SummaryThe cuticle plays a critical role in plant survival during extreme drought conditions. There are, however, surprisingly, many gaps in our understanding of cuticle biosynthesis.An Arabidopsis thaliana T-DNA mutant library was screened for mutants with enhanced transpiration using a simple condensation spot method. Five mutants, named cool breath (cb), were isolated.The cb5 mutant was found to be allelic to bodyguard (bdg), which is affected in an a/bhydrolase fold protein important for cuticle structure. The analysis of cuticle components in cb5 (renamed as bdg-6) and another T-DNA mutant allele (bdg-7) revealed no impairment in wax synthesis, but a strong decrease in total cutin monomer load in young leaves and flowers. Root suberin content was also reduced. Overexpression of BDG increased total leaf cutin monomer content nearly four times by affecting preferentially C18 polyunsaturated x-OH fatty acids and dicarboxylic acids. Whole-plant gas exchange analysis showed that bdg-6 had higher cuticular conductance and rate of transpiration; however, plant lines overexpressing BDG resembled the wild-type with regard to these characteristics. This study identifies BDG as an important component of the cutin biosynthesis machinery in Arabidopsis. We also show that, using BDG, cutin can be greatly modified without altering the cuticular water barrier properties and transpiration.

The plant cuticle is a chemically heterogeneous lipophilic layer composed of a cutin polymer matrix and waxes which covers the aerial parts of plants. This layer plays an essential role in the survival of plants by protecting them from desiccation and (a)biotic stresses. Knowledge on the gene networks and mechanisms regulating the synthesis of cuticle components during organ expansion or stress response remains limited however. Here, using five loss-of-function mutants for histone monoubiquitination, we report on the role of two RING E3 ligases, namely HISTONE MONOUBIQUITINATION 1 and 2 (HUB1 and HUB2), in the selective transcriptional activation of four cuticle biosynthesis genes in Arabidopsis thaliana. Microscopy observations showed that in hub1-6 and hub2-2 mutants irregular epidermal cells and disorganized cuticle layers were present in rosette leaves. Water loss measurements on excised rosettes demonstrated that cuticular permeability was significantly increased in the mutants. Chemical analysis of cuticle components revealed that the wax composition was changed and that cutin 16:0 dicarboxylic acid was significantly reduced in all hub mutants. Analysis of transcript levels of selected genes indicated that LACS2, ATT1 and HOTHEAD involved in cutin biosynthesis and CER1 involved in wax biosynthesis were down-regulated in the hub mutants, while the expression of LACERATA, CER3, CER6 and CER10 remained unchanged. Chromatin immunoprecipitation assays further showed that hub mutants are impaired in dynamic changes of histone H2B monoubiquitination at several loci of down-regulated genes. Taken together, these data establish that the regulation of cuticle composition involves chromatin remodeling by H2B monoubiquitination.

Cutin and suberin represent lipophilic polymers forming plant/environment interfaces in leaves and roots. Despite recent progress in Arabidopsis, there is still a lack on information concerning cutin and suberin synthesis, especially in crops. Based on sequence homology, we isolated two cDNA clones of new cytochrome P450s, CYP77A19 and CYP77A20 from potato tubers (Solanum tuberosum). Both enzymes hydroxylated lauric acid (C12:0) on position ω-1 to ω-5. They oxidized fatty acids with chain length ranging from C12 to C18 and catalysed hydroxylation of 16-hydroxypalmitic acid leading to dihydroxypalmitic (DHP) acids, the major C16 cutin and suberin monomers. CYP77A19 also produced epoxides from linoleic acid (C18:2). Exploration of expression pattern in potato by RT-qPCR revealed the presence of transcripts in all tissues tested with the highest expression in the seed compared with leaves. Water stress enhanced their expression level in roots but not in leaves. Application of methyl jasmonate specifically induced CYP77A19 expression. Expression of either gene in the Arabidopsis null mutant cyp77a6-1 defective in flower cutin restored petal cuticular impermeability. Nanoridges were also observed in CYP77A20-expressing lines. However, only very low levels of the major flower cutin monomer 10,16-dihydroxypalmitate and no C18 epoxy monomers were found in the cutin of the complemented lines.

Epoxide hydrolases (EHs) are present in all living organisms. They have been extensively characterized in mammals; however, their biological functions in plants have not been demonstrated. Based on in silico analysis, we identified AtEH1 (At3g05600), a putative Arabidopsis thaliana epoxide hydrolase possibly involved in cutin monomer synthesis. We expressed AtEH1 in yeast and studied its localization in vivo. We also analyzed the composition of cutin from A. thaliana lines in which this gene was knocked out. Incubation of recombinant AtEH1 with epoxy fatty acids confirmed its capacity to hydrolyze epoxides of C18 fatty acids into vicinal diols. Transfection of Nicotiana benthamiana leaves with constructs expressing AtEH1 fused to enhanced green fluorescent protein (EGFP) indicated that AtEH1 is localized in the cytosol. Analysis of cutin monomers in loss-of-function Ateh1-1 and Ateh1-2 mutants showed an accumulation of 18-hydroxy-9,10-epoxyoctadecenoic acid and a concomitant decrease in corresponding vicinal diols in leaf and seed cutin. Compared with wild-type seeds, Ateh1 seeds showed delayed germination under osmotic stress conditions and increased seed coat permeability to tetrazolium red. This work reports a physiological role for a plant EH and identifies AtEH1 as a new member of the complex machinery involved in cutin synthesis.

