Metabonomics Of Citrus Sinensis In Response To Chilling Stress

In order to study the effects of navel oranges with different rootstocks response to low temperature stress,and explore the differences of metabolites in leaf of navel oranges with different rootstocks,the characteristic compounds and their main metabolic pathways of navel orange leaves from different rootstocks under low temperature stress were studied and obtained.In this study,one year old Poncirus trifoliata(L.)Raf.and Citrus.sinensis(L.)Osbeck × P.trifoliata(L.)Raf.were used as rootstocks to graft one year old Newhall navel oranges(C.sinensis Osbeck)and cultured 10 months then subjected to 4°C low temperature stress test.Leaves at the upper,middle and lower parts were picked at 0,24,72,and 120 h,respectively.The volatile compounds in leaves of navel orange under low temperature stress were detected by static headspace gas chromatography mass spectrometry(SHS-GC-MS).Electrospray ionization-mass spectrometry(EESI-MS)and ultra-Performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS)detected the untargeted metabolites of two navel orange leaves under low temperature stress.Principal component analysis(PCA)and cluster analysis(Cluster Analysis,CA)were used to effectively distinguish the different stress-time samples and differential compounds.High-performance liquid chromatography(HPLC)was used to detect the change of secondary metabolites of phenolic compounds.The results of the study are as follows:1.The navel orange seedlings with Citrus.sinensis(L.)Osbeck × P.trifoliata(L.)Raf.rootstock showed slight curling at 24 hours,and the leaves were obviously curled at 72 hours.At 120 hours,the leaves curled and the top buds wilted.There was no obvious phenotypic change in the navel orange seedlings with the rootstocks of P.trifoliata(L.)Raf.at 24 h and 72 h,but the leaves curled at 120 h.Navel orange seedlings with P.trifoliata(L.)Raf.as rootstock showed greater tolerance to chilling stress.2.The compounds detected by SHS-GC-MS identified a total of 213 compounds,including terpenes,esters,alcohols,aldehydes,amino acids,and alkanecompounds.The two navel orange seedlings both contained carbamic acid,α-phellandrene,γ-terpinene,α-terpinene,pinene,(+)-limonene,D-limonene,methyl menthol,guanidine acetate,(±)-α-terbium acetate,furfural,carveol,2-methyl-naphthalene and 2-methylene-4,8,8-trimethyl-4-Ethyl-bicyclo[5.2.0]-nonane at each stage.From the heatmap,it can be seen that the components of the navel orange seedlings with citrange rootstocks at different times are more changed than with the rootstocks of P.trifoliata(L.)Raf.,and the materials with significant changes are α-phellandrene and γ-terpinene.The labeling of volatile compounds of two navel oranges under low temperature stress was terpenes.The differential compounds of navel oranges with the rootstock of P.trifoliata(L.)Raf.in the upper leaves are 4-methylene-1-(1-methylethyl)-cyclohexene,ocimene,neoisopulegol,(+)-pinene and beta-hemoelpene.The differential compounds in the middle leaves are α-phellandrene,γ-terpinene,(-)-β-pinene,terpene,and D-limonene,and the differential compounds in the lower leaves are(±)terpene,4-methylene-1-(1-methylethyl)-cyclohexene,2-terbellene,γ-terpinene and(-)-β-pinene.The differential compounds of the navel oranges with the rootstocks of citrange were(±)pinene,β-hemoetholpene,γ-terpinene,4-methylene-1-(1-methylethyl)-cyclohexene and 2-terbellene the difference compound of the middle leaf is γ-terpinene,(±)pinene,4-methylene-1-(1-methylethyl)-cyclohexene,α-phellandrene and 2-terbellene,the differential compounds of the lower leaves are α-phellandrene,γ-terpinene,neoisocamptol,β-hemoethrene and 4-methylene-1-(1-methyl ethyl)-cyclohexene.3.The EESI-MS data was analysed using PCA,the results showed that the upper and lower leaves of the two Newhall navel orange seedlings could not be distinguished at different times,while the functional leaves in the middle were differentiated at each time.The middle leaves of navel orange with the rootstocks of citrange can be completely distinguished in different stress time,the contribution rate of PC1 is 53.8%,and the navel orange leaves with the rootstocks of P.trifoliata(L.)Raf.can completely distinguish between 0,24,and 72 hours.There is partial cross-linking with the other three treatments under 120 hours of treatment,PC1 contribution rate reached 74.2%.The results indicated that the metabolism of the navel oranges with the rootstocks of citrange have major variation under the lowtemperature stress,but the navel oranges with the rootstocks of P.trifoliata(L.)Raf.could initiate some regulatory mechanisms after stress,and the metabolites did not change significantly.Based on the secondary mass spectrometry ion fragmentation and library alignment of the compounds,the identified differential compounds are4-aminobutyric acid(m/z 104),1-decanol(m/z 144),L-proline(m /z 116),malic acid(m/z 135),synephrine(m/z 168),pyroglutamic acid(m/z 130)and xylitol(m/z153).4.UPLC-Q-TOF-MS was used to detect the alcohol extracts from the middle leaves of two kinds of navel oranges,and the compounds were identified by comparison in database.A total of 35 compounds were identified,including organic acids,carbohydrates and phenolic compounds.The PCA analysis of compounds in two navel orange can be completely separated and have a high contribution rate.Among the top 20 substances in the navel oranges of which with the rootstocks of P.trifoliata(L.)Raf.,seven compounds were identified,namely kaempferol-7-O-neo-hepteoside,kaempferine-3-glucoside-2-p-coumarin,2-{2-Benzimidazol-2-yl-1-[(4-methylphenyl)methyl]ethyl}benzimidazole,hesperidin,dronabinol,isogacinoside,and fructose 2,6-bisphosphate.Five compounds were identified as the top 20 substances in PC1 in navel oranges with citrange rootstocks,namely 1-O-beta-D-Glucopyranosyl sinapate,hesperidin,rhodan,sucrose,and2-{2-benzimidazole.2-yl-1-[(4-methylphenyl)methyl]ethyl}-benzimidazole.5.The HPLC results showed that there were significant differences in the content of phenolic compounds in Newhall navel orange under low temperature stress.A total of 11 phenolic compounds were identified,including protocatechuic acid,caffeic acid,quercetin,naringenin,kaempferol,hesperetin,chlorogenic acid,ferulic acid,quercetin,myricetin and cinnamic acid.The phenolic compounds of navel orange with the rootstock of P.trifoliata(L.)Raf.had the highest values??except for caffeic acid at 24 h under low temperature stress.The contents of these 10 compounds decreased at later stress and were not significantly different from the control.It may be that the navel oranges with the rootstocks of P.trifoliata(L.)Raf.are activated by cold stress and the regulatory mechanism is initiated,then return to their normal state.The change of phenolic compounds in navel orangeleaves with citrange rootstocks was irregular,with some falling or rising straight.The above experimental results showed that the navel orange seedlings with rootstock of P.trifoliata(L.)Raf.under low temperature stress had less phenotypic and metabolite changes than those with citrange rootstocks,so they were more resistant to cold.The results of EESI-MS showed that only the metabolite changes in middle functional leaves changed significantly at different times of low temperature stress.The main differences among the middle leaves are 4-aminobutyric acid,1-decanol,L-proline,malic acid,synephrine,xylitol,pyroglutamic acid,etc.A total of 11 phenolic compounds were identified from the navel orange seedlings of two different rootstocks.The types of phenolic compounds contained were not different,but the content varied.