Molecular Dissection Of Cotton GhDREB1 In Regulation Of Low Temperature Tolerance, Plant Growth And Development

Plants are sessile organisms and hence cannot escape unfavourable environmental conditions within their life cycle, such as high salinity, drought, waterlog, high or low temperature, pathogen attack and mechanical agitation. Among them, low temperature is one of the main constraints in plant geographical distribution and crop productivity.Plants have evolved finely tuned low-temperature signaling and resistance mechanisms. When exposed to low temperature, plants respond rapidly and carry out protective processes, for example, physical adaptation (changes in plasma membrance lipid components and cell cytoskeleton remodeling), accumulation of osmoprotectants (soluble sugars, proline, betaine, etc) in cytosol, increased contents of various antioxidants (superoxide dismutase, catalase, ascorbinase) , induced synthesis of proteins (anti-freezing proteins, dehydrins and heat-shock proteins) encoded by low-temperature-responsive genes, which reprogram the biological activities and establish a new metabolism balance for stressed plants and help survive the low temperature stress. In planta, low temperature signal could be transmitted via ABA-dependent and ABA-independent pathways. However, the roles for ABA in low temperature signaling and tolerance are illusive. To date, cold signaling and tolerance mechanism conferred by CBF/DREB1 transcription factors is the most understood and important pathway, which function in an ABA-independent manner.Following cold exposure, CBF/DREB1 genes are rapidly and transiently induced via an ABA-independent pathway, and their products activate CBF regulon to enhance freezing tolerance by specifically recognizing and binding to CRT/DRE cis-element in promoter region of cold responsive genes. CBF/DREB1 homologues have been identified in many plant species and overexpression of one CBF/DREB1 gene in transgenic plants gives rise to strong constitutive expression of CBF regulon and hence increased low temperature tolerance. In addition, CBF/DREB1 overexpression also increases the salt and osmotic stress tolerance in transgenic plants. Nevertheless, severe growth retardation is observed in these transformants and the underlining molecular mechamism is still largely unknown. Cotton (Gossypium hirsutum) is one of the oldest and most important fiber and oil crops. Its growth and yield are severely inhibited by low temperature, especially at germination and emergence stages. The CBF/DREB1 functional homologue in cotton, GhDREB1, is cloned and its expression modes and function are examined in detail:(1) Transient transformation assay with onion epidermal cells revealed that GhDREB1 is exclusively localized in the nucleus, correlates well with the typical feature for canonical transcription factors.(2) The promoter region of GhDREB1 was cloned by TAIL-PCR. PlantCARE prediction analysis revealed the existence of putative low-temperature-responsive and gibberellin-responsive elements in GhDREB1 promoter sequence. GFP and GUS reporter genes under the control of GhDREB1 promoter displayed cold-induced expression in BY-2 suspension cells. On the other hand, exogenous GA3 suppressed the activity of GhDREB1 promoter in both BY-2 cells and cotton seedlings.(3) GhDREB1 enhanced the chilling tolerance of transgenic tobaccos but not freezing tolerance. Consititutive expression of stress-responsive genes and a higher proline level shoud account for the increased tolerance in transgenic plants.(4) Overexpression of GhDREB1 in tobaccos reduced the contents of GA1+3, which was responsible for the dwarf and late-flowering phenotypes of transgenic tobacco plants.(5) The GhDREB1-overexpressing Arabidopsis plants exhibited a more compact statue, bluish green leaves, prostrate growth habit, delayed flowering and male sterility of the first flowers on main inflorescence. Interestingly, the differences in plant size between wild type and transgenic Arabidopsis plants diminished gradually at the late developmental stages and the final biomass and seed yield are even somewhat higher in transgenic Arabidopsis plants.(6) Among the phytohormones tested, only GA could rescue the phenotypic changes of GhDREB1-overexpressing Arabidopsis. Examination of gibberellin responsive marker genes and endogenous GA content supported the same conclusion: GhDREB1 reduced the bioactive GA levels in transgenic Arabidopsis. However, RT-PCR results indicated that neither the GA biosynthesis related genes were suppressed nor the GA deactivation genes were upregulated. By contrast, opposite expression patteres of these genes were observed. (7) GhDREB1 significantly improved the freezing tolerance of transgenic Arabidopsis, a characteristic not found in transgenic tobaccos. In addition to the freezing tolerance, the transgenic Arabidopsis were more tolerant to salt and osmotic stress. Multiple mechanisms appeared to contribute to the increase in abiotic stress tolerance, including constitutive expression of stress-responsive genes and accumulation of the osomoprotective solutes, such as proline and inorganic ions.(8) GhDREB1 severely delayed the transition from vegetative growth stage to reproductive growth stage in transgenic Arabidopsis plants. RT-PCR results indicated that besides the GA promotion pathway, vernalization and photoperiodic pathway were also disturbed by GhDREB1 in transgenic Arabidopsis.(9) GhDREB1 indirectly suppressed the expression level of type-B ARRs and subsequent type-A ARRs in transgenic Arabidopsis, which were important componets in cytokinin signaling. Therefore, the GhDREB1-overexpressing Arabidopsis plants were less sensitive to exogenous cytokinin.