The cell wall-targeted purple acid phosphatase AtPAP25 is critical for acclimation of Arabidopsis thaliana to nutritional phosphorus deprivation

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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/tpj.12663 This article is protected by copyright. All rights reserved.

Received Date : 04-Jun-2014 Revised Date : 15-Aug-2014 Accepted Date : 29-Aug-2014 Article type : Original Article The cell-wall targeted purple acid phosphatase AtPAP25 is critical for

acclimation of Arabidopsis thaliana to nutritional phosphorus-deprivation

Hernan A. Del Vecchio1, Sheng Ying1, Joonho Park2, Vicki L. Knowles1, Satomi

Kanno3, Keitaro Tanoi3, Yi-Min She4 and William C. Plaxton1,5*

1Department of Biology and 5Department of Biomedical and Molecular Sciences, Queen’s

University, Kingston, Ontario K7L 3N6, Canada

2Department of Fine Chemistry, Seoul National University of Science and Technology, 138

Gongreung-Gil, Nowon-Gu, Seoul 139-743, Korea

3Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo

113-8657, Japan

4Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, Songjiang,

Shanghai 201602, China

Corresponding author: William C. Plaxton, Department of Biology, Queen’s University,

Kingston, Ontario K7L 3N6, Canada. Phone: 1-613-533-6150; FAX: 1-613-533-6617; E-

mail: [email protected]

Running title: Cell wall PAPs of Pi-deprived Arabidopsis

Key words: Arabidopsis thaliana (thale cress), functional genomics, cell wall proteome,

phosphorus nutrition, purple acid phosphatase.

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This article is protected by copyright. All rights reserved.

SUMMARY

Plant purple acid phosphatases (PAPs) belong to a relatively large gene family whose

individual functions are poorly understood. Three PAP isozymes upregulated in cell walls of

phosphate (Pi) starved (–Pi) Arabidopsis thaliana suspension cells were purified and

identified by MS as AtPAP12 (At2g27190), AtPAP25 (At4g36350), and AtPAP26

(At5g34850). AtPAP12 and AtPAP26 were previously isolated from the culture media of the

–Pi cell cultures and shown to be secreted by roots of Arabidopsis seedlings to facilitate Pi

scavenging from soil-localized organophosphates. AtPAP25 exists as a 55-kDa monomer

containing complex NX(S/T) glycosylation motifs at Asn172, Asn367, and Asn424. Transcript

profiling and immunoblotting with anti-AtPAP25 immune serum indicated that AtPAP25 is

exclusively synthesized under –Pi conditions. Coupled with AtPAP25’s mixed-type inhibition

by Pi (I50 = 50 µM), this indicates a tight feedback control by Pi that would prevent AtPAP25

from being synthesized or functioning as a phosphatase except when Pi levels are quite low.

Promoter:β-glucuronidase reporter assays revealed AtPAP25 expression in shoot vascular

tissue of –Pi plants. Development of an atpap25 T-DNA insertion mutant was arrested

during cultivation on a soluble Pi-deficient soil, but rescued upon Pi fertilization or

complementation with AtPAP25. Transcript profiling by qPCR indicated that Pi starvation

signaling was attenuated in the atpap25 mutant. AtPAP25 exhibited near-optimal

phosphatase activity with several phosphoproteins and phosphoamino acids as substrates.

We hypothesize that AtPAP25 plays a key signaling role during Pi-deprivation by functioning

as a phosphoprotein phosphatase, rather than as a non-specific scavenger of Pi from

extracellular organic-P esters.

INTRODUCTION

Acid phosphatases (APases; EC 3.1.3.2) catalyze the release of orthophosphate (Pi) from a

wide variety of P-monoesters and anhydrides with an acidic pH optimum. They function in

the production and recycling of Pi, a crucial but environmentally limiting macronutrient for

cellular metabolism and bioenergetics (Tran et al., 2010a). Purple APases (PAPs) are the

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largest class of non-specific plant APase, and have a characteristic pink or purple color in

solution which is caused by the occurrence of a binuclear metal ion complex at their active

site. Upregulation of PAPs is a ubiquitous plant response to Pi deficiency, a frequent abiotic

stress that restricts plant growth in natural ecosystems (Tran et al., 2010a). Intracellular

PAPs recycle Pi from dispensable P-monoesters and anhydrides during Pi stress.

Extracellular PAPs belong to an assortment of Pi starvation inducible (PSI)

phosphohydrolases that cleave Pi from external organic-P molecules, the main form of P in

soil solutions which can account for up to 80% of the soil’s total P content (Tran et al.,

2010a).

Genomic and proteomic methodologies have helped to identify PAP genes that

mediate efficient Pi acquisition and use by vascular plants. Genome annotation identified 29

putative PAP genes in Arabidopsis thaliana, several of which are induced during Pi

deprivation (Tran et al., 2010a). The variation and complexity of AtPAP1-29 expression has

been well documented (Robinson et al., 2012a; Tran et al., 2010a, 2010b; Veljanovski et al.,

2006; Wang et al., 2011; Wang et al., 2014; Zamani et al., 2014; Zhang et al., 2008; Zhu et

al., 2005). To assess the function of specific AtPAP genes in the metabolic adaptations of Pi-

starved (–Pi) Arabidopsis, we have purified, characterized, and identified native intracellular

and secreted AtPAP isozymes upregulated during Pi stress. Cell suspension cultures have

been instrumental in this respect since a large quantity of –Pi cells and the corresponding

liquid media containing secreted enzymes can be readily acquired (Tran et al., 2010b; Tran

and Plaxton, 2008; Veljanovski et al., 2006). Thus, the primary intracellular (vacuolar) PAP

upregulated by –Pi Arabidopsis was isolated and identified as AtPAP26 (At5g34850)

(Veljanovski et al., 2006). Subsequent analyses of an atpap26 loss-of-function mutant

confirmed vacuolar AtPAP26 as the predominant contributor to intracellular APase activity,

and that it plays essential roles to scavenge and remobilize Pi during nutritional Pi

deprivation or leaf senescence (Hurley et al., 2010; Robinson et al., 2012a; 2012b; Shane et

al., 2014). Additional studies revealed that AtPAP26 is also targeted to the cell wall (CW)

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and secretome of –Pi Arabidopsis (Robinson et al., 2012a). For example, AtPAP26 and the

closely related AtPAP12 (At2g27190) account for about 75% of the total APase activity

secreted by roots of Arabidopsis seedlings cultivated under –Pi conditions, as well as on

various exogenous organic-P sources such as glycerol-3-P or salmon sperm DNA (Robinson

et al., 2012a). AtPAP12 and AtPAP26 were also upregulated on root surfaces and in shoot

CW proteomes of –Pi seedlings (Robinson et al., 2012a; Wang et al., 2014). CW-associated

AtPAP12 and AtPAP26 were hypothesized to facilitate P-status maintenance by recycling Pi

from cytoplasmic P-monoesters that had leaked into CWs during Pi deprivation (Robinson et

al., 2012a). Furthermore, AtPAP10 is a PSI PAP predominantly localized to the surface of

root epidermal cells, and that also functions in the acclimation of Arabidopsis to Pi

deprivation (Wang et al., 2011; 2014).

