as immobilized P-esters, which are unavailable for assimilation by plants, and are
referred to as organic Pi (Xiao et al., 2005). The majority of this organic Pi component is believed
to consist of recalcitrant phytate, or myo-inositol hexaphosphate, derived from decomposing plant
matter (Richardson et al., 2001). Remaining organic Pi components result from the
immobilization of Pi by soil microbes, which compete with plants for the uptake of Pi
(Richardson et al., 2001). Plants therefore face two primary challenges in acquiring Pi from their
environment. The first is the solubility of free Pi, which plants alter by secretion of large amounts
of organic acids into the rhizosphere to saturate soil anion exchange capacity (Plaxton and
Podesta, 2006; Vance et al., 2003). Second, plants may also possess the ability to mobilize Pi
from organic Pi pools via the secretion of hydrolases that free esterified Pi (Richardson et al.,
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2001). However, which organic Pi pools are accessible to plants remain unclear, and for the most
part, plants appear unable to utilize phytate as a nutritional source of Pi (Richardson et al., 2001).
Consequently, most natural soils are Pi deficient and plants have evolved a number of exquisite
adaptations that allow for growth and reproduction in the face of limiting amounts of this critical
macronutrient.
1.2.2 Plant adaptations to Pi starvation
1.2.2.1 Morphological and ultrastructural adaptations to Pi starvation
Plant adaptations to Pi starvation may be grouped into two general categories, increasing
either the acquisition of Pi or the efficiency of Pi utilization (Vance et al., 2003). Both strategies
underlie the morphological adaptations displayed by -Pi plants. The primary morphological
response of -Pi plants is to reduce shoot growth in order to decrease overall Pi demand (Lai et al.,
2007). Treatment of -Pi plants with proliferative cell division signals (e.g. sugar or cytokinin)
results in increased PSI gene expression and increased Pi metabolism, indicative of increased Pi
deprivation (Karthikeyan et al., 2006; Lai et al., 2007). In addition to decreasing Pi demand by
inhibition of shoot growth, plants drastically alter their root structure architecture (RSA) in
response to Pi starvation (Williamson et al., 2001). Generally, -Pi plants decrease growth rates of
their primary root and allocate greater resources to lateral roots and root hairs in order to increase
the volume of soil explored and the surface area for Pi absorption (Vance et al., 2003). Some
species form highly clustered lateral roots, or proteoid roots, that drastically increase root surface
area (Grennan, 2008). It appears that most changes in RSA are mediated primarily by auxin
signalling with smaller contribution from ethylene (Lopez-Bucio et al., 2002). Consistent with
this, Arabidopsis defective in auxin perception and signalling or redistribution display altered
RSA during Pi starvation (Li and Xue, 2007; Nacry et al., 2005; Perez-Torres et al., 2008). The
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increase in auxin sensitivity that accompanies Pi starvation is at least partially mediated by the
transcription factor ARF19 which increases the expression of the auxin receptor TIR1 (PerezTorres et al., 2008). However, other transcription factors may play a role as transgenic
manipulation of PSI transcription factors also causes perturbations in RSA during Pi starvation,
although their auxin sensitivity is yet to be tested (Devaiah et al., 2007a; Devaiah et al., 2007b).
The morphological adaptations undertaken by -Pi plants are accompanied by a
reorganization of cellular ultrastructure. The most dramatic re-organization occurs in the vacuole,
endomembrane system, and plasma membrane (Fife et al., 1990; Wanke et al., 1998). In
particular, there is a shift from few small vacuoles to a single large one (Fife et al., 1990; Wanke
et al., 1998), while the plasma membrane develops numerous invaginations (Gaude et al., 2008;
Wanke et al., 1998). In addition, mitochondria often become condensed (Wanke et al., 1998).
This ultrastructural shift may facilitate the re-arrangements that occur in phospholipid content
during extended Pi stress (Tjellström et al., 2008). Consistent with this, PSI phospholipases either
alter their tonoplastic localization (Yamaryo et al., 2008) or associate with plasma membranes
during Pi starvation (Gaude et al., 2008).
A major question regards the location and mechanism by which plants sense low Pi status
to induce Pi starvation responses (PSR). Some evidence points to a central role for external Pi
supply (Ticconi and Abel, 2004). Growth on –Pi media induces a determinate developmental
program in Arabidopsis roots (Sanchez-Calderon et al., 2005), but does so on an individual root
cap or trichoblast cell basis (Ticconi and Abel, 2004). When cultivated on agar solidified nutrient
media with a heterogeneous distribution of Pi, the elongation zone of the primary root and the
rate of primary root growth decreased considerably when regions of low Pi were reached, and
was restored on regions of full nutrient (Linkohr et al., 2002). Further, quantitative trait loci
mapping has identified two multi-copper oxidases that are critical for inhibition of primary root
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growth while also demonstrating that physical contact between the root tip and Pi deplete media
is required for initiation of PSR leading to RSA (Svistoonoff et al., 2007). However, some lines
of evidence point to some form of intracellular Pi sensing. Recently, the protein kinase SNrK1
isoforms AKIN10/11 were shown to be differentially phosphorylated during Pi starvation and
knockout of AKIN10 resulted in differential regulation of a subset of PSI genes (Fragoso et al.,
2009). However, both SNrK1 isoforms are localized to the chloroplast which implies some
retrograde signalling to the nucleus would be required to initiate PSR.
Future work concerning morphological adaptations to Pi starvation and downstream
signalling needs to address the specificity of the transcriptional networks induced by Pi
starvation. For example, it is not clear if modifications to root architecture and morphology are
mediated by the same transcription factor networks delineating control of molecular and
biochemical adaptations to Pi starvation. Answers to such questions would aid in developing
plants that can tailor induction of specific elements of PSR, for example, induction of PSI
hydrolyases and altered root architecture without concomitant decreases in growth.
1.2.2.2 Transcriptional responses to Pi starvation
Many elements of PSR are controlled at the transcriptional level (Mukatira et al., 2001)
and -Pi plants extensively remodel their transcriptome and proteome to co-ordinate the required
metabolic and morphological adaptations. The collection of microarray data regarding
transcriptional responses to Pi starvation has shed some light on the molecular identity and
regulation underlying many classical biochemical and physiological adaptations to Pi stress. One
of the first revelations was that during exposure to low Pi, PSI gene expression is highly coordinated in a temporal and tissue specific manner, with PSI gene induction preceded by a subset
of non-specific stress responsive genes (Hammond et al., 2003; Misson et al., 2005). On the other
hand, decreases in cytoplasmic Pi, which accompany prolonged Pi starvation, are met by a highly
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specific response that differs in roots and shoots of Arabidopsis (Misson et al., 2005; Wu et al.,
2003). Despite an extensive response involving the induction of 600-1800 genes across all
tissues, there appears to only ~25% overlap between those specifically induced by the root and
shoot, implying strong tissue-specific adaptations to Pi starvation (Misson et al., 2005; Wu et al.,
2003). Transcriptional repression or mRNA turnover also plays a critical role in determining
PSR, with 250-700 genes showing decreased transcript accumulation in response to Pi starvation.
Again tissue specificity plays a key role in this response with only a 5-10% overlap in repressed
gene expression between roots and shoots (Misson et al., 2005; Wu et al., 2003).
Extensive microarray data developed from –Pi plants has revealed that transcription may
control many classical PSI biochemical and physiological adaptations. For example, although
many enzymes involving carbon anabolism are downregulated, those involved generally in
carbon catabolism become induced, specifically those catalyzing adenylate/Pi independent
bypasses of glycolytic steps such as pyrophosphate dependent phosphofructo-1-kinase (PPi-PFK)
or phosphoenolpyruvate (PEP) carboxylase (PEPC) (Misson et al., 2005). Further, many subunits
of both photosystems (PS) and the photosynthetic transport chain are downregulated during Pi
starvation (Wu et al., 2003) consistent with decreased PSII yields (Kobayashi et al., 2009).
Extended Pi starvation also results in substantial re-organization of protein synthesis. Genes
involved in protein degradation and inhibition of translation are up-regulated, with a concomitant
decrease in genes involved in protein synthesis, mRNA processing and amino-acyl tRNA
charging (Misson et al., 2005; Morcuende et al., 2007).
1.2.2.3 Transcriptional control of PSI gene expression
A major windfall from transcriptome studies of -Pi Arabidopsis was the identification of
novel PSI transcription factors (TFs) (Misson et al., 2005; Morcuende et al., 2007). Previously,
its was thought that most PSI gene expression was mediated by inhibitory TFs expressed during
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Pi sufficiency (Mukatira et al., 2001) or activated by the PSI transcription factor PHR1, a MYBcoiled coil family member related to the master PSI TF in green algae PSR1 (Rubio et al., 2001).
This view has greatly expanded to include multiple families of TFs. Of particular note was the
discovery of PSI TFs containing SPX domains, which are present in the major Pi sensing and
signalling components of yeast (Morcuende et al., 2007). Reverse genetic analysis of four
Arabidopsis SPX containing TFs revealed that, unlike in yeast, SPX TFs play diverse roles during
Pi starvation. Although three of the SPX TFs were induced by Pi starvation, one was suppressed,
and transgenic plants displayed altered regulation of PSI genes or exaggerated symptoms of Pi
deficiency (Duan et al., 2008). Transcriptional control of PSR and PSI gene expression is also
mediated by members of the WRKY, zinc finger (ZAT) and basic-helix-loop-helix (bHLH)
families (Chen et al., 2007; Devaiah et al., 2007a; Devaiah et al., 2007b). Although PHR1 has
been shown to function as a homo-dimer, and bind an imperfect palindromic sequence present in
the promoters of many PSI genes, such analyses have not been performed for any other PSI TF
(Rubio et al., 2001). This represents a major limitation of PSI TF work to date, and future work
must endeavour to identify in vivo targets, mechanism of action, oligomeric status and any
information regarding protein function or structure.
1.2.2.4 Post-Transcriptional and post-translational control of PSI gene expression
Despite the transcript-centric focus of many PSI gene expression studies, there is
emerging evidence that post-transcriptional mechanisms play a critical role in the control of PSI
gene expression and activity. The best characterized example to date is the regulatory pathway
defined by the microRNA 399 (miR399), the E2 ubiquitin conjugase AtUBC24 and the TF PHR1
(Bari et al., 2006). During Pi sufficiency, AtUBC24 is expressed and exerts its regulatory effects,
presumably decreasing the expression of critical PSI genes (Chiou et al., 2005). Upon Pi
starvation PHR1 is induced and activates expression of miR399 which leads to the targeted
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destruction of AtUBC24, and accumulation of AtUBC24 targets (Bari et al., 2006). However,
miR399 regulatory effects appear to be mediated by the PSI non-coding RNA At4 via ‘target
mimicry’ meaning that two layers of post-transcriptional regulation regulate expression of a
subset of PSI genes (Franco-Zorilla et al., 2007). To date, no attempts have been made to
determine the targets of AtUBC24 or miR399 targets that may be regulated by inhibition of
translation rather than mRNA cleavage. Such analyses are pressing, given that proteomic analysis
of -Pi corn, rice and Arabidopsis have shown that transcript abundance of some genes are not
indicative of protein accumulation during Pi stress, thus implying some form of translational
control (Fukuda et al., 2007; Li et al., 2008; Tran and Plaxton, 2008). In a similar vein, the
activity of the PSI small-ubiquitin like modifier E3 ligase AtSIZ1 has been identified as critical to
PSR (Miura et al., 2005), but none of its targets have been identified. Future work must focus on
these downstream post-transcriptional mechanisms as they may define novel PSI genes and new
targets for biotechnological engineering of Pi efficient crop plants.
