Functional Characterization Of BnTT1 Involved In Seed Development And Metabolism Of Brassica Napus By RNAi

Oilseed rape is one of the four major oil crops in the world and belongs to Brassica,Cruciferae.As one of the three cultivars of oilseed rape,Brassica napus(2n = 38,AACC)is one of the most important oil crops throughout the world,and is the largest rapeseed crop in China.Increasing yield of rapeseed,and improving oil content and quality at the same time is the core goal of contemporary rapeseed breeding.Compared with black-seeded rapeseed,yellow-seeded rapeseed has thinner testa,higher oil content,and lower pigment and hull percentages.Therefore,yellow-seed traits are an important breeding goal in rapeseed breeding.Among the three cultivars of oilseed rape,Brassica napus has the highest yield of rapeseed,but it lacks natural yellow-seed material.The seed color of Brassica napus is highly correlated with the secondary metabolic pathway of flavonoid biosynthesis.In-depth study of rape flavonoid pathway is not only helpful to figure out the formation mechanism of rapeseed coat color,but also is important to yellow-seed breeding target.In this study,we have performed functional identification of BnTT1,a regulatory factor of flavonoid biosynthesis in Brassica napus.The main results are as follows: 1 Transgenic phenotype of BnTT1 gene family RNAi linesThe growth and development of four transgenic B.napus lines were slower than those of WT.The transgenic lines at seedling stage were short,and the bolting and flowering of transgenic plants were delayed.The transgenic lines could blossom,pollinate and bear seeds normally,and transgenic plants could reach the normal plant height of ZS10 at the end of the flowering,but the number of branches decreased.The seed phenotype survey showed that the mature seeds of the transgenic lines were yellow,the grain was small,and the 1000-grain weight was low.Seed sections showed that both the seed coat of developing seeds and mature seeds of transgenic lines was thinner than the WT.2 BnTT1 gene family regulates flavonoid biosynthesis and affects B.napus seed development In our study in B.napus,expression of TT1 family genes was suppressed,and as a result,mature transgenic B.napus seeds were yellow,small,and had reduced weight and thinner seed coat.Anatomic observation of developing transgenic seeds of B.napus showed that developing transgenic seeds had thinner inner and outer integuments than the WT seeds had.In the seed coat of B.napus,we observed five cell layers,including one epidermis cell layer,one endothelium cell layer,and three palisade cell layers.The cells in the palisade layer of transgenic seeds were thinner and smaller than those in WT seeds,which resulted in a thinner palisade layer and contributed to a thinner integument,together with a thinner endothelium cell layer in developing transgenic seeds.These results suggest that BnTT1 s can affect the development and morphology of the palisade and endothelium cell layers of seeds in B.napus.Meanwhile,slight staining of vanillin and TBO indicated decreased accumulation of catechin and phenolic compounds in transgenic seed coats.RNA-seq analysis of developing transgenic seeds and WT seeds revealed a large number of DEGs involved in flavonoid biosynthesis and transportation,including regulatory and structural genes.qRT-PCR analysis showed that most of the structural genes were down-regulated in transgenic B.napus seeds,including CHS / TT4,DFR / TT3,LDOX /TT18,ANR/BAN,TT12,CFI/TT5,F3H/TT6 and GSTF12/TT19.As a consequence of this down-regulated expression,the levels of seed flavonoids such as epicatechin,quercetin and its derivatives(Qn-3-O-sophoroside,Qn-3-O-glucoside-1),isorhamnetin and its derivatives(Is-3-O-glucoside,Is-3-O-glucoside-7-O-glucoside),kaempferol derivative(Km-3-O-glucoside-7-O-glucoside),soluble PCs(Procyanidin B2 [DP 2],[DP 2]-2,[DP 3],[DP 3]-2,[DP 4]),and insoluble PCs were lower in transgenic than in WT seeds.These results suggest that BnTT1 gene family regulates flavonoid biosynthesis,and deficiency of BnTT1 down-regulates the expression of flavonoid biosynthetic genes,leading to the decreases of epicatechin and its oligomers contributed to alteration of the color of the transgenic seeds.Moreover,when CHS/TT4,DFR/TT3,LDOX/TT18,ANR/BAN,TT12,CFI/TT5,F3H/TT6,and GSTF12/TT19 were notably down-regulated in transgenic seeds,the very late biosynthetic genes such as LAC15/TT10 and some UGTs were markedly up-regulated.They are involved in the formation of flavonoid end products.Although LAC15 was markedly up-regulated,the levels of insoluble PAs were still decreased,indicating that there was a severe deficiency in the levels of the PA precursors such as epicatechin.Above component analysis and quantitative PCR evidence together showed that the BnTT1 family also regulated the accumulation of embryo flavonol in addition to seed coat pigment.Moreover,expression specificity analysis showed that BnTT1 genes were not only expressed in the seed coat but also significantly expressed in the seed embryo,suggesting that the BnTT1 gene family may be directly involved in regulating the development and metabolism of seed embryo.In addition,anatomical observation showed that transgenic seeds not only had a thinner endothelium layer,but also had a thinner palisade layer(main part of the seed coat),suggesting that BnTT1 gene family was not only involved in the development of inner integument,but also involved in the development of outer integument.3 BnTT1 genes affect the expression of genes of the common phenylpropanoid pathwayRNA-seq analysis showed that the gene expression