Molecular Mechanisms Of CmBBX24.1 And CmBBX22 Involved In Chrysanthemum Drought Tolerance

Chrysanthemum(C.morifolium)originated in China and is an ornamental flower cultivated by long-term artificial selection,it has been cultivated for more than three thousand years in China.Chrysanthemum was not only popular in our country,but also in the world.It is one of the ten famous flowers in China and the four cut flowers in the world,due to its high ornamental and great economic value.However,it often suffers from various abiotic stresses.Drought and salt act as important stress factors,which seriously affect the growth,quality,yield and geographical distribution of chrysanthemum.BBX transcription factors regulate plant growth and development,as well as in response to abiotic stresses.However,the regulation mechanism of BBX transcription factors on abiotic stress in chrysanthemum was not well understood.The study is aim to clone the BBX transcription factor and analyze its function in response to drought and salt stress,and lay the foundation for the identification of drought and salt-tolerance genes and cultivation of new varieties breeding.The main contents and conclusions are as follows:1.A BBX transcription factor was cloned and designated as CmBBX24.1.Its amino acid sequence has the structural characteristics of the fourth subfamily member and contains two B-boxs domains.The results of qRT-PCR showed that CmBBX24.1 had the highest expression in ligulate flowers and leaves.The expression of CmBBX24.1 was reduced by drought and exogenous ethylene treatment.We also find the expression of CmBBX24.1 was induced by exogenous ABA treatment.Subcellular localization and transcriptional activity experiments indicate that the CmBBX24.1 protein was localized in the nucleus and has transactivation activity.We constructed chimeric repressor vector SRDX-CmBBX24.1 and transformed into chrysanthemum by Agrobacterium-mediated leaf disk infection method.Drought tolerance of transgenic plants was assessed upon drought treatment,plants were re-watered and continually cultured for 1 week to count the plant survival rate.The transgenic plants displayed a higher death rate when compared with the WT plants.We then compared stomatal apertures between transgenic and wild type plants,results suggest that the stomatal opening in two SRDX line plants were more than wild type.In addition,transcriptome analysis of CmBBX24.1 transgenic lines and wild type showed that the expression of genes involved in ethylene synthesis,such as ACO and ACS,were up-regulated in the transgenic lines,and the ACC content of the SR-B24.1 transgenic lines was higher than wild type.2.To further clarify the regulatory mechanism of CmBBX24.1-mediated salt and drought tolerance.We used a CmBBX24.1-Boxs segment with no transcriptional activation activity as a bait protein for yeast two-hybrid screening library,and screened a zinc finger transcription factor family protein CmBBX22.CmBBX22 contains two B-Box domains belonging to the fourth subfamily of the BBX family.The CmBBX22 sequence included an 807 bp open reading frame encoding 268 residues.The interaction between CmBBX22 and CmBBX24.1 was further confirmed by yeast two-hybrid,BiFC.Subcellular localization and transcriptional activity experiments indicate that the CmBBX22 protein was localized in the nucleus and has transactivation activity.3.Tissue quantification showed that CmBBX22 had the highest expression in tubular flowers and leaves.The expression of CmBBX22 increased first and then decreased under drought conditions,and the expression reached the highest level after 12 h treatment.In addition,the expression of CmBBX22 was dramatically induced by the treatment of 200 mM NaCl within 6 h and gradually downregulated.The expression of CmBBX22 was induced by exogenous ethylene and ABA treatment.We constructed genetic transform,ation expression vectors pMDC43-CmBBX22,the pMDC43-CmBBX22 vector was transformed into chrysanthemum by Agrobacterium-mediated leaf disk infection method.After drought and salt treatment,transgenic chrysanthemum overexpressing CmBBX22 exhibited significantly reduced drought and salinity tolerance.Constitutive expression of CmBBX22 can promote the expression of genes involved in ethylene synthesis,such as ACOs,which was consistent with the up-regulation of ACO in the transcriptome of overexpressing lines and the ethylene content by gas chromatography.In CmBBX22-OE plants,ethylene content were increased significantly than wild type plants.Based on these results,our data indicate that CmBBX22 regulates drought and salinity resistance by affecting the ethylene biosynthesis in chrysanthemum.These data indicated that CmBBX22 and CmBBX24.1 involved in the drought tolerance of chrysanthemum by regulating ethylene synthesis synergically.