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

Phan Phuong Thao Doan; Jin Hee Kim; Jeongsik Kim

doi:10.3389/fpls.2022.818239

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

Front Plant Sci. 2022 Mar 17:13:818239. doi: 10.3389/fpls.2022.818239. eCollection 2022.

Authors

Phan Phuong Thao Doan¹, Jin Hee Kim², Jeongsik Kim^{1

2

3}

Affiliations

¹ Interdisciplinary Graduate Program in Advanced Convergence Technology & Science, Jeju National University, Jeju, South Korea.
² Subtropical Horticulture Research Institute, Jeju National University, Jeju, South Korea.
³ Faculty of Science Education, Jeju National University, Jeju, South Korea.

Abstract

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 SEN4 and SAG12, early and late senescence-responsive genes, depending on effectors of known positive and negative senescence regulators. Overexpression of positive senescence regulators, including ORE1, RPK1, and RAV1, increased the expression of both SEN4- and SAG12-LUC while ORE7, a negative senescence regulator decreased their expression. Consistently with overexpression, knockdown of target genes using amiRNAs resulted in opposite SAG12-LUC 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, ORE1 and RPK1 are involved in cell death responses, with more prominent and earlier involvement of ORE1 than RPK1. 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 ORE1 causes acute cell death and RPK1 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.

Keywords: Arabidopsis thaliana; ROS; cell death; leaf senescence; luciferase; protoplasts; transient expression.