The identification and duplication of a gene that controls production of plants' outermost protective coating may allow Purdue University researchers to create crops with increased drought resistance.

Scientists cloned the gene WAX2 after they discovered a fast-wilting mutant of Arabidopsis, a commonly used experimental plant. The gene is directly associated with the synthesis of the protective layer of plants, called the cuticle, and its contained waxes, according to the study published in the May issue of The Plant Cell.

The difference in the mutant Arabidopsis when compared to a wild-type, or normal, plant is the plants' ability to retain water. This is apparently because the mutation, called wax2, has a different cuticle structure than found in a plant that has the normal gene, WAX2.

"If we can alter the expression of the WAX2 gene, we might be able to produce a cuticle that is thicker or more rigid so that it's less permeable to water loss," said Matt Jenks, associate professor of horticulture and landscape architecture.

Manipulating what the gene does or when it is turned on could result in plants better able to survive in arid conditions.

Jenks and his research team isolated more than 20 mutant Arabidopsis plants that showed alterations in the amount of wax they produced. Of these, only a few lost water more quickly than the wild type.

"The mutant wax2 was the most drought susceptible," Jenks said. "Unlike previously described wax mutants, removal of the WAX2 gene product causes dramatic alteration in the cuticle membrane, and the plant no longer is able to prevent water loss."

Jenks said he believes that when the cuticle membrane structure is changed because of the wax2 malfunction of the WAX2 gene, the protective wax within the cuticle membrane is displaced, affecting transpiration. Transpiration is how plants emit waste matter though their leaf surfaces.

"It's likely that the cuticle meshwork is disrupted so the wax molecules no longer stack properly within the cuticle," he said. "The plant becomes very permeable to water and overall is less able to withstand drought conditions."

The study using the mutant wax2 also revealed unique interactions between the cuticle and other aspects of plant development.

The researchers found that the wax2 mutant has fewer stomata, the small holes in the plant's surface that regulate water loss. This mutant also has a male sterility problem that prevents pollen from activating the stigma, where reproduction begins.

"The cloning of WAX2 is providing evidence that lipids in the cuticle may serve as signals that control how plants develop," Jenks said. "Lipids in animals are known to play important roles in regulating development, but lipid signaling in plants is not well understood."

Lipids are water-insoluble molecules that aid in various cell metabolic functions.

"We want to understand the genetics and biochemistry of plant cuticle production so that ultimately we may be able to modify economically important crops to grow better during drought" he said.

The other authors of the study are postdoctoral student Xinbo Chen, visiting professor Xionglun Liu, and graduate students S. Mark Goodwin and Virginia Boroff, all of the Purdue Department of Horticulture and Landscape Architecture.

The U.S. Department of Agriculture National Research Initiative and Purdue University provided support for the research.

Writer: Susan A. Steeves, (765) 496-7481, ssteeves@aes.purdue.edu

Source: Matthew Jenks, (765) 494-1332, jenks@hort.purdue.edu

Ag Communications: (765) 494-2722; Beth Forbes, bforbes@aes.purdue.edu; http://www.agriculture.purdue.edu/AgComm/public/agnews/

Related Web sites:
Purdue Horticulture
National Science Foundation
USDA 

PHOTO CAPTION
Research conducted at Purdue University by Matt Jenks with Arabidopsis plants may lead to the development of more drought-resistant plants. Jenks is an assistant professor of horticulture. (Purdue Agricultural Communication photo/Tom Campbell)

A publication-quality photograph is available at ftp://ftp.purdue.edu/pub/uns/jenks.wax2.jpeg.

ABSTRACT

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

Insertional mutagenesis of Arabidopsis ecotype C24 was used to identify a novel mutant, designated wax2, that had alterations in both cuticle membrane and cuticular waxes. Arabidopsis mutants with altered cuticle membrane have not been reported previously. Compared with the wild type, the cuticle membrane of wax2 stems weighed 20.2 percent less, and when viewed using electron microscopy, it was 36.4 percent thicker, less opaque, and structurally disorganized. The total wax amount on wax2 leaves and stems was reduced by 78 percent and showed proportional deficiencies in the aldehydes, alkanes, secondary alcohols, and ketones, with increased acids, primary alcohols, and esters. Besides altered cuticle membranes, wax2 displayed postgenital fusion between aerial organs (especially in flower buds), reduced fertility under low humidity, increased epidermal permeability, and a reduction in stomatal index on adaxial and abaxial leaf surfaces. Thus, wax2 reveals a potential role for the cuticle as a suppressor of postgenital fusion and epidermal diffusion and as a mediator of both fertility and the development of epidermal architecture (via effects on stomatal index). The cloned WAX2 gene (verified by three independent allelic insertion mutants with identical phenotypes) codes for a predicted 632—amino acid integral membrane protein with a molecular mass of 72.3 kD and a theoretical pI of 8.78. WAX2 has six transmembrane domains, a His-rich diiron binding region at the N-terminal region, and a large soluble C-terminal domain. The N-terminal portion of WAX2 is homologous with members of the sterol desaturase family, whereas the C terminus of WAX2 is most similar to members of the short-chain dehydrogenase/reductase family. WAX2 has 32 percent identity to CER1, a protein required for wax production, but not for cuticle membrane production. Based on these analyses, we predict that WAX2 has a metabolic function associated with both cuticle membrane and wax synthesis. These studies provide new insight into the genetics and biochemistry of plant cuticle production and elucidate new associations between the cuticle and diverse aspects of plant development.

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