Effects Of H₂O₂ And MAPKs Protein Kinases On The Content Of 2-phenylethyl Chromone In Callus Of Aquilaria Sinensi

Aquilaria sinensis classified as Thymelaeacea,is one of essential plant resources for producing agarwood in China which is widely used pharmaceutically as sedative,digestive and anti-emetic in tranditional medical treatments,and is also as incense and perfume.Agarwood is the resinous wood of Aquilaria sinensis trees after they have been wounded.However,little is known about the mechanism of agarwood formation.Previous studies indicated that agarwood is the product of a plant defense response.2-（2-phenylethyl）chromones derivatives are the principal components responsible for the quality of agarwood and are also the defensive substances.H₂O₂,NADPH oxidase and MAPKs family play important roles in plant defense under various biotic and abiotic stresses.However,little is known about the involvement of H₂O₂,NADPH oxidases and MAPKs family have an effect on 2-（2-phenylethyl）chromones accumulation.In present study,the regulation of H₂O₂ and NADPH oxidases on the production of 2-（2-phenylethyl）chromones induced by salinity stress were firstly investigated by pharmacological approaches.Furthermore,we indicated that AsMAPKK5-AsMAPKll regulated the production of 2-（2-phenylethyl）chromones under salt stress.Our studies facilitate the understanding of the potential mechanisms of salt-induced agarwood formation and provide new clues for plant defense.Our previous study showed that the activity of the NaCl-treated cells decreased significantly at 120 h,reaching to 5%of the control samples.Therefore,the amount of 2-（2-phenylethyl）chromones and the content of H₂O₂ were examined by LC-MS-IT-TOF system after salt treatment within 120 h.Salt stress also induced a transient increase in AsRbohA-C expression and NADPH oxidase activity.Furthermore,exogenous H₂O₂ increased AsRbohA-C expression and NADPH oxidase activity,while DMTU inhibited AsRbohA-C expression and NADPH oxidase activity under salt stress.Moreover,DPI,the inhibitor of NADPH oxidases,reduced AsRbohA-C expression and NADPH oxidase activity,but significantly induced 2-（2-phenylethyl）chromones accumulation during salt stress.These results clearly demonstrated the central role of H₂O₂ and NADPH oxidases have an important influence in salt induced 2-（2-phenylethyl）chromones accumulation in A.sinensis calli.Our previous study indicated that the main 2-（2-phenylethyl）chromones detected from the calli were 6,7-dimethoxy-2-[2-（4’-methoxyphenyl）ethyl]chromone（AH6）and 6,7-dimethoxy-2-（2-phenylethyl）chromone（AH8）during the early inducing period.Therefore,the occurrence of AH6 and AH8 could be used as a vital indicator to investigate the formation of agarwood.To determine whether MAPKKs could be involved in 2-（2-phenylethyl）chromones accumulation,the calli were performed with NaCl and PD98059,the inhibitor of MAPKK.The results from LC-MS-IT-TOF system and AB Sciex 5500 Qtrap mass spectrometer indicated that the content of 2-（2-phenylethyl）chromones,including AH6 and AH8,was remarkably decreased after application of PD98059 under salt treatment,comparing with the samples treated with NaCl.Trypan blue staining and DAB staining of calli indicated that salt stress induced cell death and H₂O₂ accumulation in the cells of calli.A novel gene,AsMAPKK5 was isolated and characterized from A.sinensis.Agrobacterium-mediated protein expression experiments indicated that AsMAPKK5 protein localized in cytoplasm and cell wall,and transient expression of AsMAPKK5 protein resulted in cell death characterized by trypan blue staining and DAB staining,suggesting AsMAPKK5 could be involved in 2-（2-phenylethyl）chromones accumulation under salt stress.Quantitative reverse transcription（qRT-PCR）results indicated the transcript level for AsMAPKK5 was elevated by salt stress and could be inhibited by PD98059.Importantly,the expression of AsPKS3 was remarkably increased after overexpressing AsMAPKK5,comparing with the pCAMBIA1300-35S-EGFP.These results suggested that AsMAPKK5 was had an effect on 2-（2-phenylethyl）chromones accumulation during salt stress.MPK cascades in plants generally consist of three components:MAPK kinase kinases（MAPKKKs）,MAPK kinases（MAPKKs）,and MAPKs;these components are sequentially activated by phosphorylation to transfer information from sensors.In order to investigate how does AsMAPKK5 regulate the 2-（2-phenylethyl）chromones accumulation signaling pathways,we use the yeast two-hybrid to screen for AsMAPKK5 interacting proteins.The results showed that AsMAPKK5 interacted with AsMAPK11.Agrobacterium-mediated protein expression experiments indicated that AsMAPK11 protein localized cell wall,and transient expression of AsMAPKK5 protein could not result in cell death characterized by trypan blue staining and DAB staining.Furthermore,we confirm that AsMAPKK5 can physically interact with AsMAPKll using Bimolecular fluorescence complementation（BiFC）,Far Western Blotting and GST Pull-down assays,respectively.The expression level of AsMAPK11 was also increased under salt stress and inhibited by PD98059.Furthermore,the expression of AsPKS3 was remarkably increased after overexpressing AsMAPK11.These results indicated that AsMAPKK5-AsMAPK11 had an effect on 2-（2-phenylethyl）chromones accumulation during salt stress.