The CW is a dynamic structure intimately involved with plant growth and

development, responses to abiotic stress, and interactions with pathogens and symbionts.

The complexity and importance of the CW is reflected by the large number of genes

suspected to play a role in its biogenesis, assembly, and modifications (Albenne et al.,

2013). CW proteins represent about 10% of the CW mass, and encompass hundreds of

different proteins that function as CW modifying enzymes, structural proteins, or stress-

inducible defense proteins (Albenne et al., 2013). The aim of the current study was to target

the CW proteome for purification, identification, and eventual biochemical, molecular, and

functional characterization of CW PAPs upregulated by –Pi Arabidopsis suspension cells.

CW-localized AtPAP12 and AtPAP26 were shown to be strongly upregulated, corroborating

previous results obtained with –Pi Arabidopsis seedlings (Robinson et al., 2012a). However,

the previously uncharacterized AtPAP25 was discovered to be exclusively expressed and

targeted to CWs of –Pi Arabidopsis while exhibiting kinetic features consistent with its

possible function as a phosphoprotein phosphatase. Analysis of an atpap25 mutant

indicated that AtPAP25 makes a pivotal contribution to the ability of Arabidopsis to acclimate

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to Pi deficient soils, and that Pi-starvation signaling appears to have been attenuated in the

mutant.

RESULTS AND DISCUSSION

Influence of Pi-deprivation on cell wall acid phosphatase activity and immunoreactive

PAP polypeptides of Arabidopsis suspension cell cultures

When Arabidopsis suspension cells were subcultured into media containing 5 mM Pi and

harvested 7-d later, the cell culture filtrates (CCFs) contained 1.8 mM Pi, demonstrating that

the cells were still Pi-sufficient (+Pi) (Figure 1a). However, extracellular Pi became

undetectable when parallel –Pi cells were cultivated for more than 4-d in media containing 0

mM Pi; this was paralleled by a 4-fold increase in CW APase activity (Figure 1b). Resupply

of the –Pi cells with 2.5 mM Pi at day-6 triggered a steadily decrease in CW APase activity to

+Pi levels over the next several days (Figure 1b). Hydrophobic interaction chromatography

of the CW extracts was performed using butyl Sepharose to enrich CW APase activity. The

resulting samples from the –Pi cells showed increased specific APase (5.11 ±0.09 units mg-

1) relative to those from the +Pi cells (1.26 ±0.03 units mg-1). This was correlated with the

upregulation of 60 and 55 kDa immunoreactive polypeptides on immunoblots of butyl

Sepharose-enriched –Pi CW extracts probed with anti-AtPAP12 immune serum (anti-

PAP12), which respectively co-migrated with AtPAP12 and AtPAP26 isolated from the CCF

of the –Pi cell cultures (Figure 1c). These results agree with earlier work demonstrating that

AtPAP12 and AtPAP26 are also strongly upregulated in shoot CW proteomes and on root

surfaces of –Pi Arabidopsis seedlings (Robinson et al., 2012a).

Previous research established that secreted CCF proteins from the +Pi or –Pi

Arabidopsis cell cultures were not contaminated by cytoplasmic marker enzymes (Tran et

al., 2010b; Tran and Plaxton, 2008). Likewise: (i) immunoreactive cytosolic

phosphoenolpyruvate (PEP) carboxylase and aldolase polypeptides were absent on

immunoblots of the CW extracts, but detected on immunoblots of corresponding clarified

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extracts prepared from cells that had previously been treated with CaCl2 for CW protein

extraction, and (ii) PEP carboxylase and aldolase activities were detected in the clarified cell

extracts, but not in the corresponding CW proteomes (Figure S1).

Purification and identification of cell wall PAPs of Pi-deprived Arabidopsis

suspension cells

Three peaks of APase activity (corresponding to AtPAP12, AtPAP25, and AtPAP26; see

below) were resolved during SO3--cation exchange fast protein liquid chromatography

(FPLC) of the concentrated butyl Sepharose fraction obtained from the CW proteome of the

–Pi cells (Figure 2b). As reported during AtPAP26 purification from the CCF of the –Pi cells

(Tran et al., 2010b), the pooled AtPAP26 activity fractions from the butyl Sepharose column

resolved as a pair of putative glycoforms (AtPAP26-CW1 and AtPAP26-CW2) during lectin-

affinity chromatography on conA. AtPAP26-CW1 did not bind to the conA column, whereas

AtPAP26-CW2 (and AtPAP12 and AtPAP25) was bound and subsequently eluted by a

methyl-α-D-mannopyranoside gradient (denotes existence of terminal mannose residues).

The proportion of AtPAP26-CW1 to AtPAP26-CW2 activities eluting during conA

chromatography was about 2:1. AtPAP26-CW1’s failure to bind to the conA matrix was not

due to column overloading since immediate application of this sample on a second, freshly

prepared conA column led to quantitative recovery of AtPAP26-CW1 in the resultant

unbound fractions. Additional research is needed to determine glycan structures and

linkages at each N-linked glycosylation site of AtPAP26 glycoforms secreted into the CW

and apoplast by –Pi Arabidopsis. However, our earlier study of a pair of AtPAP26 glycoforms

purified from the CCF of –Pi Arabidopsis cells: (i) employed a glycan differentiation kit that

provided independent evidence that are diffentially glycosylated, and (ii) reported that

differential glycosylation appears to modulate their respective substrate specificities (Tran et

al., 2010b).

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AtPAP12, AtPAP25, AtPAP26-CW1, and AtPAP26-CW2 were purified to final PEP

hydrolyzing specific activities of 66, 5.5, 252, and 108 units mg-1, respectively (Table S1).

The concentrated final preparations of AtPAP12, AtPAP26-CW1, and AtPAP26-CW2

showed a pink color in solution, while AtPAP25 was purple. The purified PAPs were

subjected to N-terminal sequencing and peptide mass fingerprinting via matrix-assisted laser

desorption/ionization time-of-flight (MALDI-TOF) MS. Comparison with databank sequences

identified them as AtPAP12 (At2g27190), AtPAP25 (At4g36350), and two glycoforms of

AtPAP26 (At5g34850) (Figure 3 and Table S2). AtPAP25 was previously detected in the CW

proteome of Arabidopsis suspension cells (Borderies et al., 2003; Chivasa et al., 2002).