Reversible phosphorylation is also emerging as a key post-translational mechanism for
the control of PSI gene activity. The upregulation of PEPC activity represents an archetypical
adaptation to Pi starvation, with PEPC thought to play a role in (i) intracellular Pi recycling, as Pi
is a byproduct of the PEPC reaction, (ii) excretion of organic acids to solubilize otherwise
inaccessible Pi and (iii) as a metabolic bypass to the adenylate/Pi expensive pyruvate kinase
catalyzed step of glycolysis (Plaxton and Podesta, 2006; Vance et al., 2003). Consistent with this
AtPPC1 transcripts accumulate in response to Pi starvation (Gregory et al., 2009; Morcuende et
al., 2007). Further, PEPC’s sensitivity to allosteric effectors is tightly controlled by reversible
phosphorylation (Plaxton and Podesta, 2006). Both PEPC protein kinases are amongst the most
responsive PSI transcripts (Misson et al., 2005; Morcuende et al., 2007) and AtPPC1 becomes
activated in vivo by phosphorylation during Pi starvation of Arabidopsis (Gregory et al., 2009).
8
Also, many elements of phosphorylation signalling pathways are differentially regulated in
response to Pi starvation. For example, in cultivars of corn more resistant to Pi starvation there is
preferential upregulation of phosphoprotein phosphatase (PP) 2A catalytic subunits, implying a
central role for reversible phosphorylation in response to Pi starvation (Li et al., 2008). However,
apart from the Arabidopsis PEPC study of Gregory and coworkers (2009), there is little evidence
demonstrating the direct functional consequences of protein phosphorylation during Pi starvation,
and future endeavours must try to demonstrate the functional relevance of PSI phosphorylation
leading to changes in gene activity.
1.2.2.5 Metabolic adaptations to Pi starvation
In response to Pi starvation, plants increase the acquisition and efficiency of Pi utilization
by distinct re-organization of their carbon and Pi metabolism. This is achieved through the coordinated expression of high-affinity Pi transporters, upregulation of enzymes catalyzing
metabolic reactions which bypass the use of adenylate/Pi and the upregulation of intracellular and
secreted PSI hydrolyases for the scavenging of Pi from non-essential P-esters.
The upregulation of high affinity Pi transporters of the plasma membrane represent a
fundamental adaptation to Pi starvation. High affinity Pi transporters in Arabidopsis belong to the
nine member PHT1 family and consist of Pi/H+ symporters with 12 transmembrane domains
(González et al., 2005). While all nine members are responsive to Pi starvation, each appears to
have some form of tissue specific expression, with some expressed in epidermal and root hair
cells while others are expressed in stelar cells of the root (Mudge et al., 2002). Consistent with
this, knockout of Pht1;4 or Pht1;1 results in decreased Pi acquisition during Pi deficiency (Shin
et al., 2004), while knockout of Pht1;1 (pho1) also results in failure to accumulate Pi in shoots
during Pi sufficiency due to its role in xylem loading of Pi (Stefanovic et al., 2007).
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A critical adaptation to Pi starvation involves the re-organization of metabolism to utilize
non-adenylate forms of energy, such as pyrophosphate (PPi). Plants alter the organization of
respiration and vacuolar H+ pumps during Pi stress such that adenylate and Pi requiring steps are
bypassed (Plaxton and Podesta, 2006). These bypasses allow respiration and vacuolar pH
maintenance to continue while conserving Pi and adenylate by using PPi to perform cellular work
(Plaxton and Podesta, 2006). Additionally, the PEPC catalyzed bypass of pyruvate kinase allows
the formation of organic acids from glycolytic metabolites. This is critical for the anaplerotic
replenishment of TCA cycle intermediates as well as the excretion of organic acids, a common
response to Pi stress (Müller et al., 2004; Plaxton and Podesta, 2006; Vance et al., 2003). Organic
acid excretion is thought to aid in chelating some of the cations (Al3+, Cu2+, Fe2/3+) that
immobilize Pi, thus increasing Pi availability (Vance et al., 2003).
Plants increase the efficiency of Pi use during Pi starvation via upregulation of PSI
hydrolyases that are believed to scavenge Pi from non-essential P-esters. Upregulation of
hydrolases is accompanied by a marked decrease in cytoplasmic, vacuolar and plasma membrane
P-esters during Pi stress (Jouhet et al., 2003; Lee and Ratcliffe, 1993). Classical PSI hydrolases
include various non-specific phospholipases (PL), ribonucleases (RNase) and acid phosphatases
(APase) (Abel et al., 2000; Tjellström et al., 2008; Veljanovski et al., 2006). PL activity is
accompanied by replacement of phosphoheadgroups with sulfonyl or galactosyl-headgroups, thus
maintaining the polarity of lipid bilayers, and in the case of chloroplastic phospholipids, structural
requirements for photosynthesis (Kobayashi et al., 2009; Okazaki et al., 2009). Phospholipids
represent a dynamic and indispensable phosphate reserve during Pi starvation (Tjellström et al.,
2008). Knockout of PL activity or the downstream synthases required for lipid remodelling
results in decreased growth during Pi starvation (Cruz-Ramierez et al., 2006; Gaude et al., 2008;
Kobayashi et al., 2009).
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Comparatively little is known about PSI RNases or APases, although both are
upregulated intracellularly and secreted into the rhizosphere to scavenge Pi from extracellular Pesters (Abel et al., 2000; Haran et al., 2000; Tran and Plaxton, 2008). In particular, the S-type
RNases RNS1 and RNS2 protein accumulate in response to Pi starvation, in the extracellular and
intracellular space, respectively (Bariola et al., 1994; Bariola et al., 1999). Anti-sense inhibition
of RNS1/RNS2 results in the reduction of RNS activity and greater accumulation of anthocyanins
during Pi starvation, implying a failure to adequately acclimate (Bariola et al., 1999). Even less is
known about PSI APases, with no functional studies to date and little information on the relative
roles of APases induced by Pi starvation in Arabidopsis thaliana (Misson et al., 2005).
1.2.3 Purple acid phosphatases
Acid phosphatases (APases; E.C. 3.1.3.2) catalyze the hydrolysis of Pi from phosphate
monoesters and anhydrides within the acidic pH range. They are ubiquitous in the genomes of
bacteria, plants and mammals (Schenk et al., 2000). The Arabidopsis genome encodes over 50
APase genes including 10 vegetative storage protein type APases (VSP), four phosphatidic acid
APases and one histidine APase (Li et al., 2002). However, the most numerous APase class are
the purple acid APases (PAPs), which are encoded by at least 28 active genes in Arabidopsis. The
diversity of vascular plant APases represents a fascinating biological question in its own right.
This is further complicated by the fact that not all APases appear to function as metabolic
enzymes. For example, in depodded soybean leaves VSPα accumulates to almost 40% of total
soluble protein but contributes less than 1% to extractable APase activity, while a single point
mutation can increase its APase activity 20-fold (Leelapon et al., 2004; Staswick et al., 1994).
The single PAP of mammals plays a role in bone resorption, iron transport and metabolism of
reactive oxygen species (Li et al., 2002). The genetic and functional diversity of APases
necessitates greater study, with particular focus on the largest group in Arabidopsis, the PAPs.
11
1.2.3.1 Purple acid phosphatases – Structure and function
Purple acid phosphatases received their colourful nomenclature owing to their distinctive
purple color in solution. This results from a charge transfer transition at ~560 nm from the metalcoordinating tyrosine to the metal ligand Fe(III) (Li et al., 2002). PAPs belong to a superfamily of
metallophosphoesterase that includes phosphoprotein phosphatases, exonucleases and other
phosphomonoesterases, which all contain five blocks of conserved metal ligating residues,
although the location, number and identity of the residues differ between the family groups (Li et
al., 2002). Members of the PAP family all contain seven metal residues (DxG-GDXXYGNH(D/E)-VXXH-GHXH; bold letters indicate metal ligating residues, dashes indicate
separation between blocks) which are highly conserved among all bacterial, mammalian and plant
PAPs and form di-metallic active sites (Schenk et al., 2000). However, despite conservation of
metal ligating residues, mammalian, and plant PAPs differ in the composition of active site
metals. Mammalian PAPs contain a Fe(III)-Fe(II) active site while plant PAPs contain an Fe(III)Me(II) active site where the second metal is either zinc or manganese (Schenk et al., 2000),
although recent analyses of a PAP purified from tobacco cell walls implied a di-iron active site
(Kaida et al., 2008). Interestingly, mammalian and plant PAPs function in vitro with Zn(II) or
Fe(II), respectively, instead of the complementary in vivo divalent metal atom (Klablunde et al.,
1995). Given the availability of these metals in mammalian or plant cells, it implies that
specificity of divalent metals may provide a form of functional specialization (Klablunde et al.,
1995).
The structure of PAP catalytic sites and domains are also highly conserved (Klablunde et
al., 1995; Schenk et al., 2000). Bacterial, cyanobacterial, mammalian and plant PAPs all contain
catalytic domains that consist of two sandwiched β-α-β-α-β motifs, with almost perfect alignment
and order of the conserved metal ligating residues ( Schenk et al., 2000; Li et al., 2002). Despite
12
the conservation of catalytic domains, mammalian and plant PAPs differ in their oligomeric
structure (Schenk et al., 2000;Li et al., 2002). Mammalian PAPs exist as ~35-kDa monomers
consisting solely of a catalytic domain (Klablunde et al., 1995), although mammalian-like low
molecular weight (LMW) PAPs also exist in plants (del Pozo et al., 1999; Schenk et al., 2000). In
contrast, plants also posses high molecular weight (HMW) oligomeric PAPs composed of 50-60
kDa subunits that consist of an N-terminal domain with no catalytic function and a C-terminal
catalytic domain that is structurally related to monomeric PAPs (Klablunde et al., 1995; Li et al.,
2002; Schenk et al., 2000). Although most HMW PAPs exist as homodimers (Schenk et al.,
2000; Veljanovski et al., 2006), the tomato intracellular PAP consists of a heterodimer of
structurally distinct, but related, subunits (Bozzo et al., 2006). Additionally, dimeric PAP
structures form either through disulfide bridges (Schenk et al., 2000) or via non-covalent
interactions (Olczak and Olczak, 2007). However, all PAPs characterized to date are stabilized
via intra-molecular disulphide bridges, or possess conserved cysteine residues that are essential
for structure (Li et al., 2002; Schenk et al., 2000). It is not currently clear how oligomeric
structure impacts PAP function, or even why PAPs exist in two disparate oligomeric states.
