Molecular and Biochemical Characterization of AtPAP15,a Purple Acid Phosphatase with Phytase Activity,in Arabidopsis1[W][OA]
Ruibin Kuang2, Kam-Ho Chan2, Edward Yeung, and Boon Leong Lim*
School of Biological Sciences, University of Hong Kong, Pokfulam, Hong Kong, China (R.K., K.-H.C., B.L.L.);and Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4 (E.Y.)
Purple acid phosphatase (PAP) catalyzes the hydrolysis of phosphate monoesters and anhydrides to release phosphate withinan acidic pH range. Among the 29 PAP-like proteins in Arabidopsis (Arabidopsis thaliana), AtPAP15 (At3g07130) displays agreater degree of amino acid identity with soybean (Glycine max; GmPHY) and tobacco (Nicotiana tabacum) PAP (NtPAP) withphytase activity than the other AtPAPs. In this study, transgenic Arabidopsis that expressed an AtPAP15 promoter::b-glucuronidase (GUS) fusion protein showed that AtPAP15 expression was developmentally and temporally regulated, withstrong GUS staining at the early stages of seedling growth and pollen germination. The expression was also organ/tissuespecific, with strongest GUS staining in the vasculature, pollen grains, and roots. The recombinant AtPAP purified fromtransgenic tobacco exhibited broad substrate specificity with moderate phytase activity. AtPAP15 T-DNA insertion linesexhibited a lower phytase and phosphatase activity in seedling and germinating pollen and lower pollen germination ratecompared with the wild type and their complementation lines. Therefore, AtPAP15 likely mobilizes phosphorus reserves inplants, particularly during seed and pollen germination. Since AtPAP15 is not expressed in the root hair or in the epidermalcells, it is unlikely to play any role in external phosphorus assimilation.
At pH in the range of 4 to 7, purple acid phospha-tases (PAPs) catalyze the hydrolysis of a wide range ofactivated phosphoric acid monoesters and diestersand anhydrides (Klabunde et al., 1996). They aredistinguished from the other phosphatases by theirinsensitivity to L-(+) tartrate inhibition and thereforeare also known as tartrate-resistant acid phosphatases.Their characteristic pink or purple color derives from acharge transfer transition between a Tyr residue andthe “chromophoric” ferric ion in the binuclear Fe(III)-Me(II) center, where the metal (Me) is iron, zinc, ormanganese (Schenk et al., 1999). PAP proteins are alsocharacterized by seven conserved amino acid residues(shown in boldface) in the five conserved motifsDXG,GDXXY, GNH(D/E), VXXH, and GHXH, which areinvolved in the coordination of the dimetal nuclearcenter (Li et al., 2002).PAPs are widespread in mammals, fungi, bacteria,
and plants. Interestingly, while only a few copies of
PAP-like genes are present in mammalian and fungalgenomes (Mullaney and Ullah, 2003; Flanagan et al.,2006), multiple copies are present in plant genomes(Schenk et al., 2000). For example, 29 PAP-like geneshave been identified in the Arabidopsis (Arabidopsisthaliana) genome (Li et al., 2002). It is intriguing that somany PAP-like genes are required for plant metabo-lism; this diverse portfolio of PAP-like genes impliesdifferential functions for them. Plant PAPs are gener-ally considered to mediate phosphorus acquisitionand redistribution based on their ability to hydrolyzephosphorus compounds (Cashikar et al., 1997; Bozzoet al., 2004; Lung et al., 2008). However, additionalbiological roles have been reported for some plantPAPs. For example, the PAPs AtACP5 (AtPAP17),SAP1, and SAP2 (del Pozo et al., 1999; Bozzo et al.,2002) display not only phosphatase but also peroxi-dase activity, suggesting their involvement in theremoval of reactive oxygen compounds in plant or-gans. GmPAP3, isolated from salted-stressed soybean(Glycine max), reportedly mediates salt tolerance viaNaCl and oxidative stress inductions but not byphosphorus starvation (Liao et al., 2003).
Some PAP members can hydrolyze phytic acid(myoinositol hexakisphosphate [InsP6]) to inorganicphosphate and free or lower phosphoric esters ofmyoinositol. Since the major storage form of phospho-rus in plant seeds and pollen grains is phytate, PAPswith phytase activity may play a role in seed andpollen germination. However, not all PAPs exhibitphytase activity. The first plant phytase PAP, GmPHY,was isolated from the cotyledon of germinating
1 This work was supported by the University Research Commit-tee (grant no. 10206029) and by a Discovery Grant from the NaturalSciences and Engineering Research Council of Canada to E.Y.
2 These authors contributed equally to the article.* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Boon Leong Lim ([email protected]).
[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-
scription.www.plantphysiol.org/cgi/doi/10.1104/pp.109.143180
Plant Physiology�, September 2009, Vol. 151, pp. 199–209, www.plantphysiol.org � 2009 American Society of Plant Biologists 199
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soybean seedlings (Hegeman and Grabau, 2001). Atobacco (Nicotiana tabacum) root PAP phytase wasidentified more recently that is likely involved inmobilizing external organic phosphorus in soil (Lunget al., 2008).
Relatively little is known about the biochemicalproperties and physiological roles of the 29 PAP-likeArabidopsis genes (del Pozo et al., 1999; Veljanovskiet al., 2006). An enzyme assay involving the glutathi-one S-transferase (GST)-AtPAP23 fusion protein re-vealed that the Arabidopsis PAP AtPAP23 exhibitsphytase activity (Zhu et al., 2005). A GUS studyshowed that AtPAP23 is exclusively expressed in theflower of the Arabidopsis plant. In a recent report, arecombinant AtPAP15 expressed in Escherichia coliwasalso found to exhibit phytase activity; this PAP poten-tially modulates plant ascorbate synthesis throughsupply of myoinositol from the phytate hydrolysisreaction (Zhang et al., 2008). However, the possiblephysiological roles of AtPAP15 in phosphorus mobi-lization have not been examined.
