GmPAP3, a novel purple acid phosphatase-like gene in soybean induced by NaCl stress but not phosphorus deficiency

Gene 318 (2003) 103 – 111 www.elsevier.com/locate/gene

GmPAP3, a novel purple acid phosphatase-like gene in soybean induced by NaCl stress but not phosphorus deficiency Hong Liao a,b,1, Fuk-Ling Wong a,1, Tsui-Hung Phang a, Ming-Yan Cheung a, Wing-Yen Francisca Li a, Guihua Shao a,c, Xiaolong Yan b, Hon-Ming Lam a,* a Department of Biology, The Chinese University of Hong Kong, Shatin, N.T. Hong Kong S.A.R., China Laboratory of Plant Nutritional Genetics, South China Agricultural University, Guangzhou 510642, China c Institute of Crop Breeding and Cultivation, Chinese Academy of Agricultural Sciences, Beijing 100081, China b

Received 16 January 2003; received in revised form 20 May 2003; accepted 17 June 2003 Received by G. Theissen

Abstract Purple acid phosphatases (PAPs) are commonly found in plants, but the physiological functions of different classes of PAPs are not thoroughly understood. In the present study, we identified a novel gene, GmPAP3, from salt-stressed soybean using suppression subtractive hybridization (SSH) techniques. Protein sequence alignment studies and phylogenetic analysis strongly suggested that GmPAP3 belongs to the group of plant PAPs and PAP-like proteins that are distinct from those of fungi and animals. In addition, the invariable consensus metal binding residues of PAPs were all conserved in GmPAP3. Surprisingly, analysis of protein sorting signals showed that a putative mitochondrion targeting transit peptide is present on GmPAP3. Northern blot analysis revealed that NaCl stress causes a general induction of GmPAP3 expression in both roots and leaves of various cultivated (Glycine max) and wild (Glycine soja) soybean varieties. Further test using two genetically unrelated cultivated soybean varieties showed that the expression pattern of GmPAP3 is distinct from other PAP genes in soybeans. NaCl stress and oxidative stress but not phosphorus (P) starvation induces the expression of GmPAP3. These results suggest that the physiological role of GmPAP3 might be related to the adaptation of soybean to NaCl stress, possibly through its involvement in reactive oxygen species (ROS) forming and/or scavenging or stress-responding signal transduction pathways. D 2003 Elsevier B.V. All rights reserved. Keywords: PAP; Gene expression; Glycine max; Glycine soja

1. Introduction Phosphatase, a collective term given to enzymes that hydrolyze orthophosphate monoesters into inorganic phosphate, are found to be involved in various metabolic processes (Majerus, 1992; Duff et al., 1994). Purple acid

Abbreviations: PAPs, purple acid phosphatases; SSH, suppression subtractive hybridization; ROS, reactive oxygen species; P, phosphorus; Pi, orthophosphate; PQ, paraquat; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction. * Corresponding author. Tel.: +852-26096336; fax: +852-26096336. E-mail address: [email protected] (H.-M. Lam). 1 H.L. and F.-L.W. contributed equally and are considered co-first authors. 0378-1119/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0378-1119(03)00764-9

phosphatases (PAPs) belong to a family of binuclear metalcontaining acid phosphatases (Schenk et al., 1999) and are commonly found in a wide range of plant species, including red kidney bean (Beck et al., 1986), lupin (Wasaki et al., 1999), rice (Igaue et al., 1976), sweet potato (Sugiura et al., 1981; Durmus et al., 1999), soybean (LeBansky et al., 1992; Schenk et al., 1999), Arabidopsis thaliana (Patel et al., 1998; del Pozo et al., 1999), and duckweed (Nakazato et al., 1998). Similar to those isolated from mammals, plant PAPs are distinguished from other phosphatases by their acidic pH optima and their unique purple color (Schenk et al., 2000). The major reaction of PAPs is to catalyze the hydrolysis of orthophosphate monoesters, hence plant PAP activities are frequently found to increase under P-deficient conditions

104

H. Liao et al. / Gene 318 (2003) 103–111

(Cashikar et al., 1997; del Pozo et al., 1999). However, a recent study in A. thaliana showed that not all PAP genes are induced under phosphorus (P) starvation conditions (Li et al., 2002). In addition, PAPs may also perform other biochemical functions. For instance, a phosphate starvationinduced PAP-like protein isolated from A. thaliana (AtACP5) exhibits both phosphatase and peroxidase activities. The gene encoding AtACP5 (AtACP5) was not only responsive to P starvation but also to abscisic acid and salt stress, revealing the multi-functional nature of this enzyme (del Pozo et al., 1999). In soybean, a few cDNA clones encoding putative PAPs were reported previously (Schenk et al., 1999, 2000; Hegeman and Grabau, 2001). One soybean PAP protein was purified and well characterized for its biochemical properties (LeBansky et al., 1992). However, the response of these soybean PAP genes under P starvation has not been experimentally verified (Duff et al., 1994; Hegeman and Grabau, 2001). In this study, we report the cloning of a novel PAP gene (GmPAP3) identified from salt-stressed soybean using suppression subtractive techniques (Diatchenko et al., 1998). The putative mitochondrial location, NaCl and oxidative stress inducibility and insensitivity toward P starvation clearly distinguish GmPAP3 from other known plant PAPs.

