| Drought is a major yield limiting factor heavy affecting the crop grown and productivity,especially rice.C4 plants,such as maize,shows higher photosynthetic and water use efficiencies under drought stress than rice.Given virtue of C4 plant has variety different mechanisms to cope with the drought,Botanist hope to the key gene of C4 plant introduce to C3 plant rice via genetic engineering,to improve its photosynthetic efficiency,drought tolerance and yield.The key enzyme of v photosynthetic pathway,PEPCase(Phosphoenolpyruvate carboxylase)(PEPC,EC 4.1.1.31)not only increasing the C4 photosynthesis,but also the considerable part of plant resistant reaction,especially drought tolerance.Now,applying genetic engineering technology,it has got the maize C4-PEPC transgenic rice(PC),showing higher net photosynthetic(Pn),carboxylation efficiency and grain yield than wild type rice,but also its has relatively stronger Pn and oxidative resistance in drought stress.However,How PC respond to drought stress of molecular mechanisms is still limited.Calcium is one of the most important plant cell signal molecules,it is involved in nearly all aspects of plant development,especially,and it participates in many abiotic stresses.Precious work has found,it detected significant difference in the levels of endogenous Ca2+ between PC and WT,PC leaves has more endogenous Ca2+ content,and exogenous NO donors induced increase of PEPC activity,PEPC gene expression,and enhanced photosynthetic via Ca2+ signal.Therefore,we speculate that endogenous Ca2+plays an important role on drought tolerance in PC lines.In this paper,To investigated the effects of Ca2+ on drought tolerance of PC,we used PC and WT as this experimental materials,applications of PEG-6000(Polyethylene glycol-6000,PEG-6000)simulated drought.The results are as follows:1.Applying 0.5 mmol·L-1、2 mmol·L-1 and 10 mmo1·L-1 exogenous CaCl2 on PC and WT of 4-5 leaf age,Under 15%PEG-6000(Polyethylene glycol-6000,PEG)treatment for 2 days and rewater 2 days,leaf relative water content(RWC),net photosynthetic rate(Pn),PEPCase activity and PEPC gene expression in the leaves of the rice plants were investigated.Data showed,PC leaf RWC was significant enhanced under 0.5 mmol·L-1 Ca2+treatment(P<0.05)except 2 mmol·L-1 and 10 mmol·L-1 Ca2+ treatment,and those in WT were unaffected.different exogenous Ca2+ concentration treatments did not affect Pn of PC under 15%PEG treatment,and low Ca2+ concentration treatments(0.5 mmol·L-1)maintained Gs of PC under keeping RWC.Moreover,endogenous Ca2+ concentration data further showed that PC has an certain capacity of maintenance and stabilization endogenous Ca2+ level in leaves under PEG 6000 treatment,while higher exogenous Ca2+ concentration treatment(10 mmol·L-1 Ca2+)decreased PEPCase activity,PEPC expression and the soluble protein in leaves of PC.The results hint that endogenous Ca2+ plays an important role on drought tolerance in PC lines.2.To evaluate the role of the endogenous calcium to drought stress in PC,PC and wild-type rice(WT)were investigated in this study.Under drought stress,we observed that more drought tolerance was exhibited in PC than those in WT according to the higher survival rate and less decrease of net photosynthetic rate(Pn)in long-term Polyethylene glycol-6000(PEG-6000).The involvement of endogenous Ca2+ in increasing the drought tolerance in PC was confirmed in early stage of PEG-600(for 90 min).The results showed that a rapid removal of superoxide radical(O2’)production rate was appeared with a higher SOD and CAT activities of PC leaves.Increasing of CaM content may be involved in PC drought tolerance by Ca2+ signal.While other signaling molecular such as NO and H2O2 of PC and WT were also increased under PEG-6000 treatment for 90 min and the contents of PC was much higher than those of WT.These performance were limited after the application of Glycol-bis(b-aminoethylether)-N,N,N’,N’-tetraaxetic acid(EGTA)(Ca2+chelator)and Ruthenium Red(antagonist of sites of Ca2+ release).At the same time,we did not observe the changes in ABA content in PC plants both under PEG treatment and using calcium inhibitors combined with PEG treatment.Compared with WT,PEG-6000 treatment increased the activities of PEPCase in PC,and expression of PEPC as well.Furthermore,PEG-6000 treatment also upregulated expression of transcription factors OsNAC6 and bZIP60,and protein kinase gene CPK9 both in PC and WT from RT-PCR data and the increases of PC were much higher than those of WT under PEG treatment these performances were also eliminated after the application of calcium inhibitors combined with PEG treatment.The changes of activities of antioxidant enzymes,SOD and CAT were also inhibited the similar trend under the same treatments.These results confirmed the importance of endogenous calcium on PC for drought response.3.To research effect of protein phosphorylation in PC PEPCase activity,further analysis PC and WT leaves total protein kinase and PEPCK(Phosphoenolpyruvate carboxykinase,PEPCK)activities treat with PEG-6000.Results showed that PC leaves total protein kinase was increased under PEG-6000 for 30 min,and PC leaves PEPCK activity was consistented with it.And WT was not affected.And to analysis whether endogenous calcium regulates PC leaves protein kinase and PEPC,applying EGTA and RR treatment,we measure PC and WT leaves total protein kinase and PEPCK activities under PEG-6000 for 60 min.EGTA and RR pro-treatment decreased PC total protein kinase and PEPCK activities,especially EGTA Suppressed completely that.Moreover,H2O2 and NO participate in endogenous calcium-mediated PC total protein kinase and PEPCK activities.As discussed above,applying cPTIO(NO scavenger)and DMTU(H2O2 scavenger),its also inhibited PC total protein kinase and PEPCK activities.These results indicated that endogenous calcium-mediated PC total protein kinase could rapidly response to drought stress,and induced PC leaves PEPCK activity increase PEPCase activity which is required NO and H2O2.Variety of signaling molecules are the important molecular base by be involved in PC response to drought stress.Altogether,PC leaves has higher endogenous Ca2+ content is easier harmed under higher exogenous Ca2+ treatment,endogenous Ca2+ plays an critical role on regulation drought tolerance and adaptation drought environment in PC lines.Between Ca2+ and H2O2,NO signal moleculars cross talk,its increase C4-EPCase activity by transcribed gene and protein phosphorylation to induce PC rapid response to environments stress and maximum keep metabolic activity,which is important event to PC. |
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