Calreticulin (CRT) is a highly conserved and ubiquitously expressed Ca2+-binding protein in multicellular eukaryotes. CRT plays a crucial role in many cellular processes including Ca2+ storage and release, protein synthesis, and molecular chaperone activity. To elucidate the function of CRTs in plant responses against drought, a main abiotic stress limiting cereal crop production worldwide, a full-length cDNA encoding calreticulin protein namely TaCRT was isolated from wheat (Triticum aestivum L.). The deduced amino acid sequence of TaCRT shares high homology with other plant CRTs. Phylogenetic analysis indicates that TaCRT cDNA clone encodes a wheat CRT3 isoform. Southern analysis suggests that the wheat genome contains three copies of TaCRT. Subcellular locations of TaCRT were the cytoplasm and nucleus, evidenced by transient expression of GFP fused with TaCRT in onion epidermal cells. Enhanced accumulation of TaCRT transcript was observed in wheat seedlings in response to PEG-induced drought stress. To investigate further whether TaCRT is involved in the drought-stress response, transgenic plants were constructed. Compared to the wild-type and GFP-expressing plants, TaCRT-overexpressing tobacco (Nicotiana benthamiana) plants grew better and exhibited less wilt under the drought stress. Moreover, TaCRT-overexpressing plants exhibited enhanced drought resistance to water deficit, as shown by their capacity to maintain higher WUE (water use efficiency), WRA (water retention ability), RWC (relative water content), and lower MDR (membrane damaging ratio) (P ≤0·01) under water-stress conditions. In conclusion, a cDNA clone encoding wheat CRT was successfully isolated and the results suggest that TaCRT is involved in the plant response to drought stress, indicating a potential in the transgenic improvements of plant water-stress.
Calreticulin (CRT) is an abundant Ca2+-binding protein that ubiquitously expresses in all of the multicellular eukaryotes investigated (Coppolino and Dedhar, 1998; Michalak et al., 1999). CRT was first detected in the endoplasmic reticulum (ER) of rabbit skeletal muscle (Ostwald and MacLennan, 1974) and later was cloned from rabbit (Fliegel et al., 1989) and mouse (Smith and Koch, 1989). Besides its main location in ER (Opas et al., 1996), CRT has been found to reside in the nuclear envelope (Napier et al., 1995), the spindle apparatus of the dividing cells (Denecke et al., 1995), the cell surface (Gardai et al., 2005), and the plasmodesmata (Laporte et al., 2003; Chen D et al., 2005), indicating that CRT is essential for normal cell function.
Extensive studies of mammalian CRTs have elucidated a number of key physiological functions, including the regulation of Ca2+ homeostasis and Ca2+-dependent signal pathways (Michalak et al., 2002; Gelebart et al., 2005), molecular chaperone activity in the folding of many proteins (Denecke et al., 1995; Williams, 2006), modulation of nuclear-hormone receptor-mediated gene expression (Burns et al., 1994), control of cell adhesion (Opas et al., 1996), and integrin-dependent Ca2+ signalling at the extra-ER sites in mammalian cells (Coppolino et al., 1997; Krause and Michalak, 1997). In addition, CRT appears to play a role in the immune system (Guo et al., 2002) and apoptosis. For example, CRT-dependent shaping of Ca2+ signalling was found to be a critical contributor to the modulation of the T cell adaptive immune response (Porcellini et al., 2006). Surface CRT mediates muramyl dipeptide-induced RK13 cell apoptosis through activating the apoptotic pathway (Chen D et al., 2005).
CRT is highly conserved in eukaryotic cells, which is indicated by sequence analysis on the deduced amino acids of the known CRT cDNA clones from several mammalian species (Fliegel et al., 1989; Smith and Koch, 1989) and other organisms including nematode (Smith, 1992a), fruit fly (Smith, 1992b), marine snail (Kennedy et al., 1992), clawed frog (Treves et al., 1992), rainbow trout (Stephen et al., 2004), and Cotesia rubecula (Zhang et al., 2006). The protein contains three distinct structural and functional domains with loosely defined boundaries: the nearly neutral N-domain, the proline-rich P-domain, and the polyacidic C-domain. CRT also has an N-terminal signal peptide sequence and an ER retention motif in the C-domain. The P-domain is responsible for the high-affinity and low-capacity Ca2+ binding while the C-domain is responsible for the low-affinity and high-capacity Ca2+ binding. Within the P-domain, there are two types of triplicate repeated motifs that are highly conserved among various animal species. However, the C-domain is less conserved than other domains of CRT (Michalak et al., 1992, 1999). Four amino acid residues (Glu239, Asp241, GLu243, and Trp244) at the tip of the ‘extended arm’ of the P-domain are critical in the chaperone function of CRT (Martin et al., 2006). At present, no structural information is available for the C-domain which is involved in Ca2+ storage in the lumen of the ER (Nakamura et al., 2001).
