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. 2007 Feb 1;99(3):439–450. doi: 10.1093/aob/mcl285

A Wheat (Triticum aestivum) Protein Phosphatase 2A Catalytic Subunit Gene Provides Enhanced Drought Tolerance in Tobacco

Chongyi Xu 1, Ruilian Jing 1,*, Xinguo Mao 1, Xiaoyun Jia 1, Xiaoping Chang 1
PMCID: PMC2802960  PMID: 17272305

Abstract

Background and Aims

Multiple copies of genes encoding the catalytic subunit (c) of protein phosphatase 2A (PP2A) are commonly found in plants. For some of these genes, expression is up-regulated under water stress. The aim of this study was to investigate expression and characterization of TaPP2Ac-1 from Triticum aestivum, and to evaluate the effects of TaPP2Ac-1 on Nicotiana benthamiana in response to water stress.

Methods TaPP2Ac-1

cDNA was isolated from wheat by in silico identification and RT-PCR amplification. Transcript levels of TaPP2Ac-1 were examined in wheat responding to water deficit. Copy numbers of TaPP2Ac-1 in wheat genomes and subcellular localization in onion epidermal cells were studied. Enzyme properties of the recombinant TaPP2Ac-1 protein were determined. In addition, studies were carried out in tobacco plants with pCAPE2-TaPP2Ac-1 under water-deficit conditions.

Key Results TaPP2Ac-1

cDNA was cloned from wheat. Transcript levels of TaPP2Ac-1 in wheat seedlings were up-regulated under drought condition. One copy for this TaPP2Ac-1 was present in each of the three wheat genomes. TaPP2Ac-1 fused with GFP was located in the nucleus and cytoplasm of onion epidermis cells. The recombinant TaPP2Ac-1 gene was over-expressed in Escherichia coli and encoded a functional serine/threonine phosphatase. Transgenic tobacco plants over-expressing TaPP2Ac-1 exhibited stronger drought tolerance than non-transgenic tobacco plants.

Conclusions

Tobacco plants with pCAPE2-TaPP2Ac-1 appeared to be resistant to water deficit, as shown by their higher capacity to maintain leaf relative water content, leaf cell-membrane stability index, water-retention ability and water use efficiency under water stress. The results suggest that the physiological role of TaPP2Ac-1 is related to drought stress response, possibly through its involvement in drought-responding signal transduction pathways.

Key words: Triticum aestivum, protein phosphatase, TaPP2Ac-1, Nicotiana benthamiana, gene expression, drought tolerance, physiological responses

INTRODUCTION

Drought stress is one of several environmental factors greatly limiting crop production and plant distribution worldwide. Previous work has shown that drought stress can lead to water deficit in plant cells, inhibiting plant growth and development. However, plant responses to water deficit are complex involving the co-ordination and integration of multiple biochemical pathways leading to the expression of a number of genes encoding proteins which contribute to drought adaptation. Genes whose expression is increased during water deficit include those encoding the key enzyme of ABA biosynthesis (Bray, 1997), proteins involved in osmotic adaptation and tolerance of cellular dehydration (Shinozaki et al., 1997), cellular protective enzymes (Ingram and Bartels, 1996), and a range of signalling proteins such as protein kinases/protein phosphatases (Hong et al., 1997) and transcription factors (Soderman et al., 1996).

Reversible protein phosphorylation is central to the perception and response to water deficit (Hirt, 1997), and constitutes a major mechanism for the control of cellular functions, such as responses to hormonal, pathogenic, environmental stimuli and control of metabolism (Cohen, 1988). Structure, expression and functions of protein kinases have been emphasized in initial studies, but recent studies have been focusing on protein phosphatases.

Protein phosphatases in eukaryotes can be divided into two main groups based on their enzymatic specificity: Ser/Thr and Tyr phosphatases, respectively (Villafranca et al., 1996). Four major Ser/Thr-specific phosphatase activities (PP1, PP2A, PP2B and PP2C) have been detected and classified according to their subunit compositions, substrate selectivity, inhibitor sensitivity and absolute requirement for bivalent cations (Millward et al., 1999). Many Ser/Thr phosphatases (PP1, PP2A, PP2C and PP5) have been detected in plants (Stubbs et al., 2001). PP2A holoenzyme consists of a constant dimeric core, a catalytic subunit (PP2Ac) and a structural subunit (PP2Aa) associated with one member of the variable b subunit (PP2Ab) family. PP2Ac is the enzymatically active component.

Genes encoding PP2A subunits have been identified and characterized in several plant species (Terol et al., 2002). In Arabidopsis thaliana, at least five genes encoding the catalytic PP2Ac subunit, three genes encoding the scaffolding a subunit, two genes encoding the 55-kDa b regulatory subunit, nine genes encoding the b′-type regulatory subunit and five genes encoding the 72/130-kDa b′′ subunit have been identified (Smith and Walker, 1996; Haynes et al., 1999; Camilleri et al., 2002). The RCN1 and TON2 genes encoding the PP2Aa and PP2Ab′′ subunits are involved in the regulation of auxin/ABA/ethylene signalling and cytoskeleton dynamics, respectively (Larsen and Cancel, 2003). The RCN1 functions as a general positive transducer of early ABA signalling (Kwak et al., 2002). Although these 16 PP2A-related genes are expressed ubiquitously in arabidopsis, they appear to function differently (Garbers et al., 1996). Five PP2Ac isogenes have been isolated from Oryza sativa. Both OsPP2A-1 and OsPP2A-3 are ubiquitously expressed in stems, flowers and leaves. OsPP2A-1, but not OsPP2A-3, is also highly expressed in roots. Drought and high salinity up-regulated both genes in leaves, whereas heat stress repressed OsPP2A-1 in stems and induced OsPP2A-3 in all organs, indicating that the two PP2Ac genes are subject to developmental and stress-related regulation (Yu et al., 2003). Two genes of Solanum lycopersicon (formerly Lycopersicon esculentum) that encode catalytic subunits of PP2A have been isolated; functional analysis of LePP2Ac1 indicated it as a negative regulator of plant defence responses (He et al., 2004). In Medicago sativa, the pp2aMs transcript is present in leaves, stems, roots and bud flowers, but the highest mRNA level is found in stems (Pirck et al., 1993).

