GENES OF ARABIDOPSIS THALIANA INVOLVED IN WAX METABOLISM ROXANA-IULIANA TEODOR *, AL. YEPHREMOV *

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1 Analele ştiinţifice ale Universităţii Al. I. Cuza Iaşi Tomul LIV, fasc. 2, s.ii a. Biologie vegetală, 2008 GENES OF ARABIDOPSIS THALIANA INVOLVED IN WAX METABOLISM ROXANA-IULIANA TEODOR *, AL. YEPHREMOV * Abstract: The aerial surfaces of land plants are covered with cuticle that acts as a barrier providing protection against water loss, pathogen invasion and other environmental aggressions. Besides physical and chemical barriers, such as a waxy cuticle, plant defense mechanisms also involve a coordinated activation of cellular responses to limit damage. The eceriferum (cer) mutants of Arabidopsis define multiple genes required for various steps in cuticular wax biosynthesis, transport and regulation of lipid-related pathways. Although the basic biochemistry of wax production has been elucidated, very little is known about its regulation and its contribution to the natural immunity. This review presents recently cloned wax biosynthetic genes and discusses regulatory aspects of wax biosynthesis. Key words: wax, Arabidopsis mutants Role of cuticle for plants A distinctive characteristic of all epidermal cell types is the presence of cuticle covering their outer surface as a continuous lipophilic layer, which forms a barrier over the aerial organs of land plants during their primary stages of development [7; 14]. Plant cuticle is formed mainly of cutin, a polyester of hydroxy and hydroxy-epoxy fatty acid derivatives generated from cellular fatty acids [11], embedded in and covered with a complex mixture of highly hydrophobic soluble materials (aliphatic compounds comprised mainly of C24-C34 alkanes, alcohols and ketones) called cuticular waxes, as well as other minor compounds, of an extremely diverse nature [30]. As they are physically very closely associated, it is difficult to distinguish between the relative contribution of the cutin matrix and that of cuticular waxes to the physical properties and the biological roles of the cuticle. However, in biochemical experiments, the cutin and cuticular waxes are usually considered and analyzed separately, due to the soluble properties of the waxes versus the cutin polymer, which remains insoluble. Such a structure gives the cuticle a set of highly protective features and these were studied initially as to limiting nonstomatal water loss and gaseous exchanges, controlling the absorption of lipophilic compounds, and providing mechanical strength and viscoelastic properties [2; 25]. Additionally, the cuticle also functions in normal plant developmental processes, including the prevention of postgenital organ fusion and pollen pistil interactions [17; 28], as well as protecting the plant from biotic and non-biotic environmental stress factors [27]. Pruitt et al. [24] and Sieber et al. [28] have suggested that cuticle permeability also influences cell-to-cell communication by enhancing or attenuating the passage of signal molecules. For example, such signals could be required for organ adhesion, when they * Molecular Plant Genetics, Max Planck Institute for Plant Breeding Research, Carl-von-Linne Weg 10, Cologne, Germany 109

