Overexpression of pigeonpea stress-induced cold and drought regulatory gene (CcCDR) confers drought, salt, and cold tolerance in Arabidopsis

Abstract

A potent cold and drought regulatory protein-encoding gene (CcCDR) was isolated from the subtractive cDNA library of pigeonpea plants subjected to drought stress. CcCDR was induced by different abiotic stress conditions in pigeonpea. Overexpression of CcCDR in Arabidopsis thaliana imparted enhanced tolerance against major abiotic stresses, namely drought, salinity, and low temperature, as evidenced by increased biomass, root length, and chlorophyll content. Transgenic plants also showed increased levels of antioxidant enzymes, proline, and reducing sugars under stress conditions. Furthermore, CcCDR-transgenic plants showed enhanced relative water content, osmotic potential, and cell membrane stability, as well as hypersensitivity to abscisic acid (ABA) as compared with control plants. Localization studies confirmed that CcCDR could enter the nucleus, as revealed by intense fluorescence, indicating its possible interaction with various nuclear proteins. Microarray analysis revealed that 1780 genes were up-regulated in CcCDR-transgenics compared with wild-type plants. Real-time PCR analysis on selected stress-responsive genes, involved in ABA-dependent and -independent signalling networks, revealed higher expression levels in transgenic plants, suggesting that CcCDR acts upstream of these genes. The overall results demonstrate the explicit role of CcCDR in conferring multiple abiotic stress tolerance at the whole-plant level. The multifunctional CcCDR seems promising as a prime candidate gene for enhancing abiotic stress tolerance in diverse plants.

Keywords: Abiotic stress tolerance; Cajanus cajan; cDNA library; cold and drought regulatory gene; nuclear localization..

Fig. 1.

Molecular characterization of the Cajanus cajan cold and drought regulatory gene (CcCDR) and comparison of its protein product with other plant proteins. (A) Northern blot analysis of CcCDR: 4-week-old plants of pigeonpea were subjected to no stress (1); PEG (20%) stress for 6h (2); NaCl (1M) stress for 6h (3); or cold (4 °C) stress for 6h (4). About 20 μg of total RNA was used for northern blot analysis. The blot was hybridized with the cDNA fragment of CcCDR. Ethidium bromide-stained 28S rRNA is shown for equal RNA loading. (B) Southern blot analysis of CcCDR: lane 1, EcoRI-digested; lane 2, BamHI-digested; lane 3, HindIII-digested; lane 4, SalI-digested genomic DNA of pigeonpea. Positions of 2.0kb and 10.0kb fragments in the gel are indicated. (C) Comparison of the deduced amino acid sequence of CcCDR with other proteins. Multiple sequence alignment of CcCDR (GU444042) with Carica papaya maturation-associated like Src1 protein (AAL73185), Glycine max cold-induced protein Src1 (BAA19768), G. max low temperature-inducible protein (ABO70349), and G. max KS-type dehydrin (ABQ81887). Identical and conserved amino acids are represented in black and grey, respectively. (This figure is available in colour at JXB online.)

Fig. 2.

Evaluation of CcCDR-transgenics under different abiotic stresses. Two-week-old seedlings of WT, VT, and transgenics were grown under 200mM mannitol, 150mM NaCl, and cold stress (4 °C) for 7 d. Seedlings were allowed to recover on MS plates. Data on survival rate (A), total biomass (B), and root length (C) were recorded after 10 d of recovery. In each treatment, 20 seedlings of WT, VT, and two transgenic lines were used. Chlorophyll content (D) was determined from the leaf discs of control and transgenic plants after 72h of incubation in water, 200mM mannitol, and 150mM NaCl solutions independently at room temperature (20±1 °C); for cold stress, leaf discs were incubated in water at 4 °C. The mean and SE from three independent experiments are shown. * indicates significant differences in comparison with the WT at P<0.05. WS, without stress; CS1 and CS2, 35S transgenic lines; RD1 and RD2, rd29A transgenic lines; WT, wild type; VT, vector-transformed plants; FW, fresh weight. (This figure is available in colour at JXB online.)

Fig. 3.

