Responses Of Primary Metabolism In Ray And Disc Florets Of Chrysanthemum Nankingense To Sucrose Feeding Via Integrative Analysis Of Target Metabolomics And Transcriptomics

Chrysanthemum is a traditional flower in China with high economic and ornamental values.From the point of view of evolution,Chrysanthemums mainly originates from chrysanthemum vestitum,chrysanthemum indicum and chrysanthemum nankingense.So far,people have not thoroughly studied metabolism in chrysanthemums.Limited investigations are focused on seconday metabolism because seconday metabolism is more closely related to petal color and aroma.Moreover,people rarely probe flower metabolism from the angle of primary metabolism.In this study,we used Chrysanthemum nankingense(a unique wild diploid species of chrysanthemum genus)as the plant material to study primay metabolism in flower organs.We fed the detached flower buds with 200 m M sucrose and collected ray and disc floret samples at 0,12,24,36,48 and 60 hours.Via combined analysis of targeted metabolic profiling and transcriptome analysis,we investigated changes in contents of primary metabolites and expressions of related genes in ray and disc florets.More importantly,via correlation analysis,we had screened out key genes that were involved in metabolic responses to sucrose feeding,including some metabolic genes,sugar signaling-related genes,cell cycle-related genes,sugar transporter genes,and several transcription factors.These potential targeted genes can then be applied to metabolic engineering reseach,which in turn may lead to new findings in the field of genetic improvement in ornamental features of chrysanthemums.The main research results are as follows:(1)Sucrose-fed flower buds developed slower relative to control,showing differences from 12 to 60 hours.Under sucrose feeding,the development of ray florets was not significantly different from those in control,wh Ile the development of disc florets showed significant difference from 12 to 24 hours,and the stamens of disc florets were shorter in sucrose feeding conditions from 24 to 60 hours.(2)According to metabolic data,under sucrose feeding,sucrose,fructose,glucose,xylose and melibiose in ray florets showed an overall downward trend.Sucrose,fructose and glucose in disc florets first increased and then decreased to the original level.Myo-Inositol and melibiose in disc florets increased relative to those in 0 hour.Organic acids in ray florets changed lesswhereas in disc florets,except for shikimate,other organic acids showed an upward trend.Eight amino acids(Ser,Val,Asn,Ile,Glu,Lys,Gln,and Pro)incrased in ray florets,wh Ile twelve amino acids(Ser,Thr,Leu,Asn,Gln,Asp,Val,Phe,Ile,Pro,Lys and GABA)increased in disc florets.Principal component analysis showed that metabolites with greater contributions in ray florets were fructose,shikimate,melibiose,Gln,Asn,Lys,and tartrate and metabolites with greater contribution in disc florets were Asn,Thr,Gln,Phe,Val,Ser,Asp,Ile and GABA.Correlation analysis showed that,except malate,succinate,shikimate,stearate,Phe and GABA,other sugars,alcohols,organic acids and amino acids were negatively correlated with each other in ray florets,wh Ile sugars,alcohols,organic acids and amino acids in disc florets were positively correlated with each other.These data suggest that,compared with ray florets,disc florets could convert more sugars to downstream metabolites in response to sucrose feeding.(3)According to RNA-seq analysis,except for genes in volved in oxidative pentose phosphate pathway(OPPP),which was negatively regulated by sucrose feeding,other seven metabolic pathways were mainly positively regulated.Related genes in sucrose degradation pathway were all positively regulated,and Su Sy was more affected by sucrose.Relatedgenes in glycolytic pathway were mainly positively regulated with ATP-PFK,GAPDH and PK were greatly affected by sucrose.XI of the pentose related pathway was negatively regulated and XK was positively regulated.DXS in the mevalonate pathway was mainly negatively regulated and DXR was mainly positively regulated.Genes of the tricarboxylic acid cycle pathway were positively regulated by sucrose feeding,mainly through the regulation of PDH,PDHK,ICDH,SDH and NAD-MDH.The anaplerotic pathway was positively regulated by sucrose feeding with PEPC and PEPCK playing major roles.Amino acid biosynthetic pathways were positively regulated by sucrose feeding mainly via GOGAT,GS,Asp AT,GAD,GADH,PGDH,PSAT,and ADT.Taken together,these results showed that the metabolic activity of disc florets was higher under sucrose feeding than that in ray florets.Disc florets also responded to sucrose to a greater extent over time.(4)TOR and Sn RK1 were up-regulated by sucrose feeding.Cell cycle related genes(CYCA,CYCB,CYCD,CDKA,CDKD,and KRP)were positively regulated by sucrose feeding,and sugar transporters were also positively regulated to some extent.MYBs and MADS-box genes were mainly positively regulated wh Ile WRKYs was mainly negatively regulated.(5)Via correlation analysis between genes and metabolites,genes involved in sucrose degradation,glycolysis and OPPP were associated with most metabolites(succinate,sartrate,Ser,Val,Leu,Phe,Ile,Gln,Pro and GABA);metabolites and Su Sy,HK,PPi-PFK and GAPDH were mainly positively correlated;metabolites and SPS,G6 PDH and RPI were mainly negatively correlated.In the mevalonate pathway,DXS was positively correlated withmost metabolites including myo-Inositol,ribose,malate,citrate,succinate,tartrate,stearate,Ser,Val,Leu,Asp,Asn,Phe,Thr,Ile,Gln,Pro and GABA.In the tricarboxylic acid cycle pathway,PDH,ICDH,SDH and NAD-MDH were positively correlated with most metabolites(Stearate,Ser,Val,Leu,Phe,Ile,Gln,Pro and GABA).In the anaplerotic pathway and amino acid biosynthesis pathway,related genes were associated with most metabolites(Stearate,Ser,Val,Leu,Asn,Phe,Thr,Ile,Gln,Pro,and GABA);GOGAT,GAD and ADT were mainly positively correlated with metabolites;GS and AS were mainly negatively correlated with metabolites;NAD-ME was negatively associated with metabolites(ribose,malate,citrate,succinate,tartrate,Ser,Val,Leu,Ile,Lys,and Gln).(6)Eventually,we screened out the following genge including Su Sy(CHR00028875、CHR00079911 、 CHR00078809)、 ATP-PFK(CHR00073228 、 CHR00058964)、 PK(CHR00071004、CHR00078673、CHR00075072、CHR00047565)、SDH(CHR00027431、CHR00026631、CHR00014302)、PEPC(CHR00068446、CHR00056148、CHR00060124)、GOGAT(CHR00046711、CHR00046712)、ADT(CHR00027430)、TOR(CHR00022934)、Sn RK1(CHR00065459 、 CHR00081324)and MYB(CHR00065188 、 CHR00043606 、CHR00001486).According to our studies,these genes significantly responded to sugar signals and were involved in the metabolic regulation of chrysanthemum petals.In the future,these genes can be metabolically engineered to improve key agronomic traits of chrysanthemums which in turn may lead to production of new cultivars.