Biosynthesis Mechanism Analysis Of ?-ODAP In Lathyrus Sativus L. Based On Transcriptomics And Metabolomics Approach

Endogenous ?-ODAP is a key factor limiting the development and utilization of Lathyrus sativus L..Unfortunately,few studies have been done on the key genes and proteins involved in?-ODAP biosynthesis pathways and the regulatory pathways of ?-ODAP are still unclear.In this study,transcriptomics,metabolomics and multi-omics analysis were performed on the tissues with different ?-ODAP content in the germination stage of L.sativus,and functional analysis of the key geneb-cyanoalanine synthase?CASase?of ?-ODAP biosynthesis was carried out.The main findings obtained were as follows:A total of 6,589 differential genes were identified in transcriptomics.Extensive Gene Ontology and KEGG analysis suggested that the?-ODAP content was co-regulated with primary metabolism such as carbon metabolism and sulfur assimilation/metabolism at the genetic level.61 modules were obtained by WGCNA analysis with the above differential genes,two of which were related to CSase,indicating that CSase family proteins may be involved in the regulation of ?-ODAP accumulation.There are 8 LsCSase sequences in L.sativus,belonging to CYSA,CYSB,CYSC,CYSD and SCS family respectively,among which LsCASase?CYSC family?may be a key enzyme in the biosynthesis of ?-ODAP.A total of 141 differential metabolites,including amino acids,carbohydrates and purines,were detected at the time points of 2DAS,6DAS and 25DAS by metabolomics analysis.PCA analysis showed that many metabolites were co-regulated with ?-ODAP.The results of fold change,hierarchical clustering,and pathway analysis showed that?-ODAP metabolism was associated with primary metabolism such as nitrogen metabolism,sulfur metabolism,amino acid metabolism and nucleic acid metabolism.The combined analysis of metabonomics and transcriptomics was performed by mapman software to construct the network of differential genes and differential metabolites.It showed that some metabolites and genes in sulfur metabolism,TCA cycle and amino acid metabolism were co-regulated with ?-ODAP.The results of enzyme activity assay showed that LsCASase had both CAS?pH<8.0?and CS?pH>8.0?activities.The effect of temperature on the activity of the two enzyme activity was not significant,and LsCASase maintained high enzyme activity at low temperatures.Treatment with ZnSO₄,MgSO₄,FeSO₄ and ?-ME increased the two enzyme activities of LsCASase,while CuSO₄ treatment inhibited CS enzyme activity and increased CAS activity.The optimal substrate concentrations for CS activity were 0.5mM OAS,6mM Na₂S,respectively and for CAS activity were 7.5mM KCN,3mM Cys,respectively.Lys¹0303 is a key amino acid residue for the LsCASase to function normally,while M²3535 and S²3939 are key sites for the ratio of the two enzyme activities of LsCASase.Subcellular localization results indicated that LsCASase,LsSAT2,and LsSAT3 were localized to mitochondria.The results of yeast two-hybrid system,bimolecular fluorescence complementation and pull down showed that LsCASase interacted with LsSAT2 and LsSAT3.The complex formed by LsCASase and LsSAT2 or LsSAT3 may regulate the ?-ODAP biosynthesis by affecting the BIA biosynthesis of Cys and isoxazolin-5-one.Predictions of protein structure,enzyme active sites and binding sites indicated that LsCASase is a homodimer with active sites as follows:K¹⁰³,N¹³⁴,M²³⁵,G²³⁶,I²³⁷,G²³⁸,S²³⁹,G²⁴⁰,G²⁴¹,T²⁴²,T²⁸¹,G²⁸².LsSAT2 is a hexamer composed with two trimers,whose Ser binding sites were D²⁶⁵,D²⁷⁹,H²⁸⁰,E²⁸¹,G³⁰⁶,R³¹⁴and CoA binding sites were D²⁷⁹,H²⁸⁰,H³⁰⁰,G³⁰⁶,A³²⁶,K³⁴¹,A³⁴⁴,T³⁵⁷.The results of the prediction of interaction site and pull down experiments showed that T¹³¹,S¹3232 and Q²0404 of LsCASase were the key sites for its interaction with LsSAT2.The Cys synthase activity of LsCASase mutants was almost completely lost,and the CAS activity was not significantly affected.The above results indicated that ?-ODAP metabolism of L.sativus was strongly linked to primary metabolism such as sulfur metabolism,nitrogen metabolism and carbon metabolism.Between these primary metabolisms,LsCASase sits at a juncture and had been proven to form a CRC complex with LsSAT2 or LsSAT3 to regulate the synthesis of ?-ODAP in mitochondria.These results provide a solid basis for further investigation of the ?-ODAP regulation and improving the sulfur-containing nutrients and other qualities of seeds by genetic manipulation of key enzymes involved in the sulfur metabolism of L.sativus.