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Rapid Investigation of Functional Roles of Genes in Regulation of Leaf Senescence Using Arabidopsis Protoplasts
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...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8969776/ https://www.ncbi.nlm.nih.gov/pubmed/35371171 http://dx.doi.org/10.3389/fpls.2022.818239 |
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author | Doan, Phan Phuong Thao Kim, Jin Hee Kim, Jeongsik |
author_facet | Doan, Phan Phuong Thao Kim, Jin Hee Kim, Jeongsik |
author_sort | Doan, Phan Phuong Thao |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-8969776 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-89697762022-04-01 Rapid Investigation of Functional Roles of Genes in Regulation of Leaf Senescence Using Arabidopsis Protoplasts Doan, Phan Phuong Thao Kim, Jin Hee Kim, Jeongsik Front Plant Sci Plant Science 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. Frontiers Media S.A. 2022-03-17 /pmc/articles/PMC8969776/ /pubmed/35371171 http://dx.doi.org/10.3389/fpls.2022.818239 Text en Copyright © 2022 Doan, Kim and Kim. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Plant Science Doan, Phan Phuong Thao Kim, Jin Hee Kim, Jeongsik Rapid Investigation of Functional Roles of Genes in Regulation of Leaf Senescence Using Arabidopsis Protoplasts |
title | Rapid Investigation of Functional Roles of Genes in Regulation of Leaf Senescence Using Arabidopsis Protoplasts |
title_full | Rapid Investigation of Functional Roles of Genes in Regulation of Leaf Senescence Using Arabidopsis Protoplasts |
title_fullStr | Rapid Investigation of Functional Roles of Genes in Regulation of Leaf Senescence Using Arabidopsis Protoplasts |
title_full_unstemmed | Rapid Investigation of Functional Roles of Genes in Regulation of Leaf Senescence Using Arabidopsis Protoplasts |
title_short | Rapid Investigation of Functional Roles of Genes in Regulation of Leaf Senescence Using Arabidopsis Protoplasts |
title_sort | rapid investigation of functional roles of genes in regulation of leaf senescence using arabidopsis protoplasts |
topic | Plant Science |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8969776/ https://www.ncbi.nlm.nih.gov/pubmed/35371171 http://dx.doi.org/10.3389/fpls.2022.818239 |
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