Total Wax Load Research Articles

The cuticular wax composition and crystal coverage of leaves and petals differ in a consistent manner between plant species.

Both leaves and petals are covered in a cuticle, which itself contains and is covered by cuticular waxes. The waxes perform various roles in plants' lives, and the cuticular composition of leaves has received much attention. To date, the cuticular composition of petals has been largely ignored. Being the outermost boundary between the plant and the environment, the cuticle is the first point of contact between a flower and a pollinator, yet we know little about how plant-pollinator interactions shape its chemical composition. Here, we investigate the general structure and composition of floral cuticular waxes by analysing the cuticular composition of leaves and petals of 49 plant species, representing 19 orders and 27 families. We show that the flowers of plants from across the phylogenetic range are nearly devoid of wax crystals and that the total wax load of leaves in 90% of the species is higher than that of petals. The proportion of alkanes is higher, and the chain lengths of the aliphatic compounds are shorter in petals than in leaves. We argue these differences are a result of adaptation to the different roles leaves and petals play in plant biology.

Fatty alcohols, a minor component of the tree tobacco surface wax, are associated with defence against caterpillar herbivory.

Despite decades of research resulting in a comprehensive understanding of epicuticular wax metabolism, the function of these almost ubiquitous metabolites in plant-herbivore interactions remains unresolved. In this study, we examined the effects of CRISPR-induced knockout mutations in four Nicotiana glauca (tree tobacco) wax metabolism genes. These mutations cause a wide range of changes in epicuticular wax composition, leading to altered interactions with insects and snails. Three interaction classes were examined: chewing herbivory by seven caterpillars and one snail species, phloem feeding by Myzus persicae (green peach aphid) and oviposition by Bemisia tabaci (whitefly). Although total wax load and alkane abundance did not affect caterpillar growth, a correlation across species, showed that fatty alcohols, a minor component of N. glauca surface waxes, negatively affected the growth of both a generalist caterpillar (Spodoptera littoralis) and a tobacco-feeding specialist (Manduca sexta). This negative correlation was overshadowed by the stronger effect of anabasine, a nicotine isomer, and was apparent when fatty alcohols were added to an artificial lepidopteran diet. By contrast, snails fed more on waxy leaves. Aphid reproduction and feeding activity were unaffected by wax composition but were potentially affected by altered cutin composition. Wax crystal morphology could explain the preference of B. tabaci to lay eggs on waxy wild-type plants relative to both alkane and fatty alcohol-deficient mutants. Together, our results suggest that the varied responses among herbivore classes and species are likely to be a consequence of the co-evolution that shaped the specific effects of different surface wax components in plant-herbivore interactions.

Brassica napus BnaC9.DEWAX1 Negatively Regulates Wax Biosynthesis via Transcriptional Suppression of BnCER1-2.

Very-long-chain alkane plays an important role as an aliphatic barrier. We previously reported that BnCER1-2 was responsible for alkane biosynthesis in Brassica napus and improved plant tolerance to drought. However, how the expression of BnCER1-2 is regulated is still unknown. Through yeast one-hybrid screening, we identified a transcriptional regulator of BnCER1-2, BnaC9.DEWAX1, which encodes AP2\ERF transcription factor. BnaC9.DEWAX1 targets the nucleus and displays transcriptional repression activity. Electrophoretic mobility shift and transient transcriptional assays suggested that BnaC9.DEWAX1 repressed the transcription of BnCER1-2 by directly interacting with its promoter. BnaC9.DEWAX1 was expressed predominantly in leaves and siliques, which was similar to the expression pattern of BnCER1-2. Hormone and major abiotic stresses such as drought and high salinity affected the expression of BnaC9.DEWAX1. Ectopic expression of BnaC9.DEWAX1 in Arabidopsis plants down-regulated CER1 transcription levels and resulted in a reduction in alkanes and total wax loads in leaves and stems when compared with the wild type, whereas the wax depositions in the dewax mutant returned to the wild type level after complementation of BnaC9.DEWAX1 in the mutant. Moreover, both altered cuticular wax composition and structure contribute to increased epidermal permeability in BnaC9.DEWAX1 overexpression lines. Collectively, these results support the notion that BnaC9.DEWAX1 negatively regulates wax biosynthesis by binding directly to the BnCER1-2 promoter, which provides insights into the regulatory mechanism of wax biosynthesis in B. napus.

