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The cuticle, a hydrophobic layer of cutin and waxes synthesized by plant epidermal cells, is the major barrier to water loss when stomata are closed. Dissecting the genetic architecture of natural variation for maize (Zea mays L.) leaf cuticular conductance (gc) is important for identifying genes relevant to improving crop productivity in drought-prone environments. To this end, we performed an integrated genome- and transcriptome-wide association studies (GWAS and TWAS) to identify candidate genes putatively regulating variation in leaf gc. Of the 22 plausible candidate genes identified, 4 were predicted to be involved in cuticle precursor biosynthesis and export, 2 in cell wall modification, 9 in intracellular membrane trafficking, and 7 in the regulation of cuticle development. A gene encoding an INCREASED SALT TOLERANCE1-LIKE1 (ISTL1) protein putatively involved in intracellular protein and membrane trafficking was identified in GWAS and TWAS as the strongest candidate causal gene. A set of maize nested near-isogenic lines that harbor the ISTL1 genomic region from eight donor parents were evaluated for gc, confirming the association between gc and ISTL1 in a haplotype-based association analysis. The findings of this study provide insights into the role of regulatory variation in the development of the maize leaf cuticle and will ultimately assist breeders to develop drought-tolerant maize for target environments.

Summary All aerial epidermal cells in land plants are covered by the cuticle, an extracellular hydrophobic layer that provides protection against abiotic and biotic stresses and prevents organ fusion during development.Genetic and morphological analysis of the classic maize adherent1 (ad1) mutant was combined with genome‐wide binding analysis of the maize MYB transcription factor FUSED LEAVES1 (FDL1), coupled with transcriptional profiling of fdl1 mutants.We show that AD1 encodes an epidermally‐expressed 3‐KETOACYL‐CoA SYNTHASE (KCS) belonging to a functionally uncharacterized clade of KCS enzymes involved in cuticular wax biosynthesis. Wax analysis in ad1 mutants indicates that AD1 functions in the formation of very‐long‐chain wax components. We demonstrate that FDL1 directly binds to CCAACC core motifs present in AD1 regulatory regions to activate its expression. Over 2000 additional target genes of FDL1, including many involved in cuticle formation, drought response and cell wall organization, were also identified.Our results identify a regulatory module of cuticle biosynthesis in maize that is conserved across monocots and eudicots, and highlight previously undescribed factors in lipid metabolism, transport and signaling that coordinate organ development and cuticle formation.

Significance Plant cuticles provide barriers to water loss and arose as aquatic plants adapted to the dry terrestrial environment. The cuticle components, waxes and the fatty acid-based polymer cutin, are synthesized in the plant epidermis, exported across the cell wall, and deposited on the plant surface. This study suggests a role for PHYTOCHROME light receptors during cuticle development in leaves of maize and moss, diverse species that are separated by more than 400 million y of land plant evolution. We hypothesize that phytochrome-mediated light signaling contributed to the evolution of cuticles in land plants., Plant cuticles are composed of wax and cutin and evolved in the land plants as a hydrophobic boundary that reduces water loss from the plant epidermis. The expanding maize adult leaf displays a dynamic, proximodistal gradient of cuticle development, from the leaf base to the tip. Laser microdissection RNA Sequencing (LM-RNAseq) was performed along this proximodistal gradient, and complementary network analyses identified potential regulators of cuticle biosynthesis and deposition. A weighted gene coexpression network (WGCN) analysis suggested a previously undescribed function for PHYTOCHROME-mediated light signaling during the regulation of cuticular wax deposition. Genetic analyses reveal that phyB1 phyB2 double mutants of maize exhibit abnormal cuticle composition, supporting the predictions of our coexpression analysis. Reverse genetic analyses also show that phy mutants of the moss Physcomitrella patens exhibit abnormal cuticle composition, suggesting an ancestral role for PHYTOCHROME-mediated, light-stimulated regulation of cuticle development during plant evolution.