These studies were likely conducted with Pi-limited cells, as the cells were subcultured for

≥7-d in conventional MS media containing 1.25 mM Pi, which is suboptimal for maintaining

Arabidopsis suspension cells fully +Pi during one week in batch culture (Tran and Plaxton,

2008; Veljanovski et al., 2006).

Physical and immunological properties

When the purified CW PAPs were analyzed by SDS-PAGE, major protein-staining

polypeptides of approximately 60 (AtPAP12) and 55 kDa (AtPAP25 and AtPAP26-CW1/-

CW2) were observed, which cross-reacted with anti-AtPAP12 (Figure 4a and 4b). By

contrast, anti-AtPAP25 and anti-AtPAP26 were relatively monospecific for their respective

antigens (Figure 4b). SDS-PAGE followed by protein staining indicated that AtPAP12 and

AtPAP26-CW2 were highly purified, whereas AtPAP25 and AtPAP26-CW1 were purified to

apparent and near homogeneity, respectively (Figure 4a). The native Mr of AtPAP12 was

estimated by analytical gel filtration to be 120 ±10 kDa, whereas that of AtPAP25, AtPAP26-

CW1, and AtPAP26-CW2 were each 55 ±5 kDa (means ± SEM, n = 3 determinations). The

combined results indicate that purified AtPAP12 exists as a homodimer, while AtPAP25 and

AtPAP26-CW1/-CW2 are monomeric. Identical results were obtained with AtPAP12 and a

pair of AtPAP26 glycoforms isolated from the CCF of the –Pi suspension cells (Tran et al.,

2010b).

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Bioinformatic analysis of AtPAP25

The AtPAP family is encoded by 29 genes that were categorized into three discrete groups

following bioinformatics analysis of their deduced amino acid sequences (Tran et al., 2010).

Group Ia-1 is represented by AtPAP5, AtPAP6, AtPAP11, AtPAP19, and AtPAP25, whereas

Group Ia-2 is composed of AtPAP10, AtPAP12, and AtPAP26. AtPAP25 shares 51-71% and

55-60% sequence identity with Group Ia-1 and 1a-2 members of the AtPAP family,

respectively, while its sequence identity with AtPAP25 orthologs from other plants ranges

from 56-68% (Table S3). In silico analysis of the deduced AtPAP25 sequence indicated that

the mature protein has molecular mass of 53 kDa (Table S3). The 2 kDa discrepancy with

the subunit Mr of the purified native AtPAP25 as estimated by SDS–PAGE (Figure 4) is

explained by the post-translational (covalent) incorporation of glycan groups (Table S4).

Signal P incorrectly predicted that the N-terminus of the deduced AtPAP25 polypeptide

contains a 21 amino acid signal peptide. AtPAP25’s actual signal peptide of 29 amino acids

was revealed by comparing the N-terminal sequence obtained with the purified AtPAP25

with its deduced full-length amino acid sequence (Figure 3). It is notable that the signal

peptides of AtPAP12, AtPAP25, and AtPAP26 were all cleaved at an identical site, beginning

after a conserved arginine residue (Figure 3).

AtPAP25 glycosylation motifs

Glycosylation is an essential post-translational modification that can modulate enzyme

stability, localization, solubility, and kinetic properties (Varki, 1993). Secreted plant proteins

usually contain highly heterogeneous glycan pools. Although all characterized plant PAPs

are glycosylated (Olczak et al., 2003; Olczak and Olczak, 2007; Tran et al., 2010a), few

details of their N-linked glycans are available. The majority of the glycan residues of plant

PAPs studied to date consist of complex oligosaccharides containing mannosylated,

fucosylated and/or xylosylated residues (Olczak et al., 2003; Stahl et al., 1994). Plant PAPs

having 55-kDa subunit sizes usually have three to five N-glycosylation sites and

approximately 10% sugar content (Olczak et al., 2003; Olczak and Olczak, 2007; Stahl et al.,

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1994). N-linked oligosaccharides of purified AtPAP25 were characterized by high-resolution

ultra-performance liquid chromatography (UPLC) separation coupled with linear ion-trap

Fourier transform ion cyclotron resonance tandem MS (LTQ-FT MS/MS) analysis of

glycopeptides generated by either specific trypsin or nonspecific proteinase K digestion. The

three putative N-glycosylation sites of AtPAP25 (Asn172, Asn367, and Asn424; Figure 3)

possess the consensus NX(S/T) glycosylation motif (where X is any amino acid except

proline), were each determined to contain two compositions of xylomannosides;

GlcNAc(Fuc)GlcNAc(Xyl)Man3 and GlcNAc(Fuc)GlcNAc(Xyl)Man3GlcNAc (Supplemental

Table S4), but no high-mannose-type glycan. The Asn424 glycosylation site was further

modified by the addition of a terminal galactose residue to form an extended side chain of

GlcNAc(Fuc)GlcNAc(Xyl)Man3GlcNAcGal (Table S4). A site-directed mutagenesis study of

Phaseolus vulgaris (common bean) and Lupin luteus (European white lupin) PAP isozymes

(orthologous to AtPAP12 and AtPAP26, respectively) was employed to generate various

PAP mutants lacking N-glycosylation at specific asparagine residues (Olczak and Olczak,

2007). The results established that glycosylation, particularly at the conserved glycosylation

site located near the C-terminus of all known plant PAPs (e.g., corresponding to Asn424 of

AtPAP25) is an essential prerequisite for PAP stability and secretion.”

A study of the phosphotyrosylated proteome of Arabidopsis cell cultures reported that

AtPAP25 is phosphorylated at Tyr423 (Sugiyama et al., 2008). However, MS/MS analysis of

AtPAP25 failed to detect evidence for AtPAP25 phosphorylation at any site, including Tyr423

(Figure S2). Phosphorylation of AtPAP25 at Tyr423 is improbable given the bulky N-linked

oligosaccharide that is covalently bonded to the adjacent Asn424 residue (Figure 3, Table

S4).

AtPAP25 kinetic properties

Similar to other plant PAPs, AtPAP25 displayed a wide pH-activity profile with a maximum at

pH 5.6 (Figure S3), and hyperbolic PEP saturation kinetics (Km(PEP) = 0.42 mM). AtPAP25

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showed a low Vmax with PEP (5.5 units mg-1) as well as other in vitro substrates that were

identified, relative to values ranging from about 60 - 1700 units mg-1 obtained with AtPAP12

or AtPAP26 isoforms isolated from CW or CCF of the –Pi Arabidopsis suspension cells

(Tables 1 and S1) (Tran et al., 2010b). Although AtPAP25 hydrolyzed Pi from a broad variety

of P-esters and anhydrides, it exhibited a substrate selectivity consistent with its possible

function as a phosphoprotein phosphatase (Table 1). Comparable activities were obtained

with saturating levels of the phosphoamino acids P-Thr, P-Ser, and P-Tyr, as well as with the

storage phosphoproteins ovalbumin and phosvitin (Table 1), unlike AtPAP12 or AtPAP26

(Tran et al., 2010b; Veljanovski et al., 2006). However, AtPAP25 failed to use 5 mM phytate

as a substrate which demonstrates that in contrast to AtPAP15 (Zhang et al., 2008), it is not

a phytase.