Suppression of cuticular wax biosynthesis mediated by rice LOV KELCH REPEAT PROTEIN 2 supports a negative role in drought stress tolerance.

Drought tolerance is important for grain crops, including rice (Oryza sativa); for example, rice cultivated under intermittent irrigation produces less methane gas compared to rice grown in anaerobic paddy field conditions, but these plants require greater drought tolerance. Moreover, the roles of rice circadian-clock genes in drought tolerance remain largely unknown. Here, we show that the mutation of LOV KELCH REPEAT PROTEIN 2 (OsLKP2) enhanced drought tolerance by increasing cuticular wax biosynthesis. Among ZEITLUPE family genes, OsLKP2 expression specifically increased under dehydration stress. OsLKP2 knockdown (oslkp2-1) and knockout (oslkp2-2) mutants exhibited enhanced drought tolerance. Cuticular waxes inhibit non-stomatal water loss. Under drought conditions, total wax loads on the leaf surface increased by approximately 10% in oslkp2-1 and oslkp2-2 compared to the wild type, and the transcript levels of cuticular wax biosynthesis genes were upregulated in the oslkp2 mutants. Yeast two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays revealed that OsLKP2 interacts with GIGANTEA (OsGI) in the nucleus. The osgi mutants also showed enhanced tolerance to drought stress, with a high density of wax crystals on their leaf surface. These results demonstrate that the OsLKP2-OsGI interaction negatively regulates wax accumulation on leaf surfaces, thereby decreasing rice resilience to drought stress.

Characterization of Glossy Spike Mutants and Identification of Candidate Genes Regulating Cuticular Wax Synthesis in Barley (Hordeum vulgare L.).

Cuticular waxes comprise the hydrophobic layer that protects crops against nonstomatal water loss and biotic and abiotic stresses. Expanding on our current knowledge of the genes that are involved in cuticular wax biosynthesis and regulation plays an important role in dissecting the processes of cuticular wax metabolism. In this study, we identified the Cer-GN1 barley (Hordeum vulgare L.) mutant that is generated by ethyl methanesulfonate mutagenesis with a glossy spike phenotype that is controlled by a single recessive nuclear gene. A physiological analysis showed that the total cuticular wax loads of Cer-GN1 were one-third that of the progenitor wild-type (WT), and its water loss rate was significantly accelerated (p < 0.05). In addition, Cer-GN1 was defective in the glume's cuticle according to the toluidine blue dye test, and it was deficient in the tubule-shaped crystals which were observed on the glume surfaces by scanning electron microscopy. Using metabolomics and transcriptomics, we investigated the impacts of cuticular wax composition and waxy regulatory genes on the loss of the glaucous wax in the spikes of Cer-GN1. Among the differential metabolites, we found that 16-hydroxyhexadecanoic acid, which is one of the predominant C16 and C18 fatty acid-derived cutin monomers, was significantly downregulated in Cer-GN1 when it was compared to that of WT. We identified two novel genes that are located on chromosome 4H and are downregulated in Cer-GN1 (HvMSTRG.29184 and HvMSTRG.29185) that encode long-chain fatty acid omega-monooxygenase CYP704B1, which regulates the conversion of C16 palmitic acid to 16-hydroxyhexadecanoic acid. A quantitative real-time PCR revealed that the expression levels of HvMSTRG.29184 and HvMSTRG.29185 were downregulated at 1, 4, 8, 12, and 16 days after the heading stage in Cer-GN1 when it was compared to those of WT. These results suggested that HvMSTRG.29184 and HvMSTRG.29185 have CYP704B1 activity, which could regulate the conversion of C16 palmitic acid to 16-hydroxyhexadecanoic acid in barley. Their downregulation in Cer-GN1 reduced the synthesis of the cuticular wax components and ultimately caused the loss of the glaucous wax in the spikes. It is necessary to verify whether HvMSTRG.29184 and HvMSTRG.29185 truly encode a CYP704B1 that regulates the conversion of C16 palmitic acid to 16-hydroxyhexadecanoic acid in barley.

