Study On The Regulation Of Terpenoids Metabolism By ABA And Its Receptor Protein PYL In Hedychium Coronarium

Hedychium coronarium is a perennial herb which belonging to zingiberaceae.It is popular with people because of their beauty,color and fragrance.The main constituents of the scent are terpenes and their derivatives.The precursors were converted to corresponding terpenes by different terpene synthase.However,the synthesis and release of terpenoids are regulatedd by floral development and hormones,and there are significant differences in the release amount of terpenoids in different species.Abscisic acid plays an important role in the process of flower maturation and senescence in various flowers.However,there is no report on the relationship between abscisic acid and the synthesis and release of terpenoids of flowers.In this paper,terpenoid volatile content and ABA content in the flower development process,were determined by GC-MS and HPLC technology in differnent Hedychium species.And then,expression of related genes were analysised by fluorescent quantitation,suggesting that the regulation of ABA on terpenoid synthesis and release.By the method of endogenous phytohormone treatment,GC-MS,HPLC,RT-qPCR,Virus silence,subcellular localization,yeast two-hybrid,bimolecular fluorescence complementation,CO-immunoprecipitation,Western blot,co-transformation in tobacco,the regulation of fragrance release,expressions of terpene synthases and MYC2 s transcription factor by ABA and its receptor protein PYL were investigated in Hedychium coronarium.The study aims to explore the molecular mechanism of the formation of the terpenoid in Hedychium coronarium,the signal regulation of abscisic acid and the intersection or co-regulation between the two.The main results are as follows:1.The content of the terpene volatiles in the flower development process in differnent Hedychium species was determined by GC-MS technique.The results showed that the terpenoids release of Hedychium coronarium and Hedychium gardnerianum was significantly higher than that of Hedychium coccineum.Hedychium coronarium and Hedychium gardnerianum,two scented species,performed high consistency in the pattern of floral volatiles.The pattern is that floral volatiles gradually increasing with the bloom and gradually decreasing with aging,which in line with the characteristics of flowers “not open not fragrant”.Among them,the content of the volatile of the ocimene in Hedychium coronarium was slightly lower than that of Hedychium gardnerianum,while linalool was much higher.The total cotent of the terpene volatiles was: Hedychium coronarium> Hedychium gardnerianum>Hedychium coccineum.Combined with fluorescence quantitative analysis,it showed that the expression of terpene synthase gene was the same as that of the terpene,which were significantly different among different species.2.The content of ABA in the flower development process was determined by HPLC technique.The results showed that the content of ABA in the flower development process was: Hedychium coronarium>Hedychium gardnerianum>Hedychium coccineum.Hedychium coronarium shared the same tendency with Hedychium gardnerianum,which increased first and then decreased and there was a peak of ABA content before the bloom.The content of ABA did not change significantly in Hedychium coccineum.The results were in accordance with the the pattern of floral volatiles,indicating that ABA is related to the release of floral volatiles and more,ABA may be involved in the regulation of the synthesis and release of floral volatiles.Combined with fluorescence quantitative analysis,the results showed that ABA metabolism was regulated by multiple genes(NCED1,NCED2,ABA2 and CYP707 A of the key enzymes of anabolism and catabolism).3.After treatment with exogenous hormone ABA and its inhibitor,it can significantly induce and reduce the release of floral volatiles in Hedychium coronarium.Among them,ocimene and methyl benzoate increased significantly,linalool extremely and significantly increased,reaching to 40%-70% after treatment with the exogenous hormone ABA;while the content of floral volatiles such as myrcene,ocimene,methyl benzoate and alloocimene decreased significantly after the treatment with the ABA inhibitor.Combined with fluorescence quantitative analysis,the results showed that the relative expressions of ocimene,linalool and methyl benzoate synthase gene HcTPS3,HcTPS1,HcTPS5,HcBSMT1 and HcBSMT2 were significantly increased,reaching to 30%-70% after ABA treatment;while the relative expression of floral genes decreased in varying degrees,including terpene synthase related genes HcTPS3 and HcTPS1 decreasing significantly,HcTPS5 decreasing extremely and significantly after ABA inhibitor treatment.The change of relative expression of these genes directly led to the increase and the decrease of the release of floral volatiles