Chemically Induced Cuticle Mutation Affecting Epidermal Conductance to Water Vapor and Disease Susceptibility in Sorghum bicolor (L.) Moench

Abstract

Analysis of Sorghum bicolor bloomless (bm) mutants with altered epicuticular wax (EW) structure uncovered a mutation affeding both EW and cuticle deposition. l h e cuticle of mutant bm-22 was about 60% thinner and approximately one-fifth the weight of the wild-type parent P954035 (Wl-P954035) cuticles. Reduced cuticle deposition was associated with increased epidermal conductance to water vapor. The reduction in EW and cuticle deposition increased susceptibility to the funga1 pathogen Exserohilum turcicum. Evidence suggests that this recessive mutation occurs at a single locus with pleiotropic effects. l h e independently occurring gene mutations of bm-2, bm-6, bm-22, and bm-33 are allelic. These chemically induced mutants had essentially identical EW strudure, water loss, and cuticle deposition. Furthermore, 138 Fz plants from a bm-22 x WT-P954035 backcross showed no recombination of these traits. This unique mutation in a near-isogenic background provides a useful biological system to examine plant cuticle biosynthesis, physiology, and function.EW provides the outermost bamer between plants and their environment. Previous studies have implicated EW layers in tolerance to various kinds of biotic and abiotic environmental stress (Eglinton and Hamilton, 1967; Thomas and Barber, 1974;Blum, 1975; Webster, 1977; Bengston et al., 1978;Jordan et al., 1984; El-Otmani et al., 1989;Jefferson et al., 1989;Percy and Baker, 1990; Stoner, 1990;Bergman et al., 1991). Near-isogenic mutants provide a model system for the dissection of biochemical (Koorneef et al., 1989; Somerville and Browse, 1991) and biophysical (Blum, 1975;Jordan et al., 1984; Saneoka and Ogata, 1987) under drought stress and disease pressure in southwestem Mexico suggested that mutants bm-2, bm-6, bm-22, and bm-33, making up an individual allelic group, were more susceptible to drought and more susceptible to leaf blight. Since individuals in this allelic group had EW structures and total EW load similar to other bm mutants that were not susceptible to drought and disease, we suspected that these responses were due to alterations in the cuticle proper. To test this hypothesis, cuticle ultrastructures and depositions on the wild-type and bm mutants were analyzed and then compared with water loss rates and disease resistance. Our results suggest that allelic mutants bm-2, bm-6, bm-22, and bm-33 possessa mutation affecting cuticle deposition. To our knowledge, a cuticle mutation in plants has not been previously reported. MATERIALS AND METHODS Plant MaterialThe mutagenesis program was initiated using two droughtresistant inbred lines of Sorghum bicolor (L.) Moench. These inbred lines, designated P954035 and P898012, were produced in the Purdue Sorghum Improvement Program (Dr. Gebisa Ejeta, Department of Agronomy). Seeds (Mo) were exposed to the chemical mutagens diethyl sulfate (J. T. Baker, Phillipsburg, NJ) or ethyl methanesulfate (Eastman Kodak Co., Rochester, NY). Seeds treated with diethyl sulfate were submerged in either a 5.7, 7.7, or 11.5 mM solut...

Cloning and Characterization of the WAX2 Gene of Arabidopsis Involved in Cuticle Membrane and Wax Production

“…Whether these mutants, like wax2 , have altered cuticle membranes is unknown. wax2 also had reduced fertility, a condition reported for many cer mutants (Koornneef et al, 1989), increased epidermal permeability similar to that of the sorghum cuticle membrane mutant bm2 (Jenks et al, 1994), and a lower stomatal index. By comparison, the cer1 and cer6 wax mutants had higher stomatal indices (Gray et al, 2000).…”

“…First, exhaustive extraction of waxes from cuticles does not alter cuticle membrane ultrastructure, except for a slight reduction in the visibility of cuticle proper lamellae (Viougeas et al, 1995). Second, the cuticle membranes of seven wax mutants in sorghum and five wax mutants in Arabidopsis, including cer1, were ultrastructurally normal (Jenks et al, 1994;M.A. Jenks, unpublished data).…”

“…These stem wax mutants then were screened for changes in the rate of water loss from excised stems (Jenks et al, 1994). One mutant (later designated wax2 ) had a higher water-loss rate than the wild type and all other mutants.…”

“…Gravimetric measurements of cuticle membrane weight were determined for wild-type and wax2 inflorescence stem samples collected from the first internodes of 6-week-old plants. After removing soluble cuticular waxes by means of a 30-s dip in hexane, stem samples were soaked for 8 h in a 60% (w/v) solution of ZnCl in HCl to separate cuticle membranes from underlying stem tissues (Jenks et al, 1994). Bulk samples of 30 stem cuticle membranes (separated with a dissecting microscope) were placed on glass microscope cover slips, oven-dried at 50ЊC, and then placed in a desiccator containing dried silica gel for at least 12 h. Average cuticle membrane weights per surface area (stem segment circumference times height) for each line were determined from six replicate bulk samples using a microbalance.…”

“…The bm2 mutant has Ͼ 60% reduction in cuticle membrane weight and thickness (Jenks et al, 1994) and a significant alteration in its waxes (Jenks et al, 2000). The BM2 gene has not been cloned.…”