Characterization Of GhACBP4 In Anther Development Of Cotton

In recent years,the regional high temperature caused by global warming becomes a serious factor in leading to the crop male sterility and threatens agriculture production.Studies have shown that GhCKI has important functions in causing cotton male sterility under high temperature(HT).To determine the mechanism of male sterility in cotton due to high temperature stress,the GhACBP4(acyl-CoA-binding protein 4)which interacted with GhCKI through yeast two-hybrid screening was identified.Three types of phosphorylated peptides of ACBP4 were further screened out from phosphorylated protein profiles of GhCKI over-expressing Arabidopsis buds and HT-sensitive cotton H05 anthers.Therefore,this study mainly analyzes the functions of GhACBP4 in anthers.The main results are as follows.1.In the upland cotton(Gossypium hirsutum)genome,six homologous genes of GhACBP4 were found through sequence analysis.In expression pattern analysis,Gh_A13G0893/Gh_D13G1134 has the highest expression in anther as compared to other tissues.Whereas in the expression pattern of proGh_A13G0893::GUS transgenic cotton,the result showed that the gene expression was high in the anther,ovule and germinating seeds.In addition,the whole GhACBP4 homologous genes might be down-regulated in anther dehiscence stage(ADS,>24mm bud)during high temperature stress.2.The gene Gh_A13G0893 was selected for further functional verification after comparison of expression level of six homologous genes and three types of phosphorylated peptides.The male sterile plants could be found both in the overexpressors of Gh_A13G0893 and Gh_A13G0893-3'UTR-RNAi transgenic plants of cotton.The expression of six homologous genes of GhACBP4 decreased in sterile plants of Gh_A13G0893-3'UTR-RNAi,consistent with the male sterility phenotype of acbp4acbp5 double mutant in Arabidopsis.Through Transmission Electron Microscopy(TEM)to observe the sterile anthers of GhACBP4-overexpressor,the sterile pollens were lack of starch and lipid droplets,and absent of intine,and there was no lipid droplet in tapetum at tapetal degradation stage.In addition,there was absent of tryphine in the pollen exine at anther dehiscence stage.3.In the GhACBP4 overexpressors,the sterile anthers showed a decreased content of fatty acid compared with wild type.Meanwhile,the fatty acids content of YZ1 and H05 were significantly changed after high temperature stress.By using the thin layer chromatography,we also found significant lipid changes in the sterile anthers of GhACBP4-overexpressor and the anthers of H05 induced under high temperature stress.The results mean overexpressing GhACBP4 showed a similar phenotype as the wild type plants under high temperature stress.4.To find if GhCKI and GhACBP4 interacts and to localize the two proteins,yeast two-hybrid,bimolecular fluorescence complementation and bimolecular luciferase complementation experiments were performed and found that the interaction really existed between GhCKI and GhACBP4,and the two proteins were localized to peroxisome.However,if 501 and 517 amino acid sites were mutated from serine to glycine,GhACBP4 with S501 A could not interact with GhCKI,but the weak interaction was found between GhACBP4 with S517 A and GhCKI.5.In the process of prokaryotic expression,the GhACBP4 protein with histidine tag may have a variety of phosphorylation status,of which could not be phosphorylated by GhCKI in vitro.6.After comprehensive analysis,we speculate that when GhCKI transcription is activated by overexpressing GhACBP4,a large number of GhACBP4 proteins will be phosphorylated by GhCKI,and then the activity of GhACBP4 protein may be decreased,or this protein is degraded.Maybe GhACBP4 protein level will decrease with its transcription inhibited.Moreover,there may be these two case simultaneously under high temperature.Therefore,these results may cause abnormal changes in lipid transportation in the peroxisome,and lead to the pollen abortion eventually.