Cuticle lipids on heteromorphic leaves of Populus euphratica Oliv. growing in riparian habitats differing in available soil moisture

Populus euphratica is an important native tree found in arid regions from North Africa and South Europe to China, and is known to tolerate many forms of environmental stress, including drought. We describe cuticle waxes, cutin and cuticle permeability for the heteromorphic leaves of P. euphratica growing in two riparian habitats that differ in available soil moisture. Scanning electron microscopy revealed variation in epicuticular wax crystallization associated with leaf type and site. P. euphratica leaves are dominated by cuticular wax alkanes, primary‐alcohols and fatty acids. The major cutin monomers were 10,16‐diOH C_16:0 acids. Broad‐ovate leaves (associated with adult phase growth) produced 1.3‐ and 1.6‐fold more waxes, and 2.1‐ and 0.9‐fold more cutin monomers, than lanceolate leaves (associated with juvenile phase growth) at the wetter site and drier site, respectively. The alkane‐synthesis‐associated ECERIFERUM1 (CER1), as well as ABC transporter‐ and elongase‐associated genes, were expressed at much higher levels at the drier than wetter sites, indicating their potential function in elevating leaf cuticle lipids in the dry site conditions. Higher cuticle lipid amounts were closely associated with lower cuticle permeability (both chlorophyll efflux and water loss). Our results implicate cuticle lipids as among the xeromorphic traits associated with P. euphratica adult‐phase broad‐ovate leaves. Results here provide useful information for protecting natural populations of P. euphratica and their associated ecosystems, and shed new light on the functional interaction of cuticle and leaf heterophylly in adaptation to more arid, limited‐moisture environments.     

Developmental Changes in Composition and Morphology of Cuticular Waxes on Leaves and Spikes of Glossy and Glaucous Wheat (Triticum aestivum L.)

The glossy varieties (A14 and Jing 2001) and glaucous varieties (Fanmai 5 and Shanken 99) of wheat (Triticum aestivum L.) were selected for evaluation of developmental changes in the composition and morphology of cuticular waxes on leaves and spikes. The results provide us with two different wax development patterns between leaf and spike. The general accumulation trend of the total wax load on leaf and spike surfaces is first to increase and then decrease during the development growth period, but these changes were caused by different compound classes between leaf and spike. Developmental changes of leaf waxes were mainly the result of variations in composition of alcohols and alkanes. In addition, diketones were the third important contributor to the leaf wax changes in the glaucous varieties. Alkanes and diketones were the two major compound classes that caused the developmental changes of spike waxes. For leaf waxes, β- and OH-β-diketones were first detected in flag leaves from 200-day-old plants, and the amounts of β- and OH-β-diketones were significantly higher in glaucous varieties compared with glossy varieties. In spike waxes, β-diketone existed in all varieties, but OH-β-diketone was detectable only in the glaucous varieties. Unexpectedly, the glaucous variety Fanmai 5 yielded large amounts of OH-β-diketone. There was a significant shift in the chain length distribution of alkanes between early stage leaf and flag leaf. Unlike C₂₈ alcohol being the dominant chain length in leaf waxes, the dominant alcohol chain length of spikes was C₂₄ or C₂₆ depending on varieties. Epicuticular wax crystals on wheat leaf and glume were comprised of platelets and tubules, and the crystal morphology changed constantly throughout plant growth, especially the abaxial leaf crystals. Moreover, our results suggested that platelets and tubules on glume surfaces could be formed rapidly within a few days.     

Overexpression of Arabidopsis ECERIFERUM1 promotes wax very-long-chain alkane biosynthesis and influences plant response to biotic and abiotic stresses

