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JXB Advance Access originally published online on July 18, 2008
Journal of Experimental Botany 2008 59(12):3359-3369; doi:10.1093/jxb/ern186
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© 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)

RESEARCH PAPER

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

Stephan Marzin1 *, Robert Mihaly1,2 *, Janos Pauk2 and Patrick Schweizer1,{dagger}

1Leibniz-Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, D-06466 Gatersleben, Germany
2Cereal Research Non-profit Co. (CRI), Szeged POB 391, H-6701, Hungary

{dagger} To whom correspondence should be addressed. E-mail: schweiz{at}ipk-gatersleben.de

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.

Key words: DsRed, particle bombardment, RNAi, single cell

* These authors contributed equally to this work.

Received 15 February 2008; Revised 23 May 2008 Accepted 24 June 2008


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