Background and AimsPrior work has examined cuticle function, composition and ultrastructure in many plant species, but much remains to be learned about how these features are related. This study aims to elucidate relationships between these features via analysis of cuticle development in adult maize (Zea mays L.) leaves, while also providing the most comprehensive investigation to date of the composition and ultrastructure of adult leaf cuticles in this important crop plant.MethodsWe examined water permeability, wax and cutin composition via gas chromatography, and ultrastructure via transmission electron microscopy, along the developmental gradient of partially expanded adult maize leaves, and analysed the relationships between these features.Key ResultsThe water barrier property of the adult maize leaf cuticle is acquired at the cessation of cell expansion. Wax types and chain lengths accumulate asynchronously over the course of development, while overall wax load does not vary. Cutin begins to accumulate prior to establishment of the water barrier and continues thereafter. Ultrastructurally, pavement cell cuticles consist of an epicuticular layer, and a thin cuticle proper that acquires an inner, osmiophilic layer during development.ConclusionsCuticular waxes of the adult maize leaf are dominated by alkanes and alkyl esters. Unexpectedly, these are localized mainly in the epicuticular layer. Establishment of the water barrier during development coincides with a switch from alkanes to esters as the major wax type, and the emergence of an osmiophilic (likely cutin-rich) layer of the cuticle proper. Thus, alkyl esters and the deposition of the cutin polyester are implicated as key components of the water barrier property of adult maize leaf cuticles.

The hydrophobic cuticle of plant shoots serves as an important interaction interface with the environment. It consists of the lipid polymer cutin, embedded with and covered by waxes, and provides protection against stresses including desiccation, UV radiation, and pathogen attack. Bulliform cells form in longitudinal strips on the adaxial leaf surface, and have been implicated in the leaf rolling response observed in drought‐stressed grass leaves. In this study, we show that bulliform cells of the adult maize leaf epidermis have a specialized cuticle, and we investigate its function along with that of bulliform cells themselves. Bulliform cells displayed increased shrinkage compared to other epidermal cell types during dehydration of the leaf, providing a potential mechanism to facilitate leaf rolling. Analysis of natural variation was used to relate bulliform strip patterning to leaf rolling rate, providing further evidence of a role for bulliform cells in leaf rolling. Bulliform cell cuticles showed a distinct ultrastructure with increased cuticle thickness compared to other leaf epidermal cells. Comparisons of cuticular conductance between adaxial and abaxial leaf surfaces, and between bulliform‐enriched mutants versus wild‐type siblings, showed a correlation between elevated water loss rates and presence or increased density of bulliform cells, suggesting that bulliform cuticles are more water‐permeable. Biochemical analysis revealed altered cutin composition and increased cutin monomer content in bulliform‐enriched tissues. In particular, our findings suggest that an increase in 9,10‐epoxy‐18‐hydroxyoctadecanoic acid content, and a lower proportion of ferulate, are characteristics of bulliform cuticles. We hypothesize that elevated water permeability of the bulliform cell cuticle contributes to the differential shrinkage of these cells during leaf dehydration, thereby facilitating the function of bulliform cells in stress‐induced leaf rolling observed in grasses.

Plant epidermal cuticles are composed of hydrophobic lipids that provide a barrier to non-stomatal water loss, and arose in land plants as an adaptation to the dry terrestrial environment. The expanding maize adult leaf displays a dynamic, proximodistal gradient of cuticle development, from the leaf base to the tip. Recently, our gene co-expression network analyses together with reverse genetic analyses suggested a previously undescribed function for PHYTOCHROME-mediated light signaling during cuticular wax deposition. The present work extends these findings by identifying a role for a specific LIPID TRANSFER PROTEIN (LTP) in cuticle development, and validating it via transgenic experiments in Arabidopsis. Given that LTPs and cuticles both evolved in land plants and are absent from aquatic green algae, we propose that during plant evolution, LTPs arose as one of the innovations of land plants that enabled development of the cuticle.