2. Materials and methods 2.1. Plant materials Five cultivated (Glycine max L. Merr.) and five wild (Glycine soja) soybean varieties consisting of different genetic backgrounds were employed in this study. The cultivated varieties Union (UN), Hark (HK) and Nebsoy (NS) are from the U.S.A. The other cultivated varieties Wengfen7 (WF) and ‘‘85-140’’ are from P.R.C. All wild soybean varieties including Yunjin3 (YJ), Mengjin1 (MJ), Kaiyuan21 (KY), Shuangcheng4 (SC) and Huma22 (HM) are originated from P.R.C. 2.2. Molecular cloning, phylogenetic analysis, and gene expression study of a PAP-like gene from soybean To prepare samples for the construction of the subtractive library, seeds of the variety Wenfeng7 (WF) were germinated and grown in perforated plastic pots filled with thoroughly washed silicon sand and irrigated with modified Hoagland’s nutrient solution (Hoagland and Arnon, 1938) for 14 days before treated with 0.3% (w/v) NaCl for 3 days. The salt treatment was then gradually increased to 0.6%, 0.9%, 1.2% and finally to 1.5% (w/v) in 3-day intervals. After treatment in 1.5% (w/v) NaCl for 3 days, total RNA was extracted from the leaves of NaCl-treated and control seedlings using a modified phenol/chloroform/ isoamylalcohol (P:C:I = 25:24:1, v/v) mediated extraction

protocol (Jackson and Larkins, 1976). The CLONTECH PCR-Selectk cDNA Subtraction Kit (Clontech K1804-1) was employed to construct the WF subtractive library. All procedures were performed according to the manufacturer’s recommendations. The tester and driver cDNAs were originated from the leaves of NaCl-treated and control WF plants, respectively. Selected subtractive fragments were cloned into the pBluescript II KS (+) by the T-A cloning method (Sambrook and Russell, 2001). The ligation products were then transformed into the Escherichia coli strain DH5a by the CaCl2 method (Sambrook and Russell, 2001). The cDNA clone in each successful transformant was sequenced using the ABI PRISMTM dRhodamine Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer 402078). Prepared sequencing samples were applied to the Genetic Analyzer ABI prism 310 to resolve the cycle sequencing product. All sequencing data were analyzed by BlastN, BlastX, and PSI-BlastX programs provided in the website of National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). One of the subtractive fragments (GmPAP3) was identified as a PAP-like gene based on its high homology to other known PAP genes (see results). The cDNA sequence covering the intact coding region of GmPAP3 was first obtained by 5V and 3V rapid amplification of cDNA ends (RACE) techniques using the SMARTk RACE cDNA amplification Kit (Clontech K1811-1). For 5V RACE, the gene specific primers were as follows: GSP1 (5V-CTGCATACCAGAGGGGACTGGG); nested-GSP1 (5-TGGGAGGCC A A G TA G G G A G TA G ) ; a n d n e s t e d - G S P 2 ( 5 VAGACCATAACCATGGATGATATGC). For 3V RACE, the gene specific primers were as follows: GSP1 (5VGAGCTGAAGCGAGTTGAGAGGG) and nested-GSP1 (5V-TATATGGAGGGTGAAAGCATGCG). Touch-down polymerase chain reaction (PCR) reactions for RACE were performed according to the manufacturer’s instruction with modification in the annealing temperatures as follows: amplification with 5V and 3V GSP1 (70, 68, and 66 jC for 1st, 2nd, and 3rd round PCR, respectively); reamplification with 5V nested-GSP1, 5V nested-GSP2 and 3V nested-GSP1 (70 and 66 jC for 1st and 2nd round PCR, respectively). The DNA sequence of GmPAP3 was subsequently verified by direct sequencing of the PCR products generated from a WF cDNA preparation using primers flanking the full-length coding region of GmPAP3 (5VTTAATACACGTGCGCACCAAC and 5V-CATTTTCATCCTTTTCAAAACGCC). Putative protein sorting signals and subcellular localization were predicted by TargetP (Emanuelsson et al., 2000), iPSORT (Bannai et al., 2002) and PSORT (Nakai, 2000) programs. Phylogenetic relationships of GmPAP3 with other PAPs were analyzed. Multiple sequence alignment was performed using the ClustalW program (Thompson et al., 1994). The bootstrap value was calculated and the phylogenetic tree was built using the MEGA program (version 2.1) (Kumar et al., 2001).

H. Liao et al. / Gene 318 (2003) 103–111

105

generate probes for GmPAP1 (5V-ATGGGCACTCAGAGAAGC and 5V-ACCATGTCACTGGAGTCAAA), GmPAP2 (5V-AGTGCTTGCGTGATGTGC and 5V-CCAAGAATCCCACCTAATG), and GmPAP3 (T3 and T7 primers flanking the original subtractive GmPAP3 clone).

Fig. 1. Effect of NaCl on the expression of GmPAP3 in various cultivated (G. max) (A) and wild (G. soya) (B) soybean varieties. Seeds of different soybean varieties were germinated and grown as described in Materials and methods. Twenty-day-old seedlings were subjected to a stepwise NaCl treatment of 0.3%, 0.6% and 0.9% (w/v) in 3-d intervals. Expression of the GmPAP3 gene was analyzed by Northern blot analysis after NaCl treatment (see Materials and methods for details). Ten micrograms of total RNA was loaded onto each lane. + NaCl and NaCl indicate plants with and without NaCl treatment, respectively. Lanes 1 – 2: WF; lanes 3 – 4: ‘‘85-140’’; lanes 5 – 6: HK; lanes 7 – 8: UN; lanes 9 – 10: NS; lanes 11 – 12: YJ; lanes 13 – 14: MJ; lanes 15 – 16: KY; lanes 17 – 18: SC; lanes 19 – 20: HM.

