The GPAT4/6/8 clade functions in Arabidopsis root suberization nonredundantly with the GPAT5/7 clade required for suberin lamellae.

Lipid polymers such as cutin and suberin strengthen the diffusion barrier properties of the cell wall in specific cell types and are essential for water relations, mineral nutrition, and stress protection in plants. Land plant-specific glycerol-3-phosphate acyltransferases (GPATs) of different clades are central players in cutin and suberin monomer biosynthesis. Here, we show that the GPAT4/6/8 clade in Arabidopsis thaliana, which is known to mediate cutin formation, is also required for developmentally regulated root suberization, in addition to the established roles of GPAT5/7 in suberization. The GPAT5/7 clade is mainly required for abscisic acid-regulated suberization. In addition, the GPAT5/7 clade is crucial for the formation of the typical lamellated suberin ultrastructure observed by transmission electron microscopy, as distinct amorphous globular polyester structures were deposited in the apoplast of the gpat5 gpat7 double mutant, in contrast to the thinner but still lamellated suberin deposition in the gpat4 gpat6 gpat8 triple mutant. Site-directed mutagenesis revealed that the intrinsic phosphatase activity of GPAT4, GPAT6, and GPAT8, which leads to monoacylglycerol biosynthesis, contributes to suberin formation. GPAT5/7 lack an active phosphatase domain and the amorphous globular polyester structure observed in the gpat5 gpat7 double mutant was partially reverted by treatment with a phosphatase inhibitor or the expression of phosphatase-dead variants of GPAT4/6/8. Thus, GPATs that lack an active phosphatase domain synthetize lysophosphatidic acids that might play a role in the formation of the lamellated structure of suberin. GPATs with active and nonactive phosphatase domains appear to have nonredundant functions and must cooperate to achieve the efficient biosynthesis of correctly structured suberin.

Subtle interplay between trichome development and cuticle formation in plants.

Trichomes and cuticles are key protective epidermal specializations. This review highlights the genetic interplay existing between trichome and cuticle formation in a variety of species. Controlling trichome development, the biosynthesis of trichome-derived specialized metabolites as well as cuticle biosynthesis and deposition can be viewed as different aspects of a common defensive strategy adopted by plants to protect themselves from environmental stresses. Existence of such interplay is based on the mining of published transcriptomic data as well as on phenotypic observations in trichome or cuticle mutants where the morphology of both structures often appear to be concomitantly altered. Given the existence of several trichome developmental pathways depending on the plant species and the types of trichomes, genetic interactions between cuticle formation and trichome development are complex to decipher and not easy to generalize. Based on our review of the literature, a schematic overview of the gene network mediating this transcriptional interplay is presented for two model plant species: Arabidopsis thaliana and Solanum lycopersicum. In addition to fundamental new insights on the regulation of these processes, identifying key transcriptional switches controlling both processes could also facilitate more applied investigations aiming at improving much desired agronomical traits in plants.

The Arabidopsis mature endosperm promotes seedling cuticle formation via release of sulfated peptides.

In Arabidopsis mature seeds, the onset of the embryo-to-seedling transition is nonautonomously controlled, being blocked by endospermic abscisic acid (ABA) release under unfavorable conditions. Whether the mature endosperm governs additional nonautonomous developmental processes during this transition is unknown. Mature embryos have a more permeable cuticle than seedlings, consistent with their endospermic ABA uptake capability. Seedlings acquire their well-sealing cuticles adapted to aerial lifestyle during germination. Endosperm removal prevents seedling cuticle formation, and seed reconstitution by endosperm grafting onto embryos shows that the endosperm promotes seedling cuticle development. Grafting different endosperm and embryo mutant combinations, together with biochemical, microscopy, and mass spectrometry approaches, reveal that the release of tyrosylprotein sulfotransferase (TPST)-sulfated CIF2 and PSY1 peptides from the endosperm promotes seedling cuticle development. Endosperm-deprived embryos produced nonviable seedlings bearing numerous developmental defects, not related to embryo malnutrition, all restored by exogenously provided endosperm. Hence, seedling establishment is nonautonomous, requiring the mature endosperm.

