Molecular Mechanism Of Rice Pectate Lyase-Like Gene OsPSE1 In Plant Senescence

Plant senescence is a highly complex but well programmed process involving in expression of many senescence associated genes,which is essential for plant growth and development.Premature senescence directly affects crop yield and grain quality.Therefore,study of plant senescence associated genes and their molecular mechanisms have profound implications for crop improvement to increase crop yields.A rice spontaneous premature senescence mutant ospse1-CW312 was identified in a previous study.The target gene was mapped on chromosome 1 using map-based cloning.Within the mapping region,one base deletion was identified within a gene encoding a protein containing a pectin lyase C domain,which was named as OsPSE1.For a functional complementation test,the wild-type OsPSE1 was transformed into ospse1-CW312 mutant,and the plant growth of resultant transgenic plants became normal.Sequence analysis showed that one base deletion in ospse1-CW312 might cause the premature senescence compared with the wild-type OsPSE1 gene.However,the molecular mechanisms of OsPSE1on regulation of premature senescence remained unclear.In this study,I have studied the OsPSE1 gene on genetic function,spatiotemporal expression,and biochemical function and achieved following main results:(1)The stable over-expression of OsPSE1:GFP gene in ospse1-CW312 mutant recoverd the normal growth of transgenic plants and even kept the leaves stay green for delayed senescence.Fluorescence signals were observed in the roots,leaf sheaths,leaves of OsPSE1:GFP over-expressing transgenic plants.The results showed that OsPSE1:GFP is expressed in all tested tissues,and is highly accumulates in the root meristem.(2)OsPSE1 was knocked out by CRISPR/Cas9 editing in rice Zhonghua 11(ZH11).The OsPSE1 knockout transgenic plants OsPSE1-KO showed premature senescence compared with ZH11 plants during the reproductive stage,indicating that OsPSE1 is indeed a key gene for premature senescence in rice.(3)The expression level of OsPSE1 was analyzed by qRT-PCR in various tissues of wild type CW312 and ospse1-CW312 mutant.The results showed that OsPSE1 expresses in roots,stems,leaves and spikelets.The expression level is highest in roots,and the expression level of OsPSE1 is higher in wild-type plants than in mutants.(4)Four transient expression vectors for subcellular localization were constructed and transformed into rice protoplasts by PEG/Ca-mediated transfection.The results showed that the GFP:OsPSE1,ospse1:GFP,and GFP:ospse1 fusion proteins is localized in the nucleus and cytoplasm,but the localization pattern of OsPSE1:GFP fusion protein is different in the cytoplasm with scattered GFP fluorescence signals.(5)MBP-tagged fusion proteins MBP:OsPSE1,MBP:ospse1,MBP:OsPSE1~D158A,R236A158A,R236A were isolated and purified from E.coli.The pectate lyase activity of these purified proteins was masured by qualitative and quantitative methods.The results showed that MBP:OsPSE1 has the activity of pectin lyase,while the activity of MBP:ospse1 is lower than that of MBP:OsPSE1.No pectate lyase activity is detected in MBP:OsPSE1~D158A,R236A158A,R236A dead mutant of pectin lyase.This study revealed the properties of pectate lyase OsPSE1,which is associated with rice premature senescence,and lays foundation for further investigation on the molecular mechanism of OsPSE1 in the regulation of senescence in rice.