Expression Analysis Of CmWRKY1、CmWRKY15 Genes In Chrysanthemum

Chrysanthemum(Chrysanthemum morifolium)is one of the most famous cut flowers globally,has a high ornamental value,and occupies an irreplaceable position in the international commerce of flowers.Drought stress is one of the most harmful types of stress because it retards chrysanthemum growth.Black spot disease,one of the most harmful diseases of chrysanthemum,is caused by the necrotrophic fungus Alternaria.Hence,it is essential to improve chrysanthemum tolerance to achieve sustainable production.In this study,we place emphasis on the mechanism through which CmWRKY modulates the ABA-mediated pathway in response to drought and Alternaria tenuissima in chrysanthemum.The main results are as follows:1.A phylogenetic analysis showed that CmWRKY1 exhibited higher similarity to AtWRKY6 in Arabidopsis.In this study,we employed CmKY1-overexpressing transgenic chrysanthemum lines for PEG treatment.After PEG treatment,the plants were severely wilted with withered leaves.The degree of leaf withering in the wild-type plants was markedly more severe than that observed in the transgenic lines.After the recovery period,the survival rates of the wild type plants and two transgenic lines were 13.3%,51.7%and 56.7%,respectively.Considering these results together,significant differences in dehydration were evident between the CmRKY1-overexpressing and wild-type plants,with the CmWRKY1-overexpressing lines showing increased water loss.Our results indicate that CmWRKY1 overexpression enhances the tolerance of chrysanthemum to dehydration stress.The relative water content(RWC)in the wild type plants and two transgenic lines after exposure to PEG were determined.The results show that the transgenic plants exhibited higher RWCs than the WT plants after treatment with PEG.To ascertain the regulatory mechanisms of CmWRKY1 in response to dehydration stress,the expression levels of a group of ABA-associated genes,including NCED3A,PP2C,PYL2,SnRK2.2,ABF4,RAB18,ABI5,MYB2,DREBIA,ABI4,ABI1 and ABI2,were compared between transgenic and wild-type plants.After dehydration stress,all of the examined ABA-associated genes were robustly activated.In contrast,the transcript levels of some negatively ABA-responsive genes(PP2C,ABI4,ABI5,ABI1 and ABI2)were markedly reduced to varying degrees in CmRKY1-overexpressing lines in comparison with the wild-type plants.In contrast,the expression levels of the positively ABA-regulated genes NCED3A,PYL2,SnRK2.2,ABF4,MYB2 and RAB18 were upregulated in the transgenic lines.These data suggest that the RWC decreases in response to dehydration stress and that the CmWRKYl gene may enhance the dehydration tolerance of transgenic chrysanthemum plants by inhibiting water loss.CmWRKYl plays an important role in the response to drought in chrysanthemum through an ABA-mediated pathway.2.Phylogenetic analysis showed that CmWRKY15 showed high similarity to AtWRKY40 in Arabidopsis.We used transgenic chrysanthemum lines overexpressing CmWRKY15,after exposure to A.tenuissima for 2 weeks,the surface area of the lesions on transgenic plants was much larger compared to the control.Significant differences in A.tenuissima infection were clearly observable between the transgenic lines and wild type plants.The disease severity indexes(DSIs)of transgenic chrysanthemum lines and wild type plants were 57.01,56.73 and 14.63,respectively.The data revealed that the transgenic lines overexpressing CmWRKYl5 were susceptible(S)to black spot disease,while the non-transgenic plants were resistant(R).Necrosis was much more evident in the transgenic plants compared to the WT plants.Our results suggest that CmWRKYl5 overexpression enhanced the susceptibility of chrysanthemum to A.tenuissima attack.Endogenous ABA content was measured in the transgenic lines and wild type plants under normal conditions and 24 h after exposure to A.tenuissima.The results show that WT plants had a higher ABA content than transgenic lines overexpressing CmWRKYl5 under normal conditions and after exposure to A.tenuissima.These data indicate that the ABA content increased in response to A.tenuissima and that CmWRKY15 might inhibit endogenous ABA synthesis in transgenic plants.To identify the mechanisms by which CmWRKY15 responds to A.tenuissima-induced stress,the expression levels of a set of ABA-related genes,including ABF4,ABI4,ABI5,RAB18,DREBIA,DREB2A,PP2C,SnRK2.2,SnRK2.3,RCARI,MYB2,PYL2,NCED3A,NCED3B and GTG1,were compared between transgenic and WT plants.Following A.tenuissima exposure,the transcription of the ABA-upregulated genes was impaired to varying degrees in CmWRKY15-overexpressing lines compared to the WT plants.In summary,our data suggest that CmWRKY15 might facilitate A.tenuissima infection by directly or indirectly antagonistically regulating the expression of ABA-responsive genes.