Most PAPs are typified as broad spectrum APases that largely catalyze the non-specific
hydrolysis of Pi from small molecules (Li et al., 2002). However, evidence exists that PAPs may
play functional roles of great diversity. For example, expression of mammalian PAPs occur in
macrophages and spleen cells after phagocytosis and play a role in the generation of reactive
oxygen and nitrogen species via a Fenton reaction involving the Fe(II) of the active site
(Klablunde et al., 1995). Additionally, several plant PAPs display alkaline peroxidase activity
that is unaffected by APase inhibitors (Bozzo et al., 2002; Bozzo et al., 2004a; del Pozo et al.,
1999; Veljanovski et al., 2006) and the soybean PAP, GmPAP3, directly increases tolerance to
oxidative stress (Francisca Li et al., 2008). Mammalian PAPs may also act as phosphotyrosine
13
phosphatases, implying a potential role in signal transduction (Schenk et al., 2000). Recently, a
PAP from tobacco cell walls was purified and shown to be active against phosphotyrosylated
peptides (Kaida et al., 2008), while purified PAPs routinely show activity against
phosphotyrosine or other phosphoamino acids (Bozzo et al., 2004a; Kaida et al., 2008;
Veljanovski et al., 2006). Interestingly transgenic expression of the tobacco wall PAP with
phosphotyrosine phosphatase activity resulted in altered cell wall composition, implying an in
vivo regulatory role (Kaida et al., 2009).
It is also likely that PAPs function as a key element of PSR. A universal response of
plants to Pi starvation is the upregulation of APase activity. The probable function of intracellular
APases is to recycle Pi from expendable intracellular organophosphate pools, whereas secreted
APases likely scavenge Pi from the organically bound Pi that is prevalent in most soils (Fig. 1.1)
(Haran et al., 2000; Tomscha et al., 2004; Tran and Plaxton, 2008). APase induction in –Pi plants
has been correlated with de novo APase synthesis in several systems, including Arabidopsis
thaliana and tomato suspension cells and seedlings (Bozzo et al., 2002; Bozzo et al., 2004a;
Bozzo et al., 2006; Veljanovski et al., 2006). Intracellular and secreted APase isozymes purified
from –Pi Arabidopsis and tomato suspension cell cultures were all characterized as PAPs (Bozzo
et al., 2002, 2004a; del Pozo et al., 1999; Veljanovski et al., 2006; Tran and Plaxton, unpublished
data).
14
Figure 1.1 - Current model for role played by APases during Pi starvation of plant
cells. Intracellular (vacuolar) and extracellular APases are highly induced in response to
Pi starvation. APases are believed to help alleviate Pi starvation by hydrolysis of nonessential P-esters releasing Pi for essential cellular processes that require Pi.
15
Therefore, consideration of PSI APase activity, and its functional significance, requires detailed
characterization of PAPs, starting with those induced by Pi starvation. Arabidopsis thaliana’s
ease of cultivation and the wealth of available ‘post-genomic’ resources (Ülker et al., 2008)
provide a convenient model organism for studying PSI PAPs. Further, Arabidopsis and other
members of the Cruciferae do not form mycotrophic association and are therefore believed to
have evolved a more elaborate response to Pi starvation than mycotrophic plants, which account
for ~90% of known plant species (Murley et al., 1998). Characterization of Arabidopsis PSI
PAPs may therefore provide ideal ‘grist for the mill’ of biotechnological improvement of Pi
acquisition in crop species.
1.2.3.2 Purple acid phosphatases of Arabidopsis thaliana
The Arabidopsis family of PAPs is encoded by 29 genes on all five chromosomes and
numbered based on the order of their loci, although only 28 appear to be actively transcribed (Li
et al., 2002). The 29 AtPAPs have been classified into three distinct groups according to sequence
homology (Fig 1.2; (Li et al., 2002). Groups I and II are comprised of oligomeric HMW PAPs,
with group I consisting of oligmeric PAPs of slightly smaller monomer size than group II. Group
Ia-2 is comprised of AtPAP10, AtPAP12 and AtPAP26, all of which have been in found in various
extracellular compartments, while AtPAP26 has also been identified in vacuoles (Red Box, Fig
1.2; (Carter et al., 2004; Irshad et al., 2008; Tran and Plaxton, 2008; Veljanovski et al., 2006).
Group III consists of the monomeric LMW mammalian-like AtPAPs.
Expression analysis of 28 members of the AtPAP family revealed that most members do
not show differential tissue expression (Zhu et al., 2005). For example, 16 members were
ubiquitously expressed in all tissues, while another 12 members showed increasingly specific
tissue expression. Interestingly, no single family member displayed totally unique expression,
meaning that all tissues express more than one PAP isoform (Zhu et al., 2005). However,
16
Figure 1.2 – Classification scheme for Arabidopsis PAPs. The AtPAP family was
grouped according to clustering analysis of amino acid sequences (Li et al., 2002). Groups
I and II are comprised of oligomeric HMW PAPs, group III consists of the LMW
mammalian-like AtPAPs. Group Ia-2 is highlighted with a red box, and contains three
members with confirmed extracellular localization. One member of group Ia-2, AtPAP26,
is dual targeted to the vacuole and extracellular space (Carter et al., 2004; Veljanovski et al.,
2006; Tran and Plaxton, 2008).
17
cell specific expression patterns of most AtPAPs remain elusive. Evidence does exist that some
AtPAP transcripts accumulate during stress responses. Most analyses have focused on responses
to Pi starvation (discussed below), although AtPAP17 is responsive to oxidative and salt stress
(del Pozo et al., 1999). Given the diverse roles of other PAPs, greater focus is required regarding
characterization of stress responsive AtPAP expression.
There is also a paucity of information regarding the subcellular localization of most
AtPAPs, given that PAPs in other species and Arabidopsis have been found in mitochondria
(GmPAP3), cell walls (AtPAP10/NtPAP12, AtPAP12 AtPAP15, AtPAP26), the cell vacuole
(AtPAP26) and the extracellular space (AtPAP12, AtPAP26) (Carter et al., 2004; Francisca Li et
al., 2008; Haran et al., 2000; Irshad et al., 2008; Kaida et al., 2008; Tran and Plaxton, 2008). This
is especially curious given that all PAP sequences to date contain signal peptides (del Pozo et al.,
1999; Schenk et al., 2000; Veljanovski et al., 2006) and are glycosylated (Bozzo and Plaxton,
2008; Olczak and Olczak, 2007; Schenk et al., 2000) implying that all PAPs must be targeted at
least to the early secretory system where glycosylation occurs.
Although the catalytic function and regulation of plant PAPs have been described, their
physiological function in plants has not been fully established. To date, only two AtPAPs and
only one AtPAP promoter have been functionally characterized. It appears that AtPAP15 encodes
a phytase involved in ascorbate synthesis via production of myo-inositol (Zhang et al., 2008).
Consistent with this, transgenic expression of AtPAP15 in soybean increases acquisition of Pi
when grown on sand supplemented with phytate (Wang et al., 2009), while functional analyses of
AtPAP23 yielded no phenotype despite its flower specific expression pattern (Zhu et al., 2005).
The promoter of AtPAP12 was responsive to Pi starvation and expressed predominantly in the
vasculature and lateral root meristems of -Pi Arabidopsis seedlings (Haran et al., 2000).
18
1.2.3.3 Phosphate starvation inducible purple acid phosphatases: Identification and
control
Most work to date has focused on the role plant PAPs play during Pi starvation.
Molecular analysis of PSI gene expression has hinted at complex regulation of plant APase gene
expression. Pi starvation induces temporal and tissue specific expression of PSI APases (Bozzo et
al., 2006; Haran et al., 2000; Wu et al., 2003) and the concomitant down-regulation of other
APases (Misson et al., 2005). The transcription factors PHR1, WRKY75, and ZAT6 have been
implicated in the control of PSI APase gene expression (Devaiah et al., 2007a; Devaiah et al.,
2007b; Rubio et al., 2001), while other studies have revealed PSI APases that are controlled by
post-transcriptional mechanisms (Tran and Plaxton, 2008; Veljanovski et al., 2006). In contrast,
Pi re-supply to –Pi plants quickly represses PSI APase genes (Müller et al., 2004) while inducing
specific proteases that appear to target PSI APases (Bozzo et al., 2004b). Further characterization
of PSI APases is required to define the molecular mechanisms underlying this archetypical plant
response to Pi starvation, as well as to identify suitable targets for improving crop Pi acquisition.
Despite the wealth of molecular information, only two PSI AtPAPs have been identified
at the protein level. Veljanovski and coworkers (2006) purified intracellular PSI APase activity
from Arabidopsis suspension cells and recovered a single PAP isozyme which was identified as
AtPAP26 via N-terminal sequencing and BLAST homology searches. Semi-quantitative
immunoblotting revealed that AtPAP26 accumulates in –Pi suspension cells and seedlings in
response to Pi starvation, and that AtPAP26 levels are correlated with PSI APase activity. It was
notable that AtPAP26 transcript levels were unresponsive to Pi starvation, implying a form of
post-transcriptional control. Interestingly, a previous forward screen mapped the phosphatase
under producer mutant, pup3, to chromosome 5 proximal to AtPAP26, although it was annotated
as a mutant allele of AtPAP12 (Tomscha et al., 2004). The pup3 mutant displayed lower APase
19
activity in shoots, roots and root exudates and was impaired in uptake of Pi from soils containing
only organic Pi (Tomscha et al., 2004). Proteomic analysis of cell culture filtrate from –Pi
Arabidopsis suspension cells confirmed that AtPAP12 and AtPAP26 are both secreted and likely
play extracellular roles in recycling Pi (Tran and Plaxton, 2008, and unpublished data). These
findings confirmed work by Haran and coworkers (2000) indicating that AtPAP12 promotersignal peptide::GFP contructs are secreted into seedling media during Pi starvation. This implies
that the upregulation of intracellular and extracellular APase activity is critical for acclimation to
Pi starvation. Consistent with this, transgenic overexpression and secretion of plant PAPs can
improve biomass and phosphorous accumulation (Hur et al., 2007; Ma et al., 2009; Xiao et al.,
2005; Xiao et al., 2006). However, most of these approaches focused on ectopic expression of
specific phytases and would benefit from discovery of PAPs with dual intracellular and
extracellular roles, broad substrate specificity and higher activity. Therefore, it is essential to
functionally characterize PSI AtPAPs with the goal of identifying new targets for manipulation in
the production of Pi efficient crops.
1.3 Thesis Objectives
Recently, AtPAP26 was identified as a PSI PAP with both intracellular and extracellular
roles in Arabidopsis seedlings and suspension cell culture (Tran and Plaxton, 2008, and
unpublished data; Veljanovski et al., 2006). Further, these studies demonstrated that vacuolar
accumulation or secretion of AtPAP26 by –Pi Arabidopsis was likely mediated by posttranscriptional mechanisms of gene regulation. This is in contrast to other reports implicating
different AtPAP family members under transcriptional control as contributors to PSI APase
activity in Arabidopsis (del Pozo et al., 1999; Devaiah et al., 2007a; Devaiah et al., 2007b;
Misson et al., 2005; Rubio et al., 2001). As a result, the molecular identity of PSI APase activity
in Arabidopsis remains elusive. It is the primary objective of this thesis to test the hypothesis that
20
AtPAP26 represents the chief contributor to intracellular and extracellular PSI APase activity in
Arabidopsis. Further, I will test the hypothesis that AtPAP26, and therefore PSI APase activity, is
a critical adaptation that facilitates the acclimation of Arabidopsis to Pi starvation. This was
carried out using a reverse genetic approach capitalizing on the publicly available T-DNA
insertion lines of Arabidopsis thaliana (Alonso et al., 2003). Isolation of lines homozygous for
the T-DNA insert at the loci of interest will permit the generation of Arabidopsis lacking
expression of a single gene (Ülker et al., 2008). Such approaches have proven profitable in the
analysis of other PSI genes predicted by genomic approaches. The non-specific phospholipases
PLDζ2 and NPC5 and the high affinity phosphate transporters Pht1;1 and Pht1;4 were confirmed
to play a central role in PSR via T-DNA knockout (Cruz-Ramierez et al., 2006; Gaude et al.,
2008; Kobayashi et al., 2009; Shin et al., 2004). Further, single gene T-DNA knockout has been
used to show reductions in PSI PLC activity despite the large number of PLC genes induced by Pi
starvation (Nakamura et al., 2005). This is analogous to the current problem posed by PSI APase
activity and the large number of AtPAPs implicated in this process. This study also highlights the
need for using biochemical and proteomic analyses to identify stress responsive genes, as
AtPAP26 has never been identified as a PSI gene via transcript profiling. Work presented here
describes the functional characterization of AtPAP26 as a PSI APase using a reverse genetic
approach informed by previous biochemical studies while providing insight into a fundamental
biochemical adaptation -Pi plants implement that allow acclimation to inclement growth
conditions.
