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A transient assay system for the assessment of cell-autonomous gene function in dehydration-stressed barley

Drought is a serious, worldwide problem for crop production and also affects yields of barley and wheat, together with other stressors such as frost, viral diseases, or fungal pathogens. Although a number of candidate genes have been identified by transcriptome approaches in recent years, only very...

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Detalles Bibliográficos
Autores principales: Marzin, Stephan, Mihaly, Robert, Pauk, Janos, Schweizer, Patrick
Formato: Texto
Lenguaje:English
Publicado: Oxford University Press 2008
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2529241/
https://www.ncbi.nlm.nih.gov/pubmed/18641397
http://dx.doi.org/10.1093/jxb/ern186
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author Marzin, Stephan
Mihaly, Robert
Pauk, Janos
Schweizer, Patrick
author_facet Marzin, Stephan
Mihaly, Robert
Pauk, Janos
Schweizer, Patrick
author_sort Marzin, Stephan
collection PubMed
description Drought is a serious, worldwide problem for crop production and also affects yields of barley and wheat, together with other stressors such as frost, viral diseases, or fungal pathogens. Although a number of candidate genes have been identified by transcriptome approaches in recent years, only very few have been tested in functional assays for a beneficial effect on drought tolerance. Here, a transient assay system in microprojectile-bombarded barley leaves is described that allows the functional testing of dehydration stress-related candidate genes by RNA interference (RNAi) or overexpression. Cellular stress or damage in dedydrated leaves is reported by a reduced accumulation of slowly maturing, native red-fluorescing protein DsRed that is known to be sensitive to denaturing conditions. After a dehydration-stress period of 4 d during which the relative fresh weight of leaves was kept at 60–66% of initial fresh weight, a reproducible reduction of normalized DsRed fluorescence was observed. In order to obtain proof of concept, a number of barley mRNAs homologous to drought response genes were selected and targeted by transient induced gene silencing (TIGS). TIGS of four tested genes resulted in a significantly stronger decrease of normalized DsRed fluorescence in dehydration-stressed leaves, whereas they had no effect in fully turgescent control leaves. These genes encode barley drought-responsive factor HvDRF1 (DREB2-like), dehydrin 6, late embryogenesis-abundant protein HVA1, and the vacuolar sodium/proton antiporter HvHNX1. The four targeted transcripts were also found to accumulate rapidly in dehydration-stressed barley leaf segments. The results suggest a value of the TIGS system for functional pre-screening of larger numbers of drought or dehydration stress-related candidate genes in barley.
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spelling pubmed-25292412009-02-25 A transient assay system for the assessment of cell-autonomous gene function in dehydration-stressed barley Marzin, Stephan Mihaly, Robert Pauk, Janos Schweizer, Patrick J Exp Bot Research Papers Drought is a serious, worldwide problem for crop production and also affects yields of barley and wheat, together with other stressors such as frost, viral diseases, or fungal pathogens. Although a number of candidate genes have been identified by transcriptome approaches in recent years, only very few have been tested in functional assays for a beneficial effect on drought tolerance. Here, a transient assay system in microprojectile-bombarded barley leaves is described that allows the functional testing of dehydration stress-related candidate genes by RNA interference (RNAi) or overexpression. Cellular stress or damage in dedydrated leaves is reported by a reduced accumulation of slowly maturing, native red-fluorescing protein DsRed that is known to be sensitive to denaturing conditions. After a dehydration-stress period of 4 d during which the relative fresh weight of leaves was kept at 60–66% of initial fresh weight, a reproducible reduction of normalized DsRed fluorescence was observed. In order to obtain proof of concept, a number of barley mRNAs homologous to drought response genes were selected and targeted by transient induced gene silencing (TIGS). TIGS of four tested genes resulted in a significantly stronger decrease of normalized DsRed fluorescence in dehydration-stressed leaves, whereas they had no effect in fully turgescent control leaves. These genes encode barley drought-responsive factor HvDRF1 (DREB2-like), dehydrin 6, late embryogenesis-abundant protein HVA1, and the vacuolar sodium/proton antiporter HvHNX1. The four targeted transcripts were also found to accumulate rapidly in dehydration-stressed barley leaf segments. The results suggest a value of the TIGS system for functional pre-screening of larger numbers of drought or dehydration stress-related candidate genes in barley. Oxford University Press 2008-09 2008-07-18 /pmc/articles/PMC2529241/ /pubmed/18641397 http://dx.doi.org/10.1093/jxb/ern186 Text en © 2008 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
spellingShingle Research Papers
Marzin, Stephan
Mihaly, Robert
Pauk, Janos
Schweizer, Patrick
A transient assay system for the assessment of cell-autonomous gene function in dehydration-stressed barley
title A transient assay system for the assessment of cell-autonomous gene function in dehydration-stressed barley
title_full A transient assay system for the assessment of cell-autonomous gene function in dehydration-stressed barley
title_fullStr A transient assay system for the assessment of cell-autonomous gene function in dehydration-stressed barley
title_full_unstemmed A transient assay system for the assessment of cell-autonomous gene function in dehydration-stressed barley
title_short A transient assay system for the assessment of cell-autonomous gene function in dehydration-stressed barley
title_sort transient assay system for the assessment of cell-autonomous gene function in dehydration-stressed barley
topic Research Papers
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2529241/
https://www.ncbi.nlm.nih.gov/pubmed/18641397
http://dx.doi.org/10.1093/jxb/ern186
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