More recently, the multifunctional roles of CRT in plant cellular events are rapidly emerging areas of study in plant biology. CRT has been identified in a few plant species, although functional analysis lags that undertaken in the animal system. The first indication that plants contain calreticulin-like proteins came from the purified Ca2+-binding proteins of spinach leaves (Menegazzi et al., 1993). Those proteins showed high homology to CRT sequences of mammals. Subsequently, CRT cDNA clones were isolated from Arabidopsis (Huang et al., 1993), barley (Chen et al., 1994), pea (Hassan et al., 1995), tobacco (Denecke et al., 1995), maize (Napier et al., 1995; Dresselhaus et al., 1996), Brassica rapa (Lim et al., 1996), Ricinus communis L. (Coughlan et al., 1997), and rice (Li and Komatsu, 2000). As a Ca2+ sensor and molecular chaperone within the ER, plant CRT shares the same structural domain features and basic functions identified for animal CRTs (Coughlan et al., 1997; Li and Komatsu, 2000; Wyatt et al., 2002). Plant CRT is highly expressed during mitosis in tobacco (Denecke et al., 1995), embryogenesis of barley (Chen et al., 1994), Nicotiana plumbaginifolia (Borisjuk et al., 1998), and maize (Dresselhaus et al., 1996), and in flower tissues (Nelson et al., 1997) including sperm cells (Williams et al., 1997), pollen tubes as well as anthers (Nardi et al., 2006). Calreticulin in plant cells has been shown to play a role in regeneration (Li and Komatsu, 2000; Jin et al., 2005), pollen–pistil interaction (Lenartowska et al., 2002), and cell-to-cell transport via the plasmodesmata (Baluška et al., 1999; Laporte et al., 2003; Chen MH et al., 2005). Increasing evidence also indicates that this protein is involved in the plant response to a variety of stress-mediated stimuli, for example, pathogen-related signalling molecules (Jaubert et al., 2002), gravistimulation (Heilmann et al., 2001), and other stress factors (Sharma et al., 2004). However, the precise mechanism of CRT function in these plant cell processes, particularly in regulating stress response, remains to be fully ascertained.
Wheat, with a huge and very complex genome, is an important cereal crop in the world. Drought is a major constraint to wheat production worldwide. Understanding the molecular basis of drought-stress response is highly required for wheat genetic improvement of drought tolerance. Little information is available for the wheat CRT gene and its functions, although a wheat calreticulin-like sequence was reported (GI 56606826). The present study aims to investigate and clarify the function of CRT in the drought-stress response of wheat. The isolation and characterization of a full-length cDNA encoding calreticulin-like protein (designated as TaCRT) which is highly expressed in the wheat seedlings subjected to water-stress, is reported here. In addition, TaCRT’s genomic organization, subcellular localization, and its mRNA expression pattern during wheat seedling response to PEG-induced drought stress is described. The role of TaCRT in the plant response to drought stress is demonstrated by its effects when expressed in transgenic tobacco plants.
Materials and methods
Plant materials and water-stress experiments
Wheat (Triticum aestivum L.) genotype ‘Hanxuan 10’ with a higher drought-tolerant phenotype was used in this study. After being sterilized with 75% ethanol and washed with sterilized water, the wheat seeds were germinated and cultured with water in a controlled-growth chamber (20±1 °C, 12 h light/dark cycle). Seedlings at the two-leaf stage (9-d-old) were treated by PEG-6000 (–0.5 MPa) solution, which, in pilot experiments, had been shown to constitute significant stress at this developmental stage. The treated plants were stressed in the PEG-6000 solution for 1, 3, 6, 12, 24, 48, and 72 h, respectively. The control seedlings were watered as normal and without PEG treatment. Leaf samples were collected from the seedlings at different time points and frozen quickly with liquid nitrogen and stored at –70 °C for RNA isolation and other analysis.
Four genotypes of wheat and its relatives were used in Southern hybridizations: Hanxuan 10, hexaploid (AABBDD) and three diploid genotypes including T. urartu (AA) accession No. 1010004, Ae. speltoides (SS, closely related to the BB genome) accession No. IcAG 400046, and Ae. tauschii (DD) accession No. PH1878. Leaves from pot-grown plants were sampled for DNA isolation.
Tobacco plants (Nicotiana benthamiana) were grown in pots filled with vermiculite in a controlled-growth chamber with 12 h photoperiod, 25 °C, 70% humidity and 45 μmol·m−2·s−1 illumination.
Cloning of full-length TaCRT cDNA
Total RNA was extracted from the leaf samples using the Trizol Reagent (Tianwei) as described in the manufacturer's instructions. Based on the candidate EST of a TaCRT from the cDNA library established in our laboratory (Pang et al., 2007), the putative full-length TaCRT cDNA was obtained by in silico cloning. Using total RNA isolated from the leaves of 12 h PEG-treated wheat seedlings as templates, the first-strand cDNA synthesis was performed with M-MLV Reverse Transcriptase reagent (Invitrogen) according to the manufacturer's instructions. To obtain full-length TaCRT cDNA, two primers, 5′-ACCACCACTTCCTCGTCTC-3′ (sense) and 5′-TTCCCTCACACGAGACAAG-3′ (antisense) were designed based on the lateral flanking sequence of ORF of the putative complete CRT cDNA.
A total of 20 μl of the RT-PCR reaction mixture contained 1 μl of 10-fold-diluted cDNAs (0.2 μg), 0.2 μM of each primer, 1.5 mM MgCl2, and 0.4 mM dNTPs, 1× buffer and 0.1 μl of proof reading Pfu Pyrobest polymerase (5 U μl−1) (Takara). Amplification was conducted in a DNA thermocycler (GeneAmp PCR System 9600, Applied Biosystems) using 30 cycles of 2 min at 94 °C, 1 min at 61 °C, and 2 min at 72 °C; final extension at 72 °C for 10 min. As a control, a Tubulin gene fragment was amplified using sense primer 5′-AGAACACTGTTGTAAGGCTCAAC-3′ and antisense primer 5′-GAGCTTTACTGC CTCGAACATGG-3′ under conditions similar to those described for TaCRT, except for annealing at 50 °C and 21 cycles.