21
Chapter 2
The dual-targeted purple acid phosphatase isozyme AtPAP26 is
essential for efficient acclimation of Arabidopsis thaliana to nutritional
phosphate deprivation
(Manuscript in preparation)
Brenden A Hurley1, Hue T Tran1, and William C Plaxton1,2,†
1
Department of Biology, Queen's University, Kingston, Ontario K7L 3N6, Canada
2
Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada
†
Corresponding Author (Department of Biology, Queen's University, Kingston, Ontario K7L
3N6, Canada (e-mail [email protected])
22
2.1 SUMMARY
Induction of intracellular and secreted purple acid phosphatases (PAPs) is a widespread plant
response to nutritional Pi-deficiency. Although the catalytic function and regulation of plant
PAPs have been described, their physiological function in plants has not been fully established.
Recent biochemical and proteomic studies indicated that AtPAP26 is the predominant
intracellular (vacuolar) and a major secreted PAP isozyme upregulated by Pi-starved (-Pi)
Arabidopsis thaliana. The in planta function of AtPAP26 was assessed by molecular,
biochemical, and phenotypic characterization of a homozygous Salk T-DNA insertion mutant.
Loss of AtPAP26 expression resulted in the elimination of AtPAP26 transcripts and 55-kDa
immunoreactive AtPAP26 polypeptides, correlated with a 9- and 5-fold decrease in extractable
shoot and root APase activity, respectively, as well as a 40% reduction in secreted APase activity
of –Pi seedlings. The results corroborate previous findings implying that AtPAP26 is: (i) the
principal contributor to Pi starvation inducible APase activity in Arabidopsis, and (ii) controlled
post-transcriptionally mainly at the level of protein accumulation. Total shoot free Pi level was
about 40% lower in –Pi atpap26 mutants relative to wild-type controls, but unaffected under Pisufficient conditions. Moreover, shoot, root, inflorescence, and silique development of the
atpap26 mutant was impaired during Pi deprivation, but unaffected under Pi-replete conditions,
or during nitrogen or potassium-limited growth, or oxidative stress. The results suggest that the
hydrolysis of Pi from organic-phosphate esters by AtPAP26 makes an important contribution to
Pi-recycling and scavenging in –Pi Arabidopsis.
23
2.2 INTRODUCTION
Orthophosphate (Pi) is an essential plant macronutrient required for many pivotal
metabolic processes such as photosynthesis and respiration. However, the massive use of soluble
Pi fertilizers in agriculture demonstrates how free Pi levels of many soils are suboptimal for plant
growth. The world’s reserves of rock-phosphate, our major source of Pi fertilizers, are projected
to be depleted by the end of this century (Vance et al., 2003). Furthermore, Pi-runoff from
fertilized fields into nearby surface waters results in environmentally destructive processes such
as aquatic eutrophication and blooms of toxic cyanobacteria. In order to ensure agricultural
sustainability and a reduction in Pi fertilizer overuse, plant and soil science research must address
the need to bioengineer Pi-efficient transgenic crops. The design of biotechnological strategies to
enhance crop Pi acquisition necessitates our detailed understanding of Pi-starvation inducible
(PSI) gene expression and the complex physiological and biochemical adaptations of Pi-deficient
(-Pi) plants.
A well documented component of the plant Pi stress response is the upregulation of
intracellular and secreted acid phosphatases (APases) which catalyze the hydrolysis of Pi from
various P-monesters and anhydrides in the acidic pH range (Duff et al., 1994). APase induction
by –Pi plants has been correlated with de novo APase synthesis in several systems, including
Arabidopsis thaliana and tomato suspension cells and seedlings (Bozzo et al., 2002; Bozzo et al.,
2004a; Bozzo et al., 2006; Veljanovski et al., 2006). The probable function of intracellular
APases is to recycle Pi from expendable intracellular organophosphate pools. This is
accompanied by a marked reduction in cytoplasmic P metabolites during extended Pi stress
(Bozzo and Plaxton, 2008). Secreted APases belong to a group of PSI phosphohydrolases that are
induced to mobilize Pi from external organophosphates that are prevalent in many soils (Bozzo
and Plaxton, 2008; Haran et al., 2000; Tomscha et al., 2004; Tran and Plaxton, 2008). Molecular
analyses of PSI transcripts and proteins have hinted at complex control of plant APase gene
expression. Pi deprivation induces temporal and tissue specific expression of PSI APase isozymes
(Bozzo et al., 2006; Haran et al., 2000; Wu et al., 2003; Zimmerman et al., 2004) and the
concomitant down-regulation of other APases (Misson et al., 2005). The transcription factors
PHR1, WRKY75, and ZAT6 have been implicated in the control of Arabidopsis PSI APases
(Devaiah et al., 2007a; Devaiah et al., 2007b; Rubio et al., 2001), while post-transcriptional
mechanisms appear to be essential for the upregulation of the purple APase (PAP) AtPAP26
24
during Pi stress (Veljanovski et al., 2006). In contrast, Pi re-supply to –Pi plants rapidly represses
PSI APase genes (Müller et al., 2004; Veljanovski et al., 2006) while inducing proteases that
target PSI APases (Bozzo et al., 2004b). Further characterization of PSI APases is required to
define the molecular mechanisms underlying this archetypical plant response to Pi starvation, as
well as to identify additional targets for improving crop Pi acquisition.
A variety of intracellular and secreted APases that are upregulated by –Pi plants have
been characterized as PAPs, which represent a specific APase class characterized by a bimetallic
active site that endows them with a pink or purple colour in solution (Bozzo et al., 2002; Bozzo et
al., 2004a; Bozzo et al., 2006; del Pozo et al., 1999; Li et al., 2002; Veljanovski et al., 2006).
Although mammals contain a single PAP-encoding gene, vascular plant PAPs belong to a large
multigene family. Li and coworkers (2002) classified 29 putative PAP genes in Arabidopsis,
several of which appear to respond to Pi deficiency. Apart from functioning as non-specific
APases, various PSI plant PAPs (including AtPAP17 and AtPAP26) exhibit alkaline peroxidase
activity indicating their potential alternative role in the metabolism of reactive oxygen species
(Bozzo et al., 2002; Bozzo and Plaxton, 2008; Li et al., 2002; Veljanovski et al., 2006).
Moreover, AtPAP17 was induced by oxidative stress (del Pozo et al., 1999) and ectopic
expression of a soybean mitochondrial PAP (GmPAP3) conferred increased resistance to
oxidative stress in transgenic Arabidopsis (Francisca Li et al., 2008). It is therefore important to
determine the physiological roles of the various plant PAPs and which of their dual enzymatic
activities are functional in –Pi plants.
The aim of the present study was to build upon biochemical analyses indicating that
AtPAP26 is the predominant intracellular (vacuolar), as well as a major secreted APase isozyme
of –Pi Arabidopsis (Tran and Plaxton, 2008; Veljanovski et al., 2006). In particular, we sought to
test the hypothesis that AtPAP26 plays a pivotal role in the Pi metabolism of Arabidopsis during
Pi stress. This was done by employing a functional genomic approach, taking advantage of the
publicly available transferred DNA-tagged (T-DNA) insertional mutagenized populations of
Arabidopsis (Alonso et al., 2003). We identified and characterized a null atpap26 allele that
negated AtPAP26 expression. This was correlated with the elimination of PSI intracellular APase
activity, as well as a significant reduction in secreted APase activity during Pi deprivation. The
atpap26 mutant demonstrated impaired development and altered shoot Pi levels relative to wildtype seedlings when exposed to Pi deficiency, but displayed normal growth under Pi replete
25
conditions, or exposure to other stresses. The results confirm that AtPAP26 has an essential and
specific function in facilitating the acclimation of Arabidopsis to suboptimal Pi nutrition.
2.3 RESULTS AND DISCUSSION
2.3.1 Identification and validation of an atpap26 mutant allele
To assess the contribution of AtPAP26 to intracellular and secreted APase activity during
Pi deficiency, as well as its impact on the phenotype of +Pi versus –Pi Arabidopsis, a T-DNA
insertion line was identified in the Salk collection (Salk_152821) (Alonso et al., 2003). The TDNA insert is putatively located in the ninth exon of the AtPAP26 gene (locus At5g34850, Fig.
2.1A) and was verified by PCR screening of genomic DNA (gDNA) using an AtPAP26 gene
specific primer and a T-DNA left border primer (Fig. 2.1B). Homozygosity of the T-DNA mutant
was confirmed by PCR of gDNA using AtPAP26-specific primers (Fig. 2.1B). T-DNA insert
number was assessed by cultivating mutant seedlings in +Pi liquid media, followed by gel blot
analysis of extracted gDNA that had been digested with EcoRI, HindIII, and SacI. Each
restriction enzyme cleaves a single digestion site within the transposable element of the pBINpROK2 vector. All digestions yielded a single strongly hybridizing band (Fig. 2.1C), indicating a
single T-DNA insertion site.
To investigate the impact of the T-DNA insertion on AtPAP26 expression, primer pairs
were employed to amplify cDNA sequences flanking the second intron of AtPAP26 (Fig. 2.1A).
The correct cDNA sequences were amplified from shoot and root mRNA isolated from wild-type
Col-0, but not atpap26 mutant plants (Fig. 2.2A). AtPAP12, AtPAP17, and AtPPCK1 (encodes
PEP carboxylase protein kinase 1) were employed as positive controls since their mRNAs are
markedly induced in -Pi Arabidopsis (del Pozo et al., 1999; Gregory et al., 2009; Haran et al.,
2000; Li et al., 2002; Tran and Plaxton, 2008). Their transcripts showed a similar induction in –Pi
atpap26 mutant and wild-type Col-0 seedlings (Fig. 2.2A), indicating that the mutant is
unimpaired in Pi starvation signalling. It is notable that AtPAP26 transcripts were present at
relatively abundant and similar levels in Col-0 tissues irrespective of nutritional Pi status, whereas
the amount of a 55-kDa AtPAP26 immunoreactive polypeptide was about twice as abundant in
root or shoot extracts from the –Pi relative to +Pi Col-0 seedlings (Fig. 2.2B). This was paralleled
by the pronounced accumulation of 55-kDa immunoreactive AtPAP26
26
Figure 2.1 - Confirmation of T-DNA insert location and number in an atpap26 T-DNA
insertional mutant. A. Schematic representation of AtPAP26 gene (At5g34850); open boxes and
solid lines represent exons and introns, respectively. T-DNA insertion location is indicated by
atpap26 T-DNA, while arrows represent primers used for RT-PCR and genotyping. B.
