Molecular and Genetic Evidence for the Key Role of AtCaM3 in Heat-Shock Signal Transduction in Arabidopsis

The cuticle covering every plant aerial organ is largely made of cutin that consists of fatty acids, glycerol, and aromatic monomers. Despite the huge importance of the cuticle to plant development and fitness, our knowledge regarding the assembly of the cutin polymer and its integration in the complete cuticle structure is limited. Cutin composition implies the action of acyltransferase-type enzymes that mediate polymer construction through ester bond formation. Here, we show that a member of the BAHD family of acyltransferases (DEFECTIVE IN CUTICULAR RIDGES DCR]) is required for incorporation of the most abundant monomer into the polymeric structure of the Arabidopsis (Arabidopsis thaliana) flower cutin. DCR-deficient plants display phenotypes that are typically associated with a defective cuticle, including altered epidermal cell differentiation and postgenital organ fusion. Moreover, levels of the major cutin monomer in flowers, 9(10),16-dihydroxy-hexadecanoic acid, decreased to an almost undetectable amount in the mutants. Interestingly, dcr mutants exhibit changes in the decoration of petal conical cells and mucilage extrusion in the seed coat, both phenotypes formerly not associated with cutin polymer assembly. Excessive root branching displayed by dcr mutants and the DCR expression pattern in roots pointed to the function of DCR belowground, in shaping root architecture by influencing lateral root emergence and growth. In addition, the dcr mutants were more susceptible to salinity, osmotic, and water deprivation stress conditions. Finally, the analysis of DCR protein localization suggested that cutin polymerization, possibly the oligomerization step, is partially carried out in the cytoplasmic space. Therefore, this study extends our knowledge regarding the functionality of the cuticular layer and the formation of its major constituent the polymer cutin.One of the most crucial adaptations of plants to the terrestrial environment 450 million years ago was the formation of their surface, the cuticle. The cuticular layer, which is covalently attached to the cell wall, plays multiple roles in the plant interaction with its surroundings, including the regulation of epidermal permeability and nonstomatal water loss (Sieber et al., 2000). It is also recognized to be vital for plant growth and development, for example through mediating the prevention or promotion of postgenital organ fusion and the interaction between the pollen and the pistil (Lolle et al., 1998).The major component of the cuticle is cutin, a polyester insoluble in organic solvents, consisting of aliphatics (C₁₆ and C₁₈ fatty acids), aromatics (mainly ferulic and coumaric acids), and glycerol, which are likely linked by the action of different acyltransferases. Cutin insolubility could be explained either by covalent linkage to the cell wall or by cross-linking within its aliphatic domain (Pollard et al., 2008). Recently, α,ω-dicarboxylic and in-chain hydroxy fatty acids have been reported as the characteristic monomers of cutin in Arabidopsis (Arabidopsis thaliana; Bonaventure et al., 2004; Franke et al., 2005). Cutin polymerization possibly involves the formation of an oligomeric building block for lipid polyesters composed of the three components mentioned above. Oligomerization putatively occurs within the epidermal cells, and the oligomers are further relocated with the aid of ATP-binding cassette (ABC) transporters to the extracellular matrix, where the polymerization itself might occur (Pollard et al., 2008). The recently identified GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE4 (GPAT4) and GPAT8 are likely involved in oligomer formation through CoA-activated aliphatic fatty acid attachment to glycerol-3-phosphate (Li et al., 2007). However, GPATs represent only one component of the more complex machinery required for cutin oligomer and polymer formation.Recently, lipase-type enzymes have been proposed to be involved in the polymerization step that occurs in the apoplastic space of the epidermal cell extracellular matrix. The BODYGUARD (BDG) gene encodes a member of the α/β-hydrolase fold protein and is polarly localized in the outer cell walls of the Arabidopsis epidermal cells. It was suggested that BDG is involved in the completion of the apoplastic polymerization process, although the mechanism of its activity remains unclear (Kurdyukov et al., 2006a). A second gene identified in Agave americana (AgaSGNH) encodes a protein belonging to the SGNH hydrolase superfamily of lipases. Similar to BDG, AgaSGNH is polarly localized in the epidermal cell outer cell wall. It is mostly expressed in the expanding parts of young leaves where cutin biosynthesis is most active. The authors suggested that AgaSGNH is involved in cutin polymer formation through a yet unknown mechanism (Reina et al., 2007).Dicarboxylic fatty acids are the major cutin monomers in leaves and stem tissues of Arabidopsis, representing nearly half of its load. In addition to dicarboxylic acids, leaves and stems of Arabidopsis contain in-chain hydroxy fatty acids, among them 9(10),16-dihydroxy-hexadecanoic acid (up to 15% of total cutin; Nawrath, 2006). 9(10),16-Dihydroxy-hexadecanoic acid is the major cutin monomer of most angiosperms and gymnosperms (Holloway, 1982) and dominates the cutin composition of reproductive organs in many plant species, such as Vicia faba flower petals (Kolattukudy et al., 1974) and fruits of tomato (Solanum lycopersicum; Saladié et al., 2007), cherry (Prunus avium; Peschel et al., 2007), and gooseberry (Ribes uva-crispa; Kolattukudy, 2001). Early studies showed that at least half of secondary and all primary hydroxy groups of polyhydroxy fatty acids are esterified within the cutin polymer (Kolattukudy, 2001; Pollard et al., 2008). Thus, the existence of acyltransferases responsible for the acylation of either the primary or the secondary hydroxy groups of, for example, 9(10),16-dihydroxy-hexadecanoic acid, is anticipated. It is also possible that a second type of acyltransferase could utilize the CoA ester of the acid in order to incorporate it into the cutin polymeric structure.In this study, we show that the DEFECTIVE IN CUTICULAR RIDGES (DCR) gene encoding a putative acyltransferase of the Arabidopsis BAHD family is indispensable for the incorporation of 9(10),16-dihydroxy-hexadecanoic acid into the cutin polymer of reproductive and vegetative tissues. Chemical analysis shows that this acid is the most abundant Arabidopsis flower cutin monomer, representing nearly half of the cutin load. The characterization of DCR highlighted two new functions of the cuticle in decorating petal conical cells and the release of mucilage from the seed coat epidermis cells. The dramatic phenotypes of DCR mutant lines and the susceptibility of the mutant plants to water deprivation, salt, and osmotic stresses emphasize the importance of the intact cuticle in the protection against abiotic stresses. Furthermore, localization experiments of the DCR protein suggest that the process of cutin oligomerization or polymerization might take place in the cytoplasmic space. These findings shed light on cutin oligomer/polymer formation and the cuticle function in organ development.