The MYB96 transcription factor regulates cuticular wax biosynthesis under drought conditions in Arabidopsis

Abstract

Drought stress activates several defense responses in plants, such as stomatal closure, maintenance of root water uptake, and synthesis of osmoprotectants. Accumulating evidence suggests that deposition of cuticular waxes is also associated with plant responses to cellular dehydration. Yet, how cuticular wax biosynthesis is regulated in response to drought is unknown. We have recently reported that an Arabidopsis thaliana abscisic acid (ABA)-responsive R2R3-type MYB transcription factor, MYB96, promotes drought resistance. Here, we show that transcriptional activation of cuticular wax biosynthesis by MYB96 contributes to drought resistance. Microarray assays showed that a group of wax biosynthetic genes is upregulated in the activation-tagged myb96-1D mutant but downregulated in the MYB96-deficient myb96-1 mutant. Cuticular wax accumulation was altered accordingly in the mutants. In addition, activation of cuticular wax biosynthesis by drought and ABA requires MYB96. By contrast, biosynthesis of cutin monomers was only marginally affected in the mutants. Notably, the MYB96 protein acts as a transcriptional activator of genes encoding very-long-chain fatty acid-condensing enzymes involved in cuticular wax biosynthesis by directly binding to conserved sequence motifs present in the gene promoters. These results demonstrate that ABA-mediated MYB96 activation of cuticular wax biosynthesis serves as a drought resistance mechanism.

Figure 1.

Upregulation of Cuticular Wax Biosynthetic Genes in myb96–1D. (A) Venn diagrams showing the distribution of overlapping and nonoverlapping genes encoding lipid metabolic and wax biosynthetic enzymes with genes upregulated in myb96-1D. Three independent experiments were performed. The microarray data set was deposited into the ArrayExpress database with accession number E-MEXP-2965 at http://www.ebi.ac.uk/at-miamexpress. (B) List of wax biosynthetic genes upregulated in myb96-1D. Genes upregulated >2-fold in the myb96-1D mutant compared with the wild type are listed in Supplemental Data Set 1A online. The P values were corrected for multiple testing using FDR methodology. The group of genes was classified based on their biochemical functions. AGI, Arabidopsis Genome Initiative number; FC, fold changes. (C) Simplified cuticular wax biosynthetic pathway. Adapted from Kunst and Samuels (2009). Numbers indicate mean fold change of the genes belonging to individual gene groups (I to XI), as marked in (B). The question marks and dotted lines indicate unidentified enzymes and processes, respectively. FAE, fatty acid elongation. (D) qRT-PCR of wax biosynthetic gene expression. Total RNAs were extracted from 2-week-old whole plants grown on MS-agar. Transcript levels were examined via qRT-PCR. Biological triplicates were averaged and statistically analyzed using a student t test (*P < 0.01). Bars indicate se of the mean. The vertical axis is displayed on a logarithmic scale to obtain better comparison of transcript levels.

Figure 2.

Scanning Electron Microscopy Analysis of Epicuticular Wax Deposition on the Surfaces of Plant Organs in myb96-1D and myb96-1 Mutants. (A) Scanning electron microscopy images of epicuticular wax crystals on the leaves. Rosette leaves of 4-week-old wild-type (Col-0) and myb96-1D plants grown in soil were used for scanning electron microscopy analysis. Bars = 20 μm. (B) Glossy appearance indicating wax deficiency of the myb96-1 stem. Inflorescence stems of 5-week-old wild-type (Col-0) and myb96-1 plants grown in soil were photographed. (C) Scanning electron microscopy images of epicuticular wax crystals on the stems. Inflorescence stems of 5-week-old wild-type (Col-0) and myb96-1 plants grown in soil were subject to scanning electron microscopy. Bars = 20 μm.

Figure 3.

Cuticular Wax Amount and Composition in myb96-1D and myb96-1 Leaves. Rosette leaves of 4-week-old wild-type (Col-0) and mutant plants grown in soil were used for analysis of cuticular wax composition and loads. Biological triplicates, each with technical duplicates, were averaged and statistically analyzed using a student t test (*P < 0.01). Bars indicate se of the mean. FW, fresh weight. (A) Cuticular wax amount in myb96-1D and myb96-1 leaves compared with the wild type. (B) and (C) Cuticular wax composition and loads in the leaves of the myb96-1D (B) and myb96-1 mutants (C) compared with the wild type. The loads of cuticular waxes of the myb96-1 mutant were also expressed as μg/cm² in Supplemental Figure 3 online.

Figure 4.

Cuticular Wax Composition in myb96-1D and myb96-1 Stems. Inflorescence stems of 5- to 10-week-old wild-type and mutant plants grown in soil were used for analysis of cuticular wax composition and loads. Biological triplicates, each with technical duplicates, were averaged and statistically analyzed using a Student’s t test (*P < 0.01). Bars indicate se of the mean. Cuticular wax composition and loads in the stems of myb96-1D (A) and myb96-1 mutants (B). The loads of cuticular waxes of the myb96-1 mutant were also expressed as μg/cm² in Supplemental Figure 4 online. FW, fresh weight.

Figure 5.