To further elucidate physiological functions of PP2A genes in plants responding to water deficit, the catalytic subunit of the PP2A gene, TaPP2Ac-1, was isolated and characterized from the drought-tolerant wheat cultivar ‘Hanxuan10’, which emerged from a screen of approx. 10 000 wheat germplasms. In this study, the response of tobacco plants over-expressing TaPP2Ac-1 to drought-stress conditions was examined.

MATERIALS AND METHODS

Plant materials, growth conditions and treatments

Seeds of hexaploid wheat (Triticum aestivum, AABBDD) cultivar ‘Hanxuan10’ were germinated and grown in growth chambers at 20 °C with a 12-h light/dark cycle. Seedlings at the two-leaf stage (9 d old) were stressed by the addition of 16·1 % PEG-6000 ( − 0·5 MPa) to the hydroponic solution, which, in pilot experiments, had been shown to constitute significant stress at this developmental stage. Leaves were harvested for RNA isolation at 0, 1, 3, 6, 12, 24, 48 and 72 h after the treatment, frozen quickly with liquid nitrogen and stored at − 70 °C.

Five accessions of wheat and its relatives were used in Southern hybridizations: ‘Hanxuan10’, tetraploid wheat (Triticum turgidum var. dicoccum, AABB) accession no. CN11646 and three diploid accessions, Triticum monococcum ssp. urartu (AA) accession no. 1010004, Aegilops speltoides (SS, closely related to the BB genome) accession no. IcAG 400046 and Aegilops tauschii (DD) accession no. PH1878. For DNA isolation, leaves from pot-grown plants were used.

Tobacco (Nicotiana benthamiana) seeds were grown in pots in vermiculite, illuminated at 2500 lux and 25 °C with a 12-h light/dark cycle, and maintained at 80 % relative humidity.

Cloning of full-length TaPP2Ac-1 cDNA

Total RNA was extracted from the leaf samples, which were treated by adding 16·1 % PEG-6000 ( − 0·5 MPa) to the hydroponic solution for 12 h, using TRIZOL reagent (Tianwei, China), according to the manufacturer's instructions. Based on the candidate EST of a TaPP2Ac-1 from cDNA libraries, the putative full-length TaPP2Ac-1 cDNA was obtained using GenBank's nonredundant EST database. A segment, 1607 bp in length, was obtained, which included a 942-bp open reading frame. According to the full-length cDNA sequence, a pair of primers were synthesized for TaPP2Ac-1 (F: 5′-TGC CTT TTC CCA CGG TCG-3′, R: 5′-GGC TCT GGT ACA CCC CTT-3′), which amplified a 1110-bp sequence in length. Total RNA (1 mg) was used for reverse transcription. PCR amplifications were performed with 1 µL of 10-fold dilution of first-strand cDNA. Purified PCR fragments were sub-cloned into the pGEM-Teasy Vector system (Tianwei) according to the manufacturer's recommendations. Positive plasmids were confirmed by DNA sequencing using ABI3730 (PE). The TaPP2Ac-1 nucleotide sequence data reported in this article has been deposited in the GenBank database under the accession number EF101900.

Northern blotting

Total RNAs (20 µg) of the leaf samples were electrophoresed in 1·5 % formaldehyde–agarose gel and transferred to nylon membranes (Hybond N + , Amersham Biosciences). The full-length cDNA sequence of TaPP2Ac-1 labelled with α_32P-dCTP was used as the probe. The blotted membrane was hybridized overnight at 65 °C in buffer [5 × SSC, 0·1 % (w/v) N-lauroyl sarcosinate, 0·02 % (w/v) SDS, 2 % (w/v) blocking reagent and 50 % (v/v) formamide] with the labelled probe. Following hybridization, the membrane was washed twice (2 × SSC, 0·1 % SDS) at 65 °C for 30 min and twice (0·5 × SSC, 0·1 % SDS) at 65 °C for 15 min. Blots were exposed on a phosphor screen (Kodak-K) for 2 d at room temperature, and the signals were captured using the Molecular Imager FX System (Bio-Rad).

Southern blotting

Genomic DNA (15 µg) was digested with three restriction enzymes EcoRV, HindIII and NcoI. The digested fragments were fractioned by electrophoresis in 0·8 % agarose gel at 1·5 V cm−1 for 8–10 h and then transferred to a nylon membrane. The blotted membrane was hybridized by the standard procedure with full-length cDNA of TaPP2Ac-1 as the probe. Membrane washing and autoradiography were performed as in northern blotting.

Subcellular localization

The TaPP2Ac-1 gene was fused to a pBIN 35S-m-GFP expression vector downstream of the constitutive CaMV 35S promoter and upstream of GFP to create a TaPP2Ac-1-GFP fusion protein. The coding sequence including the HindIII (5′) and SalI (3′) restriction sites was amplified using the primers 5′-TAC CAA GCT TTG CCT TTT CCC ACG GTC G-3′ and 5′-TAT CGT CGA CAA GGA AAT AAT CAG GTG T-3′. The PCR product and the pBIN 35S-m-GFP plasmid were restricted with HindIII and SalI and ligated together by T4 ligase. The recombinant pBIN 35S-m-TaPP2Ac-1-GFP plasmid was transferred into living onion epidermal cells using a gene gun (HeliosTM) according to the instruction manual. The transformed cells were observed by confocal microscopy (Olympus FV500) after incubation on Murashige and Skoog medium at 25 °C for 24–48 h. The recombinant pBIN 35S-m-TaPP2Ac-1-GFP plasmid and the control pBIN 35S-m-GFP plasmid were bombarded into 30 onion epidermal segments, respectively.

