Neofunctionalization of Toll Signaling in Insects: From Immunity to Dorsoventral Patterning.

Toll signaling plays a crucial role in pathogen defense throughout the animal kingdom. It was discovered, however, for its function in dorsoventral (DV) axis formation in Drosophila. In all other insects studied so far, but not outside the insects, Toll is also required for DV patterning. However, in insects more distantly related to Drosophila, Toll's patterning role is frequently reduced and substituted by an expanded influence of BMP signaling, the pathway implicated in DV axis formation in all major metazoan lineages. This suggests that Toll was integrated into an ancestral BMP-based patterning system at the base of the insects or during insect evolution. The observation that Toll signaling has an immune function in the extraembryonic serosa, an early differentiating tissue of most insect embryos, suggests a scenario of how Toll was co-opted from an ancestral immune function for its new role in axis formation.

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 Heidelberg Screen for Pattern Mutants of Drosophila: A Personal Account.

In large-scale mutagenesis screens performed in 1979-1980 at the EMBL in Heidelberg, we isolated mutations affecting the pattern or structure of the larval cuticle in Drosophila. The 600 mutants we characterized could be assigned to 120 genes and represent the majority of such genes in the genome. These mutants subsequently provided a rich resource for understanding many fundamental developmental processes, such as the transcriptional hierarchies controlling segmentation, the establishment of cell states by signaling pathways, and the differentiation of epithelial cells. Most of the Heidelberg genes are now molecularly known, and many of them are conserved in other animals, including humans. Although the screens were initially driven entirely by curiosity, the mutants now serve as models for many human diseases. In this review, we describe the rationale of the screening procedures and provide a classification of the genes on the basis of their initial phenotypes and the subsequent molecular analyses.

Acquired stress resilience through bacteria-to-nematode interdomain horizontal gene transfer.

Natural selection drives the acquisition of organismal resilience traits to protect against adverse environments. Horizontal gene transfer (HGT) is an important evolutionary mechanism for the acquisition of novel traits, including metazoan acquisitions in immunity, metabolic, and reproduction function via interdomain HGT (iHGT) from bacteria. Here, we report that the nematode gene rml-3 has been acquired by iHGT from bacteria and that it enables exoskeleton resilience and protection against environmental toxins in Caenorhabditis elegans. Phylogenetic analysis reveals that diverse nematode RML-3 proteins form a single monophyletic clade most similar to bacterial enzymes that biosynthesize L-rhamnose, a cell-wall polysaccharide component. C. elegans rml-3 is highly expressed during larval development and upregulated in developing seam cells upon heat stress and during the stress-resistant dauer stage. rml-3 deficiency impairs cuticle integrity, barrier functions, and nematode stress resilience, phenotypes that can be rescued by exogenous L-rhamnose. We propose that interdomain HGT of an ancient bacterial rml-3 homolog has enabled L-rhamnose biosynthesis in nematodes, facilitating cuticle integrity and organismal resilience to environmental stressors during evolution. These findings highlight a remarkable contribution of iHGT on metazoan evolution conferred by the domestication of a bacterial gene.

The Arabidopsis cytochrome P450 enzyme CYP96A4 is involved in the wound-induced biosynthesis of cuticular wax and cutin monomers.

The plant cuticle is composed of cuticular wax and cutin polymers and plays an essential role in plant tolerance to diverse abiotic and biotic stresses. Several stresses, including water deficit and salinity, regulate the synthesis of cuticular wax and cutin monomers. However, the effect of wounding on wax and cutin monomer production and the associated molecular mechanisms remain unclear. In this study, we determined that the accumulation of wax and cutin monomers in Arabidopsis leaves is positively regulated by wounding primarily through the jasmonic acid (JA) signaling pathway. Moreover, we observed that a wound- and JA-responsive gene (CYP96A4) encoding an ER-localized cytochrome P450 enzyme was highly expressed in leaves. Further analyses indicated that wound-induced wax and cutin monomer production was severely inhibited in the cyp96a4 mutant. Furthermore, CYP96A4 interacted with CER1 and CER3, the core enzymes in the alkane-forming pathway associated with wax biosynthesis, and modulated CER3 activity to influence aldehyde production in wax synthesis. In addition, transcripts of MYC2 and JAZ1, key genes in JA signaling pathway, were significantly reduced in cyp96a4 mutant. Collectively, these findings demonstrate that CYP96A4 functions as a cofactor of the alkane synthesis complex or participates in JA signaling pathway that contributes to cuticular wax biosynthesis and cutin monomer formation in response to wounding.

