A chromosome level genome assembly of Pseudoroegneria Libanotica reveals a key Kcs gene involves in the cuticular wax elongation for drought resistance.

BACKGROUND: The genus Pseudoroegneria (Nevski) Löve (Triticeae, Poaceae), whose genome symbol was designed as "St", accounts for more than 60% of perennial Triticeae species. The diploid species Psudoroegneria libanotica (2n = 14) contains the most ancient St genome, exhibited strong drought resistance, and was morphologically covered by cuticular wax on the aerial part. Therefore, the St-genome sequencing data could provide fundamental information for studies of genome evolution and reveal its mechanisms of cuticular wax and drought resistance. RESULTS: In this study, we reported the chromosome-level genome assembly for the St genome of Pse. libanotica, with a total size of 2.99 Gb. 46,369 protein-coding genes annotated and 71.62% was repeat sequences. Comparative analyses revealed that the genus Pseudoroegneria diverged during the middle and late Miocene. During this period, unique genes, gene family expansion, and contraction in Pse. libanotica were enriched in biotic and abiotic stresses, such as fatty acid biosynthesis which may greatly contribute to its drought adaption. Furthermore, we investigated genes associated with the cuticular wax formation and water deficit and found a new Kcs gene evm.TU.CTG175.54. It plays a critical role in the very long chain fatty acid (VLCFA) elongation from C18 to C26 in Pse. libanotica. The function needs more evidence to be verified. CONCLUSIONS: We sequenced and assembled the St genome in Triticeae and discovered a new KCS gene that plays a role in wax extension to cope with drought. Our study lays a foundation for the genome diversification of Triticeae species and deciphers cuticular wax formation genes involved in drought resistance.

Micromorphological and chemical characterization of Drimys winteri leaf surfaces: The secondary alcohols forming epicuticular wax crystals are accompanied by alkanediol, alkanetriol and ketol derivatives.

The cuticle is a hydrophobic coating of most aerial plant surfaces crucial for limiting non-stomatal water loss. Plant cuticles consist of the lipid polyester cutin and associated waxes with compositions varying widely between plant species and organs. Here, we aimed to provide a comparative analysis of the dark-glossy adaxial and pale-glaucous abaxial sides of Drimys winteri (Winteraceae) leaves. Scanning electron microscopy showed nanotubular wax crystals lining the entire abaxial side of the leaf (including stomatal pores), while the adaxial side had patches of mixed platelet/tubule crystals and smooth areas between them. Consecutive treatments for wax removal and cutin depolymerization revealed that the waxes were deposited on a cutin network with micron-scale cavities across the entire abaxial surface including the stomata pores, and on a microscopically smooth cutin surface on the adaxial side of the leaf. Gas chromatography coupled to mass spectrometry and flame ionization detection showed that the wax mixtures on both sides of the leaf were complex mixtures of very-long-chain compounds dominated by the secondary alcohol nonacosan-10-ol and alkanediols with one hydroxyl on C-10. It is therefore very likely that the tubular wax crystals characteristic of both leaf sides are formed by these alcohols and diols. Further secondary alcohols and alkanediols, as well as ketols and alkanetriols with one functional group on C-10 were identified based on mass spectral fragmentation patterns. The similarities between all these mid-chain functionalized compounds suggest that they are derived from nonacosan-10-ol via regio-specific hydroxylation reactions, likely catalyzed by three P450-dependent monooxygenases with different regio-specificities.

Gene expression analysis of drought tolerance and cuticular wax biosynthesis in diploid and tetraploid induced wallflowers.

Whole-genome doubling leads to cell reprogramming, upregulation of stress genes, and establishment of new pathways of drought stress responses in plants. This study investigated the molecular mechanisms of drought tolerance and cuticular wax characteristics in diploid and tetraploid-induced Erysimum cheiri. According to real-time PCR analysis, tetraploid induced wallflowers exhibited increased expression of several genes encoding transcription factors (TFs), including AREB1 and AREB3; the stress response genes RD29A and ERD1 under drought stress conditions. Furthermore, two cuticular wax biosynthetic pathway genes, CER1 and SHN1, were upregulated in tetraploid plants under drought conditions. Leaf morphological studies revealed that tetraploid leaves were covered with unique cuticular wax crystalloids, which produced a white fluffy appearance, while the diploid leaves were green and smooth. The greater content of epicuticular wax in tetraploid leaves than in diploid leaves can explain the decrease in cuticle permeability as well as the decrease in water loss and improvement in drought tolerance in wallflowers. GC‒MS analysis revealed that the wax components included alkanes, alcohols, aldehydes, and fatty acids. The most abundant wax compound in this plant was alkanes (50%), the most predominant of which was C29. The relative abundance of these compounds increased significantly in tetraploid plants under drought stress conditions. These findings revealed that tetraploid-induced wallflowers presented upregulation of multiple drought-related and wax biosynthesis genes; therefore, polyploidization has proved useful for improving plant drought tolerance.

