The outermost epidermal cell wall is specialized to withstand pathogens and natural stresses, and lipid-based cuticular polymers are the major barrier against incursions. The Arabidopsis thaliana mutant bodyguard (bdg), which exhibits defects characteristic of the loss of cuticle structure not attributable to a lack of typical cutin monomers, unexpectedly accumulates significantly more cell wall-bound lipids and epicuticular waxes than wild-type plants. Pleiotropic effects of the bdg mutation on growth, viability, and cell differentiation are also observed. BDG encodes a member of the a/b-hydrolase fold protein superfamily and is expressed exclusively in epidermal cells. Using Strep-tag epitope-tagged BDG for mutant complementation and immunolocalization, we show that BDG is a polarly localized protein that accumulates in the outermost cell wall in the epidermis. With regard to the appearance and structure of the cuticle, the phenotype conferred by bdg is reminiscent of that of transgenic Arabidopsis plants that express an extracellular fungal cutinase, suggesting that bdg may be incapable of completing the polymerization of carboxylic esters in the cuticular layer of the cell wall or the cuticle proper. We propose that BDG codes for an extracellular synthase responsible for the formation of cuticle. The alternative hypothesis proposes that BDG controls the proliferation/differentiation status of the epidermis via an unknown mechanism.

Although the multilayered structure of the plant cuticle was discovered many years ago, the molecular basis of its formation and the functional relevance of the layers are not understood. Here, we present the permeable cuticle1 (pec1) mutant of Arabidopsis thaliana, which displays features associated with a highly permeable cuticle in several organs. In pec1 flowers, typical cutin monomers, such as v-hydroxylated fatty acids and 10,16-dihydroxypalmitate, are reduced to 40% of wild-type levels and are accompanied by the appearance of lipidic inclusions within the epidermal cell. The cuticular layer of the cell wall, rather than the cuticle proper, is structurally altered in pec1 petals. Therefore, a significant role for the formation of the diffusion barrier in petals can be attributed to this layer. Thus, pec1 defines a new class of mutants. The phenotypes of the pec1 mutant are caused by the knockout of ATP BINDING CASSETTEG32 (ABCG32), an ABC transporter from the PLEIOTROPIC DRUG RESISTANCE family that is localized at the plasma membrane of epidermal cells in a polar manner toward the surface of the organs. Our results suggest that ABCG32 is involved in the formation of the cuticular layer of the cell wall, most likely by exporting particular cutin precursors from the epidermal cell.

SummarySuberin is a hydrophobic polyester found in the cell walls of various plant-environment interfaces, including shoot and root peridermal tissue, and the root hypodermis and endodermis. Suberin deposits form apoplastic barriers that control water and nutrient transport, protect against pathogens and seal wounded tissue. Despite this physiological importance, and the detailed information on the suberin composition of many plants, there is a great gap in our knowledge of the molecular mechanism of suberin biosynthesis, caused in part by a lack of mutants in suberin formation. Here, we report the characterization of daisy, an Arabidopsis mutant that is defective in a fatty acid elongase condensing enzyme. The daisy mutant roots exhibit disturbed growth, and the suberin level is reduced in C 22 and C 24 very long chain fatty acid derivatives, whereas C 16 , C 18 and C 20 derivatives accumulate, compared with wild-type suberin, indicating that DAISY functions as a docosanoic acid synthase. Consistent with a significantly increased level of suberin in the roots of NaCl-stressed plants, DAISY is transcriptionally activated by NaCl application, and also by polyethylene glycol-induced drought stress and wounding. Expression analysis using RT-PCR and promoter-GUS fusions demonstrated a distinct DAISY expression pattern in the root stele, senescing sepals, siliques abscission zones and the chalazamicropyle region of seeds. Together, these results indicate that DAISY is involved in suberin biosynthesis and in the formation of protective layers in these tissues, and in the response to unfavourable environmental conditions.

