Wax Load Research Articles

Leucine-rich repeat receptor kinase BM41 regulates 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 the 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 with 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.

Integrated physiological, biochemical, and transcriptomic analyses of Bruguiera gymnorhiza leaves under long-term copper stress: Stomatal size, wax crystals and composition

Copper (Cu) is a necessary mineral nutrient for plant growth and development and is involved in several morphological, physiological, and biochemical processes; however, high concentrations of Cu can negatively impact these processes. The role of stomata in responding to various biotic and abiotic stimuli has not been studied in Bruguiera gymnorhiza, particularly in terms of their coordinated interactions at the molecular, physiological, and biochemical levels. Moreover, numerous plants employ strategies such as the presence of thick waxy cuticles on their leaf epidermis and the closing of stomata to reduce water loss. Thus, this study investigates the accumulation of Cu in B. gymnorhiza and its effect on leaf morphology and the molecular response under different Cu treatments (0, 200, and 400 mg L⁻¹, Cu0, Cu200, and Cu400, respectively) during a two years stress period. The results show that Cu stress affected accumulation and transport, increased the activities of peroxidase and ascorbate peroxidase, concentrations of soluble sugar, proline, and H2O2, and decreased the activity of catalase and content of malondialdehyde. Also, Cu-induced stress decreased the uptake of phosphorus and nitrogen and inhibited plant photosynthesis, which consequently led to reduced plant growth. Scanning electron microscopy combined with gas chromatography-mass spectrometry showed that B. gymnorhiza leaves had higher wax crystals and compositions under increased Cu stress, which forced the leaf’s stomata to be closed. Also, the contents of alkanes, alcohols, primary alcohol levels (C26:0, C28:0, C30:0, and C32:0), n-Alkanes (C29 and C30), and other wax loads were significantly higher, while fatty acid (C12, C16, and C18) was lower in Cu200 and Cu400 compared to Cu0. Furthermore, the transcriptomic analyses revealed 1240 (771 up- and 469 downregulated), 1000 (723 up- and 277 down-regulated), and 1476 (808 up- and 668 downregulated) differentially expressed genes in Cu0 vs Cu200, Cu0 vs Cu400, and Cu200 vs Cu400, respectively. RNA-seq analyses showed that Cu mainly affected eight pathways, including photosynthesis, cutin, suberin, and wax biosynthesis. This study provides a reference for understanding mangrove response to heavy metal stress and developing novel management practices.

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.

An increased wax load on the leaves of goji plants (Lycium barbarum) results in increased resistance to powdery mildew

Abstract Background Goji (Lycium barbarum) is an important economic crop that is widely cultivated in Northwest China and is known as superfood. Goji plants are often affected by powdery mildew (Arthrocladiella mougeotii) in the field, resulting in considerable reduction in production and serious economic losses. The long-term reliance on agricultural chemicals to control powdery mildew not only leads to environmental pollution, but also causes excessive residues to accumulate in fruits, endangering human health. The plant epicuticular wax is the first physical barrier between land plants and the atmospheric environment, and plays an important role in the interactions of plants with pathogenic microorganisms. However, the relationship between epicuticular wax and powdery mildew resistance in goji plants is not well understood. Results The leaf epicuticular wax crystals of ‘Ningqi I’ goji were dense and small, and the wax load was 121.74 μg/cm2, which was 3.36 times greater than that of ‘Huangguo’ goji. The ratio of alcohol components in ‘Ningqi I’ goji was 121.18% greater than that in ‘Huangguo’ goji. The incidence rate and disease index of ‘Ningqi I’ goji inoculated with powdery mildew were significantly lower than those inoculated ‘Huangguo’ goji, showing high resistance and moderate susceptibility, respectively. After mechanical wax removal, the disease indices of the two goji varieties increased significantly, indicating extreme susceptibility. Seventeen differential expression genes showing significantly greater expression in ‘Ningqi’ goji leaves than in ‘Huangguo’ goji leaves were enriched in genes involved in fatty acid elongation and the wax biosynthesis pathway. The FAR activity and relative expression levels of LbaFAR and LbaWSD1 in ‘Ningqi I’ goji were significantly greater than those in ‘Huangguo’ goji. The relative expression levels of these genes significantly increased in the overexpression tobacco lines FAR-OE and WSD1-OE, and the wax load were augmented by 46.10% and 22.23%, respectively. Conclusions The increased expression of the LbaFAR and LbaWSD1 genes in ‘Ningqi I’ leaves led to increased FAR and WSD1 activity, increasing the wax load and alcohol content of the epicuticular of leaves, and improving leaf glossiness, resulting in strengthened resistance to powdery mildew. Therefore, the production of leaf epicuticular wax mediated by the LbaFAR and LbaWSD1 genes could be the main reason for the difference in powdery mildew resistance between the two goji varieties, which can provide a theoretical basis for the selection of resistant varieties to control goji powdery mildew. Graphical Abstract