Evidence for within-species transition between drought response strategies in Nicotiana benthamiana.

Nicotiana benthamiana is predominantly distributed in arid habitats across northern Australia. However, none of six geographically isolated accessions shows obvious xerophytic morphological features. To investigate how these tender-looking plants withstand drought, we examined their responses to water deprivation, assessed phenotypic, physiological, and cellular responses, and analysed cuticular wax composition and wax biosynthesis gene expression profiles. Results showed that the Central Australia (CA) accession, globally known as a research tool, has evolved a drought escape strategy with early vigour, short life cycle, and weak, water loss-limiting responses. By contrast, a northern Queensland (NQ) accession responded to drought by slowing growth, inhibiting flowering, increasing leaf cuticle thickness, and altering cuticular wax composition. Under water stress, NQ increased the heat stability and water impermeability of its cuticle by extending the carbon backbone of cuticular long-chain alkanes from c. 25 to 33. This correlated with rapid upregulation of at least five wax biosynthesis genes. In CA, the alkane chain lengths (c. 25) and gene expression profiles remained largely unaltered. This study highlights complex genetic and environmental control over cuticle composition and provides evidence for divergence into at least two fundamentally different drought response strategies within the N. benthamiana species in < 1 million years.

A leucine-rich repeat receptor kinase as a regulator in the cuticular wax deposition in sorghum.

Cuticular wax (CW) is the first defensive barrier of plants that forms a waterproof barrier, protects the plant from desiccation, and defends against insects, pathogens, and UV radiation. Sorghum, an important grass crop with high heat and drought tolerance, exhibits a much higher wax load than other grasses and the model plant Arabidopsis. In this study, we explored the regulation of sorghum CW biosynthesis using a bloomless mutant. The CW on leaf sheaths of bloomless 41 (bm41) mutant showed significantly reduced very long-chain fatty acids (VLCFAs), triterpenoids, alcohols, and other wax components, with an overall 86% decrease in total wax content compared to the wild-type. Notably, the 28-carbon and 30-carbon VLCFAs were decreased in the mutants. Using bulk segregant analysis, we identified the causal gene of the bloomless phenotype as a leucine-rich repeat transmembrane protein kinase. Transcriptome analysis of the wild-type and bm41 mutant leaf sheaths revealed BM41 as a positive regulator of lipid biosynthesis and steroid metabolism. BM41 may regulate CW biosynthesis by regulating the expression of the gene encoding 3-ketoacyl-CoA synthase 6. Identification of BM41 as a new regulator of CW biosynthesis provides fundamental knowledge for improving grass crops' heat and drought tolerance by increasing CW.

Chemical and Transcriptomic Analyses of Leaf Cuticular Wax Metabolism in Ammopiptanthus mongolicus under Osmotic Stress.

Plant cuticular wax forms a hydrophobic structure in the cuticle layer covering epidermis as the first barrier between plants and environments. Ammopiptanthus mongolicus, a leguminous desert shrub, exhibits high tolerances to multiple abiotic stress. The physiological, chemical, and transcriptomic analyses of epidermal permeability, cuticular wax metabolism and related gene expression profiles under osmotic stress in A. mongolicus leaves were performed. Physiological analyses revealed decreased leaf epidermal permeability under osmotic stress. Chemical analyses revealed saturated straight-chain alkanes as major components of leaf cuticular wax, and under osmotic stress, the contents of total wax and multiple alkane components significantly increased. Transcriptome analyses revealed the up-regulation of genes involved in biosynthesis of very-long-chain fatty acids and alkanes and wax transportation under osmotic stress. Weighted gene co-expression network analysis identified 17 modules and 6 hub genes related to wax accumulation, including 5 enzyme genes coding KCS, KCR, WAX2, FAR, and LACS, and an ABCG transporter gene. Our findings indicated that the leaf epidermal permeability of A. mongolicus decreased under osmotic stress to inhibit water loss via regulating the expression of wax-related enzyme and transporter genes, further promoting cuticular wax accumulation. This study provided new evidence for understanding the roles of cuticle lipids in abiotic stress tolerance of desert plants.