Functional conservation of an AP2/ERF transcription factor in cuticle formation suggests an important role in the terrestrialization of early land plants.

The formation of a hydrophobic cuticle layer on aerial plant parts was a critical innovation for protection from the terrestrial environment during the evolution of land plants. However, little is known about the molecular mechanisms underlying cuticle biogenesis in early terrestrial plants. Here, we report an APETALA2/Ethylene Response Factor (AP2/ERF) transcriptional activator, PpWIN1, involved in cutin and cuticular wax biosynthesis in Physcomitrium patens and Arabidopsis. The transcript levels of PpWIN1 were 2.5-fold higher in gametophores than in the protonema, and increased by approximately 3- to 4.7-fold in the protonema and gametophores under salt and osmotic stresses. PpWIN1 harbouring transcriptional activation activity is localized in the nucleus of tobacco leaf epidermal cells. Δppwin1 knockout mutants displayed a permeable cuticle, increased water loss, and cutin- and wax-deficient phenotypes. In contrast, increased total cutin and wax loads, and decreased water loss rates were observed in PpWIN1-overexpressing Arabidopsis plants. The transcript levels of genes involved in cutin or wax biosynthesis were significantly up-regulated in PpWIN1-overexpressing Arabidopsis lines, indicating that PpWIN1 acts as a transcriptional activator in cuticle biosynthesis. This study suggests that Arabidopsis WIN1/SHN1 orthologs may be functionally conserved from early to vascular land plants.

Changes in Morphology, Metabolism and Composition of Cuticular Wax in Zucchini Fruit During Postharvest Cold Storage.

Cuticle composition is an important economic trait in agriculture, as it is the first protective barrier of the plant against environmental conditions. The main goal of this work was to study the role of the cuticular wax in maintaining the postharvest quality of zucchini fruit, by comparing two commercial varieties with contrasting behavior against low temperatures; the cold-tolerant variety ‘Natura’, and the cold-sensitive ‘Sinatra’, as well as ‘Sinatra’ fruit with induced-chilling tolerance through a preconditioning treatment (15°C for 48 h). The freshly-harvested ‘Natura’ fruit had a well-detectable cuticle with a significant lower permeability and a subset of 15 up-regulated cuticle-related genes. SEM showed that zucchini epicuticular waxes mainly consisted of round-shaped crystals and clusters of them, and areas with more dense crystal deposition were found in fruit of ‘Natura’ and of preconditioned ‘Sinatra’. The cuticular wax load per surface was higher in ‘Natura’ than in ‘Sinatra’ fruit at harvest and after 14 days at 4°C. In addition, total cuticular wax load only increased in ‘Natura’ and preconditioned ‘Sinatra’ fruit with cold storage. With respect to the chemical composition of the waxes, the most abundant components were alkanes, in both ‘Natura’ and ‘Sinatra’, with similar values at harvest. The total alkane content only increased in ‘Natura’ fruit and in the preconditioned ‘Sinatra’ fruit after cold storage, whereas the amount of total acids decreased, with the lowest values observed in the fruit that showed less chilling injury (CI) and weight loss. Two esters were detected, and their content also decreased with the storage in both varieties, with a greater reduction observed in the cold-tolerant variety in response to low temperature. Gene expression analysis showed significant differences between varieties, especially in CpCER1-like and CpCER3-like genes, involved in alkane production, as well as in the transcription factors CpWIN1-like and CpFUL1-like, associated with cuticle development and epidermal wax accumulation in other species. These results suggest an important role of the alkane biosynthetic pathway and cuticle morphology in maintaining the postharvest quality of zucchini fruit during the storage at low temperatures.

