Analysis Of Genome-wide Transcriptional Response To Dehydration And Functions Of DBBs Genes In Chrysanthemum

Chrysanthemum is one of the most important ornamental crops in the world and widely used in cut flowers, potted plant and landscape. Drought stress seriously limits its production and distribution. In order to generate a functional genomic resource and obtain a deeper understanding of the molecular mechanisms regarding chrysanthemum response to dehydration stress, we performed large-scale transcriptome sequencing of chrysanthemum plants [Chrysanthemum×morifolium(Ramat.)Kitamura]'Fall Color' under dehydration stress using the Illumina sequencing technology and selected four abiotic stress-reponsive DBB subfamily members to analyse the expression pattern and biochemical features,and further investigated the role of CmDBBs by overexpression in the model plant Arabidopsis.The main results are listed as follow: 1) Two cDNA libraries constructed from mRNAs of control and dehydration-treated seedlings were sequenced by Illumina technology. A total of more than 100 million reads were generated and de novo assembled into 98180 unique transcripts which were further extensively annotated by comparing their sequencing to different protein databases. Biochemical pathways were predicted from these transcript sequences. 2) We performed gene expression profiling analysis upon dehydration treatment in chrysanthemum and identified 8558 dehydration-responsive unique transcripts, including 307 transcription factors and 229 protein kinases and many well-known stress responsive genes. Gene ontology (GO) term enrichment and biochemical pathway analyses showed that dehydration stress caused changes in hormone response, secondary and amino acid metabolism, and light and photoperiod response. These findings suggest that drought tolerance of chrysanthemum plants may be related to the regulation of hormone biosynthesis and signaling,reduction of oxidative damage, stabilization of cell proteins and structures, and maintenance of energy and carbon supply. 3) We further searched four CmDBBs subfamily genes in the chrysanthemum transcriptome, and named CmBBX19, CmBBX22a, CmBBX22b and CmBBX22c, seperately. RT-PCR analysis showed that CmBBX19 mainly expressed in leaf, flower and stem, CmBBX22a mostly in flower,while CmBBX22b and CmBBX22c mainly expressed in leaf and root. Abiotic stresses analysis showed that the expression of CmBBX22b was induced by dehydration stress, while CmBBX22a and CmBBX22c were suppressed; CmBBX19 and CmBBX22c were induced by low temperature; But this subfamily genes weren't induced by salt stress. 4) Onion epidermal cells trans-expression system analysis showed CmDBBs localized in the nuclear, yeast transcriptional activation experiment showed CmBBX22a, CmBBX22b and CmBBX22c had transcriptional activation ability, and the activation region of CmBBX22s occupied the middle of proteins, neighther the C-terminal nor the B-box domain in the N-terminal, but CmBBX19 did not have transcriptional activation ability. CmBBX22a,CmBBX22b and CmBBX22c were rapidly degraded by 26S proteasome, but mannitol ttreatment can increase CmBBX22b stabilization. 5) We obtained CmDBBs-overexpression Arabidopsis, the flowering time of the overexpression lines delayed than wild type, both excised-leaf water loss and drought stress experiments indicated that CmBBX22b-overexpession lines enhanced drought tolerance, but CmBBX19,CmBBX22a and CmBBX22c-OE lines decreased the drought tolenrance.Our transcriptome sequences can provide a valuable resource for chrysanthemum breeding and research and offer candidate genes or markers that can be used to guide future efforts attempting to breed drought tolerant chrysanthemum cultivars. The CmBBX22b will provide gene pool for new transgenic material which can improve the stress tolerance.