One thousand replicates were used for all possible tree topology calculation. To prepare RNA samples for the NaCl inducibility test, seeds of different soybean varieties were germinated and grown as described above, except that 20-day-old seedlings were used in the stepwise NaCl treatment (0.3%, 0.6% and 0.9%). Gene expression was analyzed by Northern blot analysis using a standard procedure in a hybridization solution containing 50% formamide at 42 jC (Sambrook and Russell, 2001). Single-stranded DIG (Digoxigenin)-labeled PCR products were used as probes (Finckh et al., 1991). Three primer pairs were employed to Fig. 2. Alignment of GmPAP3 to other PAPs. The full-length amino acid sequence of GmPAP3 was aligned to biochemically characterized PAPs, including those from sweet potato, soybean, and red kidney bean. The sequence of GmPAP3 showed high homology to the PAP consensus sequence generated from the aligned known PAPs. High homology was observed throughout the entire polypeptide except at the N-terminus. All of the five conserved domains (in bold) and seven invariable residues (indicated by #) of the PAP metal binding nuclei were conserved in GmPAP3. The metal binding site of PAPs seems to resemble that of the metallophos. Six out of seven invariable residues in PAPs metal binding nuclei were also found in the metallophos metal binding consensus sequence.

106

H. Liao et al. / Gene 318 (2003) 103–111

2.3. Growth conditions for coupled effects of NaCl stress versus P starvation The coupled effects of salt stress versus P starvation were studied. To achieve severe P deficiency conditions, surfacesterilized seeds were germinated in silicon sand containing modified Hoagland’s solution with 0.2 AM (LP) or 1.0 mM KH2PO4 (HP) supplement. The nutrient solution was composed of: 4.5 mM KNO3, 3.6 mM Ca(NO3)2, 1.2 mM NH4NO3, 3.0 mM MgSO4, 1.2 mM (NH4)2SO4, 4.5 AM MnSO 4 , 4.5 AM ZnSO 4 , 1.5 AM CuSO 4 , 0.4 AM (NH4)6Mo7O24, 0.09 mM Fe-EDTA, and 1.5 AM H3BO3. The K content of LP medium was replenished with K2SO4. After germination, 1-week-old seedlings of uniform growth stage were transferred to a hydroponic system containing the same culture medium. After further growth for 12 days, 0.3%, 0.6%, and 0.9% (w/v) stepwise NaCl treatments were applied at 12-h intervals. The NaCl-treated samples were collected after 12 h treatment in 0.9% (w/v) NaCl. Four plant replicates were used for each treatment. The youngest fully expanded trifoliate leaves and roots of each treated plants were collected. Sample tissues were measured for soluble orthophosphate (Pi) content, total acid phosphatase activities and soluble Na+ content. A portion of the samples was pooled for total RNA extraction.

acetic acid buffer (pH 5.0). The extract was then centrifuged at 14,000  g for 20 min at 4 jC. A 10 Al (leaves) or a 50 Al (roots) aliquot of the supernatant was added to 490 Al (for leaves) and 450 Al (for roots) of grounding buffer, respectively. Total acid phosphatase activity was assayed using Unitrophenylphosphate (U-NPP) as the substrate (McLachlan

2.4. Growth conditions for oxidative stress Surface-sterilized seeds were germinated in silicon sand containing half strength of Hoagland’s solution. After germination, 10-day-old seedlings of uniform growth stage were transferred to a hydroponic system containing the same culture medium. After equilibrate for 24 days, 10 mM paraquat (PQ) solution was sprayed on both surfaces of trifoliate leaves. The PQ-treated leaves were collected 4 h after treatment. 2.5. Assays of soluble Pi, Na+ concentration, acid phosphatase activities and protein content For assays of soluble Pi and Na+ concentration, fresh leaf and root samples collected from the above-mentioned growth experiment were frozen in liquid nitrogen, ground in a pre-cooled mortar, and macerated in double distilled water. The extract was then centrifuged at 14,000  g for 20 min at room temperature. The soluble Pi concentration in plant tissues was determined spectrophotometrically using a classic method of P measurement (Murphy and Riley, 1963). The soluble Na+ concentration was analyzed with a Sequential Plasma Spectrometer (ATOMSCAN 16, Thermo Jarrell Ash, Australia). For measurement of acid phosphatase activities, about 0.2 g of fresh tissue was taken from the youngest fully expanded trifoliate leaves and roots of each treated plant. The samples were frozen in liquid nitrogen, ground in a cold mortar and macerated in 1.6 ml 0.45 mM sodium acetate –

