Maize glossy6 is involved in cuticular wax deposition and drought tolerance

Abstract

Cuticular waxes, long-chain hydrocarbon compounds, form the outermost layer of plant surfaces in most terrestrial plants. The presence of cuticular waxes protects plants from water loss and other environmental stresses. Cloning and characterization of genes involved in the regulation, biosynthesis, and extracellular transport of cuticular waxes onto the surface of epidermal cells have revealed the molecular basis of cuticular wax accumulation. However, intracellular trafficking of synthesized waxes to the plasma membrane for cellular secretion is poorly understood. Here, we characterized a maize glossy (gl6) mutant that exhibited decreased epicuticular wax load, increased cuticle permeability, and reduced seedling drought tolerance relative to wild-type. We combined an RNA-sequencing-based mapping approach (BSR-Seq) and chromosome walking to identify the gl6 candidate gene, which was confirmed via the analysis of multiple independent mutant alleles. The gl6 gene represents a novel maize glossy gene containing a conserved, but uncharacterized, DUF538 domain. This study suggests that the GL6 protein may be involved in the intracellular trafficking of cuticular waxes, opening the door to elucidating the poorly understood process by which cuticular wax is transported from its site of biosynthesis to the plasma membrane.

Keywords: glossy6 ( gl6 ); Cuticular waxes; DUF538; drought tolerance; glossy mutant; maize (Zea mays).

Fig. 1.

Morphological characterization of the gl6-ref mutant. (A) gl6-ref mutant exhibits a glossy phenotype. Water is sprayed on seedlings to distinguish gl6-ref mutant seedlings from wild-type (WT). (B) Adaxial leaf epicuticular wax accumulation in WT and mutant (gl6-ref) seedlings detected via SEM. ×5000 magnification. (C–E) TEM analysis of leaf epidermal wax secreting cells from WT and gl6-ref mutant. (E) Enlarged view showing the red rectangular area marked in (D). Arrows in (D, E) indicate unusual linear inclusions. Scale bars: 3 cm in (A), 5 µm in (B–D), 500 nm in (E). (This figure is available in color at JXB online.)

Fig. 2.

Total leaf epicuticular wax load and wax composition of wild-type and gl6-ref mutant. (A) Total leaf cuticular wax load of wild-type and gl6-ref mutant. (B) Epicuticular wax composition of wild-type and gl6-ref mutant. Values are means of eight biological replicates ±SD. UI, unidentified. Asterisks indicate statistically significant differences between wild-type and gl6-ref mutant (*P<0.05, **P<0.01, ***P<0.001, Student’s t-test).

Fig. 3.

Leaf surface permeability analysis of gl6-ref mutant. (A) Kinetics of chlorophyll leaching from leaves of the WT and gl6-ref mutant. (B) The total leaf chlorophyll content of WT and gl6-ref mutant. (C) Detached leaves from WT and gl6-ref mutant immediately after harvest and after 2 h at room temperature. (D) Water loss of detached leaves of the WT and gl6-ref mutant. Values in (A, B, D) are means of three biological replicates ±SD. (This figure is available in color at JXB online.)

Fig. 4.

gl6-ref mutant seedlings are sensitive to drought stress. (A) False-color infrared image of the wild-type and the gl6-ref mutant under well-watered and drought stressed conditions. (B–D) Stomatal density (B), pavement cell density (C), and stomatal index (number of stomata per total epidermal cells) (D) analysed in leaf abaxial epidermal layers from WT and gl6-ref mutant. Data are means of five individual plants. (E) Drought phenotypes of WT and gl6-ref mutant seedlings in soil following drought stress and after re-watering. (F) The survival rate of WT and gl6-ref mutant seedlings after drought stress and re-watering. (G, H) The leaf relative water content and relative electrical conductivity of WT and gl6-ref mutant seedlings under well-watered (WW), drought stressed (DS) and re-watered (RW) conditions. In (B–D, F), data are means of five replicates ±SD, and in (G, H) data are means of three replicates ±SD (***P<0.001, Student’s t-test). Scale bars: 10 cm in (A, E). (This figure is available in color at JXB online.)

Fig. 5.

Molecular cloning of the gl6 gene and its phylogenetic analysis. (A) BSR-Seq analysis of an F₂ segregated gl6-ref population mapped gl6 to the 113.5–132.4 Mb interval of chr3. (B) PAGE results of digestion–ligation–amplification (DLA) analysis using the adaptor primer (Nsp-15ctc). The rectangle indicates the specific bands produced from gl6/+ plants but not from WT plants. (C) The gene structure of gl6, Mu insertions in four alleles, and lesions in two gl6 EMS alleles. (D) Phylogenetic tree constructed using MEGA 7.0 and the GL6 protein and GL6 homologs aligned using ClustalW. Distances were estimated with a neighbor-joining algorithm, and bootstrap support is indicated to the left of branches. (This figure is available in color at JXB online.)

Fig. 6.

Subcellular localization of the GL6 protein in maize protoplasts. Confocal images show the expression of YFP, GL6 fused at its C terminus with YFP, and organelle markers. (A, B) Subcellular localization YFP and GL6–YFP. (C) Co-localization of GL6–YFP with RFP–CNX (an ER marker). (D) Co-localization of GL6–YFP with mRFP–ManI (a Golgi marker). (E) Co-localization of GL6–YFP with RFP–SYP41 (a TGN marker). (F) Co-localization of GL6–YFP with FM4-64 (a plasma membrane marker). Scale bar: 10 µm. (This figure is available in color at JXB online.)