Abstract
Temperature stress restricts plant growth and development. Antifreeze protein (AFP) can improve plants antifreeze ability. In our previous study, the AnAFP gene cloned from Ammopiptanthus nanus was confirmed to be an excellent candidate enhancing plant cold resistance. But, AnAFP protein shared similar structures with KnS type dehydrins including K, N and S domains except ice crystal binding domain A. Here, we generated AnAFPΔA, AnAFPΔK, AnAFPΔN and AnAFPΔS, and transformed them into ordinary and cold sensitive strains of E. coli, and Arabidopsis KS type dehydrin mutant to evaluate their function. Expression of AnAFPΔA decreases cold and heat tolerance in E. coli, meanwhile, AnAFP enhances heat tolerance in Arabidopsis, suggesting that domain A is a thermal stable functional domain. AnAFP, AnAFPΔA and AnAFPΔS localize in whole cell, but AnAFPΔK and AnAFPΔN only localizes in nucleus and cytoplasm, respectively, exhibiting that K and N domains control localization of AnAFP. Likewise, K domain blocks interaction between AnAFP and AnICE1. The result of RT-qPCR showed that expression of AnAFP, AnICE1 and AnCBF genes was significantly induced by high-temperature, indicating that the AnAFP is likely regulated by ICE1-CBF-COR signal pathway. Taken together, the study provides insights into understanding the mechanism of AnAFP in response to temperature stress and gene resource to improve heat or cold tolerance of plants in transgenic engineering.
Highlights
Temperature stress restricts plant growth and development
After low temperature treatment at 17 °C for 9 days, the cold-sensitive BX04 containing pINIII-AnAFPΔA showed growth defect and failed to form colonies with an average survival rate < 10%, which was similar to BX04 cells contain pINIII
The BX04 containing pINIII-AnAFPΔK, pINIII-AnAFPΔS and pINIII-AnAFPΔN grew vigorously and formed more colonies with an average survival rate > 30%, respectively, which was similar to BX04 cells with pINIII-AnAFP (Fig. 1)
Summary
Temperature stress restricts plant growth and development. Antifreeze protein (AFP) can improve plants antifreeze ability. In order to evaluate the function of these domains, four mutants of AnAFP deleting A (AnAFPΔA), K (AnAFPΔK), S (AnAFPΔS) and N (AnAFPΔN) domain were generated by overlapping PCR, respectively In this study, these four mutants and AnAFP were introduced into cold-sensitive and ordinary strains of E. coli, as well as Arabidopsis mutant of KnS type dehydrin gene AtHIRD11 to identify thermal stability of each domain under low- and high-temperature stress, respectively. These four mutants and AnAFP were introduced into cold-sensitive and ordinary strains of E. coli, as well as Arabidopsis mutant of KnS type dehydrin gene AtHIRD11 to identify thermal stability of each domain under low- and high-temperature stress, respectively Together with their subcellular localization, interacting proteins and induced endogenous expression, the regulation mechanism of the AnAFP protein in response to temperature stress was elucidated
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