| Currently,the scope of soil secondary salinization is expanding in the world with the severity gradually increasing,which seriously affects the agricultural production and ecological environment.The improvement of saline soil and vegetation restoration are the hot issues concerned by researchers around the world.As an important signaling molecule,nitric oxide(NO)participates in the process of plant seed's germination,growth,development,senescence,It also plays an important role in plant stress response.Recent studies have shown that NO can affect plant salt tolerance by changing plant metabolism,but the regulatory mechanism is not clear.Catharanthus roseus(C.roseus)has attracted much attention because of the alkaloids with anticancer activities in its body.Subsequently,numerous studies have shown that this plant also contains phenolic compounds most of which have a variety of pharmacological effects in immune regulation,anti-tumor,anti-oxidation etc.Now,It has become an important source of new drugs,lead compounds and new chemical entities.Studies have shown that both salt stress and exogenous nitrogen can promote the increase of alkaloid content in C.roseus,but there are few reports on phenolic compounds which are also secondary metabolites.In this study,on the basis of the change of the growth and physiological indexes of the C.roseus under salt stress treated by sodium nitropptusside(SNP)with gradient concentration,LC-MS technology is adopted to measure the components and content of phenolic metabolites in different parts of the C.roseus and to reveal the response of phenolic metabolites under the salt stress in NO regulation from the level of metabolome,and a foundation is laid for further exploring the mechanism that the regulation of phenolic metabolism and NO improves the salt tolerance of the C.roseus,and has practical significance to the development and utilization of saline-alkali land.The major findings are as follows:(1)The difference of the C.roseus seedling(with undifferentiated xylem)was not significant between NaCl(25 mM)treated group and the control group within short time(10 d),NaCl(25 mM)treatment even partly promoted plant growth;as the NaCl concentration exceeded 50mM,the difference was significant compared with the control group;overall,NaCl inhibited plant growth,and with the increase of concentration and the prolongation of time,the inhibitory effect was more conspicuous.Growth and morphological changes of the C.roseus seedling(xylem undifferentiation)under NaCl treatment within long time(85 d),the difference of each growth index was not significant compared with the control group within 15 d under NaCl stress,and the difference significance began after 20 d.The plant height mainly depends on the increase in the number of nodes,and the change of node pitch is not obvious,seedlings of C.roseus with xylem being formed has a certain ability of resistance to salt stress.(2)As the concentration of NaCl is below 100 mM,the average value of proline and malondialdehyde(MDA)content in the upper,middle and lower leaves of the plant were all lower than those of the control group.The leaf area,leaf length,leaf width and other growth indicators were lower than those of the control group;at the concentration of 125 mM NaCl,proline and MDA content were higher than those of the control group,and reached the maximum value;the leaf area,leaf length,leaf width and other growth indicators of the plant stopped growing,which obviously inhabited the growth of the plant.The concentration of 100-125 mM NaCl is the critical range of serious effects on plant growth.(3)In the experiment of the effect of SNP on the C.roseus seed germination under salt stress,the germination potential,germination rate,germination index,vigor index,root length,bud length under 50 mM NaCl stress were all lower than those of the normal control group.Salt stress inhibited the seed germination;0.1 mM SNP effectively relieved the inhibiting effect of salt stress on the c.roseus seed germination,and even restored the normal germination rate of seeds;SNP exceeding 0.5 mM would aggravate salt stress injury;various germination parameters,bud length,and root length were lower than those under salt stress.NO plays an important role in signal regulation under salt stress during the c.roseus seed germination.Low concentration alleviates salt stress injury,and high concentration exacerbates salt stress injury.(4)Exogenous SNP effectively increased the activity of nitrate reductase(NR)in the leaves of the catharanthus roseus under salt stress.After 4d,the activity of NR reached the maximum value,4 d>5 d>3 d>2 d>1 d;the activity of NR was the highest with a concentration of 0.5mM SNP,during which 0-0.5 mM SNP had a significant effect on improving NR,while the effect of 1.0-2 mM SNP declined.As the C.roseus plant being treated by 0.1 mM SNP under salt stress,proline content accumulation was promoted,MDA content rising caused by salt stress was reduced,content of soluble sugar reached the highest,injury caused by salt stress was relieved,and salt tolerance of plants was strengthened.(5)SNP had an insignificant slow-release effect on growth index of the C.roseus plant with xylem maturation under salt stress,in which 125 ?M SNP obviously inhibited the plant height growth,and the change of plant height mainly depended on reducing the number of nodes.At the same time,the basal stem obviously thickened,as plants increased the resistance to external stress by reducing height and increasing stem diameter.NO participated in regulating stomatal movement as a kind of signaling molecule.The number of stomata and opening number on lower epidermis of leaves of the C.roseus treated by SNP under salt stress increased.As the number of times applied increased,the range of variation increased.The increase in number of stomata treated by 0.1 mM SNP was the largest.(6)LC-MS technology was adopted to analyze the phenolic substance changes in different positions like roots,stems,leaves,flowers of the C.roseus under salt stress under the effect of SNP,and 18 kinds of phenolic substances and L-phenylalanine were identified.According to the chemical structure formula,the 18 kinds of phenolic substances were classified into 5 kinds of C6C1 phenols:vanillic acid,p-hydroxybenzoic acid,eugenic acid,protocatechuic acid,gallic acid;5 kinds of C6C3 phenols:p-hydroxycinnamic acid,chlorogenic acid,ferulic acid,cinnamic acid,p-coumaric acid;and 8 kinds of C6C3C6(flavonoids):genistein,galuteolin,naringenin,quercitrin,myricetrin,quercetin,kaempferol,rutin,among which the quercetin,chlorogenic acid,protocatechuic acids are the main phenolic substances in the body of the C.roseus,which exist in different positions.(7)The responsive accumulation of the phenolic compounds in different tissues of C.roseus were significantly different under different concentrations of SNP treatment.It is some small molecular phenolic acids that were accumulated in roots and stems;however C3C6C3 phenolic compounds were enriched mainly in surface leaves and flowers.In contrast to flower and leaf,adaptive responses of phenolic compounds in roots and stems were more sensitive to salt stress and SNP treatments.(8)The distribution of phenolic compounds in different positions is significantly different.The composition and relative content of flavonoids with the structure of C6C3C6 in roots,stems,leaves,and flowers are quite different.8 kinds of flavonoids are all detected in stems,but the genistein,galuteolin,naringenin and quercitrin are not detected in leaves.The relative content of the quercitrin is the highest in different positions.Its content increases in stems and leaves under salt stress,but sharply declines in roots and flowers.(9)Under 75 ?M SNP(3#),the plant didn't bloom,C6C1 Phenolic compounds under this treatment,there were vanillic acid,syringic acid appearing in leaves,not appearing in leaves of other flowering plants.Phenolic compounds are closely related to flower formation. |
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