AtPAP25 activity was unaffected by the addition of MgCl2, CoCl2, MnCl2, or EDTA (5

mM each). As with other PAPs, the APase activity of AtPAP25 was insensitive to 5 mM L-

tartrate, but potently inhibited by vanadate, arsenate, molybdate, Zn2+, Fe2+ and Cu2+ (Table

S5). Apart from its low Vmax, another unusual kinetic feature of AtPAP25 was its strong

feedback inhibition by Pi as reflected by a low I50(Pi) value of 50 ±6 µM (determined with 0.5

mM PEP). Interestingly, Pi functions as a mixed-type inhibitor since increasing

concentrations of Pi significantly decreased and increased AtPAP25’s Vmax and Km(PEP)

values, respectively (Figure S4). The enzyme’s competitive and uncompetitive dissociation

constants for Pi were determined to be 0.43 ±0.04 and 0.74 ±0.05 mM, respectively. By

contrast, Pi typically serves as a competitive inhibitor of plant PAPs (i.e., increases Km,

without influencing Vmax) (Tran et al., 2010a).

Influence of P-status on AtPAP25 gene and protein expression

Transcripts of AtPAP25 were detected by RT-PCR in –Pi, but not +Pi, Arabidopsis

suspension cells (Figure 5a). This effect was quantified by qPCR, which revealed striking

induction of AtPAP25 transcripts in roots and particularly shoots of –Pi seedlings (Figure 5b).

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Similarly: (i) comparison of APase activity elution profiles during cation-exchange FPLC of

CW extracts from the +Pi versus –Pi cells indicated that AtPAP25 was de novo synthesized

in response to Pi deprivation, whereas AtPAP12 and AtPAP26 were markedly upregulated

(Figure 2), and (ii) immunoblot analysis using anti-AtPAP25 indicated that unlike AtPAP12

and AtPAP26 (Figure 1C and Figure 2), AtPAP25 polypeptides were exclusively expressed

in CWs of the –Pi cells (Figure 5c). In contrast, immunoreactive AtPAP25 polypeptides were

absent on immunoblots of CW or total protein extracts of shoots or roots of –Pi Col-0

seedlings (using chromogenic or chemiluminscent detection). Likewise, immunoreactive

AtPAP25 polypeptides were not apparent on immunoblots of flower extracts from the Col-0

seedlings. This suggests that AtPAP25 polypeptide expression is very low in planta, relative

to other PSI secreted PAPs such as AtPAP12 and AtPAP26 (Robinson et al., 2012a; Tran et

al., 2010b).

To assess the tissue specificity of AtPAP25 expression, an AtPAP25 promoter: β-

glucuronidase (GUS) reporter gene fusion was generated. The expression of GUS activity

was examined in eleven independent AtPAP25:GUS transgenic lines, all of which indicated

strong expression of the AtPAP25 promoter in shoot vascular tissue during vegetative

growth (particularly during –Pi growth), as well as in anthers and developing embryos of

flowering seedlings (Figure 6). Flower specific-expression of AtPAP25 was previously

demonstrated in Arabidopsis, with AtPAP25 transcripts being particularly abundant in

stamens (Zhu et al., 2005).

Characterization of an atpap25 loss-of-function mutant

An atpap25 mutant was obtained in which a T-DNA insertion was predicted to occur in the

first exon of the AtPAP25 gene (locus At4g36350). This was confirmed by PCR screening of

gDNA using an AtPAP25-specific primer and a T-DNA right border primer (Figure 7a and

7b). Mutant homozygosity was established by PCR of gDNA using AtPAP25-specific primers

(Figure 7b). Elimination of AtPAP25 expression was confirmed by RT-PCR of cDNA

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prepared from shoots of –Pi atpap25 plants (Figure 8b). Total shoot CW APase activity of –

Pi atpap25 mutant seedlings was not significantly different from that of the Col-0 controls

(0.27 ±0.05 and 0.30 ±0.04 units mg-1, respectively). Histochemical staining of APase activity

on root surfaces of –Pi atpap25 and Col-0 seedlings using 5-Br-4-Cl-3-indoyl-P (which

serves as an effective substrate for purified AtPAP25; Table 2) was also indistinguishable

(Figure S5b). These results further indicate that AtPAP25 polypeptide levels are very low in

–Pi Col-0 plants.

No obvious differences were noted relative to Col-0 controls in the development or

appearance of atpap25 plants when 7-d hydroponically-cultivated +Pi seedlings were

transferred to sterile –Pi liquid or agar-solidified MS media, or a Pi-fertilized soil mixture

(Figures S5, 7c, 8a, and 8c). Although Pi deprivation stimulated 33Pi uptake capacity of

hydroponically-cultivated Col-0 and atpap25 seedlings, no differences in the capacity to

translocate 33Pi from roots to the shoots were apparent (Figure S6). Similarly, no phenotypic

differences relative to Col-0 were revealed when +Pi atpap25 seedlings were cultivated on

nitrogen, sulfur, or potassium deficient sois. However, atpap25 development was blocked

when 7-d old +Pi seedlings were transplanted into an organic-P containing soil mix lacking

soluble Pi and cultivated for an additional 14-d under a regular light:dark regime (Figure 7c).

Their leaves turned dark purple due to marked anthocyanin accumulation, and total leaf Pi

concentration was over 4-fold lower than that of –Pi Col-0 controls (Figure 7d). The arrested

development of the 21-d-old soil-cultivated –Pi atpap25 plants was rapidly reversed when

they were fertilized with 2 mM Pi and cultivated for an additional 14-d (Figure 8A). Likewise,

complementation with AtPAP25 resulted in normal growth of the resulting transgenic plants

on the +Pi or –Pi soil mix (Figure 8b and 8c). qPCR also revealed that AtPAP25 expression

was undetectable in leaves of –Pi atpap25 plants, but was massively induced (by 840 ±60%)

in leaves of soil-cultivated –Pi Col-0 plants, relative to +Pi controls. This corroborates qPCR

results obtained with Col-0 seedlings cultured in sterile MS liquid media (Figure 5B).