Fig. 3. Phylogenetic analysis of PAPs. Phylogenetic relationship of PAPs, including GmPAP3 was analyzed using the ClustalW and MEGA (version 2.1) programs (Thompson et al., 1994; Kumar et al., 2001). A metallophosphatase from bluegreen algae (accession no. BAA16722) was used as an outgroup. The analyzed polypeptides were divided into four major groups, including: plant PAPs and PAP-like proteins (Group I), fungal PAPs (Group II), short PAPs in animals (Group III), and short PAPs in plants (Group IV). Bootstrap values were indicated for major branches as percentages. One thousand replicates were used for all possible tree topology calculation. Except GmPAP3, the first two letters of each protein label represent the abbreviated species name, followed by GenBank accession number. Ib: Ipomoea batatas; Gm: Glycine max; La: Lupinus albus; Ao: Anchusa officinalis; Sp: Spirodela punctata; At: Arabidopsis thaliana; Pv: Phaseolus vulgaris; Ll: Lupinus luteus; Af: Aspergillus ficuum; An: Aspergillus nidulans; Mm: Mus musculus; Rn: Rattus norvegicus; Hs: Homo sapiens; Ss: Sus scrofa; Sy: Synechocystis sp.

H. Liao et al. / Gene 318 (2003) 103–111

et al., 1987). To obtain the values of specific enzyme activity, total soluble protein content was determined by a commercial kit (Bio-Rad Protein Assay, Catalog no. 5000001) based on the Bradford method (Bradford, 1976).

3. Results 3.1. Identification of the salt-inducible GmPAP3 gene in soybean Suppression subtractive hybridization (SSH) techniques (see Materials and methods) were used to identify saltinducible genes from the soybean variety WF. The full-

107

length coding regions of selective fragments were obtained by 5Vand 3V-RACE. One of the candidate genes (GmPAP3; accession no. AY151271) was found to be strongly induced by NaCl stress in both leaves and roots of all the five cultivated and five wild soybean varieties tested (Fig. 1). DNA sequence analysis indicated that GmPAP3 was a PAPlike gene due to its high homology to well characterized plant PAPs. The putative GmPAP3 peptide shares 73%, 72% and 72% homology, respectively, to three sweet potato PAPs (accession nos. T51094, T51095 and AAF19822) (Durmus et al., 1999; Schenk et al., 1999). This protein also shares 70% homology to two red kidney bean PAPs (accession nos. CAA04644 and S51031) (Klabunde et al., 1994; Vogel et al., 2002) and 68% homology to a lupin PAP

Table 1 Predicted subcellular location of PAP and PAP-like proteins Groupa

Source

Accession no. (Protein i.d.)

No. of amino acid residues

N-terminal signalsb

Putative subcellular locationc

I

Ipomoea batatas Ipomoea batatas Ipomoea batatas Glycine max Lupinus albus Arabidopsis thaliana Anchusa officinalis Spirodela punctata Arabidopsis thaliana Phaseolus vulgaris Phaseolus vulgaris Ipomoea batatas Glycine max Lupinus luteus Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Glycine max Arabidopsis thaliana

AAF19822 T51095 T51094 AAF19820 BAA82130 AAD22297 AAD20634 BAA92365 AAD26885 CAA04644 S51031 AAF19821 AY151271 CAD30328 CAA18136 AAD31353 CAB89242 CAB89243 CAB89239 AAC04486 CAB36834 AAK49438d AAF20233

465 465 465 464 638 468 470 455 469 459 459 473 512 477 466 441 437 426 427 516 474 547 532

s.p. s.p. s.p. s.p. s.p. s.p. s.p. s.p. s.p. s.p. s.p. s.p. m.t. s.p. s.p. s.p. s.p. s.p. s.p. s.p. s.p. s.p. s.p.

outside outside outside outside ER/plasma membrane outside outside outside outside ER ER outside mitochondrion outside outside outside outside outside outside outside outside Plasma membrane outside

II

Aspergillus ficuum Aspergillus nidulans

AAA91632 CAB02076

614 618

s.p. s.p.

Outside/microbody outside

III

Mus musculus Rattus norvegicus Homo sapiens Sus scrofa Glycine max Arabidopsis thaliana Ipomoea batatas Phaseolus vulgaris

Q05117 P29288 P13686 P09889 AAF60316 AAF19823 AAF60315 AAF60317

327 327 323 340 332 314 312 331

s.p. s.p. s.p. s.p. s.p. s.p. s.p. s.p.

Arabidopsis thaliana

CAB63938

338

s.p.

ER ER outside ER outside outside outside Plasma membrane outside

IV

a

The PAPs and PAP-like proteins are separated into four major groups including: (I) plant PAPs and PAP-like proteins, (II) fungal PAPs, (III) short PAPs in animals and (IV) short PAPs in plants. b N-terminal signal was predicted by TargetP (Emanuelsson et al., 2000), and iPSORT (Bannai et al., 2002); s.p.: signal peptide, m.t.: mitochondrion targeting peptide. c Putative subcellular location was predicted by PSORT (Nakai, 2000); ER: endoplasmic reticulum. d A PAP-like protein from soybean carries phytase activities (Hegeman and Grabau, 2001).

