KIN?? Regulates The Number Of Mitochondria And Lipid Droplets In Arabidopsis Pollen

Adhension and hydration of pollen on the stigma surface is the first step of plant sexual reproduction,which is crucial for crop yield.Our previous study revealed that the ?? subunit(KIN??)of Sn RK1 complex in Arabidopsis thaliana regulates the biosynthesis of mitochondrion in pollen,which affects the level of ROS(Reactive Oxygen Species)in pollen and the hydration of pollen on the stigma surface.However,the molecular mechanism of KIN??in the regulation of the biosynthesis of mitochondrion in pollen is unknown.In this study,we analyzed the RNA-seq results in the pollen of kin??-1/+ mutants,and found that the W-box elements to which WRKY transcription factors preferentially bind were enriched in the promoters of these genes whose expression levels were significantly different from the wild type.The expression levels of WRKY2 and WRKY34 were the highest in pollen.We found that WRKY2 and WRKY34 interacted with KIN?? revealed by yeast two-hybrid and Pull-down analysis.The number of mitochondria in the mature pollen of wrky2 wrky34 double mutant was significantly less than that in the wild type.The percentage of pollen that could not hydrate on the stigma was significantly higher than that in the wild type.Thus,WRKY2 and WRKY34 interact with KIN?? to regulate the number of mitochondria in Arabidopsis pollen,and thus affect pollen hydration on stigma surface.Next,the RNA-seq results of pollen from kin??-1/+ and wrky2 wrky34 double mutants were compared,and a gene with unknown function was found to be up-regulated in both mutants,named PALD(Protein Associated with Lipid Droplet).There is a W-box in the promoter of PALD.WRKY2 could bind to the W-box site in PALD promoter,and WRKY34 and KIN?? could enhance the ability of WRKY2 to inhibited the expression of PALD.Therefore,WRKY2 and WRKY34 interact with KIN?? to form a complex,which binds to the promoter of PALD and inhibits its expression.PALD was specifically expressed in mature pollen and pollen tubes,and PALD was located on the LD(Lipid Droplet)in Arabidopsis.According to its protein structure,PALD was divided into six regions(I-VI),in which region I and region V contained an amphipathic ?-helix each.Subcellular localization analysis revealed that region I-IV of PALD and the amphipathic ?-helix in region I were crucial to the subcellular localization of PALD on LD.The expression of PALD was positively correlated with LD number during pollen development.The number and diameter of LD in pollen of pald knock-out mutants were lower than those of the wild type.The number of mitochondria in the pollen of the pald mutants was significantly higher than that of the wild type,and the percentage of pollen that could not hydrate on the stigma surface was also significantly higher than that of the wild type.Therefore,PALD plays its roles in the regulation of the number of mitochondria in pollen and the hydration of pollen on the stigma surface.Previous studies found that GPT1(Glucose-6-Phosphate 1)was located in plastid and involved in LD accumulation in pollen.To further analyze the relationship between LD and mitochondrial number in pollen,we created the gpt1-2 mutant.The number of LD in pollen of the mutant was less than that in the wild type,and the number of mitochondria was more than that of the wild type,suggesting that LD in pollen was negatively correlated with the number of mitochondria.In conclusion,KIN?? interacts with WRKY2 and WRKY34 to form a complex that enhance the ability of WRKY2 to inhibited the expression of PALD,which regulates the number of mitochondria and LD in pollen and affects the hydration of pollen on the stigma surface.