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 within an acidic pH range. Among the 29 PAP-like proteins in Arabidopsis (Arabidopsis thaliana), AtPAP15 (At3g07130) displays a greater degree of amino acid identity with soybean (Glycine max; GmPHY) and tobacco (Nicotiana tabacum) PAP (NtPAP) with phytase 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, with strong GUS staining at the early stages of seedling growth and pollen germination. The expression was also organ/tissue specific, with strongest GUS staining in the vasculature, pollen grains, and roots. The recombinant AtPAP purified from transgenic tobacco exhibited broad substrate specificity with moderate phytase activity. AtPAP15 T-DNA insertion lines exhibited a lower phytase and phosphatase activity in seedling and germinating pollen and lower pollen germination rate compared with the wild type and their complementation lines. Therefore, AtPAP15 likely mobilizes phosphorus reserves in plants, particularly during seed and pollen germination. Since AtPAP15 is not expressed in the root hair or in the epidermal cells, it is unlikely to play any role in external phosphorus assimilation.
At pH in the range of 4 to 7, purple acid phosphatases (PAPs) catalyze the hydrolysis of a wide range of activated phosphoric acid monoesters and diesters and anhydrides (Klabunde et al., 1996). They are distinguished from the other phosphatases by their insensitivity to L-(+) tartrate inhibition and therefore are also known as tartrate-resistant acid phosphatases. Their characteristic pink or purple color derives from a charge transfer transition between a Tyr residue and the “chromophoric” ferric ion in the binuclear Fe(III)Me(II) center, where the metal (Me) is iron, zinc, or manganese (Schenk et al., 1999). PAP proteins are also characterized by seven conserved amino acid residues (shown in boldface) in the five conserved motifs DXG, GDXXY, GNH(D/E), VXXH, and GHXH, which are involved in the coordination of the dimetal nuclear center (Li et al., 2002). PAPs are widespread in mammals, fungi, bacteria, and plants. Interestingly, while only a few copies of 1 This work was supported by the University Research Committee (grant no. 10206029) and by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada to E.Y. 2 These authors contributed equally to the article. * Corresponding author; e-mail
[email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described 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 subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.109.143180
PAP-like genes are present in mammalian and fungal genomes (Mullaney and Ullah, 2003; Flanagan et al., 2006), multiple copies are present in plant genomes (Schenk et al., 2000). For example, 29 PAP-like genes have been identified in the Arabidopsis (Arabidopsis thaliana) genome (Li et al., 2002). It is intriguing that so many PAP-like genes are required for plant metabolism; this diverse portfolio of PAP-like genes implies differential functions for them. Plant PAPs are generally considered to mediate phosphorus acquisition and redistribution based on their ability to hydrolyze phosphorus compounds (Cashikar et al., 1997; Bozzo et al., 2004; Lung et al., 2008). However, additional biological roles have been reported for some plant PAPs. For example, the PAPs AtACP5 (AtPAP17), SAP1, and SAP2 (del Pozo et al., 1999; Bozzo et al., 2002) display not only phosphatase but also peroxidase activity, suggesting their involvement in the removal of reactive oxygen compounds in plant organs. GmPAP3, isolated from salted-stressed soybean (Glycine max), reportedly mediates salt tolerance via NaCl and oxidative stress inductions but not by phosphorus starvation (Liao et al., 2003). Some PAP members can hydrolyze phytic acid (myoinositol hexakisphosphate [InsP6]) to inorganic phosphate and free or lower phosphoric esters of myoinositol. Since the major storage form of phosphorus in plant seeds and pollen grains is phytate, PAPs with phytase activity may play a role in seed and pollen germination. However, not all PAPs exhibit phytase activity. The first plant phytase PAP, GmPHY, was isolated from the cotyledon of germinating
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soybean seedlings (Hegeman and Grabau, 2001). A tobacco (Nicotiana tabacum) root PAP phytase was identified more recently that is likely involved in mobilizing external organic phosphorus in soil (Lung et al., 2008). Relatively little is known about the biochemical properties and physiological roles of the 29 PAP-like Arabidopsis genes (del Pozo et al., 1999; Veljanovski et al., 2006). An enzyme assay involving the glutathione S-transferase (GST)-AtPAP23 fusion protein revealed that the Arabidopsis PAP AtPAP23 exhibits phytase activity (Zhu et al., 2005). A GUS study showed that AtPAP23 is exclusively expressed in the flower of the Arabidopsis plant. In a recent report, a recombinant AtPAP15 expressed in Escherichia coli was also found to exhibit phytase activity; this PAP potentially modulates plant ascorbate synthesis through supply of myoinositol from the phytate hydrolysis reaction (Zhang et al., 2008). However, the possible physiological roles of AtPAP15 in phosphorus mobilization have not been examined. In this study, AtPAP15 expressed in a plant (tobacco) system was biochemically characterized, and its temporal and spatial expression patterns in Arabidopsis were examined. The physiological roles of AtPAP15 in phosphorus mobilization were also delineated.