108

H. Liao et al. / Gene 318 (2003) 103–111

(accession no. BAA82130) (Wasaki et al., 1999). Moreover, the invariable consensus metal binding residues (Schenk et al., 1999) were all conserved in GmPAP3 (Fig. 2). Further protein sequence analysis showed that GmPAP3 (like other PAPs) may belong to the metallophos (calcineurin-like phosphoesterase) family, since six out of the seven invariable metal-binding residues in PAPs also exist in the metallophos consensus sequence (Fig. 2). Since the homology of GmPAP3 is closer to the acid phosphatase from yellow lupin (Lupinus luteus L.) (accession no. CAD30328, 88% homology) than to the previously reported soybean PAPs (GmPAP1 and GmPAP2; accession no. AAF19820 and AAK49438, 71% and 44% homology, respectively), the phylogenetic relationship of the GmPAP3 polypeptide to other PAPs was analyzed using the ClustalW

Fig. 5. Northern blot analysis of GmPAP1, GmPAP2 and GmPAP3 under NaCl stress and P starvation. A portion of the samples collected for measurements in Fig. 4 were pooled and total RNA samples were extracted as described in Materials and methods. Ten micrograms of total RNA was loaded onto each lane. (A) RNA from the youngest fully expanded trifoliate leaves; (B) RNA from roots. + NaCl: with NaCl treatment.

Fig. 4. Establishment of P starvation and NaCl stress conditions. Surfacesterilized seeds were germinated in silicon sand containing modified Hoagland’s solution with 0.2 AM (LP) or 1.0 mM KH2PO4 (HP) supplement (see Materials and methods). After germination, 1-week-old seedlings of uniform growth stage were transferred to a hydroponic system containing the same culture medium. After further growth for 12 days, 0.3%, 0.6%, and 0.9% (w/v) stepwise NaCl treatments were applied at 12-h intervals. The NaCl-treated samples were collected after 12 h treatment in 0.9% (w/v) NaCl. Soluble Pi concentration (A), total acid phosphatase activities (B), and soluble Na+ concentration (C) were measured as described in Materials and methods. Open bar and black bar indicate results without or with NaCl treatments. [SPi]: soluble Pi concentration; APA: total acid phosphatase activity; [Na+]: soluble Na+ concentration. Error bars: standard errors.

(Thompson et al., 1994) and MEGA (version 2.1) program (Kumar et al., 2001) (Fig. 3). A metallo-phosphatase from bluegreen algae (accession no. BAA16722) was used as an outgroup. It was found that the PAPs and PAP-like proteins can be separated into four major groups including: (I) plant PAPs and PAP-like proteins, (II) fungal PAPs, (III) short PAPs in animals and (IV) short PAPs in plants (Fig. 3 and Table 1). From the phylogenetic tree data, the newly identified GmPAP3 belong to group I, along with a previously identified soybean PAP (GmPAP1; accession no. AAF19820). One remaining soybean PAP is a short PAP (GmPAP2; accession no. AAF60316) and the other soybean PAP (GmPhy; accession no. AAK49438) carries phytase activities (Schenk et al., 1999, 2000; Hegeman and Grabau, 2001). The putative protein sorting signals and subcellular locations of various PAP and PAP-like proteins were analyzed (Table 1). Most PAPs contain a signal peptide and are

H. Liao et al. / Gene 318 (2003) 103–111

expected to be secreted outside the cell via endoplasmic reticulum. However, GmPAP3 represents a novel PAP-like protein due to the presence of a putative mitochondrion targeting transit peptide. 3.2. Expression of GmPAP3 was induced by NaCl stress but not P deficiency To further characterize the function of GmPAP3 in response to salinity stress and P deficiency, we determined the stress conditions that may affect the expression of GmPAP3 using two genetically distinct soybean varieties: WF (from P.R.C.) and UN (from U.S.A.). Expression of three soybean PAP genes (GmPAP3, GmPAP1, and GmPAP2) in response to NaCl stress and P starvation was compared using Northern blot analysis. Establishment of P starvation conditions was verified by measurement of the total soluble Pi concentration (Fig. 4A) and assay of the total acid phosphatase activities (Fig. 4B). Compare to the plants treated under HP conditions, significantly lower soluble Pi concentration (Fig. 4A) and higher total phosphatase activities (Fig. 4B) were observed in both leaves and roots of plants treated under LP conditions. The uptakes of Na+ (Fig. 4C) and Cl (data not shown) into the plant tissues were also experimentally confirmed. Northern blot analysis using total RNA from leaves (Fig. 5A) and roots (Fig. 5B) clearly demonstrated that P starvation induced the expression of

Fig. 6. Northern blot analysis of GmPAP1, GmPAP2 and GmPAP3 under oxidative stress. Surface-sterilized seeds were germinated in silicon sand containing modified Hoagland’s solution as described in Materials and methods. After germination, 10-day-old seedlings of uniform growth stage were transferred to a hydroponic system containing the same culture medium. After equilibrate for 24 days, 10 mM paraquat (PQ) solution was sprayed on both surfaces of trifoliate leaves. The PQ-treated leaves were collected 4 h after treatment. Ten micrograms of total RNA was loaded onto each lane. + PQ and PQ indicate with and without PQ treatment, respectively.