The Combination of Abscisic Acid (ABA) and Water Stress Regulates the Epicuticular Wax Metabolism and Cuticle Properties of Detached Citrus Fruit.

The phytohormone abscisic acid (ABA) is a major regulator of fruit response to water stress, and may influence cuticle properties and wax layer composition during fruit ripening. This study investigates the effects of ABA on epicuticular wax metabolism regulation in a citrus fruit cultivar with low ABA levels, called Pinalate (Citrus sinensis L. Osbeck), and how this relationship is influenced by water stress after detachment. Harvested ABA-treated fruit were exposed to water stress by storing them at low (30–35%) relative humidity. The total epicuticular wax load rose after fruit detachment, which ABA application decreased earlier and more markedly during fruit-dehydrating storage. ABA treatment changed the abundance of the separated wax fractions and the contents of most individual components, which reveals dependence on the exposure to postharvest water stress and different trends depending on storage duration. A correlation analysis supported these responses, which mostly fitted the expression patterns of the key genes involved in wax biosynthesis and transport. A cluster analysis indicated that storage duration is an important factor for the exogenous ABA influence and the postharvest environment on epicuticular wax composition, cuticle properties and fruit physiology. Dynamic ABA-mediated reconfiguration of wax metabolism is influenced by fruit exposure to water stress conditions.

Cuticular wax composition contributes to different strategies of foliar water uptake in six plant species from foggy rupestrian grassland in tropical mountains

The cuticle is the outermost region of the epidermal cell wall of plant aerial organs. The cuticle acts as a two-way lipid barrier for water diffusion; therefore, it plays a vital role in foliar water uptake (FWU). We hypothesised that the chemical composition of the cuticular waxes influences the FWU strategy that plants adopt in a foggy tropical ecosystem. We analysed the leaf cuticular waxes of six plant species known by their different FWU strategies, in both qualitative and quantitative approaches, to test this hypothesis. We also investigated the fine structure of the plant cuticle by scanning electron microscopy. Neither the total wax loads nor the amounts of single wax compound classes correlated to the FWU. In contrast, the qualitative chemical composition of the cuticular waxes was related to the water absorption speed but not to the maximum water absorbed. The presence of wax crystals might interfere with the FWU. Our findings suggest that a complex three-dimensional network of the cuticular compounds contributes to different strategies of FWU in six plant species from foggy tropical mountaintops.

Phenotypic characterization of the glossy1 mutant and fine mapping of GLOSSY1 in common wheat (Triticum aestivum L.).

A novel wax locus GLOSSY1 was finely mapped to an approximately 308.1-kbp genomic interval on chromosome 2DS of wheat. The epicuticular wax, the outermost layer of aerial organs, gives plants their bluish-white (glaucous) appearance. Epicuticular wax is ubiquitous and provides an essential protective function against environmental stresses. In this study, we identified the glossy1 mutant on the basis of its glossy glume from an EMS population in the elite wheat (Triticum aestivum L.) cultivar Jimai22. The mutant had a dramatically different profile in total wax load and composition of individual wax constituents relative to the wild type, resulting in the increased cuticle permeability of glumes. The glossy glume phenotype was controlled by a single, semidominant locus mapping to the short arm of chromosome 2D, within a 308.1-kbp genomic interval that contained ten annotated protein-coding genes. These results pave the way for an in-depth analysis of the underlying genetic basis of wax formation patterns and enrich our understanding of mechanisms regulating wax metabolism.