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Pi-starvation signaling is attenuated in the atpap25 mutant

Expression of AtPAP12 and AtPAP17 as well as several additional, well-documented PSI

genes were also evaluated by qPCR to determine if Pi-starvation signaling was affected in

the soil-cultivated atpap25 mutant. At4 is a strongly PSI gene in vascular tissue of

Arabidopsis root and shoots that is believed to function in Pi distribution between shoots and

roots during Pi stress (Ha and Tran, 2014; Shin et al., 2006). AtPPCK1 encodes a cytosolic

protein kinase that phosphorylates and activates phosphoenolpyruvate carboxylase in –Pi

Arabidopsis (Gregory et al., 2009), whereas AtRNS1 encodes a vacuolar and secreted (cell

wall, secretome) ribonuclease involved in Pi-recycling from expendable RNA pools (Ha and

Tran, 2014; Tran and Plaxton, 2008). Expression of At4, AtPPCK1, AtRNS1, AtPAP12, and

AtPAP17 was significantly reduced (by 35-70%) in atpap25 shoots relative to Col-0 controls,

whereas expression of AtPAP26 and AtPHO1 was not significantly affected (Figure 9).

AtPAP26 and AtPHO1 represent genes known to play essential roles in Arabidopsis Pi-

starvation responses, but whose transcripts are weakly responsive to Pi deprivation stress

(Ha and Tran, 2014; Tran et al., 2010b; Veljanovski et al., 2006).

CONCLUDING REMARKS

The current results demonstrate that AtPAP25, AtPAP12, and AtPAP26 are the predominant

AtPAP isozymes upregulated and targeted to the extracellular matrix of –Pi Arabidopsis

suspension cells. In contrast to AtPAP12 and AtPAP26 (Hurley et al., 2010; Robinson et al.,

2012a; Robinson et al., 2012b; Tran et al., 2010b; Veljanovski et al., 2006; Wang et al.,

2014), we are unaware of earlier reports describing the biochemical or functional properties

of AtPAP25, nor its involvement in the Arabidopsis Pi-starvation response. It is remarkable

that the transit peptides of AtPAP25, as well as AtPAP12 and its soybean (Glycine max)

ortholog, secreted and vacuolar AtPAP26 glycoforms, and AtPAP26 orthologs purified from

onion (Allium cepa) bulbs and –Pi tomato (Lycopersicon esculentum) cell cultures are all

cleaved at the same site, beginning after an invariant arginine residue (Figure 3) (Tran et al.,

2010a). Future studies are needed to identify the signal peptidase that hydrolyzes the transit

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peptide from precursors of vacuolar and secreted PAP isoforms during their subcellular

sorting in –Pi plant cells. AtPAP25’s restricted CW localization may be the result of

glycosylation-driven targeting, and its putative signaling role might also rely on this, adding

more complexity to its in vivo function. LTQ-FT MS/MS analysis of AtPAP25 revealed a

complex array of glycans, N-linked to three Asn residues possessing the conserved NX(S/T)

glycosylation motif. The importance of glycosylation in controlling enzyme localization,

turnover, activity, and association with binding partners may transcend PAPs and Pi

starvation. The involvement of both the individual protein and its cellular environment in

determining glycosylation site occupancy and glycan processing allows cells to generate an

assortment of glycoforms, thus endowing a single glycosylated enzyme with an assortment

of functional and dynamic activities (Varki, 1993).

GUS reporter assays indicated that AtPAP25 is specifically and strongly expressed in

shoot vascular tissue during vegetative growth of –Pi plants, as well as in stamens and

developing embryos of the corresponding flowering plants. Similar results were obtained with

AtPAP10:GUS seedlings, although the APase activity of AtPAP10 appears to be

predominantly associated with root surfaces of –Pi Arabidopsis (Wang et al., 2011). By

contrast, –Pi AtPAP12:GUS and AtPAP26:GUS plants showed widespread GUS activity

(Robinson et al., 2012a). Another uncommon feature of AtPAP25 was that it appeared to be

exclusively expressed and synthesized in response to Pi deprivation, but at low levels

compared to other PSI PAPs such AtPAP12 and AtPAP26 (Robinson et al., 2012a; Tran et

al., 2010b). Coupled with AtPAP25’s mixed-inhibition by Pi, this indicates a tight feedback

control by Pi that would prevent AtPAP25 from being synthesized or functioning as a

phosphatase except when Pi levels are very low.

Unusual kinetic properties of AtPAP25 also included its relatively low Vmax, and a

substrate selectivity suggesting that it might function as a phosphoprotein phosphatase

rather than as a non-specific scavenger of Pi from extracellular organic-P compounds, as

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demonstrated for AtPAP10, AtPAP12, and AtPAP26 (Robinson et al., 2012a; Tran et al.,

2010b; Wang et al., 2011; Shane et al., 2014; Wang et al., 2014). Similarly, NtPAP12 is a

CW PAP of +Pi tobacco cells orthologous to AtPAP10 and that appears to function as a

phosphoprotein phosphatase that participates in the control of cellulose metabolism (Kaida

et al., 2009, 2010). Extracellular protein phosphorylation is widespread in eukaryotic cells. It

will thus be important to determine the in vivo substrate(s) for AtPAP25, and whether

AtPAP25 contributes to reprogramming of the CW phosphoproteome upon nutritional Pi-

deficiency.

AtPAP25 clearly plays an important role in the Arabidopsis Pi-starvation response as

the atpap25 mutant demonstrated a dramatic arrested growth phenotype when 7-d old +Pi

seedlings were cultivated for two weeks in a peat-vermiculite soil mixture lacking soluble Pi

(Figures 7 and 8). Quantification of transcripts encoding five PSI genes indicated that Pi

starvation signaling was attenuated in shoots of the soil-cultivated atpap25 mutant (Figure

9). It is notable, however, that AtPAP26 and AtPHO1 transcript levels were unaffected in the

mutant. Although AtPAP26 and AtPHO1 play essential roles in Arabidopsis Pi starvation

responses, levels of their respective transcripts are largely unresponsive to Pi deprivation

(Ha and Tran, 2014; Hurley et al., 2010; Robinson et al., 2010a; Tran et al., 2010b;

Veljanovski et al., 2006). This suggests that the reduced expression of At4, AtPPCK1,

AtRNS1, AtPAP12, and AtPAP17 observed in the –Pi atpap25 mutant was not due to a

general restriction in cellular function, as opposed to a specific disruption of the Pi starvation

response. Additional studies are needed to test our hypothesis that AtPAP25 functions as a

phosphoprotein phosphatase in CWs of shoot vascular tissue of –Pi plants, and that this

somehow participates in the overall signaling pathway by which Arabidopsis responds to

nutritional P deprivation at the molecular level. In this regard, microarray transcriptome and

(phospho)proteomic profiling to assess global gene and (phospho)protein expression

changes in atpap25 plants would help to further establish the degree to which Pi-starvation

signaling has been impaired in the mutant. Overall, these studies are relevant to current

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applied efforts to bioengineer P-efficient crops, urgently required to reduce the overuse of

non-renewable and polluting Pi-containing fertilizers in agriculture.

