Cuticular Wax Research Articles

Cuticular Wax Triterpenes Maintain Storage Quality of Blueberries by Reducing Water Loss.

Cuticular wax contributes to maintaining postharvest storage quality against fruit water loss and softening. Triterpenoids, such as oleanolic acid (OA) and ursolic acid (UA), are the main components in blueberry cuticular wax, but their role in water migration during the storage of blueberries remains to be determined. Here, we examined the relationship between the content of OA and UA and the storage quality of blueberry fruit (25 °C). The results revealed that the UA content during eight-day postharvest storage ranged from 58 to 77 μg cm-2, which was negatively related to weight loss. Additionally, we investigated the effect of exogenous OA and UA on water migration in the blueberry fruit during storage at room temperature; the weight loss was significantly lower (by 22%) with UA treatment than in the control fruit. Our findings indicate that OA and UA effectively affect water migration in blueberry fruit during postharvest storage, which could contribute to improving postharvest preservation techniques.

Cuticular Wax Variability of A. alba, A. x borisii-regis and A. cephalonica from the Balkans: Chemophenetic and Ecological Aspects.

This is the first study on cuticular wax variability of Abies alba, A. x borisii-regis and A. cephalonica, using 18 native populations from the assumed hybrid zone in the Balkan Peninsula. Presence of 13 n-alkanes with chain-lengths ranging from C21 to C33, one primary alcohol, two diterpenes, one triterpene and one sterol was determined in n-hexane extracts of 269 needle samples. The multivariate statistical analyses at the population level entirely failed in supporting circumscription of Balkan Abies taxa and therefore in identifying hybrid populations. However, performed at the species level, these analyses revealed a certain tendency of differentiation between A. alba and A. cephalonica, while individuals of A. x borisii-regis were largely overlapped by the clouds of both parent species. Finally, the correlation analysis suggested that the observed variation of wax compounds was probably genetically conditioned and that it does not represent an adaptive response to various environmental factors.

Do the edge effects influence epicuticular wax production in Vismia guianensis (Aubl.) Choisy (Hypericaceae)?

Forest fragmentation leads to various environmental problems. It causes edge effects that can decrease biodiversity, promote the growth of lianas, and affect the production of epicuticular wax. This study focuses on examining the secondary metabolites found in the epicuticular wax of Vismia guianensis leaves and investigating the impact of the edge effect on their production. We extracted and quantified the epicuticular wax and analyzed its constituents using gas chromatography coupled with a mass spectrometer. The amount of cuticular wax extracted from V. guianensis leaves ranged from 33.69 to 631.57 μg/cm². Individuals at the forest edge had the lowest wax content, averaging 250.96 μg/cm². On the other hand, individuals located in the interior of the forest had the highest values, with an average of 340.67 μg/cm² per surface unit. The analysis of the samples identified and quantified nine long-chain alkanes (C27 to C35) in the composition of the cuticular wax. The predominant compound in the wax of all individuals was nonacosane alkane (C29). Interestingly, the individuals within the forest fragment exhibited less diversity of compounds compared to those at the forest edge.

Eceriferum Genes in Tomato (Solanum lycopersicum): Genome-Wide Identification and Expression Analysis Reveal Their Potential Functions during Domestication

Plant cuticular wax plays an important role in resistance to environmental stresses. Eceriferum (CER) genes are involved in wax synthesis. However, little information is available for tomato species. In this study, 26 SlCER genes were identified in tomato (S. lycopersicum), and they were classified into four clades. The physicochemical properties and conserved motifs of their proteins were predicted. These SlCERs were mainly expressed in leaves, flowers or fruits, and most SlCERs played roles in response to abiotic stresses, especially drought stress. Furthermore, the changes in haplotypes indicated that SlCERs might have been involved in adapting to the environments for wild species S. pimpinellifolium before domestication. These findings would lay a foundation for future functional studies of SlCERs and also provide insights for anti-stress improvement in tomato in the near future.

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.

OsFAR1 is involved in primary fatty alcohol biosynthesis and promotes drought tolerance in rice.