In land plants all aerial epidermal cells are covered by the cuticle, an extracellular hydrophobic layer. The cuticle represents a primary barrier between cells and the external environment, provides protection against abiotic and biotic stresses, and prevents organ fusion during development. Here we report the cloning and characterization of a classic mutant of maize called adherent1 (ad1), first described a century ago, and we show that AD1 encodes a 3-KETOACYL-CoA SYNTHASE involved in the deposition of cuticular wax on the epidermis of leaves and inflorescences. ad1 mutants show decreased amounts of various wax components as well as a range of organ fusion defects during vegetative and reproductive development. Accordingly, we find that AD1 is strongly expressed in the epidermis of various developing organs where it is directly regulated by the MYB transcription factor FUSED LEAVES1 (FDL1), which in turn controls a series of additional genes involved in cuticle formation. Altogether, our results identify a major pathway of cuticle biosynthesis essential for the development of maize plants, and a key regulatory module that is conserved across monocot and eudicot species.One sentence summaryThe classic maize mutant adherent1, first isolated a century ago, is affected in an enzyme responsible for cuticle formation that is regulated by the MYB transcription factor FUSED LEAVES1.

The cuticle is a hydrophobic layer on the outer surface plant shoots, which serves as an important interaction interface with the environment. It consists of the lipid polymer cutin, embedded with and covered by waxes, and provides protection against stresses including desiccation, UV radiation, and pathogen attack. Bulliform cells form in longitudinal strips on the adaxial leaf surface, and have been implicated in the leaf rolling response observed in drought stressed grass leaves. In this study, we show that bulliform cells of the adult maize leaf epidermis have a specialized cuticle, and we investigate its function along with that of bulliform cells themselves. Analysis of natural variation was used to relate bulliform strip pattering to leaf rolling rate, providing evidence of a role for bulliform cells in leaf rolling. Bulliform cells displayed increased shrinkage compared to other epidermal cell types during dehydration of the leaf, providing a potential mechanism to facilitate leaf rolling. Comparisons of cuticular conductance between adaxial and abaxial leaf surfaces, and between bulliform-enriched mutants vs. wild type siblings, provided evidence that bulliform cells lose water across the cuticle more rapidly than other epidermal cell types. Bulliform cell cuticles have a distinct ultrastructure, and differences in cutin monomer content and composition, compared to other leaf epidermal cells. We hypothesize that this cell type-specific cuticle is more water permeable than the epidermal pavement cell cuticle, facilitating the function of bulliform cells in stress-induced leaf rolling observed in grasses.One sentence summaryBulliform cells in maize have a specialized cuticle, lose more water than other epidermal cell types as the leaf dehydrates, and facilitate leaf rolling upon dehydration.

Plant cuticles are composed of wax and cutin, and evolved in the land plants as a hydrophobic boundary that reduces water loss from the plant epidermis. The expanding maize adult leaf displays a dynamic, proximodistal gradient of cuticle development, from the leaf base to the tip. Laser microdissection RNA Sequencing (LM-RNAseq) was performed along this proximodistal gradient, and complementary network analyses identified potential regulators of cuticle biosynthesis and deposition. Correlations between cuticle development and cell wall biosynthesis processes were identified, as well as evidence of roles for auxin and brassinosteroids. In addition, our network analyses suggested a previously undescribed function for PHYTOCHROME-mediated light signaling during cuticular wax deposition. Genetic analyses reveal that the phyB1 phyB2 double mutant of maize exhibits abnormal cuticle composition, supporting predictions of our coexpression analyses. Reverse genetic analyses also show that phy mutants of the moss Physcomitrella patens exhibit abnormal cuticle composition, suggesting a role for light-stimulated development of cuticular waxes during plant evolution.

Although extensive prior work has characterized cuticle composition, function, ultrastructure and development in many plant species, much remains to be learned about how these features are interrelated. Moreover, very little is known about the adult maize leaf cuticle in spite of its significance for agronomically important traits in this major crop. We analyzed cuticle composition, ultrastructure, and permeability along the developmental gradient of partially expanded adult maize leaves to probe the relationships between these features. The water barrier property is acquired at the cessation of cell expansion. Wax types and chain lengths accumulate asynchronously along the developmental gradient, while overall wax load does not vary. Cutin begins to accumulate prior to establishment of the water barrier and continues thereafter. Ultrastructurally, pavement cell cuticles consist of an epicuticular layer, a thin cuticle proper that acquires an inner, osmiophilic layer during development, and no cuticular layer. Cuticular waxes of the adult maize leaf are dominated by alkanes and wax esters localized mainly in the epicuticular layer. Establishment of the water barrier coincides with a switch from alkanes to esters as the major wax type, and the emergence of an osmiophilic (likely cutin-rich) layer of the cuticle proper.Higlight statementChemical, ultrastructural and functional analysis of cuticle development in partially expanded adult maize leaves revealed important roles for wax esters and an osmiophilic, likely cutin-rich, layer in protection from dehydration.