Land plant aerial organs are covered by a hydrophobic layer called the cuticle that serves as a waterproof barrier protecting plants against desiccation, ultraviolet radiation, and pathogens. Cuticle consists of a cutin matrix as well as cuticular waxes in which very-long-chain (VLC) alkanes are the major components, representing up to 70% of the total wax content in Arabidopsis (Arabidopsis thaliana) leaves. However, despite its major involvement in cuticle formation, the alkane-forming pathway is still largely unknown. To address this deficiency, we report here the characterization of the Arabidopsis ECERIFERUM1 (CER1) gene predicted to encode an enzyme involved in alkane biosynthesis. Analysis of CER1 expression showed that CER1 is specifically expressed in the epidermis of aerial organs and coexpressed with other genes of the alkane-forming pathway. Modification of CER1 expression in transgenic plants specifically affects VLC alkane biosynthesis: waxes of TDNA insertional mutant alleles are devoid of VLC alkanes and derivatives, whereas CER1 overexpression dramatically increases the production of the odd-carbon-numbered alkanes together with a substantial accumulation of iso-branched alkanes. We also showed that CER1 expression is induced by osmotic stresses and regulated by abscisic acid. Furthermore, CER1-overexpressing plants showed reduced cuticle permeability together with reduced soil water deficit susceptibility. However, CER1 overexpression increased susceptibility to bacterial and fungal pathogens. Taken together, these results demonstrate that CER1 controls alkane biosynthesis and is highly linked to responses to biotic and abiotic stresses.     

The Inhibitor of wax 1 locus (Iw1) prevents formation of β‐ and OH‐β‐diketones in wheat cuticular waxes and maps to a sub‐cM interval on chromosome arm 2BS

Glaucousness is described as the scattering effect of visible light from wax deposited on the cuticle of plant aerial organs. In wheat, two dominant genes lead to non‐glaucous phenotypes: Inhibitor of wax 1 (Iw1) and Iw2. The molecular mechanisms and the exact extent (beyond visual assessment) by which these genes affect the composition and quantity of cuticular wax is unclear. To describe the Iw1 locus we used a genetic approach with detailed biochemical characterization of wax compounds. Using synteny and a large number of F₂ gametes, Iw1 was fine‐mapped to a sub‐cM genetic interval on wheat chromosome arm 2BS, which includes a single collinear gene from the corresponding Brachypodium and rice physical maps. The major components of flag leaf and peduncle cuticular waxes included primary alcohols, β‐diketones and n‐alkanes. Small amounts of C19–C27 alkyl and methylalkylresorcinols that have not previously been described in wheat waxes were identified. Using six pairs of BC₂F₃ near‐isogenic lines, we show that Iw1 inhibits the formation of β‐ and hydroxy‐β‐diketones in the peduncle and flag leaf blade cuticles. This inhibitory effect is independent of genetic background or tissue, and is accompanied by minor but consistent increases in n‐alkanes and C₂₄ primary alcohols. No differences were found in cuticle thickness and carbon isotope discrimination in near‐isogenic lines differing at Iw1.     

Plant response to drought stress simulated by ABA application: Changes in chemical composition of cuticular waxes

Plant cuticles form the interface between epidermal plant cells and the atmosphere. The cuticle creates an effective barrier against water loss, bacterial and fungal infection and also protects plant tissue from UV radiation. It is composed of the cutin matrix and embedded soluble lipids also called waxes. Chemical composition of cuticular waxes and physiological properties of cuticles are affected by internal regulatory mechanisms and environmental conditions (e.g. drought, light, and humidity). Here, we tested the effect of drought stress simulation by the exogenous application of abscisic acid (ABA) on cuticular wax amount and composition. ABA-treated plants and control plants differed in total aboveground biomass, leaf area, stomatal density and aperture, and carbon isotope composition. They did not differ in total wax amount per area but there were peculiar differences in the abundance of particular components. ABA-treated plants contained significantly higher proportions of aliphatic components characterized by chain length larger than C₂₆, compared to control plants. This trend was consistent both between and within different functional groups of wax components. This can lead to a higher hydrophobicity of the cuticular transpiration barrier and thus decrease cuticular water loss in ABA-treated plants. At both ABA-treated and control plants alcohols with chain length C₂₄ and C₂₆ were predominant. Such a shift towards wax compounds having a higher average chain length under drought conditions can be interpreted as an adaptive response of plants towards drought stress.     

