Identification Of Adenylyly Cyclase ZmRPP13-LK3 And The Analysis Of CAMP In Thermotolerance In Maize

With the global warming,heat stress occurs more and more frequently in the Huang-Huai-Hai plain of China and leads to serious decrease of the maize yield and quality which becomes a prominent problem to be solved urgently in crop production.Therefore,to solve the problems of maize yield reduction caused by heat stress,it is crucial to better understand the physiological,biochemical and molecular mechanisms of thermotolerance in maize,especially the regulatory mechanism of important genes for cultivating new heat-resistant maize varieties.Adenylyl cyclase（AC）which is the enzyme that catalyzes ATP to form 3’,5’-cyclic adenylate（cAMP）.Although cAMP has been proved to be an important and multifunctional signaling molecule and functions in response to heat stress in plant,it is still unclear about which AC could respond to heat stress and what roles the cAMP plays in plant response to heat stress.To carry out the study will be helpful to reveal the role of the ACs-cAMP signaling cascade in plant response to high temperature.Therefore,by using iTRAQ quantitative proteomics and other technologies,to identify the ACs responding to heat stress and illuminate the mechanism of cAMP produced by AC in response to heat stress in maize will not only lays a theoretical foundation for revealing the mechanism of cAMP in improving the heat tolerance of maize,and also provides genetic resources for breeding the new stress-resistant crops.The main results of this study are as follows:⑴ cAMP alleviates oxidative damage caused by heat stress.In order to investigate the role of cAMP in maize tolerance to heat stress,10 μm 8-Br-cAMP（membrane permeable cAMP analogy）and 20 μm DDA（AC inhibitor,2’,5’-dideoxyadenosine）were used.The results showed that,compared with the control,8-Br-cAMP pretreatment significantly reduced heat stressincreased H2O2 and MDA content in maize leaves and roots caused by heat stress,but promoted the increases of heat-induced SOD and APX activities in maize leaves and roots.However,DDA pretreatment was the opposite results.In addition,after 7 days heat stress,the survival rate of cAMP-pretreated maize plants（86%）was 5 times of that of control maize plants（17%）.These results showed that cAMP pretreatment could maintain the stability of lipid membranes and improve maize thermotolerance by enhancing the activity of antioxidant defense enzymes to eliminate excess peroxides in maize plants,and showed that ACs-cAMP signaling cascade may play a positive role in plant thermotolerance.⑵ Identification of cAMP-regulated differentially expressed proteins under heat stress.To identify which ACs can respond to heat stress and determine the impacts of cAMP on the maize proteome under heat stress,maize plants were pretreated with 8-Br-cAMP or DDA and then subjected to heat stress.Subsequently,leaf proteins were extracted and analyzed by iTRAQ quantitative proteomics technique.310 differentially expressed proteins（DEPs）related to cAMP under heat stress were identified.KEGG pathway analysis showed that these DEPs were mainly involved in metabolism pathways,biosynthesis of secondary metabolic,protein processing in endoplasmic reticulum,oxidative phosphorylation,ribosomes,photosynthesis,ubiquitin-mediated protein degradation,plants-pathogen interaction and so on.This suggested that cAMP participates in a variety of physiological activities in plants,implying its importance in plant adaptation to stress.Besides,we found the protein expression of a putative disease resistance protein 13 like3（ZmRPP13-LK3）was increased 7 times by heat stress and was further induced by cAMP pretreatment.According to the known adenylate cyclase in maize,ZmPSiP,which also belongs to the disease resistance protein family,we thus speculate that ZMRPP13-LK3 may be a novel AC.⑶ Identification of adenylyl cyclase activity of ZmRPP13-LK3 in maize.To prove that ZmRPP13-LK3 is a novel AC,the sequence of ZmRPP13-LK3 and the conserved catalytic active centers of seven known ACs in plants was compared.The results showed there are three conserved AC catalytic centers in ZmRPP13-LK3.Subsequently,three experiments were designed to prove that ZmRPP13-LK3 is a novel AC: 1)the recombinant plasmid of ZmRPP13-LK3 was overexpressed in the cyc A mutant strain of Escherichia coli which lacked AC,and the results showed that the phenotype of the cyc A mutant strain supplemented by ZMRPP13-LK3 was consistent with the wild type;2)the inhibition of ZMRPP13-LK3 in maize protoplast and the overexpression of ZMRPP13-LK3 in HEK293 cell showed that the content of cAMP was significantly reduced and increased,respectively;3)the recombinant ZmRPP13-LK3 protein purified from HEK293 cells with ZmRPP13-LK3 expression was added into the reaction system including the substrates,cAMP production was found in the reaction system,and its amount reached to the maximum at 30 min.Taken together,the results proved that ZmRPP13-LK3 was a new AC in maize,and lay a foundation for revealing the role of ACS-cAMP signaling cascade in plant tolerance to heat stress.