| Trichomes are unicellular or multicellular hair-like structures that initiate from epidermis of many plants,and are classified into two main classes: glandular and non-glandular.Glandular trichomes include a great variety of structures and shapes,but all have the capacity to synthesize,store or secrete large quantities of specialized metabolites.As such,glandular trichomes were qualified as true cell biofactories for the production of valuable chemicals,making them suitable targets for metabolic engineering and breeding.However,it is not commensurate with their importance that little is known about the regulatory molecular mechanism underlying the development of glandular trichomes.Artemisia annua is famous for its production of the antimalarial drug artemisinin.There are only two kinds of trichomes in A.annua,one is glandular trichome where the artemisinin is produced and the other is T-shaped nonglandular trichome.They can be easily distinguished from each other according to their appearance.In addition,the genetic transformation of A.annua is effective and stable,and its genome sequencing has been completed.Jasmonate promotes the development of glandular trichomes in A.annua,which increases the content of artemisinin.Therefore,A.annua plant was used in this study.By combination of the effects of gene cloning,gene expression analysis,yeast one hybrid assay,yeast two hybrid assay,Co-Immunoprecipitation,Dual luciferase assay and functional study by transgenic A.annua plants,the main results were achieved as follows.1.The homeodomain-leucine zipper transcription factor AaHD1 was regulated by JA on both transcriptional and protein levels.Furthermore,AaHD1 positively controls glandular trichome initiation.JA signaling pathway is conserved in different plants,it mainly roles through controlling the interaction between the repressor protein JAZ and the corresponding transcription factor proteins.Consequently,AaJAZ8 was used to screen the yeast two hybrid library made by A.annua young leaf cDNA.Several transcription factors,including AaHD1,a HD-ZIP IV subfamily protein,were found interacting with AaJAZ8.Previously researches showed that HDZIP IV subfamily transcription factors typically have an epidermis-specific expression pattern and were associated with epidermal cell differentiation,including trichome development.Therefore,AaHD1 was chosen for further investigation.Expression analysis of AaHD1 in different organs,at different leaf positions and after phytohormones treatment and GUS activity identification all reveal that the expression pattern of AaHD1 is similar with the development pattern of glandular trichomes.Then we found that in the AaHD1 RNAi knock-down lines,the densities of both glandular and non-glandular trichomes were significantly decreased with the reduction of artemisinin content while compared with wild type plants.Meanwhile,in the AaHD1-overexpressing lines,the densities of both glandular and non-glandular trichomes were significantly increased with the increase of artemisinin content while compared with wild type plants.The phenotype analysis by SEM showed that the phenotype of both glandular and nonglandular trichomes had no different with wild-type plants.The results showed that AaHD1 promotes both glandular and non-glandular trichome initiation,but not their later development.Moreover,glandular trichome initiation of AaHD1 knock-down lines showed less sensitivity to JA compared with wild plants,indicating that AaHD1 plays an important role in JA-mediated glandular trichome initiation.The Dual-LUC assay revealed that the interaction between AaHD1 and AaJAZ8 suppressed the transcription activity of AaHD1.My work reveals a novel mechanism by which JA promotes glandular trichome formation via a homeodomain protein dependent pathway.2.Identification of A.annua HD-ZIP IV transcription factor,AaHD8,which interacts with AaMIXTA1 to promote the development of trichome by positively regulating the expression of AaHD1.By analyzing the promoter sequence of AaHD1,six L1-box cis-elements were found between-526 and-1326 in the AaHD1 promoter.On this basis,AaHD1 mostly was directly regulated by other HD-ZIP IV members in A.annua.Through the yeast one-hybrid analysis and Dual-LUC assay,we identified that AaHD8 promotes AaHD1 expression by directly binding to the L1-box in its promoter.Then the results showed that in the AaHD8 RNAi knock-down lines,the densities of both glandular and non-glandular trichomes were significantly decreased with the reduction of AaHD1 expression and the artemisinin content was also significantly decreased.Meanwhile,in the AaHD8-overexpressing lines,the densities of both glandular and non-glandular trichomes were significantly increased with the increase of AaHD1 expression,and the artemisinin contents were also significantly increased.Therefore,AaHD8 appears to be upstream of AaHD1,since the expression level of AaHD8 has no change in neither AaHD1-silenced nor overexpressed lines.These results reveal that AaHD8 promotes the initiation of both glandular and non-glandular trichomes development by positively regulating the expression of AaHD1.To further investigate how AaHD8 functions,we screened an A.annua young leaves cDNA library for proteins interacting with it.Several proteins were identified,including a MYB transcription factor AaMIXTA1 which has been proved to be involved in regulating glandular trichome initiation in A.annua by our group.Given the fact that both AaHD8 and AaMIXTA1 were involved in positively regulating glandular trichome initiation,we wondered whether their interaction would impact the transcriptional activation of AaHD1.Consequently,a dual-LUC assay in tobacco leaves was performed for this purpose.The luciferase activity controlled by AaHD1 promoter was elevated when AaHD8 and AaMIXTA1 were expressed alone.Activation of AaHD8 to the AaHD1 promoter was significantly enhanced by AaMIXTA1,and this enhancement was more than addition.Our results uncover a novel molecular mechanism whereby a HD-ZIP IV/MIXTA complex to promote glandular trichome initiation in A.annua. |
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