In this study, AtPAP15 expressed in a plant (tobacco)system was biochemically characterized, and its tem-poral and spatial expression patterns in Arabidopsiswere examined. The physiological roles of AtPAP15 inphosphorus mobilization were also delineated.
RESULTS
Overexpression and Purification of AtPAP15 in
Transgenic Tobacco Plants
The soluble GST-AtPAP15 protein did not show anyenzymatic activity. Therefore, a His-tagged AtPAP15protein was stably overexpressed in tobacco plantsusing an explant method. Gene expression was con-firmed by PCR (data not shown) and western-blotanalysis using specific anti-AtPAP15 antiserum (Fig.1A). Phytase activity was approximately 3-fold greaterin transgenic tobacco leaves compared with wild-typeleaves (Fig. 1B). Leaves from permanent lines wereused for further protein purification.
The purification of the AtPAP15 protein wasachieved by ion-exchange, affinity, and gel filtration chro-matography. The purification table of a representativerun is shown in Table I. A single polypeptide band ofapproximately 60 kD was detected by silver staining(Fig. 2A, lane 5), which confirmed the homogeneity ofthe protein. The apparent molecular mass of the nativeenzyme was estimated to be approximately 58 kD,using preparative-grade gel filtration chromatography(data not shown), confirming that the native enzyme isa monomeric protein. Western-blot analysis (Fig. 2B)and mass spectrometry were employed to confirm theidentity of the protein. The sequence coverage of thepeptide mass fingerprint reached 25% (134 of 532residues) and theMOWSE score was 66 (SupplementalFile S1), confirming its identity as AtPAP15. The en-zyme had been purified approximately 344-fold with
an overall recovery of 2.4%; it exhibited a phytaseactivity of 10 units mg21 protein (Table I).
Biochemical Properties of Purified AtPAP15
pH and Temperature Effects on AtPAP15 Phytase Activity
A pH activity profile of the purified protein isshown in Figure 3A. The enzyme showed an acidicpH activity profile, with maximal activity at pH 4.5.The enzyme had its highest activity at low tempera-tures (23�C–37�C; Fig. 3B) and was most stable in thesame temperature range. Negligible activity was de-tected when the temperature was higher than 65�C(Fig. 3B).
Effects of Ions and Inhibitors on Enzymatic Activityof AtPAP15
We investigated the influence of various ions on theenzymatic activity of AtPAP15. Ca2+ and Zn2+ stimu-lated the phytase activity of AtPAP15 (127.5% and129.4%, respectively), whereas Cu2+ and SO3
22 inhib-ited this activity (reduced to 17.8% and 43.1%, respec-tively). Other ions (Mn2+, Mg2+, Ni2+, SO4
22, and NO32)exhibited no effects on phytase activity.
The most prominent PAP inhibitor, molybdate, wasalso the most notable inhibitor of AtPAP15; 0.25 mM
MoO42 was sufficient to completely abolish enzymatic
activity. The presence of 5 mM F2 or PO432 ions
reduced the residual activities of the enzyme to 20%
Figure 1. Western blotting and phytase activity assays of transgenictobacco lines. A, AtPAP15 expression was confirmed by westernblotting. Lanes 1 to 6 represent molecular mass standards, the wildtype, and pBa002a-PAP15 lines (transgenic lines 5-3, 7-4, 10-2, and13-4), respectively. Thirty micrograms of protein was loaded into eachlane. The position of AtPAP15 is indicated by the arrow at right. B,Specific phytase activity (Sp. phytase act.; means 6 SE; four replicates).wt, Wild type. Values marked by different letters are significantlydifferent (P , 0.05).
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or 0%, respectively. The purified enzyme was signif-icantly resistant to treatment with tartrate. Other com-pound supplements such as EDTA and citrate exertedno influence on enzymatic activity (Fig. 4).
Substrate Specificity of Purified AtPAP15
Purified AtPAP15 exhibited broad substrate speci-ficity (Table II), with p-nitrophenyl-phosphate (pNPP),phosphoenolpyruvate (PEP), and Na-pyrophosphatebeing the most effective substrates. Compared withactivity toward pNPP, the activities toward variousdeoxyribonucleotide triphosphateswere approximately31% to 52% and the activity toward phytate was 7%.Very low activity was seen when monophosphates(AMP or GMP) were used as substrates. Negligibleactivity was detected on Glc-1-P.
Kinetic Parameters of Purified AtPAP15
The kinetic parameters of AtPAP15 were measuredwhen pNPP, PEP, or Na-phytate was used as substrate(Table III). AtPAP15 had relatively higher affinity(Km = 278 mM) and lower Vmax (13.44 units mg21) to-ward Na-phytate than toward pNPP or PEP. Thecatalytic efficiencies (kcat/Km) of the enzyme towardpNPP and PEP were approximately 10-fold higherthan that toward Na-phytate.The biochemical properties of the recombinant
AtPAP15 from plant were very different from thatpresented by Zhang et al. (2008), who reported thespecific activities of a GST:AtPAP15 toward a fewsubstrates. In our study, the GST:AtPAP15 producedfrom E. coli was inactive. Zhang et al. (2008) reportedthe specific activity of GST:AtPAP15 toward pNPP tobe only 0.546 units mg21, which was far lower than theplant-derived AtPAP15 level (165 units mg21; Table III)and that reported for the other plant PAPs (222–368units mg21 for tomato [Solanum lycopersicum] PAPs[Bozzo et al., 2002] and 266 units mg21 for KbPAP[Vogel et al., 2002]). Regarding specific activities to-ward phytate, the two recombinant AtPAPs gave 10.0(plant) and 0.7 (bacterial) units mg21, respectively.
Expression of AtPAP15 in Different Tissues andDevelopmental Stages of Arabidopsis
To determine the expression patterns of AtPAP15with respect to specific tissue and developmental
stages, transgenic Arabidopsis plants expressing anAtPAP15 promoter::GUS fusion protein were pro-duced. All three promoter regions directed the samepattern of GUS expression under normal growth con-ditions (data not shown).