RESULTS Overexpression and Purification of AtPAP15 in Transgenic Tobacco Plants
The soluble GST-AtPAP15 protein did not show any enzymatic activity. Therefore, a His-tagged AtPAP15 protein was stably overexpressed in tobacco plants using an explant method. Gene expression was confirmed by PCR (data not shown) and western-blot analysis using specific anti-AtPAP15 antiserum (Fig. 1A). Phytase activity was approximately 3-fold greater in transgenic tobacco leaves compared with wild-type leaves (Fig. 1B). Leaves from permanent lines were used for further protein purification. The purification of the AtPAP15 protein was achieved by ion-exchange, affinity, and gel filtration chromatography. The purification table of a representative run is shown in Table I. A single polypeptide band of approximately 60 kD was detected by silver staining (Fig. 2A, lane 5), which confirmed the homogeneity of the protein. The apparent molecular mass of the native enzyme was estimated to be approximately 58 kD, using preparative-grade gel filtration chromatography (data not shown), confirming that the native enzyme is a monomeric protein. Western-blot analysis (Fig. 2B) and mass spectrometry were employed to confirm the identity of the protein. The sequence coverage of the peptide mass fingerprint reached 25% (134 of 532 residues) and the MOWSE score was 66 (Supplemental File S1), confirming its identity as AtPAP15. The enzyme had been purified approximately 344-fold with 200
Figure 1. Western blotting and phytase activity assays of transgenic tobacco lines. A, AtPAP15 expression was confirmed by western blotting. Lanes 1 to 6 represent molecular mass standards, the wild type, and pBa002a-PAP15 lines (transgenic lines 5-3, 7-4, 10-2, and 13-4), respectively. Thirty micrograms of protein was loaded into each lane. 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 significantly different (P , 0.05).
an overall recovery of 2.4%; it exhibited a phytase activity 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 is shown in Figure 3A. The enzyme showed an acidic pH activity profile, with maximal activity at pH 4.5. The enzyme had its highest activity at low temperatures (23°C–37°C; Fig. 3B) and was most stable in the same temperature range. Negligible activity was detected when the temperature was higher than 65°C (Fig. 3B). Effects of Ions and Inhibitors on Enzymatic Activity of AtPAP15
We investigated the influence of various ions on the enzymatic activity of AtPAP15. Ca2+ and Zn2+ stimulated the phytase activity of AtPAP15 (127.5% and 129.4%, respectively), whereas Cu2+ and SO322 inhibited this activity (reduced to 17.8% and 43.1%, respectively). Other ions (Mn2+, Mg2+, Ni2+, SO422, and NO32) exhibited no effects on phytase activity. The most prominent PAP inhibitor, molybdate, was also 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% Plant Physiol. Vol. 151, 2009
Studies on AtPAP15 during Seed and Pollen Germination
Table I. Purification of recombinant AtPAP15 from transgenic tobacco Step
Crude HiPrep CM HiTrap HP Superdex 75
Total Protein
Phytase
munits mg21 protein
mg
29 289 1,053 10,014
94,390 6,370 88 6.6
Volume
Protein
Phytase
mL
mg mL21
250 60 1.3 1
380 170 68 6.6
or 0%, respectively. The purified enzyme was significantly resistant to treatment with tartrate. Other compound supplements such as EDTA and citrate exerted no influence on enzymatic activity (Fig. 4). Substrate Specificity of Purified AtPAP15
Purified AtPAP15 exhibited broad substrate specificity (Table II), with p-nitrophenyl-phosphate (pNPP), phosphoenolpyruvate (PEP), and Na-pyrophosphate being the most effective substrates. Compared with activity toward pNPP, the activities toward various deoxyribonucleotide triphosphates were approximately 31% to 52% and the activity toward phytate was 7%. Very low activity was seen when monophosphates (AMP or GMP) were used as substrates. Negligible activity was detected on Glc-1-P.
