The Function Analysis Of NtLTPI.38 Based On Multiomics And Its Role Study In Regulating Tobacco Salt Tolerance

Salt stress is one of the most detrimental abiotic stressors worldwide.It limits plant productivity and geographic distribution,and threatens the quality and yield of crops.It is of great important for improving plant salt tolerance by using genetic engineering through in-depth studying the molecular mechanism of tobacco or other plants in response to salt stress.Non-specific lipid transfer proteins(nsLTPs)generally have conserved structural similarity,low sequence uniformity,and played important roles in plant growth and development,and respond to external abiotic and biotic stresses.Based on the overexpressed NtLTPI.38(OE)and knockout plants(ntltpI.38)obtained before,the molecular mechanism of NtLTPI.38 gene regulating salt tolerance was explored through multi-omics integrated analysis of transcriptome,metabolome and lipidome,combined with morphological,physiological and metabolism analysis under salt stress.The main findings are as follows:1.Comprehensive LC/MS untargeted metabolomics analysis identified 699,608,and 573 differentially accumulated metabolites(DAMs)annotated by OE vs.ntltpI.38,ntltpI.38 vs.WT,and OE vs.WT,respectively.Different types of lipids and flavonoids accounted for the largest proportion of the DAM subclass in lipids and lipid-like molecules.Among them,Flavonoids had 74,51,and 54 DAMs with 47,17,and 26 upregulated metabolites in the OE vs.ntltpI.38,ntltpI.38 vs.WT,and OE vs.WT comparable groups,respectively.The results showed that the amounts of flavonoids in OE lines was the mostly upregulated than that in WT and knockout plants.KEGG enrichment analysis found that all three groups were enriched to pathways in ’glycerophospholipid metabolism’,’TCA cycle’ and ’arachidonic acid metabolism’.In the ntltpI.38 vs WT and OE vs WT compared groups,the KEGG metabolic pathway were enriched in ’glyoxylate and dicarboxylate metabolism’,’linoleic acid metabolism’ and ’purine metabolism’.2.Non-targeted LC/MS lipidomic profiles showed a total of 28,32,and 19 differential lipid metabolites were detected and annotated based on OE vs.ntltpI.38,ntltpI.38 vs.WT,and OE vs.WT,respectively.Among them,most triradylglycerols(TGs)were upregulated in OE compared with ntltpI.38 and WT,and downregulated in ntltpI.38 compared to WT.Meanwhile,all ceramides(Cer)[such as Cer(d42:4)and Cer(d44:4)]were downregulated in OE compared to WT and ntltpI.38.And,the KEGG enrichment results showed that the lipid DAMs were all enriched in"Glycerophospholipid metabolism," "GPI-anchor Biosynthesis and autophagy-other"pathways in all three comparable groups.3.A total of 4812,4253,and 4223 differentially expressed genes(DEGs)were found based on OE vs.ntltpI.38,ntltpI.38 vs.WT,and OE vs.WT.The Venn diagram showed that 205 DEGs were co-differentially expressed in all three groups.Further,GO and KEGG enrichment analyses were performed to explore the functions of DEGs.DEGs enriched in BP were related to "protein phosphorylation","protein kinase activity",and "DNA regulation of transcription".The DEGs annotated as"calcineurin B-like proteins(CBLs)",“CBL-interacting protein kinase(CIPK)",and"calcium dependent protein kinase-like protein(CDPK)",and transcription factors(TFs).In addition,genes related to Ca2+ signal transduction under salt stress,including FERONIA(FER),annexin(ANN),wall-associated kinase(WAK),respiratory burst oxidase homologue D(RbohD),and abscisic acid receptor,including PYRABACTIN RESISTANCE-LIKE(PYLs),and sucrose non-fermenting 1-related protein kinases 2.6(SnRK2.6)in our RNA-seq data.Otherwise,some DEGs were annotated as ion transport channels proteins,such as K+transporter(AKT),high-affinity K+transporter(HKT),Na+(K+)/H+antiporters(NHX),cyclic nucleotide-gated channels(CNGCs),and chloride channel(CLC).Further KEGG analysis of found that DEGs was mainly enriched in ’Glycerophospholipid metabolism’and ’glycerolipid metabolism’ which corresponded to the results of metabolomics and lipidomics.In addition,many DEGs were significantly enriched in the "starch and sucrose metabolism" pathways in all three comparison groups.4.Through the joint analysis of different omics,the metabolic regulation network diagram of NtLTPI.38 gene regulating the gene expression and substance metabolism of flavonoids and lipids was constructed.NtLTPI.38 regulats the synthesis of lipid metabolites through tricarboxylic acid cycle,glyoxylic acid and dicarboxylic acid metabolism,glycine,serine and threonine metabolism,glycerophospholipid metabolism and glyceride metabolism,and regulats the metabolism of flavonoids through tricarboxylic acid,shikimic acid metabolism,phenylalanine,tyrosine and tryptophan biosynthesis cycle.5.Further analysis after salt treatment with 200 mM NaCl showed that NTLTPI.38 overexpression increased the salt tolerance under high salt stress.After salt stress,NtLTPI.38 overexpression in tobacco increased the contents of chlorophyll,K+,Ca2+,soluble sugar and soluble protein,proline,total flavonoid,DPPH and ABA,enhanced the activity of four antioxidant enzymes,but reduced the relative conductivity,Na+content,Cl-content,H2O2 content,O2-content and MDA content of tobacco leaves.In summary,NtLTPI.38 enhanced salt tolerance in tobacco by regulating lipid and flavonoid synthesis,antioxidant activity,ion homeostasis,Ca2+ signaling and ABA signaling pathways.