Influence of Cuticular Waxes from Triticale on Rumen Fermentation: A Metabolomic and Microbiome Profiling Study.

Cuticular wax, a critical defense layer for plants, remains a relatively unexplored factor in rumen fermentation. We investigated the impact of cuticular wax on rumen fermentation using triticale as a model. In total, six wax classes were identified, including fatty acids, aldehydes, alkane, primary alcohol, alkyresorcinol, and ß-diketone, with low-bloom lines predominated by 46.05% of primary alcohols and high-bloom lines by 35.64% of ß-diketone. Low-wax addition (2.5 g/kg DM) increased the gas production by 19.25% (P < 0.05) and total volatile fatty acids by 6.34% (P > 0.05), and enriched key carbohydrate-fermenting rumen microbes like Saccharofermentans, Ruminococcus, and Prevotellaceae, when compared to non-wax groups. Metabolites linked to nucleotide metabolism, purine metabolism, and protein/fat digestion in the rumen showed a positive correlation with low-wax, benefiting rumen microbes. This study highlights the intricate interplay among cuticular wax, rumen microbiota, fermentation, and metabolomics in forage digestion, providing insights into livestock nutrition and forage utilization.

Melatonin improves the storage quality of rabbiteye blueberry (Vaccinium ashei) by affecting cuticular wax profile.

Cuticular wax is the first line of structural defense for plants against external stresses. This study investigated the effects of melatonin (MT) on chemical composition and accumulation profile of wax, as well as fruit quality of rabbiteye blueberry during storage. The results indicated a significant reduction in the overall wax content during storage. Nevertheless, MT effectively delayed the decline, with a higher amount of 9.8% and 15.17% in the treated 'Baldwin' and 'Garden Blue' compared to their respective controls at 21st day of storage. The wax composition significantly varied depending on storage time, MT treatment, and cultivars. Additionally, MT markedly improved the fruit quality of rabbiteye blueberries. Correlation analysis revealed water loss and decay rates were negatively correlated with triterpenoids and fatty acids. Taken together, this study highlights the positive effects of post-harvest MT application on shelf life and fruit quality of blueberry by modifying the wax profile during storage.

MdDEWAX decreases plant drought resistance by regulating wax biosynthesis.

Apple epidermal wax protects plants from environmental stresses, determines fruit gloss and improves postharvest storage quality. However, the molecular mechanisms underlying the biosynthesis and regulation of apple epidermal waxes are not fully understood. In this study, we isolated a MdDEWAX gene from apple, which localized in the nucleus, expressed mainly in apple fruit, and induced by drought. We transformed the MdDEWAX gene into Arabidopsis, and found that heterologous expression of MdDEWAX reduced the accumulation of cuticular waxes in leaves and stems, increased epidermal permeability, the rate of water loss, and the rate of chlorophyll extraction of leaves and stems, altered the sensitivity to ABA, and reduced drought tolerance. Meanwhile, overexpression or silencing of the gene in the epidermis of apple fruits decreased or increased wax content, respectively. This study provides candidate genes for breeding apple cultivars and rootstocks with better drought tolerance.

Identification of long-chain acyl-CoA synthetase gene family reveals that SlLACS1 is essential for cuticular wax biosynthesis in tomato.