Drought tolerance of faba bean (Vicia faba L.) can be improved by specific LED light wavelengths

A light wavelength affects both plant photomorphogenesis and stress resistance. In this study, the phenotypic plasticity (growth parameters and cuticular waxes) and physiological adaptation (photosynthetic properties and antioxidant enzyme activities) of faba bean subjected to specific LED lights, red, yellow, blue, violet, and white, were analyzed under two water conditions (normal and drought). The plants grown under red and yellow lights possessed the smaller leaf size and the higher leaf relative water content. The blue light had a positive effect on improving stomatal conductance and net photosynthetic rate of faba bean leaves, and the plants grown under the blue light also had higher antioxidant enzyme activities. The blue light also changed the dominant wax component to alkanes and significantly decreased a total wax load under the drought stress, and thus minimized the cuticle transpiration (water loss). The decreased wax deposition and the increase of C31-C33 alkanes abundance in plants grown under the yellow light also resulted in a lower leaf water loss under the drought stress. The red light increased but the violet light did not change the cuticle water loss. These results suggest that plant drought tolerance could be improved by supplementing the blue and yellow lights.

AP2/DREB Transcription Factor RAP2.4 Activates Cuticular Wax Biosynthesis in Arabidopsis Leaves Under Drought

Drought is a critical environmental stress that limits growth and development of plants and reduces crop productivity. The aerial part of land plants is covered with cuticular waxes to minimize water loss. To understand the regulatory mechanisms underlying cuticular wax biosynthesis in Arabidopsis under drought stress conditions, we characterized the role of an AP2/DREB type transcription factor, RAP2.4. RAP2.4 expression was detected in one-week-old seedlings and rosette leaves, stems, stem epidermis, cauline leaves, buds, flowers, and siliques of 6-week-old Arabidopsis. The levels of RAP2.4 transcripts increased with treatments of abscisic acid (ABA), mannitol, NaCl, and drought stress. Under drought, total wax loads decreased by approximately 11% and 10%, and in particular, the levels of alkanes, which are a major wax component, decreased by approximately 11% and 12% in rap2.4-1 and rap2.4-2 leaves, respectively, compared with wild type (WT) leaves. Moreover, the transcript levels of cuticular wax biosynthetic genes, KCS2 and CER1, decreased by approximately 15–23% and 32–40% in rap2.4-1 and rap2.4-2 leaves, respectively, relative to WT 4 h after drought treatment, but increased by 2- to 12-fold and 3- to 70-fold, respectively, in three independent RAP2.4 OX leaves relative to WT. Epicuticular wax crystals were observed on the leaves of RAP2.4 OX plants, but not on the leaves of WT. Total wax loads increased by 1.5- to 3.3-fold in leaves of RAP2.4 OX plants relative to WT. Cuticular transpiration and chlorophyll leaching occurred slowly in the leaves of RAP2.4 OX plants relative to WT. Transcriptional activation assay in tobacco protoplasts showed that RAP2.4 activates the expression of KCS2 and CER1 through the involvement of the consensus CCGAC or GCC motifs present in the KCS2 and CER1 promoter regions. Overall, our results revealed that RAP2.4 is a transcription factor that activates cuticular wax biosynthesis in Arabidopsis leaves under drought stress conditions.

Relative Humidity Influences Epicuticular Wax Load and Particulate Matter Accumulation on the Leaf Surface in <i>Asplenium nidus</i>

Particulate matter (PM) has recently been considered one of the most harmful air pollutants to public health. Plants have been known to degrade and deposit particle pollutants with epicuticular wax (EW), and this capacity can be influenced by environmental conditions including relative humidity (RH). The present study examined the effects of RH on EW generation and PM deposition upon leaf surfaces within Asplenium nidus ‘Avis’. The plants were treated in growth chambers with two levels of RH (low: 30% - 40% and high: 80% - 90%) for a period of four weeks, and subsequently exposed to a 30 μg･m-3 concentration of TiO2 particles as a PM resource for 72 hours. The EW ultrastructure on the leaf surface was observed as the thin films type, which was not morphologically changed in the condition of low or high RH treatment. For four weeks of RH treatment, the fresh weight and leaf area per plant were not significant between low and high RH treatment, while dry weight was significantly higher in the high RH condition. We also found that greater amounts of EW per fresh weight, dry weight and leaf area were generated in high RH. However, the total amounts of PM deposition (surface PM + in-wax PM) of the plants were higher within the low RH treatment with a higher proportion of surface PM. In contrast the proportion of in-wax PM was 15% higher within the high RH. These results suggest that EW generation is affected by air humidity and that proportion of PM deposition in the EW layer were influenced by the amount of total wax load.