Assessment of T-DNA location and homozygosity of mutants via PCR-based screening of
gDNA. Genomic DNA was isolated from +Pi seedlings cultivated in liquid culture for 14-d
according to (Veljanovski et al., 2006) the legend for Fig. 2.2. PCR products were amplified from
Col-0 and atpap26 gDNA in a 30 cycle PCR reaction containing the indicated primers. M denotes
a 100-bp ladder for confirmation of product size. C. Analysis of T-DNA insert number by
Southern blot analysis of atpap26 gDNA probed with NPTII. Arrows indicate the base pair length
of EcoRI and HindIII digested phage λ-DNA markers labelled with digoxigenin (Roche).
27
polypeptides on immunoblots of concentrated media proteins secreted by –Pi Col-0 seedlings
(Fig. 2.3). By contrast, immunoblotting of clarified shoot or root extracts, or secretome proteins
of the +Pi or -Pi atpap26 mutant failed to reveal any immunoreactive AtPAP26 polypeptides
(Figs. 2.2B and 2.3), whereas the upregulation and secretion of 60-kDa AtPAP12 polypeptides
during Pi stress was unaffected (Fig. 2.3A). Therefore, atpap26 defines a null allele of AtPAP26,
with abrogated expression of AtPAP26 transcript and protein.
2.3.2 Post-transcriptional control of AtPAP26 expression by Pi nutrition
Results of Fig. 2.2A corroborate previous studies of heterotrophic Arabidopsis
suspension cells documenting the marked upregulation of intracellular and secreted AtPAP26 in
response to Pi starvation, without concomitant changes in AtPAP26 transcript abundance (Tran
and Plaxton, 2008; Veljanovski et al., 2006). Transcript profiling of 28 members of the
Arabidopsis PAP family confirmed that basal levels of AtPAP26 transcripts are relatively
abundant and invariant following Pi stress, and are constitutively expressed in all tissues (Zhu et
al., 2005). Recent proteomic studies have documented a variety of intracellular and secreted
proteins that are also controlled post-transcriptionally mainly at the level of protein accumulation
in plants (i.e., Arabidopsis, maize, rice) responding to changes in environmental Pi availability
(Fukuda et al., 2007; Li et al., 2008; Tran and Plaxton, 2008). Several reports have further
emphasized the involvement of post-transcriptional processes in PSI gene regulation, particularly
those played by the microRNA (miRNA) miR399 and E2-ubiquitin conjugase AtUBC24 (pho2)
(Chiou et al., 2005), with transcriptional control of miR399 mediated by the transcription factor
PHR1 (Bari et al., 2006). Plant miRNAs have been shown to act by translational inhibition
(Brodersen et al., 2008). In addition: (i) splice variants of AtPAP10 preferentially associate with
ribosomes during Pi starvation (Li et al., 2002), (ii) reversible phosphorylation has been
implicated as an important post-translational protein modification involved in certain PSI
responses (Gregory et al., 2009), whereas (iii) turnover of extracellular PSI tomato PAPs appears
to be mediated by serine proteases that are induced and secreted upon Pi-resupply to –Pi cells
(Bozzo et al., 2004b). It stands to reason that the regulation of AtPAP26 abundance by nutritional
Pi status could be controlled by any of these processes without concomitant changes in AtPAP26
transcripts. Transcription factors identified as controlling PSI APase activity in Arabidopsis (i.e.,
PHR1, WRKY75 and ZAT6) (Devaiah et al., 2007a; Devaiah et al., 2007b; Rubio et al., 2001)
28
could potentially act on AtPAP26 via indirect mechanisms mediated by the activity of their target
genes. Greater knowledge is required regarding the transcriptional targets of PHR1, WRKY75
and ZAT6, as well as precise mechanisms by which AtPAP26 expression is controlled. In contrast
to AtPAP26, the upregulation of PAPs such as AtPAP17 and AtPAP12 during Pi deprivation
appears to be mainly controlled at the transcriptional level (del Pozo et al., 1999; Haran et al.,
2000; Li et al., 2002; Rubio et al., 2001; Tran and Plaxton, 2008) (Fig. 2.2A). As AtPAP12 is
secreted by -Pi Arabidopsis suspension cells and seedlings (along with AtPAP26) it is expected to
play an extracellular Pi-scavenging role (Haran et al., 2000; Tran and Plaxton, 2008) (Fig. 2.3A).
AtPAP17 transcripts also accumulate in response to oxidative or salt stress, similar to GmPAP3
(del Pozo et al., 1999; Francisca Li et al., 2008). AtPAP17 may thus function to detoxify reactive
oxygen species during general stress rather than play a significant Pi remobilization and
scavenging role in –Pi Arabidopsis.
2.3.3 AtPAP26 is the predominant intracellular and a major secreted APase isozyme
upregulated by -Pi Arabidopsis
Consistent with previous results (Tran and Plaxton, 2008; Veljanovski et al., 2006;
Zakhleniuk et al., 2001) Pi deprivation of Col-0 seedlings resulted in a 2- to 3-fold increases in
extractable shoot/root and secreted APase activities (Figs. 2.2C and 2.3). In contrast, -Pi atpap26
mutants exhibited 9- and 5-fold lower shoot and root APase activities, respectively (Fig. 2.2C), as
well as a 40% reduction in secreted APase activity relative to Col-0 (Fig. 2.3). It is notable that no
increase in intracellular APase activity was detected in response to Pi starvation of the atpap26
mutant (Fig. 2.2C). APase assays employing 5 mM para-nitrophenol-P (pNPP) rather than 5 mM
PEP as the substrate were also performed with shoot extracts of -Pi Col-0 and the atpap26
mutant. Consistent with the PEP-based assays (Fig. 2.2C), extracts from -Pi shoots of atpap26
mutant seedlings exhibited 4-fold lower pNPP-hydrolyzing activity relative to Col-0 controls (65
±7 and 265 ±20 µmol pNPP hydrolyzed/min/mg protein, respectively; means ± SEM of n = 4
biological replicates). Clarified extracts of –Pi shoots were also resolved by non-denaturing
PAGE and subjected to in-gel APase activity staining using β-naphthyl-P as the substrate, and
parallel immunoblotting with anti-AtPAP26 immune serum (Fig. 2.2D). Shoot extracts of –Pi
Col-0 yielded several APase activity staining bands, in agreement with previous results (Tomscha
et al., 2004). However, an abundant high molecular mass APase activity staining band that
29
Figure 2.2 – AtPAP26 is the predominant intracellular APase isozyme upregulated by –Pi
Arabidopsis and whose expression is nullified in atpap26 mutant seedlings. RNA and soluble
proteins were isolated from seedlings cultivated in 250 ml plant culture boxes containing 50 ml of
sterile 0.5X MS medium pH 5.7, 1% (w/v) sucrose and 0.2 mM Pi for 7-d prior to transfer into
30
media containing 0 (-Pi) or 1.5 mM Pi (+Pi) for an additional 7-d. The seedlings were cultivated
under continuous 125 μmol·m-2·s-1 photosynthetically active radiation (PAR) at 24 °C on an
orbital shaker set at 80 rpm. A. Levels of mRNA were analyzed by semi-quantitative RT-PCR
using gene specific primers for AtPAP12, AtPAP17, AtPAP26, AtPPCK1 and AtACT2. AtACT2
was used as a reference to ensure equal template loading. Template concentrations needed to
achieve non-saturating conditions for primer pairs as tested for roots of −Pi seedlings are
indicated in parentheses. Control RT–PCR reactions lacking reverse transcriptase did not show
any bands. B. Purified native AtPAP26 from -Pi Arabidopsis suspension cells (50 ng/lane)
(Veljanovski et al., 2006) and clarified extract proteins from shoots (2 μg/lane) and roots (4g
μg/lane) of the +Pi and –Pi seedlings were resolved by SDS-PAGE and electroblotted onto a
PVDF membrane. Following oxidation of antigenic glycosyl groups with sodium-m-periodate
(Laine, 1988), blots were probed with a 1000-fold dilution of anti-(native AtPAP26)-immune
serum and immunoreactive polypeptides detected using an alkaline-phosphatase linked secondary
antibody and chromogenic detection (Veljanovski et al. 2006). C. APase activity of clarified
extracts represent means (±SEM) of duplicate assays on n = 3 biological replicates; asterisks
denote values that are significantly different from Col-0. D. Clarified extracts from –Pi shoots of
Col-0 and atpap26 were resolved by non-denaturing PAGE and subjected to in-gel APase activity
staining (70 μg protein/lane) or immunoblotting with anti-(native AtPAP26)-immune serum (7 μg
protein/lane). O, origin; TD, tracking dye front.
31
strongly cross-reacted with anti-AtPAP26 immune serum was absent in the atpap26 mutants (Fig.
2.2D). The collective results of Figs. 2.2 and 2.3 corroborate previous biochemical studies (Tran
and Plaxton, 2008; Veljanovski et al., 2006) indicating that AtPAP26 is a principal contributor of
PSI intracellular and secreted APase activity of Arabidopsis. Similar results were obtained by
Tomscha and co-workers (2004) who characterized an Arabidopsis phosphatase-underproducer
(pup3) ethylmethane sulfonate mutant that exhibited about 40% lower APase activity in shoot and
root extracts. Although immunoblotting using anti-(recombinant AtPAP12) immune serum led
these workers to conclude that AtPAP12 was one of the PAP isozymes defective in pup3,
AtPAP26 was strongly implicated since the pup3 mutation mapped to a 2.7 Mb sequence of
chromosome 5 within the Arabidopsis genome that encompasses AtPAP26 (Tomscha et al.,
2004). That pup3 may have been defective in AtPAP26 rather than AtPAP12 is supported by the
observations that: (i) AtPAP12 transcript levels were unaffected in pup3 (Tomscha et al., 2004),
and (ii) the anti-(recombinant AtPAP12) immune serum employed by Tomscha et al. (2004)
effectively cross-reacts with both AtPAP12 and the closely related AtPAP26 (Fig. 2.3A) (Tran
and Plaxton, 2008). In several instances, AtPAP isozyme loss-of-function has resulted in no
detectable influence on total APase activity. For example, no alteration in extractable APase
activity was reported in AtPAP23 T-DNA mutants (Zhu et al., 2005). Similarly, extracts of
AtPAP15 T-DNA mutants contained 6-fold lower phytase activity, but unaltered pNPP hydrolytic
activity relative to wild-type controls, possibly due to AtPAP15’s specificity as a phytase (Zhang
et al., 2008) and/or its low abundance relative to AtPAP26.
2.3.4 AtPAP26 loss of function compromises total shoot Pi levels under –Pi, but not +Pi
growth conditions
The atpap26 mutant accumulated normal levels of Pi when cultivated for 14-d in +Pi
liquid media, whereas the free Pi concentration of shoots and roots of Col-0 and atpap26 mutant
seedlings exhibited statistically identical 10- and 30-fold decreases, respectively, following
exposure of 7-d-old +Pi seedlings to 7-d of Pi-deprivation (Fig. 2.4). Similar results have been
noted for both –Pi Arabidopsis cell suspensions and seedlings (Kobayashi et al., 2009;
Veljanovski et al., 2006). However, the total amount of Pi in shoots of the -Pi atpap26 mutants
was significantly (40%) lower than that of wild-type Col-0 controls (Fig. 2.4B).