OsFAR1 encodes a fatty acyl-CoA reductase involved in biosynthesis of primary alcohols and plays an important role in drought stress response in rice. Cuticular waxes cover the outermost surface of terrestrial plants and contribute to inhibiting nonstomatal water loss and improving plant drought resistance. Primary alcohols are the most abundant components in the leaf cuticular waxes of rice (Oryza sativa), but the biosynthesis and regulation of primary alcohol remain largely unknown in rice. Here, we identified and characterized an OsFAR1 gene belonging to the fatty acyl-CoA reductases (FARs) via a homology-based approach in rice. OsFAR1 was activated by abiotic stresses and abscisic acid, resulting in increased production of primary alcohol in rice. Heterologous expression of OsFAR1 enhanced the amounts of C22:0 and C24:0 primary alcohols in yeast (Saccharomyces cerevisiae) and C24:0 to C32:0 primary alcohols in Arabidopsis. Similarly, OsFAR1 overexpression significantly increased the content of C24:0 to C30:0 primary alcohols on rice leaves. Finally, OsFAR1 overexpression lines exhibited reduced cuticle permeability and enhanced drought tolerance in rice and Arabidopsis. Taken together, our results demonstrate that OsFAR1 is involved in rice primary alcohol biosynthesis and plays an important role in responding to drought and other environmental stresses.

Evaluation of Introgressed Lines of Sunflower (Helianthus annuus L.) under Contrasting Water Treatments

Drought stress is a major production constraint in crops globally. Crop wild relatives are important sources of resistance and tolerance for both biotic and abiotic stresses, respectively. A breeding program was initiated to introgress drought tolerance in sunflowers through hybridization between the wild species Helianthus argophyllus and the cultivated pool of H. annuus. Selection was carried out from the F2 to F5 segregating populations for the silver canopy, high cuticular wax, small leaf area, single heading and high oil content. Cuticular wax ranged between 8.72 µg g−1 and 17.19 µg g−1 in the F5 offspring. The selected F5 breeding lines were self-pollinated to obtain the F6 generation. Thereafter, this F6 was compared with the non-adapted elite sunflower germplasm in a factorial complete randomized design with different water treatments; i.e., comparing fully irrigated (100%, T0) versus 75% (T1), 50% (T2) and 25% (T3) of total irrigation. The comparison between the two types of the germplasm showed that drought-tolerant breeding lines had a comparatively lesser decrease in leaf area (0, 11, 22%) and shoot length (4, 21, 28%) than the elite germplasm, which experienced a decrease in leaf area (21%, 33% and 40%) and shoot length (17, 27 and 34%) under the various drought treatments. Moreover, drought-tolerant breeding lines had 100% more root shoot ratios than the elite germplasm (20%) in T3 when compared with control. Several drought-tolerant promising lines (D-2, D-5 and D-27) were selected due to their high leaf area, great root length and increased root to shoot ratio under T3. Some of the lines could be directly used for the development of drought-tolerant hybrids. Combining ability testing indicated that D-27 (F7) was a good general combiner for seed yield plant−1 and oil content after mating with male-line RSIN.82. Resulting hybrids could help to minimize seed yield loss due to water stress and to achieve profitable cultivation of sunflowers in arid regions of Pakistan.

Genetic variation in ZmWAX2 confers maize resistance to Fusarium verticillioides.

Fusarium verticillioides (F. verticillioides) is a widely distributed phytopathogen that incites multiple destructive diseases in maize, posing a grave threat to corn yields and quality worldwide. However, there are few reports of resistance genes to F. verticillioides. Here, we reveal that a combination of two single nucleotide polymorphisms (SNPs) corresponding to ZmWAX2 gene associates with quantitative resistance variations to F. verticillioides in maize through a genome-wide association study. A lack of ZmWAX2 compromises maize resistance to F. verticillioides-caused seed rot, seedling blight and stalk rot by reducing cuticular wax deposition, while the transgenic plants overexpressing ZmWAX2 show significantly increased immunity to F. verticillioides. A natural occurrence of two 7-bp deletions within the promoter increases ZmWAX2 transcription, thus enhancing maize resistance to F. verticillioides. Upon Fusarium stalk rot, ZmWAX2 greatly promotes the yield and grain quality of maize. Our studies demonstrate that ZmWAX2 confers multiple disease resistances caused by F. verticillioides and can serve as an important gene target for the development of F. verticillioides-resistant maize varieties.

Stone fruit phenolic and triterpenoid compounds modulate gene expression of Monilinia spp. in culture media