Comparative Analysis of Leaf Trichomes, Epidermal Wax And Defense Enzymes Activities in Response to Puccinia horiana in Chrysanthemum and Ajania Species

White rust caused by Puccinia horiana is a destructive disease of chrysanthemum plants. To better understand the resistance mechanisms of composite species to this disease, the leaf cuticular traits, antioxidant and defensive enzymes activities of immune (Chrysanthemum makinoi var. wakasaense) and highly susceptible (Ajania shiwogiku var. kinokuniense) species were compared. Trichome density of two species was markedly different, negatively associated with plant resistance to P. horiana. Total wax load in C. makinoi var. wakasaense was two times more than that in A. shiwogiku var. kinokuniense. The wax composition in immune one was abundant in esters and primary alcohols. Superoxide dismutase (SOD, EC 1.15.1.1), peroxidase (POD, EC 1.11.1.7), polyphenoloxidase (PPO, EC 1.14.18.1 or EC 1.10.3.2) and phenylalanine ammonia lyase (PAL, EC 4.3.1.5) activities were investigated. In C. makinoi var. wakasaense, the activity of SOD and POD increased rapidly after inoculation, which might be non-host induced reactive oxygen species (ROS) activated antioxidant enzymes, however SOD and POD remained a low and steady level in the highly susceptible one after inoculation. Quick increase in PPO activities after inoculation was observed in both species, however it remained higher in C. makinoi var. wakasaense at the late period of inoculation. PAL in C. makinoi var. wakasaense was induced after pathogen inoculation, but not in A. shiwogiku var. kinokuniense, suggesting that these two enzymes might contribute to the resistance to P. horiana.

Differential response of cuticular wax and photosynthetic capacity by glaucous and non-glaucous wheat cultivars under mild and severe droughts

Cuticular wax is known to play an important role in non-stomatal transpiration. However, support is lacking regarding the waxy phenotype for wheat breeding against drought. In this study, four wheat cultivars with different wax phenotypes (glaucous and non-glaucous types) were used to evaluate their responses to drought stress and impact on photosynthetic capability of wheat. Xinong 291 and HY 2912, with the glaucous trait, demonstrated higher diketone ratios and contents compared with Pubing 201 and Jinmai 47, which are the non-glaucous type. The cultivars HY 2912 and Jinmai 47 had 35% higher biomass than did Xinong 291 and Pubing 201 under severe drought condition. HY 2912 exhibited the highest wax load with or without drought stress. Jinmai 47 showed the highest ratio of alkane content. Among glaucous cultivars, drought-resistant HY 2912 may promote growth by decreasing water loss, increasing the diketone content, increasing the total wax load, and maintaining mesophyll and stomatal conductance. Among non-glaucous cultivars, drought-resistant Jinmai 47 may enhance growth via stomatal closure and increased mesophyll conductance and alkane ratios. The glaucous trait was not always associated with drought resistance, and correlation analysis revealed that the diketone ratio was positively related to the intercellular CO2 concentration. These results suggest that the mechanism of drought resistance in wheat is systematically regulated by wax alteration, stomatal conductance and mesophyll conductance. Therefore, wax content and composition as well as mesophyll and stomatal regulation should be considered in the breeding and selection of drought-resistant wheat cultivars.

Expression Analysis and Functional Characterization of CER1 Family Genes Involved in Very-Long-Chain Alkanes Biosynthesis in Brachypodium distachyon.