profile of the phenylpropanoid pathway was significantly changed.All the expression of the three enzyme genes involved in the common phenylpropanoid pathway was down-regulated in different degrees.qRT-PCR analysis showed that the total expression of PAL,C4 H and 4CL genes was significantly down-regulated,but the members of each gene have different expression pattern,suggesting functional division between members.Meanwhile,the impact of BnTT1 gene family silencing on the expression of genes involved in common phenylpropanoid pathway was selective and specific,for example,among the 4CL gene family members,only the expression of 4CL3 was influenced,suggesting that BnTT1 selectively and specifically regulate the gene members involved in flavonoid biosynthesis.4 Silencing BnTT1 gene family upregulates lignin biosynthesis in seed coatLignin pathway is the branching pathway of phenylpropanoid pathway.DEG analysis showed that gene expression of lignin pathway was also changed,mainly including β-glucosidase gene family,peroxidase gene family,HCT,CAD,F5H1,UGT84 A,etc.qRT-PCR survey showed that the gene expression of C3 H,CAD,CCoAOMT,F5 H,F5H1 and UGT84 A were slightly up-regulated in the transgenic seeds,while COMT1 and CCoAOMT1 genes were slightly down-regulated.As a consequence of these changes in gene expression,the lignin content of transgenic seeds also changed slightly but significantly,showing a certain degree of rise,suggesting that the impact of BnTT1 gene family silencing on the lignin synthesis and metabolism is relatively minor.It is possible that BnTT1 silencing leads to drastic decrease of flux to flavonoid branch pathwasy,thus correspondingly increases the flox to the comptetive lignin branch pathway,which promotes the lignin pathway gene expression and synthesis and deposition of lignin in the seed coat.5 BnTT1 gene family affects fatty acid biosynthesisAs a class of aliphatic carboxylic acids,FAs are widespread in plant seeds and play important roles throughout the lifespan of plants.To day,functions of TT1 in affecting seed FA biosynthesis and altering the accumulation of FA composition remain unknown.In this study,we found that suppression of BnTT1 gene expression resulted in significant alteration of FA composition,including decrease of C18:1 and increases of C18:2 and C18:3.Meanwhile,the total FA content remained unchanged.Accompanying this compositional variation,DEGs showed corresponding alterations,in which the expression of many FA biosynthetic genes encoding key enzymes in plastids and the endoplasmic reticulum were altered by BnTT1 suppression,including ω-6 FA desaturase gene FAD6,ω-3 FA desaturase gene FAD3,stearoyl-ACP desaturase genes,etc.Markedly increased expression of FAD6 and FAD3 could be the reason for the clear increases of C18:2 and C18:3 in transgenic seeds,respectively.Stearoyl-ACP desaturase genes such as AACPD2,AACPD3,and AACPD5 were slightly up-regulated,except SSI2,leading to a slight decrease of C18:0.The expression of KASII was slightly down-regulated in transgenic seeds,whereas KASI exhibited no significant change,which could help to explain the slight increase of C16:0.No clear upregulation in the expression of ACETYL was detected by qRT-PCR,which was consistent with the unchanged total FA content in transgenic seeds.Several key transcriptional factors regulate FA biosynthesis during seed maturation,including ethylene-responsive transcription factor WRI1;B3 domain-containing transcription factors ABI3,LEC2,and FUS3;nuclear transcription factor Y subunit B-6 L1L;and nuclear transcription factor Y subunit B-9 LEC1.RNA-seq and qRT-PCR analysis showed that expression of ABI3,L1 L and LEC1 was significantly up-regulated in developing transgenic seeds.Hence,it was possible that the aberrant expression of ABI3,LEC1,and L1 L altered transcript levels of FAD6,FAD3,KASII,and AACPDs,which further resulted in the alteration of FA composition and seed development.These results suggest that BnTT1 genes may also be involved in the metabolism of fatty acids in addition to the secondary metabolism of flavonoids.6 Effects of BnTT1 gene on the growth and development of B.napus plantInvestigation of transgenic plants from seedling to flowering showed that the growth and development of the transgenic lines were delayed.Transgenic lines at the seedling stage were significantly shorter than WT,and the bolting and flowering time of transgenic lines was delayed 5 to 10 days compared with those of WT.Mature transgenic plants had no significant difference in plant type and plant height compared with WT,but the number of branches decreased.Those results showed that the BnTT1 genes could affect the growth and development of B.napus plants,but expression analysis showed that the expression of BnTT1 genes was not detected in vegetative organs of B.napus such as roots,stems and leaves,suggesting that the growth retardation of the transgenic lines should not be directly affected by the silencing of the BnTT1 gene family.In addition,T1 generation plants from tissue culture had not those phenotypic differences compared with the control group.Hence,we proposed that it is the indirect effect of BnTT1 silencing.As the seed development was affected by BnTT1 silencing,the transgenic seeds were significantly smaller than WT seeds,and nutrient reserve of transgenic seeds were abnormal,leading to delayed seed germination and later growth and development of T2 and T3 generation plants,further more,affected by this negative effect,the bolting and flowering time of transgenic plants were also later than those of WT,and the branches are also reduced accordingly.