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.

Epicuticular wax accumulation and regulation of wax pathway gene expression during bioenergy Sorghum stem development.

Bioenergy sorghum is a drought-tolerant high-biomass C4 grass targeted for production on annual cropland marginal for food crops due primarily to abiotic constraints. To better understand the overall contribution of stem wax to bioenergy sorghum's resilience, the current study characterized sorghum stem cuticular wax loads, composition, morphometrics, wax pathway gene expression and regulation using vegetative phase Wray, R07020, and TX08001 genotypes. Wax loads on sorghum stems (~103-215 µg/cm2) were much higher than Arabidopsis stem and leaf wax loads. Wax on developing sorghum stem internodes was enriched in C28/30 primary alcohols (~65%) while stem wax on fully developed stems was enriched in C28/30 aldehydes (~80%). Scanning Electron Microscopy showed minimal wax on internodes prior to the onset of elongation and that wax tubules first appear associated with cork-silica cell complexes when internode cell elongation is complete. Sorghum homologs of genes involved in wax biosynthesis/transport were differentially expressed in the stem epidermis. Expression of many wax pathway genes (i.e., SbKCS6, SbCER3-1, SbWSD1, SbABCG12, SbABCG11) is low in immature apical internodes then increases at the onset of stem wax accumulation. SbCER4 is expressed relatively early in stem development consistent with accumulation of C28/30 primary alcohols on developing apical internodes. High expression of two SbCER3 homologs in fully elongated internodes is consistent with a role in production of C28/30 aldehydes. Gene regulatory network analysis aided the identification of sorghum homologs of transcription factors that regulate wax biosynthesis (i.e., SbSHN1, SbWRI1/3, SbMYB94/96/30/60, MYS1) and other transcription factors that could regulate and specify expression of the wax pathway in epidermal cells during cuticle development.

The R2R3-MYB transcription factor EVER controls the emission of petunia floral volatiles by regulating epicuticular wax biosynthesis in the petal epidermis.

The epidermal cells of petunia (Petunia × hybrida) flowers are the main site of volatile emission. However, the mechanisms underlying the release of volatiles into the environment are still being explored. Here, using cell-layer-specific transcriptomic analysis, reverse genetics by virus-induced gene silencing and clustered regularly interspaced short palindromic repeat (CRISPR), and metabolomics, we identified EPIDERMIS VOLATILE EMISSION REGULATOR (EVER)-a petal adaxial epidermis-specific MYB activator that affects the emission of volatiles. To generate ever knockout lines, we developed a viral-based CRISPR/Cas9 system for efficient gene editing in plants. These knockout lines, together with transient-suppression assays, revealed EVER's involvement in the repression of low-vapor-pressure volatiles. Internal pools and annotated scent-related genes involved in volatile production and emission were not affected by EVER. RNA-Seq analyses of petals of ever knockout lines and EVER-overexpressing flowers revealed enrichment in wax-related biosynthesis genes. Liquid chromatography/gas chromatography-MS analyses of petal epicuticular waxes revealed substantial reductions in wax loads in ever petals, particularly of monomers of fatty acids and wax esters. These results implicate EVER in the emission of volatiles by fine-tuning the composition of petal epicuticular waxes. We reveal a petunia MYB regulator that interlinks epicuticular wax composition and volatile emission, thus unraveling a regulatory layer in the scent-emission machinery in petunia flowers.