Cuticular wax accumulation and composition affects drought resistance in plants. Brachypodium distachyon plants subjected to water deficit and polyethylene glycol treatments resulted in a significant increase in total wax load, in which very-long-chain (VLC) alkanes were more sensitive to these treatments than other wax compounds, implying that VLC alkanes biosynthesis plays a more important role in drought resistance in B. distachyon. ECERIFERUM1 (CER1) has been reported to encode a core enzyme involved in VLC alkanes biosynthesis in Arabidopsis (Arabidopsis thaliana), but few corresponding genes are investigated in B. distachyon. Here, we identified eight CER1 homologous genes in B. distachyon, namely BdCER1-1 to BdCER1-8, and then analyzed their sequences feature, expression patterns, stress induction, and biochemical activities. These genes had similar protein structure to other reported CER1 and CER1-like genes, but displayed closer phylogenetic relationship to the rice OsGL1 genes. They were further found to exhibit various tissue expression patterns after being induced by abiotic stresses. Among them, BdCER1-8 gene showed extremely high expression in leaves. Heterologous introduction of BdCER1-8 into the Arabidopsis cer1 mutant rescued VLC alkanes biosynthesis. These results indicate that BdCER1 genes are likely to be involved in VLC alkanes biosynthesis of B. distachyon. Taken together, BdCER1-8 seems to play an explicit and predominant role in VLC alkanes biosynthesis in leaf. Our work provides important clues for further characterizing function of CER1 homologous genes in B. distachyon and also an option to improve drought resistance of cereal crops.

Genome-Wide Analysis of Coding and Long Non-Coding RNAs Involved in Cuticular Wax Biosynthesis in Cabbage (Brassica oleracea L. var. capitata).

Cuticular wax is a mixture of very long chain fatty acids (VLCFAs) and their derivatives, which determines vital roles for plant growth. In cabbage, the cuticular wax content of leaf blades is an important trait influencing morphological features of the head. Understanding the molecular basis of cuticular wax biosynthesis can help breeders develop high quality cabbage varieties. Here, we characterize a cabbage non-wax glossy (nwgl) plant, which exhibits glossy green phenotype. Cryo-scanning electron microscope analysis showed abnormal wax crystals on the leaf surfaces of nwgl plants. Cuticular wax composition analyzed by GC-MS displayed severely decreased in total wax loads, and individual wax components in nwgl leaves. We delimited the NWGL locus into a 99-kb interval between the at004 marker and the end of chromosome C08 through fine mapping. By high-throughput RNA sequencing, we identified 1247 differentially expressed genes (DEGs) and 148 differentially expressed lncRNAs in nwgl leaves relative to the wild-type. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that the DEGs and cis-regulated target genes for differentially expressed lncRNAs were significantly enriched in wax and lipid biosynthetic or metabolic processes. Our results provide the novel foundation to explore the complex molecular basis of cuticular wax biosynthesis.

Three endoplasmic reticulum-associated fatty acyl-coenzyme a reductases were involved in the production of primary alcohols in hexaploid wheat (Triticum aestivum L.)

BackgroundThe cuticle covers the surface of the polysaccharide cell wall of leaf epidermal cells and forms an essential diffusion barrier between the plant and the environment. The cuticle is composed of cutin and wax. Cuticular wax plays an important role in the survival of plants by serving as the interface between plants and their biotic and abiotic environments, especially restricting nonstomatal water loss. Leaf cuticular waxes of hexaploid wheat at the seedling stage mainly consist of primary alcohols, aldehydes, fatty acids, alkane and esters. Primary alcohols account for more than 80% of the total wax load. Therefore, we cloned several genes encoding fatty acyl-coenzyme A reductases from wheat and analyzed their function in yeast and plants. We propose the potential use of these genes in wheat genetic breeding.ResultsWe reported the cloning and characterization of three TaFARs, namely TaFAR6, TaFAR7 and TaFAR8, encoding fatty acyl-coenzyme A reductases (FAR) in wheat leaf cuticle. Expression analysis revealed that TaFAR6, TaFAR7 and TaFAR8 were expressed at the higher levels in the seedling leaf blades, and were expressed moderately or weakly in stamen, glumes, peduncle, flag leaf blade, sheath, spike, and pistil. The heterologous expression of three TaFARs in yeast (Saccharomyces cerevisiae) led to the production of C24:0 and C26:0 primary alcohols. Transgenic expression of the three TaFARs in tomato (Solanum lycopersicum) and rice (Oryza sativa) led to increased accumulation of C24:0–C30:0 primary alcohols. Transient expression of GFP protein-tagged TaFARs revealed that the three TaFAR proteins were localized to the endoplasmic reticulum (ER), the site of wax biosynthesis. The three TaFAR genes were transcriptionally induced by drought, cold, heat, powdery mildew (Blumeria graminis) infection, abscisic acid (ABA) and methyl jasmonate (MeJa) treatments.ConclusionsThese results indicated that wheat TaFAR6, TaFAR7 and TaFAR8 are involved in biosynthesis of very-long-chain primary alcohols in hexaploid wheat and in response to multiple environmental stresses.