In plants, extracellular matrix polymers built from polysaccharides and cuticular lipids have structural and protective functions. The cuticle is found to be ten times thinner in Arabidopsis thaliana (L.) Heynh than in many other plants, and there is evidence that it is unusual in having a high content of alpha-,omega-dicarboxylic fatty acids (FAs) in its polyesters. We designated the new organ fusion mutant hth-12 after it appeared to be allelic to adhesion of calyx edges (ace) and hothead (hth), upon molecular cloning of the gene by transposon tagging. This mutant is deficient in its ability to oxidize long-chain omega-hydroxy FAs to omega-oxo FAs, which results in leaf polyesters in decreased alpha-,omega-dicarboxylic FAs and increased omega-hydroxy FAs. These chemical phenotypes lead to disorder of the cuticle membrane structure in hth-12. ACE/HTH is a single-domain protein showing sequence similarity to long-chain FA omega-alcohol dehydrogenases from Candida species, and we hypothesize that it may catalyze the next step after cytochrome P450 FA omega-hydroxylases in the omega-oxidation pathway. We show that ACE/HTH is specifically expressed in epidermal cells. It appears very likely therefore that the changes in the amount of alpha-,omega-dicarboxylic FAs in hth-12 reflect the different composition of cuticular polyesters. The ACE/HTH gene is also expressed in root epidermal cells which do not form a polyester membrane on the exterior surface, thereby making it possible that the end products of the pathway, alpha-,omega-dicarboxylic FAs, are generally required for the cross-linking that ensures the integrity of the outer epidermal cell wall.

It is commonly thought that deep phylogenetic conservation of plant microRNAs (miRNAs) and their targets 1,2 indicates conserved regulatory functions. We show that the blind (bl) mutant of Petunia hybrida 3 and the fistulata (fis) mutant of Antirrhinum majus 4,5 , which have similar homeotic phenotypes, are recessive alleles of two homologous miRNA-encoding genes. The BL and FIS genes control the spatial restriction of homeotic class C genes 6,7 to the inner floral whorls, but their ubiquitous early floral expression patterns are in contradiction with a potential role in patterning C gene expression. We provide genetic evidence for the unexpected function of the MIRFIS and MIRBL genes in the center of the flower and propose a dynamic mechanism underlying their regulatory role. Notably, Arabidopsis thaliana, a more distantly related species, also contains this miRNA module but does not seem to use it to confine early C gene expression to the center of the flower.The spatial partitioning of floral homeotic gene expression is crucial for wild-type flower development. Several transcription factors participate in this control, which aims at transcriptional silencing of the so-called 'C genes' outside their genuine expression domain in the inner two whorls of the flower, where they govern reproductive organ (stamen and carpel) development 6,7 . The functions of orthologous repressor genes, constituting the A function of the floral ABCs 6 , are, in part, comparable between different species 8 , as are some of the cis-acting regulatory regions within the large second intron of their structurally and functionally related target C genes AGAMOUS (AG) in Arabidopsis thaliana 7 , pMADS3 in P. hybrida 9 and PLENA and FARINELLI (PLE and FAR) in A. majus 10 . There are also exceptions to these overall similarities among species. For instance, orthologs of the A. thaliana APETALA2 (AP2) gene have no role in C gene regulation in P. hybrida 11 or A. majus 12 , raising the question of whether other genes fulfill this role. Candidates are the BL gene in P. hybrida and FIS in A. majus, which, when mutated, show markedly similar homeotically converted stamenoid petals in their second floral whorls 4,5 (Fig. 1).By a combination of transposon tagging and map-based cloning strategies, we cloned the BL and FIS genes and found that they encode homologous bona fide miRNAs (miRBL and miRFIS), related in their core sequences to members of the large miR169 family 13,14 (Fig. 2). The bl-1 and fis-1 alleles lie within large genomic deletions and thus represent null alleles; bl-2 and fis-2 are transposon induced and genetically unstable alleles (Fig. 2a). miRNA-encoding genes are relatively small targets for mutation, and therefore, recessive mutants are infrequent; bl and fis thus offer a rare opportunity to study and compare the function of potential orthologs in two plant species. miRNAs control gene expression by recognizing short complementary sequences in their transcripts (miRNA-recognition elements, or MREs), which are then post-transcrip...