Over-expression and purification of the recombinant TaPP2Ac-1 protein in Escherichia coli

The TaPP2Ac-1 coding sequence including BamHI (5′) and SacI (3′) restriction sites was amplified using the primers 5′-TCG GAT CCG ATG GAG CCC ATG AGC GTG GA-3′ and 5′-TGT CGA CAA GGA AAT AAT CAG GTG TTC TCC-3′. The PCR product and the plasmid pET-22b ( + ) were restricted with BamHI and SacI and ligated together by T4 ligase. The recombinant plasmid was transformed into E. coli BL21 (DE3) (Novagen) for over-expression of the TaPP2Ac-1-His fusion protein. After inducing expression with 0·3 mm isopropyl b-d-thiogalactopyranoside (IPTG), the cells from two 1-L cultures were harvested by centrifugation at 4000 g and 4 °C for 25 min, resuspended in 100 mL Tris–HCl (25 mm, pH 7·4) at the temperature of ice-cold, and then lysed by sonication. The lysate was centrifuged at 20 000 g and 4 °C for 20 min, the supernatant was collected for purification by Chelating Sepharose Fast Flow (Amersham), according to the manufacturer's instructions. The recombinant purified fusion protein was concentrated by Amicon Ultra-15 Centrifugal Filter Units (Millipore). Protein content was measured using the method of Bradford (1976). The purified protein was stored at − 20 °C.

Serine/threonine phosphatase activity assay of the recombinant TaPP2Ac-1 protein

The purified recombinant TaPP2Ac-1 fusion protein was assayed for phosphatase activity by Ser/Thr protein phosphatase assay system (Promega) according to the manufacturer's instructions. Chemically synthesized phosphopeptide RRA(pT)VA was used as a substrate. For the time-course analysis of TaPP2Ac-1 activity, reactions were initiated by adding 100 ng of enzyme protein and 5 µL of 1 mm RRA(pT)VA to the 50-μL reaction mixture. After incubating the reaction mixture at 30 °C for various periods of time, the reaction was terminated by adding 50 µL of molybdate dye/additive mixture and monitored by absorbance at 600 nm (A600) at various time points. To analyse the phosphatase activity at various enzyme concentrations, 100, 200, 300, 400, 500, 600 and 700 ng of TaPP2Ac-1 protein were included in the 50 µL assay buffer, and the reaction mixtures were incubated for 10 min. Blank incubations were performed without the TaPP2Ac-1 fusion protein. One unit of protein phosphatase activity released 1 pmol phosphate min−1 from phosphopeptide at 30 °C.

Construction of plasmid pCAPE2-TaPP2Ac-1 and Agrobacterium-mediated transformation of tobacco plants

pCAPE1 and pCAPE2-GFP, which were derived from the binary vector PEBV (the tobravirus pea early browning virus), were kindly donated by Dr Daowen Wang (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences). The TaPP2Ac-1 coding sequence including SacI (5′) and SalI (3′) restriction sites was amplified using the primers 5′-TCC GAG CTC ATG GAG CTA TTG AGC GTG-3′ and 5′-TAT CGT CGA CTC AGT GAT GGT GAT GGT GAT GAA GGA AAT AAT CAG GT-3′. In the reverse primer, the nucleotide sequences of six histidines were appended before the stop codon (TGA), allowing validation by commercial his-tag monoclonal antibody if the exogenous TaPP2Ac-1 gene was expressed in the host N. benthamiana. The PCR product and the pCAPE2-GFP plasmid were cleaved with SacI and SalI and ligated together. GFP in the pCAPE2-GFP plasmid was replaced by the full-length TaPP2Ac-1 cDNA and the recombinant pCAPE2-TaPP2Ac-1 vector was constructed. The binary vectors derived from PEBV were introduced into Agrobacterium tumefaciens strain GV3101 by electroporation (Gene Pulser II, Bio-Rad, Herlev, Denmark) as described by Shen and Forde (1989). Individual clones were cultured in 30 mL of Luria-Broth liquid medium supplemented with 100 µg mL−1 rifampicin, 50 µg mL−1 kanamycin, 0·1 m 2-morpholinoethanesulfonic acid and 0·2 mm acetosyringone at 28 °C for 16–18 h with shaking. At OD600≈2·0 the bacteria were harvested by centrifugation (3500 g, 15 min, room temperature). Cells were resuspended in about 30 mL of infiltration medium (Luria-Broth liquid medium) (0·2 m MgCl2, 0·1 m 2-morpholinoethanesulfonic acid and 2 mm acetosyringone), and incubated at room temperature for 3 h without shaking. Agrobacterium cultures harbouring a pCAPE1 and a pCAPE2 derivative were mixed 1 : 1 prior to infiltration. The mixture was infiltrated to the abaxial side of the basal pair of 4-week-old leaves of tobacco using a 5-mL syringe for studying its function for a 40-d period during which the plants were subjected to increasing water deficit.

Analysis of TaPP2Ac-1 expression

Total DNA was isolated from leaf tissues of wild-type (WT) plants and positive transformant tobacco plants carrying pCAPE2-TaPP2Ac-1 by the CTAB method (Reichardt and Rogers, 1994) and 100 ng of DNA was amplified using the primers 5′-TTG CCT TTT CCC ACG GTC G-3′ and 5′-ATG AAG GAA ATA ATC AGG T-3′ for the full-length cDNA of TaPP2Ac-1.

Total RNA was isolated from leaves of WT plants and tobacco plants carrying pCAPE2-TaPP2Ac-1. For RT-PCR analysis, first-strand cDNA was synthesized from 500 ng of total RNA. PCR products of the constitutively expressed tubulin gene were used as a quantitative control. The PCR procedure was as follows: 94 °C for 2 min; 28 cycles (TaPP2Ac-1) or 20 cycles (tubulin) of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 45 s; 72 °C for 5 min. The PCR primers for the full-length cDNA of TaPP2Ac-1 were as previously described for the detection of DNA level, and tubulin was amplified using the primers 5′-AGA ACA CTG TTG TAA GGC TCA AC-3′ and 5′-GAG CTT TAC TGC CTC GAA CAT GG-3′. Tubulin and TaPP2Ac-1 were amplified separately in equivalent experiments. Six microlitres of PCR product of each reaction was fractioned in an agarose gel.

