Nadia Efremova

The Epidermis-Specific Extracellular BODYGUARD Controls Cuticle Development and Morphogenesis in Arabidopsis

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 α/β-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.

A conserved microRNA module exerts homeotic control over Petunia hybrida and Antirrhinum majus floral organ identity

It is commonly thought that deep phylogenetic conservation of plant microRNAs (miRNAs) and their targets indicates conserved regulatory functions. We show that the blind (bl) mutant of Petunia hybrida and the fistulata (fis) mutant of Antirrhinum majus, 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 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.

Genetic and biochemical evidence for involvement of HOTHEAD in the biosynthesis of long-chain alpha-,omega-dicarboxylic fatty acids and formation of extracellular matrix.

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 α-,ω-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 ω-hydroxy FAs to ω-oxo FAs, which results in leaf polyesters in decreased α-,ω-dicarboxylic FAs and increased ω-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 ω-alcohol dehydrogenases from Candida species, and we hypothesize that it may catalyze the next step after cytochrome P450 FA ω-hydroxylases in the ω-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 α-,ω-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, α-,ω-dicarboxylic FAs, are generally required for the cross-linking that ensures the integrity of the outer epidermal cell wall.

A Member of the PLEIOTROPIC DRUG RESISTANCE Family of ATP Binding Cassette Transporters Is Required for the Formation of a Functional Cuticle in Arabidopsis

This work shows that the full-length ATP binding cassette (ABC) transporter ABCG32 of Arabidopsis thaliana is involved in the formation of the cuticular layer of the epidermal cell wall of petals. It demonstrates that several ABC transporters, most likely with differing substrate specificities, are necessary to build a functional multilayered cuticle. 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 ω-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.

Molecular and genetic interactions between STYLOSA and GRAMINIFOLIA in the control of Antirrhinum vegetative and reproductive development

STYLOSA (STY) in Antirrhinum and LEUNIG (LUG) in Arabidopsis control the spatially correct expression of homeotic functions involved in the control of floral organ identity. We show here that the sty mutant also displays alteration in leaf venation patterns and hypersensitivity towards auxin and polar auxin transport inhibitors, demonstrating that STY has a more general role in plant development. STY and LUG are shown to be orthologues that encode proteins with structural relation to GRO/TUP1-like co-repressors. Using a yeast-based screen we found that STY interacts with several transcription factors, suggesting that STY, like GRO/TUP1, forms complexes in vivo. Proteins of the YABBY family, characterised by containing a partial HMG domain, represent a major group of such interactors. In vivo association of STY with one of the YABBY proteins, 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.

The DAISY gene from Arabidopsis encodes a fatty acid elongase condensing enzyme involved in the biosynthesis of aliphatic suberin in roots and the chalaza-micropyle region of seeds

Suberin 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 chalaza-micropyle 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.

Dissection of the complex phenotype in cuticular mutants of Arabidopsis reveals a role of SERRATE as a mediator.

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.

Misexpression of FATTY ACID ELONGATION1 in the Arabidopsis epidermis induces cell death and suggests a critical role for phospholipase A2 in this process.

Very-long-chain fatty acids (VLCFAs) are important functional components of various lipid classes, including cuticular lipids in the higher plant epidermis and lipid-derived second messengers. Here, we report the characterization of transgenic Arabidopsis thaliana plants that epidermally express FATTY ACID ELONGATION1 (FAE1), the seed-specific β-ketoacyl-CoA synthase (KCS) catalyzing the first rate-limiting step in VLCFA biosynthesis. Misexpression of FAE1 changes the VLCFAs in different classes of lipids but surprisingly does not complement the KCS fiddlehead mutant. FAE1 misexpression plants are similar to the wild type but display an essentially glabrous phenotype, owing to the selective death of trichome cells. This cell death is accompanied by membrane damage, generation of reactive oxygen species, and callose deposition. We found that nuclei of arrested trichome cells in FAE1 misexpression plants cell-autonomously accumulate high levels of DNA damage, including double-strand breaks characteristic of lipoapoptosis. A chemical genetic screen revealed that inhibitors of KCS and phospholipase A2 (PLA2), but not inhibitors of de novo ceramide biosynthesis, rescue trichome cells from death. These results support the functional role of acyl chain length of fatty acids and PLA2 as determinants for programmed cell death, likely involving the exchange of VLCFAs between phospholipids and the acyl-CoA pool.Lipids produced by the epidermis serve a number of protective functions, and also act as messengers which activate plant defense responses. The fatty acid elongases which catalyze the formation of very long-chain fatty acids, may be instrumental in the remodeling of the various classes of epidermal lipids, and they also provide a means with which to further investigate the defense mechanisms. In a recent publication, we reported that the epidermal mis-expression of FATTY ACID ELONGASE1 (FAE1) in the Arabidopsis plant both increased the levels of very long-chain fatty acids in various lipid classes, and unexpectedly induced a cell-type specific cell death program in trichome cells, giving the plants a glabrous appearance. Using these plants as a model system for a fatty acid-induced cell death (lipoapoptosis), and a platform for the chemical genetic screen, we identified trichome death inhibitors in the glycerophospholipid fatty acyl remodeling pathway: phospholipase A2 inhibitors, aristolochic acid and bromoenol lactone, as well as the putative lysophospholipid acyltransferase inhibitor, clofibrate. Herein, and due to space limitations, we will briefly discuss these results and the different ways in which the appearance of increasing chain-length fatty acids is likely to regulate the cellular life-or-death switch. The death receptor hypothesis implies the existence of a bioactive lipid ligand(s), the functionality of which is determined by phosphorylation, acyl chain length and saturation.

Functional conservation and maintenance of expression pattern of FIDDLEHEAD-like genes in Arabidopsis and Antirrhinum

In Arabidopsis, loss of function of the epidermis-specific FDH gene coding for a putative β-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 β-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.

The MADS-domain protein MPF1 of Physalis floridana controls plant architecture, seed development and flowering time

Floral and vegetative development of plants is dependent on the combinatorial action of MADS-domain transcription factors. Members of the STMADS11 subclade, such as MPF1 of Physalis, are abundantly expressed in leaves as well as in floral organs, but their function is not yet clear. Our studies with transgenic Arabidopsis that over-express MPF1 suggest that MPF1 interacts with SOC1 to determine flowering time. However, MPF1 RNAi-mediated knockdown Physalis plants revealed a complex phenotype with changes in flowering time, plant architecture and seed size. Flowering of these plants was delayed by about 20% as compared to wild type. Expression of PFLFY is upregulated in the MPF1 RNAi lines, while PFFT and MPF3 genes are strongly repressed. MPF1 interacts with a subset of MADS-domain factors, namely with PFSOC1 in planta, and with PFSEP3 and PFFUL in yeast, supporting a regulatory role for this protein in flowering. The average size of seeds produced by the transgenic MPF1 RNAi plants is increased almost twofold. The height of these plants is also increased about twofold, but most axillary buds are stunted when compared to controls. Taken together, this suggests that members of the STMADS11 subclade act as positive regulators of flowering but have diverse functions in plant growth.