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

The phytohormone abscisic acid (ABA) is a key regulator of seed development, root growth, stomatal aperture in higher plants and it is also involved in adaptation of plants to various stresses. The oxidative cleavage of cis-epoxycarotenoids catalyzed by nine-cis-epoxycarotenoid dioxygenase (NCED) is considered to be the rate-limiting step in ABA biosynthesis in higher plants. The AhNCED1 gene plays a vital role in the regulation of ABA biosynthesis in peanut plants in response to drought stress. Two binary vectors, p35S::ORF and pAtNCED3p::ORF, were established which harbored the AhNCED1 gene, respectively, driven by the CaMV 35S promoter originated from pCAMBIA1301 and the AtNCED3 gene promoter from wild type Arabidopsis. Wild type and 129B08/nced3 mutant Arabidopsis plants were separately transformed with Agrobacterium harboring p35S::ORF or pAtNCED3p::ORF vectors, generating 35S::ORF-WT and A3p::ORF-B08 transgenic plants, respectively, after hygromycin screening and PCR detection. The stable expression of AhNCED1 gene in Arabidopsis plants was confirmed by duplex RT-PCR performance. Wild type, 129B08/nced3 mutant and transgenic Arabidopsis plants were subsequently tested for sensitivity to exogenous ABA and tolerance to osmotic stress. The results showed that the ABA sensitivity of 129B08/nced3 mutant declined, and that of Arabidopsis plants ectopically expressing the AhNCED1 gene increased. Under sorbitol stress, the relative germination rate of 129B08/nced3 mutant seeds was far lower than that of wild type seeds; however, the relative germination rate of A3p::ORF-B08 transgenic seeds was close to that of wild type seeds, significantly higher than that of 129B08/nced3 mutant seeds. The relative germination rate of 35S::ORF-WT transgenic seeds was higher than that of wild type seeds at the treatment of 300 mmol L–1 sorbitol. Under 300 mmol L–1 sorbitol stress, leaf of 129B08/nced3 mutant plants was highly chlorotic, and root formation and seedling growth were severely inhibited. In contrast, at this concentration of sorbitol, leaf of A3p::ORF-B08 transgenic seedlings was only slightly chlorotic, just similar to that of wild-type plants; the growth of 35S::ORF-WT transgenic seedlings was nearly not affected. Germination assay and phenotypic evaluation revealed that 129B08/nced3 mutant was hypersensitive to the nonionic osmotic stress induced by sorbitol, and that ectopic expression of peanut AhNCED1 gene reverted the hypersensitivity of 129B08/nced3 mutant Arabidopsis to sorbitol and conferred enhanced osmotic stress tolerance of transgenic Arabidopsis plants. These observations and an improved understanding of the roles of AhNCED1 gene in stresses adaptation could provide the basis for engineering greater stress tolerance in crops.