Protein isolation and western blotting of leaves of tobacco

Total protein was extracted from approx. 0·1 g tobacco leaf tissues using 100 µL buffer containing HEPES–NaOH (pH 7·5), 5 mm EDTA, 5 mm EGTA, 10 mm Na3VO4, 10 mm NaF, 5 % glycerol, 10 mm DTT, 1 mm phenylmethylsulfonyluoride, 10 µg mL−1 leupeptin, 10 µg mL−1 aprotitin and 10 µg mL−1 antipain. To attain enough protein, the homogenate was placed on ice for 1–2 h and centrifuged at 14 000 g for 1 h at 4 °C. The supernatant was immediately frozen in liquid nitrogen and further fractionated by centrifugation at 14 000 g for 40 min at 4 °C. The resulting supernatant was sampled for proteins. Proteins were quantified according to the Bradford method (Bradford, 1976). Protein samples were electrophoretically separated on 12·5 % polyacrylamide gels according to Laemmli (1970) and subsequently transferred to nitrocellulose membranes (pore size: 0·45 µm) using TBST (2 mm Tris, 192 mm glycine, 20 % methanol complemented with 0·1 % SDS) as transfer buffer. The membrane was detected with Ponceau, blocked with 5 % skim milk and blotted with commercial his-tag monoclonal antibody diluted in TBS complemented with 5 % skim milk and 0·5 % Tween 20. After extensive washing procedures, the bound primary antibody was detected with horseradish peroxidase-conjugated goat antimouse IgG secondary antibody using the ECL technique according to the manufacturer's recommended procedures (Amersham).

Drought stress of tobacco plants carrying pCAPE2-TaPP2Ac-1

Wild tobacco plants and tobacco plants with Agrobacterium mixtures carrying the pCAPE1 and pCAPE2-derived plasmids were cultured under normal conditions for 2 weeks before exposure to drought stress. When GFP fluorescence was observed in tobacco roots, drought stress was imposed by withholding water in a growth chamber (20 °C, 50–60 % relative humidity, continuously illuminated at 2500 lux) until a lethal effect of dehydration was observed on most of the control plants.

Physiological responses to water stress in tobacco plants with pCAPE2-TaPP2Ac-1

Leaf relative water content (RWC) was estimated according to the method of Turner (1981): RWC (%) = (fresh weight − dry weight)/(turgid weight − dry weight) × 100. Leaf cell membrane stability index (MSI) was determined according to the method of Sairam (1994), with some modification. MSI (%) = (1 − electrical conductivity before incubating/electrical conductivity after incubating) × 100. Water retention ability (WRA) was measured according to the following method: leaves of uniform position were taken and weighed (fresh weight), desiccated for 24 h under controlled conditions (65 % relative humidity and 25 °C), and weighed again. After 24 h, leaves were dried at 90 °C for 8 h and their dry weights were recorded. WRA (%) = (desiccated weight − dry weight)/(fresh weight − dry weight) × 100. Water use efficiency (WUE (%) = rate of net photosynthesis/rate of transpiration × 100) was measured by the LI-6400 photosynthesis system according to the manufacturer's instructions. Rate of transpiration and net photosynthesis of leaves of uniform position were recorded. The average of every physiological index was determined from ten independent plants from each sample group of three replicates.

RESULTS

Molecular characterization of TaPP2Ac-1

The reconstituted cDNA of TaPP2Ac-1 was 1110 bp in full length, including a 942-bp open reading frame, a 46-bp 5′-untranslated region and a 122-bp 3′-untranslated region. The open reading frame encodes a peptide of 314 amino acid residues with a predicted molecular mass of 36 kDa and a predicted pI value of 5·03. The deduced amino acid sequence of TaPP2Ac-1 shows high homology with O. sativa, A. thaliana and N. tabacum serine/threonine protein phosphatase (PP2A) catalytic subunits (Fig. 1). TaPP2Ac-1 has 95 % identity and 91 % similarity to O. sativa PP2A-4 (Q9SBW3) and PP2A-2 (Q9XF94), 92 % identity and 89 % similarity to A. thaliana PP2A-4 (P48578) and PP2A-3 (Q07100), and 90 % identity and 89 % similarity to N. tabacum PP2A (Q9XGH7) and PP2A-5 (Q04860). Conserved motifs, which may be involved in substrate binding or catalysis, were found by sequence alignment and the PROSITE motif search program among the O. sativa, A. thaliana and N. tabacum serine/threonine protein phosphatase PP2A catalytic subunits. The serine/threonine-specific protein phosphatase signature LRGNHE was identified at residues 118–123 of TaPP2Ac-1 (region 1); tyrosine kinase phosphorylation sites KCPDTNY and KIFTDLFDY were displayed in residues 78–84 and 148–156 (region 2); protein kinase C phosphorylation sites SVR, SPR and TRR were shown by sequences from residues 183–185, 216–218 and 305–307 (region 3); casein kinase II phosphorylation sites SDPD and SILE were also conserved in the sequences from residues 205–208 and 278–281 (region 4); the YRCG motif involved in okadaic acid binding appeared in residues 274–277 (region 5); and DYFL residues regulating PP2A activity occurred in the C-terminus (region 6).

Fig. 1.

Fig. 1.

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Primary structure analysis of TaPP2Ac-1 cDNA: alignment of TaPP2Ac-1 and PP2Ac homologues in plants. The conserved prosite motifs common to nearly all the PP2Acs are underlined. Regions 1–6 represent a serine/threonine specific protein phosphatase signature, a tyrosine kinase phosphorylation site, a protein kinase C phosphorylation site, a casein kinase II phosphorylation site, an okadaic acid binding site and a PP2A activity regulating site, respectively. Alignments were performed using the Megalign program of DNAStar. OsPP2A-4 and OsPP2A-2 (GenBank accessions Q9SBW3 and Q9XF94), AtPP2A-3 and AtPP2A-4 (Q07100 and P48578), NtPP2A and NtPP2A-5 (Q9XGH7 and Q04860). Abbreviations on the left side of each sequence: Os, Oryza sativa; At, Arabidopsis thaliana; Nt, Nicotiana tabacum.