in Hedychium coronarium,which were approximately identical with the result of GC-MS.4.It can significantly induce and reduce the content of endogenous ABA and expression of relative genes in ABA signal element in Hedychium coronarium after treatment with exogenous hormone ABA and its inhibitor.The results showed that the endogenous ABA content increased 18 times after exogenous ABA treatment,decreased by 82.7% after sodium tungstate treatment,decreased by 92.4% after NDGA treatment and decreased by 98.1% after sodium tungstate and NDGA treatment.Combined with fluorescence quantitative analysis,it showed that the relative expression of signal transduction factors,like HcPP2C-1,HcPP2C-2 and HcABF were significantly increased by the treatment of ABA,while HcPP2C-1,HcPP2C-2,HcSnRK2-1 ?HcSnRK2-3 were significantly decreased by ABA inhibitor.On the other hand,the relative expression of transcription factor HcMYC2-1 was significantly decreased and increased after treatment with exogenous hormone ABA and its inhibitor.5.The full length sequence of cDNA of ABA signal receptor protein PYL gene from the flowers of Hedychium coronarium was cloned by using RT-PCR technology.Sequence analysis showed that the cDNA length of HcPYL was 621 bp,encoding 206 amino acids,which contained a conserved PYR/PYL/RCAR domain,belonging to the SRPBCC domain superfamily.This family has a deep hydrophobic ligand binding pocket,which can bind to ABA and regulate the signal transduction.Phylogenetic analysis showed that HcPYL and MaPYL1 are a subfamily.MEME online analysis of the conserved motif found that all of the different species include Motif1,Motif2 and Motif3 motifs,which indicated that the HcPYL protein was highly conserved.Homology analysis showed that the PYR/PYL/RCAR protein domains of the amino acid sequences of different species contained a high degree conservation of most domain of Motif1,Motif3 and all domain of Motif2.At the same time,CL2(SGLPA)Loop rings and CL3(HRL)Loop rings were conserved in different species,which indicated that the mechanism of PYL protein binding ABA was similar.Combined with fluorescence quantitative analysis,it was found that the expression pattern of the HcPYL gene increasd first and then deceased in Hedychium coronarium,which reached the highest at the blossom stage,in line with the release laws of floral volatiles.6.Plant virus silencing vector of HcPYL was constructed to infect the petals of Hedychium coronarium through agrobacterium tumefaciens for instant interference.The results showed that using pCa BS-? virus vector as a control after silencing of HcPYL,ocimene,methyl benzoate and linalool decreased by 39%,36% and 45.8%,respectively.myrcene,eucalyptol,alloocimene and farnesene were reduced by 36%-47%.Combined with the quantitative analysis,it showed that silencing of HcPYL,the relative expression of HcPYL decreased by 65.9%.ABA related genes and transcription factors such as,HcMYC2-1 and HcMYC2-2 decreased by 48.2% and 41.2%,HcNCED1 and HcNCED2 by 58.6% and 39.7%,HcCYP707 A by 37.2%,while HcABA2 increased by 33.1%;on the other hand,the relative expression of floral genes HcTPS3 decreased by 83.4%,HcBSMT1 and HcBSMT2 by 40.3% and 59.6%,HcTPS1,HcTPS5 and HcTPS8 by 41.2%,53.2% and 84.7%.The results showed that HcPYL had some effect on the volatile components of Hedychium coronarium.7.Two kinds of GFP transient expression vector of HcPYL were constructed(pEAQ-GFP and p35S-GFP),then transformed into tobacco leaves by Agrobacterium tumefaciens and mediated into Arabidopsis thaliana protoplasts by PEG,respectively.The results of these two methods showed that HcPYL was localized in the nucleus and cytoplasm.8.The bait vector of HcPYL and the prey vector of HcMYC2-1 were constructed.The yeast two-hybrid system was carried out to demonstrate that HcPYL can interact with HcMYC2-1.Then,C-terminal vector of HcPYL and N-terminal vector of HcMYC2-1 of bimolecular fluorescence complementation was constructed,then co-transformed to tobacco leaves by Agrobacterium.After three days,GFP fluorescence is observed very weak and almost does not exist by fluorescence microscope.rthermore,it was found that the interaction between HcPYL and HcMYC2-1 was significantly enhanced by exogenous ABA treatment.These results indicated that ABA could enhance the interaction between HcPYL and HcMYC2-1 in tobacco,and it might be related to the conformation of PYL by ABA.9.The GFP vector of HcPYL and the His vector of HcMYC2-1 were constructed for Co-IP,then co-transformed to tobacco leaves by Agrobacterium.After three to four days,total protein was extracted for Co-IP and Western blot,to further verify the interaction between HcPYL and HcMYC2-1 protein,while the degree of interaction could be enhanced under ABA treatment.The result was in agreement with the previous yeast two-hybrid and bimolecular fluorescence complementation experiments.