Genome-Wide Analysis Of Sphingolipid Metabolism Pathway In Brassica Napus

Brassica napus is a premium oilseed crop primarily grown for oil purpose all the world over.Environmental regimes such as drought,salinity and temperature etc.are the constraints severely restricting its production.However,the underlying mechanism through which Brassica napus maintains its production in spite of these extremes remains unknown.Recent studies regarding sphingolipids indicate that they play key roles in stress responses.Therefore,the focus of this study was to build sphingolipid metabolism pathway in Brassica napus based on sequence similarities with Arabidopsis orthologs,monitor the transcript abundance of the pathway genes and corresponding sphingolipid changes in response to different abiotic stresses including dehydration,salt,ABA and cold stress(4℃)at two time points.Based on sphingolipid profile and gene expression in response to stresses,key genes were selected for further functional characterization in Brassica napus.This study identified 127 sphingolipid metabolism pathway genes in Brassica napus in comparison with 39 orthologs in Arabidopsis which were categorized into catabolic,anabolic and sphingolipid modifying genes.These genes presented different expression pattern in different tissues of Brassica napus as well as actively regulated at different time points of the stresses.The analysis of sphingolipidome revealed the identification of 178 sphingolipid molecular species which were classified into 7 different classes including long chain bases(LCBs),long chain base phosphates(LCBPs),ceramides(Cer),ceramide-1-phosphates(C1P),hydroxyceramides(hCer),glucosylceramides(GlcCer),and glycosylinositolphosphorylceramide(GIPC).We quantified 4 LCBs and their corresponding phosphorylated forms,30Cer,25 hCer,40 C1 P,40 GlcCer and 35 GIPC molecular species whose physiological levels altered at different time points of the stresses.Interestingly,all the complex sphingolipids quantified in this study lacked molecular species with d18:2 background probably due to lack of sphingolipid (?)4 LCB desaturase enzyme which could be a unique feature of Brassica napus.Apart from this,one of the challenging goals in sphingolipid quantification is to develop a method to detect phosphorylated ceramides hence,we established a method to detect 40 molecular species of C1 P which were altered in response to the abiotic stresses.Based on the mRNA transcripts and sphingolipidome data,ceramide kinase(BnaCERK)gene was selected and evaluated for its role in seed germination and root architecture phenotypes in response to drought stress.The BnaCERK-overexpression lines had relatively increased germination rate than wild type and CRISPR/Cas9 mutant lines.The BnaCERK-overexpression lines had relatively lower endogenous abscisic acid(ABA)levels and higher gibberellic acid(GA)contents than WT and mutant lines.Moreover,exogenous application of ABA further retarded the germination phenotype of mutant lines compared to WT and BnaCERK-overexpression lines,while exogenous supply of GA restored the germination phenotype of bnacerk mutant lines almost to the level of WT under drought stress induced by PEG 15%.These findings suggested that knockout of ceramide kinase gene may affect ABA/GA balance leading to seed germination hypersensitivity under drought stress.Genetic manipulation of BnaCERK gene also altered root architecture phenotype under drought stress.The bnacerk mutant lines had a greater number of lateral roots than WT and BnaCERK-overexpression lines both under control and drought stress.However,the increase was noticeably far more than observed under normal condition.Furthermore,the overexpression lines had relatively lengthier primary roots and shorter lateral roots than WT and mutant lines of BnaCERK gene under drought stress.Analysis of gene expression revealed higher transcription rate of indole acetic(IAA)biosynthesis genes and higher physiological levels of IAA contents in mutants than WT and overexpression lines suggesting that BnaCERK gene could affect the root architecture phenotype by interfering with transcription machinery of IAA biosynthesis genes leading to varying IAA contents.The palmitic acid synthesized in plastid is exported to endoplasmic reticulum(ER)by fatty acid exporter 1(FAX1)for the synthesis of very long chain fatty acids(VLCFAs)and complex cellular lipids such as phospholipids,sulpholipids and galactolipids.However,the role of FAX1 in maintaining the homeostasis of complex sphingolipids including GIPC,GlcCer and hCer was not known.In the present study we demonstrated that the physiological levels of complex sphingolipids in the bnafax1 knockout mutants were relatively lower than wild type(WT)and overexpression lines.The low levels of these sphingolipids in the mutant lines was mainly due to the low levels of the respective molecular species having amide linked VLCFAs to their long chain base(LCB)backbone.The reduced fresh weight and root length phenotypes of bnafax1 mutants could be attributed to disturbance in the homeostasis of these complex sphingolipids due to loss of function of Bna FAX1.In summary,this study provided a whole sketch of sphingolipid metabolism pathway,sphingolipidome and its dynamics under different abiotic stresses.Furthermore,the BnaCERK gene significantly altered seed germination and root architecture phenotypes and Bna FAX1 gene caused alteration in the levels of complex sphingolipids.Hence,these genes could be used as a potential candidate in future molecular breeding programs for improved yield and quality of Brassica napus.