The cuticle proteome of a planktonic crustacean.

The cuticles of arthropods provide an interface between the organism and its environment. Thus, the cuticle's structure influences how the organism responds to and interacts with its surroundings. Here, we used label-free quantification proteomics to provide a proteome of the moulted cuticle of the aquatic crustacean Daphnia magna, which has long been a prominent subject of studies on ecology, evolution, and developmental biology. We detected a total of 278 high-confidence proteins. Using protein sequence domain and functional enrichment analyses, we identified chitin-binding structural proteins and chitin-modifying enzymes as the most abundant protein groups in the cuticle proteome. Structural cuticular protein families showed a similar distribution to those found in other arthropods and indicated proteins responsible for the soft and flexible structure of the Daphnia cuticle. Finally, cuticle protein genes were also clustered as tandem gene arrays in the D. magna genome. The cuticle proteome presented here will be a valuable resource to the Daphnia research community, informing genome annotations and investigations on diverse topics such as the genetic basis of interactions with predators and parasites.

A very long chain fatty acid responsive transcription factor, MYB93, regulates lateral root development in Arabidopsis.

Lateral roots (LRs) are critical to root system architecture development in plants. Although the molecular mechanisms by which auxin regulates LR development have been extensively studied, several additional regulatory systems are hypothesized to be involved. Recently, the regulatory role of very long chain fatty acids (VLCFAs) has been shown in LR development. Our analysis showed that LTPG1 and LTPG2, transporters of VLCFAs, are specifically expressed in the developing LR primordium (LRP), while the number of LRs is reduced in the ltpg1/ltpg2 double mutant. Moreover, late LRP development was hindered when the VLCFA levels were reduced by the VLCFA synthesis enzyme mutant, kcs1-5. However, the details of the regulatory mechanisms of LR development controlled by VLCFAs remain unknown. In this study, we propose a novel method to analyze the LRP development stages with high temporal resolution using a deep neural network and identify a VLCFA-responsive transcription factor, MYB93, via transcriptome analysis of kcs1-5. MYB93 showed a carbon chain length-specific expression response following treatment of VLCFAs. Furthermore, myb93 transcriptome analysis suggested that MYB93 regulated the expression of cell wall organization genes. In addition, we also found that LTPG1 and LTPG2 are involved in LR development through the formation of root cap cuticle, which is different from transcriptional regulation by VLCFAs. Our results suggest that VLCFA is a regulator of LRP development through transcription factor-mediated regulation of gene expression and the transportation of VLCFAs is also involved in LR development through root cap cuticle formation.

A GDSL-motif Esterase/Lipase Affects Wax and Cutin Deposition and Controls Hull-Caryopsis Attachment in Barley.

The cuticle covering aerial organs of land plants is well known to protect against desiccation. Cuticles also play diverse and specialized functions, including organ separation, depending on plant and tissue. Barley shows a distinctive cuticular wax bloom enriched in ß-diketones on leaf sheaths, stem nodes and internodes and inflorescences. Barley also develops a sticky surface on the outer pericarp layer of its grain fruit leading to strongly adhered hulls, 'covered grain', important for embryo protection and seed dispersal. While the transcription factor-encoding gene HvNUDUM (HvNUD) appears essential for adherent hulls, little is understood about how the pericarp cuticle changes during adhesion or whether changes in pericarp cuticles contribute to another phenotype where hulls partially shed, called 'skinning'. To that end, we screened barley lines for hull adhesion defects, focussing on the Eceriferum (= waxless, cer) mutants. Here, we show that the cer-xd allele causes defective wax blooms and compromised hull adhesion, and results from a mutation removing the last 10 amino acids of the GDS(L) [Gly, Asp, Ser, (Leu)]-motif esterase/lipase HvGDSL1. We used severe and moderate HvGDSL1 alleles to show that complete HvGDSL1 function is essential for leaf blade cuticular integrity, wax bloom deposition over inflorescences and leaf sheaths and pericarp cuticular ridge formation. Expression data suggest that HvGDSL1 may regulate hull adhesion independently of HvNUD. We found high conservation of HvGDSL1 among barley germplasm, so variation in HvGDSL1 unlikely leads to grain skinning in cultivated barley. Taken together, we reveal a single locus which controls adaptive cuticular properties across different organs in barley.

