A Medicago truncatula EF-hand family gene, MtCaMP1, is involved in drought and salt stress tolerance

Abstract

Background: Calcium-binding proteins that contain EF-hand motifs have been reported to play important roles in transduction of signals associated with biotic and abiotic stresses. To functionally characterize genes of EF-hand family in response to abiotic stress, an MtCaMP1 gene belonging to EF-hand family from legume model plant Medicago truncatula was isolated and its function in response to drought and salt stress was investigated by expressing MtCaMP1 in Arabidopsis.

Methodology/principal findings: Transgenic Arabidopsis seedlings expressing MtCaMP1 exhibited higher survival rate than wild-type seedlings under drought and salt stress, suggesting that expression of MtCaMP1 confers tolerance of Arabidopsis to drought and salt stress. The transgenic plants accumulated greater amounts of Pro due to up-regulation of P5CS1 and down-regulation of ProDH than wild-type plants under drought stress. There was a less accumulation of Na(+) in the transgenic plants than in WT plants due to reduced up-regulation of AtHKT1 and enhanced regulation of AtNHX1 in the transgenic plants compared to WT plants under salt stress. There was a reduced accumulation of H2O2 and malondialdehyde in the transgenic plants than in WT plants under both drought and salt stress.

Conclusions/significance: The expression of MtCaMP1 in Arabidopsis enhanced tolerance of the transgenic plants to drought and salt stress by effective osmo-regulation due to greater accumulation of Pro and by minimizing toxic Na(+) accumulation, respectively. The enhanced accumulation of Pro and reduced accumulation of Na(+) under drought and salt stress would protect plants from water default and Na(+) toxicity, and alleviate the associated oxidative stress. These findings demonstrate that MtCaMP1 encodes a stress-responsive EF-hand protein that plays a regulatory role in response of plants to drought and salt stress.

Figure 1. Sequence analysis of EF-hand family proteins.

The highly conserved EF-hand motifs of MtCaMP1 and other known EF-hand family proteins were aligned in panel A. Alignments were performed using the ClustalX2.1 software. X, Y, Z, -Y, -X and -Z represent conserved amino acids of Ca²⁺-binding loop. Phylogenetic tree of these proteins was constructed by MEGA 5 in panel B. The corresponding ID of AtCBL4 (SOS3), ZmCBL4, AtCBL1, OsCDPK7, NtCDPK2, AtCPK21, GmCaM4, AtCML18 (CaM15) and At4G32060 are NP_001190377.1, NP_001150076.1, NP_567533.1, BAB16888.1, CAC82998.1, NP_192381.1, NP_001237902.1, NP_186950.1 and NP_194934.1, respectively.

Figure 2. Ca²⁺-dependent electrophoretic mobility shift assay of glutathione S-transferasefucata (GST) and GST-MtCaMP1 recombinant protein.

Purified protein was run on a 10% non-denaturing polyacrylamide gel in the presence of Ca²⁺ or EGTA. CaCl2 at 5 mM or EGTA was added into the sample buffer.

Figure 3. Expression patterns of MtCaMP1 in M. truncatula.

Expression of MtCaMP1 in response to drought stress for varying periods was analyzed in panel A. Effect of 200 mM NaCl and 20% PEG6000 on expression of MtCaMP1 was shown in panel B. Expression patterns of MtCaMP1 in different organs were shown in panel C. Data are means±SE with three biological replicates.

Figure 4. Analysis of MtCaMP1 expression level in wild-type and transgenic plants.

Expression level of MtCaMP1 in wild-type (WT) and transgenic plants (line1–5) was monitored by RT-PCR.

Figure 5. Effect of drought stress on wild-type and transgenic plants.

Phenotypes of wild-type and transgenic plants after withholding water for 14 days were shown in panel A. Survival rates were scored after recovery of watering for 7 days (panel B). Water loss rates were determined at 0, 0.5, 1, 2, 3, 4, 5 and 6 h after drought treatment (panel C). Data are mean±SE with three replicates. Asterisks represent statistically significant differences between wild-type and transgenic lines. * P≤0.05, ** P≤0.01.

Figure 6. Effect of salt stress on wild-type and transgenic plants.

Phenotypes of wild-type and transgenic plants after treatment with NaCl for 14 days were shown in panel A. Survival rates were counted after recovery of watering for 7 days (panel B). Data are mean±SE with three replicates. Asterisks represent statistically significant differences between wild-type and transgenic lines. * P≤0.05, ** P≤0.01.

Figure 7. Effect of drought and salt stress on contents of H₂O₂ and malondialdehyde.

H₂O₂ contents in wild-type and transgenic plants in control and treatment with drought and salt stress (panel A). Malondialdehyde contents in wild-type and transgenic plants in control and treatment with drought and salt stress (panel B). Four-week-old seedlings withheld water or irrigated with 200 mM NaCl for 6 days were used in the experiments. Data are means ± SE of three biological replicates. Asterisks represent statistically significant differences between wild-type and transgenic lines. * P≤0.05, ** P≤0.01.

Figure 8. Drought and salt stress-induced changes in Pro contents.

Effect of drought and salt stress on contents of Pro was shown in panel A. The expression levels of genes encoding Pro synthetase (P5CS1) and dehydrogenase (ProDH) under stress were shown in panel B and C, respectively. Data are mean±SE with three replicates. Asterisks represent statistically significant differences between wild-type and transgenic lines. * P≤0.05, ** P≤0.01.

Figure 9. Effect of NaCl on contents of Na⁺ and K⁺, and expression of AtHKT1 and AtNHX1.

Contents of Na⁺ and K⁺ in shoots of wild-type and transgenic plants treated with and without NaCl were shown in panel A and B, respectively. Na⁺/K⁺ ratio was shown in panel C. The expression levels of AtHKT1and AtNHX1 were shown in panel D and E, respectively. Four-week-old seedlings irrigated with or without 200 mM NaCl for 6 days were tested in this experiment. Data are mean±SE with three replicates. Asterisks represent statistically significant differences between wild-type and transgenic lines. * P≤0.05, ** P≤0.01.