Characterization of drought stress regulator <em>CBF/DREB</em> genes in <em>Hordeum vulgare</em>

Abstract

Improving crop yield under water deficit environments is an urgent task for crop breeding worldwide. Many efforts have been made to identify key regulators of plant drought stress responses. Most of these studies have been performed with the model plant <em>Arabidopsis thaliana</em> under controlled laboratory conditions. Little work has been done to investigate the functions of these key regulators in the improvement of drought tolerance in crops, especially under field conditions. In the present study, ten barley <em>CBF/DREB</em> gene transcripts (<em>CBF1, CBF2, CBF3, CBF4, CBF6, CBF11, DREB1, DRF1.1, DRF1.3 </em>and<em> DRF2</em>) were isolated from different barley cultivars by using gene-specific primers. Phylogenetic analysis showed that all candidate <em>CBF/DREB</em> genes can be grouped into three phylogenetic subgroups, designated <em>HvCBF1, HvCBF4 </em>and<em> HvDREB1</em>. The phylogenetic distribution of barley <em>CBF/DREB</em> genes in the reconciled tree coincides with their responses to drought. A strongly constitutive expression of the <em>HvDREB1, HvDRF1.1</em> and <em>HvDRF1.3</em> genes upon drought has been identified in this study, which is contrary to the published data in the literature. This may be due to the age and physiological state of the plants. By analyzing the <em>CBF</em> expression patterns upon drought in leaf tissues of plants grown in the field, we found that barley cultivars respond to drought at different time points. Monitoring the expression of <em>HvCBF1, HvCBF2, HvCBF3, HvCBF4, HvCBF6 </em>and<em> HvCBF11</em> transcripts upon drought in field grown material revealed that rapid dehydration triggered the reduction of the above-mentioned transcripts. This was further validated by experiments performed under controlled laboratory conditions. Furthermore, tissue- and age-dependent expression has been identified in barley <em>HvCBF1, HvCBF3, HvCBF4, HvCBF6 </em>and<em> HvCBF11 </em>genes, indicating that the <em>CBF</em> transcription factors are developmentally regulated. Importantly, in contrast to the common thought, our analyses of methylation patterns in the <em>HvCBF1</em> promoter showed that DNA methylation within the promoter does not always lead to down-regulation of the corresponding gene. Finally, a more efficient and non-invasive method to analyze the barley root architecture and development has been initiated with help and permissions from Prof. Dr. Philip Benfey, Duke University, Durham NC, USA.