Intense GUS staining was observed at the earlystages of seedling establishment and in the cotyledon,radicle, and hypocotyl (Fig. 5, A–D). The signalwas stronger and more diffuse in the cotyledonleaves but weaker and more restricted in youngerimmature leaves (Fig. 5E). In mature, fully expandedrosette leaves of soil-grown Arabidopsis transgeniclines, GUS staining was predominantly detected inthe vasculature (Fig. 5F). In roots, intense stainingcould be observed at all stages but was mainlyrestricted to the vascular cylinder (Fig. 5G). No ex-pression could be found in the root hairs (Fig. 5G) or
Table I. Purification of recombinant AtPAP15 from transgenic tobacco
Step Volume Protein PhytaseTotal
ProteinPhytase Yield Purification
mL mg mL21 munits mg21 protein mg munits % fold
Crude 250 380 29 94,390 2,745 100.0 1HiPrep CM 60 170 289 6,370 1,843 67.1 10HiTrap HP 1.3 68 1,053 88 93 3.8 36Superdex 75 1 6.6 10,014 6.6 66 2.4 344
Figure 2. SDS-PAGE analysis of proteins at different purification steps.Protein was detected by silver staining (A) and western blotting (B).Lane 1, Molecular mass standards; lane 2, leaf crude protein (10 mg);lane 3, activity fraction from HiPrep 16/10 CM FF column (10 mg); lane4, activity fraction fromHiTrap HP (Ni2+) column (1 mg); lane 5, activityfraction from Superdex 75 gel filtration column (0.5 mg). The position ofAtPAP15 is indicated by the arrows at right.
Studies on AtPAP15 during Seed and Pollen Germination
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in the root cap cells (Fig. 5H). GUS staining wasconsistently detected in the vasculature of both thestems and the roots. Cross-sectional analysis revealedthat the staining was mainly localized to the xylemand phloem tissues in leaf, hypocotyl, and root sec-tions (Fig. 5, I–L).
In reproductive organs, GUS staining was obviousin the flowers (Fig. 5M); however, little or no stainingwas evident in the developing seeds or siliques (datanot shown). Blue color was observed in the anther andalso the stigma papillae (Fig. 5N). Pollen squash anal-ysis indicated that the majority of the GUS stainingwas from pollen grains (Fig. 5O). To further analyzeGUS expression in flowers, AtPAP15 promoter::GUStransgenic Arabidopsis anthers were sectioned to re-veal the stage at which GUS expression was evident.Cross-sections of Arabidopsis anthers revealed little orno GUS gene expression in the early stage of pollendevelopment (Fig. 6, A–D). This expression increasedat later stages of pollen development and was obviousin mature pollen grains (Fig. 6, E and F). Staining couldalso be detected in in vitro-germinated pollen (Fig. 5P).
The promoter::GUS data indicated that AtPAP15expression is developmentally and temporally regu-lated, with strong GUS staining at early stages ofseedling growth and at late stages of pollen develop-ment. The expression was also organ/tissue specific,
with the vasculature, pollen grains, and seedlingsshowing the strongest staining.
AtPAP15::GUS Expression in Response to a Variety
of Stimuli
To analyze AtPAP15::GUS expression in response toa variety of stimuli, two independent homozygoustransgenic lines were used for in situ analysis of GUSactivity. In general, the overall GUS staining patternsin shoots and roots were not affected by these treat-ments. The only exception was at the root tip, where anincrease of GUS activity was observed under salicylicacid, abscisic acid, NaCl, sorbitol, or mannitol treat-ment (Fig. 7). In contrast, no alterations in GUS stain-ing were observed under nutrient stresses, such asphosphorus, nitrogen, or potassium starvation (datanot shown).
Molecular Characterization of AtPAP15 T-DNA Insertion
Mutants and Their Complementation
Six T-DNA insertion lines at the AtPAP15 locus areavailable from the Arabidopsis Biological ResourceCenter, of which we chose SAIL_529_D01 (T9) andSALK_061597 (T2), which insert into exon 1 and exon2, respectively, to be used in this study. T2 and T9seeds from the distribution stock were germinated,and segregation of the T-DNA-encoded antibiotic re-sistance marker was tracked. Genomic DNAwas thenextracted from the antibiotic-resistant T2 progeny, andthis DNA was screened for T-DNA insertion by PCRusing gene-specific primers and primers anchored inthe T-DNA borders. Six (T2-1, -3, -5, -6, -8, and -9) andthree (T9-2, -7, and -9) individual plants were selectedas homozygotes by the absence of the AtPAP15 PCRproduct (data not shown). Gene silencing of the ho-mozygous plants was confirmed by reverse transcrip-tion (RT)-PCR and western blotting. In RT-PCR assays,a single amplified DNA fragment with the predictedsize (approximately 1.6 kb) was absent in T-DNAmutants when compared with wild-type plants (Fig.
Figure 3. Biochemical properties of the purified AtPAP15. A, pHprofile. B, Temperature and thermostability profiles. Each value repre-sents the mean 6 SD of three experiments.
Figure 4. Effects of inhibitors on purified AtPAP15 activity. SD valueswere calculated on the basis of three independent experimental trials.
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8A). Protein was extracted from seedlings for western-blot analysis via AtPAP15 antiserum. No AtPAP15expression was observed in these insertion mutantscompared with wild-type plants (Fig. 8B). A slightlysmaller protein band was recognized in all lines by theanti-PAP15 antiserum (Fig. 8, B and C). This band isnot AtPAP15 because it was present in all T-DNAlines, which were shown not to express AtPAP15mRNA by RT-PCR (Fig. 8A). This band, which wasnot present in tobacco (Fig. 2B), could be an AtPAPprotein with high amino acid sequence homology toAtPAP15. Western blotting of wild-type plants usingthe preimmune serum did not give any band (Sup-plemental Fig. S1).To verify that the phenotypic difference was due to
the disruption of AtPAP15, complementation lines
were produced by introducing the construct AtPAP15in the NOS promoter-containing pCAMBIA1300 intothe mutants. Four homozygous T3 generation linesbearing one copy of the complemented gene wereselected based on segregation of hygromycin resis-tance; these lines were verified by PCR screening andwestern blotting (Fig. 8C). Two lines (PC1 and PC2)were chosen for subsequent studies (Fig. 8, A and B).
