Cloning And Functional Research Of Cold Response Key Genes HbICE2 In Hevea Brasiliensis

Cold stress can disadvantageously affect the plants growth and development,and ultimately limit ecological distribution.As an important tropical species,H.brasiliensis is highly susceptible to low temperature stress.Cold stress not only affects rubber production,but also threatens the survival of rubber trees in China.Thus,a better understanding of the mechanisms governing tolerance and adaptation to cold stress could inspire new strategies for improving natural rubber yield and expanding the geographical range within which rubber trees can be successfully grown.In this study,we cloned the key components of INDUCER OF CBF EXPRESSION(ICE)-C-REPEAT BINDING FACTOR(CBF)cold acclimation signaling pathway,including HbICE2,HbCBF2 and HbCBF3.In addition,the role of HbICE2 in the cold response was further studied.Furthermore,the mechanism of JA regulates cold tolerance in the rubber tree was investigated.The main results are as follows:1.Based on the rubber tree genome sequences,one ICE and two CBF genes,designated as HbICE2,HbCBF2 and HbCBF3,with the Open Reading Frame of 1641 bp,705 bp and 729 bp,were isolated and characterized from rubber tree.Phylogenetic analysis and amino acid alignment suggested that these genes in rubber tree shared high homology with the ICE or CBF genes from other plants.2.Transactivation ability test assay indicated that HbICE2 exhibits transactivation activity,and the N-terminus is required for transcription-activating activity in yeast.3.The experiments for the subcellular localization of HbICE2 protein with 35S::ICE1-GFP showed that this protein localized in the nucleus.4.The chilling tolerance assays of HbICE2-OE transgenic plants and WT seedlings-show that transgenic lines were more tolerant to freezing than WT plants.5.The assays for physiological parameters indicated that compared with the wild type,HbICE2-OE transgenic plants had higher proline and chlorophyll content,lower electrolyte leakage and less malondialdehyde(MDA)accumulation when subjected to freezing stress.6.Differentially expressed genes(DEGs)analysis showed that cold treatment changed genes expression profiles involved phytohormones perception and transduction.Nine out of 50 DEGs associated with phytohormones are genes related to jasmonic acid signaling,suggesting JA signaling pathways might work to cope with cold tolerance in rubber tree.7.JA treatment could induce cold tolerance in H.brasiliensis.In addition,JA-treated rubber trees displayed a higher proline content and chlorophyll content,and reduced electrolyte leakage and MDA metabolism compared with control plants subjected to cold temperature stress.8.RT-qPCR analysis revealed that MeJA treatment enhanced the CBF transcriptional regulatory pathway under cold stress in H.brasiliensis,and also show that cold stress enhanced the expression level of HbJAZs9.Yeast two-hybrid assay reveals that HbICE2 interacted with both HbJAZl and HbJAZ12,suggesting HbICE2 mediates JA-regulated cold tolerance in H.brasiliensis through interaction with HbJAZs.In summary,HbICE2 was found to influence parameters related to cold stress such as ion leakage,proline content,and MDA metabolism,thereby enhancing osmotic protection.In addition,MeJA,enhanced cold tolerance in the rubber tree.Furthermore,several HbJAZs,known repressors of JA signaling,interact with HbICE2 and HbICE1,implicating them in MeJA-induced cold tolerance in H.brasiliensis via regulation of CBF cold response pathway-associated genes.