|Other Abstract||~Drought is one of the most serious environmental factors. Mitochondria plays an important role in plant growth and development and adaptation to stresses. There are multiple pathways in plant mitochondrial electron transport chain, including the main cytochrome pathway and two branch pathways (alternating pathway and uncoupling pathway). Alternative oxidase (AOX) and Uncoupling protein (UCP) have similar functions in plant thermogenesis, defense, and energy dissipation. However, the function and relationship between UCP and AOX in plant tolerance to drought stress are still unclear. In this study, Col-0, UCP1 mutant (ucp1), AOX1a mutant (aox1a) and ucp1/aox1a double mutant were used to explore the function and molecular mechanism of UCP1 and AOX1a in Arabidopsis tolerance to drought stress.
The main results are as follows:
1.The UCP1 T-DNA insertion mutant CS821384 contains two non-homologous insertions in addition to the target insertion (the 5'UTR of UCP1). After crossing CS821384 with Col-0, the single insertion and homozygous ucp1 mutant was isolated and confirmed by PCR. RT-PCR results showed that there was no UCP1 gene expression in ucp1. The ucp1 and aox1a were crossed, and the ucp1/aox1a double mutant was generated and confirmed by PCR .
2.Phenotype and biomass analysis results showed that the fresh weight and dry weight of 28-days-old ucp1 plants were significantly lower than that of Col-0,decreasing by 11.3 % and 14.5 %, respectively. The total chlorophyll content in ucp1 was decreased by 3.9 % than Col-0However, the mutation of AOX1a did not affect the growth and photosynthetic pigment content.
3.Under drought stress, the expression of UCP family genes (UCP1, UCP2, UCP3, UCP4, UCP5, UCP6) and AOX family genes (AOX1a, AOX1b, AOX1c, AOX1d) was increased significantly. The increase of UCP1 and AOX1a was most significant, and reached the maximum (increased by 6.5 times and 35.6 times, respectively) at 24 h. GUS staining results further showed that UCP1 and AOX1a were expressed throughout the life cycle of Arabidopsis, and drought significantly induced the expression in leaf tips, veins and main roots. Western blot analysis showed that drought induced a significant increase in the protein levels of UCP1 and AOX1/2 in Arabidopsis leaves.With the increased duration of drought, the total respiration rate, CP capacity, AP capacity, the ratio of AP to CP capacity and the UCP activity were increased significantly, and they all reached the maximum values at 24 h.
4.The results of natural drought, lethality rate and water loss rate showed that ucp1/aox1a had the highest lethality rate and water loss rateunder drought stress, ucp1/aox1a leaves accumulated the most ROS, which mainly in chloroplasts. This indicates that the dysfunction of UCP1 and AOX1a reduces the drought tolerance in Arabidopsis.
5.Under drought stress, the loss of function of UCP1 and AOX1a increased the carbonylation degree of chloroplast proteins carbonylation degre, which resulted in the degradation of chloroplast protein complexes and photosynthetic pigments, and decreased the photosynthetic rate. In the double mutant ucp/aox1a, these parameters were decreased most significantly. qRT-PCR results showed that mutations of UCP1 and AOX1a decreased the drought-induced transcriptions of antioxidant enzyme genes (CSD1, sAPX) and the genes in the malate-OAA pathway (chlMDH, MMDH, ICDH). In ucp/aox1a under drought stress, the expression of CSD1, sAPX, chlMDH, MMDH, ICDH was decreased by 68.2 %，88.5 %，89.6 %，91.2 % and 87.5 % compared to that in Col-0.
6.Under CK and drought conditions, the expression of AOX1a in ucp1 was decreased, and the expression of UCP1 in aox1a was also significantly reduced. It implies that the expression of UCP1 and AOX1a is dependent upon each other in Arabidopsis. The mutation of UCP1 and AOX1a decreased the expressions of genes encoding the mitochondrial electron transport protein complex (PFK4, COX15) under drought stress, but the total respiration rate and CP capacity were increased significantly, suggesting that the mitochondrial electron transport proteincomplex might be regulated at the post translational level.
In summary, drought stress induces the transcription, translation and activity of UCP1 and AOX1aloss of function of UCP1 and AOX1a reduces the expression of antioxidant enzyme genes and causes ROS accumulation. Under drought stress, expressions of genes in the Mal-OAA pathway is decreased in ucp1/aox1a, affecting the output and consumption of excessive reducing power in the chloroplasts and leading to ROS accumulation and oxidative damage. Thus, the degree of chloroplast protein carbonylation is increased, the photosynthetic protein complexes are degraded, and the photosynthetic rate is decreased. The mutation of UCP1 and AOX1a affects the respiration rate of plants under drought stress. In conclusion, the dysfunction of UCP1 and AOX1a reduces Arabidopsis tolerance to drought stress, and there is a synergistic relationship between the AP and UCP pathway, but the molecular mechanism needs to be further studied.|