109

GmPAP2 (in both leaves and roots) and GmPAP1 (in roots). However, no P starvation induced gene expression was observed in GmPAP3. On the other hand, NaCl effect on gene expression was most prominent in GmPAP3. A strong induction of GmPAP3 was observed in both leaves and roots. No significant NaCl inductions of GmPAP1 and GmPAP2 were observed. Indeed, a slight repression of GmPAP2 by NaCl was found in roots treated under LP conditions. 3.3. Expression of GmPAP3 was induced by oxidative stress Since GmPAP3 is predicted to be mitochondrial located based on the context of N-terminal amino acid residues, we further tested if GmPAP3 gene expression would also be induced by oxidative stress that is tightly related to the function of mitochondria under abiotic stresses. The herbicide paraquat (PQ) was previously shown to induce the production of activated oxygen species in treated plant (Tsang et al., 1991; Jiang and Zhang, 2002). Among the three soybean PAP genes tested, only GmPAP3 was induced by PQ treatments (Fig. 6). A slight repression was observed for GmPAP1 and no effects were found in GmPAP2 (Fig. 6).

4. Discussion In a search for salt-inducible genes in soybean using suppression subtractive hybridization techniques, we cloned a novel PAP-like gene. We named this gene GmPAP3 to distinguish it from the two other PAP-like genes and one PAP-like phytase gene reported previously in soybean (Schenk et al., 1999, 2000; Hegeman and Grabau, 2001). Two major characteristics of this PAP suggest that it is indeed a member of the PAP family: (1) it exhibits high homology to classical PAPs such as those from sweet potato (73%) (Durmus et al., 1999), red kidney bean (70%) (Klabunde et al., 1994; Vogel et al., 2002), and lupin (68%) (Wasaki et al., 1999); and (2) it possesses all metal binding amino acid residues (Fig. 2) that are conserved in all PAPs (Strater et al., 1995; Klabunde et al., 1996; Schenk et al., 1999). Nevertheless, there are many lines of evidence indicating that GmPAP3 is a novel gene that in many ways differs from the previously identified PAP-like genes in higher plants. First, unlike most PAPs that contain a signal peptide and are expected to be secreted outside the cell via endoplasmic reticulum, GmPAP3 represents a novel type of PAP-like protein that possesses a mitochondrial transit peptide and is predicted to be located in the mitochondria (Table 1). To the best of our knowledge, this is the first report suggesting a mitochondrion-located PAP in plants. The putative location of GmPAP3 in the mitochondria may imply a unique physiological role distinguishing from other plant PAPs.

110

H. Liao et al. / Gene 318 (2003) 103–111

Another unique feature of GmPAP3 is that it is strongly induced by NaCl stress but unresponsive to P deficiency. Our results showed that NaCl stress causes a general induction of GmPAP3 expression in both roots and leaves of various soybean varieties (Fig. 1). However, P starvation exhibits no induction effect on the expression of this gene (Fig. 5). The expression pattern of GmPAP3 is clearly distinguished from two other PAP genes found in soybean. It appears that GmPAP1 (in roots) and GmPAP2 (in leaves and roots) are induced under P starvation conditions to synthesize more PAP enzymes to cope with P deficiency. This was consistent with the previous finding that under P deficient conditions, PAP activity will be increased in plants to catalyze the hydrolytic breakdown of orthophosphate monoesters (Duff et al., 1994; Plaxton and Carswell, 1999). The lack of response of GmPAP3 to P deficiency is intriguing, for this may mean that this protein probably is involved in biochemical reactions other than catalyzing the hydrolysis of polyphosphates to cope with P deficiency. Purified GmPAP1 enzyme was able to utilize a range of phosphate ester and anhydride substrates, from activated substrates like U-nitrophenylphosphate (U-NPP) to unactiˆ vated substrate like h-glycerophosphate (Schenk et al., 1999). It is interesting to find that while GmPAP1 was induced by P starvation in roots, no strong induction was observed in the leaves (Fig. 5). A slight repression of GmPAP1 was also observed in the leaves under oxidative stress (Fig. 6). The complexity of gene regulation of GmPAP1 may be partly related to the multifunction of its gene product. In another study, a soybean phytase (GmPhy) with sequence similarity to PAPs was also shown to have enzymatic activity on a variety of substrates, suggesting that the major metabolic function of PAP-like proteins is to catalyze the hydrolysis of polyphosphates (Hegeman and Grabau, 2001). The NaCl inducibility and putative location in mitochondria of GmPAP3 may suggest that its physiological role could be related to the adaptation to NaCl stress. Mitochondria are the source of reactive oxygen species (ROS), particularly in non-photosynthetic tissues (Puntarulo et al., 1991; Bohnert et al., 1999; Dat et al., 2000). It was estimated that about 1% of the total O2 consumption in plant tissue goes to ROS production (Casolo et al., 2000). Under NaCl stress, reduction in CO2 fixation leads to higher leakage of electrons to O2 and results in the production of more ROS (Dat et al., 2000). Restoration of oxidative balance may represent a major way to cope with NaCl stress (Gueta-Dahan et al., 1997; Kennedy and De Filippis, 1999; Meneguzzo et al., 1999). A peroxidative role of a plant PAP from red kidney bean (KBPAP) was proposed based on the observation that the Fe(III) of KBPAP could be reduced to Fe(II) in the presence of ascorbic acid, which could reduce oxygen to water and thereby reduce the concentration of free radicals (Klabunde et al., 1995). The possible relationship between GmPAP3 and oxidative stress

was demonstrated by the induction of the GmPAP3 gene expression by oxidative stress (Fig. 6). This induction is unique to GmPAP3 among the three soybean PAP genes tested. Further works are needed to physically demonstrate the subcellular localization of GmPAP3 and delineate its possible function related to NaCl induced cellular stress responses.

