Characterization of increased cuticular wax mutant and analysis of genes involved in wax biosynthesis in Dianthus spiculifolius

Abstract

Cuticular wax formation on the surface of plant leaves is associated with drought-stress tolerance. The identification of wax biosynthesis-related genes will contribute to the genetic improvement of drought resistance in plants. In this study, we characterize a novel Dianthus spiculifolius mutant with increased cuticular wax. The mutant exhibited stronger drought resistance as indicated by less leaf wilting and death, higher leaf relative water content and water retention capacity, and slower water loss and chlorophyll extraction than did the wild type during drought treatment. In the mutant leaves, 2 730 upregulated and 2 151 downregulated differentially expressed genes (DEGs) were identified by transcriptome sequencing. A wax biosynthesis pathway of the identified DEGs was significantly enriched. Finally, three key genes (DsCER1, DsMAH1, and DsWSD1) involved in wax biosynthesis were identified and verified by qPCR. These results suggest that differential expression of DEGs involved in wax biosynthesis may be associated with the increase in cuticular wax in the mutant. Taken together, our results help elucidate wax formation patterns in D. spiculifolius. Furthermore, the DEGs involved in wax biosynthesis identified here may be valuable genetic resources for improving plant stress tolerance through increased accumulation of cuticular wax.

Fig. 1. Phenotype of cuticular wax mutants of D. spiculifolius.

a Wild type (WT) and cuticular wax mutants (‘Greyish-green, GG)’ of D. spiculifolius grown in an open field and a greenhouse, respectively. Leaf morphology (b) and color (c) of WT and GG plants. Comparison of chlorophyll (d) and carotenoid (e) content of WT and GG leaves. Asterisks indicate significant differences between WT and GG plants (**P < 0.01; Student’s t test). FW fresh weight

Fig. 2. Scanning electronic microscopy (SEM) of WT and GG D. spiculifolius leaf surfaces.

SEM images of the leaf surfaces (a, b, e, f) and leaf freeze-fracture cross-sections (c, d) of WT and GG plants. Comparison of stomatal density (g), and stomatal aperture (h) of WT and GG leaves. Asterisks indicate significant differences between WT and GG plants (**P < 0.01; Student’s t test). Error bars represent SD (n = 3). Scale bars = 20 μm (a, b), 5 μm (c, d), and 100 μm (e, f)

Fig. 3. Drought tolerance assay of WT and GG D. spiculifolius plants.

a Morphological appearance of WT and GG under normal and drought stress. Three-month-old plants were drought stressed (30 °C) for 8 days and rewatered for 4 days. Comparison of relative water content (b) and water retention capacity (c) of normal and stress-treated plants. Water loss (d) and chlorophyll leaching assays (e) of WT and GG under drought stress. The leaves of plants acclimated to darkness for 12 h to assure stomatal closure were subjected to water loss measurements and chlorophyll leaching assays. The leaves were weighed at the indicated time points or soaked in 80% ethanol for the indicated time points. Extracted chlorophyll contents at individual time points are expressed as percentages of that at 6 h after initial immersion. Asterisks indicate significant differences between WT and GG plants (*P < 0.05; **P < 0.01; Student’s t test). Error bars represent SE (n = 6)

Fig. 4. Volcano plots showing the number of differentially expressed genes (DEGs) in GG vs. WT D. spiculifolius plants.

Abundance of each gene was normalized as RPKM. DEGs are shown in red (upregulated) and blue (downregulated), while gray indicates genes that were not differentially expressed (no-DEGs). We used a false discovery rate ≤ 0.05 and the absolute value of log₂Ratio ≥ 1 as the threshold to judge the significance of the differences in gene expression

Fig. 5. Scatterplot of KEGG pathways enriched for differentially expressed genes (DEGs) in GG vs. WT D. spiculifolius plants.

The rich factor is the ratio of the number of DEGs annotated in a given pathway term to the number of all genes annotated in the pathway term. A greater rich factor means greater intensity. The Q value is the corrected P value and ranges from 0 to 1, and a lower Q value indicates greater intensity. The size of the circles indicates the number of genes. The top 20 enriched pathway terms in the KEGG database are listed. The red arrow indicates the significantly enriched pathway associated with wax biosynthesis

Fig. 6. qPCR verification of key genes involved in the wax biosynthesis pathway in D. spiculifolius.

a Simplified cuticular wax biosynthetic pathway from the KEGG database. Upregulated genes in GG vs. WT D. spiculifolius plants are marked with red borders. The structural information about the wax compounds is displayed in the black box. b–d qPCR analysis of key genes (DsCER1, DsWSD1, and DsMAH1) involved in wax biosynthesis. The DsActin gene was used as an internal control, and the transcript level in WT plants was set as 1.0. Asterisks indicate significant differences between WT and GG plants (**P < 0.01; Student’s t test). Error bars represent SE (n = 3)