Phenolic and triterpenoid compounds are essential components in stone fruit skin and flesh tissues. They are thought to possess general antimicrobial activity. However, regarding brown rot disease, investigations were only confined to a limited number of phenolics, especially chlorogenic acid. The activity of triterpenoids against Monilinia spp., as an essential part of the peach cuticular wax, has not been studied before. In this work, the anti-fungal effect of some phenolics, triterpenoids, and fruit surface compound (FSC) extracts of peach fruit at two developmental stages were investigated on Monilinia fructicola and Monilinia laxa characteristics during in vitro growth. A new procedure for assaying anti-fungal activity of triterpenoids, which are notoriously difficult to assess in vitro because of their hydrophobicity, has been developed. Measurements of colony diameter, sporulation, and germination of second-generation conidia were recorded. Furthermore, the expression of twelve genes of M. fructicola associated with germination and/or appressorium formation and virulence-related genes was studied relative to the presence of the compounds. The study revealed that certain phenolics and triterpenoids showed modest anti-fungal activity while dramatically modulating gene expression in mycelium of M. fructicola on culture medium. MfRGAE1 gene was overexpressed by chlorogenic and ferulic acids and MfCUT1 by betulinic acid, at 4- and 7- days of mycelium incubation. The stage II FSC extract, corresponding to the period when the fruit is resistant to Monilinia spp., considerably up-regulated the MfLAE1 gene. These findings effectively contribute to the knowledge of biochemical compounds effects on fungi on in vitro conditions.

Leaf epicuticular wax as a determinant trait for drought resistance in cereals

The wax coating is an element of the lipid protective layer, the cuticle, which covers the above-ground organs of plants and is the main barrier that prevents non-stomatal water loss. The cuticle helps protect plant surfaces from pathogens and ultraviolet radiation and influences interactions between plants and insects. The accumulation of cuticular wax is one of the mechanisms of adaptation to drought stress. As a result, there are more and more reports on the relationship between cuticular wax and plant resistance to drought. This article deals with the relationship between cuticular waxes and drought resistance in cereals. The paper presents the achievements to date on (i) the relationship between wax biosynthesis and plant response to drought stress using glaucous and nonglaucous near-isogenic lines and on (ii) investigating the role of genes for cuticular wax biosynthesis and transport and the transcription factors that regulate them using mutants with disrupted wax coat formation.

Hydroclimate variations over the last 17,000 years as estimated by leaf waxes in rodent middens from the south-central Atacama Desert, Chile

Leaf cuticular waxes are one of the most important environment-plant interaction structural systems that enable desert plants to withstand extreme climatic conditions. We present a long chain n-alkyl lipids study in fresh plant leaves and rodent palaeomiddens collected along an elevational gradient in the south-central Atacama Desert of Chile, covering six different vegetation belts: Steppe (4500-4000 m asl), Puna (4000-3300 m asl), pre-Puna (3300-2400 m asl), Absolute Desert (2400-1000 m asl) and Coastal Desert (1000-0 m asl). The 28 rodent palaeomiddens analyzed from Quebrada Incahuasi (25.6 °S, 3600 m asl) span the last 17,000 years. Modern-day distribution of long-chain n-alkanes and n-alkanoic acids varies among the dominant plant associations of the Atacama Desert. These plants show a species-specific chemotaxonomy linked to the climatic conditions. Furthermore, differences in average chain length (ACL) and carbon preference index (CPI) suggest that these plant communities are highly adapted to extreme environmental conditions. The sum of leaf wax n-alkanes was highest under wet conditions, while n-alkanoic acids (between n-C24 and n-C28) increased with hyperaridity. Similarly, analysis of n-alkane time series from palaeomiddens showed that the greatest changes in leaf wax n-alkane distributions (ACL and CPI) corresponded to the greatest increases in moisture during the Central Andean Pluvial Event (CAPE; between 18 and 9 ka cal BP) and the Late Holocene. The shift in the palaeomidden n-alkane distributions is corroborated by the relative abundance of rainfall-dependent extra-local taxa. This is the first study to report leaf wax content obtained from ancient rodent middens, and shows promising results as a robust hydroclimate proxy for the Atacama Desert region.

Bacterium-enabled transient gene activation by artificial transcription factors for resolving gene regulation in maize.

Understanding gene regulatory networks is essential to elucidate developmental processes and environmental responses. Here, we studied regulation of a maize (Zea mays) transcription factor gene using designer transcription activator-like effectors (dTALes), which are synthetic Type III TALes of the bacterial genus Xanthomonas and serve as inducers of disease susceptibility gene transcription in host cells. The maize pathogen Xanthomonas vasicola pv. vasculorum was used to introduce 2 independent dTALes into maize cells to induced expression of the gene glossy3 (gl3), which encodes a MYB transcription factor involved in biosynthesis of cuticular wax. RNA-seq analysis of leaf samples identified, in addition to gl3, 146 genes altered in expression by the 2 dTALes. Nine of the 10 genes known to be involved in cuticular wax biosynthesis were upregulated by at least 1 of the 2 dTALes. A gene previously unknown to be associated with gl3, Zm00001d017418, which encodes aldehyde dehydrogenase, was also expressed in a dTALe-dependent manner. A chemically induced mutant and a CRISPR-Cas9 mutant of Zm00001d017418 both exhibited glossy leaf phenotypes, indicating that Zm00001d017418 is involved in biosynthesis of cuticular waxes. Bacterial protein delivery of dTALes proved to be a straightforward and practical approach for the analysis and discovery of pathway-specific genes in maize.

