Study On The Function Of Seed Coat Color Regulating Genes TT20 And TT21 In Arabidopsis Thaliana

The transparent seed coat character of Arabidopsis thaliana is a kind of character widely and deeply studied at present.It is generally believed that the appearance of transparent seed coat of Arabidopsis thaliana is mainly related to proanthocyanidins produced by flavonoid metabolic pathway.Proanthocyanidins mainly exist in the seed coat cells of plant seeds.Under normal circumstances,proanthocyanidins are easy to be oxidized into brown substances under the action of oxidase,resulting in the brown seed coat of Arabidopsis thaliana.When the genes of some metabolites or enzymes in the flavonoid metabolic pathway are mutated,or the genes related to the transport and polymerization of proanthocyanidins are mutated,the seed coat color of plants will change and the transparent seed coat character will appear.At present,most of the structural and regulatory genes in the plant flavonoid synthesis pathway have been found,but there are still many questions,such as the transport pathway of proanthocyanidins,the polymerization and oxidation process of proanthocyanidins,which still need to be deeply studied.Therefore,exploring more transparent seed coat mutations,cloning related genes and clarifying their regulation mechanism can not only enrich and expand the flavonoid synthesis pathway,but also lay a foundation for plant improvement of flavonoid related traits through metabolic engineering.In this study,a homozygous double mutant tt20tt21 with transparent seed coat character was screened.Through subcellular localization,GUS staining experiment and metabolomic analysis,we carried out a series of analysis on the screened homozygous double mutant tt20tt21,and obtained the following results:(1)Comparing the seed coat color of double mutant tt20tt21 with that of wild-type Arabidopsis,single mutants tt20 and tt21,we determined that the seed would have transparent seed coat character only when both of the two genes of TT20 and TT21 were mutated.In addition,We found that whether the two genes were mutated at the same time or not,it had no significant effect on the development of Arabidopsis.Therefore,we can determine that the simultaneous mutation of the gene TT20 and TT21 mainly affects the formation of seed coat color;(2)In order to understand the localization of TT20 and TT21 proteins in cells,we designed GFP subcellular localization experiment.It was found that the proteins expressed by the gene TT20 and TT21 were localized in the periphery of chloroplast in the form of spots;(3)In order to understand the expression of the gene TT20 and TT21 in plants,the promoters of the gene TT20 and TT21 were connected to GUS gene,and the location of gene expression was determined by detecting GUS fluorescence signal.The results showed that the gene TT20 and TT21 accumulated in large quantities in reproductive organs of plant,especially seed coat,suggesting that they may play an important role in the formation of seed coat color;(4)In order to analyze the formation mechanism of transparent phenotype of homozygous double mutant tt20tt21,we analyzed the metabolites in mature dry seeds of WT and tt20tt21 by extensive targeted metabonomics.The results showed that abnormal accumulation of epicatechin,the precursors of proanthocyanidins synthesis,and proanthocyanidins may be the main reason for the color change of seed coat in the double mutant.Then,we enriched the detected differential metabolites in KEGG pathway.The results showed that the top five pathways with the highest enrichment were flavonoid and flavonol anabolic pathway,arginine biosynthetic pathway,flavonoid synthesis,lysine degradation metabolism and tyrosine metabolism.Therefore,we can basically confirm that the flavonoid synthesis pathway in tt20tt21 mutant seeds is affected,especially the excessive accumulation of downstream proanthocyanidins and their precursors,which leads to the transparent seed coat phenotype of the mutant.(5)In order to further confirm the results of mass spectrometry,we detected the proanthocyanidins in mature seeds by chemical staining.The results showed that proanthocyanidins metabolites were accumulated in the seed coat of both WT and tt20tt21 mutant,which proved the accuracy of mass spectrometry.In conclusion,we obtained a double mutant tt20tt21 with transparent seed coat phenotype,and found a new set of the gene TT20 and TT21 that regulate seed coat color in Arabidopsis.Through this study,we found that the gene TT20 and TT21 function redundantly to regulate the seed coat color of Arabidopsis,and determined that the the gene TT20 and TT21 are mainly expressed in the reproductive organs of plants,and TT20 and TT21 proteins are located on the chloroplast membrane.Metabolic analysis showed that simultaneous mutation of the gene TT20 and TT21 leds to the abnormal metabolic process of proanthocyanidins downstream of flavonoid synthesis pathway,resulting in the excessive accumulation of proanthocyanidins,and finally affected the oxidation process of proanthocyanidins.