Evaluation of transgenic plants expressing Cajanus cajan cold and drought regulatory protein (CcCDR) under different abiotic stress conditions. Two-week-old seedlings of control and CcCDR-transgenics were subjected to 200mM mannitol, 150mM NaCl, and cold (4 °C) for 7 d. For each treatment, 20 seedlings were used. Treated seedlings were allowed to recover for 7 d at 20±1 °C. Later, seedlings were transferred to soil and allowed to grow for 3 weeks under normal conditions, and were photographed. (This figure is available in colour at JXB online.)

Fig. 4.

Biochemical characterization of transgenic plants expressing CcCDR. Two-week-old seedlings of control and transgenic plants were grown under 200mM mannitol, 150mM NaCl, and cold stress (4 °C) for 3 d, for estimation of proline (A), reducing sugars (B), MDA (C), catalase (D), and SOD (E). The mean and SE from three independent experiments are shown. For each treatment, 20 seedlings were used. * indicates significant differences in comparison with the WT at P<0.05. WT, wild type; VT, vector transformed; CS1 and CS2, 35S transgenic lines; RD1 and RD2, rd29A transgenic lines. (This figure is available in colour at JXB online.)

Fig. 5.

Hydrogen peroxide and superoxide detection in transgenic plants subjected to different abiotic stresses. Two-week-old A. thaliana seedlings grown on MS medium were subjected to mannitol (200mM), NaCl (150mM, and cold (4 °C) for 72h. For each treatment, 20 seedlings were used. (A) NBT staining for superoxide detection (B) DAB staining for H₂O₂ detection. WT, wild type; VT, vector control; CS1 and CS2, 35S transgenic lines; RD1 and RD2, rd29A transgenic lines. (This figure is available in colour at JXB online.)

Fig. 6.

Physiological characterization of CcCDR-transgenic plants. (A) Cell membrane stability measurement of transgenics, WT, and VT seedlings treated with 150 mM NaCl. (B) Comparison of relative water contents of detached rosettes of controls and transgenic plants. (C) Measurement of osmotic potential of transgenic lines, WT, and VT plants under well-watered conditions. (D) Germination rates of transgenic (CS) and WT seeds on ABA-containing medium. Seeds were placed on MS medium containing 0, 0.5, 1.0, 1.5, or 2.0 μM ABA and the germination rate was calculated after 7 d. Seeds were considered to have germinated when the radicle tip had expanded by 1mm. (E) Size of stomatal apertures of transgenics (CS) and the WT after ABA treatment. Leaves of transgenics and the WT were treated with ABA- (2.0 μM and 10.0 μM) containing solution for 2h. Stomatal apertures in epidermal peels were observed under a confocal microscope and 50 stomatal apertures were measured for each treatment. The mean and SE from three independent experiments are shown. For each treatment, 20 seedlings were used. * indicates significant differences in comparison with the WT at P<0.05. WT, wild type; VT, vector transformed; CS1 and CS2, 35S transgenic lines; RD1 and RD2, rd29A transgenic lines. (This figure is available in colour at JXB online.)

Fig. 7.

Subcellular localization and expression profile of CcCDR in transgenic Arabidopsis. (A) Subcellular localization of CcCDR. Plasmid constructs harbouring CaMV35S-GFP and CaMV35S-CcCDR::GFP were introduced into Arabidopsis independently by floral infiltration. Individual cells of callus derived from roots of transgenic plants were observed under a confocal microscope. Images 1 and 4 are dark field, 2 and 5 are combined, 3 and 6 are bright field. Ths scale bar=50 μm. N represents the nucleus. (B) Transcriptome profile of wild-type and rd29A-CcCDR-transgenic plants under drought stress by microarray analysis. Two-week-old seedlings were subjected to 200mM mannitol stress for 72h. The heat map represents normalized expression intensity values of differentially regulated genes (fold change ≥2 and P-value <0.05) in the RD2 transgenic line (T) as compared with the wild type (WT). Dark color shows overexpressed genes and light color shows underexpressed genes. (C) Expression profiles of stress-responsive genes under drought stress by qRT-PCR analysis. Comparisons of the relative transcript levels of LEA, ZNF, bZIP, CDPK, MYB, MAPK, DREB2A, CBF4, and CBF1 in rd29A-CcCDR-transgenic (T) and wild-type (WT) plants and under 200mM mannitol stress for 72h. Actin was used as an internal control. The vertical column indicates the relative transcript level. The mean and SE from three independent experiments are shown. * indicates significant differences in comparison with the control at P<0.05. (This figure is available in colour at JXB online.)