Yield
Purification
munits
%
fold
2,745 1,843 93 66
100.0 67.1 3.8 2.4
1 10 36 344
stages, transgenic Arabidopsis plants expressing an AtPAP15 promoter::GUS fusion protein were produced. All three promoter regions directed the same pattern of GUS expression under normal growth conditions (data not shown). Intense GUS staining was observed at the early stages of seedling establishment and in the cotyledon, radicle, and hypocotyl (Fig. 5, A–D). The signal was stronger and more diffuse in the cotyledon leaves but weaker and more restricted in younger immature leaves (Fig. 5E). In mature, fully expanded rosette leaves of soil-grown Arabidopsis transgenic lines, GUS staining was predominantly detected in the vasculature (Fig. 5F). In roots, intense staining could be observed at all stages but was mainly restricted to the vascular cylinder (Fig. 5G). No expression could be found in the root hairs (Fig. 5G) or
Kinetic Parameters of Purified AtPAP15
The kinetic parameters of AtPAP15 were measured when 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) toward Na-phytate than toward pNPP or PEP. The catalytic efficiencies (kcat/Km) of the enzyme toward pNPP and PEP were approximately 10-fold higher than that toward Na-phytate. The biochemical properties of the recombinant AtPAP15 from plant were very different from that presented by Zhang et al. (2008), who reported the specific activities of a GST:AtPAP15 toward a few substrates. In our study, the GST:AtPAP15 produced from E. coli was inactive. Zhang et al. (2008) reported the specific activity of GST:AtPAP15 toward pNPP to be only 0.546 units mg21, which was far lower than the plant-derived AtPAP15 level (165 units mg21; Table III) and that reported for the other plant PAPs (222–368 units mg21 for tomato [Solanum lycopersicum] PAPs [Bozzo et al., 2002] and 266 units mg21 for KbPAP [Vogel et al., 2002]). Regarding specific activities toward phytate, the two recombinant AtPAPs gave 10.0 (plant) and 0.7 (bacterial) units mg21, respectively. Expression of AtPAP15 in Different Tissues and Developmental Stages of Arabidopsis
To determine the expression patterns of AtPAP15 with respect to specific tissue and developmental Plant Physiol. Vol. 151, 2009
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); lane 4, activity fraction from HiTrap HP (Ni2+) column (1 mg); lane 5, activity fraction from Superdex 75 gel filtration column (0.5 mg). The position of AtPAP15 is indicated by the arrows at right. 201
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with the vasculature, pollen grains, and seedlings showing the strongest staining. AtPAP15::GUS Expression in Response to a Variety of Stimuli
To analyze AtPAP15::GUS expression in response to a variety of stimuli, two independent homozygous transgenic lines were used for in situ analysis of GUS activity. In general, the overall GUS staining patterns in shoots and roots were not affected by these treatments. The only exception was at the root tip, where an increase of GUS activity was observed under salicylic acid, abscisic acid, NaCl, sorbitol, or mannitol treatment (Fig. 7). In contrast, no alterations in GUS staining were observed under nutrient stresses, such as phosphorus, nitrogen, or potassium starvation (data not shown). Molecular Characterization of AtPAP15 T-DNA Insertion Mutants and Their Complementation
Figure 3. Biochemical properties of the purified AtPAP15. A, pH profile. B, Temperature and thermostability profiles. Each value represents the mean 6 SD of three experiments.