The sequence was aligned by the PROF program (http://www.embl-heidelberg.de/predictprotein/predictprotein.html). The secondary structure of TaPP2Ac-1 indicated that the sequence formed 10 α-helixes and 13 β-pleated sheets. The tertiary structure was predicted using Swiss-model (http://www.expasy.org/swissmod/SWISS-MODEL.html), resulting in a model similar to the 3D structure of human PP2Ac (Evans et al., 1999).

The phylogenetic relationship of the putative amino acid sequences of TaPP2Ac-1 and other plant PP2Acs was analysed using ClustalW (Thompson et al., 1994) and Phylip (Fig. 2). The plant PP2Acs can be separated into three major groups. The five arabidopsis PP2Ac genes fell into groups II and III. The newly identified TaPP2Ac-1 belonged to group II, and is related most closely to rice OsPP2A-4. It has been reported that this rice gene responded to drought and salinity stresses (Yu et al., 2005).

Fig. 2.

Fig. 2.

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Phylogenetic tree of PP2Acs. Alignment of phylogenetic relationships among PP2Ac from various plant species, including wheat, rice, arabidopsis, tobacco, rape, Medicago, sunflower, Hevea, Acetabularia and Eremothecium. The amino acid sequences analysed were divided into three major groups. The numbers on the branches indicate the numbers of times the partition of the species into the two sets were separated by that branch occurred among the trees, out of 100 00 trees. Database accession numbers are indicated for each protein. TaPP2Ac-1 is circled. Abbreviations: Eg, Eremothecium gossypii; Ap, Acetabularia peniculus; Ms, Medicago sativa; Bn, Brassica napus; Ha, Helianthus annuus; Hb, Hevea brasiliensis, others are as in Fig 1.

Expression pattern of TaPP2Ac-1 in wheat seedlings subjected to drought stress

The TaPP2Ac-1 EST was originally isolated from a cDNA library using wheat seedlings treated for 12 h in PEG-6000 ( − 0·5 MPa), and the full-length cDNA was obtained from total RNA isolated from the same sample. The expression pattern of TaPP2Ac-1 in wheat seedlings is shown in Fig. 3. TaPP2Ac-1 transcripts accumulated slightly after 1 h of water deficit, dramatically increased at 3 h, were maintained at this higher level at 6 h and 12 h, and gradually declined from 24 h on, but the levels remained higher than the basal level (CK). The pattern indicated that the expression of TaPP2Ac-1 was differentially regulated at different times after the initiation of water stress.

Fig. 3.

Fig. 3.

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Expression pattern of TaPP2Ac-1 mRNAs with time in response to water stress (CK = basal level). Total RNAs were extracted from wheat seedling leaves at various intervals after treatment with 16·1 % PEG-6000. Twenty micrograms of total RNA was loaded into 1·5 % formaldehyde-agarose gel, and then blotted onto a nylon membrane. Hybridization was carried out with the 32P-labelled TaPP2Ac-1 cDNA probe.

Determination of the gene copy numbers

To determine the copy numbers of TaPP2Ac-1 in common wheat and related tetraploid and diploid species, genomic Southern hybridization analyses were carried out. The genomic DNAs from the five species were digested with three restriction enzymes EcoRV, HindIII and NcoI, and hybridized with the full-length cDNA of TaPP2Ac-1 derived from the PCR product of plasmid pGEM-Teasy-TaPP2Ac-1. The probe had no restriction sites for EcoRV, HindIII and NcoI. As shown in Fig. 4, three bands in hexaploid wheat, two bands in tetraploid wheat and one band in diploids. The appearance of three bands in the S genome (HindIII-digested) not withstanding, most likely caused by a site in an intron, one copy of TaPP2Ac-1 is present in each of the three genomes of common wheat.

Fig. 4.

Fig. 4.

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Southern blotting analysis of genomic DNA from common wheat and related tetraploid and diploid species digested with three restriction enzymes. Fragments were separated in a 0·8 % agarose gel, blotted onto nylon membrane and hybridized with an [α-32P]-labelled probe generated from the full-length cDNA of TaPP2Ac-1. ABD, Common wheat; AB, Tetraploid wheat; A, T. urartu; S, Ae. speltoides; D, Ae. tauschii.

Subcellular localization of the TaPP2Ac-1 protein

To test the cellular localization of TaPP2Ac-1, the TaPP2Ac-1 cDNA was ligated into the upstream of the GFP coding region of pBIN 35S-m-GFP expression vector driven by the CaMV 35S promoter and expressed as a GFP fused protein. Twenty-four hours after transfer into onion epidermal segments by gene gun, fluorescence based on GFP expression was observed by confocal laser-scanning microscopy throughout the cytoplasm and in nuclei. Thirty segments were analysed with identical results (Fig. 5). Evidently, TaPP2Ac-1 was distributed in the nuclear and cytoplasmic compartments.

Fig. 5.

Fig. 5.

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Subcellular localization of TaPP2Ac-1 fused with GFP. GFP and the TaPP2Ac-1-GFP fusion protein were expressed transiently in onion epidermis cells of 24 h after bombardment using a gene gun and visualized with a laser-scanning confocal microscope. (A and B) Images of the pBIN 35S-m-GFP expression vector and pBIN 35S-m-TaPP2Ac-1-GFP expression vector in onion epidermal cells, respectively. a and d, Bright-field images; b and e, images of green fluorescence of GFP in cells under the confocal microscope; c and f, overlaid images of (a) and (b) and (d) and (e). Scale bar = 50 µm. Each construct was bombarded into at least 30 onion epidermal cells.

The TaPP2Ac-1 gene encodes a functional serine/threonine phosphatase

The regulation of expression of TaPP2Ac-1 by water deficit (Fig. 3) suggested its involvement in plant responses to environmental stresses. To determine whether the TaPP2Ac-1 cDNA coded for an enzymatically active, functional catalytic subunit of PP2A, recombinant TaPP2Ac-1 protein was produced and its enzyme properties characterized.

