| Rapeseed(Brassica napus L.)is a valuable crop cultivated worldwide.In China,it is the largest source of edible vegetable oil.China is among the leading countries for producing 8million tons of rapeseed oil from 1.4 billion hectare of cultivation area.Protein rich vegetable oil and rapeseed meal is very useful for human and animal consumption.Silique development of rapeseed is an important parameter to increase the seed yield and quality of rapeseed oil,as siliques walls play essential roles in seeds filling.Light is a critical factor for the synthesis and transport of these metabolites that are later transported into seeds,including phenolic acids,flavonoids,glucosinolates(GSLs),lipids and other polar compounds.Among these metabolites,glucosinolates are bioactive compounds that play crucial roles in plant defense mechanism against biotic and abiotic stresses.Meanwhile,their higher accumulation in seeds caused pungent taste and reduced the nutritional quality of rapeseed oil.So,it is necessary to attain the balanced level of GSL production and accumulation in rapeseed.The aim of this study was to investigate the dynamic changes of metabolites in siliques,effects of light on siliques metabolites,and comparing the accumulation and transportation of glucosinolates in siliques under shading condition.The major conclusions are as follow:1.Metabolite profiling of siliquesWe performed the metabolome study of siliques using UPLC-HESI-MS/MS method in negative mode and data were acquired in a mass range(m/z)from 100 to 1500.We identified3,245 discernible peak chromatograms with MS-DIAL software.On the basis of available standards,exacting MS,MS~2 spectral data and retention times,249 peaks of which were identified and classified into six groups,contained 32 phenolic acids,38 flavonoids,22glucosinolates,34 lipids,56 hydroxycinnamic acids,and 67 unknown compounds,respectively.Among them,89 compounds were quantified using external calibration curves of respective chemical standards,including phenolic acids(29),flavonoids(38),and glucosinolates(22),respectively,others were relatively quantified by the base peak area,such as lipids,hydroxycinnamic acids and unknown compounds.2.Comparative analysis of metabolites in siliques under normal and shading conditions In this study,shading treatment to the siliques was provided through aluminum foil after 20days of pollination(20DAP).The phenotypes of siliques and seeds(at 35DAP,45DAP and maturity)had morphological changes after shading,including chlorosis of siliques,size reduction,and wrinkled shapes of seed,indicating that light plays important roles in formation of storage reserves of siliques and seeds.As a result,each class of metabolites showed fluctuations after shading treatment in siliques.Among these,total contents of flavonoids,glucosinolates and hydroxycinnamic acids increased significantly after shading in rapeseed.Significantly differential metabolites were determined using the standards of fold change value≤0.5 or≥2.0 and p-value≤0.05 for each group.Then,62 common differential metabolites were identified after shading treatment,including 12 GSL,7 lipids,3 phenolic acids,3 flavonoids,3 hydroxycinnamic acids and 34 unknown compounds.Our study suggest that metabolites of siliques should be significantly affected by light deficiency.3.Effects of shading on GSL accumulation in siliques of rapeseedAs known,the siliques are the main sites for GSLs biosynthesis in rapeseed,that are later transported from silique walls(source)to the seeds(sink).In this study,we found that the total GSLs had significantly higher accumulation in siliques after shading when compared with the normal condition.Among 12 differential GSLs,10 of which were final products of GSLs biosynthesis pathway,including 4 aliphatic GSL(glucoalyssin(1.28),glucoalyssin(1.70),glucoberteroin and glucoerucin),4 indolic GSL(tryptophan,glucobrassicin,4-hyroxyglucobrassici and 4-methoxyglucobrassicin)and 2 aromatic GSL(gluconasturtiin and glucotropaeolin),respectively.Similar to their total content,each individual GSL was also widely accumulated in siliques after shading treatment.The results indicated that deficiency of light could be associated with GSL accumulation and transport from siliques to seeds.4.Effects of shading on the expression levels of GSL family genes in rapeseedAs a‘model’for secondary metabolites,the GSLs pathway has been studied extensively in plants.Then the expression profiles of the GSL family genes were investigated using RNA-seq to examine the effects of shading on GSL biosynthesis and transport pathways.The results showed that most of GSL genes were suppressed after shading treatment.To confirm this,we randomly selected the associated genes of differential GSLs to do validation with q RT-PCR analysis,such as AOP2,GSL-OH,from aliphatic pathways,IGMT from indolic pathways and St5a from aromatic GSL pathway and GSLs transporter GTRs genes from transport pathway.As expected,the expression patterns of these genes were down-regulated after shading treatment,showed highly consistent results with the accumulation patterns of GSLs in silique walls.Therefore,we infer that light plays crucial roles in GSL transport by regulating the expression of GSLs family genes.5.Comparative analysis of GTR family genes between higher and lower GSL rapeseed To elucidate the roles of GTR genes,we investigated the GTR family genes analysis from Arabidopsis thaliana,Brassica rapa,Brassica oleracea and B.napus.A total of 28 GTR genes were identified,including 2 from A.thaliana,7 from B.rapa,7 from B.oleracea,and12 from B.napus,respectively,and divided into two subgroups(GTR1 and GTR2).Furthermore,the different GTR family gene members had different expression patterns in siliques and seeds,and showed the structural variations in sequences between high GSL(GH06)and low GSL(ZS11)varieties of B.napus.In addition,we further explored that light could affect the Bna GTR genes through Cis regulatory elements.A total of 7 regulatory elements in the promoter regions of Bna GTRs with light-related responses elements should play important roles in activating Bna GTRs in B.napus.Our results provide deep insights into understanding the roles of light in mechanism of GSL accumulation and transport from siliques to seeds in rapeseed.6.m GWAS analysis of GSLs affected by shading in rapeseedWe examined the GSLs significantly affected by shading in nine materials(GH06,ZY821,L1262,L1263,L1266,L1267,L1188,P130 and P95)of rapeseed including gluconasturtiin(m169),glucobrassicin(m165)and glucoalyssin(m157).These GSLs showed a significant accumulation pattern in siliques but had a decreased content in seeds.Their m GWAS analysis revealed total 17789 SNP markers,out of which we screened 306 significant SNP associated with m157,m165 and m169.Maximum numbers of these SNP markers were present on Chr C02 and Chr A09 i.e.,83 and 65,respectively.A total of 9886 genes were identified associated with the 100 kb upstream and downstream region of these SNP markers when mapped with referenced genome of high GSL variety(GH06).We further screened 14candidate genes from GSL pathway divided as 7 structural genes and 7 transcription factors from GSL pathway.Furthermore,the presence of 18 cis regulatory elements in their promoter regions confirmed their roles under shading treatment which ultimately affected their accumulation and transport in seed of rapeseed. |