in the root cap cells (Fig. 5H). GUS staining was consistently detected in the vasculature of both the stems and the roots. Cross-sectional analysis revealed that the staining was mainly localized to the xylem and phloem tissues in leaf, hypocotyl, and root sections (Fig. 5, I–L). In reproductive organs, GUS staining was obvious in the flowers (Fig. 5M); however, little or no staining was evident in the developing seeds or siliques (data not shown). Blue color was observed in the anther and also the stigma papillae (Fig. 5N). Pollen squash analysis indicated that the majority of the GUS staining was from pollen grains (Fig. 5O). To further analyze GUS expression in flowers, AtPAP15 promoter::GUS transgenic Arabidopsis anthers were sectioned to reveal the stage at which GUS expression was evident. Cross-sections of Arabidopsis anthers revealed little or no GUS gene expression in the early stage of pollen development (Fig. 6, A–D). This expression increased at later stages of pollen development and was obvious in mature pollen grains (Fig. 6, E and F). Staining could also be detected in in vitro-germinated pollen (Fig. 5P). The promoter::GUS data indicated that AtPAP15 expression is developmentally and temporally regulated, with strong GUS staining at early stages of seedling growth and at late stages of pollen development. The expression was also organ/tissue specific, 202
Six T-DNA insertion lines at the AtPAP15 locus are available from the Arabidopsis Biological Resource Center, of which we chose SAIL_529_D01 (T9) and SALK_061597 (T2), which insert into exon 1 and exon 2, respectively, to be used in this study. T2 and T9 seeds from the distribution stock were germinated, and segregation of the T-DNA-encoded antibiotic resistance marker was tracked. Genomic DNA was then extracted from the antibiotic-resistant T2 progeny, and this DNA was screened for T-DNA insertion by PCR using gene-specific primers and primers anchored in the T-DNA borders. Six (T2-1, -3, -5, -6, -8, and -9) and three (T9-2, -7, and -9) individual plants were selected as homozygotes by the absence of the AtPAP15 PCR product (data not shown). Gene silencing of the homozygous plants was confirmed by reverse transcription (RT)-PCR and western blotting. In RT-PCR assays, a single amplified DNA fragment with the predicted size (approximately 1.6 kb) was absent in T-DNA mutants when compared with wild-type plants (Fig.
Figure 4. Effects of inhibitors on purified AtPAP15 activity. SD values were calculated on the basis of three independent experimental trials. Plant Physiol. Vol. 151, 2009
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Table II. Substrate specificity of AtPAP15 purified from transgenic tobacco Enzymatic activities were assayed at 37°C for 1 h in 100 mM NaOAc buffer (pH 4.5) containing various substrates (final concentration, 1 mM). Each value represents the mean of three experiments and is expressed as a percentage relative to the measurement using pNPP as substrate. Substrate
Relative Activity
SD
were produced by introducing the construct AtPAP15 in the NOS promoter-containing pCAMBIA1300 into the mutants. Four homozygous T3 generation lines bearing one copy of the complemented gene were selected based on segregation of hygromycin resistance; these lines were verified by PCR screening and western blotting (Fig. 8C). Two lines (PC1 and PC2) were chosen for subsequent studies (Fig. 8, A and B).