The coding region of TaPP2Ac-1 was cloned into the pET-22b ( + ) vector and expressed as a His-tagged recombinant protein in E. coli. As shown in Fig. 6A, IPTG induction resulted in the over-expression of a TaPP2Ac-1–his fusion protein (lane 2). The fusion protein was purified by Chelating Sepharose Fast Flow and yielded the approx. 36-kDa TaPP2Ac-1 protein (lane 3). After purification, the protein was suitable for biochemical characterization.

Fig. 6.

Fig. 6.

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Expression, purification and enzyme assay of the recombinant TaPP2Ac-1 protein in E. coli. (A) Total protein was isolated from bacterial cells before (lane 1) and after (lane 2) IPTG induction. The purified recombinant TaPP2Ac-1 protein appears as a single protein band (lane 3). Proteins were fractioned by SDS–PAGE and visualized by addition of Coomassie Brilliant Blue R 250 staining (Sambrook et al., 1989). Lane M, low range SDS–PAGE molecular weight standards. The molecular masses of proteins are shown on the left. (B) Time course of TaPP2Ac-1 phosphatase activity. Using RRA(pT)VA as a substrate, TaPP2Ac-1 activity was measured as absorption at 600 nm (A600), indicating the production of free phosphate. The concentration of the TaPP2Ac-1 protein was 20 ng µL−1. (C) Phosphatase activity as a function of enzyme concentration. All experiments were repeated three times, and the data from one representative assay are shown here.

Phosphatase assays, using a RRA(pT)VA peptide as a substrate, were carried out by measuring the release of free phosphate and the appearance of phospho-molybdate adducts by absorbance at 600 nm (A600). The expression and isolation of the TaPP2Ac-1 protein were found to be reproducible in three preparations from cells isolated from 2 L of culture. The phosphatase activity was consistently approx. 70 units µg−1. The TaPP2Ac-1 protein hydrolysed RRA(pT)VA rapidly, and the activity was linear during the first 15 min of the reaction (Fig. 6B). As a function of enzyme concentration, RRA(pT)VA hydrolysis increased proportionally (Fig. 6C), indicating that TaPP2Ac-1 is a highly active serine/threonine phosphatase.

Generation of transgenic tobacco plant

To test involvement of the target gene in response to water deficit, a virus-induced gene over-expression system, PEBV, was assembled. PEBV is a bipartite, rod-shaped virus with an RNA genome consisting of RNA1 and RNA2. RNA1 expression cassettes encode all viral proteins required for replication and movement and can produce infection without RNA2. An intron was inserted into RNA1 in the open reading frame encoding the RNA-dependent RNA polymerase. This generated infectious clones of the RNA virus in E. coli (Johansen, 1996). RNA2 expression cassettes encode the coat protein and GFP under control of the TRV coat protein promoter. The RNA1 and RNA2 expression cassettes were transferred to a binary Agrobacterium vector pCAMBIA1300 (KmR) and cloned under control of the 35S promoter and the NOS terminator. The pCAMBIA1300-derived constructs with the RNA1 cassette, containing an intron, and RNA2-GFP cassette, were named pCAPE1 and pCAPE2-GFP, respectively. A construct with the full-length cDNA of TaPP2Ac-1, which replaced the GFP cassette in pCAPE2-GFP, was generated and named pCAPE2-TaPP2Ac-1 (Fig. 7).

Fig. 7.

Fig. 7.

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PEBV binary vectors. PEBV expression cassettes of RNA1 and RNA2 were inserted between the right and left borders (RB and LB) of a pCAMBIA1300-derived plasmid. Transcriptional control was exerted by a 35S promoter and a NOS terminator (T). (A) The pCAPE1 containing full-length cDNA of PEBV RNA1 with an intron inserted to stabilize the plasmid in bacteria. (B) The pCAPE2-GFP containing full-length cDNA of PEBV RNA2 with the GFP coding sequence. CP, Coding region of PEBV coat protein. (C) The pCAPE2-TaPP2Ac-1 with a 942-bp full-length TaPP2Ac-1 cDNA of wheat inserted in the RNA2 cDNA.

Agrobacterium cultures carrying pCAPE1 and the pCAPE2-derived constructs (pCAPE2-GFP and pCAPE2-TaPP2Ac-1) at a ratio of 1 : 1 were used for inoculating tobacco plants. Each construct was inoculated into at least 20 plants in each of three independent experiments. Non-inoculated wild-type tobacco plants were used as blank controls (indicated as WT). Agrobacterium cultures carrying pCAPE1 and pCAPE2-derived constructs were mixed and infiltrated into the abaxial side of the basal pair of 4-week-old tobacco plants. Tobacco plants inoculated with Agrobacterium cultures carrying pCAPE1 and pCAPE2-GFP constructs were used as positive controls (indicated as GFP). Positive controls were assayed 14–21 d post-inoculation to confirm whether the agro-inoculation was successful (Fig. 8). Tobacco plants carrying pCAPE1 and pCAPE2-TaPP2Ac-1 construct were indicated as TaPP2Ac-1. Fifty-eight plants were confirmed to carry TaPP2Ac-1 by PCR amplification (Fig. 9A). TaPP2Ac-1 cDNA expression in leaves of tobacco was analysed by RT-PCR; PCR amplification of the same 58 TaPP2Ac-1 plants produced the expected 1-kb fragment of TaPP2Ac-1. No amplification of DNA was detected in WT plants (Fig. 9B).

Fig. 8.

Fig. 8.

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Determination of green fluorescence in roots of tobacco plants with pCAPE2-GFP. GFP was expressed in the root epidermal cells 2 weeks after agroinfection and detected with a laser-scanning confocal microscope. (A, B) Images of epidermal cells from wild type and tobacco plants inoculated with pCAPE2-GFP, respectively. a and d, Bright-field images; e, green fluorescence of GFP in root epidermal cells imaged under the confocal microscope; c and f, overlaid images of (a) and (b) and (d) and (e), respectively. Scale bar = 50 µm. At least 30 tobacco plants with pCAPE2-GFP were observed.