Acknowledgements The authors thank the technical assistance by K.-W. Yim, S.-W. Tong and X. Wang. This research was supported by the Hong Kong Research Grant Council Earmarked Grant (CUHK-4180/99M to H.-M.L.), by HK AoE Funding on Plant and Fungal Biotechnology (to H.-M.L.), by the National Natural Science Foundation of China (39925025/ 30230220 to X.Y.), and by the National Key Basic Research Special Funds of China (G1999011700 to X.Y.).

References Bannai, H., Tamada, Y., Maruyama, O., Nakai, K., Miyano, S., 2002. Extensive feature detection of N-terminal protein sorting signals. Bioinformatics 18, 298 – 305. Beck, J.L., McConachie, L.A., Summors, A.C., Arnold, W.N., Dejersey, J., Zerner, B., 1986. Properties of a purple acid phospatase from red kidney bean: a zinc – iron metalloenzyme. Biochim. Biophys. Acta 869, 61 – 68. Bohnert, H.J., Su, H., Shen, B., 1999. Molecular mechanisms of salinity tolerance. In: Kazuo, S., Kazuko, Y.S. (Eds.), Molecular Responses to Cold, Drought, Heat and Salt Stress in Higher Plants. R.G. Landes, Austin, pp. 29 – 60. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248 – 254. Cashikar, A.G., Kumaresan, R., Rao, N.M., 1997. Biochemical characterization and subcellular localization of the red kidney bean purple acid phosphatase. Plant Physiol. 114, 907 – 915. Casolo, V., Braidot, E., Chiandussi, E., Macri, F., Vianello, A., 2000. The role of mild uncoupling and non-coupled respiration in the regulation of hydrogen peroxide generation by plant mitochondria. FEBS Lett. 474, 53 – 57. Dat, J., Vandenabeele, S., Vranova, E., Van Montagu, M., Inze, D., Van Breusegem, F., 2000. Dual action of the active oxygen species during plant stress responses. Cell. Mol. Life Sci. 57, 779 – 795. del Pozo, J.C., Allona, I., Rubio, V., Leyva, A., Pena, A.D.L., Aragoncillo, C., Paz-Ares, J., 1999. A type 5 acid phosphatase gene from Arabidopsis thaliana is induced by phosphate starvation and by some other types of phosphate mobilising/oxidative stress conditions. Plant J. 19, 579 – 589. Diatchenko, L., Chenchik, A., Siebert, P., 1998. Suppression subtractive hybridization: A method for generating subtracted cDNA libraries starting from poly (A+) or total RNA. In: Siebert, P., Larrick, J. (Eds.), RTPCR Method for Gene Cloning and Analysis. Eaton Publishing, Natick, MA, pp. 213 – 239. Duff, S.M.G., Sarath, G., Plaxton, W.C., 1994. The role of acid phosphatases in plant phosphorus metabolism. Physiol. Plant. 90, 791 – 800. Durmus, A., Eicken, C., Spener, F., Krebs, B., 1999. Cloning and comparative protein modeling of two purple acid phosphatase isozymes from sweet potatoes (Ipomoea batatas). Biochim. Biophys. Acta 1434, 202 – 209. Emanuelsson, O., Nielsen, H., Brunak, S., von Heijne, G., 2000. Predicting

H. Liao et al. / Gene 318 (2003) 103–111 subcellular localization of proteins based on their N-terminal amino acid sequence. J. Mol. Biol. 300, 1005 – 1016. Finckh, U., Lingenfelter, P.A., Myerson, D., 1991. Producing singlestranded DNA probes with the Taq DNA polymerase: a high yield protocol. Biotechniques 10, 35 – 38. Gueta-Dahan, Y., Yaniv, Z., Zilinskas, B.A., Ben-Hayyim, G., 1997. Salt and oxidative stress: similar and specific responses and their relation to salt tolerance in Citrus. Planta 203, 460 – 469. Hegeman, C.E., Grabau, E.A., 2001. A novel phytase with sequence similarity to purple acid phosphatases is expressed in cotyledons of germinating soybean seedlings. Plant Physiol. 126, 1598 – 1608. Hoagland, D.R., Arnon, D.I., 1938. The water-culture method for growing plants without soil. Calif. Agric. Expt. Circ. 347, 1 – 39. Igaue, I., Watabe, H., Takahashi, K., Takahashi, M., Morota, A., 1976. Violet-colored acid phosphatase isozymes associated with cell wall preparariom rice plant cultured cells. Agric. Biol. Chem. 40, 823 – 825. Jackson, A.O., Larkins, B.A., 1976. Influence of ionic strength, pH, and chelation of divalent metals on isolation of polyribosomes from tobacco leaves. Plant Physiol. 57, 5 – 10. Jiang, M., Zhang, J., 2002. Water stress-induced abscisic acid accumulation triggers the increased generation of reactive oxygen species and upregulates the activities of antioxidant enzymes in maize leaves. J. Exp. Bot. 53, 2401 – 2410. Kennedy, B.F., De Filippis, L.F., 1999. Physiological and oxidative response to NaCl of the salt tolerant Grevillea ilicifolia and the salt sensitive Grevillea arenaria. J. Plant Physiol. 155, 746 – 754. Klabunde, T., Stahl, B., Suerbaum, H., Hahner, S., Karas, M., Hillenkamp, F., Krebs, B., Witzel, H., 1994. The amino acid sequence of the red kidney bean Fe(III) – Zn(II) purple acid phosphatase. Determination of the amino acid sequence by a combination of matrix-assisted laser desorption/ionization mass spectrometry and automated Edman sequencing. Eur. J. Biochem. 226, 369 – 375. Klabunde, T., Strater, N., Krebs, B., Witzel, H., 1995. Structural relationship between the mammalian Fe(III) – Fe(II) and Fe(III) – Zn(II) plant purple acid phosphatases. FEBS Lett. 367, 56 – 60. Klabunde, T., Strater, N., Witzel, H., Frohlich, R., Krebs, B., 1996. Mechanism of Fe(III) – Zn(II) purple acid phosphatases based on crystal structures. J. Mol. Biol. 259, 737 – 748. Kumar, S., Tamura, K., Jakobsen, I.B., Nei, M., 2001. MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17, 1244 – 1245. LeBansky, B.R., Mcknight, T., Griffing, L.R., 1992. Purification and characterization of a secreted purple phosphatase from soybean suspension culture. Plant Physiol. 99, 391 – 395. Li, D., Zhu, H., Liu, K., Liu, X., Leggewie, G., Udvardi, M., Wang, D., 2002. Purple acid phosphatases of Arabidopsis thaliana. J. Biol. Chem. 277, 27772 – 27781. Majerus, P.W., 1992. Inositol phosphate biochemistry. Ann. Rev. Biochem. 61, 225 – 250. McLachlan, K.D., Elliott, D.E., De Marco, D.G., Garran, J.H., 1987. Leaf acid phosphatase isozymes in the diagnosis of phosphorus status in field-grown wheat. Aust. J. Agric. Res. 38, 1 – 13. Meneguzzo, S., Navari-Izzo, F., Izzo, R., 1999. Antioxidative responses of