Long-chain acyl-CoA synthetases (LACSs), belonging to the acyl-activating enzyme superfamily, play crucial roles in lipid biosynthesis and fatty acid catabolism. Here, we identified 11 LACS genes in the tomato reference genome, and these genes were clustered into six subfamilies. Gene structure and conserved motif analyses indicated that LACSs from the same subfamily shared conserved gene and protein structures. Expression analysis revealed that SlLACS1 was highly expressed in the outer epidermis of tomato fruits and leaves. Subcellular localization assay results showed that SlLACS1 was located in the endoplasmic reticulum. Compared with wild-type plants, the wax content on leaves and fruits decreased by 22.5-34.2 % in SlLACS1 knockout lines, confirming that SlLACS1 was involved in wax biosynthesis in both leaves and fruits. Water loss, chlorophyll extraction, water-deficit, and toluidine blue assays suggested that cuticle permeability was elevated in SlLACS1 knockout lines, resulting in reduction in both drought stress resistance and fruit shelf-life. Overall, our analysis of the LACSs in tomato, coupled with investigations of SlLACS1 function, yielded a deeper understanding of the evolutionary patterns of LACS members and revealed the involvement of SlLACS1 in wax accumulation contribute to drought resistance and extended fruit shelf-life in tomato.

Leaf cuticular wax composition of a genetically diverse collection of lettuce (Lactuca sativa L.) cultivars evaluated under field conditions.

Cuticular waxes of plants impart tolerance to many forms of environmental stress and help shed dangerous human pathogens on edible plant parts. Although the chemical composition of waxes on a wide variety of important crops has been described, a detailed wax compositional analysis has yet to be reported for lettuce (Lactuca sativa L.), one of the most widely consumed vegetables. We present herein the leaf wax content and composition of 12 genetically diverse lettuce cultivars sampled across five time points during their vegetative growth phase in the field. Mean total leaf wax amounts across all cultivars varied little over 28 days of vegetative growth, except for a notable decrease in total waxes following a major precipitation event, presumably due to wax degradation from wind and rain. All lettuce cultivars were found to contain a unique wax composition highly enriched in 22- and 24-carbon length 1-alcohols (docosanol and tetracosanol, respectively). In our report, the dominance of these shorter chain length 1-alcohols as wax constituents represents a relatively rare phenotype in plants. The ecological significance of these dominant and relatively short 1-alcohols is still unknown. Although waxes have been a target for improvement of various crops, no such work has been reported for lettuce. This study lays the groundwork for future research that aims to integrate cuticular wax characteristics of field grown plants into the larger context of lettuce breeding and cultivar development.

Elevated CO2 concentration enhances drought resistance of soybean by regulating cell structure, cuticular wax synthesis, photosynthesis, and oxidative stress response.

The atmospheric [CO2] and the frequency and intensity of extreme weather events such as drought are increased, leading to uncertainty to soybean production. Elevated [CO2] (eCO2) partially mitigates the adverse effects of drought stress on crop growth and photosynthetic performance, but the mitigative mechanism is not well understood. In this study, soybean seedlings under drought stress simulated by PEG-6000 were grown in climate chambers with different [CO2] (400 µmol mol-1 and 700 µmol mol-1). The changes in anatomical structure, wax content, photosynthesis, and antioxidant enzyme were investigated by the analysis of physiology and transcriptome sequencing (RNA-seq). The results showed that eCO2 increased the thickness of mesophyll cells and decreased the thickness of epidermal cells accompanied by reduced stomatal conductance, thus reducing water loss in soybean grown under drought stress. Meanwhile, eCO2 up-regulated genes related to wax anabolism, thus producing more epidermal wax. Under drought stress, eCO2 increased net photosynthetic rate (PN), ribulose-1,5-bisphosphate carboxylase/oxygenase activity, and alerted the gene expressions in photosynthesis. The increased sucrose synthesis and decreased sucrose decomposition contributed to the progressive increase in the soluble saccharide contents under drought stress with or without eCO2. In addition, eCO2 increased the expressions of genes associated with peroxidase (POD) and proline (Pro), thus enhancing POD activity and Pro content and improving the drought resistance in soybean. Taken together, these findings deepen our understanding of the effects of eCO2 on alleviating drought stress in soybean and provide potential target genes for the genetic improvement of drought tolerance in soybean.

1-MCP treatment improves the postharvest quality of Jinxiu yellow peach by regulating cuticular wax composition and gene expression during cold storage.