A glossy mutant in onion (Allium cepa L.) shows decreased expression of wax biosynthesis genes.

Cuticular wax is a characteristic feature of land plants that provides protection against both biotic and abiotic stresses. In this study, a glossy mutant lacking an epicuticular wax layer was identified in the γ-irradiated M2 mutant population of the onion cultivar Bhima Super. The inheritance of the mutant's glossy phenotype was determined to be recessive and single locus. Scanning electron microscopy analysis showed poor accumulation of wax crystals in the glossy mutant, concentrated near the stomata. The plant height, number of leaves per plant, and stomatal parameters of the mutant were similar to the wild-type. RNA-seq was used to comprehend the expression variations of waxy cuticle-related genes in the glossy mutant and its wild-type waxy cultivars. Differential gene expression analysis of the RNA-seq data revealed that the genes involved in wax biosynthesis, such as AcCER1, AcCER26, AcMAH1, and AcWSD1, were downregulated by 2.72, 1.74, 2.59 and 2.12-fold, respectively, in the glossy mutant respectively. The expression patterns of these four unigenes were validated using semi-quantitative RT-PCR. The glossy mutant displayed a substantial 3.5-fold reduction in cuticular wax load compared to the wild-type due to the significant downregulation of these wax biosynthesis genes. These findings represent early advancements in understanding the molecular mechanisms of wax biosynthesis in onions. Furthermore, they provide a foundation for utilizing the glossy mutant trait in breeding programmes to enhance stress and pest resilience.

Isolation and characterization of the gene HvFAR1 encoding acyl-CoA reductase from the cer-za.227 mutant of barley (Hordeum vulgare) and analysis of the cuticular barrier functions.

The cuticle is a protective layer covering aerial plant organs. We studied the function of waxes for the establishment of the cuticular barrier in barley (Hordeum vulgare). The barley eceriferum mutants cer-za.227 and cer-ye.267 display reduced wax loads, but the genes affected, and the consequences of the wax changes for the barrier function remained unknown. Cuticular waxes and permeabilities were measured in cer-za.227 and cer-ye.267. The mutant loci were isolated by bulked segregant RNA sequencing. New cer-za alleles were generated by genome editing. The CER-ZA protein was characterized after expression in yeast and Arabidopsis cer4-3. Cer-za.227 carries a mutation in HORVU5Hr1G089230 encoding acyl-CoA reductase (FAR1). The cer-ye.267 mutation is located to HORVU4Hr1G063420 encoding β-ketoacyl-CoA synthase (KAS1) and is allelic to cer-zh.54. The amounts of intracuticular waxes were strongly decreased in cer-ye.267. The cuticular water loss and permeability of cer-za.227 were similar to wild-type (WT), but were increased in cer-ye.267. Removal of epicuticular waxes revealed that intracuticular, but not epicuticular waxes are required to regulate cuticular transpiration. The differential decrease in intracuticular waxes between cer-za.227 and cer-ye.267, and the removal of epicuticular waxes indicate that the cuticular barrier function mostly depends on the presence of intracuticular waxes.

Effect of Cymbopogan citratus Fibre on Physical and Impact Properties of Thermoplastic Cassava Starch/Palm Wax Composites.