%
pNPP Na-pyrophosphate PEP 3-PGA ADP dGTP Glc-3-P dTTP dCTP dATP P-Tyr P-Ser Glc-6-P CMP P-Thr Rib-5-P ATP Fru-6-P Na-phytate AMP GMP Glc-1-P
100 95 92 69 50 52 43 37 35 31 28 25 20 14 13 11 10 10 7 4 4 0
0 1 4 8 3 4 3 8 4 2 2 2 3 0 2 2 5 1 1 0 0 1
Growth Analysis of T-DNA Insertion and Its Complementation under a Variety of Stimuli
8A). Protein was extracted from seedlings for westernblot analysis via AtPAP15 antiserum. No AtPAP15 expression was observed in these insertion mutants compared with wild-type plants (Fig. 8B). A slightly smaller protein band was recognized in all lines by the anti-PAP15 antiserum (Fig. 8, B and C). This band is not AtPAP15 because it was present in all T-DNA lines, which were shown not to express AtPAP15 mRNA by RT-PCR (Fig. 8A). This band, which was not present in tobacco (Fig. 2B), could be an AtPAP protein with high amino acid sequence homology to AtPAP15. Western blotting of wild-type plants using the preimmune serum did not give any band (Supplemental Fig. S1). To verify that the phenotypic difference was due to the disruption of AtPAP15, complementation lines
Based on results of the above GUS assay, AtPAP15 is highly expressed in early stages of germination, implying that it may play a role(s) in seed germination or seedling growth. However, the insertion mutants and their complementation lines did not differ in growth performance, phenotypes, or seed germination rate when planted in soil (data not shown). Therefore, the phosphatase activities of 2-d-old seedlings were measured. It was observed that both phytase activity and acid phosphatase (APase) activity were significantly lower in the T-DNA mutants than in the wild type, whereas the complementation line showed comparable value to the wild type (Table IV). Since GUS studies indicated that AtPAP15 expression was substantial during pollen germination, the enzyme activities in germinating pollen were also assayed, which showed similar results (Table IV). In general, the specific activities of phytase and phosphatase in pollen were higher than that in the seedlings among all lines, possibly due to a lower complexity of total protein in pollen. An in vitro pollen germination experiment was then conducted. As shown in Figure 9, the pollen germination rate was significantly reduced in the mutants (30%–35%) compared with wild-type plants (78%). Complementation of AtPAP15 mutants with pCAMBIA1300-PAP15 resulted in recovery of the mutagens relative to the wild-type response (65%). These results indicated that the pollen germination phenotype of the AtPAP15 mutant correlates to AtPAP15 deficiency.
DISCUSSION
Only two PAP-like genes (Flanagan et al., 2006) are present in animal genomes, compared with the
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 values were calculated on the basis of three independent experimental trials. Substrate
Na-phytate pNPP PEP Plant Physiol. Vol. 151, 2009
Vmax
Km
kcat
kcat/Km
units mg21
mM
min21
min21 mM21
531 6 78 9,714 6 3,581 11,917 6 3,509
1,602 6 39 14,480 6 1,857 15,229 6 1,460
13.4 6 0.6 165.4 6 9.0 134.7 6 2.6
278 6 28 703 6 333 801 6 294
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Kuang et al. Figure 5. Histochemical localization of GUS activity in transgenic Arabidopsis plants containing the AtPAP15 promoter::GUS construct. A, One-day-old seed. B, Two-day-old seedling showing intense GUS staining in the radicle. C and D, Three-day-old (C) and 7-d-old (D) seedlings showing strong GUS activity in roots, hypocotyls, and cotyledon leaves. E, Ten-day-old plant. F, A mature soil-grown rosette leaf with GUS staining in the vasculature. G and H, A representative main root (G) and the root tip (H) of a 7-d-old seedling. I to L, Cross-sections of the leaf (I), hypocotyls (J), main root (K), and lateral root (L) of a 7-d-old seedling. M, Flower. N, Stigma papillae of the stigma. O, Mature pollen grains. P, An in vitrogerminated pollen grain. Bars = 50 mm.
greatly larger numbers in plants (Li et al., 2002). It is intriguing that so many different PAPs are required for plant metabolism and how different the physiological functions of these PAPs are. In this study, we focused on AtPAP15, a PAP with phytase activity. Not all PAPs exhibit phytase activity; for instance, no phytase activity has been reported for mammalian PAPs. In addition, some plant PAPs, including KbPAP (Cashikar et al., 1997), two secreted tomato PAPs (Bozzo et al., 2002), and an intracellular tomato PAP (Bozzo et al., 2004), lack phytase activity.