Impaired Cuticle Functionality and Robust Resistance to Botrytis cinerea in Arabidopsis thaliana Plants With Altered Homogalacturonan Integrity Are Dependent on the Class III Peroxidase AtPRX71.

Pectin is a major cell wall component that plays important roles in plant development and response to environmental stresses. Arabidopsis thaliana plants expressing a fungal polygalacturonase (PG plants) that degrades homogalacturonan (HG), a major pectin component, as well as loss-of-function mutants for QUASIMODO2 (QUA2), encoding a putative pectin methyltransferase important for HG biosynthesis, show accumulation of reactive oxygen species (ROS), reduced growth and almost complete resistance to the fungal pathogen Botrytis cinerea. Both PG and qua2 plants show increased expression of the class III peroxidase AtPRX71 that contributes to their elevated ROS levels and reduced growth. In this work, we show that leaves of PG and qua2 plants display greatly increased cuticle permeability. Both increased cuticle permeability and resistance to B. cinerea in qua2 are suppressed by loss of AtPRX71. Increased cuticle permeability in qua2, rather than on defects in cuticle ultrastructure or cutin composition, appears to be dependent on reduced epidermal cell adhesion, which is exacerbated by AtPRX71, and is suppressed by the esmeralda1 mutation, which also reverts the adhesion defect and the resistant phenotype. Increased cuticle permeability, accumulation of ROS, and resistance to B. cinerea are also observed in mutants lacking a functional FERONIA, a receptor-like kinase thought to monitor pectin integrity. In contrast, mutants with defects in other structural components of primary cell wall do not have a defective cuticle and are normally susceptible to the fungus. Our results suggest that disrupted cuticle integrity, mediated by peroxidase-dependent ROS accumulation, plays a major role in the robust resistance to B. cinerea of plants with altered HG integrity.

Suberin plasticity to developmental and exogenous cues is regulated by a set of MYB transcription factors.

Suberin is a hydrophobic biopolymer that can be deposited at the periphery of cells, forming protective barriers against biotic and abiotic stress. In roots, suberin forms lamellae at the periphery of endodermal cells where it plays crucial roles in the control of water and mineral transport. Suberin formation is highly regulated by developmental and environmental cues. However, the mechanisms controlling its spatiotemporal regulation are poorly understood. Here, we show that endodermal suberin is regulated independently by developmental and exogenous signals to fine-tune suberin deposition in roots. We found a set of four MYB transcription factors (MYB41, MYB53, MYB92, and MYB93), each of which is individually regulated by these two signals and is sufficient to promote endodermal suberin. Mutation of these four transcription factors simultaneously through genome editing leads to a dramatic reduction in suberin formation in response to both developmental and environmental signals. Most suberin mutants analyzed at physiological levels are also affected in another endodermal barrier made of lignin (Casparian strips) through a compensatory mechanism. Through the functional analysis of these four MYBs, we generated plants allowing unbiased investigation of endodermal suberin function, without accounting for confounding effects due to Casparian strip defects, and were able to unravel specific roles of suberin in nutrient homeostasis.

ABCG transporters export cutin precursors for the formation of the plant cuticle.

The plant cuticle is deposited on the surface of primary plant organs, such as leaves, fruits, and floral organs, forming a diffusion barrier and protecting the plant against various abiotic and biotic stresses. Cutin, the structural polyester of the plant cuticle, is synthesized in the apoplast. Plasma-membrane-localized ATP-binding cassette (ABC) transporters of the G family have been hypothesized to export cutin precursors. Here, we characterize SlABCG42 of tomato representing an ortholog of AtABCG32 in Arabidopsis. SlABCG42 expression in Arabidopsis complements the cuticular deficiencies of the Arabidopsis pec1/abcg32 mutant. RNAi-dependent downregulation of both tomato genes encoding proteins highly homologous to AtABCG32 (SlABCG36 and SlABCG42) leads to reduced cutin deposition and formation of a thinner cuticle in tomato fruits. By using a tobacco (Nicotiana benthamiana) protoplast system, we show that AtABCG32 and SlABCG42 have an export activity for 10,16-dihydroxy hexadecanoyl-2-glycerol, a cutin precursor in vivo. Interestingly, also free ω-hydroxy hexadecanoic acid as well as hexadecanedioic acid were exported, furthering the research on the identification of cutin precursors in vivo and the respective mechanisms of their integration into the cutin polymer.

