| White birch cells are rich in triterpenoids which have important medicinal value. Several studies indicated that many signaling molecules can promote the accumulation of triterpenoids in white birch so far, but the interactions among these signaling molecules and the effect on intrinsic signals have not been reported. In this study, white birch(Betula platyphylla Suk.) cell suspension cultures were as material, using SA, MeJA, CaCl2and combination of various inhibitors of Ca2+as treatment, the accumulation dynamics of triterpenoids and ingredients and the changes of antioxidant enzyme activities were investigated and gene expression of key enzymes for triterpenoid biosynthesis were detected, to explicit the role and mechanism of Ca2+in triterpenoids synthesis after the induction of SA and MeJA in white birch cell.The content of JA and SA, and the effect on the activities of Ca2+-ATPase and NOS were detected after the treatment of exogenous inducers, to make clear the feedback effect of endogenous SA, JA and NO, the catalytic synthesis of JA/SA and signal cross talk triggering Ca2+/ROS which reveal the mechanism of the signal molecules in triterpenoid biosynthesis. The results were as follows:1ã€After7days sespention culture for, the cells, were treated with SA, CaCl2and their combinations with inhibitor, to investigate the role of Ca2+in the SA-induced triterpenoids synthesis in white birch. The synthesis of triterpenoids substances (total triterpenoids, oleanolic acid, botulin and betulinic acid) and the related genes (FPS, SS, SE, BPW, BPY, CAS, CAM) expression were found up-regulated after SA treatment in suspension cells, and such effect had been more significant by adding Ca2+, which indicated that calcium signaling molecules mediated the inducement of SA on triterpenoid substances synthesis pathway in white birch cells. Studies on the inhibitors of Ca2+showed that Ca2+is a necessary role to enhance the inducement of SA on the synthesis of triterpenoids. However, the induction of the SA had not been inhibited in certain times, while the synthesis of metabolites had been promoted instead, which suggested that other signaling molecules or other pathway exist, participating in the induction of SA on the biosynthesis of triterpenoid in white birch cells. The results of cell viability, antioxidative enzyme activity, plant defense enzyme sctivity and gene expression patterns related to NO, JA, ethylene signaling indicated that SA could induced the ROS signaling and in response to stress, NO, JA and ethylene signaling pathways were involved in signal transduction. In this process, the Ca2+promoted the stress signals transmitted to the downstream.2^The results treated with MeJA, CaCl2and their combinations with inhibitors showed that MeJA treatment had significantly promoted synthesis of the total triterpenoids and oleanolic acid in white birch cells, and reached the highest content of46.90mg/g and1.31mg/g at48h respectively. The effect of MeJA on synthetic triterpenoids were decreased by treatment of MeJA+EGTA and MeJA+LaCl3, while still significant higher than the control. The results of stress-tolerant enzymes showed, CaCl3and MeJA+CaCl2treatment had improved CAT, PAL and APX activity in different degree, while SOD activity was inhibited by the Ca2+; Activities of stress-tolerant enzymes had been affected by two kinds of Ca2+inhibitors. Quantitative PCR results showed that, in the expression investigation of key enzyme genes in triterpenoid each pathway, FPS, SS, SE, BPW, BPY, had been significantly up-regulated in early time of MeJA treatment (2h). In early time of treatment by MeJA+LaCl3(2h), expression levels of BPY, BPX2, SS and SE were higher than those in MeJA treated separately, except the reduction of BPW; Compared with MeJA treatment, expression levels of BPW, BPX2, SS, SE and FPS were down-regulated by MeJA+EGTA treatment for2h. Which Suggesting that Ca2+participated in the inducement of MeJA in the startup of defense system and triterpenoids synthesis, but the two Ca2+inhibitors, EGTA and LaCl2, had different effects and mechanisms in triterpenoids synthesis.3ã€In the studies of MeJA and SA, the addation of exogenous MeJA and S A into media affected the synthesis of endogenous signaling molecules (JA, SA and NO),and Ca2+-ATPase activity showed, that adding SA had promoted the JA production at12～120h; The addition of exogenous MeJA promoted the signal molecule JA, except for the early treatment; Combining MeJA with SNP or SA treatment, had less effective inducement on JA synthesis than the combination treatment of SA and SNP, which suggesting that SA had a better effect on the synthesis of endogenous JA; Except for all treatments at120h and MeJA+SNP treatment at6h, other treatments at each time had little effect or inhibition on the accumulation of free SA, especially at24h of all treatments, which result showed a significant inhibition on the accumulation of free SA. Exogenous MeJA can induce the accumulation of cell-binding SA effectively, while exogenous SA and other treatments did not show the promoting the accumulation of endogenous binding state of SA. The results showed that there were the feedback effects from endogenous SA, JA and NO, and the catalytic synthesis and antagonismof endogenous JA/SA after inductions by MeJAand SA. All of them participated in the regulation of triterpenoids synthesis.4ã€The results of MeJA, SA and its interaction treatments on suspension cells showed that, endogenous NOS and Ca2+-ATPase early respond to each treatment, and peaked at early inducement(2,6h). While the levels of endogenous JA and SA changed later than the increase of the NOS and Ca2+-ATPase activities. Ca2+or Ca2+-ATPase plays an important role in mediating the endogenous signaling processes by exogenous SA and MeJA, and NO was the upstream signal of JA and SA. The three of them participated in regulation of triterpenoids synthesis in white birch.The results of this study showed that SA and MeJA could induce changes in intracellular reactive oxygen species and startup of defense system. H2O2played a key role and an important point of contact in mediating the expression of defense genes and synthesis of secondary product stimulated by SA, MeJA and NO. According to the analysis above, Ca2+participated in mediating triterpenoids synthesis process induced by SA and MeJA respectively, in white birch suspension cells. And Ca2+signal transduction was not the only way. The synthesis of endogenous SA and JA signaling molecule was as a response to the experiment treatments. Accompanied by oxidative bursting and defense enzyme starting, study showed that NO synthesis was an early event in which the exogenous SA and MeJA induced the synthesis of endogenous hormone and startup defense system, and was located in upstream of SA and JA.The feedback effects of endogenous SA, JA and NO was induced by MeJA and SA, and resulted in JA/SA self-catalytic synthesis and antagonism, and can trigger the signal response and interaction between Ca2+and ROS. Interactive cross talk of signaling molecules participated in and regulated the synthesis and dynamic accumulation of triterpenoids materia. |