Cymbopogan citratus fibre (CCF) is an agricultural waste plant derived from a natural cellulosic source of fibre that can be used in various bio-material applications. This paper beneficially prepared thermoplastic cassava starch/palm wax blends incorporated with Cymbopogan citratus fibre (TCPS/PW/CCF) bio-composites at different CCF concentrations of 0, 10, 20, 30, 40, 50 and 60 wt%. In contrast, palm wax loading remained constant at 5 wt% concentration using the hot moulding compression method. TCPS/PW/CCF bio-composites were characterised in the present paper via their physical and impact properties. The addition of CCF significantly improved impact strength by 50.65% until 50 wt% CCF loading. Furthermore, it was observed that the inclusion of CCF resulted in a little decrement in biocomposite solubility compared to neat TPCS/PW biocomposite from 28.68% to 16.76%. Water absorption showed higher water resistance in the composites incorporating 60 wt.% fibre loading. The TPCS/PW/CCF biocomposites with different fibre contents had 11.04-5.65% moisture content, which was lower than the control biocomposite. The thickness of all samples decreased gradually with increasing fibre content. Overall, these findings provide evidence that CCF waste can be utilised as a high-quality filler in biocomposites due to its diverse characteristics, including improving the properties of biocomposites and strengthening their structural integrity.

Functional characterization of BoGL5 by an efficient CRISPR/Cas9 genome editing system in broccoli

CRISPR/Cas9 has been widely used for precise and efficient genome editing in a number of organisms, notably in major crops, for gene function characterization and trait improvement. However, the application of this genome editing tool is very limited in Brassica oleracea, especially in broccoli (Brassica oleracea var. italica). Here, we report that the genome of broccoli, a worldwide important Brassicaceae vegetable, can also be precisely edited by a CRISPR/Cas9 system with multiple single-guide RNA-expressing cassettes. The gene BoGL5, previously identified as a candidate for the glossy green mutant, was chosen as the target gene. We generated 14 T0 transgenic plants, among which 14.3% and 50% produced mutations at the expected position in targeted regions 1 and 2, respectively. Two of them with a 100% mutation rate showed a glossy green phenotype and dramatically reduced cuticular wax load. These results indicate that CRISPR/Cas9 can be used as a promising technique for gene functional verification and trait improvement in broccoli.

Spent Coffee Grounds Biochar Composite Phase Change Material Design Challenges in a Lab-Scale Solar Water Heater System for Thermal Energy Storage

Thermal energy storage systems that use composite phase change materials (CPCM), such as paraffin wax and nonbiodegradable high-density polyethylene, are gaining attention in recent years due to the effort to resolve energy issues. There is a need to undertake research and development on how to prepare durable CPCMs from thermo-chemically treated biowastes, a renewable resource. Raw spent coffee grounds (SCG) have been experimented on previously, but more research needs to be conducted on CPCMs prepared from pyrolyzed SCG-biochar (SCGB) for use in a water tank. This research investigated a biodegradable CPCM made from SCGB and carnauba wax in a lab-scale solar water heater system. The carnauba wax loading of 60% was chosen due to the minimized thermal wax leakage from the PCM. Thermal characterization results revealed that the latent heat of SCGB CPCM is 88.47 J/g which was found to be competitive compared to other biodegradable CPCMs reported earlier. The results also show further potential for using SCGB and carnauba wax as a CPCM in a thermal energy storage system.

Cranberry fruit epicuticular wax benefits and identification of a wax-associated molecular marker