Growth Analysis of T-DNA Insertion and ItsComplementation under a Variety of Stimuli
Based on results of the above GUS assay, AtPAP15 ishighly expressed in early stages of germination, im-plying that it may play a role(s) in seed germination orseedling growth. However, the insertion mutants andtheir complementation lines did not differ in growthperformance, phenotypes, or seed germination ratewhen planted in soil (data not shown).
Therefore, the phosphatase activities of 2-d-oldseedlings were measured. It was observed that bothphytase activity and acid phosphatase (APase) activitywere significantly lower in the T-DNAmutants than inthe wild type, whereas the complementation lineshowed comparable value to the wild type (TableIV). Since GUS studies indicated that AtPAP15 expres-sion was substantial during pollen germination, theenzyme activities in germinating pollen were alsoassayed, which showed similar results (Table IV).In general, the specific activities of phytase and phos-phatase in pollen were higher than that in the seed-lings among all lines, possibly due to a lowercomplexity of total protein in pollen.
An in vitro pollen germination experiment was thenconducted. As shown in Figure 9, the pollen germinationrate was significantly reduced in themutants (30%–35%)compared with wild-type plants (78%). Complementa-tion of AtPAP15 mutants with pCAMBIA1300-PAP15resulted in recovery of the mutagens relative to thewild-type response (65%). These results indicated thatthe pollen germination phenotype of the AtPAP15 mu-tant correlates to AtPAP15 deficiency.
DISCUSSION
Only two PAP-like genes (Flanagan et al., 2006)are present in animal genomes, compared with the
Table II. Substrate specificity of AtPAP15 purified fromtransgenic tobacco
Enzymatic activities were assayed at 37�C for 1 h in 100 mM NaOAcbuffer (pH 4.5) containing various substrates (final concentration,1 mM). Each value represents the mean of three experiments and isexpressed as a percentage relative to the measurement using pNPP assubstrate.
Substrate Relative Activity SD
%
pNPP 100 0Na-pyrophosphate 95 1PEP 92 43-PGA 69 8ADP 50 3dGTP 52 4Glc-3-P 43 3dTTP 37 8dCTP 35 4dATP 31 2P-Tyr 28 2P-Ser 25 2Glc-6-P 20 3CMP 14 0P-Thr 13 2Rib-5-P 11 2ATP 10 5Fru-6-P 10 1Na-phytate 7 1AMP 4 0GMP 4 0Glc-1-P 0 1
Table III. Enzyme kinetics of AtPAP15
Enzymatic activity was estimated over a range of substrate concentrations (0, 0.05, 0.1, 0.2, 0.4, 0.5,and 1 mM). Four time points (0, 20, 40, and 60 min) were tested at each substrate concentration. SD valueswere calculated on the basis of three independent experimental trials.
Substrate Vmax Km kcat kcat/Km
units mg21 mM min21 min21 mM21
Na-phytate 13.4 6 0.6 278 6 28 531 6 78 1,602 6 39pNPP 165.4 6 9.0 703 6 333 9,714 6 3,581 14,480 6 1,857PEP 134.7 6 2.6 801 6 294 11,917 6 3,509 15,229 6 1,460
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greatly larger numbers in plants (Li et al., 2002).It is intriguing that so many different PAPs arerequired for plant metabolism and how differentthe physiological functions of these PAPs are. Inthis study, we focused on AtPAP15, a PAP withphytase activity. Not all PAPs exhibit phytase activ-ity; for instance, no phytase activity has been re-ported for mammalian PAPs. In addition, some plantPAPs, including KbPAP (Cashikar et al., 1997), twosecreted tomato PAPs (Bozzo et al., 2002), and anintracellular tomato PAP (Bozzo et al., 2004), lackphytase activity.
Our results confirmed that AtPAP15 is an acidicphosphatase with phytase activity. Phytate (InsP6),which is primarily complexed with metal ions, is theprincipal storage form of phosphorus in seeds (Oteguiet al., 2002) and pollen (Jackson and Linskens, 1982).The globoid in the protein storage vacuole of theArabidopsis seed embryo is primarily composed ofcalcium/magnesium/potassium-phytate salts (phytin);thus, phytin breakdown in these cells releases phos-phorus, calcium, magnesium, potassium, and carbonfor embryo growth and division (Otegui et al., 2002). Inpollen, small electron-dense globoids rich in calcium,
Figure 5. Histochemical localization of GUS activityin transgenic Arabidopsis plants containing theAtPAP15 promoter::GUS construct. A, One-day-oldseed. B, Two-day-old seedling showing intense GUSstaining in the radicle. C and D, Three-day-old (C)and 7-d-old (D) seedlings showing strong GUS ac-tivity in roots, hypocotyls, and cotyledon leaves. E,Ten-day-old plant. F, A mature soil-grown rosette leafwith GUS staining in the vasculature. G and H, Arepresentative main root (G) and the root tip (H) of a7-d-old seedling. I to L, Cross-sections of the leaf (I),hypocotyls (J), main root (K), and lateral root (L) of a7-d-old seedling. M, Flower. N, Stigma papillae of thestigma. O, Mature pollen grains. P, An in vitro-germinated pollen grain. Bars = 50 mm.
Figure 6. Cross-sections of anthers (3 mm)of representative transgenic Arabidopsisplants containing the AtPAP15 promoter::GUS construct. The following develop-mental stages are shown: stage 1, pollenmother cells (A); stage 2, meiosis (B); stage3, tetrads (C); stage 4, vacuolate micro-spores (D); stage 5, binucleate and trinu-cleate microspores (E); stage 6, maturemicrospores (F). Right panels show sec-tions stained to reveal general histology.Left panels show sections of GUS-stainedsamples. Bars = 50 mm.