The RT-PCR generated a 1446 bp DNA fragment namely TaCRT. This DNA fragment was subcloned into pGEM-T easy Vector system (Tianwei) and introduced in E. coli JM109 according to the manufacturer's recommendations. The plasmid DNA isolated from the positive E. coli cells was digested with EcoRI, and the inserted DNA (TaCRT cDNA) was sequenced in both directions using the BigDye Terminator method on an autosequencer (Model XL3730, Applied Biosystems).
Database searches of the nucleotide and deduced amino acid sequences were performed through the NCBI/GenBank/Blast Sequence alignment and similarity among species were determined by the megAlign program in DNAStar. Signal sequence was predicted with SignalP (http://genome.cbs.dtu.dk/services/SignalP). The functional region and activity sites were identified using PROSITE (http://expasy.hcuge.ch/sprot/ prosite.html) and SMART motif search program (http://coot.embl-heidelberg.de/SMART).
Subcellular localization of TaCRT
A full-length cDNA clone of TaCRT was inserted between the upstream GFP and the downstream constitutive CaMV 35S promoter in a pJIT163-GFP expression vector for constructing a 35S-TaCRT-GFP fusion protein. Restriction sites HindIII (5′) and SalI (3′) were added to the 5′ and 3′ ends of the coding region by the PCR method. The PCR product obtained was digested with HindIII and SalI, and then ligated into the HindIII and SalI sites of the pJIT163-GFP plasmid polylinker to create a recombinant plasmid pJIT163-TaCRT-GFP for expressing the fusion protein. The positive plasmids were confirmed by restriction analysis, and further verified by sequencing. The recombinant plasmid pJIT163-TaCRT-GFP was transformed into living onion epidermal cells by GeneGun (Helios™) according to the instruction manual. The subcellular location of the TaCRT was detected by monitoring the transient expression of GFP in onion epidermal cells. The transformed cells were incubated in Murashige and Skoog (MS) medium at 28 °C for 24–48 h and then were observed with a laser scanning confocal microscope (Olympus FV500) and the images obtained were recorded automatically. The recombinant pJIT163-TaCRT-GFP plasmid and the control pJIT163-GFP plasmid were bombarded into 20 onion epidermal segments, respectively. Twenty fluorescent cells of these segments were observed with identical results.
Southern blot analysis
Genomic DNA of hexaploid wheat and its diploid relatives was extracted according to the procedure described by Stewart and Via (1993). Aliquots of genomic DNA (15 μg) was digested overnight with EcoRI, EcoRV, and HindIII, respectively. The digested DNA was fractionated in 0.8% agarose gel followed by blotting to nylon membranes (Hybond N+, Amersham) overnight in 20× SSC. After UV cross-linking, the membrane was hybridized to a [α_32P]dCTP-labelled full-length TaCRT cDNA probe in 50× Denhardt's solution at 65 °C. The membrane was washed with 2× SSC, 0.1% SDS; 0.2× SSC, 0.1% SDS, and 0.1× SSC, 0.1% SDS for 15 min at 65 °C, respectively. The hybridized blot was exposed to a phosphor screen (Kodak-K) for 2 d and visualized with the Molecular Imager FX (Bio-Rad).
Northern blot analysis
Total RNA of each sample was isolated from the leaf tissue of the wheat plants stressed by PEG-6000 (–0.5 MPa) solution using the Trizol Reagent methods as described above. Twenty micrograms of total RNA from each sample was separated in 1.5% agarose gels with 2% formaldehyde followed by transferring to nylon membranes (Hybond N+, Amersham) overnight in 20× SSC. The blotted membranes were subsequently prehybridized for 1 h at 65 °C in 50× Denhardt's solution with the addition of 500 μl of 10 mg ml−1 of denatured salmon DNA (Promega) and then hybridized with the [α_32P]dCTP-labelled full-length TaCRT cDNA probe under the same conditions overnight. Washing and autoradiography of the hybridized membranes was performed as described for the Southern blot.
Quantitative real-time PCR
Treated with deoxyribonuclease I, the RNA samples used for northern blot also served as templates for the first-strand cDNA synthesis by the Superscript First-Strand Synthesis System kit (Invitrogen). Quantitative real-time PCR (qRT-PCR) was performed in triplicate with an ABI PRISM® 7000 system using the SYBR Green PCR master mix kit (Applied Biosystems) according to the manufacturer's instructions. The relative amount of gene expression was calculated using the expression of Tubulin as internal standard. Oligonucleotides of qRT-PCR primers were as following: TaCRT forward primer 5′-GAAGCCCCCCAAATCTT-3′ and reverse primer 5′-CCTCACACGAGACAAGAAACAC-3′. Tubulin forward primer 5′- TGTGCCCCGTGCTGTTCTTATG-3′ and reverse primer 5′- CCCTTGGCCCAGTTGTTACCC-3′.
The relative quantity of gene expression was detected using 2–ΔΔCT method (Livak et al., 2001). ΔΔCT=(CT,Target–CT,Tubulin)Time x –(CT,Target–CT,Tubulin)Time 0. The CT (cycle threshold) values for both the target and internal control genes were means of triplicate independent PCRs. Time x is any treated time point (1, 3, 6, 12, 24, 48, or 72 h) and Time 0 represents the untreated time (0 h).