BackgroundAs the global climate changes, periods of abiotic stress throughout the North American cranberry growing regions will become more common. One consequence of high temperature extremes and drought conditions is sunscald. Scalding damages the developing berry and reduces yields through fruit tissue damage and/or secondary pathogen infection. Irrigation runs to cool the fruit is the primary approach to controlling sunscald. However, it is water intensive and can increase fungal-incited fruit rot. Epicuticular wax functions as a barrier to various environmental stresses in other fruit crops and may be a promising feature to mitigate sunscald in cranberry. In this study we assessed the function of epicuticular wax in cranberries to attenuate stresses associated with sunscald by subjecting high and low epicuticular wax cranberries to controlled desiccation and light/heat exposure. A cranberry population that segregates for epicuticular wax was phenotyped for epicuticular fruit wax levels and genotyped using GBS. Quantitative trait loci (QTL) analyses of these data identified a locus associated with epicuticular wax phenotype. A SNP marker was developed in the QTL region to be used for marker assisted selection.ResultsCranberries with high epicuticular wax lost less mass percent and maintained a lower surface temperature following heat/light and desiccation experiments as compared to fruit with low wax. QTL analysis identified a marker on chromosome 1 at position 38,782,094 bp associated with the epicuticular wax phenotype. Genotyping assays revealed that cranberry selections homozygous for a selected SNP have consistently high epicuticular wax scores. A candidate gene (GL1-9), associated with epicuticular wax synthesis, was also identified near this QTL region.ConclusionsOur results suggest that high cranberry epicuticular wax load may help reduce the effects of heat/light and water stress: two primary contributors to sunscald. Further, the molecular marker identified in this study can be used in marker assisted selection to screen cranberry seedlings for the potential to have high fruit epicuticular wax. This work serves to advance the genetic improvement of cranberry crops in the face of global climate change.

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.

CsCER6 and CsCER7 Influence Fruit Glossiness by Regulating Fruit Cuticular Wax Accumulation in Cucumber.

Fruit glossiness is an important external fruit quality trait that greatly affects the marketability of fresh cucumber (Cucumis sativus) fruits. A few reports have suggested that the extent of cuticular wax loading influences the glossiness of the fruit surface. In the present study, we tested the wax contents of two inbred cucumber lines, comparing a line with waxy fruit (3401) and a line with glossy fruit (3413). Wax content analysis and dewaxing analysis demonstrate that fruit cuticular wax loads negatively correlate with fruit glossiness in cucumber. Identifying genes that were differentially expressed in fruit pericarps between 3401 and 3413 and genes induced by abscisic acid suggested that the wax biosynthesis gene CsCER6 (Cucumis sativus ECERIFERUM 6) and the regulatory gene CsCER7 may affect wax accumulation on cucumber fruit. Expression analysis via RT-qPCR, GUS-staining, and in situ hybridization revealed that CsCER6 and CsCER7 are abundantly expressed in the epidermis cells in cucumber fruits. Furthermore, the overexpression and RNAi lines of CsCER6 and CsCER7 showed dramatic effects on fruit cuticular wax contents, fruit glossiness, and cuticle permeability. Our results suggest that CsCER6 and CsCER7 positively regulate fruit cuticular wax accumulation and negatively influence fruit glossiness.

Dynamic change of tea (Camellia sinensis) leaf cuticular wax in white tea processing for contribution to tea flavor formation

Despite some studies on tea leaf cuticular wax, their component changes during dehydration and withering treatments in tea processing and suspected relation with tea flavor quality formation remain unknown. Here, we showed that tea leaf cuticular wax changed drastically in tea leaf development, dehydration, or withering treatment during tea processing, which affected tea flavor formation. Caffeine was found as a major component of leaf cuticular wax. Caffeine and inositol contents in leaf cuticular wax increased during dehydration and withering treatments. Comparisons showed that tea varieties with higher leaf cuticular wax loading produced more aroma than these with lower cuticular wax loading, supporting a positive correlation between tea leaf cuticular wax loading and degradation with white tea aroma formation. Dehydration or withering treatment of tea leaves also increased caffeine and inositol levels in leaf cuticular wax and triggered cuticular wax degradation into various molecules, that could be related to tea flavor formation. Thus, tea leaf cuticular waxes not only protect tea plants but also contribute to tea flavor formation. The study provides new insight into the dynamic changes of tea leaf cuticular waxes for tea plant protection and tea flavor quality formation in tea processing.

