Metabolomics Analysis Reveals Exogenous Glycine Promoting Phenolic Compounds Accumulation In Lettuce(Lactuca Sativa L.)

Lettuce?Lactuca sativa L.?is a minimally processed food product available throughout the entire year and consumed worldwide.Lettuce is also a significant source of natural health-promoting compounds,including glycosylated flavonoids,phenolic acids,carotenoids,ascorbic acid,tocopherols and sesquiterpene lactones?e.g.,lactucin and lactucopicrin?.Glycine,the simplest amino acid in nature,is one of the most abundant free amino acids in horticultural soil and is regarded as a model amino acid in plant organic nitrogen research.In this study,a novel lettuce metabolite library and novel non-targeted metabolomics strategy were devised,and the draft metabolome of lettuce was presented.Moreover,the effects of exogenous glycine nitrogen on primary and secondary metabolites,transcriptional levels and antioxidant activity of lettuce leaves were investigated hydroponically.The main results are as follows:1.Phenolic compounds are the main differential metabolites among different lettuce accessions.The total amount of polyphenols in 45 lettuce varieties was evaluated.The results showed that the content of polyphenols in looseleaf lettuce was the highest,followed by cos lettuce and butterhead lettuce,and the lowest content was detected in crisphead type.The total polyphenols content of red or purple varieties of lettuce was higher than that in green leaves.We used combined GC×GC-TOF/MS and UPLC-IMS-QTOF/MS to detect and relatively quantify metabolites in 30 lettuce cultivars representing large genetic diversity.Compared with online databases,the published literature and standards enabled putative identification of 171 metabolites.Among them,37 metabolites were identified as level 1?identified compounds?,and 86 metabolites were level 2?putatively annotated?.Sixteen of these 171 metabolites,including caffeoylquinic acid hexose isomer 1 and isomer 2,dihydrocaffeic acid hexose isomer 2 and isomer3,dihydroxybenzoic acid,caffeoyl-hexose isomer 1,quercetin 3-glucoside-6?-acetate isomer 1 and isomer2,quercetin diacetyl-hexoside,quercetin 3-?6?-malonyl?-glucoside 7-glucoside,quercetin 3-?6?-malonyl?-glucoside 7-glucuronide,quercetin hexoside glucuronide isomer 3,luteolin di-glucoside,luteolin 7-glucuronide,luteolin glucuronide-hexoside,and geniposide,were present at significantly different levels in leaf and head type lettuces.A combination of the results and metabolic network analysis techniques suggested that the significant metabolomic variations between the leaf and head types of lettuce are related to phenolic compound biosynthesis,and leaf and head types have evolved different polyphenol metabolic strategies to adapt to the environment during evolution or human selection.2.Exogenous glycine supply promotes the accumulation of glycosylated flavones and flavonols in lettuce.Lettuces were hydroponically cultured in media containing 9 mmol/L nitrate or 9 mmol/L glycine for 4weeks.Primary and secondary metabolites were analyzed using GC/MS and UPLC-IMS-QTOF/MS.Significant differences in flavonoid biosynthesis,phenolic biosynthesis,and the tricarboxylic acid?TCA?cycle response were observed between N sources.Compared with nitrate,glycine promoted accumulation of glycosylated flavonoids?quercetin 3-glucoside,quercetin 3-?6??-malonyl-glucoside?,luteolin 7-glucuronide,luteolin 7-glucoside?,ascorbic acid and amino acids?L-valine,L-leucine,L-glutamine,asparagine,L-serine,L-ornithine,4-aminobutyric acid,L-phenylalanine?but reduced phenolic acids?dihydroxybenzoic acid hexose isomers 1 and 2,chicoric acid,chicoric acid isomer 1?and TCA intermediates?fumaric,malic,citric and succinic acids?.These results provide strong evidence to support that supplying glycine as a nitrogen source dramatically alters the nutritional value of lettuce.3.Glycine increases the transcriptional levels of key genes involved in phenolic compounds biosynthesis results in promoting phenolic compounds accumulation.Lettuces were hydroponically cultured in nitrogen deficiency media or media containing 9 mmol/L