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. 2017 Jan;15(1):107-121.
doi: 10.1111/pbi.12595. Epub 2016 Aug 1.

Expression of the MYB transcription factor gene BplMYB46 affects abiotic stress tolerance and secondary cell wall deposition in Betula platyphylla

Huiyan Guo  1   2 , Yucheng Wang  1 , Liuqiang Wang  1 , Ping Hu  1 , Yanmin Wang  1   3 , Yuanyuan Jia  1 , Chunrui Zhang  1 , Yu Zhang  1 , Yiming Zhang  1 , Chao Wang  1 , Chuanping Yang  1
Affiliations

Expression of the MYB transcription factor gene BplMYB46 affects abiotic stress tolerance and secondary cell wall deposition in Betula platyphylla

Huiyan Guo et al. Plant Biotechnol J. 2017 Jan.

Abstract

Plant MYB transcription factors control diverse biological processes, such as differentiation, development and abiotic stress responses. In this study, we characterized BplMYB46, an MYB gene from Betula platyphylla (birch) that is involved in both abiotic stress tolerance and secondary wall biosynthesis. BplMYB46 can act as a transcriptional activator in yeast and tobacco. We generated transgenic birch plants with overexpressing or silencing of BplMYB46 and subjected them to gain- or loss-of-function analysis. The results suggest that BplMYB46 improves salt and osmotic tolerance by affecting the expression of genes including SOD, POD and P5CS to increase both reactive oxygen species scavenging and proline levels. In addition, BplMYB46 appears to be involved in controlling stomatal aperture to reduce water loss. Overexpression of BplMYB46 increases lignin deposition, secondary cell wall thickness and the expression of genes in secondary cell wall formation. Further analysis indicated that BplMYB46 binds to MYBCORE and AC-box motifs and may directly activate the expression of genes involved in abiotic stress responses and secondary cell wall biosynthesis whose promoters contain these motifs. The transgenic BplMYB46-overexpressing birch plants, which have improved salt and osmotic stress tolerance, higher lignin and cellulose content and lower hemicellulose content than the control, have potential applications in the forestry industry.

Keywords: Betula platyphylla; BplMYB46; abiotic stress; secondary wall deposition.

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Conflict of interest statement

The authors have no conflict of interest to declare.