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magnesium, and phosphorus were only found withincytoplasmic vesicles of vegetative cells but not in germcells (Butowt et al., 1997). These globoids, which weresuggested to contain phytin, were found to increaseand decrease in size and number during pollen mat-uration and germination, respectively (Butowt et al.,1997). During seed and pollen germination, phytasesare required to hydrolyze phytate to less phosphory-lated compounds myoinositol and inorganic phos-phate. This phosphorous supply is essential forseedling development (Reddy et al., 1989). GUS stain-ing studies demonstrated that AtPAP15 was stronglyexpressed during seed and pollen germination. Itsexpression was significantly increased in 2-d-old com-pared with 1-d-old seedlings (Fig. 5, A and B), and inpollen, AtPAP15 was not expressed during pollendevelopment but was strongly expressed during pol-len germination (Fig. 6). These observations are con-sistent with its possible role in the release of internalphosphorus reserves.The seedlings of T-DNA lines only exhibited 35% to
55% of the phytase activity of wild-type seedlings, butthis reduction did not affect the germination rate of the
seedlings. The expression of redundant phytase genesand the abundance of phosphorus in the seeds mayexplain the normal seed germination rate of theT-DNA lines. During pollen germination, pollen ofT-DNA lines only exhibited 25% to 57% phytase activityand 59% to 71% APase activity compared with thewild type (Table IV), and this reduction is correlatedwith a lower in vitro germination rate of the pollen(Fig. 9). These results indicated that AtPAP15 is a keyphytase/phosphatase during pollen germination andimplicated a physiological role in the mobilization ofphosphorus reserves in pollen. It is unlikely thatAtPAP15 is secreted from pollen for external phos-phorus assimilation. During in vitro germination ofLilium longiflorum pollen, in-gel acid phosphatase ac-tivity staining of germination medium only generateda single 32-kD tartrate-resistant acid phosphatase;therefore, it is unlikely to be an AtPAP15-like protein(approximately 60 kD; Ibrahim et al., 2002).
PAPs with phytase activity have been identified insoybean (Hegeman and Grabau, 2001), Medicago (Xiaoet al., 2005), and tobacco (Lung et al., 2008). AtPAP15 isnot the only Arabidopsis PAP with phytase activity.
Figure 7. GUS staining of AtPAP15::GUStransgenic Arabidopsis seedling root tipsin response to various stimuli. Transgenicplants (T3) were grown on MS agar platesfor 7 d and then transferred into treatedliquid medium and grown for 24 h asdescribed in “Materials and Methods.”GUS activity was detected in situ after 8h of incubation. Four replicates were per-formed with 20 plants for each treatment.The conditions were optimized to providelow signals in control and thereby allowvisualization of changes above back-ground. ABA, Abscisic acid; GA3, gibber-ellic acid; H2O2, hydrogen peroxide; MeJA,methyl jasmonate; SA, salicylic acid.
Figure 8. Molecular characterization of AtPAP15T-DNA insertionmutants and their complementation.A, RT-PCR with gene-specific primers for analyzingthe transcription of AtPAP15 in 5-d-old seedlings ofT-DNA lines. The wild type (Wt), complementationline (PC2), and genomic DNA (GDNA) were used ascontrols. Actin2 primers were used as a reference. M,Molecular mass markers. B, Western-blot analysis ofseedling protein (80 mg protein lane21) on a 10%(w/v) SDS-PAGE gel with anti-AtPAP15 antiserum.Wild-type and complementation lines (PC2) wereused as controls. The nonspecific band can be usedas a loading control. C, Western-blot analysis of com-plementation lines (PC1-4) with anti-AtPAP15 antise-rum using proteins from T-DNA and the emptyvector control protein EV1-2 (40 mg protein lane21).
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Phylogenetic analysis of the 29 PAP-like sequences inArabidopsis indicated that AtPAP13, AtPAP15, andAtPAP23 were clustered in the subgroup 1b-1 (Li et al.,2002). All of the subgroup 1b-1 PAPs shared nine Cysresidues at conserved positions, which were not foundin PAPs from other subgroups. The presence of thesenine Cys residues (Cys-160, -237, -240, -375, -414, -441,-456, -467, and -522 with respect to the amino acidsequence of AtPAP15) may be a signature of plantPAPs with phytase activity.
AtPAP23 also exhibits phytase activity (Zhu et al.,2005), and its GUS expression profile was markedlydifferent from that of AtPAP15. The promoter ofAtPAP23 is only highly active in flowers but not inshoot apical meristems during the early stages offlower differentiation. In mature flowers, AtPAP23 isonly active in petals, stamen filaments, the base of thestamen filaments, and the pistil, areas where AtPAP15is not expressed. In contrast to AtPAP15, AtPAP23 isnot expressed in the stigma or the anthers. It may bethat AtPAP23 is specialized for phosphorus supplyduring flower development, whereas AtPAP15 is re-sponsible for phosphorus reserve mobilization duringpollen development and germination. This wouldaccount for the differential expression of the twoPAPs in terms of their temporal and spatial profiles.The differential expression ofAtPAP15 andAtPAP23 inArabidopsis is one example where multiple PAP genesare required for their various functions in plants.
AtPAP15 expression was not restricted to the reservestorage organs; its expression was also strong in theplant vascular tissues (Fig. 5, E–L), where phytateamounts are scarce. Furthermore, it is noteworthy thatphytate was not the only substrate of AtPAP15 (TableII). Rather, it was active toward many organic phos-phorus compounds. Vascular tissues allow the trans-port of energy compounds from the shoots to the rootsand the transport of nutrients from the roots to theshoots. AtPAP15 may mediate the remobilizationof inorganic phosphates from organic phosphorus
compounds and may help maximize the phosphorusefficiency of the plant by redistributing surplusphosphorus from the mature to the growing tissues.This possibility is consistent with the lack of AtPAP15expression in root tips and hairs (Fig. 5, G and H),where very large amounts of inorganic phosphateare consumed for building biomolecules such as DNAand RNA.