Elucidation of Physiological, Transcriptomic and Metabolomic Salinity Response Mechanisms in Medicago sativa.

Alfalfa (Medicago sativa L.) is a widely grown perennial leguminous forage crop with a number of positive attributes. However, despite its moderate ability to tolerate saline soils, which are increasing in prevalence worldwide, it suffers considerable yield declines under these growth conditions. While a general framework of the cascade of events involved in plant salinity response has been unraveled in recent years, many gaps remain in our understanding of the precise molecular mechanisms involved in this process, particularly in non-model yet economically important species such as alfalfa. Therefore, as a means of further elucidating salinity response mechanisms in this species, we carried out in-depth physiological assessments of M. sativa cv. Beaver, as well as transcriptomic and untargeted metabolomic evaluations of leaf tissues, following extended exposure to salinity (grown for 3-4 weeks under saline treatment) and control conditions. In addition to the substantial growth and photosynthetic reductions observed under salinity treatment, we identified 1233 significant differentially expressed genes between growth conditions, as well as 60 annotated differentially accumulated metabolites. Taken together, our results suggest that changes to cell membranes and walls, cuticular and/or epicuticular waxes, osmoprotectant levels, antioxidant-related metabolic pathways, and the expression of genes encoding ion transporters, protective proteins, and transcription factors are likely involved in alfalfa's salinity response process. Although some of these alterations may contribute to alfalfa's modest salinity resilience, it is feasible that several may be disadvantageous in this context and could therefore provide valuable targets for the further improvement of tolerance to this stress in the future.

Leaf Cuticular Waxes of Bermudagrass Response to Environment-Driven Adaptations of Climate Effect Inferred from Latitude and Longitude Gradient in China.

Bermudagrass (Cynodon dactylon) is a widely used warm season lawn grass. Cuticular wax covering the surface of plant leaves plays an important role in helping plants resist biotic and abiotic stresses. We analyzed the changes of cuticle wax in 25 bermudagrass populations from different longitude and latitude gradients, in order to verify how environmental conditions affect the structure and chemical composition of cuticle wax. Five wax components were identified, including alkanes, esters, alkenes, aldehydes and primary alcohols. The wax characteristics were divided into two principal components, explaining 58.2 % and 66.7 % of the total variability in latitude and longitude, even some populations had a certain correlation with each other. Pearson correlation analysis further showed that the total wax coverage, wax component content and antioxidant enzyme activity of bermudagrass populations on the latitudinal gradient had different responses to environmental factors. Finally, nineteen key genes involved in wax biosynthesis, redox and photosynthesis were identified and verified by RT-qPCR. The results showed that the responses of bermudagrass in different populations to climate change were quite different, which was of great significance for the evolution of bermudagrass populations.

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.

Genome-Wide Investigation and Functional Analysis Reveal That CsKCS3 and CsKCS18 Are Required for Tea Cuticle Wax Formation.

Cuticular wax is a complex mixture of very long-chain fatty acids (VLCFAs) and their derivatives that constitute a natural barrier against biotic and abiotic stresses on the aerial surface of terrestrial plants. In tea plants, leaf cuticular wax also contributes to the unique flavor and quality of tea products. However, the mechanism of wax formation in tea cuticles is still unclear. The cuticular wax content of 108 germplasms (Niaowang species) was investigated in this study. The transcriptome analysis of germplasms with high, medium, and low cuticular wax content revealed that the expression levels of CsKCS3 and CsKCS18 were strongly associated with the high content of cuticular wax in leaves. Hence, silencing CsKCS3 and CsKCS18 using virus-induced gene silencing (VIGS) inhibited the synthesis of cuticular wax and caffeine in tea leaves, indicating that expression of these genes is necessary for the synthesis of cuticular wax in tea leaves. The findings contribute to a better understanding of the molecular mechanism of cuticular wax formation in tea leaves. The study also revealed new candidate target genes for further improving tea quality and flavor and cultivating high-stress-resistant tea germplasms.

