Structure - Function Analyses of Cutin and the Cuticle in Arabidopsis
The aerial portions of plants are covered with a continuous extracellular layer of hydrophobic material, the cuticle, that plays an important role in protecting these organisms from water and solute loss, UV irradiation, frost damage, as well as pathogen and insect attack. The cuticle consists of two major components, cutin and wax. Cutin, a polyester of interesterified ω-hydroxylated fatty acids, dicarboxylic acids and glycerol, is the major structural component of the cuticle. Waxes, a complex mixture of hydrophobic material containing very long-chain fatty acids and their derivatives, are deposited within the cuticle and on its surface contributing to the layered cuticular structure (Figure 1).
In order to study the biosynthesis and functions of cutin and the cuticle in Arabidopsis two experimental systems have been developed during the last years:
1) Transgenic Arabidopsis plants expressing a fungal cutinase
In order to discover phenotypes of plants having less cutin in the cuticle transgenic Arabidopsis plants that express and secrete a fungal cutinase into the cell wall and degrade their cutin in vivo have been generated. Cutinase-expressing Arabidopsis plants show an altered ultrastructure (Figure 2) and a higher permeability of the cuticle, resulting in increased solute loss and toxin uptake as well as an increase in herbicide and drought sensitivity. In addition, cutinase-expressing plants exhibit a strong organ fusion phenotype leading to distortions of the growth habit of the plant (Figure 3) (Sieber et al., 2000) .
Another very striking phenotype of cutinase-expressing Arabidopsis plants is their strong resistance to the necrotrophic fungus Botrytis cinerea, while the resistance to many other pathogens is not altered. The development of Botrytis stops after the initial germination of the spores, so that the plants do not develop any symptoms of disease (Figure 4). (Chassot et al., 2007). The resistance mechanism is not yet elucidated in detail. However, an increased amount of antifungal components are present on the surface of the cutinase-expressing plants. A mutant screen for Arabidopsis plants which are strongly resistant to Botrytis lead to isolation of the bre1 (botrytis resistant1) mutant of Arabidopsis (Bessire et al., 2007).
2) Mutants of Arabidopsis that have a strongly permeable cuticle
Arabidopsis plants with a permeable cuticle take up rapidly colorants, such as calcofluor white and toluidine blue. Arabidopsis mutants having a permeable cuticle have been isolated based on the calcofluor stain (Figure 5). Interestingly, all permeable cuticle (pec) mutants showed an increased resistance to Botrytis cinerea. In addition, one of the bre mutants, bre1, had a strongly permeable cuticle. Map-based cloning strategies were undertaken to identify BRE1 and several different PEC genes to elucidate the functions of the corresponding proteins.
The bre1 mutant carries a knockout mutation in the LACS2 gene (LACS=long chain acyl-CoA synthethase), which encodes an enzyme that functions in the activation of fatty acids. The bre1/lacs2 mutant has only 20% of the cutin of wild type in leaves that is not deposited in a distinct structural layer of cuticular proper. The cuticle is therefore strongly permeable. The formation of CoA esters by LACS2 is thus very important step in cutin monomer biosynthesis (Bessire et al., 2007).
The pec1 mutant carries a knockout of an ABC transporter of the Pleiotropic Drug Resistance (PDR) family that is responsible for the mutant phenotype. Only minor changes in the amount and composition of cuticular components can be observed in the leaves of the pec1 mutant although the permeability of the cuticle is significantly increased indicating a potential defect in cuticle crosslinking.
Combined approaches using genetics, biochemistry, cell biology, physiology and molecular biology are undertaken to enrich our understanding of the formation and function of cutin and the cuticle. In the long-term, this knowledge might then lead to the generation of crop plants that survive better in their environment.