32
Figure 2.3 - Immunological AtPAP detection and corresponding APase activities of secreted
media proteins of +Pi and –Pi Col-0 and atpap26 mutant seedlings. APase activity was
assayed from the liquid culture media collected from Arabidopsis seedlings cultivated as
described in the legend for Fig. 2.2. All APase activities represent the means of n = 3 biological
replicates and are reproducible to within ±20% of the mean value. Growth medium containing
secreted proteins was passed through Whatman #1 filter paper and concentrated >250-fold with
Amicon Ultra-15 ultrafiltration devices (30,000 Mr cutoff; Millipore) at 4 oC. Concentrated
secreted proteins (15 µg/lane) as well as homogeneous secreted native AtPAP12 (Tran and
Plaxton, unpublished data) and native AtPAP26 from –Pi Arabidopsis suspension cells
(Veljanovski et al., 2006) (20 ng each) were subjected to SDS-PAGE and immunoblotting as
described in the legend for Fig. 2.2, except that the immunoreactive polypeptides were visualized
using a horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminscent
detection (ECL Plus, GE Healthcare).
33
Figure 2.4 - Influence of Pi-deprivation on shoot and root Pi levels of atpap26 mutant and
Col-0 seedlings. Free Pi was determined colorimetrically from perchloric acid extracts of
seedlings cultivated as in the legend for Fig. 2.2. A. Free Pi concentrations in roots and shoots of
+Pi or -Pi seedlings. B. Total free Pi in shoots of +Pi and –Pi seedlings. All values represent
means (±SEM) of n = 3 biological replicates; the asterisk denotes a value that is significantly
different from Col-0.
34
2.3.5 AtPAP26 is essential for acclimation of Arabidopsis to Pi starvation
Shoot development of 14-d-old atpap26 and Col-0 seedlings cultivated on +Pi and –Pi
agar-solidified nutrient media is depicted in Figs. 5A and 5B. No differences were noted in the
appearance or fresh weights of +Pi Col-0 versus atpap26 shoots. However, atpap26 shoot
development was markedly reduced during growth on –Pi media (Fig. 2.5A), exhibiting a 30%
decrease in fresh weight relative to Col-0 (Fig. 2.5B). Shoot development of atpap26 mutants was
also characterized in 21-d-old plants cultivated on +Pi or -Pi agar-solidified media, as well as on
+Pi media deficient in nitrogen (-N) or potassium (-K), or supplemented with 1 µM paraquot
(PQ). Similar to 14-d-old plants (Fig. 2.5B), rosette fresh weight of 3-week-old –Pi atpap26
mutants was significantly reduced (by about 20%) relative to –Pi Col-0, but unaffected under +Pi
conditions (Fig. 2.5C). Likewise, no phenotypic differences relative to Col-0 were apparent when
+Pi atpap26 mutant seedlings were subjected N- or K-deficiency, or PQ treatment (Figs. 2.5C). It
was notable that loss of AtPAP26 function did not influence the sensitivity of Arabidopsis to PQmediated oxidative stress, suggesting that the in vitro alkaline peroxidase activity of purified
AtPAP26 from –Pi Arabidopsis (Veljanovski et al., 2006) has little intracellular relevance during
Pi stress. This is consistent with AtPAP26’s vacuolar localization (Carter et al., 2004) in which an
acidic pH of about pH 5.5 closely aligns with the enzyme’s APase pH-activity optimum, but is far
below AtPAP26’s peroxidase pH-activity optimum of pH 9.0 (Veljanovski et al., 2006). It
therefore appears that while AtPAP26 is indispensable for the acclimation of Arabidopsis to Pi
deprivation, it is expendable in +Pi Arabidopsis, or during other macronutrient deficiencies or
oxidative stress. Impaired growth of –Pi Arabidopsis resulting from disrupted expression of genes
encoding PSI metabolic enzymes has previously been reported, albeit in the context of altered Pi
recycling from membrane phospholipids (resulting from loss of the PSI phospholipase C isoform
NPC5 or monogalactosyldiacylglycerol synthase 2) (Gaude et al., 2008; Kobayashi et al., 2009).
Similarly, loss of PSI high-affinity Pi transporters disrupted the shoot development of –Pi, but not
+Pi Arabidopsis (Shin et al., 2004).
Root development of the atpap26 mutant was assessed via cultivation of seedlings on
vertically orientated agar plates for 14-d. Cultivation of Col-0 and atpap26 mutant seedlings on
–Pi plates resulted in decreased primary root growth, and a greater allocation to secondary or
lateral roots (Fig. 2.6). However, the root growth of the atpap26 mutant was disrupted in –Pi, but
not +Pi, seedlings (Fig. 2.6A).
35
Figure 2.5 - Influence of nutrient deprivation or oxidative stress on shoot growth of atpap26
mutant and Col-0 seedlings. A. Seedlings were cultivated under continuous illumination (125
μmol·m-2·s-1 PAR) on agar-solidified 0.5X MS media containing 1% sucrose and 1.5 mM or 50
μM Pi for 14-d. Plates shown are representative of four replicates; scale bar = 1 cm. B. Shoot
fresh weight of seedlings cultivated as in panel A. C. Shoot fresh weight of seedlings cultivated as
in panel A on media containing 1.5 mM Pi for 7-d, then grown for an additional 14-d on media
containing 1.5 mM or 50 μM Pi, as well as +Pi media lacking nitrogen (-N) (Scheible et al.,
2004) or potassium (-K) (Cao et al., 2007), or containing 1 μM paraquot (PQ). All values in B
and C represent means (±SEM) of n = 20 seedlings from four different plates; asterisks denote
values that are significantly different from Col-0.
36
in particular, primary, secondary, and total root length of -Pi atpap26 mutant seedlings was
significantly (20-24%) reduced relative to Col-0 controls (Fig. 2.6A-D). However, there was no
consequence of AtPAP26’s elimination on: (i) ratio of secondary:primary root length (Fig. 6E), or
(ii) root hair number or length within 5 mm of the root tip (results not shown). Although altered
root development is a common phenomenon within functional analyses of Arabidopsis PSI genes,
this response can be grouped into two categories based on the function of the PSI gene in
question. For example, disruption of genes involved in transcriptional reorganization or hormonemediated responses to Pi starvation generally cause a shift in root structure architecture in
addition to decreased primary root growth. Over-expression or knockout of PSI transcription
factors frequently causes decreased primary root growth with concomitant increases in lateral root
and root hair growth relative to wild-type plants (Devaiah et al., 2007a; Devaiah et al., 2007b).
Similar results have been observed with genes that mediate auxin signalling during Pi starvation,
such as PLDζ2, where knockouts have altered root structure architecture during Pi starvation (Li
et al., 2006). However, disruption of PSI genes encoding metabolic enzymes often results in a
reduction of total root growth during Pi stress without changes secondary:primary root growth
ratio. For example, knockout of monogalactosyldiacylglycerol synthase 2 resulted in an overall
20% decrease in root growth, without changes in root structure architecture (Kobayashi et al.,
2009). Both the observed upregulation of three well documented PSI genes (Fig. 2.2A) and
overall reduced root growth without concomitant changes in root structure architecture are
consistent with AtPAP26’s function in Pi scavenging and recycling, rather than Pi signalling,
during Pi starvation.
PSI gene expression is influenced by sugar levels (Karthikeyan et al., 2006), while
exogenous sucrose may exacerbate Pi starvation through increased cell proliferation signalling
(Lai et al., 2007). Thus, plant cultivation under sterile conditions in the presence of exogenous
sucrose may produce artefactual phenotypes not seen under more physiologically relevant
conditions. We thus established whether soil-grown atpap26 mutant plants displayed any obvious
phenotype. Plants were cultivated in a nutrient-depleted peat-perlite soil mixture supplemented
with 0.25X Hoagland’s solution containing either 1.5 mM or 50 μM Pi (Fig. 2.7). Both genetic
backgrounds attained all stages of germination, vegetative, and reproductive growth (Boyes et al.,
2001) at the same time; rosette diameter and fresh weight of the +Pi or -Pi atpap26 mutants was
also unaffected relative to Col-0 (results not shown). Pi may have been indirectly available to the
–Pi seedlings in the form of soil bound P-esters (Hayes et al., 2000; Tomscha et al., 2004), thus
37
reducing the severity of Pi stress during this treatment. Although, AtPAP26 does play an
extracellular role there could be sufficient redundancy in secreted PSI PAP isozymes (particularly
AtPAP12) to negate any phenotype in null AtPAP26 backgrounds (Irshad et al., 2008; Tran and
Plaxton, 2008, and unpublished data). However, the atpap26 mutant exhibited significantly
reduced development of reproductive tissue during cultivation under -Pi conditions (Fig. 2.7A),
with a 18% reduction in the height of the primary inflorescence which yielded 26% fewer siliques
than Col-0 at senescence (Fig. 2.7B and C).
2.3.6 Concluding remarks
Results of the present study corroborate parallel biochemical analyses of Arabidopsis
suspension cells and seedlings (Tran and Plaxton, 2008, and unpublished data; Veljanovski et al.,
2006) indicating that AtPAP26 : (i) encodes the principal intracellular, as well as a major secreted
APase isozyme upregulated by -Pi Arabidopsis, and (ii) is controlled post-transcriptionally at the
level of protein accumulation. The latter finding highlights the need to effectively integrate
transcript profiling studies with parallel biochemical and proteomic analyses of plant stress
responses, as the combined datasets will provide a more robust depiction of how alterations in
gene expression may be linked to adaptive changes in plant metabolism. This is especially
pertinent for our understanding of plant Pi starvation responses in which most studies to date have
focused on identifying genes whose transcripts differentially accumulate during Pi stress
(Devaiah et al., 2007a; Devaiah et al., 2007b; Misson et al., 2005), despite accumulating
evidence that transcript abundance does not always predict intra- or extracellular proteome
remodelling that ensue plant nutrient deprivation (Fukuda et al., 2007; Li et al., 2008; Tran and
Plaxton, 2008; Yoshimoto et al., 2007). Further, several studies have uncovered genes such as
miR399 and AtUBC24 that function in the post-transcriptional control of plant gene expression in
response to altered Pi nutrition (Chiou et al., 2005; Bari et al., 2006). Constitutive transcriptional
expression of key Pi-metabolizing enzymes such as AtPAP26 is expected to help ‘prime the
system’, thereby accelerating their rate of biosynthesis upon any subsequent exposure to
suboptimal environmental Pi levels. It is equally important to note that AtPAP26 displays
significant product inhibition by Pi (I50 = 2.1 mM) (Veljanovski et al., 2006). The intracellular Pi
concentration of shoots and roots of the +Pi seedlings (≈ 5 - 10 mM, assuming 1 gFW ≈ 1 ml)
(Fig. 2.4A) should exert significant feedback inhibition of AtPAP26’s APase activity in vivo.
Conversely, the >10-fold reductions in intracellular Pi levels of –Pi seedling tissues will
38
Figure 2.6 - Effect of Pi deprivation on root growth and architecture of atpap26 mutant and
Col-0 seedlings. A. Seedlings were cultivated vertically for 14-d on agar-solidified 0.5X MS
media containing 1% sucrose and 1.5 mM or 50 μM Pi, and then scanned at 1200 dpi. Seedlings
shown are representative of 16 seedlings; scale bar = 1 cm. B-D. Descriptive statistics of root
architectural features of seedlings grown as in A. All values represent means (±SEM) of n = 16
seedlings from four different plates; asterisks denote values that are significantly different from
Col-0.
