AUTHOR=Doan Phan Phuong Thao , Kim Jin Hee , Kim Jeongsik 

TITLE=Rapid Investigation of Functional Roles of Genes in Regulation of Leaf Senescence Using Arabidopsis Protoplasts

JOURNAL=Frontiers in Plant Science

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2022.818239

DOI=10.3389/fpls.2022.818239

ISSN=1664-462X

ABSTRACT=<p>Leaf senescence is the final stage of leaf development preceding death, which involves a significant cellular metabolic transition from anabolism to catabolism. Several processes during leaf senescence require coordinated regulation by senescence regulatory genes. In this study, we developed a rapid and systematic cellular approach to dissect the functional roles of genes in senescence regulation through their transient expression in Arabidopsis protoplasts. We established and validated this system by monitoring the differential expression of a luciferase-based reporter that was driven by promoters of <italic>SEN4</italic> and <italic>SAG12</italic>, early and late senescence-responsive genes, depending on effectors of known positive and negative senescence regulators. Overexpression of positive senescence regulators, including <italic>ORE1</italic>, <italic>RPK1</italic>, and <italic>RAV1</italic>, increased the expression of both <italic>SEN4</italic>- and <italic>SAG12-LUC</italic> while <italic>ORE7</italic>, a negative senescence regulator decreased their expression. Consistently with overexpression, knockdown of target genes using amiRNAs resulted in opposite <italic>SAG12-LUC</italic> expression patterns. The timing and patterns of reporter responses induced by senescence regulators provided molecular evidence for their distinct kinetic involvement in leaf senescence regulation. Remarkably, <italic>ORE1</italic> and <italic>RPK1</italic> are involved in cell death responses, with more prominent and earlier involvement of <italic>ORE1</italic> than <italic>RPK1</italic>. Consistent with the results in protoplasts, further time series of reactive oxygen species (ROS) and cell death assays using different tobacco transient systems reveal that <italic>ORE1</italic> causes acute cell death and <italic>RPK1</italic> mediates superoxide-dependent intermediate cell death signaling during leaf senescence. Overall, our results indicated that the luciferase-based reporter system in protoplasts is a reliable experimental system that can be effectively used to examine the regulatory roles of Arabidopsis senescence-associated genes.</p>