Our results confirmed that AtPAP15 is an acidic phosphatase with phytase activity. Phytate (InsP6), which is primarily complexed with metal ions, is the principal storage form of phosphorus in seeds (Otegui et al., 2002) and pollen (Jackson and Linskens, 1982). The globoid in the protein storage vacuole of the Arabidopsis seed embryo is primarily composed of calcium/magnesium/potassium-phytate salts (phytin); thus, phytin breakdown in these cells releases phosphorus, calcium, magnesium, potassium, and carbon for embryo growth and division (Otegui et al., 2002). In pollen, small electron-dense globoids rich in calcium,
Figure 6. Cross-sections of anthers (3 mm) of representative transgenic Arabidopsis plants containing the AtPAP15 promoter:: GUS construct. The following developmental stages are shown: stage 1, pollen mother cells (A); stage 2, meiosis (B); stage 3, tetrads (C); stage 4, vacuolate microspores (D); stage 5, binucleate and trinucleate microspores (E); stage 6, mature microspores (F). Right panels show sections stained to reveal general histology. Left panels show sections of GUS-stained samples. Bars = 50 mm.
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Studies on AtPAP15 during Seed and Pollen Germination Figure 7. GUS staining of AtPAP15::GUS transgenic Arabidopsis seedling root tips in response to various stimuli. Transgenic plants (T3) were grown on MS agar plates for 7 d and then transferred into treated liquid medium and grown for 24 h as described in “Materials and Methods.” GUS activity was detected in situ after 8 h of incubation. Four replicates were performed with 20 plants for each treatment. The conditions were optimized to provide low signals in control and thereby allow visualization of changes above background. ABA, Abscisic acid; GA3, gibberellic acid; H2O2, hydrogen peroxide; MeJA, methyl jasmonate; SA, salicylic acid.
magnesium, and phosphorus were only found within cytoplasmic vesicles of vegetative cells but not in germ cells (Butowt et al., 1997). These globoids, which were suggested to contain phytin, were found to increase and decrease in size and number during pollen maturation and germination, respectively (Butowt et al., 1997). During seed and pollen germination, phytases are required to hydrolyze phytate to less phosphorylated compounds myoinositol and inorganic phosphate. This phosphorous supply is essential for seedling development (Reddy et al., 1989). GUS staining studies demonstrated that AtPAP15 was strongly expressed during seed and pollen germination. Its expression was significantly increased in 2-d-old compared with 1-d-old seedlings (Fig. 5, A and B), and in pollen, AtPAP15 was not expressed during pollen development but was strongly expressed during pollen germination (Fig. 6). These observations are consistent with its possible role in the release of internal phosphorus reserves. The seedlings of T-DNA lines only exhibited 35% to 55% of the phytase activity of wild-type seedlings, but this reduction did not affect the germination rate of the
seedlings. The expression of redundant phytase genes and the abundance of phosphorus in the seeds may explain the normal seed germination rate of the T-DNA lines. During pollen germination, pollen of T-DNA lines only exhibited 25% to 57% phytase activity and 59% to 71% APase activity compared with the wild type (Table IV), and this reduction is correlated with a lower in vitro germination rate of the pollen (Fig. 9). These results indicated that AtPAP15 is a key phytase/phosphatase during pollen germination and implicated a physiological role in the mobilization of phosphorus reserves in pollen. It is unlikely that AtPAP15 is secreted from pollen for external phosphorus assimilation. During in vitro germination of Lilium longiflorum pollen, in-gel acid phosphatase activity staining of germination medium only generated a 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 in soybean (Hegeman and Grabau, 2001), Medicago (Xiao et al., 2005), and tobacco (Lung et al., 2008). AtPAP15 is not the only Arabidopsis PAP with phytase activity. Figure 8. Molecular characterization of AtPAP15 T-DNA insertion mutants and their complementation. A, RT-PCR with gene-specific primers for analyzing the transcription of AtPAP15 in 5-d-old seedlings of T-DNA lines. The wild type (Wt), complementation line (PC2), and genomic DNA (GDNA) were used as controls. Actin2 primers were used as a reference. M, Molecular mass markers. B, Western-blot analysis of seedling protein (80 mg protein lane21) on a 10% (w/v) SDS-PAGE gel with anti-AtPAP15 antiserum. Wild-type and complementation lines (PC2) were used as controls. The nonspecific band can be used as a loading control. C, Western-blot analysis of complementation lines (PC1-4) with anti-AtPAP15 antiserum using proteins from T-DNA and the empty vector control protein EV1-2 (40 mg protein lane21).
