Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Doan, Phan Phuong Thao, Kim, Jin Hee, Kim, Jeongsik
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
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
_version_ 1784679330214838272
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
work_keys_str_mv AT doanphanphuongthao rapidinvestigationoffunctionalrolesofgenesinregulationofleafsenescenceusingarabidopsisprotoplasts
AT kimjinhee rapidinvestigationoffunctionalrolesofgenesinregulationofleafsenescenceusingarabidopsisprotoplasts
AT kimjeongsik rapidinvestigationoffunctionalrolesofgenesinregulationofleafsenescenceusingarabidopsisprotoplasts