Cuticular Wax Research Articles

Melatonin treatment delays the softening of blueberry fruit by modulating cuticular wax metabolism and reducing cell wall degradation

The effects of postharvest melatonin (MT) treatment on cuticular wax and cell wall metabolism in blueberry fruit (Vaccinium spp.) were evaluated. The results revealed that MT treatment maintained the cuticular wax rod-like structure and delayed wax degradation. The gas chromatography-mass spectrometry analysis results revealed that MT application changed the cuticular wax composition in blueberries, and 25 metabolic components were screened. The metabolic regulation of wax quality in blueberry fruit may therefore be influenced by MT. Additionally, MT slowed down pectin and cellulose degradation by reducing the activities of cell wall degrading enzymes like pectin methyl esterase polygalacturonase, β-galactosidase, and cellulose in the later stages of storage. It also downregulated the transcriptional expression of related genes like VcPE, VcPG, VcBG6, and VcGAL1. Thus, MT prevented softening and senescence by postponing the degradation of the cell wall in postharvest blueberry fruit.

Effects of konjac glucan-nan/low-acyl gellan edible coatings loaded thymol-β-cyclodextrin microcapsules on postharvest blueberry

This study developed an edible antimicrobial coating using a blend of konjac glucomannan (KGM) and low acyl gellan gum (LAG) hydrogel to incorporate thymol nanoparticles (TKL). The optimized TKL formulation (TKL60) comprised 0.22% thymol microcapsules (TMs), 0.075% total polysaccharide content (KGM:LAG = 1:2), and 99.63% distilled water. When applied to blueberries, TKL60 significantly extended their shelf life to 42 d at 2 ± 0.5 °C, tripling that of control fruit. TKL60 reduced decay rate, weight loss, and respiration rate, delayed softening and senescence during cold storage. It preserved the outer epidermis by retaining cuticular waxes, curbing lipid oxidation, and sustaining defense-related enzyme activities. Flavor analysis revealed altered volatile compound concentrations in TKL60-treated berries, including decreased terpenes and benzaldehyde, and increased esters and aldehydes like 2-methylbutanol, 3-methylbutanol, and linalool. Discriminant Analysis highlighted TKL60′s efficacy in delaying aroma deterioration by over 21 d. TKL60 exhibits potential as a substitute for synthetic coatings and chemical insecticides.

Formation of plant resistance to pathogens involving epigenetic factors and phytohormones

The development of resistance to plant pathogens is determined by the presence of resistance genes and regulation of their activity due to phytohormones, the activity of the nuclear pore complex, and epigenetic modifications at the post-transcriptional and post-translational levels of DNA and histones, respectively. Changes in gene expression due to such modifications can be inherited in generations and contribute to the selection of resistant plants in populations.The nuclear pore complex is composed of nucleoporins, a nuclear pore basket, cytoplasmic filaments and is able to selectively transport transcription factors to the nucleus from the cytoplasm and mRNA in the opposite direction, affecting gene expression. Methylation is the most common and well-studied among the epigenetic modifications. It is described for DNA and histones and ensures genome stability and the availability of transcription factors. Incorporation of methyl groups to amino acid residues is not always a factor in gene silencing for histones. The number of the incorporated groups as well as an amino acid they are attached to are crucial. Histone acetylation is associated with the transition to transcriptional activity.The general control histone acetyltransferase TaGCN5 is able to promote expression of the enoyl-CoA reductase gene in allohexaploid wheat, which is involved in the biosynthesis of cuticular wax as one of the resistance factors. The reverse process of deacetylation acts both as a positive regulator, through ethylene response factors, and a negative one, as it interferes with acetylation and methylation of histone amino acids. Jasmonic and salicylic acids, along with combinations of the mentioned phytohormones, mediate formation of systemic acquired resistance in addition to ethylene. Auxin is a positive phytohormone for pathogens due to its ability to influence the structure of the cell wall. At the same time, pathogens, due to effectors, are able to inhibit the plant’s immune responses, so there is a constant “arms race” resulting in the selection of more effective means of penetration and development in the plant as well as its protective reactions. Investigation of the mechanisms of resistance formation and identifying the main factors of resistance, such as either presence of a gene sequence and/or factors regulating its expression, are of great importance.

Comprehensive analysis of KCS gene family in pear reveals the involvement of PbrKCSs in cuticular wax and suberin synthesis and pear fruit skin formation.

