A Functional Study Of NtCAF1 Affecting The Development Of Epidermal Trichomes In Arabidopsis

The trichome is a specialized epidermal appendage of plant epidermal cells,which plays an important role in plant response to a variety of biotic and abiotic stresses.In the model plant Arabidopsis thaliana,the molecular mechanism of trichome formation has been tentatively elucidated,but some unknown regulatory factors have not been discovered.In previous studies,the glandular hair density of transgenic tobacco Ch IFN-? was more than doubled.The whole gene expression profile of transgenic tobacco Ch IFN-? was detected by Agilent gene microarray.Seven genes related to glandular hair development were screened out,and four candidate genes Cy P71,LBM1,Isp H and CAF1 were obtained by fluorescence quantitative PCR,of which Cy P71,LBM1 and Isp H were screened and genetically transformed into Arabidopsis thaliana with the changes of plant trichomes.It was found that the trichomes of overexpressed plants increased significantly.Therefore,candidate gene CAF1(CCR4-Assocaited Factor 1)was selected as the target gene in this study.Through genetic transformation of Arabidopsis thaliana,its effects on the growth and development of plant trichomes were studied.The main research contents and results are as follows:1.The protein three-dimensional structure of At CAF1a(AT4G34350)and wild-type tobacco Nt CAF1(KJ003855)were compared by SWISS-MODEL,an online bioinformatics analysis software.The results showed that the protein domains of Nt CAF1 and At CAF1 a were similar,with a highly conserved domain CAF1 Super Family,which was responsible for the catalysis of m RNA adenylate,and the CCC4-CAF1 complex was highly conserved in eukaryotes.2.The overexpression vector p SH737-Nt CAF1 of the target gene was constructed and transformed into Agrobacterium tumefacien LBA4404,and Arabidopsis thaliana was genetically transformed by dipping flowers,of which mature seeds sowed on the resistant plate and then moved to the marl.GUS staining results showed that the expression of GUS reporter gene could be activated in roots,stems and leaves of transgenic Arabidopsis thaliana.Compared with the wildtype at the same time,the transgenic Nt CAF1 plants grew a little better and had larger leaves.These results suggested that Nt CAF1 could promote the development of vegetative organs in Arabidopsis thaliana.3.By observing the trichome,it was found that the number of trichome of Nt CAF1 in Arabidopsis thaliana was higher than that of WT.Statistical analysis found that compared with WT Arabidopsis at seedling stage,bolting stage and fruiting period,the number of trichome of transgenic Nt CAF1 Arabidopsis thaliana plants increased significantly,being 1.4,2.42 and 1.58 times that of WT,respectively,while the number of trichome of ?caf1a mutant plants decreased significantly,being 0.68,0.71 and 0.82 times that of WT,respectively.The results showed that the expression of CAF1 gene was positively correlated with the development of Arabidopsis thaliana trichome.4.ACT2 was selected as the reference gene,and fluorescence quantitative PCR was used to measure the expression levels of the key genes that regulate trichome development in Arabidopsis thaliana plants(GL1?GL2?TTG1?EGL3?CPC)of transgenic Nt CAF1,WT and ?caf1a mutants.The results showed that compared with WT Arabidopsis thaliana at different stages,the expressions of GL1,GL2,TTG1 and EGL3 genes in the transgenic Nt CAF1 plants were significantly increased,while the expression of CPC gene was decreased.The expression levels of GL1,GL2,TTG1 and EGL3 in ?caf1a mutant were decreased,while the expression levels of CPC gene were increased.Meanwhile,the expression level of CAF1 in the leaves of transgenic Nt CAF1 Arabidopsis plants was significantly higher than that of WT.These results indicate that increasing the transcriptional activity of CAF1 can directly affect the expression of MBW triple protein complex(GL1?EGL3 and TTG1).5.Some studies reported that CAF1 in tomato was overexpressed in pepper,and it was found that the expressions of AOC and AOS,the key genes regulating JA synthesis,were increased in pepper.Therefore,in order to further reveal the reasons for the upregulated expression of key genes regulating trichome development in transgenic Nt CAF1 plants,the expression levels of key JA synthesis genes AOS and AOC in transgenic Nt CAF1,WT and ?caf1a mutant Arabidopsis thaliana at different periods were detected.It was found that in the leaves of transgenic Nt CAF1 plants,the expressions of AOC and AOS,the key genes of JA synthesis,were significantly increased in the leaves of transgenic Nt CAF1 plants,while the expressions of AOC and AOS were significantly decreased in the ?caf1a mutant plants.The content of endogenous jasmonic acid and salicylic acid of Nt CAF1,WT and ?caf1a Arabidopsis leaves at bolting stage and fruiting period was texted by LC-MS,and the results showed that in bolting stage,compared with WT,flowers turn Nt CAF1 JA and SA in Arabidopsis thaliana plants significantly increased(p < 0.05),1.15 times and 1.52 times respectively,?caf1a JA and SA in Arabidopsis thaliana plants decreased significantly(p < 0.05),1.26 times and 1.18 times respectively;Compared with WT,the contents of Me SA,JA and Me JA in the leaves of transgenic Nt CAF1 Arabidopsis thaliana were significantly increased(P <0.05)by1.44,0.72 and 1.09 times,respectively;the contents of Me SA,JA and Me JA in ?caf1a Arabidopsis thaliana leaves were significantly decreased(P <0.05)by 0.27,0.34 and 0.13 times,respectively.However,there was no significant difference in SA content between the transgenic Nt CAF1 and?caf1a mutants in Arabidopsis leaves.These results indicate that changes in the transcription level of CAF1 can directly or indirectly affect the contents of endogenous hormones(JA,Me JA,SA,and MESA)in Arabidopsis thaliana.In this study,the content of endogenous hormones in Arabidopsis thaliana plants was detected by LC-MS,and it was found that the content of JA was significantly increased in Arabidopsis thaliana plants overexpressing CAF1,while the content of JA was significantly decreased in Arabidopsis thaliana plants expressing ?caf1a mutants.Indicate,CAF1 exists in the upstream of JA.Therefore,CAF1 affects the content of JA through an unknown pathway,the accumulation of JA content enhances the transcription of MYB-BHLH-WD40 complex,promotes the expression of GL2,and thus regulates the development of Arabidopsis thaliana trichome.