EXPERIMENTAL PROCEDURES

Plant material and growth conditions

Heterotrophic Arabidopsis (Arabidopsis thaliana, cv. Landsberg erecta) suspension cells

were maintained at 21 oC in the dark as previously described (Tran et al., 2010b; Veljanovski

et al., 2006). For large-scale –Pi subculture, 50 ml aliquots of 7-d Pi-replete (+Pi; 5 mM

K2HPO4) cultures were used to inoculate 2.6 l Fernbach flasks containing 450 ml of fresh MS

media lacking Pi. Cells were collected after 7-d by filtration, frozen in liquid N2, and stored at

-80 °C.

For routine plant growth and mutant isolation, Arabidopsis (Col-0 ecotype) seeds

were sown in a standard soil mixture (Sunshine Aggregate Plus Mix 1; SunGro, Vancouver,

Canada) and stratified at 4 oC for 3-d. Plants were cultivated in growth chambers at 23 °C

(16/8 h photoperiod at 100 µmol m-2 s-1 PAR) and fertilized biweekly by subirrigation with

0.25x Hoagland’s media. To assess the influence of Pi deprivation on soil-grown plants,

seedlings were established for 7-d in a 24-well microtitre plate (1 seedling per well)

containing 0.5 ml of 0.5x MS media supplemented with 1% (w/v) sucrose and 0.2 mM Pi,

then transplanted into a 75-85% sphagnum peat moss/perlite soil mix lacking all nutrients

(Sunshine Mix 2; SunGro). Plants were grown for an additional 14-d and fertilized biweekly

with 0.25x Hoagland’s media (pH 6.0) containing either 0 or 2 mM KH2PO4. Whenever Pi

was eliminated, it was replaced by 2 mM KH2SO4 and 0.5 mM MES. For liquid cultures, 5 mg

of seeds were surface sterilized, stratified, placed in 250 ml Magenta boxes containing 50 ml

of 0.5x MS media, pH 5.7, 1% (w/v) sucrose and 0.2 mM KH2PO4, and placed on an orbital

shaker (80 rpm) at 24 oC under continuous illumination (100 µmol m-2 s-1). After 7-d the

medium was replaced with fresh media containing 0 or 1.5 mM filter-sterilized KH2PO4. The

14-d seedlings were blotted dry, snap frozen in liquid N2, and stored at -80 oC. Stratified

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seeds were also placed on vertically oriented 1% (w/v) agar (Micropropagation Type I Agar

from Caisson Labs, Cat. #A038) plates containing 0.5x MS media and 1% (w/v) sucrose

supplemented with 50 µM or 1.5 mM KH2PO4, or 1.5 mM DL-glycerol-3-P (Sigma Chemical

Co.) and cultivated for up to 21-d as described for the liquid grown seedlings.

Enzyme activity assays and kinetic studies

All enzyme assays were linear with respect to time and concentration of enzyme assayed,

and optimized with respect to pH and substrate concentration. One unit of activity is defined

as the amount resulting in the use of 1 µmol min-1 of substrate at 25 °C. Aldolase and PEP

carboxylase activities were determined using coupled spectrophotometric assays (Tran and

Plaxton, 2008). For routine measurements of APase activity, the hydrolysis of PEP to

pyruvate was coupled to the lactate dehydrogenase reaction and assayed at 25 °C by

monitoring the oxidation of NADH at 340 nm using a Molecular Devices Spectromax Plus

Microplate spectrophotometer. Standard APase assay conditions were: 50 mM Na-acetate

(pH 5.6) containing 5 mM PEP, 0.2 mM NADH, and 3 units of rabbit muscle lactate

dehydrogenase (final volume = 0.2 ml). Assays were corrected for any background NADH

oxidation by omitting PEP from the assay mixture. Substrate selectivity studies were

performed by quantifying the Pi released by the APase reaction as previously described

(Veljanovski et al., 2006). AtPAP25’s: (i) apparent Km(PEP) and I50(Pi) values were

calculated using a non-linear regression enzyme kinetics program (Brooks, 1994), and (ii)

competitive and uncompetitive inhibition constants for Pi were determined using the

graphical method described by Cornish-Bowden (1974). All kinetic parameters are the

means of at least three independent experiments and are reproducible to within ±10% of the

mean value.

Protein electrophoresis and immunoblotting

SDS-PAGE, subunit Mr estimation via SDS–PAGE, immunoblotting onto poly(vinylidene)

difluoride (PVDF) membranes, imaging of antigenic polypeptides using an alkaline

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phosphatase-tagged secondary antibody, and chromogenic detection were carried out as

previously described (Veljanovski et al., 2006). All immunoblot results were replicated at

least three times with representative results shown in the figures.

RT-PCR and qPCR analysis

Total RNA was extracted and purified as described previously (Gregory et al., 2009;

Robinson et al., 2012a; Tran et al., 2010b). RNA samples were evaluated for purity via their

A260/A280 ratio and integrity by resolving 1 µg of total RNA on a 1% (w/v) denaturing agarose

gel. RNA (5 µg) was reverse transcribed with Superscript III (Invitrogen, Carlsbad, CA,

USA), and noncompetitive RT-PCR accomplished using gene specific primers listed in Table

S6 or as previously described (Gregory et al., 2009; Hurley et al., 2010). Conditions were

optimized for all RT-PCR reactions to ensure linearity of response for comparison between

samples. All PCR products were verified by sequencing.

Quantitative real-time PCR (qPCR) was performed as previously described

(Robinson et al., 2012b) using gene-specific primers listed in Table S6. Relative expression

was determined by normalizing transcript abundance with AtActin2 (NM_112764) transcripts.

Relative expression (fold-change) was calculated using the 2–∆∆Ct method as previously

described (Robinson et al., 2012b). All qPCR experiments were repeated at least three times

using cDNA prepared from three independent samples.

GUS analysis

The AtPAP25 promoter (982 bp sequence upstream of the start codon of the AtPAP25 gene)

was amplified from genomic DNA using primers listed in Table S6. Amplified promoter region

of AtPAP25 was mixed in a 3:1 molar ratio with pBI101 N1 linearized by HindIII and XbaI,

incubated with In-Fusion reaction mix, and transformed according to manufacturer’s

(Clontech) protocol to yield AtPAP25:GUS. The construct was transferred into

Agrobacterium tumefaciens strain LBA4404 and transformed into Arabidopsis plants via the

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floral dip method as previously described (Robinson et al., 2012a). Transformed plants were

selected on 0.8% (w/v) agar plates containing 0.5x MS media, 1% (w/v) sucrose, and 30 µg

ml-1 kanamycin, and transferred to soil for self-pollination and propagation. For analysis of

mature plants, seeds were planted in soil and grown for 28-d while being fertilized bi-weekly

with 0.25x Hoagland’s media containing 0 or 2 mM Pi. Histochemical staining of GUS

activity was performed as previously described (Robinson et al., 2012a). Tissues were

incubated at 37 °C overnight in GUS staining buffer (100 mM NaPi, pH 7.0, containing 1 mM

X-Gluc, 0.5 mM EDTA, 0.1% (v/v) Triton X-100, 2 mM K3Fe(CN)6, and 2 mM K4Fe (CN)6).

