Isolation and Characterization of DREB/CBF Gene Orthologs in Lettuce

Lettuce (Lactuca sativa L.) is one of the most consumed leafy vegetables in the world. Its productivity is dramatically decreased by environmental stresses, such as heat, drought, and high salinity. Improved stress tolerance is desirable in new lettuce cultivars. Isolation and characterization of genes involved in stress in this plant would help us understand the molecular mechanisms of stress response and to breed more tolerant lettuce using transgenic technology. DREBs/CBFs encoding DRE/CRT-binding factor proteins play important roles in plant responses to abiotic stress. In this study, the DREB1 and DREB2 orthologs LsDREB1A and LsDREB2A, respectively, were isolated from lettuce and their expressions and functions were analyzed. LsDREB1A and LsDREB2A encoded proteins with the conserved AP2 (apetala 2) domain, and they were classified into A-1 and A-2 subgroups of the DREB subfamily, respectively. Southern blot analysis indicated that there might be more than ten DREB homologs in the lettuce genome. Quantitative real-time PCR experiments revealed that the expression level of LsDREB1A was highest under cold conditions, followed by salt and abscisic acid stresses, while the expression of LsDREB2A was significantly induced by drought and high salinity treatment, but not cold, heat, or abscisic acid treatments. These results suggested that LsDREB1A and LsDREB2A play different roles during drought, cold, and high salinity stress responses. LsDREB2A could specifically recognize the DRE sequence in vitro as determined by an electrophoresis mobility shift assay. In a yeast one-hybrid assay, both LsDREB1A and LsDREB2A proteins specifically bound to the DRE sequence and activated the expression of both the His3 and LacZ reporter genes. These results indicated that both proteins might function as transcription factors. Overexpression of LsDREB1A and LsDREB2A increased tolerance to salt stress in transgenic Arabidopsis plants, suggesting that these genes might show promise for producing transgenic lettuce that is tolerant to salt stress. This work was supported by Grant Number 1116 from the Salt Science Research Foundation and by JSPS KAKENHI Grant Number 25292022.