The Role And Mechanism Of A Wheat BZIP Gene TaGBF In Regulating Photomorphogenesis And Stress Tolerance

Being sessile organisms, plants are forced to develop complex responses to deal with endogenous and exogenous environmental stimulations.Light is an essential environmental facor regulating plant growth and development. Light signals are absorbed by photoreceptors and transducted by a series of light signaling components to modulate the expression of light-regulated genes and induce photomorphogenesis.In addition to light, brassinosteroids (BR) play a key role in photomorphogenesis. The crosstalk between light signaling and BR signaling transduction pathway is becoming a hotspot nowadays.Abiotic stresses such as salinity and osmotic stresses are unfavorable environmental conditions affecting plant growth and development. Plants respond to abiotic stresses via both ABA-dependent and ABA-independent signaling pathway. Some light signaling components including CRY1and HY5are involved in abiotic stress responses.A new wheat introgression line SR3with high yield and tolerance to abiotic stresses was generated with somatic hybridization in our lab. During the study of the stress tolerance of SR3with SSH and microarray, a bZIP gene TaGBF and a potential TaGBF-interacting protein gene TaCKB were cloned and studied preliminarily.This study illustrates that TaGBF is an important modulator integrating light, BR and abiotic stress responses signaling pathway during plant growth and development Research was carried out as follows:1) The expression pattern of TaGBF in different tissues and light conditions, regulation of photomorphogenesis and stomatal development in Arabidopsis by TaGBF, genetic relationship analysis in light signaling pathway and protein interaction between TaGBF and important components in light signaling pathway.2) Expression of TaGBF in response to BR, sensitivity to BR affected by TaGBF, genetic relationship between TaGBF and BZR1, protein interaction between TaGBF and important components in BR signaling pathway.3) Expression of TaGBF in response to abiotic stresses, generation of TaGBF overexpressing lines and RNAi lines in wheat, phenotype of wheat TaGBFox lines and Arabidopsis TaGBFox lines under abiotic stresses, expression of stress responsive genes in stress signaling pathways.4) Cloning and characterization of TaCKB, a gene encoding potential TaGBF-interacting protein. The expression pattern of TaCKB in different tissues and abiotic stresses, subcellular localization of TaCKB, phenotype of Arabidopsis TaCKBox lines under abiotic stresses, flowering time, seed dormancy signaling pathway regulating TaCKBox lines, and the interaction between TaCKB and TaGBF in yeast-two-hybrid assy. The main results are summarized as follows:1. The function of TaGBF in light signaling pathway.TaGBF belongs to bZIP family group G in plant.The expression of TaGBF in wheat was relatively higher in roots, leaves, shoots and flowers, and was induced by blue light.Overexpression of TaGBF promoted photomorphogenesis under blue light, characterized by shorter hypocotyl length and larger cytoledon area. In addition to photomorphogenesis, TaGBF promoted stomatal development under blue light.Genetic analysis indicated that TaGBF functioned downstream of CRY1in light signaling pathway.Yeast-two-hybrid assay showed that TaGBF interacted with AtCOP1, and didn’t interact with AtHY5.The results showed that TaGBF functions downstream of CRY1, as a positive blue light signaling regulator.2. TaGBF mediated the crosstalk between blue light and BR signaling pathway.Expression of AtGBFs was induced by BR signaling. Exogenous brassinolide induced the expression of TaGBF. These results suggested that GBF responded to BR.The hypocotyl length of Arabidopsis TaGBFox lines was significantly shorter than wild type under blue light. Exogenous brassinolide partly suppressed the short hypocotyl phenotype of TaGBFox lines, suggesting the antagonistic function of blue light and BR signaling pathway in regulating photomorphogenesis.The responses of TaGBFox lines to both BR and BRZ were changed under blue light. In darkness, the hypocotyls of TaGBFox lines were hypersensitive to inhibitor of BR synthesis. Expression of BR responsive genes were significantly downregulated in TaGBFox lines. These results indicated overexpression of TaGBF changed BR responses.Genetic analysis indicated that bzr1-1D partly suppressed the short hypocotyl phenotype of TaGBFox lines under blue light, further supporting the antagonistic function of blue light and BR signaling pathway in regulating photomorphogenesis. Bzr1-1D partly suppressed the responses of TaGBFox lines to inhibitor of BR synthesis.These results revealed that TaGBF is a key crosstalk integrating blue light and BR signaling pathway.3. The function of TaGBF in abiotic stress responses.Expression of TaGBF was induced by abiotic stresses such as salinity and osmotic stresses in SR3.TaGBF overexpressing lines and RNAi lines were generated in wheat. Wild type wheat seedlings showed mild wilting under salt stress, wheareas their leaves were still green. TaGBFox wheat seedlings exhibited serious wilting and yellow leaves, with shorter roots than wild type. These results indicated that overexpression of TaGBF heightened the sensitivity of wheat to salt stress.Overexpression of TaGBF in Arabidopsis decreased the germination rate and inhibited cytoledon development from turning green under salt and osmotic stresses. These results indicated that overexpression of TaGBF heightened the sensitivity of Arabidopsis to abiotic stress.In TaGBFox Arabidopsis seedlings, a set of stress responsive genes were screened. Among ABA-dependent stress responsive genes, AtMYC2was down-regulated, whereas AtAB15was up-regulated. TaGBF responded to abiotic stresses via ABA-dependent signaling pathway. 4. Cloning and functional analysis of TaCKB in abiotic stress tolerance and flowering.Expression of TaCKB was induced by salt stress in wheat.TaCKB which encodes a casein kinase2(CK2) regulatory subunit was cloned from wheat.Two-yeast-hybrid assay indicated that TaCKB interacted with TaGBF.Overexpression of TaCKB in Arabidopsis increased the germination rate under salt stress. TaCKB increased PAC(inhibitor of GA synthesis) tolerance at germination stage, but didn’t affect ABA response. This indicated that TaCKB regulated seed germination via GA signaling pathway. Seed dormancy depends on the balance between ABA and GA signaling. How does TaCKB interact with TaGBF to regutate both GA and ABA signaling pathway is worth to be investigated further.