OsCDC48/48E Complex-mediated Premature Senescence And Plant Survival In Rice

The AAA-ATPase family?ATPase Associated with a variety of Cellular Activities,AAA?related gene OsCDC48?Cell division cycle 48?was previously identified and cloned in this laboratory.The mutant psd128,in the early developmental stage,has showed early senescence,which result in a lethal phenotype upon heading.Agronomic traits of psd128?plant height,ear length,1000-grain weight and seed setting rate?were significantly lower than wild type.Genetic analysis confirmed that psd128 premature aging was caused by a single recessive gene.Further study has found that single-base mutation in OsCDC48 gene of psd128 leads to premature senescence,indicating that this gene is associated with plant cell life activities and molecular mechanisms underlying aging in rice.Based on the previous laboratory research,this study carried out related experiments on the mechanism of premature aging of psd128.The main results are as follows:1)Protein sequence alignment revealed that OsCDC48 and OsCDC48E were highly homologous with a similarity of 97%.Their expression patterns were highly similar and both were constitutively expressed.In addition,these two proteins were localized in the cytoplasm and nucleus.2)The mutant psd128 is more susceptible to 11 races of Xanthomonasoryzaepv.oryzae?Xoo?than the wild type?WT?.The hormone levels and the expression of hormone metabolism-related genes were significantly altered in psd128 compared to WT,indicating that the susceptibility of psd128 to Xoo could be associated with plant hormone metabolism.3)The psd128 plants were shorter in height with reduced number of tillers.The tissue section analysis showed that the decrease in cell number was the major reason for plant height shortness.Flow cytometric analysis showed that cell number reduction is due to the impaired function of cell cycle in psd128 as reflected by a significant change in cell cycle-related gene expression between psd128 and WT.4)Transcriptome analysis indicated 374 differentially expressed genes?DEGs?,including cell cycle related genes and AAA-ATPase related genes,between the mutant BC₂F₃-IR64?mt?and wild type?WT?,which further implied the compromised cell cycle progression in the premature senescence and plant lethality occurrence.5)OsCDC48 encodes a member of AAA-ATPases.The total ATPase activity of psd128 was significantly higher than that of WT,but the ATPase activity of the mutant protein OsCDC48-PSD128 lacking the C-terminal,which involved 27 amino acids,was significantly decreased compared with OsCDC48.This result indicated that the deletion of 27 amino acids at the C-terminus affect ATPase activity.6)Upon the Yeast double-hybrid,pull-down,and bimolecular fluorescence complementation experiments both OsCDC48 and OsCDC48E proteins showed intra and intermolecular interaction to form homomultimers and heteromultimers,which jointly regulate rice growth and development.In contrast,OsCDC48-PSD128 could neither interact with itself nor with OsCDC48E.7)Knockout of OsCDC48E resulted in premature senescence and plant death,indicating that both OsCDC48E and OsCDC48 complemented each other to regulate the growth and development in rice.Taken together,our results demonstrated that the C-terminal region of OsCDC48 was essential for maintaining the full ATPase activity and OsCDC48/48E complex might function in form of heteromultimers to modulate cellular processes and plant survival in rice.