Analyses Of The Lipid-related Transcriptome And Phosphoproteomics And The Role Of Fatty Acid Export 6(FAX6)in Brassica Napus

Oilseed rape,including Brassica napus L.,Brassica rapa L.and Brassica juncea L.,belong to brassica of the family cruciferae.Brassica napus(AACC,2n=38)is an allotetraploid of Brassica oleracea(CC,2n=18)and Brassica campestris(AA,2n=20)after hybridization and doubling under natural conditions.Oilseed rape is one of China’s four major oil-bearing crops and widespreadly planted in the middle and lower reaches of the Yangtze river in China.Canola oil is of high nutritional value with high concentrations of oleic acid(Cl8:1)and linoleic acid(Cl8:2)and really loved by the people.Improve seed oil content and yield of oilseed rape has important theoretical and practical significance for meeting people’s demand for rapeseed oil and safeguarding national vegetable oil safety.However,the mechaninms and regulatory-model of oil biosynthesis and branch formation are not clear.This paper focuses on the following three aspects of this scientific problem.1.Transcriptome profiling analysis reveals the role of silique in controlling seed oil content in Brassica napus.RNA sequencing technique(RNA-Seq)was performed to explore differentially expressed genes in siliques of two Brassica napus lines(HFA and LFA which contain high and low oil contents in seeds,respectively)at 15 and 25 days after pollination(DAP).The RNA-Seq results showed that 65746 and 66033 genes were detected in siliques of low oil content line at 15 and 25 DAP,and 65236 and 65211 genes were detected in siliques of high oil content line at 15 and 25 DAP,respectively.By comparative analysis,the differentially expressed genes(DEGs)were identified in siliques of these lines.The DEGs were involved in multiple pathways,including metabolic pathways,biosynthesis of secondary metabolic,photosynthesis,pyruvate metabolism,fatty metabolism,glycophospholipid metabolism,and DNA binding.Also,DEGs were related to photosynthesis,starch and sugar metabolism,pyruvate metabolism,and lipid metabolism at different developmental stage,resulting in the differential oil accumulation in seeds.Furthermore,RNA-Seq and qRT-PCR data revealed that some transcription factors positively regulate seed oil content.Thus,our data provide the valuable information for further exploring the molecular mechanism of lipid biosynthesis and oil accumulation in B.nupus.2.The ABA-BnSnRK2;2/BnCDPK6-BnLECl-[BnNFYCs]cascade regulate lipid biosynthesis in rapessed seed.Based on the quantitative phosphoproteomic analysis of 25 days after pollination(DAP)seeds in two Brassica napus with inverse oil content and CoIP/MS analysis,we found that ABA mediated phosphorylation of BnLEC1 through kinase of BnSnRK2;2 and BnCDPK6 response to sugar signal played vital role in oil synthesis.ABA was highly biosynthesized during key period for seed oil synthesis.Exogenous ABA enhanced the kinase activity of BnSnRK2;2 by heightening phosphorylation its Thr175 residue located in activation domain,and then promoted the phosphorylation of BnLEC1 at Ser17,Ser153 and Ser158 residues.Moreover,BnCDPK6 could also phosphorylate these three Ser residues of BnLECl.BnLECl-[BnNF-YC 1/4/9A/9B]heterodimer were assembled only when the Ser153 and Ser158 of BnLEC1 were phosphorylated and translocated into nucleus when the Ser1 7 of BnLEC1 were phosphorylated,and then promoted oil biosynthesis through heightening the expression of a subset of genes involved in glycolysis and lipid biosynthesis.The nonphosphorylated BnLEC1 were rapidly degraded.Consistent with seed-specific ovexpression of BnSnRK2;2,BnCDPK6 and BnLECl,overdose ABA causes an decreased protein content and increased oil content and long chain fatty acids(LCFAs)ratio in rapeseed seed.Futhermore,exogenous glucose promote both the expressions of genes in ABA biosynthesis and BnCDPK6 leading to the up-regulated phosphorylation of BnLEC 1.Taken together,we uncovered a homeostatic mechanism that ABA-BnSnRK2;2/BnCDPK6-BnLECl-[BnNF-YCs]signalling cascade promoted flow of carbon source to lipid biosynthesis but not protein response to intracellular sugar state,providing important cues for future plant breeding.3.Fatty acid export protein BnFAX6 regulate axillary bud releasing.BnFAX6 were preferentially expressed in axillary bud and seeds during critical period of oil synthesis.Moreover,the expression of BnFAX6 was enhanced after the loss of the shoot tip.Overexpression BnFAX6 increased oil content in seed and leaf through enhancing the flow of photosynthetic products to fatty acid biosynthesis and heightening the expression of a subset of genes involved in glycolysis and lipid biosynthesis.Furthermore,enhancing the expression of BnFAX6 resulted in semi-dwarf and increased branching phenotypes with more siliques,contributing to increased yield per plant relative to wild type,the process of which depended on the role of leaf.Metabonomics analysis showed that,consistent with decapitation,overexpression of BnFAX6 resulted in most metabolites levels rose rapidly,especially lipid including linoleic acid and related derivatives in axillary bud.However,the level of various kinds of sugar including sucrose decreased rapidly,leading to the flow of the carbon from photosynthetic products to fatty acid biosynthesis in axillary bud.Increasing linoleic acid levels in axillary bud represses the expression of the key transcriptional regulator(BnBRC1,BnBRANCHED1)responsible for maintaining bud dormancy,and resulted in rapid bud release.Taken together,BnFAX6 as a key valve command the flow of carbon source of photosynthetic products from sugar to fatty acid and derivatives derived from fatty acid,regulating the oil accumulation and branch formation.