Suberin is a lipid and phenolic cell wall heteropolymer found in the roots and other organs of all vascular plants. Suberin plays a critical role in plant water relations and in protecting plants from biotic and abiotic stresses. Here we describe a transcription factor, AtMYB41 (At4g28110), that can activate the steps necessary for aliphatic suberin synthesis and deposition of cell wall-associated suberin-like lamellae in both Arabidopsis thaliana and Nicotiana benthamiana. Overexpression of AtMYB41 increased the abundance of suberin biosynthetic gene transcripts by orders of magnitude and resulted in the accumulation of up to 22 times more suberin-type than cutin-type aliphatic monomers in leaves. Overexpression of AtMYB41 also resulted in elevated amounts of monolignols in leaves and an increase in the accumulation of phenylpropanoid and lignin biosynthetic gene transcripts. Surprisingly, ultrastructural data indicated that overexpression led to the formation of suberin-like lamellae in both epidermal and mesophyll cells of leaves. We further implicate AtMYB41 in the production of aliphatic suberin under abiotic stress conditions. These results provide insight into the molecular-genetic mechanisms of the biosynthesis and deposition of a ubiquitous cell wall-associated plant structure and will serve as a basis for discovering the transcriptional network behind one of the most abundant lipid-based polymers in nature.

Remarkable progress on the biology of plant secondary metabolism has recently been realized. The application of advanced biochemistry, molecular, cellular, and genomic methodologies has revealed biological paradigms unique to the biosynthesis of secondary metabolites, including alkaloids, flavonoids, glucosinolates, phenylpropanoids, and terpenoids. The use of model plant systems has facilitated integrative research on the biosynthesis and regulation of each group of natural products. The model legume, Medicago truncatula Gaertn., plays a key role in studies on phenylpropanoid and flavonoid metabolism. Mint ( Mentha × piperita L.) and various conifers are the systems of choice to investigate terpenoid metabolism, whereas members of the mustard family (Brassica spp.) are central to work on glucosinolate pathways. Arabidopsis thaliana (L.) Heynh. is also used to study the biosynthesis of most secondary compounds, except alkaloids. Unlike other categories of secondary metabolites, the many structural types of alkaloids are biosynthetically unrelated. The biology of each group is unique, although common paradigms are also apparent. Opium poppy ( Papaver somniferum L.) produces a large number of benzylisoquinoline alkaloids and has begun to challenge Madigascar periwinkle ( Catharanthus roseus (L.) G. Don), which accumulates monoterpenoid indole alkaloids, as the most versatile model system to study alkaloid metabolism. An overview of recent progress on the biology of plant alkaloid biosynthesis, with a focus on benzylisoquinoline alkaloid pathways in opium poppy and related species, highlights the emergence of opium poppy as an important model system to investigate secondary metabolism.