Construction of expression vector of pCAPE2-TaCRT and Agrobacterium-mediated transformation of tobacco plants
PEBV (the tobravirus pea early browning virus) has already been developed as an expression vector for the reporter gene GFP in both P. sativum and N. benthamiana (MacFarlane and Popovich, 2000). The PEBV-based pCAPE vector system including two plasmids, pCAPE1 (assistant plasmid) and pCAPE2-GFP (control plasmid) were used to construct a plasmid vector to express TaCRT in tobacco plants. For developing a vector pCAPE2 to express TaCRT, GFP in pCAPE2-GFP was replaced by the ORF sequence of TaCRT. The restriction sites of SacI and SalI were introduced in the 5′ and 3′ ends of the ORF sequence of TaCRT cDNA by PCR with forward primer 5′-AGCGAGCTCACCACCACTTCCTCGTCTC-3′ and reverse primer 5′-TATCGTCGACTCAGTGGTGGTGATGATGGTGTCATGGTAGTCATC-3′. The underlined sequences of the primers correspond to the SacI and SalI restriction sites, respectively. In the reverse primer, the nucleotide sequences of six histidines (boxed region) were added before the stop codon (TGA) so that the expressed protein could have a His tag, allowing validation by a commercial His-tag monoclonal antibody if the exogenous TaCRT gene was expressed in the host N. benthamiana. The PCR product of TaCRT and the vector pCAPE2-GFP were both cleaved with SacI and SalI. The digested vector pCAPE2 and the PCR fragment of TaCRT were then ligated together to generate a new recombinant vector pCAPE2-TaCRT.
Assistant vector pCAPE1, control vector pCAPE2-GFP, and the recombinant vector pCAPE2-TaCRT were transformed into Agrobacterium tumefaciens strain GV3101 by electroporation (Gene Pulser II, Bio-Rad) as described by Shen and Forde (1989), respectively. The transformed agrobacteria were grown overnight at 28 °C under kanamycin and rifampicin selection. The positive clones were confirmed by restriction analysis, plasmidic PCR, and sequencing. An individual clone was grown in 20 ml of Luria Broth (LB) liquid medium including 0.1 M 2-morpholinoethanesulphonic acid (MES), 0.2 mM acetosyringone (As), 100 μg ml−1 rifampicin and 50 μg ml−1 kanamycin at 28 °C for 16–18 h with shaking (230 rpm). At OD600≈2.0, the bacteria were collected by centrifugation (7 500 rpm, 15 min). Cells were resuspended in about 20 ml infiltration medium (0.1 M MES, 2 mM As, 0.2 M MgCl2), and adjusted to a final OD600 of 2.0, and then incubated at room temperature for 3 h without shaking. Agrobacterium cultures carrying pCAPE2-GFP and pCAPE2-TaCRT were mixed, respectively, with Agrobacterium carrying pCAPE1 at a ratio of 1:1, and infiltrated into the abaxial side of the third pair of leaves on 5-week-old tobacco plants using a 5 ml syringe without needle. Twenty plants in each of three independent experiments were inoculated, respectively. The positive transgenic plants were confirmed by detecting GFP fluorescence of pCAPE2-GFP transformants, PCR and RT-PCR assays of pCAPE2-TaCRT transformants. At least 58 tobacco plants expressing pCAPE2-TaCRT were obtained and subsequently analysed for their phenotypes.
Western blot analysis
Protein isolation and quantification were performed using methods adapted from Laemmli et al. (1970) and Bradford (1976). Twenty micrograms of protein from each sample, including His-tagged TaCRT protein and control protein, were electrophoretically separated in two pieces of 12.5% SDS–PAGE gels with Bio-Rad Mini-Protean gel rigs following the procedure described by Kyse-Anderson (1984). One of the gels was stained with Coomassie Brilliant Blue G250. The other was subsequently transferred electrophoretically onto a nitrocellulose membrane (pore size: 0.45 μm) using a semi-dry transfer blotter (Bio-Rad) in transfer buffer TBST (2 mM TRIS, 192 mM glycine, 20% methanol, and 0.1% SDS). The membranes were blocked in TBS buffer with 3% BSA for 1 h and thereafter blotted with a commercial His-tag monoclonal antibody for 3 h at a 1:2000 dilution. After extensive washing, the bound primary antibody was detected with a horseradish peroxidase-conjugated goat antimouse IgG secondary antibody using the ECL technique according to the manufacturer's protocol (Amersham).
Physiological analysis of tobacco plants expressing TaCRT under drought stress
Tobacco plants expressing pCAPE2-TaCRT and pCAPE2-GFP, respectively, 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 (25 °C, 50–60% relative humidity, continuously illuminated at 45 μmol·m−2·s−1) until a lethal effect of dehydration was observed on most of the control plants (wild type). Ten independent plants from each sample group of three replicates were examined for all of the following physiological indexes.
Leaf relative water content (RWC) was estimated according to the method of Turner (1981). RWC(%)=(fresh weight–dry weight)/(turgid weight–dry weight)×100.
Water retention ability (WRA) was measured according to the formula: WRA(%)=(desiccated weight–dry weight)/(fresh weight–dry weight)×100. The leaves with the same age were taken and weighed (fresh weight), then desiccated for 24 h under controlled conditions (65% relative humidity and 25 °C), and weighed again (desiccated weight). The leaves were finally oven-dried over a period of 24 h at 90 °C to a constant weight (dry weight).
Water use efficiency (WUE) was calculated with the formula: WUE(%)=net photosynthetic rate (Pn)/transpiration rate (Tr)×100. Fully expanded leaves with the same age were selected for measuring Pn and Tr by the Li-6400 photosynthesis system (Li-Cor Inc.) according to the manufacturer's instructions.