Mutations in LACERATA (LCR), FIDDLEHEAD (FDH), and BODYGUARD (BDG) cause a complex developmental syndrome that is consistent with an important role for these Arabidopsis genes in cuticle biogenesis. The genesis of their pleiotropic phenotypes is, however, poorly understood. We provide evidence that neither distorted depositions of cutin, nor deficiencies in the chemical composition of cuticular lipids, account for these features, instead suggesting that the mutants alleviate the functional disorder of the cuticle by reinforcing their defenses. To better understand how plants adapt to these mutations, we performed a genome-wide gene expression analysis. We found that apparent compensatory transcriptional responses in these mutants involve the induction of wax, cutin, cell wall, and defense genes. To gain greater insight into the mechanism by which cuticular mutations trigger this response in the plants, we performed an overlap meta-analysis, which is termed MASTA (MicroArray overlap Search Tool and Analysis), of differentially expressed genes. This suggested that different cell integrity pathways are recruited in cesA cellulose synthase and cuticular mutants. Using MASTA for an in silico suppressor/enhancer screen, we identified SERRATE (SE), which encodes a protein of RNA–processing multi-protein complexes, as a likely enhancer. In confirmation of this notion, the se lcr and se bdg double mutants eradicate severe leaf deformations as well as the organ fusions that are typical of lcr and bdg and other cuticular mutants. Also, lcr does not confer resistance to Botrytis cinerea in a se mutant background. We propose that there is a role for SERRATE-mediated RNA signaling in the cuticle integrity pathway.

GRAMINIFOLIA (GRAM), is supported by enhanced phenotypic defects in sty gram double mutants, for instance in the control of phyllotaxis, floral homeotic functions and organ polarity. Accordingly, the STY and GRAM protein and mRNA expression patterns overlap in emerging lateral organ primordia. STY is expressed in all meristems and later becomes confined to the adaxial domain and (pro)-vascular tissue. This pattern is similar to genes that promote adaxial identity, and, indeed, STY expression follows, although does not control, adaxial fate. We discuss the complex roles of STY and GRAM proteins in reproductive and vegetative development, performed in part in physical association but also independently.

In Arabidopsis, loss of function of the epidermis-specific FDH gene coding for a putative b-ketoacyl-CoA synthase results in ectopic organ fusions in mutants. Corresponding mutants are not available for Antirrhinum majus, however, organ fusions can be induced in both species by chloroacetamide inhibitors of b-ketoacyl-CoA synthases using a chemical genetics approach. We isolated the ortholog of FDH from Antirrhinum majus, the ANTIRRHINUM FIDDLEHEAD (AFI ) gene, and showed that AFI complements fdh when expressed in the epidermis under control of the FDH promoter. Like FDH, the AFI gene exhibits protodermis-and epidermis-specific expression, and its promoter directs the expression of reporter genes to the epidermis in transgenic Antirrhinum and Arabidopsis. We demonstrate down-regulation of the FDH promoter in the epidermis of the ovary septum, thereby supporting the assumption that FDH-like genes may directly facilitate the cell-cell interactions that need to occur during carpel fusion and pollen tube growth. Up-regulation of FDH in the stomium, on the other hand, provides evidence for its possible involvement in cell separation during anther dehiscence. Down-regulation of the FDH and AFI promoters in the septum is observed in transgenic Arabidopsis but not in Antirrhinum plants. This probably reflects differences in the ontogeny of the ovary septum between the two species. We also show that epidermis-specific FDH-like genes may not be able to efficiently elongate fatty acid chains when misexpressed in seeds.