Improvement of Tolerance to Osmotic Stress in Arabidopsis Plants Ectopically Expressing Peanut AhNCED1 Gene

Abstract

Gene AhNCED1 plays a vital role in regulation of abscisic acid (ABA) biosynthesis in peanut (Arachis hypogaea L.) plants in response to drought stress. Two binary vectors harboring AhNCED1 gene, p35S::ORF and pAtNCED3p::ORF, were established, which were driven by the CaMV 35S promoter from pCAMBIA1301 and the AtNCED3 gene promoter from wild type Arabidopsis, respectively. Wild type and 129B08/nced3 mutant Arabidopsis plants were separately transformed with Agrobacterium harboring p35S::ORF or pAtNCED3p::ORF vector, resulting in 35S::ORF-WT and A3p::ORF-B08 transgenic plants, accordingly. The stable expression of AhNCED1 gene in Arabidopsis plants was confirmed with duplex reverse transcription PCR (RT-PCR). The wild type, 129B08/nced3 mutant, and the transgenic Arabidopsis plants were subsequently tested for sensitivity to exogenous ABA and tolerance to osmotic stress. The results showed that the ABA sensitivity was declined in the 129B08/nced3 mutant and increased in the Arabidopsis plants ectopically expressing AhNCED1 gene. Under sorbitol stress, the relative germination rate of 129B08/nced3 mutant seeds was far lower than that of the wild type seeds. However, the relative germination rate of A3p::ORF-B08 transgenic seeds was close to that of the wild type seeds, which was significantly higher than that of 129B08/nced3 mutant seeds. The relative germination rate of 35S::ORF-WT transgenic seeds was higher than that of the wild type seeds in the treatment of 300 mmol L⁻¹ sorbitol. When treated with 300 mmol L⁻¹ sorbitol, the leaves of 129B08/nced3 mutant plants were highly chlorotic, and root formation and seedling growth were severely inhibited. In contrast, at this concentration of sorbitol, the leaves of A3p::ORF-B08 transgenic seedlings were only slightly chlorotic, just similar to those of the wild type plants. There was almost no influence on the growth of 35S::ORF-WT transgenic seedlings under the sorbitol treatment. Based on the results of germination assay and phenotypic evaluation, the 129B08/nced3 mutant was hypersensitive to the nonionic osmotic stress induced by sorbitol, and ectopic expression of peanut AhNCED1 gene reverted the hypersensitivity of 129B08/nced3 mutant Arabidopsis to sorbitol and conferred enhanced osmotic stress tolerance in transgenic Arabidopsis plants.