Molecular Mechanisms Of CmSCL4 And CmTCP14 Involved In Chrysanthemum Drought Tolerance

Chrysanthemum(Chrysanthemum morifolium),as one of the ten famous flowers in China and the four cut flowers in the world,possesses high ornamental and great economic value.However,it often suffers from various abiotic stresses and as one of the most important stress factors,drought impacts the growth,development and the flowers quality of chrysanthemum.GRAS and TCP transcription factors have been demonstrated to be involved in a large variety of growth and development processes,as well as in response to biotic and abiotic stresses.However,there is little information about GRAS and TCP transcription factors participating in drought tolerance in chrysanthemum so far and our study are aimed to isolate the sequences and elucidate the molecular functions of the corresponding genes and laid the foundation for the identification of drought-tolerance genes and cultivation of new varieties breeding.The main contents and conclusions are as follows:1.According to the transcriptome data,we obtained a GRAS gene.By phylogentic analysis,this GRAS gene was classified into AtSCL4/7 subfamily and designated as CmSCL4.Quantitative polymerase chain reaction(qPCR)analysis revealed that CmSCL4 was dramatically induced by the treatment of 20%PEG6000 within 3 h and gradually downregulated afterward.We cloned the promoter of CmSCL4 which contains sereral drought-induced MYB binding sites.The transcriptional abundance of CmSCL4 was relatively higher in the stem compared with leaf and root.Subcellular localization indicated that CmSCL4 was specifically localized in the nuclei of the N.benthamiana leaf epidermal cells.Y1H assay revealed that CmSCL4 had transactivation activity.2.We constructed genetic transformation expression vectors pMDC43-CmSCL4 and pSRDX-CmSCL4,and introduced the pMDC43-CmSCL4 into Arabidopsis,pMDC43-CmSCL4 and PSRDX-CmSCL4 into 'Jinba' via floral dip and Agrobacterium tumefaciens mediated leaf disc methods,respectively.The transgenic Arabidopsis lines have higher survival rate and lower water loss rate compared with WT.Transcriptional aboundances of ABA synthesis and signaling genes indicated that in transgenic lines the expression levels of AtAB13 and AtABI4 were higher than in WT either before drought or treated for 6 h.The seed germination and early seedling development of CmSCL4 overexpressed lines were severely suppressed when sown on 1/2 MS plates containing 0.1?mol ABA,while there was no difference between them on 1/2 MS plates.These results showed overexpression of CmSCL4 enhanced the sensitivity to ABA in Arabidopsis.Under 20%PEG6000 treatment,the pMDC43-CmSCL4 transgenic chrysanthemum lines had lower wounding leaves rate than WT and pSRDX-CmSCL4.Meanwhile,the relative water content(RWC)of pMDC43-CmSCL4 lines was higher than WT and the pSRDX-CmSCL4 lines had the lowest RWC under 20%PEG6000 treatment for 3 days.The relative expression level of CmABI4 in PMDCA3-CmSCL4 lines was higher than in WT and pSRDX-CmSCL4 lines.Thus,we made the conclusion that CmSCL4 participates in ABA signaling pathway to positively regulate the drought tolerance of chrysanthemum probably by regulating the expression of CmABI4.3.To further clarify the regulatory mechanism of CmSCL4-mediated drought tolerance,yeast two-hybrid screening assay was performed with the truncated region of 181-561 aa of CmSCL4 which exhibited no transactivation activity.By screening the cDNA library of chrysanthemum,several clones encoding a MYB transcriptional factor were identified and the full-length cDNA was further conformed by transcriptome data and RACE method.Sequence analysis revealed that this MYB transcriptional factor contains the conserved MYB-CC and SANT domains and was designated as CmRIMYB1 hereafter.Expression level of CmR1MYB1 was upregulated upon 20%PEG6000 treatment,reaching the highest level at 3 h and gradually decreased afterwards.The interaction between CmSCL4 and CmR1MYB1 was further confirmed by yeast two-hybrid,BiFC and pull-down assays and the MYB-CC domain of CmRIMYB1 was indispensable for the interaction.We also generated the 35S:CmR1MYB1 transgenic Arabidopsis and chrysanthemum plants.The transgenic Arabidopsis plants were more sensitive to ABA treatment and the drought tolerance was also significantly enhanced.qRT-PCR analysis showed that expression levels of the ABA response gene AtABI3 and AtABI4 were obviously upregulated in transgenic lines,whereas no significant differences were detected for AtABI1,AtABI2 and AtABI5.Moreover,leaves of wild type appeared more wilted than the transgenic plants upon 20%PEG6000 treatment Taken together,these results revealed that CmSCL4 regulates drought tolerance by interacting with CmRIMYB1 in a ABA-dependent manner.4.A full-length cDNA encoding a TCP family transcriptional factor was identified from our transcriptome data combined with RACE method and designated as CmTCP14 hereafter.Expression level of CmTCP14 was initially decreased upon 20%PEG6000 treatment,reaching the lowest level at 3 h and gradually upregulated afterwards.qPCR analysis revealed that CmTCP14 was predominately expressed in stem and barely detected in the ligulate flower.CmTCP14 was specifically distributed in the nuclei and exhibits no obvious transactivation activity in yeast cells.35S::CmTCP14 transgenic Arabidopsis plants displayed significantly enhanced drought tolerance and delayed flowering and senescence progress.To further clarify the CmTCP14-mediated drought tolerance mechanism,yeast two-hybrid screening assay was performed with our chrysanthemum cDNA library and four cDNA fragments encoding DELLA proteins were identified.We further isolated the full length of the proteins and found they all displayed obvious transactivation activity and extensively localized in the nuclei.Moreover,the mechanism of CmTCP14 interaction with CmDELLAs involved in drought reaiatance and other processes need to be further uncovered.