The maize lilliputian1 (lil1) gene, encoding a brassinosteroid cytochrome P450 C-6 oxidase, is involved in plant growth and drought response

Abstract

Background and aims: Brassinosteroids (BRs) are plant hormones involved in many developmental processes as well as in plant-environment interactions. Their role was investigated in this study through the analysis of lilliputian1-1 (lil1-1), a dwarf mutant impaired in BR biosynthesis in maize (Zea mays).

Methods: We isolated lil1-1 through transposon tagging in maize. The action of lil1 was investigated through morphological and genetic analysis. Moreover, by comparing lil1-1 mutant and wild-type individuals grown under drought stress, the effect of BR reduction on the response to drought stress was examined.

Key results: lil1-1 is a novel allele of the brassinosteroid-deficient dwarf1 (brd1) gene, encoding a brassinosteroid C-6 oxidase. We show in this study that lil1 is epistatic to nana plant1 (na1), a BR gene involved in earlier steps of the pathway. The lill-1 mutation causes alteration in the root gravitropic response, leaf epidermal cell density, epicuticular wax deposition and seedling adaptation to water scarcity conditions.

Conclusions: Lack of active BR molecules in maize causes a pleiotropic effect on plant development and improves seedling tolerance of drought. BR-deficient maize mutants can thus be instrumental in unravelling novel mechanisms on which plant adaptations to abiotic stress are based.

Fig. 1.

The lil1-1 mutant is an allele of brd1. (A) Schematic representation of the maize cytochrome P450 CYP85A1 C-6 oxidase gene structure (Maize B73 RefGen_v3). The lil1-1 mutant allele carries a Mutator1 (Mu1) insertion, indicated as a triangle, in the second exon at 877 bp from the ATG start codon. Positions of the brd1-m1 mutation at 1145 bp (Makarevitch et al., 2012) and of primers (arrows) used in this work are also indicated. (B) Representative phenotypes of 10-day old wild-type, homozygous lil1-1 and homozygous brd1-m1 seedlings. Scale bar = 2 cm.

Fig. 2.

Morphological features of lil1-1 mutants. (A, B) Longitudinal sections of shoot apices of 2-week-old wild-type (A) and homozygous lil1-1 mutant (B) plants. Samples were stained with Toluidine Blue O. Red asterisks mark leaves primordia. (C, D) Transverse section of leaf blades of 2-week-old wild-type (C) and homozygous lil1-1 mutant (D) plants. (E–G) Primary root of 4-day old seedlings. Altered gravitropic response in homozygous lil1-1 mutant roots growing upwards (E) or parallel to the surface (F) compared with wild-type (WT) root, which shows a positive gravitropic response (G).

Fig. 3.

The lil1-1 mutant is epistatic to the na1-1 mutant . (A) Representative wild-type (WT), na1-like (na1) and lil1-like (lil1) phenotypes of 10-day old F₂ plants obtained from selfing F₁ heterozygous na1-1/+ lil1-1/+ plants. (B–D) Characterization of WT, homozygous na1-1, homozygous lil1-1 and double-homozygous na1-1 lil1-1 genotypic classes. (B) Whisker plot representing seedling height of 10-day old plants. (C) Numbers of guard cells (GC) and pavement cells (PC). (D) Stomatal index of fourth fully expanded leaves of 50-day old plants. Error bars show the standard error. In (B), (C) and (D) different letters denote differences between values that are statistically significant (P < 0.05) as calculated by one-way ANOVA. In (C) GC (black letters) or PC (red letters) values were compared. In (D) abaxial (black letters) or adaxial (red letters) values were compared.

Fig. 4.

Permeability and cuticular wax analysis. (A) Chlorophyll leaching assay on the sixth fully expanded leaf of 40-day old wild-type (WT), homozygous na1-1 and homozygous lil1-1 mutant plants. Values represent the mean of five leaves per genotype. Error bars show the standard error. FW, fresh weight. (B, C) Representative adult leaf phenotype of WT (B) and homozygous lil1-1 mutant (C) plants misted with water. (D–I) Scanning electron microscope images of epicuticular waxes of the fourth adaxial leaf surface of WT (D, G) and homozygous lil1-1 mutant plants (E, F, H, I). Mutant samples were taken from a smooth (E, H) and a crinkly sector (F, I) of the leaf.

Fig. 5.

The lil1-1 mutant plants are more tolerant to drought stress. (A) Representative phenotype of 14-day old wild-type (WT) and homozygous lil1-1 mutant plants grown under well-watered conditions (0 Days) and (B) after 6 days of drought stress initiation by withholding irrigation (6 Days). (C) Relative soil water content (RWSC) measured at 0, 3, 6 and 9 days after drought stress indicates that the water loss from the soil of pots in which WT plants were grown was identical to that of pots in which mutant (lil1-1) plants were grown. (D) Leaf relative water content (RWC) of WT and homozygous lil1-1 mutant plants at 0 and 9 days of drought treatment. (E, F) Physiological parameters were measured at 0, 3 and 4 days. (E) Stomatal conductance (g_s). (F) Transpiration rate (E). (G) Net photosynthesis rate (A). Error bars in (C–G) show standard error. Comparison was made at each time point between wild-type and homozygous lil1-1 genotypes. ****P < 0.0001 (two tailed Student’s t-test); ns, not significant.