Stocky1, a Novel Gene Involved in Maize Seedling Development and Cuticle Integrity

Abstract

The cuticle is the plant's outermost layer that covers the surfaces of aerial parts. This structure is composed of a variety of aliphatic molecules and is well-known for its protective role against biotic and abiotic stresses in plants. Mutants with a permeable cuticle show developmental defects such as organ fusions and altered seed germination and viability. In this study, we identified a novel maize mutant, stocky1, with unique features: lethal at the seedling stage, and showing a severely dwarfed phenotype, due to a defective cuticle. For the first time, the mutant was tentatively mapped to chromosome 5, bin 5.04. The mutant phenotype investigated in this work has the potential to contribute to the elucidation of the role of the cuticle during plant development. The possibility of controlling this trait is of relevance in the context of climate change, as it may contribute to tolerance to abiotic stresses.

Keywords: maize cuticle; plant cuticle; seedling development.

Figure 1

Phenotype of des mutant seedlings: pictures of wild type (WT) and mutant seedlings obtained from the EMS mutagenized population at 10 days after sowing (DAS): WT, des*-4, des*-19/stocky1, des*-47, des*-85, des*-217, des*-256. Scale bar: 1 cm. The mutants show a dwarf phenotype, and are delayed in the first phases of seedling development.

Figure 2

Phenotypic characterization of stocky1 seedlings: (A) Representative stocky1 seedling at 12 DAS showing the first three developing leaves; the third leaf (arrowhead) is bent, rolled and twisted, and has been released manually from the second one (arrow), which is ripped in the adhesion regions; the lower and upper dashed lines indicate, respectively, the proximal and distal part from which cross sections shown in C, E, G, H, have been cut. Cross sections shown in B, D, F, are from WT seedlings and were cut in the same regions for comparison; all sections were stained with Calcofluor White and Auramine-O, the latter specific for the cuticle, which stains in bright yellow-green when excited at about 450 nm by the fluorescence microscope. (B) WT proximal region showing normally developed leaf primordia [P] rolled up around the stem [S] and outlined by a bright yellow cuticle (arrowheads); leaf trichomes [T] and xylem vessels (arrows) are also visible by autofluorescence. (C) stocky1 proximal region with leaf primordia [P], in some parts not completely separated from each other (asterisk) and with a less defined cuticle (arrowhead); the stem [S] is larger than in WT seedlings. (D) Distal part of a WT seedling with rolled and well-separated developing leaves which show a continuous cuticle, more clearly visible in the enlargement in F (arrows). (E) Distal part of a stocky1 seedling in which developing leaves are fused together in some regions (arrows); in these regions, the cuticle of the fused leaves is not visible, as shown in the enlargement in G (asterisk); in other fused regions, the cuticle appears discontinuous (H, arrows). (I) TB permeability quantification in the wild type and in the stocky1 mutant showing a higher TB uptake in the latter; error bars represent SD from three biological replicates; black asterisk indicates a statistically significant difference (* p < 0.01) obtained with t-Test.

Figure 3

Effect of hormones on stocky1 growth in embryo-rescue experiment. Immature embryos were collected between 17 and 21 DAP, and were cultured for 10 days before taking root and shoot length measures. The embryos were cultured on MS media; hormones were added at a concentration of 10⁻⁵ M: (A) Graph showing the average length of WT and stocky1 seedlings shoot 10 days after culture (DAC). (B) Graph showing the average length of WT and stocky1 seedlings root 10 DAC. Error bars represent SD from biological replicates, WT (n ≥ 30) and stocky1 (n ≥ 12). Different letters on top of SD indicate a statistically significant difference (p < 0.05) obtained with Tukey HSD test after one-way ANOVA.

Figure 4

Genetic map of the stocky1 mutant using SSR molecular markers.