Figures

Figure 1
Figure 1
Subcellular localization and transactivation assay of BplMYB46. (a) The 35S : BplMYB46‐ GFP fusion gene and 35S: GFP control plasmid were transformed into onion epidermal cells using particle bombardment. The transformed cells were imaged by confocal microscopy after transformation for 24 h. DAPI: DAPI staining of nucleus; GFP: GFP fluorescence detection; merge: the merged images of bright‐field, GFP and DAPI staining. (b) Various truncated sequences of the CDS of BplMYB46 were fused in‐frame, respectively, to the GAL4 DNA‐binding domain in pGBKT7 and transformed into AH109 yeast cells. The transformed cells were plated onto SD/‐Trp (growth control) or SD/‐Trp/‐His/‐Ade/X‐a‐Gal medium. The pGBKT7 empty vector was used as a negative control.
Figure 2
Figure 2
Expression patterns of BplMYB46. (a) Expression of BplMYB46 in response to NaCl, ABA and mannitol treatment. Well‐watered plants were used as controls to normalize the expression level at each time point. (b) The positions of the birch stem internodes analysed for gene expression. Arrows indicate the different stem internodes used in the analysis. (c) BplMYB46 mRNA levels in the roots, leaves and 1st, 4th, 8th, 12th, 16th and 18th stem internodes from 6‐month‐old birch plants. The BplMYB46 mRNA level in birch leaves was set to 1 to normalize its transcription level to that of roots and stem internodes. The error bars indicate the standard deviation (SD) from three biological replicates.
Figure 3
Figure 3
Analyses of BplMYB46 binding motifs. (a) Schematic diagram of the effector and reporter constructs used in Y1H analysis. (b) Analysis of binding of BplMYB46 to MYBCORE and AC‐box using Y1H. (c) Schematic diagram of the effector and reporter constructs used for coexpression in tobacco plants. (d, e) GUS staining (d) and GUS activity (e) Assays of the binding of BplMYB46 to the AC‐box and MYBCORE motifs in tobacco plants. The error bars indicate the standard deviation (SD) from three biological replicates.
Figure 4
Figure 4
Salt and osmotic stress tolerance. (a) Comparison of growth phenotypes among OE, WT and SE birch plants under control, salt and osmotic stress conditions. (b–d) analysis of relative fresh weight (b), root length (c) and chlorophyll contents (d). Asterisk indicates P < 0.05. The error bars indicate the standard deviation (SD) from three biological replicates. ANOVA was used to determine statistically significant differences between results.
Figure 5
Figure 5
Detection of ROS scavenging and analysis of proline biosynthesis. (a) Detection of ROS using DAB in situ staining. (b) Assay of H2O2 levels. (c) Analyses of ROS production in intact guard cells by H2DCF‐DA staining. (d) SOD activity assay. (e) POD activity assay. (f) Expression analysis of POD and SOD genes under NaCl and mannitol treatment using qRT ‐ PCR. The expressions of the genes in WT plants were used as calculators to normalize their expressions in OE and SE lines. (g) Analysis of proline levels in OE, WT and SE lines. (h) Expression analysis of proline biosynthesis and degradation‐related genes following NaCl and mannitol treatment using qRT ‐ PCR. The expressions of the genes in WT plants were used to normalize their expressions in OE and SE lines. Asterisk indicates P < 0.05. The error bars indicate the standard deviation (SD) from three biological replicates. ANOVA was used to determine statistically significant differences between results.
Figure 6
Figure 6
Analysis of cell death. (a) Assay of cell death among OE, WT and SE lines under NaCl and mannitol treatment using propidium iodide (PI) fluorescence staining. (b) Electrolyte leakage assay. Asterisk indicates P < 0.05. The error bars indicate the standard deviation (SD) from three biological replicates. ANOVA was used to determine statistically significant differences between results.
Figure 7
Figure 7
Analysis of water loss rate, stomatal aperture and BpMYB61 expression in OE, WT and SE BplMYB46 lines. (a) Water loss rates and (b, c) measurement of stomatal aperture under control conditions, 50 mm NaCl and 50 mm mannitol. (d) Expression of BpMYB61 (GenBank accession number: KT344120) in OE, WT and SE BplMYB46 lines under control conditions, 50 mm NaCl and 50 mm mannitol. Asterisk indicates P < 0.05. The error bars indicate the standard deviation (SD) from three biological replicates. ANOVA was used to determine statistically significant differences between results.
Figure 8
Figure 8
Microstructure of xylem vessels and fibres, and the expression of genes involved in secondary wall deposition. (a–c) Sections stained with phloroglucinol‐HCl (20×) in OE (a), WT (b) and SE plants (c); ve, vessel; xf, xylem fibre; bar = 50 μm. (d–f) Lignin autofluorescence in sections observed by microscopy (63×) in OE (d), WT (e) and SE (f), bar = 15 μm. (g–i) Sections stained with toluidine blue (20×) in OE (g), WT (h) and SE (i); ve, vessel; xf, xylem fibre, bar = 50 μm. (j–l) Sections analysed by scanning electron microscopy (3000×) in OE (j), WT (k) and SE (l); the arrow refers to the wall thickness of the xylem fibre, bar = 5 μm. (m) Secondary wall thickness of xylem fibres in OE, WT and SE. (n) Ratio of the vessel area to the total area (%) in OE, WT and SE. (o) Relative expression of genes related to secondary wall deposition in transgenic lines overexpressing BplMYB46 (OE) and RNAi‐silenced BplMYB46 (SE) lines. The sections were, respectively, analysed from the same stem positions of different plants of the same transgenic line. The error bars indicate the standard deviation (SD) from three biological replicates. ANOVA was used to determine statistically significant differences between results. a, b and c represent significant difference (P < 0.05) in OE, SE and WT birch plants. GenBank accession numbers: Pal1,2: KP711309 and KP711310; CcoAOMT1‐4: KP711311, KP711312, KT223488 and KT223489; 4CL1‐3: KP711313, KP711314 and KP711315; POD3,4,9,10: KP711298, KP711299, KP711304 and KP711305; LAC1,2: KP711316 and KT223490; CCR : JQ783349; CESA1‐3: KP711317, KP711318 and KP711319; FRA: KU168419; IRX : KU168420; MYB1‐3: KP711285, KP711286 and KP711287.
Figure 9
Figure 9
The regulation of target genes by BplMYB46, as determined by ChIP and GUS activity analyses. (a) Real‐time quantitative PCR analysis showing the enrichment of the promoter sequences of genes after chromatin immunoprecipitation. ChIP+: The sonicated chromatin was immunoprecipitated with GFP antibody; mock: the sonicated chromatin was immunoprecipitated with anti‐hemagglutinin (HA); input: the sonicated chromatin used as positive control. Three biological replicates were performed. Chromatin from whole seedlings was isolated from pROK2‐35S: BplMYB46‐GFP birch plants produced by Agrobacterium tumefaciens‐mediated transient transformation. The tubulin sequence was used as an internal control. After normalization against tubulin, the values of the enrichment of the promoter sequences of target genes in ChIP‐ were set to 1. The error bars indicate the standard deviation (SD) from three biological replicates. (b) Schematic diagram of the reporter and effector constructs used for coexpression in tobacco plants. (c) Relative GUS activity of the truncated promoters of genes. Asterisk indicates P < 0.05. The error bars indicate the standard deviation (SD) from three biological replicates. ANOVA was used to determine statistically significant differences between results. The GenBank accession numbers of promoters used in (a) and (c): KX373440–KX373458.
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