Endo-β-mannanase cDNAs were cloned and characterized from ripening tomato (Lycopersicon esculentum Mill. cv Trust) fruit, which produces an active enzyme, and from the tomato cv Walter, which produces an inactive enzyme. There is a two-nucleotide deletion in the gene from tomato cv Walter, which results in a frame shift and the deletion of four amino acids at the C terminus of the full-length protein. Other cultivars that produce either active or inactive enzyme show the same absence or presence of the two-nucleotide deletion. The endo-β-mannanase enzyme protein was purified and characterized from ripe fruit to ensure that cDNA codes for the enzyme from fruit. Immunoblot analysis demonstrated that non-ripening mutants, which also fail to exhibit endo-β-mannanase activity, do so because they fail to express the protein. In a two-way genetic cross between tomato cvs Walter and Trust, all F1 progeny from both crosses produced fruit with active enzyme, suggesting that this form is dominant and homozygous in tomato cv Trust. Self-pollination of a plant from the heterozygous F1 generation yielded F2 plants that bear fruit with and without active enzyme at a ratio appropriate to Mendelian genetic segregation of alleles. Heterologous expression of the two endo-β-mannanase genes in Escherichia coliresulted in active enzyme being produced from cultures containing the tomato cv Trust gene and inactive enzyme being produced from those containing the tomato cv Walter gene. Site-directed mutagenesis was used to establish key elements in the C terminus of the endo-β-mannanase protein that are essential for full enzyme activity.

The accuracy of the sensitive gel-diffusion assay for endo-beta-mannanase activity was improved when protein was added to fruit extracts or into the substrate-gel matrix in which the enzyme assays were conducted. Mixing of commercially available protease inhibitors with fruit enzyme extracts also resulted in increased assayable activity. These treatments were less effective when applied to extracts from tomato seeds, which contained over three times more endogenous protein than fruit extracts. Thus the presence of added or higher amounts of endogenous proteins served as the protectant for endo-beta-mannanase during the course of the gel-diffusion assay, which required an incubation at 32 degrees C for at least 18 h. There was no difference in assayable endo-beta-mannanase activity in the presence and absence of added protein when measured rapidly by viscometry. An effective modification was made to the galactomannan substrate gel assay for endo-beta-mannanase, which is the most efficient method for assaying large numbers of extracts, to improve its accuracy when the enzyme is obtained from tissues containing a low endogenous protein content. This involved incorporating an optimal concentration of gelatin into the galactomannan assay matrix gel. Much higher enzyme activities were recorded, with up to a 10-fold increase for tomato fruit extracts, compared to the same samples assayed on gels with no gelatin added. This increased activity was also obtained using extracts from the fruit of cantaloupe, peach, and nectarine. When incorporated into esterified pectin substrate gels, gelatin also increased the assayable activity of pectin methylesterase. Thus the incorporation of protein (gelatin) into substrate gels during the assay also should be widely more useful for other cell-wall-mobilizing enzymes and hydrolases.

High amounts of endo-β-mannanase (EC 3.2.1.78) activity were extracted from tomato (Lycopersicon esculentum Mill.) fruits when a high-salt-containing buffer was used. Two pI forms of the fruit enzyme were identified, one being much more basic than the many seed isoforms. The number of isoforms increased if a protease inhibitor was not used during extraction. The enzyme was found in the ripe fruits of many other species, and was particularly active in those of muskmelon ( Cucumis melo L. Cantalupensis Group) and watermelon (Citrullus lanatus (Thunb.) Matsum. and Nak.). In most fruits, enzyme activity was localized in the skin and the epidermal and subepidermal regions. The enzyme in several fruits had a molecular weight of ≈40,000 and reacted immunologically with the tomato seed endo-β β β β β-mannanase antibody. niques, we have confirmed in this paper that the enzyme is present in tomato fruit, but, unlike that in seeds, requires a high salt buffer for its extraction. Endo- β-mannanase has not been reported to be present in the fruits of other

Fruits of the tomato cultivar Walter undergo normal development to the red-ripe stage but, unlike those of the cultivar Trust, they do not produce any active endo-beta-mannanase. Reasons for this failure to produce the enzyme were sought. The cv. Walter contains genes for endo-beta-mannanase, as shown by Southern blot analysis, and transcripts for the enzyme are present in ripening fruits, as revealed using Northern hybridization. Moreover, the enzyme protein is detectable by Western blots using an endo-beta-mannanase-specific antibody from tomato. In addition, the inactive enzyme is localized appropriately in the wall regions of the outer layers of the fruit (skin and outer pericarp). Mixing inactive fruit extracts of cv. Walter, in excess, with extracts from cv. Trust fruits, which contain active enzyme, leads to an increase rather than a reduction in enzyme activity, showing that there are no inhibitors of endo-beta-mannanase in cv. Walter fruits. Similar results were obtained with fruits of the tomato cv. Heinz 1439. In contrast to the situation in fruits, the seeds of both cvs Walter and Heinz 1439 produce active enzyme, especially following germination.

