Chrysanthemum Heat Shock Transcription Factors CmHSFA4,CmHSFB1 Cloning And Function Analysis

Chrysanthemum(Chrysanthemum morifolium)is one of the ten most famous cut flowers globally,has a high ornamental value,and occupies an irreplaceable position in the international commerce of flowers.Salinity and aphids affect the yield and quality of chrysanthemum seriously.It is essential to improve chrysanthemum tolerance to achieve sustainable production.HSF transcription factors have involved in varieties of plants stress resistance however little information about HSFs in chrysanthemum are available.In this study,we isolated two HSFs from chrysanthemum and analysed their expression feature,transcriptional activity and subcellular localization.To identify the function of HSFs,the overexpression chrysanthemum lines of HSFs were generated through Agrobacterium tumefaciens mediated leaf disc transformation.The main content and conclusions are as follows.1.CmHSFA4 was cloned from chrysanthemum with the method of homologous cloning.The ORP of CmHSFA4 was 1242bp contained a 1077 bp open reading frame(ORF).The amino acid sequence analysis showed that,CmHSFA4 belongs to HSF A4 Class with high homology to Arabidopsis thaliana AtHSFA4a.To investigate the location and the transactivation activity of CmHSFA4,we constructed expression vectors by double digestion and the LR reaction using Gateway technology(Invitrogen).CmHSFA4 localized to the nucleus in onion epidermal cells.The results of yeast one-hybrid suggested that CmHSFA4 functioned as a transactivator and AHA motif is essential.Salinity induced Na+toxicity and oxidative stress hamper plant growth.Chrysanthemum overexpressing CmHSFA4 displayed enhanced salinity tolerance by limiting Na+ accumulation while maintaining K+ concentration,which is consistent with the up-regulation of ion-transporters CmSOSl and CmHKT2.Additionally,the transgenic plants reduced H2O2 and O2’-accumulation under salinity,which could be due to up-regulation of ROS-scavenger activities such as SOD,APX and CAT as well as CmHSP70,CmHSP90.Together,these results suggest that CmHSFA4 conferred salinity tolerance in chrysanthemum as a consequence of Na+/K+ ion and ROS homeostasis.2.CmHSFB1 was cloned from chrysanthemum with the method of homologous cloning.The ORF of CmHSFB1 was 1154bp contained a 858 bp open reading frame(ORF).The amino acid sequence analysis showed that,CmHSFB1 belongs to HSFB1 Class with high homology to Arabidopsis thaliana AtHSFBl.To investigate the location and the transactivation activity of CmHSFB1,we constructed expression vectors by double digestion and the LR reaction using Gateway technology(Invitrogen).CmHSFB1 localized to the nucleus in onion epidermal cells.The results of yeast one-hybrid suggested that no transcriptional activation of CmHSFB1.The expression of CmHSFBl was strongly induced by aphid stress,and tissue-specific expression analysis showed a high level of CmHSFBl in leaf and stem.Overexpression of CmHSFB1 in chrysanthemum increased the tolerance to aphids.CmHSFBl overexpressing chrysanthemum accelerated aphid-induced H2O2 and O2·O-production.Additionally,the overexpression of CmHSFBl exhibited elevated expression of ROS-generating and ROS-responsive genes,suggesting that aphids counter the oxidative stress associated with CmHSFBl.