Study On The Regulation Of Leaf Senescence By PPRs And WHIRLY3 In Arabidopsis Thaliana

Aging is the last stage of life of plants.It is regulated by various internal and external factors.Leaf senescence is a typical kind of aging and has direct relations with crop yield and quality.During leaf aging,a series of physiological and biochemical changes,including loss of chlorophyll,protein degradation,etc,will be happened.However,the molecular mechanism of leaf senescence is still not very clear.Pentatricopeptide repeats(PPR)family is one of the largest protein families in terrestrial plants,they play important roles in the regulation of plant growth and response to various environmental stresses.PPRs is one of the members of PPR family.A previous study found that PPRs has negative correlation with Arabidopsis leaf senescence,and PPRs protein is localized in plastids of onion epidemic cell by biolist assay.WHIRLY3 is one of members of the plant WHIRLY family.Previous observations of the WHIRLY1 and WHIRLY3 double knockout plants showed a phenotype with a variegation pale yellow leaves,and also dysfunctions of chloroplasts.Based on the genomic structure of PPRs and WHIRLY3 genes in the chromosome,they present two potential targets of a small noncoding microRNA840a(miR840a).Previous studies showed that overexpressing miR840a showed early plant senescence phenotype,while miR840a knockdown mutant showed retarded leaf senescence phenotype.Therefore,in this research,we studied the relationships of PPRs,WHIRLY3 or both with leaf senescence regulations.The main results are as follows:1.We obtained PPRs homozygous T-DNA insertion mutant lines by using three primer screening method on the genome level and confirmed its expression by using semi-quantitative PCR on transcription level.The gene GAPC is used as an internal control.We performed senescence phenotype observations of PPRs or WHIRLY3 single mutant lines.It was showed that the rosette leaves of PPRs mutant showed indiscrimination leaf senescence phenotype compared with wild-type.At the same time,the antisense PPR and WHIRLY3 knockout mutant respectively showed no obvious phenotype changes compared with wild-type.The senescence related physiological parameters such as chlorophyll concent and Fv/Fm values proved the phenotype observations.2.After crossing aPPR and kowh3 or kowhy1/3 lines,we obtained aPPRkowhy3 double and aPPR kowhy1/3 triple mutant lines via Basta resistance selection and PCR verifications.The senescence phenotype of rosette leaves of aPPR kowhy1/3,aPPRkowhy3 and wild-type plants were compared.The result showed that both aPPR kowhy1/3 and aPPRkowhy3 accelerate leaf senescence of process compared with wild-type plants,but the effect of aPPR kowhy1/3 is stonger.The expression of senescenceassocaited genes was also confirmed in these two mutants and showed that the expression of some senescence-associated genes such as SAG12 and SAG101 in aPPR kowhy1/3 mutant is more prominent then aPPRkowhy3 mutant,which proved the senescence phenotype difference of these two mutants.3.To further study the relations of PPRs and WHIRLY3 protein,their interactions were investigated by yeast two hybrid and BIFC.The result showed that PPRs could not interact with WHIRLY3,but PPRs can interact with itself.