DEWAX2 Transcription Factor Negatively Regulates Cuticular Wax Biosynthesis in Arabidopsis Leaves.

The aerial parts of terrestrial plants are covered with hydrophobic wax layers, which represent the primary barrier between plant cells and the environment and act to protect plants from abiotic and biotic stresses. Although total wax loads are precisely regulated in an environmental- or organ-specific manner, regulatory mechanisms underlying cuticular wax biosynthesis remain largely unknown. In this study, we characterized DEWAX2 (DECREASE WAX BIOSYNTHESIS2) which encodes an APETALA 2 (AP2)/ethylene response element-binding factor (ERF)-type transcription factor and is predominantly expressed in young seedlings, and rosette and cauline leaves. Total wax loads increased by approximately 12% and 16% in rosette and cauline leaves of dewax2, respectively, but were not significantly altered in the stems of dewax2 relative to the wild type (WT). The excess wax phenotype of dewax2 leaves was rescued upon expression of DEWAX2 driven by its own promoter. Overexpression of DEWAX2 decreased total wax loads by approximately 15% and 26% in the stems and rosette leaves compared with those of the WT, respectively. DEWAX2:eYFP (enhanced yellow fluorescent protein) was localized to the nucleus in Arabidopsis roots and hypocotyls. DEWAX2 possessed transcriptional repression activity in tobacco protoplasts. Transcriptome and quantitative real-time PCR analyses showed that the transcript levels of CER1, ACLA2, LACS1, LACS2 and KCS12 were down-regulated in DEWAX2 overexpression lines compared with the WT. Transient transcriptional assays showed that DEWAX2 represses the expression of its putative target genes. Quantitative chromatin immunoprecipitation-PCR revealed that DEWAX2 binds directly to the GCC motifs of the LACS1, LACS2, KCS12 and CER1 promoters. These results suggest that DEWAX2-mediated transcriptional repression may contribute to the total wax load in Arabidopsis leaves.

Studies of genetic characteristics of two heart-shaped glossy cabbage mutants

Abstract Cabbage (Brassica oleracea L.var. capitata), an important crop of the Brassicaceae family, is a highly valued source of anti-cancer compounds, vitamin C and high-quality protein. Glossiness of the leaf surface is an important character that has an important influence on the quality of commercial products. In this study, two heart-shaped glossy cabbage mutants were examined to determine their characteristics and potential practical applications. Firstly, mutant-type (MT) plants showed a shiny and attractive appearance, and the total wax load in MT leaves was significantly less than that of wild-type (WT) leaves, when assessed by scanning electron micrographs. Then, the results of χ2 tests on the separated proportion proved that the glossy wax-less trait is controlled by a single recessive gene. Unexpectedly, the sulforaphane content, which has been identified as a naturally potent anticarcinogen, showed an increasing trend in MT plants than the corresponding WT plants. Lastly, our work also showed that some genes that are involved in wax biosynthesis and export pathways are down-regulated in MT leaves, which could cause the glossy phenotype of MT plants. These analyses will shed light on the benefits of incorporating the two mutants and other glossy mutants into future breeding programs to develop varieties with morphological markers linked to desirable traits.