The HD-ZIP II gene PaHAT14 increases cuticle deposition by down-regulating ERF gene PaERF105 in Phalaenopsis.

To analyze the gene involved in orchid floral development, a HD-Zip II gene PaHAT14, which specifically and highly expressed in perianth during early flower development was identified from Phalaenopsis. Transgenic Arabidopsis plants expressing 35S::PaHAT14 and 35S::PaHAT14+SRDX (fused with the repressor motif SRDX) exhibited similar altered phenotypes, including small leaves, early flowering, and bending petals with increased cuticle production. This suggests that PaHAT14 acts as a repressor. In contrast, transgenic Arabidopsis plants expressing 35S::PaHAT14+VP16 (fused with the activation domain VP16) exhibited curled leaves, late flowering, and folded petals with decreased cuticle production within hardly opened flowers. Additionally, the expression of the ERF gene DEWAX2, which negatively regulates cuticular wax biosynthesis, was down-regulated in 35S::PaHAT14 and 35S::PaHAT14+SRDX transgenic Arabidopsis, while it was up-regulated in 35S::PaHAT14+VP16 transgenic Arabidopsis. Furthermore, transient overexpression of PaHAT14 in Phalaenopsis petal/sepal increased cuticle deposition due to the down-regulation of PaERF105, a Phalaenopsis DEWAX2 orthologue. On the other hand, transient overexpression of PaERF105 decreased cuticle deposition, whereas cuticle deposition increased and the rate of epidermal water loss was reduced in PaERF105 VIGS Phalaenopsis flowers. Moreover, ectopic expression of PaERF105 not only produced phenotypes similar to those in 35S::PaHAT14+VP16 Arabidopsis but also compensated for the altered phenotypes observed in 35S::PaHAT14 and 35S::PaHAT14+SRDX Arabidopsis. These results suggest that PaHAT14 promotes cuticle deposition by negatively regulating downstream gene PaERF105 in orchid flowers.

Cuticle deposition ceases during strawberry fruit development.

BACKGROUND: Ideally, the barrier properties of a fruit's cuticle persist throughout its development. This presents a challenge for strawberry fruit, with their rapid development and thin cuticles. The objective was to establish the developmental time course of cuticle deposition in strawberry fruit. RESULTS: Fruit mass and surface area increase rapidly, with peak growth rate coinciding with the onset of ripening. On a whole-fruit basis, the masses of cutin and wax increase but on a unit surface-area basis, they decrease. The decrease is associated with marked increases in elastic strain. The expressions of cuticle-associated genes involved in transcriptional regulation (FaSHN1, FaSHN2, FaSHN3), synthesis of cutin (FaLACS2, FaGPAT3) and wax (FaCER1, FaKCS10, FaKCR1), and those involved in transport of cutin monomers and wax constituents (FaABCG11, FaABCG32) decreased until maturity. The only exceptions were FaLACS6 and FaGPAT6 that are presumably involved in cutin synthesis, and FaCER1 involved in wax synthesis. This result was consistent across five strawberry cultivars. Strawberry cutin consists mainly of C16 and C18 monomers, plus minor amounts of C19, C20, C22 and C24 monomers, ω-hydroxy acids, dihydroxy acids, epoxy acids, primary alcohols, carboxylic acids and dicarboxylic acids. The most abundant monomer is 10,16-dihydroxyhexadecanoic acid. Waxes comprise mainly long-chain fatty acids C29 to C46, with smaller amounts of C16 to C28. Wax constituents are carboxylic acids, primary alcohols, alkanes, aldehydes, sterols and esters. CONCLUSION: The downregulation of cuticle deposition during development accounts for the marked cuticular strain, for the associated microcracking, and for their high susceptibility to the disorders of water soaking and cracking.

Decoding drought resilience: a comprehensive exploration of the cotton Eceriferum (CER) gene family and its role in stress adaptation.