Fig. 9.

Fig. 9.

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Expression analysis of TaPP2Ac-1 in tobacco plants. (A) PCR analysis of DNA from tobacco plants with pCAPE2-TaPP2Ac-1. Lane 1, 0·2 kb molecular marker; lane 2, Positive control (plasmid pCAPE2-TaPP2Ac-1); lane 3, Negative control (untransformed tobacco plants); lanes 4–11, tobacco plants with pCAPE2-TaPP2Ac-1. (B) RT-PCR analysis of TaPP2Ac-1 expression in wild-type tobacco plants and plants with pCAPE2-TaPP2Ac-1. Lane 1, Wild type tobacco (control); lanes 2–6, tobacco plants inoculated with pCAPE2-TaPP2Ac-1. One kilobase of RT-PCR product was obtained using TaPP2Ac-1-specific primers for PCR. The constitutively expressed Tubulin gene was used as a quantitative control (500 bp RT-PCR product). (C) Western hybridization of TaPP2Ac-1 protein. Microsome fractions were isolated from the leaves of WT and TaPP2Ac-1 tobacco plants. Ten micrograms of total protein was separated in a 12·5 % SDS–PAGE gel and immunoblotted. Commercial his-tag monoclonal antibodies were used for the immunoblot. Lane M, low molecular mass marker; lane 1, immunoblot of WT tobacco plants; lane 2, immunoblot analysis of tobacco plants with pCAPE2-TaPP2Ac-1. The figures only show some of the results.

Over-expression of TaPP2Ac-1 was confirmed by western blot analysis using commercial his-tag monoclonal antibodies against the C-terminus of TaPP2Ac-1 (see Materials and methods). Figure 9C shows that TaPP2Ac-1 was present in the leaves of the TaPP2Ac-1 plants, differentiating them from wild tobacco plants. The apparent molecular mass of TaPP2Ac-1 of 36 kDa determined by SDS–PAGE almost corresponded to the molecular mass of TaPP2Ac-1 that was detected in E. coli by expressing the TaPP2Ac-1-His-tagged fusion protein (Fig. 6A). The results indicated that the protein band of 36 kDa detected by immunoblotting was an actual TaPP2Ac-1 protein. In summary, TaPP2Ac-1 was expressed in transformed tobacco plants.

Response of transgenic tobacco plants to water stress

Plants carrying the pCAPE2–TaPP2Ac-1 construct, after confirmation of successful inoculation, were used to test drought responses. Phenotypically WT, GFP and TaPP2Ac-1 plants showed no differences under well-watered conditions (F-test, P = 0·05). Twelve days after water stress by withholding water, lower leaves of WT and GFP plants showed slight wilting but TaPP2Ac-1 plants grew normally. On the 18th day, the leaves of the TaPP2Ac-1 plant appeared normal, whereas WT and GFP plants showed signs of wilting in nearly half their leaves, with the lower leaves showing clear signs of senescence. On the 22nd day, approx. 82 % (WT) and 80 % (GFP) of the mature leaves in all 20 plants in an experiment had seriously wilted. However, only the lower leaves of the TaPP2Ac-1 plants showed slight wilting (Fig. 10A and B). These results showed that the TaPP2Ac-1 plants appeared more tolerant to drought-stress than WT and GFP plants. Under drought stress, GFP plants were very similar to WT plants in performance, indicating that the constructs carrying GFP had no effect on functional expression of TaPP2Ac-1 in tobacco. Considering that leaf number in all three lines was identical, i.e. the TaPP2Ac-1 plants grew at the same rate as wild-type and GFP-expressing plants, the results indicate enhanced drought tolerance rather than drought avoidance.

Fig. 10.

Fig. 10.

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The transgenic TaPP2Ac-1 tobacco plants enhance drought tolerance under water deficit. Agrobacterium cultures carrying pCAPE1 and pCAPE2-derived constructs were mixed 1 : 1 and infiltrated into the abaxial side of the basal pair of 4-week-old tobacco plants. The sample sizes of WT, GFP and TaPP2Ac-1 were all 60 plants. (A) After inoculation with pCAPE1 and pCAPE2-derived constructs, tobacco plants were grown under well-watered conditions for 2 weeks for drought treatments. The photographs were taken at 0, 12, 18 and 22 d after withholding water. WT, Wild-type tobacco plant; GFP, tobacco plants with pCAPE1 and pCAPE2-GFP; TaPP2Ac-1, tobacco plants with pCAPE1 and pCAPE2-TaPP2Ac-1. (B) Quantitative analysis of the wilted tobacco leaves after withholding water for 12, 18 or 22 d. Each value represents the average of at least 20 plants ± s.e. of three replicates. (C) Means and s.e. for RWC, MSI, WRA and WUE in leaves of ten plants of WT, GFP and TaPP2Ac-1 in three independent experiments grown under drought-stressed conditions. After infiltrating, WT (no vectors; white columns), GFP (blank vectors; grey columns) and TaPP2Ac-1 (vectors with TaPP2Ac-1; black columns) were grown under well-watered conditions for 2 weeks, and then the plants were exposed to drought stress for 22 d. The inset depicts the rate of transpiration after 22 d water stress that was used for the determination of the WUE ratio.

To understand mechanisms underlying the different performances of WT and TaPP2Ac-1 lines in a stressed environment, a series of physiological indicators, such as RWC, MSI, WRA and WUE, were measured. Under well-watered conditions and following 22 d of water stress, RWC, MSI, WRA and WUE were determined in WT, GFP and TaPP2Ac-1 plants (Fig. 10C). In TaPP2Ac-1 plants all physiological indicators exceeded those measured in WT or GFP plants (F-test, P = 0·05). There was no significant difference between GFP and WT plants. All TaPP2Ac-1 lines exhibited a more pronounced tolerance to water deficit than GFP and WT plants.