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Table IV. Enzyme activities in germinating seedlings and pollen Enzymatic activity was estimated with 1 mM Na-InsP6 (phytase activity) or pNPP (APase activity) as substrate. Protein was extracted from 2-d-old seedlings; pollen grains were germinated in liquid medium for 8 h and then collected for protein extraction. Values marked by different letters are significantly different (P , 0.05) in the same column. There are four replicates for each line. Line
Seedlings Phytase
Pollen
APase
Phytase 21
munits mg
Wild type T2-3 T2-5 T9-2 T9-7 PC1 PC2
1.03a 0.36b 0.53b 0.48b 0.57b 1.18a 1.48a
61.3ab 49.6b 52.6b 44.1b 47.6b 76.6a 90.9a
APase
protein
17.24a 8.91b 6.28b 5.32b 4.33b 16.24a 15.17a
186.3a 132.2b 126.5b 110.3b 116.1b 178.5a 190.4a
Phylogenetic analysis of the 29 PAP-like sequences in Arabidopsis indicated that AtPAP13, AtPAP15, and AtPAP23 were clustered in the subgroup 1b-1 (Li et al., 2002). All of the subgroup 1b-1 PAPs shared nine Cys residues at conserved positions, which were not found in PAPs from other subgroups. The presence of these nine Cys residues (Cys-160, -237, -240, -375, -414, -441, -456, -467, and -522 with respect to the amino acid sequence of AtPAP15) may be a signature of plant PAPs with phytase activity. AtPAP23 also exhibits phytase activity (Zhu et al., 2005), and its GUS expression profile was markedly different from that of AtPAP15. The promoter of AtPAP23 is only highly active in flowers but not in shoot apical meristems during the early stages of flower differentiation. In mature flowers, AtPAP23 is only active in petals, stamen filaments, the base of the stamen filaments, and the pistil, areas where AtPAP15 is not expressed. In contrast to AtPAP15, AtPAP23 is not expressed in the stigma or the anthers. It may be that AtPAP23 is specialized for phosphorus supply during flower development, whereas AtPAP15 is responsible for phosphorus reserve mobilization during pollen development and germination. This would account for the differential expression of the two PAPs in terms of their temporal and spatial profiles. The differential expression of AtPAP15 and AtPAP23 in Arabidopsis is one example where multiple PAP genes are required for their various functions in plants. AtPAP15 expression was not restricted to the reserve storage organs; its expression was also strong in the plant vascular tissues (Fig. 5, E–L), where phytate amounts are scarce. Furthermore, it is noteworthy that phytate was not the only substrate of AtPAP15 (Table II). Rather, it was active toward many organic phosphorus compounds. Vascular tissues allow the transport of energy compounds from the shoots to the roots and the transport of nutrients from the roots to the shoots. AtPAP15 may mediate the remobilization of inorganic phosphates from organic phosphorus 206
compounds and may help maximize the phosphorus efficiency of the plant by redistributing surplus phosphorus from the mature to the growing tissues. This possibility is consistent with the lack of AtPAP15 expression in root tips and hairs (Fig. 5, G and H), where very large amounts of inorganic phosphate are consumed for building biomolecules such as DNA and RNA. Arabidopsis does not secrete phytases from its roots (Richardson et al., 2001). AtPAP15 is unlikely to be secreted into the root exudates, as it was not expressed in the root hairs or in epidermal cells. In contrast, tobacco secretes phytase PAPs from its roots during phosphorus deficiency (Lung and Lim, 2006; Lung et al., 2008). Hence, while most plants secrete phosphatases to acquire external phosphorus, not all of them secrete phytases. Although phytate is the most abundant organic phosphorus compound in soil (Turner et al., 2002), it is strongly fixed to soil components. Only soluble phytate can be hydrolyzed by phytases (Tang et al., 2006); therefore, for plants secreting PAP phytases from their roots, the availability of soluble phytate (substrate) and not that of the enzyme is the major limiting factor for soil phytatephosphorus assimilation. Organic acids can enhance the availability of soluble phytate (Tang et al., 2006); thus, plants capable of secreting large amounts of organic acids from their roots are benefited by secretion of phytases. A recent report proposed that AtPAP15 helps to supply the plant with myoinositol for ascorbate synthesis (Zhang et al., 2008). However, myoinositol is not considered a major precursor for ascorbate, and there are multiple ascorbate biosynthetic pathways in plants, implying that AtPAP15 may not be an essential enzyme in ascorbate biosynthesis (Valpuesta and Botella, 2004). The role of AtPAP15 in the mobilization of internal phosphorus stores was delineated in this study. The temporal and spatial expression of AtPAP15 was developmentally, but not environmentally, regulated, since its expression was not induced by nutrient starvation.