BACKGROUND: The cuticular wax serves as a primary barrier that protects plants from environmental stresses. The Eceriferum (CER) gene family is associated with wax production and stress resistance. RESULTS: In a genome-wide identification study, a total of 52 members of the CER family were discovered in four Gossypium species: G. arboreum, G. barbadense, G. raimondii, and G. hirsutum. There were variations in the physicochemical characteristics of the Gossypium CER (GCER) proteins. Evolutionary analysis classified the identified GCERs into five groups, with purifying selection emerging as the primary evolutionary force. Gene structure analysis revealed that the number of conserved motifs ranged from 1 to 15, and the number of exons varied from 3 to 13. Closely related GCERs exhibited similar conserved motifs and gene structures. Analyses of chromosomal positions, selection pressure, and collinearity revealed numerous fragment duplications in the GCER genes. Additionally, nine putative ghr-miRNAs targeting seven G. hirsutum CER (GhCER) genes were identified. Among them, three miRNAs, including ghr-miR394, ghr-miR414d, and ghr-miR414f, targeted GhCER09A, representing the most targeted gene. The prediction of transcription factors (TFs) and the visualization of the regulatory TF network revealed interactions with GhCER genes involving ERF, MYB, Dof, bHLH, and bZIP. Analysis of cis-regulatory elements suggests potential associations between the CER gene family of cotton and responses to abiotic stress, light, and other biological processes. Enrichment analysis demonstrated a robust correlation between GhCER genes and pathways associated with cutin biosynthesis, fatty acid biosynthesis, wax production, and stress response. Localization analysis showed that most GCER proteins are localized in the plasma membrane. Transcriptome and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) expression assessments demonstrated that several GhCER genes, including GhCER15D, GhCER04A, GhCER06A, and GhCER12D, exhibited elevated expression levels in response to water deficiency stress compared to control conditions. The functional identification through virus-induced gene silencing (VIGS) highlighted the pivotal role of the GhCER04A gene in enhancing drought resistance by promoting increased tissue water retention. CONCLUSIONS: This investigation not only provides valuable evidence but also offers novel insights that contribute to a deeper understanding of the roles of GhCER genes in cotton, their role in adaptation to drought and other abiotic stress and their potential applications for cotton improvement.

Parallel evolution of angiosperm-like venation in Peltaspermales: a reinvestigation of Furcula.

Leaf venation is a pivotal trait in the success of vascular plants. Whereas gymnosperms have single or sparsely branched parallel veins, angiosperms developed a hierarchical structure of veins that form a complex reticulum. Its physiological consequences are considered to have enabled angiosperms to dominate terrestrial ecosystems in the Late Cretaceous and Cenozoic. Although a hierarchical-reticulate venation also occurs in some groups of extinct seed plants, it is unclear whether these are stem relatives of angiosperms or have evolved these traits in parallel. Here, we re-examine the morphology of the enigmatic foliage taxon Furcula, a potential early Mesozoic angiosperm relative, and argue that its hierarchical vein network represents convergent evolution (in the Late Triassic) with flowering plants (which developed in the Early Cretaceous) based on details of vein architecture and the absence of angiosperm-like stomata and guard cells. We suggest that its nearest relatives are Peltaspermales similar to Scytophyllum and Vittaephyllum, the latter being a genus that originated during the Late Triassic (Carnian) and shares a hierarchical vein system with Furcula. We further suggest that the evolution of hierarchical venation systems in the early Permian, the Late Triassic, and the Early Cretaceous represent 'natural experiments' that might help resolve the selective pressures enabling this trait to evolve.

Revisiting plant cuticle biophysics.

The plant cuticle is located at the interface of the plant with the environment, thus acting as a protective barrier against biotic and abiotic external stress factors, and regulating water loss. Additionally, it modulates mechanical stresses derived from internal tissues and also from the environment. Recent advances in the understanding of the hydric, mechanical, thermal, and, to a lower extent, optical and electric properties of the cuticle, as well as their phenomenological connections and relationships are reviewed. An equilibrium based on the interaction among the different biophysical properties is essential to ensure plant growth and development. The notable variability reported in cuticle geometry, surface topography, and microchemistry affects the analysis of some biophysical properties of the cuticle. This review aimed to provide an updated view of the plant cuticle, understood as a modification of the cell wall, in order to establish the state-of-the-art biophysics of the plant cuticle, and to serve as an inspiration for future research in the field.

LTP2 hypomorphs show genotype-by-environment interaction in early seedling traits in Arabidopsis thaliana.