Cutin and suberin are cell wall‐associated glycerolipid polymers that are specific to plants. Cutin forms the framework of the cuticle sealing the aerial epidermis, while suberin is present in the periderm of barks and underground organs. Suberised walls are also found in the root endodermis. Barriers based on cutin and suberin restrict the transport of water and solutes across cell walls and limit pathogen invasions. Chemical analysis shows that both polymers are polyesters composed mostly of fatty hydroxyacids, diacids and epoxyacids esterified to each other and to glycerol. Suberin, whose best‐known form is cork, usually differs from cutin (which has C16 and C18 fatty acids) by a higher content of C20–C24 aliphatics and aromatics. In the last 10 years, the identification of mutants of Arabidopsis or other model plants affected in cutin and/or suberin content has allowed the construction of a more complete picture of the polyester biosynthesis pathways, which currently include acyltransferases with unique specificities, fatty acid hydroxylases, acyl‐CoA synthetases, fatty acid elongases, fatty acyl‐CoA reductases, feruloyl transferases, ABC transporters and extracellular transacylases. Key Concepts The epidermal cells of plant aerial organs and periderm/endoderm cells synthesise the protective cell wall lipid polymers cutin and suberin respectively. Cutin and suberin are both polyesters containing glycerol and oxygenated fatty acids. Cutin structure is not completely understood and suberin structure remains controversial. Oxygenated fatty acid monomers are produced by fatty acid oxidases of the cytochrome P450 superfamily. Acylation of oxygenated fatty acids to glycerol is catalysed by special glycerol‐3‐phosphate acyltransferases. Cutin acylglycerol building blocks are exported to the cell wall and polymerised by extracellular transacylases. How suberin precursors are assembled is still unknown.

are able to form extraordinarily stable vesicular membranes against a number of chemical, physical and mechanical stressors. In this study, we demonstrated that PLFE can also form free-standing ''planar'' membranes on micro-pores (~100 micrometer) of polydimethylsiloxane (PDMS) thin films embedded in printed circuit board (PCB)-based fluidics. Using electrochemical impedance spectroscopy (EIS), we found that the dielectric properties of PLFE planar membranes suspended on the PDMS films are distinctly different from those obtained from diester lipid and triblock copolymer membranes. In addition to resistance (R) and capacitance (C) that were seen in all the membranes examined, PLFE planar membranes showed an inductance (L) component. Furthermore, PLFE planar membranes displayed a relatively large membrane resistance, suggesting that, among the membranes examined, PLFE planar membrane would be a better matrix for studying channel proteins and transmembrane events. PLFE planar membranes also exhibited a sharp decrease in phase angle with the frequency of the input AC signal at~1 MHz, which could be utilized to develop sensors for monitoring PLFE membrane integrity in fluidics. Since the stability of free-standing planar lipid membranes increases with increasing membrane packing tightness and PLFE lipid membranes are more tightly packed than those made of diester lipids, PLFE free-standing planar membranes are expected to be considerably stable. All these salient features make PLFE planar membranes particularly attractive for model studies of channel proteins and transmembrane events and for high-throughput drug screening. Terrestrial plants regulate environmental interactions via insoluble polymers assembled in their epidermal and/or peridermal cell walls. The plant cuticles with waterproofing and antimicrobial capabilities represent a unique class of biological assemblies composed of ester-linked and insoluble constituents such as biopolyester cutins, lipid waxes, and chemically recalcitrant cutans. Solid-state nuclear magnetic resonance (NMR) offers a powerful technique to probe the structural and dynamical properties of these geochemically important and structurally amorphous biological systems that can also motivate the engineering of water-resistant bioinspired materials from renewable sources. Based on key genes and biosynthetic pathways identified in Arabidopsis thaliana leaf cuticles, our current solid-state NMR study delineates compositional variations and multiple-timescale (ms-ns) dynamics for several genetically tailored and insoluble plant cuticle systems, linking macromolecular organization with protective performance and operational design. 2454-Pos Board B591Injectable Reverse Thermal Gel Biopolymers may Act as an Extracellular Matrix and Cell Vehicle for Cardiac Tissue Engineering Background: Recent investigations demonstrated that tissue engineering represents a promising strategy to repair diseased hearts. We hypothesized that temperature-responsive materials could be developed as extracellular ma...