Comparative Analysis Of Different Subcellular Localization Of WHIRLY2 In Regulating Arabidopsis Leaf Senescence

Plant senescence is a genetically controlled natural decay and death procedure that is affected or induced by internal and external environmental factors.Whether senescence occurs early or late has great influence to agriculture production.In this study,we intend to characterize the cellular and molecular functions of WHIRLY2(WHY2)on plant leaf senescence.WHY2 proteins belong to the single-stranded DNA binding protein WHIRLY family,which specifically and widely exist in the plant kingdom.The research contents and results are as follows:(1)The changes of distribution of WHY2 in mitochondria,chloroplast and nucleus were detected in leaves by subcellular organelle separation and protein immuno-detection during the period of leaf senescence.The results show that WHY2 can localize not only in mitochondria,but also in chloroplast and nucleus.And the total amount of WHY2 in the leaf cells showed a significant decrease when leaves developed from mature(6-week-old)to senescence(8-week-old)stage,while its content in the nucleus was significant higher than in 4-week-old developing leaves,though the content of WHY2 in mitochondria and chloroplast showed a significant decrease.The results show that WHY2 may be more likely to be distributed into the nucleus in senescent leaves.(2)By constructing transgenic plants with different subcellular localization of WHY2 and observing their phenotype changes compared with wild type,it was found that only transgenic plants with overexpressing the full-length WHY2 protein had premature senescence phenotype,and the transgenic plants with overexpressing WHY2 protein deleted mitochondrial or chloroplast transit signal peptide did not show any apparent phenotype changes compared to wild type plants.Contrary to the phenotype of transgenic plants with overexpressing full-length WHY2 protein,kowhy2 plants showed delayed leaf senescence phenotype and increased the chlorophyll content.Interestingly,overexpressing full-length WHY2 protein in seedlings before 3 weeks did not result premature leaf senescence phenotype but the leaves of 4-week-old seedlings showed rapidly aging.It indicated that WHY2 is likely to play different functions in leaves of Arabidopsis plants at two different developmental stages,before 3 weeks and after 4 weeks.(3)In order to further confirm the subcellular localization of WHY2 manipulated with or without organelle transit signal peptide,we investigated the localization of WHY2 with different subcellular localization signal peptide deletion by biolistic or protoplast transient transformation.The result showed that full-length WHY2 protein is located in mitochondria of both onion epidermal cells and Arabidopsis protoplast cells,and WHY2 protein without the mitochondrial transit signal peptide localized in both chloroplast and the nucleus in onion epidermal cells,but mostly located in the nucleus in Arabidopsis protoplast cells,and less in chloroplast.This result is consistent with the results of immuno-detection using polyclonal antibodies against WHY2 protein,indicating that WHY2 protein can indeed utilize its N-terminal variable subcellular localization signal to regulate its localization.(4)Finally,the interaction between WHY2 and WHIRLY1(WHY1),another member of the WHIRLY family,or themselves and their functions in leaf senescence were explored.The results showed that WHY1 was able to interact with WHY2 proteins in the nucleus and plastids.Phenotype analysis showed that overexpressing WHY2 in kowhyl and kowhyl/3 backgrounds did not show a stronger early senescence phenotype compared to overexpressing WHY2 in wild type background,while it showed an early senescence phenotype compared to kowhyl.It hinted that WHY2 with WHY1 only partially involved in regulating the leaf senescence,perhaps they both also play another function during plant development,such as silique development and responsive to stress.These results will give us a new insight to understand the regulation mechanism of WHIRLY proteins on leaf senescence and provide some important clues for controlling leaf senescence process for economic crops such as rice,corn,barley,Chinese cabbage and so on.