Molecular Mechanism Of TaCOLD1-mediated Regulation In Wheat Plant Height

Wheat is one of the most widely grown food crops in the world,providing food for 40 percent of the world's population.In the 1960s,“Green Revolution”,marked by the development of new varieties of dwarf wheat,led to an unprecedented increase in wheat yield.Plant height is one of the most important trait affecting wheat plant architecture and yield.Reducing plant height properly can improve the lodging resistance of crops and make crops use light and water more efficiently,ultimately increasing crop yield.At present,the widely used dwarf gene in wheat breeding is Rht1,which represses GA signaling to reduce wheat plant height.However,the dwarf gene resources of wheat are still relatively single.Therefore,finding new dwarf gene resources and elucidating the molecular mechanisms of them will promote the utilization of dwarf genes and facilitate the breeding of high yield and high resistance wheat.In this paper,we studied the interaction and regulatory relationship between proteins in regulating wheat plant height and provided theoretical basis for using these genes in breeding.In this study,we identified a light-regulated gene TaCOLD1 from wheat gene expression profiling data.The expression of this gene was obviously transient suppressed when the dark grown common wheat transferred to light.We cloned three highly homologous genes from hexaploid wheat,whose encoding proteins are homologous to cold sensor COLD1 protein in rice.The 187^th amino acid of TaCOLD1 is methionine,which is consistent with the residue in COLD1^ind.The amino acids of this site in COLD1protein sequences in diploid,tetraploid and hexaploid wheat materials are all methionine,which indicated that TaCOLD1 might be evolutionarily conserved.Subcellular localization analysis of TaCOLD1 protein in wheat protoplasts demonstrated that TaCOLD1 protein localizes to the endoplasmic reticulum and plasma membrane.Previous study has shown that COLD1 proteins have functional differentiation between japonica rice and indica rice and COLD1^jap is more active than COLD1^ind.Overexpression of TaCOLD1^M187K(consistent with the key amino acid in COLD1^jap)in the common wheat variety Kenong199(KN199,which contains a dwarf gene Rht-B1b)background resulted in a significant reduction in wheat plant height.Compared with KN199,each segment of the transgenic wheat was shorter and the cell length in the first segment under panicle was obviously shorter.We cloned three genes encoding the heterotrimeric G protein?subunits:TaG?-7A,TaG?-1B and TaG?-7D from hexaploid wheat.Chinese Spring nullisomic-tetrasomic lines demonstrated that the three genes localize in chromosomes 7A,1B and 1D,respectively.The protein sequences analysis showed that there are only six amino acids differences between TaG?-7A and TaG?-7D,while the C-terminal part of TaG?-1B is missing compared with TaG?-7A/7D.Luciferase complementary imaging(LCI)assays,in vitro pull-down assays and bimolecular fluorescence complementation(Bi FC)assays demonstrated that the intracellular hydrophilic loop of TaCOLD1(amino acid residues 178-296)could interact with TaG?-7A,but not TaG?-1B.In addition,TaCOLD1^M187K significantly enhanced its interaction with TaG?-7A.Moreover,LCI assays showed that the C-terminal region of TaG?was required for the interaction of TaG?and TaCOLD1.Previous study has shown that Ta DEP1 protein(an atypical heterotrimeric G protein?subunit)regulates wheat plant height.In this paper,we cloned Ta DEP1 gene from hexaploid wheat.LCI assays showed that Ta DEP1 interacted with TaG?-7A,but not TaG?-1B.Similarly,the C terminus of TaG?was required for its interaction with Ta DEP1.The GGL and v WFC domains of Ta DEP1 mediated its physical interaction with TaG?-7A.We discovered that both TaCOLD1 and mutated TaCOLD1^M187K could repress the physical interaction between TaG?and Ta DEP1 through competitive interaction assays.This study revealed that TaCOLD1 might act as a novel regulator of wheat plant height through interfering with the formation of heterotrimeric G protein complex in common wheat and provide a new candidate gene for wheat architecture improvement.