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. 2013 Jun 19;14(6):12827-42.
doi: 10.3390/ijms140612827.

Overexpression of Arachis hypogaea AREB1 gene enhances drought tolerance by modulating ROS scavenging and maintaining endogenous ABA content

Xiao-Yun Li  1 , Xu Liu, Yao Yao, Yi-Hao Li, Shuai Liu, Chao-Yong He, Jian-Mei Li, Ying-Ying Lin, Ling Li
Affiliations

Overexpression of Arachis hypogaea AREB1 gene enhances drought tolerance by modulating ROS scavenging and maintaining endogenous ABA content

Xiao-Yun Li et al. Int J Mol Sci. .

Abstract

AhAREB1 (Arachis hypogaea Abscisic-acid Response Element Binding Protein 1) is a member of the basic domain leucine zipper (bZIP)-type transcription factor in peanut. Previously, we found that expression of AhAREB1 was specifically induced by abscisic acid (ABA), dehydration and drought. To understand the drought defense mechanism regulated by AhAREB1, transgenic Arabidopsis overexpressing AhAREB1 was conducted in wild-type (WT), and a complementation experiment was employed to ABA non-sensitivity mutant abi5 (abscisic acid-insensitive 5). Constitutive expression of AhAREB1 confers water stress tolerance and is highly sensitive to exogenous ABA. Microarray and further real-time PCR analysis revealed that drought stress, reactive oxygen species (ROS) scavenging, ABA synthesis/metabolism-related genes and others were regulated in transgenic Arabidopsis overexpressing AhAREB1. Accordingly, low level of ROS, but higher ABA content was detected in the transgenic Arabidopsis plants' overexpression of AhAREB1. Taken together, it was concluded that AhAREB1 modulates ROS accumulation and endogenous ABA level to improve drought tolerance in transgenic Arabidopsis.

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Figures

Figure 1
Figure 1
Drought tolerance and abscisic acid (ABA) sensitivity of AhAREB1-overexpressed plants. (A) Quantification of relative primary root growth length of seedlings treated with 5 μmol/L ABA at 14 day after stratification. Bars indicate standard deviation, n = 30; (B) Photographs of seedling at 20 day after transfer to control agar plates (Mock) or plates containing 5 μmol/L ABA; (C) Germination rate of seedlings on MS agar plate containing indicated concentration of ABA; (D) The green cotyledon of seedlings was analyzed after treatment with 0.5 or 1.5 μmol/L ABA; (E) The survival rate of wild-type (WT) and transgenic plants (A22 and A38 lines) were calculated after water deprivation; (F) Photographs showing plants after control and drought stress treatment. Watering was withheld from four-week-old plants for 10 d; thereafter, plants were rewatered for 4 d before the photograph was taken; (G) Real-time PCR detected the expression of AhAREB1 genes in transgenic plants (A22, A38 and A39) and WT plants at normal conditions and dehydration for 0.5 h, respectively. All experiments were performed in triplicate, and a representative result is shown. The error bars represent standard deviations (n = 20). WT represented wild-type plants; A22 represented A22 transgenic lines; A38 represented A38 transgenic lines; A39 represented A39 transgenic lines; abi5 represented the abscisic acid-insensitivity mutant, abi5-1; T-abi5 represented the plants that transformed AhAREB1 into the abi5 line. WTD, A22D, A38D and A39D represented the plants of WT, A22, A38 and A39 that were kept dehydrated for 0.5 h.
Figure 2
Figure 2
Expression analyses of related genes in A38 lines and wild-type. The transcript level of RD29A, RD29B, RD26, RD20, ABA1, AtABCG40, NCED3, CYP707A2, CYP707A3, CAT2, CCS and CSD3 were examined by quantitative PCR analysis in 14 day-old plants. A38 and WT seedlings were grown on MS agar medium with or without dehydration treatment at the time points indicated. 18SrRNA gene expression level was used as an internal control. Error bars represent standard deviation among the three reduplicate experiments. WT represents wild-type plants under normal conditions; WTD represented wild-type plants that were treated with dehydration for 0.5 h or 1.5 h; A38 represented A38 lines under normal conditions; A38D represented A38 lines that were treated with dehydration for 0.5 h or 1.5 h. * indicated that values of the WTD, A38 and A38D were significantly different from those of WT with p < 0.05, after dehydration for 0.5 h or 1.5 h, respectively. # indicated that values of A38D were significantly different from A38 at p < 0.05 after dehydration for 0.5 h or 1.5 h, respectively.
Figure 3
Figure 3
Constitutive expressions of AhAREB1 decreases ROS levels, but enhanced ABA content. (A,B) Twenty day-old plants were grown on agar plates, then dehydrated for 0.5 h. Cellular levels of H2O2 and O2− were stained with 3,3-diaminobenzidin (DAB) and nitro blue tetrazolium (NBT) to visualize H2O2 and O2−, respectively; (C) Twenty day-old plants were grown on agar plates and then dehydrated for 0.5 h. Catalase (CAT) and superoxide dismutase (SOD) activities were calculated in WT, A22, A38, abi5 and T-abi5 plants. All experiments were repeated at least three times, and about 20 plants collected from seedlings were inspected in each experiment; (D) Endogenous ABA content was detected in whole plants of WT, A22, A38, abi5 and T-abi5. Fourteen day-old plants were grown on soil for 14 days, then dehydrated for 10 days and were subsequently collected to measure endogenous ABA. Data are presented as the mean ± standard deviation. WT represented wild-type plants; A22 represented A22 transgenic lines; A38 represented A38 lines; abi5 represented the abscisic acid-insensitivity mutant, abi5-1; T-abi5 represented the plants that transformed AhAREB1 into abi5 lines.
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