This study aimed to analyze the effect of 1-methylcyclopropene (1-MCP) treatment on the postharvest quality, epidermal wax morphology, composition, and gene expression of Jinxiu yellow peach during cold storage. The results showed that 1-MCP treatment could maintain the postharvest quality of peach fruit as compared to control (CK) during cold storage. The wax crystals of peach fruit were better retained by 1-MCP, and they still existed in 0.6 and 0.9 µL/L 1-MCP treated fruit at 36 days. The total wax content in all the fruit increased first and then decreased during cold storage. Meanwhile, n-alkanes and primary alcohols were the main wax components. Compared to CK, 1-MCP treatment could delay the reduction of wax content during cold storage. The correlation analysis indicated that the postharvest quality of yellow peach was mainly affected by the contents of fatty acids and triterpenoids in cuticular wax. The transcriptomics results revealed PpaCER1, PpaKCS, PpaKCR1, PpaCYP86B1, PpaFAR, PpaSS2, and PpaSQE1 played the important roles in the formation of peach fruit wax. 1-MCP treatment upregulated PpaCER1 (18785414, 18786441, and 18787644), PpaKCS (18774919, 18789438, and 18793503), PpaKCR1 (18790432), and PpaCYP86B1 (18789815) to deposit more n-alkanes and fatty acids during cold storage. This study could provide a new perspective for regulating the postharvest quality of yellow peach in view of the application of cuticular wax. PRACTICAL APPLICATION: 'Jinxiu' yellow peach fruit is favorable among consumers because of its high commercial value. However, it ripens and deteriorates rapidly during storage, leading to serious economic loss and consumer disappointment. The effect of 1-methylcyclopropene (1-MCP) treatment on the postharvest quality, epidermal wax morphology, composition, and genes regulation of 'Jinxiu' yellow peach during cold storage was assessed. Compared to control, 1-MCP treatment could retain the storage quality of yellow peach by affecting cuticular wax composition and gene expression. This study could provide new perspective for regulating the postharvest quality of yellow peach in view of the application of cuticular wax.

Relationship between skin greasiness and cuticular wax in harvested "Hongro" apples.

We investigated the ripening and skin greasiness of "Hongro" apples during storage at 20 °C. Postharvest treatment using 100 µLL-1 ethylene accelerated ripening and increased greasiness, whereas treatment using 1 µLL-1 1-methylcyclopropene delayed ripening and reduced greasiness. Scanning electron microscopy showed changes in cuticular wax structure linked to greasiness. Metabolic analysis identified specific metabolites related to greasiness, which varied upon postharvest treatment. Greasiness was positively associated with ethylene production and butyl-9,12-octadecadienoate content. Random forest modeling predicted greasiness levels with high accuracy, with root mean square error values of 0.322 and 0.362 for training and validation datasets, respectively. These findings illuminate the complex interplay between postharvest treatment, apple ripening, wax composition, and skin greasiness. The application of predictive models exemplifies the potential for technology-driven approaches in agriculture and aids in the development of postharvest strategies to control greasiness and maintain fruit quality.

PpyLTP36 and PpyLTP39 are involved in the transmembrane transport of cuticular wax and are associated with the occurrence of pear fruit russeting.

Non-specific lipid-transfer proteins (nsLTPs) are a group of small, cysteine-rich proteins that are involved in the transport of cuticular wax and other lipid compounds. Accumulating evidence suggests that dynamic changes in cuticular waxes are strongly associated with fruit russeting, an undesirable visual quality that negatively affects consumer appeal in pears. Currently, the regulatory role of nsLTPs in cuticular wax deposition and pear fruit skin russeting remains unclear. Here, we characterized the variations of cuticular waxes in non-treated (russeted) and preharvest bagging treated (non-russeted) pear fruits throughout fruit development and confirmed that the contents of cuticular waxes were significantly negatively correlated with the occurrence of pear fruit russeting. Based on RNA-Sequencing (RNA-Seq) and quantitative real-time PCR (qRT-PCR) analyses, two nsLTP genes (PpyLTP36 and PpyLTP39) were identified, which exhibited high expression levels in non-russeted pear fruit skins and were significantly repressed during fruit skin russeting. Subcellular localization analysis demonstrated that PpyLTP36 and PpyLTP39 were localized to the plasma membrane (PM). Further, transient Virus-Induced Gene Silencing (VIGS) analyses of PpyLTP36 and PpyLTP39 in pear fruits significantly reduced cuticular wax deposition. In conclusion, PpyLTP36 and PpyLTP39 are involved in the transmembrane transport of cuticular wax and are associated with pear fruit skin russeting.