Figure 9. Pollen germination of AtPAP15 T-DNA insertion lines with control of the wild type (Wt) and complementation lines (PC1 and PC2; T3). Pollen was germinated in vitro for 8 h and then observed by microscopy, and the germination rate was analyzed. Means + SE of three replicates, approximately 500 pollen grains per replicate, are shown. Values marked by different letters are significantly different (P , 0.05). Plant Physiol. Vol. 151, 2009
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MATERIALS AND METHODS
Phytase Activity Assay
AtPAP15 Cloning, Vector Construction, and Tobacco Transformation
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 spectrophotometrically with molybdenum blue (Murphy and Riley, 1962). Background readings due to Pi contamination (time 0) were subtracted before calculation. The protein concentration was estimated by the standard Bradford protein assay using the Protein Assay Dye Reagent Concentrate (BioRad). Bovine serum albumin was used as a standard.
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 cDNA was 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#-TATGTCGACATGACGTTTCTACTACTTCTAC-3#) and reverse (5#-GACTAGTTCAGTGGTGGTGGTGGTGGTGGCAATGGTTAACAAGGCGGT-3#) primers by Pfx polymerase (Roche). A 63 His 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 mobilized into Agrobacterium tumefaciens strain GV3101 by freeze-thaw transformation (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-APconjugated 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 supplemented 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 ¨ KTA procedures (Table I). All chromatography was carried out on an A 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 Gel Filtration Chromatography The native molecular mass of the purified enzyme was estimated by gel ¨ KTA purifier FPLC system filtration (Superdex 75, 10 3 300 mm) using the A (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).
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Biochemical Characterization of a Plant-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 following 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 substrate 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 concentrations (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 Construct and 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#-ATATTCTAGACATGCTCTGGTATATATTAAACTCC-3#; F2, 5#-ATATTCTAGACGATGCTACGTAGATGAAACG-3#; F3, 5#-ATATTCTAGAAGTG CCTAAACGAGTCCATTTA-3#) and a reverse
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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. Transformation 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 carbenicillin. 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 localization 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 Photoshop CS2.
Analysis of AtPAP15::GUS Expression in Response to a Variety of Stimuli Homozygous pBa002a-AtPAP15 GUS lines (T3) were used as plant materials. For phytohormone treatment and for salt and osmotic stresses, seeds were germinated on MS 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), and mannitol (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, MS medium was 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.
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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 germination 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 phenylmethylsulfonyl fluoride. Internal substrates, phosphate, and salts were removed 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 programs. 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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