Arabidopsis does not secrete phytases from its roots(Richardson et al., 2001). AtPAP15 is unlikely to besecreted into the root exudates, as it was not expressedin the root hairs or in epidermal cells. In contrast,tobacco secretes phytase PAPs from its roots duringphosphorus deficiency (Lung and Lim, 2006; Lunget al., 2008). Hence, while most plants secrete phos-phatases to acquire external phosphorus, not all ofthem secrete phytases. Although phytate is the mostabundant organic phosphorus compound in soil(Turner et al., 2002), it is strongly fixed to soil compo-nents. Only soluble phytate can be hydrolyzed byphytases (Tang et al., 2006); therefore, for plants se-creting PAP phytases from their roots, the availabilityof soluble phytate (substrate) and not that of theenzyme is the major limiting factor for soil phytate-phosphorus assimilation. Organic acids can enhancethe availability of soluble phytate (Tang et al., 2006);thus, plants capable of secreting large amounts oforganic acids from their roots are benefited by secre-tion of phytases.
A recent report proposed that AtPAP15 helps tosupply the plant with myoinositol for ascorbate syn-thesis (Zhang et al., 2008). However, myoinositol is notconsidered a major precursor for ascorbate, and thereare multiple ascorbate biosynthetic pathways inplants, implying that AtPAP15 may not be an essentialenzyme in ascorbate biosynthesis (Valpuesta andBotella, 2004). The role of AtPAP15 in the mobilizationof internal phosphorus stores was delineated inthis study. The temporal and spatial expression ofAtPAP15was developmentally, but not environmentally,regulated, since its expression was not induced bynutrient starvation.
Table IV. Enzyme activities in germinating seedlings and pollen
Enzymatic activity was estimated with 1 mM Na-InsP6 (phytaseactivity) or pNPP (APase activity) as substrate. Protein was extractedfrom 2-d-old seedlings; pollen grains were germinated in liquidmedium for 8 h and then collected for protein extraction. Valuesmarked by different letters are significantly different (P , 0.05) in thesame column. There are four replicates for each line.
LineSeedlings Pollen
Phytase APase Phytase APase
munits mg21 protein
Wild type 1.03a 61.3ab 17.24a 186.3aT2-3 0.36b 49.6b 8.91b 132.2bT2-5 0.53b 52.6b 6.28b 126.5bT9-2 0.48b 44.1b 5.32b 110.3bT9-7 0.57b 47.6b 4.33b 116.1bPC1 1.18a 76.6a 16.24a 178.5aPC2 1.48a 90.9a 15.17a 190.4a
Figure 9. Pollen germination of AtPAP15 T-DNA insertion lines withcontrol of the wild type (Wt) and complementation lines (PC1 and PC2;T3). Pollen was germinated in vitro for 8 h and then observed bymicroscopy, and the germination rate was analyzed. Means + SE of threereplicates, approximately 500 pollen grains per replicate, are shown.Values marked by different letters are significantly different (P , 0.05).
Kuang et al.
206 Plant Physiol. Vol. 151, 2009
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MATERIALS AND METHODS
AtPAP15 Cloning, Vector Construction, andTobacco Transformation
The Columbia ecotype of Arabidopsis (Arabidopsis thaliana) was used
throughout this study. Total RNA was extracted using the TRIzol method
(Invitrogen) from 3-week-old Arabidopsis plants. Total cDNAwas transcribed
using Moloney murine leukemia virus reverse transcriptase (Promega)
according to the manufacturer’s instructions. The open reading frame (ORF)
of AtPAP15 was amplified by forward (5#-TATGTCGACATGACGTTTCTAC-
TACTTCTAC-3#) and reverse (5#-GACTAGTTCAGTGGTGGTGGTGGTGG-
TGGCAATGGTTAACAAGGCGGT-3#) primers by Pfx polymerase (Roche). A
63His tag was appended to the reverse primer to create a C-terminal His tag.
The AtPAP15 ORF was then subcloned into a pBa002a-derived plant
expression vector carrying a Basta-resistant gene and a cauliflower mosaic
virus 35S promoter. The expression construct of pBa002a-PAP15 was mobi-
lized into Agrobacterium tumefaciens strain GV3101 by freeze-thaw transfor-
mation (Hofgen and Willmitzer, 1988). Permanent transgenic tobacco lines
were produced from Nicotiana tabacum var. Samsun by the explant method
developed by Horsch et al. (1985). The presence of the AtPAP15 transgene was
verified by PCR screening. Western-blot analysis was used to confirm protein
expression in the leaf protein extracts. Leaves were harvested from T2
transformants after 4 weeks of growth.
Generation of Specific Anti-AtPAP15 Antisera by
Affinity Purification
The signal peptide of AtPAP15 was predicted by the SignalP 3.0 server
(Bendtsen et al., 2004). The ORF of AtPAP15 excluding its predicted signal
sequence (residues 1–19) was subcloned into the pGex2T Escherichia coli
overexpression vector. Soluble recombinant GST-AtPAP15 was expressed in
the E. coli strain Rosetta and purified according to the manufacturer’s
instructions. One hundred fifty micrograms of soluble protein was used to
immunize rabbits for polyclonal antibody production. For western blotting,
the primary rabbit antibody and the secondary goat anti-rabbit IgG-AP-
conjugated antibody (1:10,000; Sigma) were diluted at 1:2,000 and 1:10,000,
respectively. Nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate
(Invitrogen) were used as substrates.
Protein Extraction and Purification
Fifty grams of transgenic tobacco leaf tissue was ground in liquid nitrogen
and extracted with 50 mM sodium acetate buffer (pH 5.0) freshly supple-
mented with 1 mM phenylmethylsulfonyl fluoride and 5 mM dithiothreitol. The
leaf tissue extracts were then centrifuged at 12,000g twice, and the supernatant
was used for protein concentration and enzyme activity measurements. The
enzyme was further purified to homogeneity by three chromatographic
procedures (Table I). All chromatography was carried out on an AKTA
purifier FPLC system (GE Healthcare), sequentially with a cation-exchange
column (HiPrep 16/10 CM FF, 16 3 100 mm), a Ni2+ affinity column (HiTrap
HP, 1 mL), and a gel filtration column (Superdex 75, 10 3 300 mm) following
the manufacturers’ instructions. The purified protein was concentrated and
stored at 280�C for further assays. The purified protein was fractionated by
10% (w/v) SDS-PAGE (MiniII; Bio-Rad) and visualized by silver staining; the
protein was further confirmed by matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry analysis (Lung et al., 2008) and western
blotting. Apparent molecular mass of AtPAP15 was calculated by plotting log
molecular mass against migration distance, using the molecular mass markers
for calibration.