Genome sequence and salinity adaptation of the desert shrub Nitraria sibirica (Nitrariaceae, Sapindales)

The genetic bases of halophytes for salinity tolerance are crucial for genetically breeding salt-tolerant crops. All natural Nitrariaceae species that exclusively occur in arid environments are highly tolerant to salt stress, but the underlying genomic bases to this adaptation remain unknown. Here we present a high-quality, chromosome-level genome sequence of Nitraria sibirica, with an assembled size of 456.66Mb and 23,365 annotated genes. Phylogenomic analyses confirmed N. sibirica as the sister to all other sampled representatives from other families in Sapindales, and no lineage-specific whole-genome duplication was found except the gamma triplication event. Still, we found that the genes involved in K + retention, energy supply, and Fe absorption expanded greatly in N. sibirica. Deep transcriptome analyses showed that leaf photosynthesis and cuticular wax formation in roots were enhanced under salt treatments. Furthermore, many transcription factors involved in salt tolerance changed their expressions significantly and displayed tissue- and concentration-dependent signaling in response to salt stress. Additionally, we found vacuolar Na + compartmentalization is an ongoing process under salt treatment, while Na + exclusion tends to function at high salt concentrations. These genomic and transcriptomic changes conferred salt tolerance in N. sibirica and pave the way for future breeding of salt-tolerant crops.

Post-processing of a lavender flowers solvent extract using supercritical CO2 fractionation

BackgroundSupercritical Fluid Extraction (SFE) was performed using an unconventional material, the solid extract of lavender flowers obtained by liquid solvent extraction and subsequent solvent elimination. MethodsSystematic extraction experiments were carried out at 8 MPa and 40 °C, and the extract was fractionated in semi-continuous mode in the SFE plant. To understand mass transfer phenomena driving the process, CO2 mass flow rates ranging between 0.60 and 1.50 kg/h were used, and yield vs. time curves were obtained. Significant findingsFractionation produced a cuticular waxes selective precipitation in the first separator and the floral fragrance in the second separator. The most abundant species in the extract were τ-cadinol (13%), lavandulol (10.5%), β-caryophyllene (10%), viridiflorene (8.5%), isocaryophyllene (6%), cedrenalol (4.5%), linalool (4%) and 1,8-cineol (4%). The fragrance contained no waxes, indicating that the fractionation was successful. At higher mass flow rates (from 0.90 to 1.50 kg/h), an asymptotic extraction yield of 5.2% w/w was obtained; whereas, at lower mass flow rates, the extraction yield was lower (2.3% w/w) since the vegetable bed was not completely wetted by the extraction fluid. The overall results indicated that an external mass transfer resistance controlled the process.

Uptake, Translocation, and Terminal Residue of Chlorantraniliprole and Difenoconazole in Rice: Effect of the Mixed-Application with Adjuvant

Plant oil adjuvants are widely used to improve the utilization rate of pesticides. In this study, the uptake, translocation, and terminal residue of chlorantraniliprole and difenoconazole spraying with plant oil adjuvant in rice (Oryza sativa L.) were evaluated. After being mixed with the tank-mixed plant oil adjuvant, the cuticular wax of rice leaf was destroyed, which decreased the hydrophobicity of the rice leaf and facilitated the wetting, spreading, and penetration of pesticides onto the rice leaf. Additionally, the adjuvant promoted the translocation of difenoconazole from leaves to stems, but had little effect on the translocation of difenoconazole from leaves to roots, while inhibiting chlorantraniliprole translocation. Although adjuvant increased the initial deposition of chlorantraniliprole and difenoconazole on rice, the terminal residue was not significantly affected. The findings can promote the safe use of chlorantraniliprole and difenoconazole in rice production, especially when used with plant oil adjuvants. In the future, studies on more rice cultivars will be necessary to determine the generality of the conclusions.

Chemotaxonomic Differentiation of Pinus Species Based on n-Alkane and Long-Chain Alcohol Profiles of Needle Cuticular Waxes.

n-Alkane and long-chain alcohol (LCOH) profiles of needle cuticular waxes of nine provenances of three Pinus species (P. taeda. P. pinaster and P. pinea) were determined and their chemotaxonomic importance was studied. n-Alkanes concentrations were very low in all Pinus spp. A lack of ability of these compounds to differentiate Pinus spp. and their provenances was observed. LCOH concentrations were much higher, being nonacosan-10-ol (10-C29 -OH) the most abundant one in all Pinus spp. Total LCOH concentrations varied (P<0.001) between Pinus spp. with P. taeda presenting the highest (P<0.05) value and P. pinea the lowest one. Differences in LCOH profiles were large (P<0.001) and allowed separation of Pinus spp. in the Principal Component Analysis (PCA). Although a noticeable separation of individuals was not observed. cluster analysis on LCOH concentrations allowed a clear distinction between species, indicating their potential to be used as chemotaxonomic markers to differentiate provenances of different Pinus spp.