39
Figure 2.7 - Inflorescence development and reproductive parameters of atpap26 mutant and
Col-0 seedlings. Seedlings were grown on agar-solidified 0.5X MS media containing 1% sucrose
and 1.5 mM Pi for 10-d prior to transfer to a peat-perlite soil mix (SunGro Sunshine professional
growing mix 2) lacking fertilizer. Plants were cultivated in a growth cabinet under a 16-h-light/8h-dark cycle at 200 μmol·m-2·s-1 PAR and 22 °C and 70% relative humidity and watered twice
weekly with 0.25X Hoagland’s solution containing either 1.5 mM or 50 μM Pi. A. Pi-deficient
Col-0 and atpap26 seedlings (50 μM Pi) at developmental stage 6.5-6.7 (Boyes et al. 2001).
Seedlings are representative of n = 16 seedlings; scale bars = 5 cm. B. Primary inflorescence
height at whole plant senescence (stage 9.70) of Col-0 and atpap26. C. Silique number at stage
9.70 of Col-0 and atpap26. All values in B and C represent means (±SEM) of n = 16 different
plants; asterisks denote values that are significantly different from Col-0.
40
effectively relieve AtPAP26 from Pi inhibition and should thus make an important contribution to
its enhanced in vivo APase activity during Pi stress. As the predominant APase isozyme
upregulated by –Pi Arabidopsis, it was not unexpected that the development atpap26 mutant
seedlings was specifically impaired during their cultivation on –Pi media (Figs. 2.5-2.7).
Although AtPAP26 was necessary for efficient acclimation of Arabidopsis to Pi starvation, it was
expendable in +Pi seedlings or during acclimation to other forms of nutrient deprivation, or
oxidative stress (Fig. 2.6). Shoots and roots of atpap26 mutants displayed no differences in free
Pi concentration, but accumulated less total shoot Pi relative to Col-0 during Pi stress (Fig. 2.4).
This implies that AtPAP26 functions in the recycling of internal P-ester pools to increase the
efficiency of Pi utilization in –Pi Arabidopsis.
Several studies have reported that overexpression and secretion of PAPs can improve
plant biomass and Pi accumulation (Hur et al., 2007; Ma et al., 2009; Xiao et al., 2005; Xiao et
al., 2006). However, these attempts have either focussed around high specificity phytases or
PAPs with extracellular roles, but unclear intracellular function. On the other hand, AtPAP26 not
only has a demonstrated intracellular role, but is a dual targeted enzyme that is also secreted by
Arabidopsis suspension cells and seedlings (Fig. 2.3) (Tran and Plaxton, 2008, and unpublished
data; Veljanovski et al., 2006). Further, intracellular and secreted AtPAP26 isoforms of –Pi
Arabidopsis are highly active against a broad range of P-ester substrates (Tran and Plaxton, 2008,
and unpublished data; Veljanovski et al., 2006), making it an ideal candidate for overexpression
studies and biotechnological strategies aimed at improving crop Pi acquisition and utilization. It
will thus be of some interest to examine the Pi metabolism and growth characteristics of AtPAP26
overexpressors cultivated on unfertilized soil and/or with various P-esters as their sole source of
exogenous P.
2.4 ACKNOWLEDGEMENTS
We thank Prof. Wayne Snedden (Queen's University) for advice throughout this project and
comments on the manuscript. We are also indebted to Prof. Thomas McKnight (Texas A & M
University) for the gift of anti-(recombinant AtPAP12) immune serum. Financial support was
generously provided by grants from the Natural Sciences and Engineering Research Council of
Canada (NSERC) and Queen’s Research Chairs program to WCP. BH and HT were the
beneficiaries of NSERC post-graduate scholarships.
41
2.5 EXPERIMENTAL PROCEDURES
2.5.1 Plant material and growth conditions
The Col-0 ecotype of Arabidopsis was used throughout this study. General conditions for plant
growth were as described previously (Veljanovski et al., 2006), with specific details described in
the various figure legends. Shoots and roots of +Pi and –Pi plants were frozen in liquid N2 and
stored at -80 °C until required.
2.5.2 Isolation of T-DNA insertion mutants
A putative AtPAP26 mutant (Salk_152821) was identified from the Salk T-DNA lines (Alonso et
al., 2003) via analyses of the SiGnAL database (http://signal.salk.edu/cgi-bin/tdnaexpress). Seeds
were obtained from the Arabidopsis Biological Resource Center (ABRC) at Ohio State
University. Homozygous mutant plants were isolated by PCR screening of genomic DNA from
the
T4
generation
(LP,
5’-ATTGCTGAAAACTTAAGCGGG-3’;
TACCGAATATCAAATGTGCGG-3’;
LBb1,
RP,
5’-
5’-GCGTGGACCGCTTGCTGCAACT-3’).
Homozygous lines were also analyzed by DNA gel blot to determine T-DNA insert number.
Genomic DNA was isolated according to (Zhang and Zeevaart, 1999) except that isopropanol
precipitated nucleic acids were pelleted by centrifugation at 15,000 g for 10 min, followed by 2
washes with 70% (v/v) ethanol. Pellets were dissolved in 10 mM Tris-HCl (pH 8.0). Gel blot
analysis was performed as described in (Quan et al., 2007). The 795 bp neomycin
phosphotransferase coding sequence (NPTII CDS) was used to generate a digoxigenin (dig)
labelled probe using Roche’s dig labelling kit. Southern blot hybridization was performed at 70
°C for 18 h using hybridization solution (250 mM NaPi, pH 7.2, 1 mM EDTA, 20% (w/v) SDS,
0.5% (w/v) milk powder) with dig-labelled NPTII CDS probes followed by three 20 min washes
at 60 °C in 20 mM sodium phosphate (pH 7.2) containing 1 mM EDTA and 1% (w/v) SDS. The
blot was incubated for 90 min in 1% (w/v) milk powder and probed for 30 min with 1:5000 antidig-IgG conjugated to alkaline phosphatase (Roche) followed by two 15 min washes in detection
wash solution. Bands were visualized by chemiluminescence using CDP-Star (Roche).
42
2.5.3 RNA isolation and semi-quantitative RT –PCR
Total RNA was extracted and purified as described previously (Veljanovski et al. 2006). RNA
samples were assessed for purity via their A260/A280 ratio and integrity by resolving 1 μg of total
RNA on a 1.2% (w/v) denaturing agarose gel. Normalization of RNA for RT was performed for
each sample by density measurement of 28S ribosomal RNA bands from the above gel (scanned
using ImageJ software from the National Institutes for Health, U.S.A.). RNA (5 μg) was reverse
transcribed with Superscript III (Invitrogen) and non-competitive RT–PCR was performed as
described in Gennidakis et al. (2007). Gene specific primers used to amplify AtPPCK1, AtPAP17,
AtPAP26, and AtACT2 were previously described (Veljanovski et al. 2006, Gregory et al. 2009).
Transcripts for AtPAP12 were amplified using the gene specific primers AtPAP12LP: 5’CACGTTCTTCGTCTCGGATT-3’ and AtPAP12RP: 5’-CCCTTGCGTTACATGAACCT-3’.
The amount of input cDNA necessary for nonsaturating amplification for each primer pair was
established by performing PCR using 0.05-1.2 ng of total RNA during first-strand cDNA
synthesis.
2.5.4 Protein extraction
Tissues were homogenized (1:2; w/v) in ice-cold extraction buffer composed of 20 mM sodium
acetate (pH 5.6), 1 mM EDTA, 1 mM DTT, 1 mM 2,2’ dipyridyl disulfide, 1 mM
phenylmethlysulfonyl fluoride, 5 mM thiourea, and 1% (w/v) insoluble polyvinyl
(polypyrrolidone). Homogenates were centrifuged in a microcentrifuge at 4 oC and 14,000xg for
5 min, and the supernatants reserved as clarified extract.
2.5.5 APase assays and determination of protein concentration
APase activity was routinely measured by coupling the hydrolysis of PEP to pyruvate to the
lactate dehydrogenase reaction and continuously monitoring NADH oxidation 340 nm using a
Molecular Devices Spectromax Plus Microplate spectrophotometer and the following optimized
assay conditions: 50 mM Na-acetate (pH 5.6), 5 mM PEP, 10 mM MgCl2, 0.2 mM NADH, and 3
units of desalted rabbit muscle lactate dehydrogenase in a final volume of 0.2 ml. Assays were
corrected for background NADH oxidation by omitting PEP from the reaction mixture. APase
assays were also carried out in an assay mix containing 50 mM sodium acetate (pH 5.6), 5 mM
pNPP, and 10 mM MgCl2 by continuously monitoring the formation of paranitrophenol at 405
nm (ε = 18,000 M-1cm-1). All APase assays were linear with respect to time and concentration of
43
enzyme assayed. One unit of activity is defined as the amount of enzyme resulting in the
hydrolysis of 1 µmol of substrate·min–1 at 25 °C. Protein concentrations were determined using a
modified Bradford assay (Bozzo et al., 2002) with bovine γ–globulin as the protein standard.
2.5.6 Protein electrophoresis and immunoblotting
SDS-PAGE, immunoblotting onto poly(vinylidene difluoride) (PVDF) membranes (Immobilon
transfer; 0.45-µm pore size; Millipore Canada) and visualization of antigenic polypeptides using
an alkaline-phosphatase-tagged secondary antibody were conducted as previously described (Tran
and Plaxton, 2008; Veljanovski et al., 2006). Densitometric analysis of immunoblots was
performed using an LKB Ultroscan XL laser densitometer and GELSCAN software (Version 2.1;
Pharmacia LKB Biotech). Derived A660 values were linear with respect to the amount of the
immunoblotted extract. All immunoblot results were replicated a minimum of three times, with
representative results shown in the various Figs. Non-denaturing PAGE was carried out using 7%
separating gels according to (Gennidakis et al., 2007). In-gel APase activity staining was
performed by equilibrating the gels in 100 mM Na-acetate (pH 5.6) containing 10 mM MgCl2 for
30 min, and then incubating in equilibration buffer containing 1 mg·ml-1 Fast Garnet GBC and
0.03% (w/v) β-naphthyl-P.
2.5.7 Analysis of root structure architecture
Seedlings were removed from vertically grown agar plates and carefully spread on a glass plate
with a fine brush then scanned at 1200 dpi. Root structure architecture was analyzed by tracing
primary and secondary roots with the ImageJ plugin NeuronJ (Meijering et al., 2004).
2.5.8 Phosphate extraction and assays
Tissue was powdered under liquid N2, ground in 10% (w/v) perchloric acid, and centrifuged at
17,500xg for 10 min. Supernatants were neutralized with 5 M KOH/1 M triethanolamine.
Neutralized extracts were centrifuged, and supernatants used for soluble Pi determinations as
previously described (Bozzo et al., 2006).
2.5.9 Statistics
All means are presented as means ± standard error of measurement (SEM). Data were analyzed
using the one tailed Student's t-test, and deemed significant if p < 0.05.
