Studies On The Regulation Of Gene Expression Of Sporopollenin Synthesis By The Arabidopsis Transcription Factor MS188

Pollen wall can protect pollen grains from external environmental damage and help promote recognition and hydration between pollen and stigma to complete the fertilization process.The mature pollen wall can be divided into exine and intine,and exine can be further divided into sexine and nexine.As model plant,Arabidopsis pollen sexine is composed of a T-shaped structure composed of sporopollenin and pollen coat composed mainly of oils and fats.Sporopollenin is a complex formed by the polymerization of various compounds.Its complex composition and tough nature make it extremely resistant to extreme environments.The current study suggests that the main components of sporopollenin are long-chain lipid derivatives and aromatic compounds.In recent years,several genes involved in the biosynthesis of sporopollenin have been identified through molecular genetic studies.The acyl-CoA synthase ACOS5,the cytochrome P450 hydroxylase family members CYP703A2 and CYP704B1,the chalcone synthase family member PKSA and PKSB,tetraketide-a-pyrone reductase TKPR1/2 and acyl-CoA reductase MS2 are all involved in the synthesis of sporopollenin components,and it is speculated through biochemical experiments that these genes form a path of formation of sporopollenin precursor component.However,there is currently only a small amount of research on how these genes are precisely regulated to exert their biological function.Tapetum cells provide the raw materials for the development of pollen wall and it is considered that the sporopollenin precursors are synthesized in tapetum by the above genes and transported to the surface of the microspore to form sexine layer.Our lab found a genetic regulatory pathway 'DYT1-TDF1-AMS-MS 188-MS 1' that exists in the tapetum cells.MS 188 is a member of MYB super transcription factor family.In ms188 mutant,the T-shaped structure of sexine is obviously absent,while intine layer is normal,suggesting that MS 188 may regulate the synthesis of sporopollenin.The bHLH family transcription factor AMS has been reported to bind the promoter of PKSB and TKPR1 in vivo,and MS 188 can interact with AMS to regulate and activate the expression of CYP703A2.In this paper,we first observed the protein localization of the sporopollenin synthase by constructing GFP-tagged transgenic plants and found that the sporopollenin synthase were significantly present in the tapetum cells and locules,suggestting that the sporopollenin precursors might also be synthesized and processed in the locules.Subsequently,we analyzed the chip data of the inflorescences of dytl,tdfl,ams,and ms 188 mutants,and verified with quantitative PCR experiments that the sporopollenin synthesis genes PKSA,PKSB,MS2,and CYP703A2 were downstream of the transcription factor MS188,while ACOS5,CYP704B1 TKPR1 and TKPR2 are downstream of AMS.MS 188 can bind to the promoters of PKSA,PKSB,MS2,ACOS5,and TKPR1 through chromatin immunoprecipitation(ChIP)and electrophoretic mobility shift assays(EMSA),and MS 188 was also able to bind to the promoter of CYP704B1 in vivo.Further,a transient over expression assay using tobacoo(Nicotiana benthamiana)leaf and Arabidopsis protoplast was performed to verify that MS 188 could activate the expression of PKSA,PKSB,MS2,TKPR1,ACOS5 and CYP704B1;however,AMS could not significantly activate the expression of TKPR1 and ACOS5,and the activation of CYP704B1 was weaker than that of MS 188,indicating that MS 188 is a core transcription factor that regulates the synthesis of sporopollenin genes.When AMS co-exists with MS 188,it can strongly activate the expression of CYP704B1.Combined with the results of AMS and MS 188 co-activation of CYP703A2,we speculated that MS188 might regulate the sporopollenin biosynthesis with AMS.We constructed the MS188-SRDX dominant suppressor transgenic plants and found that the expression of ACOS5,CYP704B1,and TKPR1/2 was significantly suppressed when the expression AMS was not affected in ms188 mutant.When AMS was absent,normal expression of MS188 significantly restored the expression of PKSA,PKSB,ACOS5,CYP704B1,and TKPR2,whereas the expression of MS2 was identical to that of CYP703A2 and only partially recovered.These results suggest that other MYB and bHLH family transcription factors are also involved in the regulation of sporopollenin synthesis genes by MS 188 and AMS in the tapetum.We conducted a preliminary verification by yeast two-hybrid experiment and found that bHLH10 can interact with MS 188,which supports our hypothesis.Based on the above results,in this paper we discussed the possible mechanism of the multi-transcription factor complex formed by the transcription factor MS 188 and the feed forward loop formed by AMS and MS188 in the tapetum of Arabidopsis thaliana in regulating the fast expression of sporopollenin synthesis genes.The study of these mechanisms has helped us to understand more deeply the biological process of the regulation of pollen wall formation by the tapetum transcription factor and the importance of this process in the plant evolution to protect the plant's pollen and normal reproduction.