nitrate or 9 mmol/L glycine for 30 days,leaf samples were used for transcriptomics and phenolic profiling.Exogenous glycine supply induces the expression levels of key genes involved in phenolic biosynthesis and increases phenolic compounds accumulation in lettuce.Compared to the leaf sample of lettuce cultivated in nitrate solution?9 mmol/L?,9 mmol/L glycine increased the expression levels of phenolic biosynthesis genes?including LsPAL1,Ls4CL2,LsHCT,LsOMT1,LsCCoAOMT1,LsCHS,LsF3H,LsF3'H,LsFLS1 and LsDFR?and most of the UDP-glycosyltransferase genes?p<0.05?,and significantly promoted the total amount of polyphenols and flavones,and the relative abundance of apigenin,luteolin and quercetin glycosides and most of the phenolic acids in lettuce leaves.In comparison to the lettuce grown in nitrogen-deficient solution,glycine?9 mmol/L?supply significantly increased the expression levels of some key enzyme genes?including LsOMT1,LsCCoAOMT1,LsCHS,LsF3H,LsFLS1,LsDFR and LsUGFT78D2?and some of the UDP-glycosyltransferase genes,and significantly promoted the accumulation of caffeic acid derivatives,luteolin glycosides,and some quercetin glycosides,but inhibited the total amount of polyphenols and flavones.4.Exogenous glycine supply enhances the accumulation of antioxidants and antioxidant activity in lettuce.In this study,two lettuce cultivars,cv.‘Shenxuan 1'and cv.‘Lollo Rossa',were hydroponically cultured in media containing 4.5,9,or 18 mmol/L glycine or 9 mmol/L nitrate?control?for 4 weeks.The levels of health-promoting compounds in lettuce leaf were evaluated by GC/MS and UPLC-IMS-QTOF/MS,and antioxidant activity of the lettuce leaf extracts was assessed by ferric ion reducing antioxidant power method,cellular antioxidant activity assay,and H₂O₂ scavenging capability method.Glycine significantly reduced fresh weight compared to control lettuce,while 9 mmol/L glycine significantly increased fresh weight compared to 4.5 or 18 mmol/L glycine.Compared to controls,glycine?18 mmol/L for‘Shenxuan 1';9mmol/L for‘Lollo Rossa'?significantly increased the levels of most antioxidants?including total polyphenols,?-tocopherol?and antioxidant activity,suggesting appropriate glycine supply promotes antioxidant accumulation and activity.Glycine supply induced most glycosylated quercetin derivatives and luteolin derivatives detected and decreased some phenolic acids compared to nitrate treatment.In summary,using combined GC×GC-TOF/MS and UPLC-IMS-QTOF/MS platforms,the novel integrated non-targeted metabolomics approach was developed,and the novel lettuce metabolite library was devised containing 171 metabolites within 37 identified?metabolite identification level 1?,which significantly improves the identification accuracy of metabolites in lettuce.Phenolic compounds were investigated as the significant metabolomic variations between different lettuce accessions by using differential metabolites analysis and metabolic network approach.Also,by comparing the lettuce plants hydroponically cultured in nitrate or glycine solutions,this study reveals that glycine supply promotes the accumulation of glycosylated apigenin 3-,luteolin 7-,and quercetin 3-and?-tocopherol in lettuce leaves and enhances their antioxidant activity in vitro and cell line.The main reasons attributed to flavonoids accumulations in lettuce were the increasing transcriptional levels of key genes involved in phenolic biosynthesis.These findings provide a potential strategy to obtain vegetable products of the high value of flavonoid glycosides,ascorbic acid,and amino acids;this strategy may have potential relevance to human nutrition.Moreover,the standardization of the novel non-targeted metabolomics strategy devised in this study can be used to characterize metabolic variations between lettuce cultivars or other plants further.The combination of metabolomics and other‘omics'approaches is the useful way to elucidate gene functions further and may help to more precisely assess the contribution of genetic factors to metabolic variation and the contribution of metabolic variation to complex traits in different lettuce cultivars.