Activity of endo-beta-mannanase increases during ripening of tomato (Lycopersicon esculentum Mill.) fruit of the cultivar Trust. beta-Mannoside mannohydrolase is also present during ripening, but its pattern of activity is different from that of endo-beta-mannanase. The increase in endo-beta-mannanase activity is greatest in the skin, and less in the outer and inner pericarp regions. This enzyme is probably bound to the walls of the outermost cell layers of the fruit during ripening, and it requires a high-salt buffer for effective extraction. The enzyme protein, as detected immunologically on Western blots, is present during the early stages of ripening, before any enzyme activity is detectable. The mRNA for the enzyme is also present at these stages; endo-beta-mannanase may be produced and sequestered in a mature-sized inactive form during early ripening. Most non-ripening mutants of tomato exhibit reduced softening and lower endo-beta-mannanase activity, but a cause-and-effect relationship between the enzyme and ripening is unlikely because some cultivars which ripen normally do not exhibit any endo-beta-mannanase activity in the fruit.

The benzylisoquinoline alkaloids of opium poppy, including the narcotic analgesics morphine and codeine, accumulate in the multinucleate cytoplasm of specialized laticifers that accompany vascular tissues throughout the plant. In mature opium poppy plants, immunofluorescence labeling using specific antibodies showed that four alkaloid biosynthetic enzymes, (S)-norcoclaurine 6-O-methyltransferase (6OMT), (S)-coclaurine N-methyltransferase (CNMT), (S)-3'-hydroxy-N-methylcoclaurine-4'-O-methyltransferase (4'OMT) and salutaridinol-7-O-acetyltransferase (SAT) were restricted to sieve elements of the phloem adjacent or proximal to laticifers. The identity of sieve elements was confirmed by (i) the specific immunogold labeling of the characteristic cytoplasm of this cell type, (ii) the co-localization of a sieve element-specific H(+)-ATPase with all biosynthetic enzymes and (iii) the strict association of sieve plates with immunofluorescent cells. The localization of laticifers was demonstrated antibodies specific to major latex protein (MLP), which is characteristic of this cell type. In situ hybridization using antisense RNA probes for 6OMT, CNMT, 4'OMT and SAT showed that the corresponding gene transcripts were found in the companion cell paired with each sieve element. Seven benzylisoquinoline alkaloid biosynthetic enzymes, (S)-N-methylcoclaurine 3'-hydroxylase (CYP80B1), berberine bridge enzyme, codeinone reductase, 6OMT, CNMT, 4'OMT and SAT were localized by immunofluorescence labeling to the sieve elements in the root and hypocotyl of opium poppy seedlings. The abundance of these enzymes increased rapidly between 1 and 3 days after seed germination. The localization of seven biosynthetic enzymes to the sieve elements provides strong support for the unique, cell type-specific biosynthesis of benzylisoquinoline alkaloids in the opium poppy.

The three-dimensional crystal structure of tomato (Lycopersicon esculentum) beta-mannanase 4a (LeMAN4a) has been determined to 1.5 A resolution. The enzyme adopts the (beta/alpha)(8) fold common to the members of glycohydrolase family GH5. The structure is comparable with those of the homologous Trichoderma reesei and Thermomonospora fusca beta-mannanases: There is a conserved three-stranded beta-sheet located near the N terminus that stacks against the central beta-barrel at the end opposite the active site. Three noncanonical beta-helices surround the active site. Similar helices are found in T. reesei but not T. fusca beta-mannanase. By analogy with other beta-mannanases, the catalytic acid/base residue is E204 and the nucleophile residue is E318. The active site cleft of L. esculentum beta-mannanase most closely resembles that of the T. reesei isozyme. A model of substrate binding in LeMAN4a is proposed in which the mannosyl residue occupying the -1 subsite of the enzyme adopts the (1)S(5) skew-boat conformation.