2 would be moving across the cuticle, or for mediating signaling between trichomes and stomata, when moving within the developing epidermis [18; 13]. Arabidopsis mutants offer information on the regulation of cuticle development Mutants with deficient or altered wax coatings have been identified due to their nonglaucous or glossy phenotype. Thus, there are no reports of mutants that lack wax completely, indicating the vital function that waxes play for the normal development of plants. On the other hand, changes in a minor wax component are less likely to lead to a clearly discernable phenotype and therefor have not been reported. Similarly, plants that overproduce waxes are not easily detected by visual screenings. In Arabidopsis, as well as in other species, the mutants that have been identified to be defective in wax and/or cutin formation facilitated the identification of enzymes associated with the cutin and wax pathways. Some of the enzymes catalyzing various steps in the wax pathway have been characterized or their function has been proposed based on the phenotype of the corresponding mutants. The identification of eceriferum (cer) mutant lines described mainly by Koornneef et al. [12] and by McNevin et al. [20] has led to the isolation and characterization of various genes associated with cuticular wax metabolism in Arabidopsis. CER1 intended to encode an aldehyde decarbonylase [1]. Several genes playing a role in the fatty acid elongation pathway that generates very long chain fatty acid (VLCFA) wax precursors have also been characterized. They include the FATTY ACID ELONGATION1 homologs (FAE1) [9], FIDDLEHEAD (FDH) [38; 24], 3-KETOACYL-CoA SYNTHASE [33], CUT1/CER6, and CER60 [21; 5]. CER6 has been suggested to be the key condensing enzyme for wax biosynthesis in Arabidopsis, due to its expression throughout all stages of stem and leaf development, as well as in the inflorescence [8]. CER2 encodes a CoA-dependent acyltransferase, a component of the fatty acid elongase complex, apparently located in the nucleus [37; 14]. The cer2 mutant shows reduced levels of the decarbonylation pathway products and it accumulates C26 and C28 acyl groups, primary alcohols, and wax esters but the precise function of the gene is still unknown. Furthermore, its nuclear localization is very intriguing for a protein of the fatty acid elongase complex. Many of the cer mutants remain still to be characterized and the isolation of their corresponding genes might bring valuable information on the mechanisms of wax metabolism. Several reports have also provided insights into the biosynthesis of cutin monomers in plants. Chen et al. [3] reported the isolation of the WAX2 gene and showed that the protein it encodes for has 32% similarity to CER1 and contains certain regions with homology to sterol desaturases and short-chain dehydrogenases/reductases. It was suggested therefore that WAX2 plays a metabolic role in both wax and cutin synthesis, thus pointing to a link between wax and cutin metabolism. ADHESION OF CALYX EDGES/HOTHEAD (ACE/HTH) is proposed to be an oxidase catalyzing the formation of dioic acids from ω-hydroxy acyl-coas [13; 16]. The Arabidopsis LACERATE (LCR) gene [36] encodes a cytochrome P450; enzyme activity assays using the recombinant LCR protein showed that it could efficiently catalyze the formation of ω-hydroxy fatty acids (ranging from C12 to C18:1). Expression of LCR gene is predominant in inflorescence and 110

3 siliques, as well as in roots and young seedling tissue and it is the first cytochrome P450 ω- hydroxylase for which a mutant has been isolated. Results of microarray analysis conducted in our group (Yephremov et al., unpublished data) on three independent cuticular mutants has revealed that a palmytoil protein thyoesterase (PPT) is almost ten fold up-regulated in three mutants, as compared to wild type. In humans, PPT is a lysosomal long-chain fatty acyl hydrolase that removes fatty acyl groups from modified cysteine residues in proteins, and the defective enzyme causes infantile neuronal ceroid lipofuscinosis, a recessive hereditary neuro-degenerative disorder [34]. In plants, acyl-acyl carrier protein (ACP) thioesterases play an essential role in chain termination during de novo fatty acid synthesis and in the channeling of carbon flux between the lipid biosynthesis pathways [10]. Epidermal differentiation and implicitly cuticle formation is essential for the general development of the whole plant, starting from the very early embryo stage. This fact is supported by the characterization of the abnormal leaf shape 1 (ale1) mutant of Arabidopsis, which shows impaired cuticle formation, adhesion of endosperm and embryo, as well as fusion of cotyledons and leaves. The corresponding ALE1 gene encodes a member of the subtilisin-like serine protease family and it is preferentially expressed during seed development, showing a weak transcript expression in young embryo and a strong one within the endosperm cells closely surrounding the developing embryo [32]. Three aminoacid residues (aspartic acid, histidine and serine) are consistently conserved in the catalytic regions of subtilisin-like serin proteases. In animals, such proteases activate precursors of hormones, growth factors, or receptors involved in the control of various developmental processes, including embryonic patterning and proper epidermal differentiation. Although many members of this family of proteases were reported in plants [29; 26], little is known, with few exceptions, about their precise role. In addition to the developmental factors controling the synthesis of cuticular lipids, environmental signals such as light intensity, photoperiod [19; 35], humidity [31], chilling [22; 23] and seasonal variation [6; 4] have also been shown to ifluence wax biosynthesis. In 1984, Sutter [31] described the dramatic response of wax production to environmental cues, during tissue culture, observing that when the relative humidity is high, wax production is low. When tissue-culture-grown plants are transfered to an environment with less humidity (growth chabinet or greenhouse), production of wax is stimulated and within a rather short period of time of a few days only, the plant synthesizes a complete protective layer of wax. Conclusion The fact that cuticular wax is ubiquitously present is testimony to its essential role in the adaptation of plants to the aerial environment, with all its implications. On the other hand, the fact that environmental cues have an influence upon wax composition and quantity is evidence that wax production is an actively regulated process. An active regulatory netword is indicated also by the high diversity of proteins that have been shown, through the Arabidopsis mutants, to be involved in the process of wax biosynthesis. Although the biosynthesis of plant cuticular components has been studied for over four decades, we still know little about the factors regulating the partitioning of fatty 111

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