The Role Of CER3 And Other Genes In Pollen Hydration And Stem Wax Synthesis In Arabidopsis

Pollen and stigma recognition is the premise for successful fertilization of compatible pollen on the stigma,and this recognition is also one of the most critical processes for the sexual reproduction of angiosperms to form mature fruits.In order to study the intercellular communication and interactions between associated substances,we screened seeds of lines from the ABRC mutant libraries of Arabidopsis thaliana,and finally identified a mutant line Salk086910 with severely reduced male fertility.After hybridization with the wild-type plants,PCR amplification and screening in Kanamycin-containing MS medium,we found that the mutant line was not a T-DNA insertion mutant.By using map-based cloning method,the mutant gene was found to be located in a 600 kb region of chromosome V between markers MMN10 and MHJ24 in the Arabidopsis genome.Combined with mutant phenotypes: the stalk surface appears bright green and lacks wax,we think the target gene is the CER3 gene in the mapping region.The upstream and downstream genes of this gene were amplified by PCR and sequenced.Finally,we found that 6 genes were deleted from the promoter region of the gene CER3 to the second intron of the AT5G57840 gene.The in vitro pollen germination experiment showed that the mutant pollen germination activity was normal,while the mutant pollen could not germinate on the mutant stigma leading to the male sterility.At the same time,pollen on the mutant stigma were found not to hydrate,and callose was found in the stigma surface cells.Transmission electron microscopy observation of developing anthers revealed abnormal formation of lipid material in the tapetum.Scanning electron microscopy observations revealed that mutant stems lacked wax.The results of the genetic complementation experiment of the CER3 gene indicated that the mutant fertility was restored,but the stem wax was not returned to normal.In situ hybridization experiments showed that the CER3 gene was highly expressed in the tapetum and microspores of the stage 9 anther.Further experimental analysis revealed that the CER3 protein was localized on the cytoplasm membrane.These results indicate that CER3 may be involved in the synthesis or transport of lipids in the tapetum and is essential for pollen and stigma signaling.