Cutin and Suberin Polyesters

International audienceCutin 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

Histone H2B monoubiquitination is involved in the regulation of cutin and wax composition in Arabidopsis thaliana

International audienceThe 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 downregulated genes. Taken together, these data establish that the regulation of cuticle composition involves chromatin remodeling by H2B monoubiquitination

BODYGUARD is required for the biosynthesis of cutin in Arabidopsis

International audienceThe 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

CYP77A19 and CYP77A20 characterized from Solanum tuberosum oxidize fatty acids in vitro and partially restore the wild phenotype in an Arabidopsis thaliana cutin mutant

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Arabidopsis thaliana EPOXIDE HYDROLASE1 (AtEH1) is a cytosolic epoxide hydrolase involved in the synthesis of poly-hydroxylated cutin monomers

International audienceEpoxide 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, Ateh1seeds 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

Argebios : integrated analysis of T-DNA mutant collections for resistance to biotic stress in rice

National audienceWe created a network, called ARGEBIOS (Agropolis Reverse GEnetics for BlOtic Stress), of four laboratories involved in the area of resistance to biotic stress resistance in rice to collectively analyse compromised phenotypes of T-DNA tagged lines identified by reverse genetics approach. The ARGEBIOS integrated approach aims at identifying novel signalling and effector components involved in broad-spectrum resistance to biotic stress in rice and in specific pathways leading to resistance to bacterial, fungal and viral pathogens. Candidate genes were identified from transcriptome and proteome data generated in the four laboratories and from available data in the literature. Mutant T2 rice lines in candidate genes are analysed for compromised resistance to host and non-host rice pathogens and genotyped to verify the linkage between the identified phenotype and the T-DNA insertion. So far 150 mutant lines were analysed for compromised resistance to the three rice pathogens and non-host resistance to M. grisea a d mutant phenotypes were identified in up to 20% of T-DNA screened lines. C. Brugidou's team is studying the molecular interactions between rice and rice yellow mottle virus (RYMV) to elucidate the mechanisms controlling sensitivity, tolerance and the resistance of rice to the virus. Using a combined transcriptome, proteome and bioinformatic approach, they identified deregulation of host genome expression at the beginning of viral infection process. 550 genes were identified as highly deregulated in Indica and Japonica cultivars and are involved in all functional categories. They are using the ARGEBIOS set-up to functionally validate their implication in the viral infection processes. JB Morel's team is focusing on defining the signalling components involved in durable host resistance to M. grisea strains in both Japonica and Indica subspecies. The screen of T2 lines is based upon a compatible interaction enabling to reveal both EDS (enhanced disease susceptibility) and EDR (enhanced disease resistance) typ of mutants. P. Piffanelli's team interest lies on the mechanisms leading to cross-species non-host resistance to M. grisea strains in rice. A high-throughput inoculation protocol and phenotypic analysis for compromised non-host resistance of rice Nipponbare to M.grisea strains attacking other monocots (e.g. wheat, Digitaria, barley) was set-up at CIRAD to identify signalling components involved in type I and type II cross-species resistance mechanisms. V. Verdier's team focuses on the molecular mechanisms leading to resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo). Screening assays were performed on 30 day-old rice plants by leaf clip inoculation. Symptoms were scored by measuring lesion lengths. Most of the strains induced a susceptible reaction while strain PX0339 induced a moderate resistant reaction (MR) and was selected to screen the T-DNA mutants looking at EDS-type phenotypes. Mutant phenotypes were identified in 13% of T-DNA lines. We are now undergoing high-throughput g notyping of the identified lines following a protocol combining Southern blot and PCR analyses to confirm the linkage between T-DNA and observed phenotypes. The comparative study with fungal, bacterial and viral pathogens will enable to pinpoint shared signalling pathways likely to be potential targets to engineer durable field resistance to rice pathogens