Stained tissues were cleared with 70% ethanol before imaging with a dissecting microscope.

Mutant isolation and complementation

A potential atpap25 mutant line (GK-004D10-014797) was obtained from The European

Arabidopsis Stock Centre (NASC). Initial screening was performed by growing plants on

0.8% (w/v) agar plates containing 0.5x MS media, 1% (w/v) sucrose, and 5 µg ml-1

sulfadiazine. Resistant plants were subjected to a second round of selection with

sulfadiazine and then transferred to soil for self-pollination and propagation. gDNA was

extracted from leaves and PCR-screened for homozygous mutants using T-DNA left-border

and gene specific primers listed in Table S6.

For complementing the atpap25 mutant phenotype, AtPAP25 was amplified by using

primers (Table S6) designed to introduce BglII and PmlI restriction enzyme sites at the 5’

and 3’ ends of its coding region, respectively. AtPAP25 was cloned into the pCAMBIA3301

(CAMBIA, Canberra, Australia) vector, thus allowing the gene to be driven by the CaMV 35S

promoter. The constructs were introduced into Agrobacterium strain GV3101 and

transformed into the atpap25 knock-out mutant using the floral dip method as previously

described (Robinson et al., 2012a). Seeds of the T0 generation were harvested and sown in

soil, and two-week-old seedlings of T1 plants screened by spraying with 0.5% (v/v)

phosphinothricin (PhytoTechnology Laboratories, USA). The surviving transformants (T1)

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were confirmed by PCR to amplify AtPAP25 and Bar genes (primers listed in Table S6). T2

seeds were placed on agar-solidified 0.5x MS media containing 7 mg l-1 phosphinothricin

and transgenic lines with a 3:1 (resistant:sensitive) segregation ratio were selected to

produce T3 seeds. The T3 lines displaying 100% phosphinothricin resistance (due to

expression of Bar gene) were considered homozygous and used for further experiments.

ADDITIONAL EXPERIMENTAL PROCEDURES

Additional experimental procedures are described in Methods S1.

ACKNOWLEDGEMENTS

We are grateful to Prof. Tom 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.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the online version of this article.

Figure S1. Immunological detection of PEP carboxylase and cytosolic aldolase in clarified

extracts from Arabidopsis suspension cells.

Figure S2. AtPAP25 is not phosphorylated at Tyr423.

Figure S3. AtPAP25 pH-activity profile.

Figure S4. Influence of Pi on PEP saturation kinetics of AtPAP25.

Figure S5. Comparison of development of Col-0 and atpap25 seedlings cultivated in liquid

or agar-solidified MS media.

Figure S6. Imaging of 33P-accumulation by shoots of Col-0 and atpap25 seedlings.

Table S1. Purification of native cell wall PAPs from 400 g of 7-d-old –Pi Arabidopsis

suspension cells

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Table S2. MALDI-TOF MS analysis of tryptic peptides derived from native AtPAP isoforms

isolated from cell wall extracts of –Pi Arabidopsis.

Table S3. Comparison of the amino acid sequence identity of AtPAP25 with AtPAP12 and

AtPAP26, as well as with AtPAP25 orthologs from other vascular plant sources.

Table S4. Identification of AtPAP25 glycopeptides by UPLC LTQ-FT LC MS/MS.

Table S5. Effect of various substances on the activity of purified AtPAP25.

Table S6. Primers used for cloning and PCR analysis.

Methods S1. Additional experimental procedures.

This material is available as part of the online article from http://www.blackwell-synergy.com

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Table 1 Substrate specificity of purified AtPAP25

Substrate Relative Activity

β-naphthyl-P

PEP

Ovalbumin

5-Br-4-Cl-3-indoyl-P

Phosphothreonine

para-nitrophenyl-P

Phosphoserine

Phosvitin

ATP

Phenyl-P

Phosphotyrosine

PPi

AMP

Glucose-6-P

6-P-Gluconate

Glucose-1-P

α-naphthyl-P

111 ±9

100 ±6

94 ±7

91 ±8

81 ±4

82 ±6

77 ±6

71 ±5

64 ±3

56 ±4

50 ±5

48 ±3

41 ±2

32 ±3

30 ±3

14 ±1

9 ±1

APase activity was determined with saturating concentrations of the storage

phosphoproteins ovalbumin and phosvitin (5 mg ml-1 each), or the indicated phosphorylated

compounds (5 mM each) using the spectrophotometric Pi assay described in the Methods.

Activity is expressed relative to the rate of Pi hydrolysis obtained with 5 mM PEP, set at

100%. All values represent means ±SE of n = 3 separate determinations.

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Figure legends

Figure 1. Upregulation of APase in CWs of Arabidopsis suspension cells becoming Pi

deficient.

At d-0, 50 ml aliquots of cells cultured for 7-d in 5 mM Pi were subcultured into 450 ml of

fresh MS media containing 0 or 5 mM Pi (–Pi and +Pi, respectively). Time-courses for cell

culture filtrate (CCF) Pi concentration (a) and CaCl2-extractable CW APase activity (b) were

determined. All values represent means ±SE of n = 3 separate flasks. Where invisible, the

error bars are too small to be seen. (c) Pooled butyl Sepharose eluates from +Pi versus –Pi

CW extracts (1 µg per lane) were subjected to immunoblotting with anti-AtPAP12. Native

AtPAP12 and AtPAP26 (50 ng/lane) purified from CCF of –Pi Arabidopsis suspension cells

(Tran et al., 2010b) served as positive controls.

Figure 2. Fractogel SO3--cation exchange FPLC of CW extracts from Arabidopsis

suspension cells.

APase isoforms of butyl Sepharose-enriched CW extracts of the +Pi (a) versus –Pi (b) cells

were resolved via Fractogel SO3- cation-exchange FPLC. APase activity and A280 values are

indicated with a dotted and solid line, respectively. The column was developed with a 0-1 M

KCl gradient (indicated by the dashed line).

Figure 3. Deduced amino acid sequence alignment of CW-targeted AtPAP isozymes

upregulated by Pi-deficient Arabidopsis.

Identical amino acids are denoted by an asterisk; the arrow indicates their predicted transit

peptide cleavage site. N-terminal sequences obtained by automated Edman degradation of

AtPAP12, AtPAP25, and AtPAP26-CW1 purified from CWs of –Pi cells are indicated in bold

font above the deduced sequences. Amino acid sequences obtained during MS/MS of tryptic

and proteinase K peptides derived from purified AtPAP25 are underlined. The boxes denote

the positions of AtPAP25’s Asn172, Asn367, and Asn424 N-linked glycosylation sites.