Fruit cuticle lipid composition during development in tomato ripening mutants

Recent studies suggest that fruit cuticle is an important contributing factor to tomato (Solanum lycopersicum) fruit shelf life and storability. Moreover, it has been hypothesized that variation in fruit cuticle composition may underlie differences in traits such as fruit resistance to desiccation and microbial infection. To gain a better understanding of cuticle lipid composition diversity during fruit ontogeny and to assess if there are common features that correlate with ripening, we examined developmental changes in fruit cuticle wax and cutin monomer composition of delayed‐ripening tomato fruit mutants, ripening inhibitor (rin) and non‐ripening (nor) and delayed‐ripening landrace Alcobaça. Previous reports show that fruit ripening processes such as climacteric ethylene production, cell wall degradation and color change are significantly delayed, or do not occur, in these lines. In the study presented here, however, we show that fruits from rin, nor and Alcobaça have cuticle lipid compositions that differ significantly from normal fruits of Ailsa Craig (AC) even at very early stages in fruit development, with continuing impacts throughout ripening. Moreover, rin, nor and the Alcobaça lines show quite different wax profiles from AC and each other throughout fruit development. Although cutin monomer composition differed much less than wax composition among the genotypes, all delayed‐ripening lines possessed higher relative amounts of C₁₈ monomers than AC. Together, these results reveal new genetic associations between cuticle and fruit development processes and define valuable genetic resources to further explore the importance of cuticle in fruit shelf life.     

Overexpression of the ABC transporter gene <Emphasis Type="Italic">TsABCG11</Emphasis> increases cuticle lipids and abiotic stress tolerance in <Emphasis Type="Italic">Arabidopsis</Emphasis>

The cuticle, composed primarily of wax and cutin, covers most plant aerial surfaces and plays a vital role in interactions between plants and their environment. Some ATP-binding cassette G subfamily (ABCG) members are involved in cuticular lipid molecule exportation to outside in the plant surface. Thellungiella salsugineum, a relative of Arabidopsis thaliana with a heavy cuticle, has extreme stress tolerance. TsABCG11, an ABCG transporter was cloned (GenBank accession number JQ389853), and its structure was studied. qRT-PCR showed that TsABCG11 expression varied in different organs of T. salsugineum and was upregulated under ABA, NaCl, drought and cold conditions. The rosette leaves from 4-week-old TsABCG11 overexpressed (OE) Arabidopsis plants displayed lower rates of water loss and decreased chlorophyll-extracted rates compared to wild-type plants. TsABCG11-OE plants also exhibited significantly increased total cuticular wax and cutin monomer amounts, mainly due to prominent changes in the C₂₉, C₃₁, and C₃₃ alkanes in the wax and C18:2 dioic in cutin monomers, respectively. TsABCG11-OE seedlings exhibit lower root growth inhibition under 100 mM of NaCl or 1 µM of ABA than the wild type. Four-week-old TsABCG11-OE plants exhibited higher photosynthetic rates and water-use efficiency under cold stress (4 °C) than control plants. These results indicate that TsABCG11 plays an important role in cuticle lipid exportation and is involved in abiotic stresses, probably having a close relationship with extreme stress tolerance in T. salsugineum.     

Fruit cuticle lipid composition and water loss in a diverse collection of pepper (Capsicum)

Pepper (Capsicum spp.) fruits are covered by a relatively thick coating of cuticle that limits fruit water loss, a trait previously associated with maintenance of postharvest fruit quality during commercial marketing. To shed light on the chemical‐compositional diversity of cuticles in pepper, the fruit cuticles from 50 diverse pepper genotypes from a world collection were screened for both wax and cutin monomer amount and composition. These same genotypes were also screened for fruit water loss rate and this was tested for associations with cuticle composition. Our results revealed an unexpectedly large amount of variation for the fruit cuticle lipids, with a more than 14‐fold range for total wax amounts and a more than 16‐fold range for cutin monomer amounts between the most extreme accessions. Within the major wax constituents fatty acids varied from 1 to 46%, primary alcohols from 2 to 19%, n‐alkanes from 13 to 74% and triterpenoids and sterols from 10 to 77%. Within the cutin monomers, total hexadecanoic acids ranged from 54 to 87%, total octadecanoic acids ranged from 10 to 38% and coumaric acids ranged from 0.2 to 8% of the total. We also observed considerable differences in water loss among the accessions, and unique correlations between water loss and cuticle constituents. The resources described here will be valuable for future studies of the physiological function of fruit cuticle, for the identification of genes and QTLs associated with fruit cuticle synthesis in pepper fruit, and as a starting point for breeding improved fruit quality in pepper.     