Leaf membrance damage rate (MDR) was determined according to the method of Sairam (1994) with a few modifications. A conductivity meter (DDS-1, YSI) was used to measure the MDR of the sample. MDR(%)=initial electrical conductivity/electrical conductivity after being boiled×100.
Cloning of calreticulin cDNA
Previously, RNA from 12 h PEG-treated wheat (cv. Hanxuan 10) seedling was used to construct a cDNA library by the method of suppression subtractive hybridization (SSH). A total of 1833 high quality ESTs including 133 known function ESTs were obtained from the cDNA library (Pang et al., 2007). A 535 bp EST, which is highly homologous to CRT genes, was chosen as a query probe for in silico cloning. Based on the sequence data of wheat dbEST, a 1680 bp extended sequence was obtained. Subsequently, a 1446 bp cDNA fragment was amplified from the drought-treated wheat seedlings by RT-PCR using a pair of primers designed from the extended sequence. This fragment was fully sequenced and identified as a wheat CRT (TaCRT) cDNA clone (GenBank accession no. EF452301).
Characterization of TaCRT cDNA sequence
The TaCRT cDNA clone was 1446 bp in length, which consists of a 35 bp 5′-untranslated region (UTR), a 115 bp 3′ UTR, and a 1296 bp open reading frame (ORF). The ORF of TaCRT encodes 431 deduced amino acid residues with a calculated molecular mass of 50.573 kDa and a predicted pI of 6.84. Like other known plant CRTs, the deduced coding sequence of TaCRT exhibits the same zonal characteristics of the three-domain structure typical of animal CRTs (Michalak et al., 1992, 1999). The three domains of TaCRT are the globular N-domain (residues 34 to 218), the proline-rich central domain (P-domain) (residues 219 to 319) which is folded into an ‘extended arm’, and the polyacidic C-domain (residues 320 to 431) (Fig. 1A).
The highly conserved N-terminal domain was nearly neutral with a 33-amino-acid-long signal sequence. The mature peptide of TaCRT has 398 amino acids and a molecular mass of 46.866 kDa. The N-domain also contained two well-conserved CRT family signature motifs of KFEQKIECGGGYVKLM and LMFGPDICG described as CRT motif 1 and CRT motif 2 (Fig. 1A). In the N-domain, the conserved cysteines (Cys) are residues 119, 151, and 177, of which the latter two are believed to form a disulphide bond, involved in folding of the native protein. These amino acids correspond to Cys120 and Cys146 of the bovine brain calreticulin, which were involved in forming an intramolecular disulphide bridge in the intact calreticulin (Matsuoka et al., 1994). A similar Cys position was also found in the amino acid sequence of barley calreticulin (Cys136 and Cys162) (Chen et al., 1994).
The N-domain is followed by the central P-domain, which is a highly conserved proline-rich region and contains 17 of the total 27 proline residues. Within this region, there are two types of triplicate repeats (A and B) that are highly conserved among CRTs from various animal and plant species. Repeat A has the structure of PXXIXDXDAXKPEDWDE while the consensus sequence for repeat B is GXWKPPXIXNPXYX (Fig. 1A). These two triplicate repeats are also highly conserved but not invariant. The two types of repeats could enable TaCRT to form the extended hairpin loop configuration as described previously in other CRTs (Ellgaard et al., 2001).
In front of the C-domain, there are 30 short hydrophobic residues (amino acid 320 to 349) (Fig. 1A). The predicted C-terminal domain of TaCRT is less conserved, but highly acidic. 32% of acidic acids in TaCRT are found in this domain, which is common to this protein family. The C-domain terminates with the amino acid motif HDEL, an ER-retention sequence.
To investigate the sequence homology of TaCRT to other known CRTs, a rooted phylogenetic tree was established (Fig. 1B) based on 14 CRT protein sequences from eight species including wheat, Arabidopsis, rice, maize, barley, tobacco, human, and mouse. As shown in Fig. 1B, these CRTs were clustered into two distinct groups, plant CRT and animal CRT groups. In plant CRTs, the CRT1 and CRT2 isoforms formed a subgroup distinct from the CRT3 subgroup. TaCRT showed the highest identity with rice CRT3 by 88%, and was clustered into the plant CRT3 isoform subgroup while another wheat CRT-like protein (AY836753) belonged to the plant CRT1/2 isoform subgroup. The isoform-specific clades of plant CRTs were similar when an analogous analysis was performed by corresponding CRT nucleotide sequences (data not shown). All these bioinformatics analyses of TaCRT clearly indicate that this cDNA clone encodes a wheat CRT protein (e.g. wheat CRT3 isoform).
Genomic organization of the TaCRT gene
To study the genomic organization and the copy number of the TaCRT gene in hexaploid wheat, Southern blot analysis of genomic DNA was performed using the full-length sequence of TaCRT cDNA as a probe (Fig. 2). The hybridization patterns reveal that TaCRT is most likely to exist as three-copy genes in the genomes of hexaploid wheat, as three-hybridized bands were always evident in each lane of genomic DNA digested with EcoRI, EcoRV, and HindIII, respectively (Fig. 2, lane 1). Similarly, the B genome contains at least two copies of this gene for each of the three enzymes resulting in two bands in the blot (Fig. 2, lane 3). In the A and D genomes, there was only one-hybridized band of TaCRT digested by EcoRI or HindIII restriction (Fig. 2, lanes 2, 4), while two copies were generated by EcoRV (Fig. 2, lanes 2, 4) probably caused by a site in the intron. This analysis indicates that the TaCRT gene has three copies in hexaploid wheat genomes and one or two copies in its ancestors.
