Overexpression of <i>Kalanchoë TIM23</i> Improves Heat and Drought Tolerance in <i>Arabidopsis thaliana</i> and Knockout of <i>TIM23</i> Reduces Growth in <i>Kalanchoë fedtschenkoi</i>

Heat and drought stress are the leading causes of crop loss worldwide. The development of crops with elevated levels of heat and drought tolerance, which could sustain the production of food, feed, fiber, and biofuels on marginal lands, is therefore highly desirable. Heat stress usually causes protein dysfunction, and maintaining proteins in their functional conformations and preventing aggregation of non-native proteins are particularly important for plant survival under heat and drought stresses. Mitochondrial protein translocation and quality control pathways play an important role in refolding or removal of damaged proteins. Translocase of the inner mitochondria membrane 23 (TIM23) is responsible for importing a wide variety of mitochondrial proteins synthesized in the cytoplasm. However, the role of TIM23 in heat and drought stress tolerance has not been investigated in plants previously. In this study, we cloned two TIM23 genes (KfeTIM23-1 and KfeTIM23-2) from Kalanchoë fedtschenkoi, which performs crassulacean acid metabolism (CAM) photosynthesis as an adaptation to water-limited environments. For gain-of-function analysis, two types of transgenic Arabidopsis thaliana plants were created by overexpressing KfeTIM23-1 and KfeTIM23-2, respectively, and these transgenic plants, along with wild-type A. thaliana plants, were then subject to drought stress and heat stress treatments. It was found that the overexpression of KfeTIM23-1 significantly enhanced heat and drought tolerance in A. thaliana. For loss-of-function analysis, one knockout mutant of KfeTIM23-1 was created by using CRISPR/Cas9 technology. The DNA sequence of the KfeTIM23-1 knockout mutant showed an insertion that disrupted the original amino acid sequence and consequently did not allow for the correct protein translation. As a result, a dwarfed phenotype was observed in the mutant line in comparison with the wild-type K. fedtschenkoi plants, suggesting that there may be a decrease in importing proteins into the mitochondria for the maintenance of CAM pathway. This research has a great potential for accelerating the genetic improvement of heat and drought tolerance in bioenergy and food crops.