⑷ ABA-increased cAMP content and ZmRPP13-LK3 expression under heat stress.This study found that the content of ABA was significantly increased in maize leaves under heat stress.To determine the impacts of ABA on the expression of ZmRPP13-LK3 under heat stress,ABA deficient mutant vp5 and its wild-type Vp5 were used in this study.The results showed that the increase of ZmRPP13-LK3 expression and cAMP content in mutant-type vp5 was significantly less than that in wild-type Vp5 under heat stress.Both heat stress and exogenous ABA could induce the expression of ZmRPP13-LK3 in maize leaves,while the expression of ZmRPP13-LK3 induced by heat stress was decreased by exogenous application of tungstate which was an ABA synthesis inhibitor.In addition,in order to further confirmed the roles heat stress and ABA in regulating the expression of ZmRPP13-LK3,the impacts of heat and ABA on the activity of ZmRPP13-LK3 promoter was analyzed.The results showed that the activity of ZmRPP13-LK3 promoter could be induced by heat stress and ABA.⑸ ZmRPP13-LK3 is located in the mitochondria and interact with ZmABC2.To better reveal the role of ZmRPP13-LK3 in maize thermotolerance,the subcellular localization of ZmRPP13-LK3 and its interaction proteins were analyzed.Confocal results showed that ZmRPP13-LK3 was localized in mitochondria.Yeast two-hybrid and BiFC assays proved that ZmRPP13-LK3 could interact with the ABC transporter ZmABC2.The results suggested that cAMP produced by ZmRPP13-LK3 may be exported from mitochondria through ZmABC2 to regulate the reverse signaling pathway from mitochondria to nucleus.⑹ ZmRPP13-LK3-dependent cAMP involved in ABA-regulated maize thermotolerance.To furthervalidate the role of cAMP produced by ZmRPP13-LK3 in maize thermotolerance,the expression levels of s HSP17.2,s HSP17.4,HSP70 and HSP82 were analyzed by using maize protoplast transient expression system.The results showed that the inhibition of ZmRPP13-LK3 expression siginificantly reduced the expression levels of these genes in maize under stress.In addition,8-Br-cAMP pretreatment significantly promoted the expression of heat tolerancerelated genes WRKY106,RD29 B and ABA1 in the ABA signaling pathway induced by heat stress.These results indicated that cAMP produced by ZmRPP13-LK3 could be involved in ABAregulated maize thermotolerance.⑺ cAMP-mediated thermotolerance in maize roots.To determine the impacts of cAMP on the maize roots under heat stress,maizes were pretreated with 8-Br-cAMP or DDA and then subjected to heat stress,and subsequently,root proteins were extracted to be analyed by iTRAQ quantitative proteomics technique.There 268 differentially expressed proteins（DEPs）related to cAMP under heat stress were identified,including ZmRPP13-LK3.KEGG pathway analysis showed that these DEPs were mainly involved in many important biological processes,such as ion absorption,autophagy or ubiquitin proteasome system degradation of proteins,rapid transport of stress-related cargo molecules through vesicles,and adaptive response of defense proteins.In addition,cAMP pretreatment increased the absorption of Ca²⁺,PO₄^3-and NO₃^-,reduced the absorption of K⁺ and no impacted the absorption of Mg²⁺ in maize roots under heat stress.In summary,this study demonstrated that the ZmRPP13-LK3 was a novel maize AC which located in mitochondria and could respond to heat stress.ZmRPP13-LK3 catalyzed ATP to produce cAMP and cAMP worked as the signaling molecule in the downstream of the ABA signaling pathway to regulate the expression of heat-resistant genes such as HSPS,WRKY106,RD29 B and ABA1 as well as the absorption of mineral nutrients by roots.These research results not only let us have a better understanding of the mechanism of the ZmRPP13-LK3-cAMP signaling pathway in plant thermotolerance,but also lay a foundation for the promotion and application of cAMP as a chemical regulator improving maize production under heat stress as well as provide excellent genetic resources for maize breeding.