Subcellular localization of the TaCRT protein
To address the subcellular localization of TaCRT in living cells, a construct containing TaCRT fused in-frame with the GFP (TaCRT::GFP) driven by the CaMV 35S promoter was transiently expressed in living onion epidermal cells. As shown in Fig. 3, confocal microscopic examination revealed that cells transferred with the unconjugated GFP (control) exhibited a diffused distribution of green fluorescence throughout the cell (Fig. 3A). By contrast, when fused with the TaCRT, the GFP signal was confined to the cytoplasm and nucleus (Fig. 3B), suggesting that TaCRT is distributed in the cytoplasmic and nuclear compartments.
Expression pattern of TaCRT in response to PEG-induced drought stress
The cloning of TaCRT cDNA was based on an EST sequence in the cDNA library constructed from 12 h water-stressed seedlings of wheat, suggesting that the TaCRT gene might be expressed in response to drought stress. To reveal the temporal expression pattern of TaCRT in wheat seedling plants subjected to drought stress, two-leaf seedlings (9-d-old) were stressed in the PEG-6000 (–0.5 MPa) solution for different periods of time. The control was wheat seedlings watered as normal in the same condition.
Northern blot and qRT-PCR both revealed similar expression patterns as shown in Fig. 4 with triplicate independent experiments, respectively. No expression of the TaCRT gene was detected in the control plants (CK). The TaCRT transcript began to appear in the seedlings 1 h after stress treatment and peaked at 12 h. However, the level of the transcript decreased gradually in the treated plants from 12 h to 72 h under the stress. Even at 72 h after the stress treatment, TaCRT expression is about 2-fold more than that at 1 h in the PEG-treated wheat seedlings (Fig. 4A). A similar expression pattern was obtained by qRT-PCR using the RNA samples from at least 25 seedlings for each time period (Fig. 4B). Clearly, TaCRT expression was significantly induced by PEG stress, suggesting that CRT may function as an important chaperone protein for wheat drought-stress response.
Heterologous overexpression analysis of TaCRT in tobacco plants
In order to verify TaCRT function, a virus-induced expression construct of TaCRT was developed (Fig. 5). Agrobacterium cultures carrying pCAPE2-GFP (Fig. 5B) and pCAPE2-TaCRT (Fig. 5C) were mixed respectively with pCAPE1 (Fig. 5A) at a ratio of 1:1 for the transformation of host tobacco plants. The transformed tobacco plants containing both pCAPE2-GFP and pCAPE1 were used as positive controls, while wild-type tobacco plants were used as blank controls. A total of 70 and 65 independent transgenic lines expressing pCAPE2-TaCRT or pCAPE2-GFP were generated, respectively. Green fluorescence was clearly and strongly detected in the root tissues of tobacco plants containing pCAPE2-GFP at 17 d of post-inoculation (Fig. 6). The result indicated that the agro-inoculation was successful.
The pCAPE2-TaCRT transformants were identified with tissue PCR amplification (Fig. 7A). The TaCRT fragments were apparently obtained from 58 of the pCAPE2-TaCRT transformants, but not from the wild-type plants and the control plants transformed with pCAPE2-GFP. The TaCRT transcripts in leaves of tobacco were detected with RT-PCR (Fig. 7B). Again, the target TaCRT fragment was only obtained from the pCAPE2-TaCRT transformants.
To testify further if TaCRT protein was expressed correctly, a western blot was performed. As described in the Materials and methods, 20 μg of total protein from each sample was electrophoretically separated on SDS-PAGE and transferred to a nitrocellulose membrane. Using the commercial His-tag monoclonal antibody, a protein band which was almost consistent with the predicted molecular mass of TaCRT, was apparently detected in the pCAPE2-TaCRT inoculated tobacco plants, but not from the wild-type plants (Fig. 7C). The immunoblot result further showed that TaCRT was overexpressed in the transformed tobacco plants.
Phenotype of the TaCRT-overexpressing tobacco plants under drought stress
TaCRT-expressing plants exhibited no obvious phenotypic difference from the GFP-expressing and WT plants under well-watered conditions as revealed by four physiological traits related to plant water status, including MDR, WUE, RWC, and WRA (F-test, P=0.05). To examine the performance of the TaCRT-expressing tobacco plants under drought stress, water was withheld from TaCRT and GFP transgenic, as well as WT tobacco, plants after confirmation of successful transformation and expression. In the first week of drought treatments, both transgenic and WT tobacco plants showed a similar phenotype. However, after 3 weeks of drought treatment, phenotypic differences were clearly observed among the TaCRT transgenic and other plants (Fig. 8A). Both WT and GFP transgenic plants displayed severe leaf-wilting, while only 40% of the TaCRT transgenic plants showed slight leaf-wilting, which demonstrated less wilting than the corresponding WT and GFP transgenic plants. GFP transgenic plants were very similar to WT plants in performance, indicating that the GFP had no effect on functional expression of TaCRT in tobacco plants. The results indicated that TaCRT was involved in the response to water stress and its over-expression may contribute to drought tolerance to some extent.
The tissue samples from the TaCRT-expressing tobacco plants and WT plants were also analysed for detecting their cellular-physiological status under the water stress. On day 22 of drought stress, MDR, WUE, RWC, and WRA were measured in WT and TaCRT transgenic plants. Corresponding to the previous phenotypic results, leaves of TaCRT transgenic plants displayed a lower MDR, higher WUE and WRA at statistically significant level (P ≤0·01) compared with the wild-type plants (Fig. 8B). The transgenic plants also showed a slightly higher RWC although the increase was not significant. In plant cells, lower MDR, and higher WUE, RWC, and WRA were believed to be responsible for stronger tolerance against osmotic stress. Therefore, analysis of these cellular-physiological data again indicated that TaCRT transgenic plants performed better than WT and the overexpression of TaCRT gene could enhance plant drought tolerance.
