Functional Analysis Of Brassica Napus BnTTG1-1 And Foxtail Millet(Setaria Italica)SiTTG1 Transcription Factors On Seed Accumulation And Other Biological Processes

TRANSPARENT TESTA GLABRA1 of Arabidopsis thaliana(AtTTG1)fucntions as a WD40 repeat transcription factor in the nucleus,and plays important roles in the regulation of trichome initiation,anthocyanin biosynthesis,and storage reserves accumulation.To determine the functions of the homologous gene BnTTG1 and SiTTG1,we cloned the full length coding sequence(CDS)from the Brassica napus cultivar “QINYOU Seven” and the foxtail millet(Setaria italica)cultivar “YUGU1”,analyzed the protein sequence using bioinformatics methods and subcellular localization in the tobacco leaves.The overexpression vectors of 35S:BnTTG1-1 and 35S:SiTTG1 were transformed into the mutant ttg1-13,respectively,and then the homozygous transgenic lines of ttg1-13 35S:BnTTG1-1 and ttg1-1335S:SiTTG1 were successfully obtained.We focused on the functions of BnTTG1-1 and Si TTG1 on lipid accumulation and abiotic stress responses in Arabidopsis,and also analyzed their roles in trichome initiation,anthocyanin biosynthesis,and other biological processes.The main results are listed as follows:(1)The bioinformatics analysis of the transcription factor TTG1 showed that the protein sequences of QINYOU.BnTTG1 and YUGU.SiTTG1 were identical to the BnTTG1-1 and SiTTG1 protein sequences,respectively.Therefore,QINYOU.BnTTG1 and YUGU.Si TTG1 were designated as BnTTG1-1 and SiTTG1 in this study,respectively.The protein sequences similarities of BnTTG1-1 and AtTTG1,SiTTG1 and AtTTG1 reached up to 99% and 60%,respectively.And they had highly conserved WD40 repeat regulatory domains.These suggested that BnTTG1-1,SiTTG1,and AtTTG1 may have similar functions.(2)The results of subcellular localization of tobacco leaves(Nicotiana benthamiana)showed that both BnTTG1-1 and SiTTG1 were localized in the nucleus of tobacco leaf cells.AtTTG1 is a WD40 repeat transcription factor,thus supporting that BnTTG1-1 and Si TTG1 also function as transcription factors.(3)In the mutant ttg1-13,overexpression of either BnTTG1-1 or SiTTG1 gene not only restored the phenotypes of the mutant without trichome initiation and anthocyanin biosynthesis,but also restored seed phenotypes,including yellow seed coat and the lack ofseed coat mucilage.These indicated that BnTTG1-1,SiTTG1,and At TTG1 exibits similar functions in regulating multiple biological processes of plant growth and development,including trichome formation,anthocyanin biosynthesis,and seed coat mucilage deposition.(4)Analysis of seed storage reserves accumulation showed that the contents of seed fatty acids and storage proteins in ttg1-13 mutant were significantly higher than that of the wild type,and the ectopic expression of either BnTTG1-1 or Si TTG1 gene in the the mutant background fully restored the fatty acid and storage protein contents to the wild type level.These results indicated that BnTTG1-1 and SiTTG1 have similar functions with AtTTG1 in regulating the accumulation of seed storage reserves.(5)Analysis of responses to the abiotic stresses showed that loss of function of AtTTG1 is sensitive to the stressed environments,such as higher concentrations of glucose and NaCl.Overexpression of either BnTTG1-1 or SiTTG1 fully restored the sensitivity of the mutant to these two abiotic stresses during seed germination and seedling establishment.These suggested that BnTTG1-1 and SiTTG1 have similar functions with AtTTG1 in regulating seed germination and seedling establishment under abiotic stress.(6)Expression analysis of genes related to the above mentioned biological processes showed that the expression of AtGL2 and AtMUM4,which promotes seed coat mucilage biosynthesis,were significantly reduced in ttg1-13 mutant;this is consistent with the phenotype of mucilage deficiencyin ttg1-13 seed coat.The genes At LEC2,AtBCCP2,AtMOD1,and AtFAE1 helpful for the seed fatty acid accumulation,and At2S2-At2S5 promoting the storage protein biosynthesis,were significantly up-regulated in ttg1-13;these are consistent with the higher fatty acids and storage protein contents in ttg1-13 seeds.Under abiotic stress,the expression levels of AtABA1,AtNCED3 and AtABI2 related to ABA biosynthesis and signal transduction pathways,and other stress-responsive genes inclusive of AtRD22,At KIN1,AtADH,AtERD15,and AtRAB18 in mutant ttg1-13 seedling were lower than that of the wild type seedlings;this is consistent with the sensitivity of ttg1-13 seedlings to abiotic stresses.The expression levels of all the referred genes above in ttg1-1335S:SiTTG1 seedlings were restored to the wild type levels.These taken together further indicated that SiTTG1 has a similar function to AtTTG1 in terms of many biological processes,such as seed coat mucilage biosynthesis,storage reserves accumulation,and responses to abiotic stresses.In summary,both BnTTG1-1 and SiTTG1 transcription factors are functionally similar to AtTTG1 in regulating plant growth and development,demonstrating that TTG1 functions are highly conserved in dicotyledon and monocotyledon.This study not only helps us understand the functional similarities and differences of TTG1 genes indicotyledon and monocotyledon,but also provides theoretical basis and excellent genetic resources for either traditioinal and mocular breeding practices.