DISCUSSION

TaPP2Ac-1 is the first gene of the PP2A family to be isolated from common wheat. The amino acid sequence of TaPP2Ac-1 shows high homology to PP2Ac of several other plant species (Fig. 1), such as OsPP2A-2 and OsPP2A-4 of rice (>91 %) (Yu et al., 2005), AtPP2A-3 and AtPP2A-4 of arabidopsis (>89 %) (Pérez-Callejón et al., 1998), and NtPP2A and NtPP2A-5 of tobacco (>86 %) (Suh et al., 1998). The result indicated that PP2Ac isogenes are highly conserved in higher plants.

Protein phosphatase 2A is involved in response to heat-shock stress (Cairns et al., 1994), and hyperosmotic stress (Cho et al., 1993). The expression of the MsPP2A β-subunit from Medicago sativa was induced by abscisic acid, indicating a specific function for this protein in stress responses (Toth et al., 2000). Although a number of PP2Acs have been described in several plant species, there are only a few detailed reports on temporal expression patterns of PP2Acs in response to abiotic stress. Analysis of TaPP2Ac-1 transcripts in wheat seedlings exposed to water stress treatment revealed that the expression of TaPP2Ac-1 was indeed up-regulated in water-stressed conditions (Fig. 3). Morever, there were distinct differences among WT, GFP and TaPP2Ac-1 plants after drought stress in RWC, MSI, WRA and WUE (Fig. 10). Drought-stressed TaPP2Ac-1 tobacco plants also showed less wilting under water-deficit conditions than non-transformed controls. Together, these results suggest that the TaPP2Ac-1 gene plays an important role in increasing plant resistance to drought stress.

Five isoforms of the PP2A catalytic subunit gene have been identified in arabidopsis and rice, respectively (Casamayor et al., 1994; Perez-Callejon et al., 1998; Yu et al., 2003, 2005). He et al. (2004) reported two tomato PP2Ac genes. Two PP2Ac cDNA clones of N. tabacum were described by Suh et al. (1998). Obviously, at least two subfamilies of PP2Ac isogenes with two distinctive genomic structures exist in higher plants. However, only one full-length cDNA encoding the catalytic subunit of PP2A from wheat was isolated. The present Southern blotting result suggested that one copy of TaPP2Ac-1 was present in each of the three wheat genomes (Fig. 4). It will be important to identify the presence and complexity of the entire PP2Ac family in wheat in future work.

Plant virus vectors are important tools for studying the over-expression of proteins (Kumagai et al., 1995). Compared with stable transformation of plants, the advantages of virus vectors are replication and spreading of the inserted fragments throughout the entire plant as part of the recombinant virus. This generates consistent phenotypes comparable with stable transformation, while the interval between cloning and phenotypic analysis is significantly shortened (Constantin et al., 2004). One additional reason for choosing PEBV in this study was that this virus belongs to the same genus as TRV, an efficient vector in N. benthamiana and tomato. Moreover, PEBV has already been developed as an expression vector for the reporter gene GFP in both N. benthamiana and Pisum sativum. According to Lu et al. (2003), agro-inoculation provides a most efficient method for introducing cDNA-derived viral RNA into plants. Infiltrating exogenous TaPP2Ac-1 into tobacco plants with PEBV mediated by Agrobacterium GV3101 was successful and TaPP2Ac-1 over-expression was observed, further confirming the validity of this tool for studying gene functions.

PP2A is a key enzyme in living cells that is involved in signal transduction (Millward et al., 1999), control of potassium channel activity in guard cells (Li et al., 1994), and regulation of maturation-promoting factor activity (Minshull et al., 1996). In vitro, PP2A regulates the activities of metabolic enzymes, including phosphoenolpyruvate carboxylase and nitrate reductase. In rice, okadaic acid-dependent Amy3 induction is regulated transcriptionally by a signal transduction pathway involving PP2A (Luan et al., 1993), and the transcription of rice chitinase (Rcht2) is also regulated by PP2A (Kim et al., 1998). PP2A, as a positive regulator, might be involved in the cold signal transduction pathway mediated by C/DRE (Kim et al., 2002). It has been shown in arabidopsis that the transcript of a PP2A-associated protein named TAP46 was induced by chilling, but not by heat or anaerobic stress (Harris et al., 1999), suggesting a positive function in the cold response for this PP2A. In the present study, tobacco plants with pCAPE2-TaPP2Ac-1 exhibited stronger drought tolerance than non-transgenic tobacco plants. This may suggest that TaPP2Ac-1 is involved in drought signal transduction as a positive regulator.

Water stress ultimately affects the amount or activity of transcription factors which interact with cis-elements of water stress-responsive genes, through reversible phosphorylation, and activation of transcription factors that induce or modulate ABA biosynthesis and ABA-induced events. Reversible protein phosphorylation emerges as a major mechanism in signal transduction by which the conformation, and thereby activity, of proteins, including transcription factors, is modulated in eukaryotic cells (Neill and Burnett, 1999). Kwak et al. (2002) identified a PP2A regulatory subunit gene, rcn1, in arabidopsis, and reported that disruption of RCN1 conferred abscisic acid insensitivity and negatively affected PP2A catalytic function. They concluded that PP2A acted as a positive regulator in ABA signalling.

Additional studies will be required to understand the function of TaPP2Ac-1 in wheat in detail. This will include further validation of its role in signal transduction during water stress. Also, the connection of this function to hormonal regulation, and especially its relationship with ABA signalling pathways, will have to be analysed. In addition, the TaPP2A system, for which multiple isoforms are expected in the three sub-families in the three wheat genomes, will have to be characterized. Based on results with other species, indicating the involvement of PP2A complexes in cold-tolerant phenotypes, the detection of multiple TaPP2A signal response machineries for abiotic stress defence mechanisms appears possible.

ACKNOWLEDGEMENTS

We thank Dr Daowen Wang (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China) for kindly providing the PEBV vector and technical advice. We thank Robert A. McIntosh (Plant Breeding Institute, University of Sydney, NSW, Australia) and Hans J. Bohnert (Department of Plant Biology, University of Illinois, USA) for kindly advising and revising the manuscript. The research is supported by the National Transgenic Plants Program of China (JY03-A-14).

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