Determination of Native Molecular Mass by GelFiltration Chromatography
The native molecular mass of the purified enzyme was estimated by gel
filtration (Superdex 75, 10 3 300 mm) using the AKTA purifier FPLC system
(GE Healthcare) as described above. It was calculated from a plot of Kav
(partition coefficient) against log molecular mass, which was calibrated using
five protein standards: IgG (molecular mass, 150 kD), albumin (molecular
mass, 55 kD), ovalbumin (molecular mass, 45 kD), chymotrypsinogen A
(molecular mass, 20 kD), and ribonuclease A (molecular mass, 15 kD).
Phytase Activity Assay
Phytase activity was estimated colorimetrically by monitoring the release
of inorganic phosphate from phytic acid (Na-InsP6; Sigma-Aldrich). One unit
of phytase activity was defined as the release of 1 mmol of phosphate per
minute under the described conditions. Fifty-microliter samples were reacted
at 37�C for 1 h in a 100 mM NaOAc (pH 4.5) assay buffer containing 1 mM
Na-InsP6; the reaction was terminated by addition of an equal volume of 4%
(w/v) TCA. The liberated inorganic orthophosphate was quantified spectro-
photometrically with molybdenum blue (Murphy and Riley, 1962). Back-
ground readings due to Pi contamination (time 0) were subtracted before
calculation. The protein concentration was estimated by the standard Brad-
ford protein assay using the Protein Assay Dye Reagent Concentrate (Bio-
Rad). Bovine serum albumin was used as a standard.
Biochemical Characterization of aPlant-Derived AtPAP15
pH Profile
The pH optimum for phytase activity was measured at 37�C at different
pH values (2.5–8.0) using 1 mM Na-InsP6 as substrate in the following buffers:
Gly-HCl (pH 2.5–4.0), NaOAc (pH 4.5–5.5), Tris-maleate (pH 6.0–7.5), and
Tris-HCl (pH 8.0).
Temperature and Thermal Stability Profiles
The optimum temperature for phytase activity was measured over a
temperature range of 23�C to 85�C in 100 mM NaOAc buffer (pH 4.5)
containing 1 mM Na-InsP6. For the thermal stability assays, the phytase assay
was carried out at 37�C after the enzyme was preincubated at different
temperatures (23�C–85�C) for 15 min.
Substrate Specificity
To determine substrate specificity, enzymatic activities toward the fol-
lowing substrates were tested: AMP, ADP, ATP, CMP, dGTP, dTTP, dCTP,
dATP, Fru-6-P, Glc-1-P, Glc-6-P, glycerol 3-phosphate, GMP, O-phospho-Tyr,
O-phospho-Ser, O-phospho-Thr, 3-phosphoglyceric acid, PEP, pNPP, Rib-5-P,
sodium phytate, and sodium pyrophosphate. The final concentration of sub-
strate in 100 mM NaOAc (pH 4.5) was 1 mM.
Effect of Ions and Inhibitors on Enzymatic Activity
The effects of different anions and cations on the phytase activity were
tested by supplementing the reaction mixture with 5 mM of various salts:
AlCl3, CaCl2, CaCl2, CuCl2, MgCl2, MnCl2, NiCl2, KCl, NaCl, NaNO3, Na2SO4,
Na2SO3, and ZnCl2. The enzyme was incubated with inhibitors at various
concentrations (0, 0.25, 0.5, 1, 2.5, and 5 mM) for 10 min prior to the enzyme
assay. Inhibitor solutions, including EDTA, sodium citrate, NaMoO4, sodium
potassium tartrate, NaF, and KH2PO4, were prepared in 100 mM NaOAc
(pH 4.5).
Enzyme Kinetics
The enzyme activity was estimated over a range of substrate concentra-
tions (0, 0.05, 0.1, 0.2, 0.4, 0.5, and 1 mM) with Na-InsP6, pNPP, and PEP as
substrates. Four time points (0, 20, 40, and 60 min) were tested for each
substrate concentration. The kinetics constants Km and Vmax were calculated
from a Lineweaver-Burk plot.
Construction of an AtPAP15 Promoter::GUS Constructand Arabidopsis Transformation
To generate AtPAP15 promoter::GUS fusion constructs, AtPAP15 promoter
sequences of various lengths (0.47, 1.1, and 1.6 kb) were first amplified by
PCR using forward primers (F1, 5#-ATATTCTAGACATGCTCTGGTATATAT-
TAAACTCC-3#; F2, 5#-ATATTCTAGACGATGCTACGTAGATGAAACG-3#;F3, 5#-ATATTCTAGAAGTG CCTAAACGAGTCCATTTA-3#) and a reverse
Studies on AtPAP15 during Seed and Pollen Germination
Plant Physiol. Vol. 151, 2009 207
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primer (PR, 5#-ATATCTCGAGCGTTCCAGAGGGTGGT-3#). The amplified
fragments were cloned into the pGEM-T vector and sequenced. The AtPAP15
promoter fragments were released by XhoI and XbaI digestion and were
subcloned into pBa002a-GUS to make transcriptional fusion sequences with
the reporter gene GUS. Binary vectors containing the transgene inserts were
mobilized into A. tumefaciens GV3101 by freeze-thaw transformation. Trans-
formation of Arabidopsis was performed by the floral dip method (Clough
and Bent, 1998). Transgenic plants were selected on Murashige and Skoog
(MS) medium supplemented with 10 mg L21 Basta and 100 mg L21 carbeni-
cillin. The presence of the AtPAP15 promoter and the GUS gene in the
transgenic plantlets was identified by genomic PCR by a GUS-specific primer
(5#-AATATCTGCATCGGCGAACT-3#) and a promoter-specific primer. An
empty vector and a vector carrying the cauliflower mosaic virus 35S sequence
were used as negative and positive controls, respectively.
