Abstract
The cuticle of land plants is the first physical barrier to protect their aerial parts from biotic and abiotic stresses. DEWAX, an AP2/ERF-type transcription factor, negatively regulates cuticular wax biosynthesis. In this study, we investigated the resistance to Botrytis cinerea in Arabidopsis thaliana and Camelina sativa overexpressing DEWAX and in Arabidopsis dewax mutant. Compared to wild type (WT) leaves, Arabidopsis DEWAX OX and dewax leaves were more and less permeable to toluidine blue dye, respectively. The ROS levels increased in DEWAX OX leaves, but decreased in dewax relative to WT leaves. Compared to WT, DEWAX OX was more resistant, while dewax was more sensitive to B. cinerea; however, defense responses to Pseudomonas syringae pv. tomato DC3000:GFP were inversely modulated. Microarray and RT-PCR analyses indicated that the expression of defense-related genes was upregulated in DEWAX OX, but downregulated in dewax relative to WT. Transactivation assay showed that DEWAX upregulated the expression of PDF1.2a, IGMT1, and PRX37. Chromatin immunoprecipitation assay revealed that DEWAX directly interacts with the GCC-box motifs of PDF1.2a promoter. In addition, ectopic expression of DEWAX increased the tolerance to B. cinerea in C. sativa. Taken together, we suggest that increased ROS accumulation and DEWAX-mediated upregulation of defense-related genes are closely associated with enhanced resistance to B. cinerea in Arabidopsis and C. sativa.
Highlights
To investigate the cuticle permeability of wild type (WT), dewax, and two DEWAX OX lines, rosette leaves of 4-week-old plants were stained with 0.05% toluidine blue
Based on the previous reports that Arabidopsis bdg and lacs2 mutants with a permeable cuticle showed increased ROS levels (L’Haridon et al, 2011), ROS assay was performed in the leaves of WT, dewax, and DEWAX OX lines
Both in situ assay of hydrogen peroxide and fluorometric measurements of ROS levels showed that the levels of ROS were lower in dewax leaves, but higher in DEWAX OX1 and OX2 leaves than in WT leaves (Figures 1B,C)
Summary
The hydrophobic cuticle covering the aboveground parts of land plants forms a physical and chemical barrier to infection by bacterial and fungal pathogens, as well as drought stress (Nawrath, 2006; Pollard et al, 2008; Beisson et al, 2012; Li-Beisson et al, 2013; Yeats and Rose, 2013). Several Arabidopsis mutants with increased cuticular permeability, including lacerate (lcr), bodyguard (bdg), long-chain acyl-CoA synthetase-2 (lacs-2) and -3, fiddlehead (fdh), permeable cuticle (fec1), and myeloblast transcription factor 96 (myb96), display enhanced resistance to B. cinerea (Yephremov et al, 1999; Wellesen et al, 2001; Kurdyukov et al, 2006; Bessire et al, 2007, 2011; Chassot et al, 2007; Voisin et al, 2009; Seo et al, 2011). The results suggest that DEWAX-mediated upregulation of defense-related genes in addition to an increase in ROS levels is important in the enhanced tolerance to B. cinerea in Arabidopsis and C. sativa overexpressing DEWAX
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