Cloning and heat tolerance analysis of the thermomorphogenesis gene KcPIF4 in Karelinia caspica

Abstract [Objective] The desert plant Karelinia caspica has strong heat tolerance. The aim is to clone and study the heat-tolerant genes of K . caspica, and apply them to economic crops such as cotton to enhance heat resistance. This provides a theoretical basis and genetic resources for molecular breeding of heat-tolerant crops. [Methods] KcPIF4 gene was cloned and bioinformatics analysis was performed. Subcellular localization of the protein was analyzed through transient expression in tobacco. qRT-PCR was used to analyze the expression of KcPIF4 in roots, stems, and leaves of K . caspica seedlings at different time under 45 ℃ treatment. Changes in phenotype of Arabidopsis overexpressing KcPIF4 at 45 ℃ were observed, and relevant physiological and biochemical indicators were measured. [Results] The full length CDS of KcPIF4 is 1 593 bp, encoding 530 amino acids and containing a bHLH conserved domain. The motifs showed differences compared to homologous proteins from various plants. The protein was localized at the membrane of the guard cell of stomata. qRT-PCR showed that the expression of KcPIF4 in K . caspica leaves was first increased and then decreased, and increased again with prolonged treatment time, reaching its peak at 240 min, 4.4 times higher than the control. Overexpression of KcPIF4 in Arabidopsis produced less chlorosis and wilting in the leaves compared to the wild type at 45 ℃, with higher level of CAT, SOD, POD, chlorophyll a, chlorophyll b, and carotenoids, as well as lower level of MDA content and relative conductivity compared to the wild type. [Conclusion] KcPIF4 functions in tolerance-responseadaptation to high temperature stress, enhancing Arabidopsis tolerance to high temperature. This result suggests that the protein has high temperature resistance.