A reference-grade genome of the xerophyte Ammopiptanthus mongolicus sheds light on its evolution history in legumes and drought-tolerance mechanisms.

Plants that grow in extreme environments represent unique sources of stress-resistance genes and mechanisms. Ammopiptanthus mongolicus (Leguminosae) is a xerophytic evergreen broadleaf shrub native to semi-arid and desert regions; however, its drought-tolerance mechanisms remain poorly understood. Here, we report the assembly of a reference-grade genome for A. mongolicus, describe its evolutionary history within the legume family, and examine its drought-tolerance mechanisms. The assembled genome is 843.07 Mb in length, with 98.7% of the sequences successfully anchored to the nine chromosomes of A. mongolicus. The genome is predicted to contain 47 611 protein-coding genes, and 70.71% of the genome is composed of repetitive sequences; these are dominated by transposable elements, particularly long-terminal-repeat retrotransposons. Evolutionary analyses revealed two whole-genome duplication (WGD) events at 130 and 58 million years ago (mya) that are shared by the genus Ammopiptanthus and other legumes, but no species-specific WGDs were found within this genus. Ancestral genome reconstruction revealed that the A. mongolicus genome has undergone fewer rearrangements than other genomes in the legume family, confirming its status as a "relict plant". Transcriptomic analyses demonstrated that genes involved in cuticular wax biosynthesis and transport are highly expressed, both under normal conditions and in response to polyethylene glycol-induced dehydration. Significant induction of genes related to ethylene biosynthesis and signaling was also observed in leaves under dehydration stress, suggesting that enhanced ethylene response and formation of thick waxy cuticles are two major mechanisms of drought tolerance in A. mongolicus. Ectopic expression of AmERF2, an ethylene response factor unique to A. mongolicus, can markedly increase the drought tolerance of transgenic Arabidopsis thaliana plants, demonstrating the potential for application of A. mongolicus genes in crop improvement.

Citrus oil gland and cuticular wax inspired multifunctional gelatin film of OSA-starch nanoparticles-based nanoemulsions for preserving perishable fruit.

Inspired by the citrus oil gland and cuticular wax, a multifunctional material that stably and continuously released the carvacrol and provided physical defenses was developed to address issues of fresh-cut fruits to microbial infestation and moisture loss. The results confirmed that low molecular weight and loose structure of starch nanoparticles prepared by the ultrasound-assisted Fenton system were preferable for octenyl succinic anhydride modification compared to native starch, achieving a higher degree of substitution (increased by 18.59 %), utilizing in preparing nanoemulsions (NEs) for encapsulating carvacrol (at 5 % level: 81.58 %). Furthermore, the NEs-based gelatin (G) film improved with surface hydrophobic modification by myristic acid (MA) successfully replicated the citrus oil gland and cuticular wax, providing superior antioxidant (enhanced by 3-4 times) and antimicrobial properties (95.99 % and 84.97 % against Staphylococcus aureus and Escherichia coli respectively), as well as the exceptional UV shielding (nearly 0 transmittance in the UV region), mechanical (72 % increase in tensile strength), and hydrophobic (WCA 133.63°). Moreover, the 5%NE-G@MA film inhibited foodborne microbial growth (reduced by 50 %) and water loss (controlled below 15 %), extending the shelf life of fresh-cut navel orange and kiwi. Thus, the multifunctional film was a potential shield for preserving perishable fresh-cut products.

Chemical characterization and discovery of novel quality markers in Citrus aurantium L. fruit from traditional cultivation areas in China using GC-MS-based cuticular waxes analysis.

Citrus aurantium L. fruit is a commonly used Chinese medicine whose therapeutic effects tends to be affected by growing conditions. In order to gain insights into the effects of growing location on the cuticular wax composition of C. aurantium L. fruit, we analyzed the differences in the wax composition of its fruits collected from different regions. The findings showed that the cuticular waxes in the fruit peels were mainly composed of fatty acids, which differed quantitatively in the chemical profiles of C. aurantium L. samples from different geographical conditions. Particularly, the concentrations of linoleic acid and stearic acid in the total component content of the fruit peel were above 1%, with a greater level in the geo-authentic samples. Thus, GC-MS-based wax analysis was first used for the chemical characterization and quantification of cuticular waxes, which could be considered as a rapid way for evaluating the quality of medicinal fruits.