ABC-type transporters and cuticle assembly: Linking function to polarity in epidermis cells

The aerial organs of plants are covered with a cuticle, a continuous layer overlaying the outermost cell walls of the epidermis. The cuticle is composed of two major classes of the lipid biopolymers: cutin and waxes, collectively termed cuticular lipids. Biosynthesis and transport of cuticular lipids occur predominantly in the epidermis cells. In the transport pathway, cuticular lipids are exported from their site of biosynthesis in the ER/plastid to the extracellular space through the plasma membrane and cell wall. Growing evidence suggests that ATP-binding cassette (ABC) transporters are implicated in transport of cuticular lipids across the plasma membrane of epidermal cells. The Arabidopsis ABC-type transporter protein CER5 (WBC12) was reported to act as a wax monomers transporter. In recent works, our group and others showed that a CER5-related protein, DESPERADO (DSO/WBC11), is required for cutin and wax monomers transport through the plasma membrane of Arabidopsis epidermis cells. Unlike the cer5 mutant, DSO loss-of-function had a profound effect on plant growth and development, particularly dwarfism, postgenital organ fusions, and altered epidermal cell differentiation. The partially overlapping function of CER5 and DSO and the fact that these proteins are half-size ABC transporters suggest that they might form a hetero-dimeric complex while transporting wax components. An intriguing observation was the polar localization of DSO in the distal part of epidermis cells. This polar expression might be explained by DSO localization within lipid rafts, specific plasma membrane microdomains which are associated with polar protein expression. In this review we suggest possible mechanisms for cuticular lipids transport and a link between DSO function and polar expression. Furthermore, we also discuss the subsequent transport of cuticular constituents through the hydrophobic cell wall and the possible involvement of lipid transfer proteins in this process.Key words: ABC transporter, cuticular lipids, polar expression, plasma membrane, epidermis     

Organ fusion and defective cuticle function in a lacs1 lacs2 double mutant of Arabidopsis

As the outermost layer on aerial tissues of the primary plant body, the cuticle plays important roles in plant development and physiology. The major components of the cuticle are cutin and cuticular wax, both of which are composed primarily of fatty acid derivatives synthesized in the epidermal cells. Long-chain acyl-CoA synthetases (LACS) catalyze the formation of long-chain acyl-CoAs and the Arabidopsis genome contains a family of nine genes shown to encode LACS enzymes. LACS2 is required for cutin biosynthesis, as revealed by previous investigations on lacs2 mutants. Here, we characterize lacs1 mutants of Arabidopsis that reveals a role for LACS1 in biosynthesis of cuticular wax components. lacs1 lacs2 double-mutant plants displayed pleiotropic phenotypes including organ fusion, abnormal flower development and reduced seed set; phenotypes not found in either of the parental mutants. The leaf cuticular permeability of lacs1 lacs2 was higher than that of either lacs1 or lacs2 single mutants, as determined by measurements of chlorophyll leaching from leaves immersed in 80% ethanol, staining with toluidine blue dye and direct measurements of water loss. Furthermore, lacs1 lacs2 mutant plants are highly susceptible to drought stress. Our results indicate that a deficiency in cuticular wax synthesis and a deficiency in cutin synthesis together have compounding effects on the functional integrity of the cuticular barrier, compromising the ability of the cuticle to restrict water movement, protect against drought stress and prevent organ fusion.     