Isogenic individuals can display seemingly stochastic phenotypic differences, limiting the accuracy of genotype-to-phenotype predictions. The extent of this phenotypic variation depends in part on genetic background, raising questions about the genes involved in controlling stochastic phenotypic variation. Focusing on early seedling traits in Arabidopsis thaliana, we found that hypomorphs of the cuticle-related gene LIPID TRANSFER PROTEIN 2 (LTP2) greatly increased variation in seedling phenotypes, including hypocotyl length, gravitropism and cuticle permeability. Many ltp2 hypocotyls were significantly shorter than wild-type hypocotyls while others resembled the wild-type. Differences in epidermal properties and gene expression between ltp2 seedlings with long and short hypocotyls suggest a loss of cuticle integrity as the primary determinant of the observed phenotypic variation. We identified environmental conditions that reveal or mask the increased variation in ltp2 hypomorphs and found that increased expression of its closest paralog LTP1 is necessary for ltp2 phenotypes. Our results illustrate how decreased expression of a single gene can generate starkly increased phenotypic variation in isogenic individuals in response to an environmental challenge.

Divergent evolution of the alcohol-forming pathway of wax biosynthesis among bryophytes.

The plant cuticle is a hydrophobic barrier, which seals the epidermal surface of most aboveground organs. While the cuticle biosynthesis of angiosperms has been intensively studied, knowledge about its existence and composition in nonvascular plants is scarce. Here, we identified and characterized homologs of Arabidopsis thaliana fatty acyl-CoA reductase (FAR) ECERIFERUM 4 (AtCER4) and bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase 1 (AtWSD1) in the liverwort Marchantia polymorpha (MpFAR2 and MpWSD1) and the moss Physcomitrium patens (PpFAR2A, PpFAR2B, and PpWSD1). Although bryophyte harbor similar compound classes as described for angiosperm cuticles, their biosynthesis may not be fully conserved between the bryophytes M. polymorpha and P. patens or between these bryophytes and angiosperms. While PpFAR2A and PpFAR2B contribute to the production of primary alcohols in P. patens, loss of MpFAR2 function does not affect the wax profile of M. polymorpha. By contrast, MpWSD1 acts as the major wax ester-producing enzyme in M. polymorpha, whereas mutations of PpWSD1 do not affect the wax ester levels of P. patens. Our results suggest that the biosynthetic enzymes involved in primary alcohol and wax ester formation in land plants have either evolved multiple times independently or undergone pronounced radiation followed by the formation of lineage-specific toolkits.

3-ketoacyl-CoA synthase 19 contributes to the biosynthesis of seed lipids and cuticular wax in Arabidopsis and abiotic stress tolerance.

Very-long-chain fatty acids (VLCFAs) are essential precursors for plant membrane lipids, cuticular waxes, suberin, and storage oils. Integral to the fatty acid elongase (FAE) complex, 3-ketoacyl-CoA synthases (KCSs) function as crucial enzymes in the VLCFA pathway, determining the chain length of VLCFA. This study explores the in-planta role of the KCS19 gene. KCS19 is predominantly expressed in leaves and stem epidermis, sepals, styles, early silique walls, beaks, pedicels, and mature embryos. Localized in the endoplasmic reticulum, KCS19 interacts with other FAE proteins. kcs19 knockout mutants displayed reduced total wax and wax crystals, particularly alkanes, while KCS19 overexpression increased these components and wax crystals. Moreover, the cuticle permeability was higher for the kcs19 mutants compared to the wild type, rendering them more susceptible to drought and salt stress, whereas KCS19 overexpression enhanced drought and salt tolerance. Disrupting KCS19 increased C18 species and decreased C20 and longer species in seed fatty acids, indicating its role in elongating C18 to C20 VLCFAs, potentially up to C24 for seed storage lipids. Collectively, KCS19-mediated VLCFA synthesis is required for cuticular wax biosynthesis and seed storage lipids, impacting plant responses to abiotic stress.

NAC transcription factor SlNOR-like1 plays a dual regulatory role in tomato fruit cuticle formation.

The plant cuticle is an important protective barrier on the plant surface, constructed mainly by polymerized cutin matrix and a complex wax mixture. Although the pathway of plant cuticle biosynthesis has been clarified, knowledge of the transcriptional regulation network underlying fruit cuticle formation remains limited. In the present work, we discovered that tomato fruits of the NAC transcription factor SlNOR-like1 knockout mutants (nor-like1) produced by CRISPR/Cas9 [clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9] displayed reduced cutin deposition and cuticle thickness, with a microcracking phenotype, while wax accumulation was promoted. Further research revealed that SlNOR-like1 promotes cutin deposition by binding to the promoters of glycerol-3-phosphate acyltransferase6 (SlGPAT6; a key gene for cutin monomer formation) and CUTIN DEFICIENT2 (SlCD2; a positive regulator of cutin production) to activate their expression. Meanwhile, SlNOR-like1 inhibits wax accumulation, acting as a transcriptional repressor by targeting wax biosynthesis, and transport-related genes 3-ketoacyl-CoA synthase1 (SlKCS1), ECERIFERUM 1-2 (SlCER1-2), SlWAX2, and glycosylphosphatidylinositol-anchored lipid transfer protein 1-like (SlLTPG1-like). In conclusion, SlNOR-like1 executes a dual regulatory effect on tomato fruit cuticle development. Our results provide a new model for the transcriptional regulation of fruit cuticle formation.