GDSL-domain proteins have key roles in suberin polymerization and degradation.

Plant roots acquire nutrients and water while managing interactions with the soil microbiota. The root endodermis provides an extracellular diffusion barrier through a network of lignified cell walls called Casparian strips, supported by subsequent formation of suberin lamellae. Whereas lignification is thought to be irreversible, suberin lamellae display plasticity, which is crucial for root adaptative responses. Although suberin is a major plant polymer, fundamental aspects of its biosynthesis and turnover have remained obscure. Plants shape their root system via lateral root formation, an auxin-induced process requiring local breaking and re-sealing of endodermal lignin and suberin barriers. Here, we show that differentiated endodermal cells have a specific, auxin-mediated transcriptional response dominated by cell wall remodelling genes. We identified two sets of auxin-regulated GDSL lipases. One is required for suberin synthesis, while the other can drive suberin degradation. These enzymes have key roles in suberization, driving root suberin plasticity.

Trafficking Processes and Secretion Pathways Underlying the Formation of Plant Cuticles.

Cuticles are specialized cell wall structures that form at the surface of terrestrial plant organs. They are largely comprised lipidic compounds and are deposited in the apoplast, external to the polysaccharide-rich primary wall, creating a barrier to diffusion of water and solutes, as well as to environmental factors. The predominant cuticle component is cutin, a polyester that is assembled as a complex matrix, within and on the surface of which aliphatic and aromatic wax molecules accumulate, further modifying its properties. To reach the point of cuticle assembly the different acyl lipid-containing components are first exported from the cell across the plasma membrane and then traffic across the polysaccharide wall. The export of cutin precursors and waxes from the cell is known to involve plasma membrane-localized ATP-binding cassette (ABC) transporters; however, other secretion mechanisms may also contribute. Indeed, extracellular vesiculo-tubular structures have recently been reported in Arabidopsis thaliana (Arabidopsis) to be associated with the deposition of suberin, a polyester that is structurally closely related to cutin. Intriguingly, similar membranous structures have been observed in leaves and petals of Arabidopsis, although in lower numbers, but no close association with cutin formation has been identified. The possibility of multiple export mechanisms for cuticular components acting in parallel will be discussed, together with proposals for how cuticle precursors may traverse the polysaccharide cell wall before their assimilation into the cuticle macromolecular architecture.

Plant surface metabolites as potent antifungal agents.

Triunsaturated fatty acids are substrates for the synthesis of the defense hormone jasmonate which plays roles in resistance to numerous fungal pathogens. However, relatively little is known about other potential roles of di-unsaturated and triunsaturated fatty acids in resistance to fungal pathogens - in particular those that can attack plants at the seedling stage. We examined the roles of polyunsaturated fatty acids (PUFAs) in Arabidopsis thaliana during attack by the necrotrophic pathogen, Botrytis cinerea. We found that PUFA-deficient Arabidopsis mutants (fad2-1, fad2-3 and fad3-2 fad7-2 fad8 [fad trip]) displayed an unexpectedly strong resistance to B. cinerea at the cotyledon stage. Preliminary analyses revealed no changes in the expression of defense genes, however cuticle permeability defects were detected in both fad2-1 and fad trip mutants. Analysis of B. cinerea development on the surface of cotyledons revealed arrested hyphal growth on fad2-3 and fad trip mutants and 28% reduction in fungal adhesion on fad2-3 cotyledons. Surface metabolite analysis from the cotyledons of PUFA mutants led to the identification of 7-methylsulfonylheptyl glucosinolate (7MSOHG), which over-accumulated on the plant surface. We linked the appearance of 7MSOHG to defects in cuticle composition and permeability of mutants and show that its appearance correlates with resistance to B. cinerea.

The Root Cap Cuticle: A Cell Wall Structure for Seedling Establishment and Lateral Root Formation.