Arabidopsis CER8 encodes LONG-CHAIN ACYL-COA SYNTHETASE 1 (LACS1) that has overlapping functions with LACS2 in plant wax and cutin synthesis

Plant cuticle is an extracellular lipid-based matrix of cutin and waxes, which covers aerial organs and protects them from many forms of environmental stress. We report here the characterization of CER8 / LACS1 , one of nine Arabidopsis long-chain acyl-CoA synthetases thought to activate acyl chains. Mutations in LACS1 reduced the amount of wax in all chemical classes on the stem and leaf, except in the very long-chain fatty acid (VLCFA) class wherein acids longer than 24 carbons (C₂₄) were elevated more than 155%. The C₁₆ cutin monomers on lacs1 were reduced by 37% and 22%, whereas the C₁₈ monomers were increased by 28% and 20% on stem and leaf, respectively. Amounts of wax and cutin on a lacs1-1 lacs2-3 double mutant were much lower than on either parent, and lacs1-1 lacs2-3 had much higher cuticular permeability than either parent. These additive effects indicate that LACS1 and LACS2 have overlapping functions in both wax and cutin synthesis. We demonstrated that LACS1 has synthetase activity for VLCFAs C₂₀–C₃₀, with highest activity for C₃₀ acids. LACS1 thus appears to function as a very long-chain acyl-CoA synthetase in wax metabolism. Since C₁₆ but not C₁₈ cutin monomers are reduced in lacs1 , and C₁₆ acids are the next most preferred acid (behind C₃₀) by LACS1 in our assays, LACS1 also appears to be important for the incorporation of C₁₆ monomers into cutin polyester. As such, LACS1 defines a functionally novel acyl-CoA synthetase that preferentially modifies both VLCFAs for wax synthesis and long-chain (C₁₆) fatty acids for cutin synthesis.     

The role of ABC transporters in cuticular lipid secretion

The aerial surfaces of plants are enveloped by a waxy cuticle, which among other functions serves as a barrier to limit non-stomatal water loss and defend against pathogens. The cuticle is a complex three-dimensional structure composed of cutin (a lipid polyester matrix) and waxes (very long chain fatty acid derivatives), which are embedded within and layered on top of the cutin matrix. Biosynthesis of cuticular lipids is believed to take place solely within aerial epidermal cells. Once synthesized, both the waxes and the cutin precursors must leave the cytoplasm, pass through the hydrophilic apoplastic space, and finally assemble to form the cuticle. These processes of secretion and assembly are essentially unknown. Initial steps toward our understanding of these processes were the characterization of CER5/ABCG12/WBC12 and more recently ABCG11/WBC11, a pair of ABC transporters required for cuticular lipid secretion. ABCG12 is involved in wax secretion, as mutations in this gene result in a lower surface-load of wax and a concomitant accumulation of lipidic inclusions within the epidermal cell cytoplasm. Mutations in ABCG11 result in a similar wax phenotype as cer5 and similar cytoplasmic inclusions. In contrast to cer5, however, abcg11 mutants also show significantly reduced cutin, post-genital organ fusions, and reduced growth and fertility. Thus, for the first time, a transporter is implicated in cutin accumulation. This review will discuss the secretion of cuticular lipids, focusing on ABCG12, ABCG11 and the potential involvement of other ABC transporters in the ABCG subfamily.     

RDR1 and SGS3, Components of RNA-Mediated Gene Silencing, Are Required for the Regulation of Cuticular Wax Biosynthesis in Developing Inflorescence Stems of Arabidopsis

The cuticle is a protective layer that coats the primary aerial surfaces of land plants and mediates plant interactions with the environment. It is synthesized by epidermal cells and is composed of a cutin polyester matrix that is embedded and covered with cuticular waxes. Recently, we have discovered a novel regulatory mechanism of cuticular wax biosynthesis that involves the ECERIFERUM7 (CER7) ribonuclease, a core subunit of the exosome. We hypothesized that at the onset of wax production, the CER7 ribonuclease degrades an mRNA specifying a repressor of CER3, a wax biosynthetic gene whose protein product is required for wax formation via the decarbonylation pathway. In the absence of this repressor, CER3 is expressed, leading to wax production. To identify the putative repressor of CER3 and to unravel the mechanism of CER7-mediated regulation of wax production, we performed a screen for suppressors of the cer7 mutant. Our screen resulted in the isolation of components of the RNA-silencing machinery, RNA-DEPENDENT RNA POLYMERASE1 and SUPPRESSOR OF GENE SILENCING3, implicating RNA silencing in the control of cuticular wax deposition during inflorescence stem development in Arabidopsis (Arabidopsis thaliana).