| Rapeseed(Brassica napus L.)is one of the most important oil crops in the world.And it is also the largest oil source of domestic vegetable oil in China.At present,it is an important topic to study the regulation mechanism on seed oil accumulation of B.napus.Arabidopsis thaliana,a member of the Cruciferae,is a close relative of B.napus.Thus,A.thaliana serves as a good model material to study the molecular mechanism and regulatory network of plant seed oil accumulation.Transcription factor(TF)and phytohormones have been reported that they are involved in regulating the expression of key enzyme genes in the pathway of oil biosynthesis,and they play a crucial role in the process of seed oil accumulation.The purpose of this study was to reveal the effects of MYB89 TF and melatonin on seed fatty acid(FA)accumulation,and their underlying molecular mechanisms in A.thaliana and B.napus.The main results of this study are listed as follows:(1)Analysis of AtMYB89 expression patterns.The result of subcellular localization indicated that AtMYB89 was localized in the nucleus in tobacco leaves.To investigate the expression of AtMYB89,we measured its transcript levels in various tissues of wild-type(Col-0)plants by using the GUS staining,quantitative real-time PCR(q RT-PCR)and in situ hybridization,and we found that the AtMYB89 was expressed in roots,stems,leaves,flowers and seeds.And AtMYB89 was highly expressed in developing seeds at 10 day after pollination(DAP)to 16 DAP.These results suggested that AtMYB89 may be relevant to the accumulation of seed FA.(2)AtMYB89 represses seed FA accumulation.The contents of seed total FA and each FA compositions in T-DNA insertion mutant of AtMYB89 were significantly higher than those of Col-0 at different stages of seed development.Using the double-stranded RNA to inhibit the expression of AtMYB89 in the Col-0 background can also result in a significant increase of the seed total FA and each FA compositions.Furthermore,overexpression of AtMYB89 in the myb89-1 background could restore the phenotype of higher seed FA contents in myb89-1 seeds.Then the results of transcriptome analysis and q RT-PCR confirmed that a series of genes involved in seed FA accumulation were significantly regulated by AtMYB89,including At WRI1,At BCCP1 and At L1 L.Meanwhile,glucocorticoid receptor(GR)inducible system,chromatin immunoprecipitation assays(Ch IP)and q RT-PCR demonstrated that AtMYB89 inhibits seed FA accumulation by directly repressing the expression of the key genes,such as At WRI1,At ENO1 and At BCCP1,and indirectly regulating some other key genes,including At L1 L,At HD-L and At ROD1.(3)Analysis of Bna C01.MYB89 expression patterns.There were two Bn MYB89 homologs in B.napus,Bna C01.MYB89(XP＿013728631.1)and Bna A04.MYB89(XP＿013746758.2).The Bna C01.MYB89 gene was cloned from ZS11 varieties of B.napus.The results of subcellular localization indicated that Bna C01.MYB89 was specifically localized in the nucleus.The q RT-PCR indicated that Bna C01.MYB89 was expressed in roots,stems,leaves,flowers and seeds.And Bna C01.MYB89 was highly expressed in developing seeds.These results showed that Bna C01.MYB89 may be relevant to the accumulation of seed metabolites,such as seed FA.(4)Bna C01.MYB89 represses seed FA accumulation.Overexpression of Bna C01.MYB89 in the myb89-1 background could completely restore the higher seed FA contents and the expression level of FA biosynthesis genes to the Col-0 level.These results suggested that Bna C01.MYB89 and AtMYB89 have similar functions in regulating seed FA accumulation.(5)AtSNAT1 and AtCOMT involved in melatonin biosynthesis in A.thaliana siliques.The results of melatonin contents determination showed that snat1-1,comt-1,and comt-2contained much less melatonin than Col-0 plants in siliques,and the double mutant of AtSNAT1 and AtCOMT genes accumulated much less than their corresponding single mutants.In addition,The results of melatonin contents determination of the complementary transgenic lines showed that the phenotype of the lower melatonin content in both snat1-1 and comt-1were completely restored to Col-0 levels.These results mean that both of AtSNAT1 and AtCOMT involved in melatonin biosynthesis in A.thaliana siliques.(6)Analysis of AtSNAT1 and AtCOMT expression patterns.The results of subcellular localization indicated that AtSNAT1 was localized in chloroplast,AtCOMT was localized in cytoplasm and nucleus in tobacco leaves.The results obtained from the GUS staining and q RT-PCR indicated that AtSNAT1 was expressed in roots,leaves and flowers,and AtCOMT was expressed in roots,cotyledons and flowers among Col-0 plants.Additionally,both of them were also highly expressed in developing seeds.The results suggested that these two genes may be involved in the accumulation of seed metabolites.(7)Melatonin represses seed FA and anthocyanin accumulation in A.thaliana.The determination results of seed FA and anthocyanin showed that the contents of total FA and anthocyanin in mature seeds of each single and double mutant of AtSNAT1 and AtCOMT genes were significantly higher than Col-0.The contents of seed total FA in snat1-1 comt-1was significantly higher than that of each single mutant,and there were no significant difference in anthocyanin contents among comt-1,comt-2 and snat1-1 comt-1,but their anthocyanin contents were significantly higher than snat1-1.In addition,the results of FA and anthocyanin contents determination of the complementary transgenic lines showed that the higher seed FA and anthocyanin contents in both snat1-1 and comt-1 was completely restored to Col-0 levels.On the other hand,exogenous application of melatonin can significantly repress FA and anthocyanin accumulation in mature seeds of Col-0,snat1-1,comt-1 and snat1-1 comt-1.But,under treatment of exogenous melatonin,the seed anthocyanin contents of comt-1 and snat1-1 comt-1 were still significantly higher than Col-0and snat1-1.Then the transcriptome analysis and q RT-PCR demonstrated that a series of genes involved in FA biosynthesis were significantly regulated by melatonin,including At WRI1,At BCCP1 and AtCAC3.And the expression of anthocyanin biosynthesis genes were also significantly altered by melatonin,such as At4CL1,AtCHI and At KAN4.These results suggested that melatonin represses seed FA and anthocyanin accumulation by regulating the expression of FA and anthocyanin biosynthesis genes,respectively.And AtCOMT,independent of melatonin,has distinct effects on the biosynthesis of anthocyanin.(8)Exogenous melatonin represses seed FA accumulation in B.napus.We measured the contents of the FA compositions in mature seeds of B.napus inbred lines,LXX1 and LXX4.Under exogenous melatonin treatment,the seed FA contents of LXX1 and LXX4 plants were lower than that of them without exogenous application of melatonin,respectively.We used q RT-PCR to detect the expression levels of FA biosynthesis genes,including Bn WRI1,Bn BCCP1,Bn CAC3 and Bn MCAMT,in developing seeds at 28 DAP between LXX1 and LXX4(with or without exogenous melatonin treatment),the results showed that all of these four genes were significantly downregulated.In summary,these results suggested that melatonin represses seed FA accumulation by repressing the expression of these four genes in B.napus.Taken together,this study mainly revealed the regulatory mechanism of MYB89 and melatonin in seed FA accumulation.Then we identified the downstream genes of MYB89 and melatonin.And we compared the differences and similarities of the seed FA accumulation between MYB89 and melatonin in A.thaliana and B.napus,respectively.These findings not only deepen our understanding of the mechanisms regulating seed FA accumulation in A.thaliana and B.napus,but also provided excellent gene resources for high-oil content breeding of oilseed crops,such as B.napus,by biotechnology. |
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