Relationship between Leaf Traits and PM-Capturing Capacity of Major Urban-Greening Species

High concentrations of airborne particulate matter (PM) in urban areas are of great concern to human health. Urban greening has been shown to be an effective and eco-friendly way to alleviate particle pollution, and attention to its role in mitigating particle pollution has increased worldwide. The species-specific PM-capturing capacity of ten urban-greening species in Seoul was evaluated by leaf functional traits (average leaf area (ALA), specific leaf area (SLA), and leaf width-to-length ratio (W/L)), microstructures (roughness, stomata, and trichomes), and physicochemical traits (contact angle (θw), surface free energy (rs), the work of adhesion for water (Wa), and epicuticular wax loads (EWL)). The relationships between leaf traits and PM adsorption by leaves were revealed by Pearson’s correlations and principal component analysis (PCA). A gravimetric method was used to quantify, by particle size, the PM adsorbed on leaf surfaces or embedded in leaf epicuticular wax layers. The key factors for PM adsorption on leaf surfaces were the SLA, the mean roughness value (Ra), and stomatal size. The SLA and Ra of adaxial leaf surfaces were negatively correlated with PM accumulation on leaf surfaces, while stomatal length and width were positively correlated with surface PM load. The rs and EWL positively affected the in-wax PM load. Species-specific PM deposition was the result of complicated mechanisms of various leaf traits. Three evergreen shrub species, Buxus sinica (Rehder & E.H. Wilson) M.Cheng var. insularis (Nakai) M.Cheng, Taxus cuspidata Siebold & Zucc., and Euonymus japonicus Thunb., were efficient in capturing both surface PM and in-wax PM. The PCA revealed that the high PM accumulation efficiency of these three species might be attributable to the interaction between stomatal size and EWL. Aesculus turbinata Blume, Chionanthus retusus Lindl. & Paxton, and Rhododendron schlippenbachii Maxim. had intermediate PM adsorption ability, which might be a result of interactions among stomatal density, the Wa of adaxial surfaces, and ALA. Magnolia denudata Desr., Styphnolobium japonicum (L.) Schott, Liriodendron tulipifera L., and Ginkgo biloba L. had low PM accumulation efficiency. These four species exhibited correlations among SLA, the Ra of adaxial leaf surfaces, and W/L, which had negative effects on PM adsorption.

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.

Temperature has a major effect on the cuticular wax composition of bilberry (Vaccinium myrtillus L.) fruit.

Cuticle is the first layer protecting plants against external biotic and abiotic factors and is responsive to climatic factors as well as determined by genetic adaptations. In this study, the chemical composition of bilberry fruit cuticular wax was investigated through a latitudinal gradient from Latvia (56°N 24°E) through Finland (65°N 25°E) to northern Norway (69°N 18°E) in two seasons 2018 and 2019. Changes in the major cuticular wax compounds, including triterpenoids, fatty acids, alkanes, aldehydes, ketones, and primary alcohols, were detected by GC-MS analysis. Generally, a decreasing trend in the proportion of triterpenoids from southern to northern latitudes, accompanied with an increase in proportion of fatty acids, aldehydes, and alkanes, in bilberry fruit cuticular wax was observed. A correlation analysis between climatic factors with proportion of wax compounds indicated that temperature was the main factor affecting the cuticular wax composition in bilberries. A controlled phytotron experiment with southern and northern bilberry ecotypes confirmed the major effect of temperature on bilberry fruit cuticular wax load and composition. Elevated temperature increased wax load most in berries of northern ecotypes. The level of triterpenoids was higher, while levels of fatty acids and alkanes were lower, in wax of bilberry fruits ripened at 18°C compared to 12°C in both northern and southern ecotypes. Based on our results, it can be postulated that the predicted increase in temperature due to climate change leads to alterations in fruit cuticular wax load and composition. In northern ecotypes, the alterations were especially evident.