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Figure 4. SDS-PAGE and immunoblot analysis of CW PAPs purified from Pi-starved

Arabidopsis suspension cells.

(a) Purified PAPs (5 µg each) were subjected to 10% SDS-PAGE and stained with

Coomassie Blue R-250. ‘MW’ denotes several molecular mass standards. (b) Immunoblot

analysis was performed following SDS-PAGE as indicated. For anti-AtPAP12 and AtPAP26

immunoblots, 250 ng of each purified PAP was loaded, while for anti-AtPAP25 blots 100 ng

of AtPAP12, 200 ng of AtPAP26-CW1, 300 ng of AtPAP26-CW2, and 55 ng of AtPAP25

were loaded.

Figure 5. AtPAP25 is strongly induced during nutritional Pi deprivation.

Analysis of gene expression in suspension cell cultures and shoots and roots of

hydroponically-cultivated seedlings was performed as described in the Experimental

Procedures. (a) Levels of mRNA were analyzed by semi-quantitative RT-PCR. AtPAP12 and

AtPPCK1 served as positive controls for PSI genes (Gregory et al., 2009; Tran et al.,

2010b), whereas AtACT2 was used as a reference to ensure equal template loading. All

PCR products were taken at cycle numbers determined to be non-saturating. Control RT-

PCR reactions lacking reverse transcriptase did not show any bands. The AtPAP12,

AtPAP25, AtPAP26, AtPPCK1, and AtACT2 transcripts amplified as 244, 610, 627, 483 and

220 bp fragments of the 5′ region of their respective cDNAs, as expected. (b) Levels of

AtPAP25 transcripts were quantified by qPCR. AtPAP25 transcript levels in shoots and roots

of +Pi seedlings were used as the calibrator, whereas AtACT2 was used as an internal

control for normalization. All values represent means ±SE of n = 3 independent

determinations using cDNAs prepared from three biological replicates. (c) Pooled butyl

Sepharose APase activity fractions from CW extracts of +Pi and –Pi suspension cells were

subjected to SDS-PAGE and immunoblotted with anti-AtPAP25 (1 µg protein per lane).

AtPAP25 purified from CWs of the –Pi suspension cells (50 ng) served as a positive control.

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Figure 6. AtPAP25:GUS is highly expressed in shoot vasculature of Pi-deficient Arabidopsis.

(a) AtPAP25:GUS transgenic lines were cultivated aseptically in 24-well microtitre plates in

liquid 0.5x MS media containing 1% (w/v) sucrose and 0.2 mM Pi for 7-d, before being

transferred into media containing 0 or 1.5 mM KH2PO4 (–Pi and +Pi, respectively) and grown

for another 3- and 7-d. (b) AtPAP12-GUS expression was also examined in several aerial

tissues of 28-d old +Pi versus –Pi soil-grown plants. (c) ‘Germinating Seed’ is a

representative image of seeds that had been placed on moistened filter paper and allowed to

germinate for 1-d prior to GUS staining. All images in panels a-c are representative of those

obtained with 11 independent transgenic lines.

Figure 7. Development of an atpap25 mutant is blocked when transferred into a soluble Pi-

deficient soil mixture.

(a) Schematic representation of the AtPAP25 gene (At4g36350); white boxes and solid lines

represent exons and introns, respectively. T-DNA insertion location is indicated by ‘atpap25

T-DNA’, while arrows represent primers used for PCR and genotyping. (b) Confirmation of T-

DNA insert location in atpap25 via PCR-based screening of gDNA template isolated from

leaves of +Pi seedlings. PCR products were amplified from the Col-0 and atpap25 gDNA in

a PCR reaction using AtPAP25-specific primers (primer pair A + B) and the overlapping

region between AtPAP25 and the T-DNA insert-specific primers (primer pair B + C). The lack

of multiple amplification products for AtPAP25 DNA in atpap25 demonstrates that the mutant

is homozygous. NC, negative control. (c) Influence of nutritional P-status on development of

soil-grown wild-type (Col-0) and atpap25 mutant seedlings. Seedlings were cultivated for 7-d

in liquid media containing 0.2 mM Pi and 1% (w/v) sucrose, then transferred into a soluble

Pi-deficient soil mixture and grown for an additional 14-d under a regular light/dark diurnal

cycle. Fertilization occurred biweekly with 0.25x Hoagland’s media containing 0 or 2 mM Pi

(–Pi and +Pi, respectively); scale bars = 1 cm. Results are representative of a minimum of 5

independent trials with at least 5 plants per trial. (d) Total Pi and anthocyanin concentrations

of rosette leaves of 21-d old seedlings cultivated the in the –Pi soil mixture. All values are

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means ±SE of n = 5 different pooled leaf samples; asterisks denote values that are

significantly different (p < 0.05).

Figure 8. Rescue of atpap25 plants cultivated on soluble Pi-deficient soil by Pi-resupply or

complementation with AtPAP25.

(a) The 21-d old –Pi atpap25 plants were fertilized with 0.25x Hoagland’s medium containing

0 or 2 mM Pi, and cultivated for an additional 14-d. (b) The AtPAP25 gene was transformed

into the atpap25 mutant background under control of the CaMV 35S promoter. Leaf mRNA

levels of two independent homozygous lines of atpap25 plants that had been complemented

with AtPAP25 were compared with those of atpap25 and Col-0 plants by semi-quantitative

RT-PCR using gene-specific primers for AtPAP25, Bar, and AtACT2. The AtPAP25, Bar,

and AtACT2 transcripts amplified as 256, 459, and 106 bp fragments of the 5′ region of their

respective cDNAs, as expected. (c) Plants were cultivated for 3 weeks as described in the

legend for Figure 7c. Fertilization occurred biweekly with 0.25x Hoagland’s media containing

0 or 2 mM Pi (–Pi and +Pi, respectively). Results are representative of those obtained with

two independent lines of atpap25 plants that had been complemented with AtPAP25. Scale

bars in panels a and c = 1 cm.

Figure 9. Pi-starvation signaling is disrupted in the atpap25 mutant.

Plants were cultivated for 3 weeks as described in the legend for Figure 7c, and levels of

shoot transcripts for several genes determined by qPCR. Transcript levels in leaves of +Pi

seedlings were used as the calibrator, whereas AtACT2 was used as an internal control for

normalization. All values represent means ±SE of n = 3 independent determinations using

cDNAs prepared from three biological replicates; asterisks denote values that are

significantly different (p < 0.05) between Col-0 and atpap25.

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The cell wall-targeted purple acid phosphatase AtPAP25 is critical for acclimation of Arabidopsis thaliana to nutritional phosphorus deprivation