In the present study, the cloning of a full-length cDNA of TaCRT containing an ORF of 1296 bp and encoding a protein of 431 amino acids and one termination codon from wheat seedlings has been described. The deduced amino acid sequence alignment (Fig. 1A) indicates that TaCRT has high sequence identity (54–88%) with other plant CRTs, whereas the similarity between animal and plant calreticulins is lower, only about 45%. Mammalian and plant CRTs form their own distinct clusters (Fig. 1B). The similarities among these 14 CRTs have been further confirmed by their nucleotide sequence alignment and phylogenetic analyses (data not shown). TaCRT has a higher sequence identity with rice CRT3, maize CRT3, and Arabidopsis CRT3, respectively. It indicates that TaCRT cDNA clone encodes a wheat CRT3 isoform. The result of sequence analysis showed an early duplication event in wheat genomes, from which the CRT1 (or CRT2 if present) and CRT3 isoforms derive. This early divergence of CRTs into two groups (CRT1/CRT2 and CRT3) was also found in Arabidopsis, rice, maize, and Brassica rapa (Persson et al., 2003).
Although plant CRTs have different isoforms, several conserved regions exist in their sequences. Like other plant CRTs, the deduced amino acid sequence of TaCRT also contains the three typical domain organization (Fig. 1A) proposed for animal CRTs (Michalak et al., 1999). In addition, the putative TaCRT protein consists of conservative amino acid residues involved in two well-conserved CRT family signature motifs and two types of triplicate repeats. The remarkable similarity between TaCRT and other known CRTs in both the deduced amino acid sequences and the protein zonal structures reinforces the viewpoint that calreticulin protein is highly conserved in all eukaryotic organisms (Chen et al., 1994; Michalak et al., 1999; Persson et al., 2003).
As a major Ca2+-binding protein, CRT was originally detected in the ER (Ostwald and MacLennan, 1974). Sequence analysis reveals that all plant calreticulins so far cloned including TaCRT contain a typical ER retention signal HDEL sequence, with the exception of Euglena calreticulin which has KDEL (Navazio et al., 1998). Consistent with the signal, plant CRT has been located primarily within the lumen of ER. However, CRT was also found outside the ER compartment in plant cells, including the cytoplasm of certain cells and nucleus (Dedhar, 1994), nuclear envelope (Napier et al., 1995), spindle apparatus of dividing cells (Denecke et al., 1995), plasmodesmata in root cells (Baluška et al., 1999), the cell surface (Coppolino and Dedhar, 1998) and Golgi compartment (Borisjuk et al., 1998). Calreticulin is totally absent from the vacuole, the major Ca2+ store in plant cells (Opas et al., 1996). Our GFP transient expression assay clearly indicates that TaCRT was mainly located in the cytoplasmic and nuclear compartments in onion epidermal cells (Fig. 3). All these suggest that CRT has multiple subcellular locations which might result from covalent modification such as phosphorylation or Ca2+-binding (Sharma et al., 2004). The precise mechanism(s) how the protein relocates from the ER to the outside of the ER, despite having the ER-retention signal, remains to be ascertained. The multiple location of TaCRT indicates that it may be involved in multiple cellular processes. The in vivo studies in wheat plants currently being conducted in our laboratory aim to characterize further the intracellular distribution of TaCRT.
Plant calreticulin does not strictly follow the rule ‘one protein, one gene’ assessed for its animal counterpart, but is encoded by a low copy-number gene family (Chen et al., 1994; Napier et al., 1995; Coughlan et al., 1997; Nelson et al., 1997; Michalak et al., 1999; Li and Komatsu, 2000). Several investigations have established that plants contain two or multiple CRT isoforms (Chen et al., 1994; Nelson et al., 1997; Persson et al., 2003). Southern blot analysis in this study suggested that the TaCRT was most probably a low-copy gene. Examining the genomic architecture of the TaCRT gene in wheat will provide more information about the intron/exon organization and evolution of the gene.
CRTs have been implicated in plant growth and development (Menegazzi et al., 1993), where they regulated calcium signalling, and assisted protein folding (Nardi et al., 2006). It has also been shown that a wide range of developmental and environmental stimuli differentially affect the expression of CRT in plant cells (Borsjuk et al., 1998). Sharma et al. (2004) found the increased expression of rice CRT under cold stress, suggesting its potential role in regulating plant stress responses. The growing data support the functional diversity of CRT protein in both animal and plant systems. Here, we are examining TaCRT function in wheat drought-stress responses. Results from our northern blot showed that no TaCRT expression was detected in the unstressed wheat seedlings, and the transcription of TaCRT was greatly induced by PEG-stress (Fig. 4). The varied expression levels of TaCRT in the PEG-stressed seedlings indicated that cells under drought stress respond quickly by elevating the transcription levels. Persson et al. (2003) revealed that different isoforms of CRT in Arabidopsis responded differently to applied external stimuli. CRT1 and CRT2 isoforms showed a slower induction under salt or tunicamycin whereas the response of CRT3 appeared to be more rapid. The CRT2 was reported in response to both tunicamycin and dithiothreitol in Arabidopsis. Although the time and level of the induced expression of CRTs are different, these reports clearly demonstrated that CRT expression was increased under stress conditions, supporting the up-regulation of TaCRT described here. The up-regulated expression of CRTs may be a conserved self-protection mechanism acquired during long-term evolution and should facilitate the survival of the plants under unfavourable osmotic conditions.
