Mechanism Of Trehalose Promoting Catharanthus Roseus Antagonizing Low Temperature Stress Based On Metabonomics

Vinblastine and catharanthine have high-efficiency anti-cancer activity,and Catharanthus roseus is an important source of plants.With the increasing demand for raw materials for cancer drugs,Catharanthus roseus has been growing from south to north.With the increase of latitude,the decrease of temperature becomes an important stress condition in the process of planting and breeding Catharanthus roseus.Trehalose,as a signal molecule,can effectively regulate plant response to low temperature stress,and the metabolic mechanism of trehalose to antagonize the low temperature stress of Catharanthus roseus is still to be studied.In order to solve the mechanism of trehalose promoting the antagonism of Catharanthus roseus against low temperature,it is the theoretical basis for the promotion and planting improvement of Catharanthus roseus in the north.The results are as follows(1)Compared with CK,the leaves of LT treatment showed wilting and plant dwarfing,and the activity of chlorophyll and antioxidant enzymes decreased significantly after LT+T(p<0.05),and the chlorophyll content recovered to the similar control group after trehalose.The contents of MDA,proline,total phenol and total flavonoids in leaves increased significantly after low temperature stress(p<0.05).With the increase of trehalose concentration,the contents of these compounds showed a trend of continuous decrease,and significantly lower than LT(p<0.05).(2)A total of 59 primary metabolites were identified by GC-MS,and 23 compounds with significant differences were identified by PLS-DA,VIP value and p value.The metabolic pathways of different metabolites were enriched and analyzed.Finally,the metabolism of galactose,sulfur,starch and sucrose,amino sugar and nucleotide sugar and citric acid cycle were obtained Cycle and fructose and mannose metabolism are the six most seriously affected metabolic pathways.(3)Twenty four phenolic compounds were identified from Catharanthus roseus leaves by UPLC-MS.The contents of syringic acid,chlorogenic acid,caffeic acid,rosmarinic acid,apigenin,quercetin,luteolin,catechin,hesperidin,liquiritigenin,galangin and protocatechin decreased significantly(p<0.01)under low temperature stress,but increased significantly after adding trehalose.On the contrary,the contents of p-hydroxycinnamic acid and ferulic acid in C6C3 type compounds and daidzein,naringenin,naringin,rutin,isoliquiritigenin,Isoquercitrin,genistein,abscisic acid and kaempferol in C6C3C6 type compounds were significantly accumulated after LT treatment(p<0.05),while the contents were significantly decreased after adding trehalose(p<0.05).Pearson correlation analysis showed that primary metabolites had the potential to affect osmotic adjustment,and phenolic metabolites might affect antioxidant capacity.(4)Determination of seven monoindole alkaloids in Catharanthus roseus leaves by UPLC-MS.Vindoline,vincristine and vinblastine,the downstream compounds of alkaloid metabolism pathway,had no significant changes after low temperature treatment compared with trehalose(p>0.05).Tryptamine,loganin,vinblastine and tabersonine were significantly increased after low temperature treatment(p<0.05),while trehalose was significantly decreased(p<0.05).(5)According to the known phenols metabolism pathway of Catharanthus roseus,6 enzyme genes(CrPAL,CrC4H,CrICS,CrCM,CrDXR and CrOMT)were selected,and six key enzyme genes(CrTDC,CrSTR,CrLAMT,CrSGD,CrGES and CrNMT)were selected for real time PCR to determine the expression level of the genes.Except CrNMT,the expression of these key enzyme genes were significantly up-regulated(p<0.01)after low temperature stress,and down regulated(p<0.01)after trehalose addition.In conclusion,the response of Catharanthus roseus seedlings to low temperature stress and trehalose is completed by a variety of internal physiological,biochemical and metabolic regulation.Low temperature stress can cause certain damage to Catharanthus roseus seedlings,resulting in growth and metabolism blocked.Trehalose can improve the antioxidant capacity and osmotic adjustment ability of Catharanthus roseus seedlings by regulating the primary metabolism and secondary metabolism(phenols metabolism and alkaloids metabolism)of Catharanthus roseus seedlings,so as to alleviate the damage caused by low temperature stress.