The Arabidopsis RING E3 Ubiquitin Ligase AtAIRP3/LOG2 Participates in Positive Regulation of High-Salt and Drought Stress Responses

The Arabidopsis RING E3 Ubiquitin Ligase AtAIRP3/LOG2 Participates in Positive Regulation of... Really Interesting New Gene (RING) E3 ubiquitin ligases have been implicated in cellular responses to the stress hormone abscisic acid ( ABA ) as well as to environmental stresses in higher plants. Here, an ABA-insensitive RING protein3 ( atairp3 ) loss-of-function mutant line in Arabidopsis ( Arabidopsis thaliana ) was isolated due to its hyposensitivity to ABA during its germination stage as compared with wild-type plants. AtAIRP3 contains a single C3HC4-type RING motif, a putative myristoylation site, and a domain associated with RING2 (DAR2) domain. Unexpectedly, AtAIRP3 was identified as LOSS OF GDU2 (LOG2), which was recently shown to participate in an amino acid export system via interaction with GLUTAMINE DUMPER1. Thus, AtAIRP3 was renamed as AtAIRP3/LOG2. Transcript levels of AtAIRP3/LOG2 were up-regulated by drought, high salinity, and ABA , suggesting a role for this factor in abiotic stress responses. The atairp3/log2-2 knockout mutant and 35S : AtAIRP3-RNAi knockdown transgenic plants displayed impaired ABA -mediated seed germination and stomata closure. Cosuppression and complementation studies further supported a positive role for AtAIRP3/LOG2 in ABA responses. Suppression of AtAIRP3/LOG2 resulted in marked hypersensitive phenotypes toward high salinity and water deficit relative to wild-type plants. These results suggest that Arabidopsis RING E3 AtAIRP3/LOG2 is a positive regulator of the ABA -mediated drought and salt stress tolerance mechanism. Using yeast ( Saccharomyces cerevisiae ) two-hybrid, in vitro, and in vivo immunoprecipitation, cell-free protein degradation, and in vitro ubiquitination assays, RESPONSIVE TO DEHYDRATION21 was identified as a substrate protein of AtAIRP3/LOG2. Collectively, our data suggest that AtAIRP3/LOG2 plays dual functions in ABA -mediated drought stress responses and in an amino acid export pathway in Arabidopsis. Glossary ABA abscisic acid ER endoplasmic reticulum T-DNA transfer DNA IP immunoprecipitation RT reverse transcription qRT quantitative reverse transcription RNAi RNA interference CaMV cauliflower mosaic virus cDNA complementary DNA SD/−Trp/−Leu/−His three-minus selection medium 3-AT 3-amino-1,2,4,-triazole SD/−Leu/−Trp/−His/−Ade four-minus selection medium PCD programmed cell death MS Murashige and Skoog http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png

The Arabidopsis RING E3 Ubiquitin Ligase AtAIRP3/LOG2 Participates in Positive Regulation of High-Salt and Drought Stress Responses

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Publisher
American Society of Plant Biologist
Copyright
Copyright © 2015 by the American Society of Plant Biologists
ISSN
1532-2548
eISSN
0032-0889
D.O.I.
10.1104/pp.113.220103
Publisher site
See Article on Publisher Site

Abstract

Really Interesting New Gene (RING) E3 ubiquitin ligases have been implicated in cellular responses to the stress hormone abscisic acid ( ABA ) as well as to environmental stresses in higher plants. Here, an ABA-insensitive RING protein3 ( atairp3 ) loss-of-function mutant line in Arabidopsis ( Arabidopsis thaliana ) was isolated due to its hyposensitivity to ABA during its germination stage as compared with wild-type plants. AtAIRP3 contains a single C3HC4-type RING motif, a putative myristoylation site, and a domain associated with RING2 (DAR2) domain. Unexpectedly, AtAIRP3 was identified as LOSS OF GDU2 (LOG2), which was recently shown to participate in an amino acid export system via interaction with GLUTAMINE DUMPER1. Thus, AtAIRP3 was renamed as AtAIRP3/LOG2. Transcript levels of AtAIRP3/LOG2 were up-regulated by drought, high salinity, and ABA , suggesting a role for this factor in abiotic stress responses. The atairp3/log2-2 knockout mutant and 35S : AtAIRP3-RNAi knockdown transgenic plants displayed impaired ABA -mediated seed germination and stomata closure. Cosuppression and complementation studies further supported a positive role for AtAIRP3/LOG2 in ABA responses. Suppression of AtAIRP3/LOG2 resulted in marked hypersensitive phenotypes toward high salinity and water deficit relative to wild-type plants. These results suggest that Arabidopsis RING E3 AtAIRP3/LOG2 is a positive regulator of the ABA -mediated drought and salt stress tolerance mechanism. Using yeast ( Saccharomyces cerevisiae ) two-hybrid, in vitro, and in vivo immunoprecipitation, cell-free protein degradation, and in vitro ubiquitination assays, RESPONSIVE TO DEHYDRATION21 was identified as a substrate protein of AtAIRP3/LOG2. Collectively, our data suggest that AtAIRP3/LOG2 plays dual functions in ABA -mediated drought stress responses and in an amino acid export pathway in Arabidopsis. Glossary ABA abscisic acid ER endoplasmic reticulum T-DNA transfer DNA IP immunoprecipitation RT reverse transcription qRT quantitative reverse transcription RNAi RNA interference CaMV cauliflower mosaic virus cDNA complementary DNA SD/−Trp/−Leu/−His three-minus selection medium 3-AT 3-amino-1,2,4,-triazole SD/−Leu/−Trp/−His/−Ade four-minus selection medium PCD programmed cell death MS Murashige and Skoog

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