The root cap surrounding the tip of plant roots is thought to protect the delicate stem cells in the root meristem. We discovered that the first layer of root cap cells is covered by an electron-opaque cell wall modification resembling a plant cuticle. Cuticles are polyester-based protective structures considered exclusive to aerial plant organs. Mutations in cutin biosynthesis genes affect the composition and ultrastructure of this cuticular structure, confirming its cutin-like characteristics. Strikingly, targeted degradation of the root cap cuticle causes a hypersensitivity to abiotic stresses during seedling establishment. Furthermore, lateral root primordia also display a cuticle that, when defective, causes delayed outgrowth and organ deformations, suggesting that it facilitates lateral root emergence. Our results show that the previously unrecognized root cap cuticle protects the root meristem during the critical phase of seedling establishment and promotes the efficient formation of lateral roots.

The ß-Ketoacyl-CoA Synthase HvKCS1, Encoded by Cer-zh, Plays a Key Role in Synthesis of Barley Leaf Wax and Germination of Barley Powdery Mildew.

The cuticle coats the primary aerial surfaces of land plants. It consists of cutin and waxes, which provide protection against desiccation, pathogens and herbivores. Acyl cuticular waxes are synthesized via elongase complexes that extend fatty acyl precursors up to 38 carbons for downstream modification pathways. The leaves of 21 barley eceriferum (cer) mutants appear to have less or no epicuticular wax crystals, making these mutants excellent tools for identifying elongase and modification pathway biosynthetic genes. Positional cloning of the gene mutated in cer-zh identified an elongase component, ß-ketoacyl-CoA synthase (CER-ZH/HvKCS1) that is one of 34 homologous KCSs encoded by the barley genome. The biochemical function of CER-ZH was deduced from wax and cutin analyses and by heterologous expression in yeast. Combined, these experiments revealed that CER-ZH/HvKCS1 has a substrate specificity for C16-C20, especially unsaturated, acyl chains, thus playing a major role in total acyl chain elongation for wax biosynthesis. The contribution of CER-ZH to water barrier properties of the cuticle and its influence on the germination of barley powdery mildew fungus were also assessed.

The Cuticle Mutant eca2 Modifies Plant Defense Responses to Biotrophic and Necrotrophic Pathogens and Herbivory Insects.

We isolated previously several Arabidopsis thaliana mutants with constitutive expression of the early microbe-associated molecular pattern-induced gene ATL2, named eca (expresión constitutiva de ATL2). Here, we further explored the interaction of eca mutants with pest and pathogens. Of all eca mutants, eca2 was more resistant to a fungal pathogen (Botrytis cinerea) and a bacterial pathogen (Pseudomonas syringae) as well as to a generalist herbivorous insect (Spodoptera littoralis). Permeability of the cuticle is increased in eca2; chemical characterization shows that eca2 has a significant reduction of both cuticular wax and cutin. Additionally, we determined that eca2 did not display a similar compensatory transcriptional response, compared with a previously characterized cuticular mutant, and that resistance to B. cinerea is mediated by the priming of the early and late induced defense responses, including salicylic acid- and jasmonic acid-induced genes. These results suggest that ECA2-dependent responses are involved in the nonhost defense mechanism against biotrophic and necrotrophic pathogens and against a generalist insect by modulation and priming of innate immunity and late defense responses. Making eca2 an interesting model to characterize the molecular basis for plant defenses against different biotic interactions and to study the initial events that take place in the cuticle surface of the aerial organs.

The roles of the cuticle in plant development: organ adhesions and beyond.

Cuticles, which are composed of a variety of aliphatic molecules, impregnate epidermal cell walls forming diffusion barriers that cover almost all the aerial surfaces in higher plants. In addition to revealing important roles for cuticles in protecting plants against water loss and other environmental stresses and aggressions, mutants with permeable cuticles show major defects in plant development, such as abnormal organ formation as well as altered seed germination and viability. However, understanding the mechanistic basis for these developmental defects represents a significant challenge due to the pleiotropic nature of phenotypes and the altered physiological status/viability of some mutant backgrounds. Here we discuss both the basis of developmental phenotypes associated with defects in cuticle function and mechanisms underlying developmental processes that implicate cuticle modification. Developmental abnormalities in cuticle mutants originate at early developmental time points, when cuticle composition and properties are very difficult to measure. Nonetheless, we aim to extract principles from existing data in order to pinpoint the key cuticle components and properties required for normal plant development. Based on our analysis, we will highlight several major questions that need to be addressed and technical hurdles that need to be overcome in order to advance our current understanding of the developmental importance of plant cuticles.