Histochemical Analysis of GUS Expression
Eight to 15 T0 transgenic lines from each construct line were examined for
GUS activity. All transgenic lines displayed identical patterns of GUS staining
but with different intensities. Further analyses were performed on two
homozygous T3 transgenic lines with strong GUS expression.
For the histological examination of tissues and the histochemical localiza-
tion of GUS staining in seedlings and anthers, fixed plant samples of different
developmental stages of pBa002a-AtPAP15 F2-GUS lines were dehydrated
through a graded ethanol series and embedded in Historesin (Yeung, 1999).
Serial sections, 3 mm thick, were cut using a Ralph knife on a Reichert-Jung
2040 Autocut rotary microtome. Histological sections were stained by the
periodic acid-Schiff’s reaction for total carbohydrates and counterstained with
1% (w/v) amido black 10B for protein (Yeung, 1984). For localization of the
GUS staining products within cells, some sections were examined without
staining (Bassuner et al., 2007). Processed sections were viewed with a Leitz
Aristoplan photomicroscope, and the images were captured using a Leica
DFC480 digital camera. Composite plates were assembled using Photo-
shop CS2.
Analysis of AtPAP15::GUS Expression in Response to aVariety of Stimuli
Homozygous pBa002a-AtPAP15 GUS lines (T3) were used as plant mate-
rials. For phytohormone treatment and for salt and osmotic stresses, seeds
were germinated onMS agar plates for 7 d prior to treatment and incubated in
liquid medium for another 12 to 48 h. The treatments were as follows: abscisic
acid (100 mM), gibberellic acid (100 mM), methyl jasmonate (50 mM), salicylic
acid (100 mM), hydrogen peroxide (4 mM), sodium chloride (250 mM), sorbitol
(300 mM), andmannitol (300 mM). For nutrient starvation analysis, plants were
first germinated on MS agar plates for 5 d, then transferred to MS medium
lacking a specific nutrient (phosphorus, nitrogen, or potassium), and grown
for another 2, 4, or 6 d. For all treatments, MSmediumwas used in parallel as a
control. After harvest, plants seedlings were GUS stained.
Confirmation of T-DNA Insertion Mutants and Their
Complementation with AtPAP15
T-DNA insertion lines were obtained from the Arabidopsis Biological
Resource Center (Ohio State University). Seeds of two AtPAP15 insertion
mutants, SALK_061597 (T2) and SAIL_529_D01 (T9), were germinated in the
presence of kanamycin (50 mg L21) or Basta (5 mg L21) to follow the
segregation of the antibiotic resistance marker. Antibiotic-resistant T2 plants
were screened for T-DNA insertion by PCR using gene-specific primers and
primers anchored in the T-DNA borders. Interruption of the AtPAP15 gene
was further confirmed by RT-PCR and western-blot analysis.
To generate the construct for complementation, an AtPAP15 construct,
including the NOS promoter and the AtPAP15 cDNA, was subcloned into a
pCAMBIA1300 plant expression vector. Empty pCAMBIA1300 vectors were
employed as controls. The construct was transferred into A. tumefaciens strain
GV3101, and T-DNA mutants were transformed using the floral dip method
as described previously. T1 plants were selected using MS plates with
hygromycin (20 mg L21) and verified by PCR screening. Homozygous T3
lines were later selected by hygromycin resistance and verified by PCR and
western-blot analysis.
Pollen Germination Analysis
T-DNA homozygous (T2 and T9), wild-type, and complementation lines
were planted in soil for pollen collection. In vitro Arabidopsis pollen germi-
nation experiments were conducted as described previously (Fan et al., 2001).
After an 8-h incubation in a climatic chamber, the pollen germination rate was
analyzed with a microscope. Experiments were repeated three times, and at
least three replicates were carried out. For each replicate, no less than 500
pollen grains were counted. The pollen grains with emerging tubes longer
than their diameter were considered as germinated.
Enzyme Assay of Seedlings and Pollen
during Germination
To measure phytase and APase activity during seed germination, around
100 seeds of different lines were surface sterilized with 20% Clorox and then
sown in sterilized MQ water. Seeds were collected after 48 h, and protein was
extracted using 100 mM sodium acetate buffer (pH 4.5) and 1 mM phenyl-
methylsulfonyl fluoride. Internal substrates, phosphate, and salts were re-
moved from the protein fraction by Microcon (molecular weight cutoff 10,000;
Millipore). Phytase and APase activity were detected using 1 mM Na-phytate
and pNPP as substrates, respectively. For pollen, different lines were planted
in soil for pollen collection. The in vitro Arabidopsis pollen germination
experiment was carried out as described above. After 8 h of germination, the
pollen was collected by centrifugation and the protein was extracted for
enzyme assays as described above. Experiments were repeated twice, and at
least four replicates were carried out.
Data Analysis
All data were analyzed by one-way ANOVA using the LSD at the level of 5%
(P , 0.05) to identify the significant differences between the observations,
with the aid of the statistical program SPSS 10.0.
For in silico analysis, homology searches in GenBank were done using the
BLAST server (http://www.ncbi.nlm.nih.gov/BLAST/). Multiple alignments
of protein sequences were performed using the ClustalX and N-J plot pro-
grams. For prediction of protein expression, Genevestigator was employed
(http://www.genevestigator. ethz.ch).
Sequence data from this article can be found in the GenBank/EMBL data
libraries under accession number At3g07130.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Specificity of the antiserum.
Supplemental File S1. Data on peptide mass fingerprint.
ACKNOWLEDGMENTS
We thank Dr. W.K. Yip’s laboratory at the University of Hong Kong for
kindly providing plant vectors and technical support for plant cultures. We
also thank Dr. Clive Lo at the University of Hong Kong for his support with
photomicroscopy.
Received June 18, 2009; accepted July 20, 2009; published July 24, 2009.
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Studies on AtPAP15 during Seed and Pollen Germination
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