Carotenoid retention during post-harvest storage of Capsicum annuum: the role of the fruit surface structure.

In this study, a chilli pepper (Capsicum annuum) panel for post-harvest carotenoid retention was studied to elucidate underlying mechanisms associated with this commercial trait of interest. Following drying and storage, some lines within the panel had an increase in carotenoids approaching 50% compared with the initial content at the fresh fruit stage. Other lines displayed a 25% loss of carotenoids. The quantitative determination of carotenoid pigments with concurrent cellular analysis indicated that in most cases, pepper fruit with thicker (up to 4-fold) lipid exocarp layers and smooth surfaces exhibit improved carotenoid retention properties. Total cutin monomer content increased in medium/high carotenoid retention fruits and subepidermal cutin deposits were responsible for the difference in exocarp thickness. Cutin biosynthesis and cuticle precursor transport genes were differentially expressed between medium/high and low carotenoid retention genotypes, and this supports the hypothesis that the fruit cuticle can contribute to carotenoid retention. Enzymatic degradation of the cuticle and cell wall suggests that in Capsicum the carotenoids (capsanthin and its esters) are embedded in the lipidic exocarp layer. This was not the case in tomato. Collectively, the data suggest that the fruit cuticle could provide an exploitable resource for the enhancement of fruit quality.

Cuticle Development And The Underlying Transcriptome-Metabolome Associations During Early Seedling Establishment.

The plant cuticle is a complex extracellular lipid barrier that has multiple protective functions. We investigated cuticle deposition by integrating metabolomics and transcriptomics data gathered from six different maize seedling organs of four genotypes, the inbred lines B73 and Mo17, and their reciprocal hybrids. These datasets captured the developmental transition of the seedling from heterotrophic skotomorphogenic growth to autotrophic photomorphogenic growth, which is a transition that is highly vulnerable to environmental stresses. Statistical interrogation of these data reveals that the predominant determinant of cuticle composition is seedling organ type, whereas the seedling genotype has a smaller effect on this phenotype. Gene-to-metabolite associations assessed by integrated statistical analyses identified three gene networks connected with the deposition of different elements of the cuticle: a) cuticular waxes; b) monomers of lipidized cell wall biopolymers, including cutin and suberin; and c) both of these elements. These gene networks reveal three metabolic programs that appear to support cuticle deposition, including processes of chloroplast biogenesis, lipid metabolism, and molecular regulation (e.g., transcription factors, post-translational regulators and phytohormones). This study demonstrates the wider physiological metabolic context that can determine cuticle deposition and lays the groundwork for new targets for modulating properties of this protective barrier.

Combined leaf gas-exchange system for model assessment.

Leaf gas-exchange measurements are useful in assessing plant environmental responses. However, uncertainties in the leaf gas-exchange model potentially limit its application. The main challenge in the model-dependent calculations is to detect violations of assumptions. Here, we developed a system that integrates into one instrument the direct measurement of leaf intercellular CO2 concentration and the standard open-flow (OF) and novel open-diffusion (OD) systems for flux measurement. In the OD system, a gas-permeable membrane between the leaf ambient air and outside air creates CO2 and H2O differentials, rather than the air flow in the OF chamber. We measured hypostomatous and amphistomatous leaves of several species with different photosynthetic capacities [sunflower (Helianthus annuus), grape (Vitis vinifera), lemon (Citrus limon), and cherry (Prunus avium)]. The CO2 and H2O differentials in the OD system strictly depend on the flux measured by the OF system. The lower permeability of the membrane resulted in a larger differential per flux, indicating that the OD system can increase the resolution for a small flux. An analysis of the conductance model along with observations suggested that cuticle and leaf intercellular conductances and the unsaturation of leaf humidity contributed to discrepancies between the direct measurement and standard calculation. The combined system developed here provides an opportunity to address these overlooked concepts in leaf gas exchange.