Connecting the Molecular Structure of Cutin to Ultrastructure and Physical Properties of the Cuticle in Petals of Arabidopsis.

The plant cuticle is laid down at the cell wall surface of epidermal cells in a wide variety of structures, but the functional significance of this architectural diversity is not yet understood. Here, the structure-function relationship of the petal cuticle of Arabidopsis (Arabidopsis thaliana) was investigated. Applying Fourier transform infrared microspectroscopy, the cutin mutants long-chain acyl-coenzyme A synthetase2 (lacs2), permeable cuticle1 (pec1), cyp77a6, glycerol-3-phosphate acyltransferase6 (gpat6), and defective in cuticular ridges (dcr) were grouped in three separate classes based on quantitative differences in the ν(C=O) and ν(C-H) band vibrations. These were associated mainly with the quantity of 10,16-dihydroxy hexadecanoic acid, a monomer of the cuticle polyester, cutin. These spectral features were linked to three different types of cuticle organization: a normal cuticle with nanoridges (lacs2 and pec1 mutants); a broad translucent cuticle (cyp77a6 and dcr mutants); and an electron-opaque multilayered cuticle (gpat6 mutant). The latter two types did not have typical nanoridges. Transmission electron microscopy revealed considerable variations in cuticle thickness in the dcr mutant. Different double mutant combinations showed that a low amount of C16 monomers in cutin leads to the appearance of an electron-translucent layer adjacent to the cuticle proper, which is independent of DCR action. We concluded that DCR is not only essential for incorporating 10,16-dihydroxy C16:0 into cutin but also plays a crucial role in the organization of the cuticle, independent of cutin composition. Further characterization of the mutant petals suggested that nanoridge formation and conical cell shape may contribute to the reduction of physical adhesion forces between petals and other floral organs during floral development.

Cuticular Defects in Oryza sativa ATP-binding Cassette Transporter G31 Mutant Plants Cause Dwarfism, Elevated Defense Responses and Pathogen Resistance.

The cuticle covers the surface of the polysaccharide cell wall of leaf epidermal cells and forms an essential diffusion barrier between plant and environment. Homologs of the ATP-binding cassette (ABC) transporter AtABCG32/HvABCG31 clade are necessary for the formation of a functional cuticle in both monocots and dicots. Here we characterize the osabcg31 knockout mutant and hairpin RNA interference (RNAi)-down-regulated OsABCG31 plant lines having reduced plant growth and a permeable cuticle. The reduced content of cutin in leaves and structural alterations in the cuticle and at the cuticle-cell wall interface in plants compromised in OsABCG31 expression explain the cuticle permeability. Effects of modifications of the cuticle on plant-microbe interactions were evaluated. The cuticular alterations in OsABCG31-compromised plants did not cause deficiencies in germination of the spores or the formation of appressoria of Magnaporthe oryzae on the leaf surface, but a strong reduction of infection structures inside the plant. Genes involved in pathogen resistance were constitutively up-regulated in OsABCG31-compromised plants, thus being a possible cause of the resistance to M. oryzae and the dwarf growth phenotype. The findings show that in rice an abnormal cuticle formation may affect the signaling of plant growth and defense.

Adaptation of Root Function by Nutrient-Induced Plasticity of Endodermal Differentiation.

Plant roots forage the soil for minerals whose concentrations can be orders of magnitude away from those required for plant cell function. Selective uptake in multicellular organisms critically requires epithelia with extracellular diffusion barriers. In plants, such a barrier is provided by the endodermis and its Casparian strips--cell wall impregnations analogous to animal tight and adherens junctions. Interestingly, the endodermis undergoes secondary differentiation, becoming coated with hydrophobic suberin, presumably switching from an actively absorbing to a protective epithelium. Here, we show that suberization responds to a wide range of nutrient stresses, mediated by the stress hormones abscisic acid and ethylene. We reveal a striking ability of the root to not only regulate synthesis of suberin, but also selectively degrade it in response to ethylene. Finally, we demonstrate that changes in suberization constitute physiologically relevant, adaptive responses, pointing to a pivotal role of the endodermal membrane in nutrient homeostasis.