Cuticular wax, cutin and suberin polyesters covering the surface of some fleshy fruit are tightly associated with skin color and appearance.β-Ketoacyl-CoA synthase (KCS) is a rate-limiting enzyme participating in the synthesis of very-long-chain fatty acids (VLCFAs), the essential precursors of cuticular waxes and aliphatic monomers of suberin. However, information on the KCS gene family in pear genome and the specific members involved in pear fruit skin formation remain unclear. In the present study, we performed an investigation of the composition and amount of cuticular waxes, cutin and aliphatic suberin in skins of four sand pear varieties with distinct colors (russet, semi-russet, and green) and demonstrated that the metabolic shifts of cuticular waxes and suberin leading to the significant differences of sand pear skin color. A genome-wide identification of KCS genes from the pear genome was conducted and 35 KCS coding genes were characterized and analyzed. Expression profile analysis revealed that the KCS genes had diverse expression patterns among different pear skins and the transcript abundance of PbrKCS15, PbrKCS19, PbrKCS24, and PbrKCS28 were consistent with the accumulation of cuticular waxes and suberin in fruit skin respectively. Subcellular localization analysis demonstrated that PbrKCS15, PbrKCS19, PbrKCS24 and PbrKCS28 located on the endoplasmic reticulum (ER). Further, transient over-expression of PbrKCS15, PbrKCS19, and PbrKCS24 in pear fruit skins significantly increased cuticular wax accumulation, whereas PbrKCS28 notably induced suberin deposition. In conclusion, pear fruit skin color and appearance are controlled in a coordinated way by the deposition of the cuticular waxes and suberin. PbrKCS15, PbrKCS19, and PbrKCS24 are involved in cuticular wax biosynthesis, and PbrKCS28 is involved in suberin biosynthesis, which play essential roles in pear fruit skin formation. Moreover, this work provides a foundation for further understanding the functions of KCS genes in pear.

A BrLINE1-RUP insertion in BrCER2 alters cuticular wax biosynthesis in Chinese cabbage (Brassica rapa L. ssp. pekinensis).

Glossiness is an important quality-related trait of Chinese cabbage, which is a leafy vegetable crop in the family Brassicaceae. The glossy trait is caused by abnormal cuticular wax accumulation. In this study, on the basis of a bulked segregant analysis coupled with next-generation sequencing (BSA-seq) and fine-mapping, the most likely candidate gene responsible for the glossy phenotype of Chinese cabbage was identified. It was subsequently named Brcer2 because it is homologous to AtCER2 (At4g24510). A bioinformatics analysis indicated a long interspersed nuclear element 1 (LINE-1) transposable element (named BrLINE1-RUP) was inserted into the first exon of Brcer2 in HN19-G via an insertion-mediated deletion mechanism, which introduced a premature termination codon. Gene expression analysis showed that the InDel mutation of BrCER2 reduced the transcriptional expression levels of Brcer2 in HN19-G. An analysis of cuticular waxes suggested that a loss-of-function mutation to BrCER2 in Chinese cabbage leads to a severe decrease in the abundance of very-long-chain-fatty-acids (> C28), resulting in the production of a cauline leaf, inflorescence stem, flower, and pistil with a glossy phenotype. These findings imply the insertion of the LINE-1 transposable element BrLINE1-RUP into BrCER2 can modulate the waxy traits of Chinese cabbage plants.

High-density GBS-based genetic linkage map construction and QTL identification associated with leaf cuticular wax, adaxial stomatal density and leaf water retention capacity in banana

The drought tolerance quantitative trait locus (QTL) in Banana has not been studied to date. In this study, the susceptible parent Musa acuminata ssp. burmannicoides Colla –'Calcutta-4' and the resistant parent Musa balbisiana collection 'Bee hee kela' were crossed to produce an F1 mapping population of 96 individuals for the purpose of mapping drought tolerance traits in banana. Consequently, an entirely novel mapping population was developed using progenitor lines with distinct genomic compositions (A genome and B genome). We employed, the GBS (Genotype By Sequencing) technique to develop a genetic linkage map. The map contained a total of 1015 SNP markers, 11 linkage groups, and a total distance of 4828.88cM. The average distance between markers was 4.9cm. The two primary methods that a plant loses water are through stomata and cuticle transpiration, both of which is not beneficial for the plant. Thus, in the present investigation, the AB mapping population's adaxial stomatal count is measured using a microscopic technique, and leaf cuticular wax was estimated using a spectroscopic method. In addition, the leaf's water retention capacity was estimated. Genetic analysis showed that the drought tolerance traits exhibited moderate to moderately high PCV, GCV, and GAM values, as well as high heritability. Using the GBS-based genetic linkage map and phenotyping information, the QTL analysis identified five main QTLs for drought tolerance on chromosomes 2, 5, and 8. They were designated as wax13_1, wax15_1, wax15_2, lwrc_1, and asd_1. The PVE (per cent) of the identified QTLs varied between 15.9 and 19.8 percent. To our knowledge, this is the first report on the employing of SNP markers to identify QTLs associated with drought tolerance traits in banana. This study's findings will be helpful in fine-mapping and marker-assisted selection in banana for drought related traits.

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.