Drought Induction of Cuticular Wax Biosynthetic Genes. Two-week-old plants grown on MS-agar plates were treated with growth hormones, such as 20 μM ABA (6 h) and 100 μM SA (6 h), and stress conditions, including cold (4°C, 24 h), 150 mM NaCl (Na, 6 h), and 5 μM flagellin22 (flg22, 24 h), before whole-plant materials were harvested for extraction of total RNAs. Drought stress (DR) was induced in 20-d-old plants grown in soil by halting watering. Transcript levels were examined as described in Figure 1D. Bars indicate se of the mean (t test, *P < 0.01). M, mock treatment. (A) Effects of growth hormones and stresses on gene expression. Statistical significance of the measurements was determined using a Student’s t test by comparing with mock treatment. (B) Effects of ABA on gene expression in myb96-1. Statistical significance of the measurements was determined using a Student’s t test by comparing Col-0 and mutant plants treated with ABA.

Figure 6.

Cuticular Wax Accumulation in the Leaves under Drought Conditions. (A) Accumulation of cuticular waxes under drought. Biological triplicates, each with technical duplicates, were averaged. Statistical significance of the measurements was determined using a Student’s t test (t test, *P < 0.01) by comparing Col-0 and mutant plants treated with drought (DR). Bars indicate se of the mean. FW, fresh weight; M, mock. (B) Cuticular wax composition in the leaves under drought. Three-week-old leaves were used. Biological triplicates, each with technical duplicates, were averaged. Statistical significance of the measurements was determined using a Student’s t test (t test, *P < 0.01) by comparing Col-0 and mutant plants treated with drought (DR). Bars indicate se of the mean.

Figure 7.

Effects of MYB96 Induction on Epicuticular Wax Deposition. (A) Induction of wax biosynthetic genes. Two-week-old transgenic plants expressing the pER8-MYB96 gene fusion under the control of a β-estradiol–inducible promoter were incubated in MS liquid cultures supplemented with 10 μM β-estradiol. Whole plants were harvested at the indicated time points (h) after β-estradiol application for total RNA extraction. Transcript levels were examined by qRT-PCR. Biological triplicates were averaged. Bars indicate se of the mean. (B) Elevation of epicuticular wax crystals after β-estradiol induction of MYB96. One-week-old plants grown in soil were sprayed every 3 d with 10 μM β-estradiol solution. Epicuticular wax crystals were examined by scanning electron microscopy 3 weeks after induction. Bars = 10 μm.

Figure 8.

Cuticular Transpiration in myb96-1D and myb96-1 Leaves. Four-week-old plants grown under either normal (mock) or drought conditions were acclimated in the dark for 6 h and used for subsequent treatments. (A) Water loss assays. Whole rosettes of the dark-acclimated plants were excised and soaked in water for 60 min in the dark. They were dried and weighed at the indicated time points. Three measurements were averaged at each time point. Bars indicate se of the mean. (B) Chlorophyll leaching assays. Whole rosettes of the dark-acclimated plants were soaked in 80% ethanol for the indicated time periods (min). Extracted chlorophyll contents at individual time points were expressed as percentages of that at 24 h after initial immersion. Three measurements were averaged. Bars indicate se of the mean.

Figure 9.

Binding of MYB96 to Consensus Motifs in the Promoters of Wax Biosynthetic Genes (A) MYB binding consensus sequences (BSs). Core binding sequences, marked in bold, were mutated, resulting in mBSs, to verify specific binding. (B) In vitro binding of MYB96 to the consensus sequences. The (−) lanes are controls without recombinant MBP-MYB96 proteins. Excess amounts (50× or 100×) of unlabeled BS or mBS DNA fragments were added as competitors. (C) ChIP assays. Total protein extracts from 35S:96-MYC transgenic plants grown on MS-agar plates for 2 weeks were immunoprecipitated with an anti-MYC antibody. Fragmented genomic DNA was eluted from the protein-DNA complexes and subjected to quantitative PCR analysis. Three measurements were averaged for individual assays. Statistical significance of the measurements was determined using a Student’s t test (t test, *P < 0.01) by comparison with the value of Col-0. Bars indicate se of the mean. In each measurement, the measurement values in Col-0 were set to 1 after normalization against eIF4a for quantitative PCR analysis. NB, nonbinding sequence. (D) Expression constructs used. The promoter sequence elements of the KCS genes listed in (A) were fused to minimal 35S promoter (Min 35S) and GUS reporter gene. In the effector construct, the MYB96 gene was fused to the CaMV 35S promoter. Nos-T, Nos terminator. (E) Transcriptional activation activity assays in Arabidopsis protoplasts. The expression constructs in (D) were expressed transiently in Arabidopsis protoplasts, and GUS activities were determined fluorimetrically. Luciferase gene expression was used to normalize the GUS activities. Three measurements were averaged (t test, *P < 0.01). Bars indicate se of the mean.

Figure 10.

Schematic Working Model of MYB96 Function in Cuticular Wax Biosynthesis under Drought Conditions. MYB96 transcription is upregulated in response to ABA-mediated drought signals. The MYB96 protein activates the cuticular wax biosynthetic genes by binding directly to the gene promoters, resulting in accumulation of cuticular waxes. MYB96 may also contribute to drought resistance via the RD22 pathway by regulating stomatal aperture (Seo et al., 2009).