To investigate the in vivo role of TaCRT in plant drought resistance, TaCRT was over-expressed in tobacco plants by the PEBV-based plant expression vector. Plant virus-based vectors have proved to be important tools for studying gene function because of their relatively convenient engineering, which does not require the development of stable transformants and the interval between cloning and phenotypic analysis is significantly short (Lindboet et al., 2001). The PEBV-based binary vector system (Fig. 5) was used as a plant gene expression vector since it has been confirmed as an efficient vector in N. benthamiana and tomato. Moreover, PEBV has already been developed as an expression vector for the reporter gene GFP in N. benthamiana. The host plant used here for the gene expression was tobacco (N. benthamiana), one of the most-studied hosts for developmental and genetic analysis by far. Via Agrobacterium mediation, a total of 58 and 68, respectively, pCAPE2-TaCRT and pCAPE2-GFP transformed plants were successfully obtained. The transgenic tobacco plants were successfully confirmed by PCR, RT-PCR assays, and western blot (Fig. 7).
MDR, RWC, WUE, and WRA are the typically physiological indices for the evaluation of drought tolerance and resistance in crop plants. Plants with higher RWC, WUE, WRA, and lower MDR have higher tolerance and are more resistant to drought stress. Transgenic tobacco plants over-expressing TaCRT displayed no obvious phenotypic difference in appearance, time of flowering, or seed production compared with the wild-type and control plants when grown to maturity in soil under normal conditions. More importantly, under treatment, the transgenic tobacco plants over-expressing TaCRT exhibited delayed and less wilting compared with wild-type plants and control plants expressing GFP alone. Further physiological analysis revealed that the TaCRT transgenic tobacco plants had significantly lower MDR, and higher WUE and WRC (P ≤0·01) than wild-type and control plants expressing GFP alone (Fig. 8). Phenotypic and physiological characterization demonstrated that TaCRT transgenic plants performed better under drought stress conditions. The results presented here are consistent with a report that the expression of the high capacity calcium-binding domain of CRT increases the bioavailable calcium stores in plants, and transgenic Arabidopsis plants expressing the C-domain of maize CRT demonstrated significant resistance to drought, salt and heavy metals stress (Wyatt et al., 2002).
Compared with the rapid growth of information coming from the animal world, current knowledge on the relevance of CRTs in plant physiology is rather limited and slow to appear. TaCRT over-expressing transgenic tobacco plants displayed an enhanced resistance to drought stress, however, the pathway and molecular mechanism through which TaCRT regulates plant drought resistance remain unclear. It has been postulated that various physiological, developmental, and environmental stress conditions cause unfolding or misfolding of proteins in the ER (Borisjuk et al., 1998). A typical cellular strategy to cope with unfolded or misfolding proteins in the ER is to trigger the increased synthesis of the chaperone proteins by the induction or release of molecular chaperones and folding enzymes located in the lumen of the ER, which, in turn, prevents unfolding or misfolding of proteins. CRT is such an important protein among those molecular chaperones (Gelebart et al., 2005). It is possible that over-expression of TaCRT provides large amounts of such chaperones to maintain the correct folding of proteins in the ER under stressed cellular conditions. Another plausible explanation would be that overexpression of TaCRT increased the Ca2+ capacity of rapidly exchanging Ca2+ stores and releases in ER, which subsequently created new calcium homeostasis and alleviated ER stress. Considering that several signalling pathways are involved in the regulation of plant stress responses, TaCRT over-expression may directly or indirectly activate the special signalling transduction, resulting in the enhancement of the related metabolism to protect cells from injury caused by drought stress. It is, therefore, conceivable that wheat calreticulin could be one of the positive regulators in regulating drought response by modulating the Ca2+ homeostasis and signalling that govern the availability of calreticulin and modulate gene expression.
Our data indicated no adverse effects of TaCRT over-expression in mature plants. However, Jin et al. (2005) reported that over-expression of Chinese cabbage calreticulin 1, BrCRT1, inhibited plant growth, but enhanced organogenesis in transgenic tobacco. The growth inhibition and stunting in seedlings were due to large amounts of CRT expressed in these plants which may adversely affect global gene expression (Jin et al., 2005). These different effects of CRT over-expression on plant performance suggest that different CRT genes may participate in different regulatory pathways, supporting functional diversity among the CRT genes.
In summary, a full-length cDNA clone (TaCRT) encoding wheat calreticulin, a wheat CRT3 isoform, has been identified. The TaCRT transcripts were induced to express by drought stress in wheat seedlings. Over-expression of TaCRT in heterologous tobacco plants enhanced the transgenic plant drought resistance. All these results demonstrate that TaCRT plays an important role in plant drought resistance, providing very useful information for the functional analysis of CRT and its implications in plant genetic improvement. Further studies, including characterizing the regulation of the signal transduction network that controls TaCRT production, will extend our understanding of the biological role and function of CRTs in plant development and growth as well as the responses to various biotic and abiotic stresses.
green fluorescent protein
membrane damaging ratio
pea early browning virus
quantitative real-time PCR
relative water content
calreticulin of Triticum aestivum
water retention ability
water use efficiency
This work was supported by the National Basic Research Program of China (2004CB117202) and the National Transgenic Plants Program of China (JY03-A-14). The authors thank Dr Daowen Wang and Professor Zhensheng Li (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China) for providing tobacco seeds, the PEBV-based pCAPE vector system and pJIT163-GFP expression vector.