44
Chapter 3
General Discussion
3.1 Overview
Plants are unable to escape unfavorable conditions due to their sessile nature, and must
therefore evolve adaptations to cope with the bevy of biotic and abiotic stresses imposed upon
them. This might be one reason for the large size of plant genomes and gene families. Nutrient
limitation represents a ubiquitous form of abiotic stress, and Pi limitation is especially common
given Pi’s low availability in most soils. Although, Pi is a critical macronutrient and required for
optimal growth plants often maintain nominal growth rates even when Pi is limited. Therefore,
plants have evolved exquisite and specific adaptations for the tolerance of Pi limitation. A well
characterized strategy dealing with Pi starvation is the de novo synthesis and expression of
APases (Bozzo et al., 2006; Veljanovski et al., 2006). This thesis demonstrated the critical role of
APase induction in response to Pi starvation. The research presented in this thesis was obtained
using a reverse genetics approach with the goal of elucidating the in vivo function of AtPAP26.
Although the majority of the work focussed on AtPAP26’s role as a PSI PAP, and its role in
recycling Pi during time of scarcity, it revealed novel and interesting aspects of plant adaptations
to Pi starvation.
This research capitalised on the extensive ‘post-genomic’ resources available for
Arabidopsis (Ülker et al., 2008). It allowed for the identification of an AtPAP26 null allele,
leading to the isolation of Arabidopsis with abrogated expression of AtPAP26, verified by RTPCR, immunoblotting and APase activity assays. One drawback of this approach was being
limited by the total number of available insertion lines. Although one null allele was identified
45
and shown used to generate a line with a single T-DNA insert at the locus in question, the ability
to analyze multiple lines would have added to the impact of this work.
After characterization of a null AtPAP26 allele, completely lacking expression of
intracellular or secreted AtPAP26, atpap26 mutants were characterized for their biochemical and
molecular phenotype. RT-PCR analysis confirmed that AtPAP26 transcript accumulation in Col-0
is unaffected by Pi status although AtPAP26 protein accumulates in the roots and shoots of –Pi
Arabidopsis (Figure 2B, 3) (Tran and Plaxton, 2008; Veljanovski et al., 2006). Elimination of
AtPAP26 expression resulted in almost a complete abrogation of soluble intracellular APase
activity, and a failure to upregulate intracellular or secreted APase activity in response to Pi
starvation. It follows that this archetypical Pi starvation response is controlled primarily at the
post-transcriptional level, and emphasizes the paucity of information regarding posttranscriptional responses to stress in plants.
The effect of AtPAP26 loss of function on the growth of -Pi Arabidopsis seedlings was
also considered. Absence of AtPAP26 expression resulted in compromised growth of ground,
vegetative and reproductive tissues during Pi starvation. Further, AtPAP26 was dispensable
during +Pi growth, during growth limited by nitrogen or potassium, and under oxidative stress
conditions. Therefore, AtPAP26 and PSI APase activity appear to be indispensable for
acclimation to Pi starvation and maintenance of nominal growth rates. The work presented in this
thesis would gain impact if complementation studies were included. Rescue of compromised
growth by expression of AtPAP26 coding sequence in transgenic atpap26 mutants would clearly
demonstrate AtPAP26’s role in increasing Pi utilization efficiency during Pi starvation.
Finally, the Pi metabolism of atpap26 mutants was evaluated by determining the size and
distribution of the free Pi pools as well as the total free Pi content of shoots. Shoots of atpap26
mutants accumulated significantly less total free Pi during Pi starvation. This implies that the
46
growth of atpap26 mutants was constrained both by total Pi content and lower efficiency of Pi
utilization. One reservation about these results regards the extent of Pi starvation imposed in this
study. The 10-30 fold decreases in free Pi are indicative of severe Pi starvation, and analysis of
seedlings at differing stages of Pi stress may have revealed different distribution or size of free Pi
pools. It would be interesting to expand this survey to include determination of total Pi, via acid
hydrolysis of P-esters (Bozzo et al., 2006), or by metabolite analysis via
31
P-NMR or GC-MS
(Jouhet et al., 2003; Lee and Ratcliffe, 1993). The additional resolution provided by these
analyses may reveal the mechanism by which AtPAP26 improves Pi utilization efficiency during
Pi starvation.
3.2 Future Directions
A major implication of the work presented in this thesis regards the extreme paucity of
information regarding post-transcriptional control mediating PSI changes in gene expression. It
appears that AtPAP26 is the predominant molecular contributor of PSI APase activity. Further,
accumulation of AtPAP26 during Pi starvation appears to be independent of transcript
accumulation. It follows that characterization of post-transcriptional control of PSI genes in
general, and AtPAP26 in particular, may provide key insights into the control of Pi starvation
responses and provide new biotechnological targets for targeted manipulation thereof.
Post-transcriptional control of gene expression has been established as a key point of
control in yeast responses to oxidative stress as well as glucose or amino acid starvation (Ashe et
al., 2000; Shenton et al., 2006; Smirnova et al., 2005). Similar results have been shown in
hypoxic roots of corn, heat stressed wheat and dehydration stressed tobacco and Arabidopsis
(Fennoy and Bailey-Serres, 1995; Gallie et al., 1997; Kawaguchi et al., 2003; Kawaguchi et al.,
2004). In all cases, the pool of actively translating ribosomes greatly decreases under stress and is
accompanied by differential phosphorylation of translation initiation factors and ribosomal
47
proteins indicative of a translationally incompetent state (Pierrat et al., 2007; Webster et al.,
1991). Intuition would suggest that Pi starvation would also cause a global translation block,
given the Pi/adenylate expensive nature of translation. Consistent with this, Pi starvation causes a
broad repression of genes involved in RNA synthesis, processing, aminoacyl tRNA activation and
protein synthesis while some ribosomal kinases are induced (Morcuende et al., 2007). A general
survey of the expression and phosphorylation status of the translational machinery during
extended Pi starvation of Arabidopsis would be extremely informative. This can be performed via
immunoblotting using anti-(phospho site specific) IgG that are commercially available for
mammalian translational machinery. It would also be helpful to determine whether PSI genes,
particularly AtPAP26, become differentially associated with polysomes during Pi stress.
Interestingly, splice variants of AtPAP10, AtPAP26’s subfamily 1a-2 member (Fig. 1.2), appear
to become differentially translated during Pi stress despite little variation in overall transcript
abundance (Li et al., 2002).
Alternatively, AtPAP26 abundance may be regulated at the level of protein stability,
either through proteasome mediated degradation or the upregulation of a specific protease.
Secreted PSI PAPs in tomato are selectively degraded by proteases induced following Pi-resupply
to –Pi cells (Bozzo et al., 2004b). Similar methodology could be applied to vaculolar preparations
from +/-Pi Arabidopsis suspension cells to determine if +Pi conditions induce an AtPAP26
targeted protease. On the other hand, ubiquitination and proteasome mediated degradation has
been highlighted as a key regulatory mechanism regulating PSI gene expression through the
PHO2/AtUBC24 and microRNA 399 saga (Bari et al., 2006). However, the target specificity of
the unique E2 ubiquitin conjugase, AtUBC24, and its cognate E3 ligases have never been
explored, and it is unclear how and which genes are controlled by these processes. Use of the
proteasome inhibitor MG132 and highly specific antibodies were critical for determining the
48
post-translational control of sucrose synthase stability in corn leaves (Hardin and Huber, 2004).
Such an experimental approach would be beneficial in determining control of AtPAP26
accumulation, and is relatively easy to implement. Elucidation of mechanisms controlling
AtPAP26 gene expression may shed light on general mechanisms of PSI gene regulation that are
currently unknown.
A pressing concern regarding AtPAP26 function relates to its shared extracellular and
intracellular roles (Tran and Plaxton, 2008). Currently it is not clear how AtPAP26 is
differentially targeted between the extracellular and vacuolar compartments. Interestingly, the PSI
high affinity Pi transporter, PHT1;1, is dependent on PHF1, a SEC12 related early secretory
protein, for transport to the plasma membrane (González et al., 2005). Expression of PHF1 is Pi
dependant, and analogous to PHO86/84 system in -Pi yeast. Whether such a system exists for
sub-cellular localization of AtPAP26 is unknown, and highlights the need for detailed sub-cellular
localization studies of AtPAP26. This could be achieved via transient expression of
AtPAP26::GFP constructs in Arabidopsis suspension cell culture. Differential glycosylation of
AtPAP26 could also play a role in determining its subcellular location. Purification of secreted
AtPAP26 from –Pi Arabidopsis suspension cells has revealed a pair of extracellular AtPAP 26
isoforms that are differentially glycosylated at the three predicted N-glycosylation sites (Tran and
Plaxton, unpublished data). Secretion of PAPs in insect cells is dependent upon the presence and
glycosylation of N- glycosylation sites (Olczak and Olczak, 2007). Therefore, it would be
informative to determine the role these glycosylation sites play in the secretion, structure and
function of AtPAP26. Point-site directed mutagenesis of the three N-glycosylation sites in the
aforementioned AtPAP26::GFP constructs would aid in elucidating their targeting function.
Additionally, development of untagged glycosylation mutants could be used to develop material
for kinetic analysis of differentially glycosylated AtPAP26.
49
It is not clear how different PAP/APase isozymes contribute to overall APase activity in
the vacuole or extracellular compartment. Multiple APases have been discovered in the vacuole,
cell wall and secretome of Arabidopsis (Carter et al., 2004; Irshad et al., 2008; Tran and Plaxton,
2008). Currently, the growth characteristics of atpap26 mutants are being evaluated on Pi
deprived soil, such that the only Pi available is likely in the form of soil esterified Pi (Tomscha et
al., 2004). Impaired growth and decreased secreted APase activity in the atpap26 mutants would
confirm AtPAP26’s functional significance in extracellular Pi recycling. Isolation of AtPAP12,
another secreted PSI PAP (Haran et al., 2000; Tran and Plaxton, 2008, and unpublished data), TDNA knockout lines is currently underway. Similar experiments will be performed using
AtPAP12 null backgrounds. Further, double knockouts of AtPAP12/AtPAP26 will be developed
to determine if these two 1a-2 family members play complementary roles in extracellular Pi
scavenging in –Pi Arabidopsis. It would also be informative to determine AtPAP26’s contribution
to cell wall associated APase activity. Root-surface staining with BCIP, followed by
immunoblotting of intact roots using AtPAP26 and AtPAP12 specific antibodies, of Col-0 and
atpap26 mutants would determine whether AtPAP26 plays a role in cell wall associated APase
activity (Bozzo et al., 2006). Further, salt washes of sedimented cell walls can release ionically
bound APases and their associated activity (Kaida et al., 2008), and may be analyzed by APase
activity assays, immunoblotting and activity stained native PAGE for confirmation of results
obtained from root staining and immunoblotting experiments.
In parallel to the knockout work over-expression studies are being carried out. The full
length coding sequences of AtPAP12 and AtPAP26 have been cloned into pMDC32, a
constitutive expression vector with 2 x cauliflower mosaic virus 35s promoters, and used to
generate transgenic Arabidopsis (Curtis and Grossniklaus, 2003). In addition to characterizing
their growth and Pi acquisition on esterified Pi, over-expressers will be used for complementation
50
studies of atpap26 mutants. It would also be interesting to test whether ectopic expression of
AtPAP26 in crop plants would increase their ability to acquire Pi during growth on unfertilized
soil, in a manner similar to experiments performed with AtPAP15 and Medicago PAPs and
phytases (Ma et al., 2009; Wang et al., 2009).
The aforementioned work will greatly expand our knowledge of PSI APases, their
function and regulation. It may also help shed light on general mechanisms of PSI gene
regulation. In both cases, this will help develop targets and strategies for biotechnological efforts
in producing crop species with increased Pi efficiency.
51
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