111

shoots and roots of wheat to increasing NaCl concentrations. J. Plant Physiol. 155, 274 – 280. Murphy, J., Riley, J., 1963. A modified single solution for the determination of phosphate in natural waters. Anal. Chim. Acta 27, 31. Nakai, K., 2000. Protein sorting signals and prediction of subcellular localization. Adv. Protein Chem. 54, 277 – 344. Nakazato, H., Okamoto, T., Nishikoori, M., Washio, K., Morita, N., Haraguchi, K., Thompson Jr., G.A., Okuyama, H., 1998. The glycosylphosphatidylinositol-anchored phosphatase from Spirodela oligorrhiza is a purple acid phosphatase. Plant Physiol. 118, 1015 – 1020. Patel, K., Lockless, S., Thomas, B., McKnight, T.D., 1998. Secreted purple acid phosphatase from Arabidopsis thaliana. Plant Physiol. 119, 373 – 375. Plaxton, W.C., Carswell, M.C., 1999. Metabolic aspects of the phosphate starvation response in plants. In: Lerner, H.R. (Ed.), Plant Responses to Environmental Stresses from Photosynthesis to Genomes Reorganization. Marcel Dekker, New York, pp. 349 – 371. Puntarulo, S., Galleano, M., Sanchez, R.A., Boveris, A., 1991. Superoxide anion and hydrogen peroxide metabolism in soybean embryonic axes during germination. Biochim. Biophys. Acta. 1074, 277 – 283. Sambrook, J., Russell, D.W., 2001. Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, New York. Schenk, G., Ge, Y.B., Carrington, L.E., Wynne, C.J., Searle, I.R., Carroll, B.J., Hamilton, S., de Jersey, J., 1999. Binuclear metal centers in plant purple acid phosphatases: Fe – Mn in sweet potato and Fe – Zn in soybean. Arch. Biochem. Biophys. 370, 183 – 189. Schenk, G., Guddat, L.W., Ge, Y., Carrington, L.E., Hume, D.A., Hamilton, S., de Jersey, J., 2000. Identification of mammalian-like purple acid phosphatases in a wide range of plants. Gene 250, 117 – 125. Strater, N., Klabunde, T., Tucker, P., Witzel, H., Krebs, B., 1995. Crystal structure of a purple acid phosphatases containing a dinuclear Fe(III) – Zn(II) active site. Science 268, 1489 – 1492. Sugiura, Y., Kawabe, H., Tanaka, H., Fujimoto, S., Ohara, A., 1981. Purification, enzymatic properties, and active site environment of a novel manganese (III)-containing acid phosphatase. J. Biol. Chem. 256, 10664 – 10670. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673 – 4680. Tsang, E.W.T., Bowler, C., Herouart, D., Van Camp, W., Villarroel, R., Genetello, C., Van Montagu, M., Inze, D., 1991. Differential regulation of superoxide dismutases in plants exposed to environmental stress. Plant Cell 3, 783 – 792. Vogel, A., Borchers, T., Marcus, K., Meyer, H.E., Krebs, B., Spener, F., 2002. Heterologous expression and characterization of recombinant purple acid phosphatase from red kidney bean. Arch. Biochem. Biophys. 401, 164 – 172. Wasaki, J., Omura, M., Osaki, M., Ito, H., Matsui, H., Shinano, T., Tadano, T., 1999. Structure of a cDNA for an acid phosphatase from phosphate-deficient lupin (Lupinus albus L.) roots. Soil Sci. Plant Nutr. 45, 439 – 449.