The ABCG transporter PEC1/ABCG32 is required for the formation of the developing leaf cuticle in Arabidopsis.

The cuticle is an essential diffusion barrier on aerial surfaces of land plants whose structural component is the polyester cutin. The PERMEABLE CUTICLE1/ABCG32 (PEC1) transporter is involved in plant cuticle formation in Arabidopsis. The gpat6 pec1 and gpat4 gapt8 pec1 double and triple mutants are characterized. Their PEC1-specific contributions to aliphatic cutin composition and cuticle formation during plant development are revealed by gas chromatography/mass spectrometry and Fourier-transform infrared spectroscopy. The composition of cutin changes during rosette leaf expansion in Arabidopsis. C16:0 monomers are in higher abundance in expanding than in fully expanded leaves. The atypical cutin monomer C18:2 dicarboxylic acid is more prominent in fully expanded leaves. Findings point to differences in the regulation of several pathways of cutin precursor synthesis. PEC1 plays an essential role during expansion of the rosette leaf cuticle. The reduction of C16 monomers in the pec1 mutant during leaf expansion is unlikely to cause permeability of the leaf cuticle because the gpat6 mutant with even fewer C16:0 monomers forms a functional rosette leaf cuticle at all stages of development. PEC1/ABCG32 transport activity affects cutin composition and cuticle structure in a specific and non-redundant fashion.

Characterization and genetic mapping of eceriferum-ym (cer-ym), a cutin deficient barley mutant with impaired leaf water retention capacity.

The cuticle covers the aerial parts of land plants, where it serves many important functions, including water retention. Here, a recessive cuticle mutant, eceriferum-ym (cer-ym), of Hordeum vulgare L. (barley) showed abnormally glossy spikes, sheaths, and leaves. The cer-ym mutant plant detached from its root system was hypersensitive to desiccation treatment compared with wild type plants, and detached leaves of mutant lost 41.8% of their initial weight after 1 h of dehydration under laboratory conditions, while that of the wild type plants lost only 7.1%. Stomata function was not affected by the mutation, but the mutant leaves showed increased cuticular permeability to water, suggesting a defective leaf cuticle, which was confirmed by toluidine blue staining. The mutant leaves showed a substantial reduction in the amounts of the major cutin monomers and a slight increase in the main wax component, suggesting that the enhanced cuticle permeability was a consequence of cutin deficiency. cer-ym was mapped within a 0.8 cM interval between EST marker AK370363 and AK251484, a pericentromeric region on chromosome 4H. The results indicate that the desiccation sensitivity of cer-ym is caused by a defect in leaf cutin, and that cer-ym is located in a chromosome 4H pericentromeric region.

Acetylation of cell wall is required for structural integrity of the leaf surface and exerts a global impact on plant stress responses.

The epidermis on leaves protects plants from pathogen invasion and provides a waterproof barrier. It consists of a layer of cells that is surrounded by thick cell walls, which are partially impregnated by highly hydrophobic cuticular components. We show that the Arabidopsis T-DNA insertion mutants of REDUCED WALL ACETYLATION 2 (rwa2), previously identified as having reduced O-acetylation of both pectins and hemicelluloses, exhibit pleiotrophic phenotype on the leaf surface. The cuticle layer appeared diffused and was significantly thicker and underneath cell wall layer was interspersed with electron-dense deposits. A large number of trichomes were collapsed and surface permeability of the leaves was enhanced in rwa2 as compared to the wild type. A massive reprogramming of the transcriptome was observed in rwa2 as compared to the wild type, including a coordinated up-regulation of genes involved in responses to abiotic stress, particularly detoxification of reactive oxygen species and defense against microbial pathogens (e.g., lipid transfer proteins, peroxidases). In accordance, peroxidase activities were found to be elevated in rwa2 as compared to the wild type. These results indicate that cell wall acetylation is essential for maintaining the structural integrity of leaf epidermis, and that reduction of cell wall acetylation leads to global stress responses in Arabidopsis.