Cuticle Development And The Underlying Transcriptome-Metabolome Associations During Early Seedling Establishment.

The plant cuticle is a complex extracellular lipid barrier that has multiple protective functions. We investigated cuticle deposition by integrating metabolomics and transcriptomics data gathered from six different maize seedling organs of four genotypes, the inbred lines B73 and Mo17, and their reciprocal hybrids. These datasets captured the developmental transition of the seedling from heterotrophic skotomorphogenic growth to autotrophic photomorphogenic growth, which is a transition that is highly vulnerable to environmental stresses. Statistical interrogation of these data reveals that the predominant determinant of cuticle composition is seedling organ type, whereas the seedling genotype has a smaller effect on this phenotype. Gene-to-metabolite associations assessed by integrated statistical analyses identified three gene networks connected with the deposition of different elements of the cuticle: a) cuticular waxes; b) monomers of lipidized cell wall biopolymers, including cutin and suberin; and c) both of these elements. These gene networks reveal three metabolic programs that appear to support cuticle deposition, including processes of chloroplast biogenesis, lipid metabolism, and molecular regulation (e.g., transcription factors, post-translational regulators and phytohormones). This study demonstrates the wider physiological metabolic context that can determine cuticle deposition and lays the groundwork for new targets for modulating properties of this protective barrier.

The impact of the GLOSSY2 and GLOSSY2-LIKE BAHD-proteins in affecting the product profile of the maize fatty acid elongase.

The maize glossy2 and glossy2-like genes are homologs, which encode proteins that belong to the BAHD family of acyltransferases. In planta genetic studies have demonstrated that these genes may be involved in the elongation of very long chain fatty acids (VLCFAs) that are precursors of the cuticular wax fraction of the plant cuticle. VLCFAs are synthesized by a fatty acyl-CoA elongase complex (FAE) that consists of four component enzymes. Previously, we functionally identified the maize FAE component enzymes by their ability to complement haploid Saccharomyces cerevisiae strains that carry lethal deletion alleles for each FAE component enzyme. In this study we used these complemented haploid strains and wild-type diploid strains to evaluate whether the co-expression of either GLOSSY2 or GLOSSY2-LIKE with individual maize FAE component enzymes affects the VLCFA product-profile of the FAE system. Wild-type diploid strains produced VLCFAs of up to 28-carbon chain length. Co-expression of GLOSSY2 or GLOSSY2-LIKE with a combination of maize 3-ketoacyl-CoA synthases stimulated the synthesis of longer VLCFAs, up to 30-carbon chain lengths. However, such results could not be recapitulated when these co-expression experiments were conducted in the yeast haploid mutant strains that lacked individual components of the endogenous FAE system. Specifically, lethal yeast mutant strains that are genetically complemented by the expression of maize FAE-component enzymes produce VLCFAs that range between 20- and 26-carbon chain lengths. However, expressing either GLOSSY2 or GLOSSY2-LIKE in these complemented strains does not enable the synthesis of longer chain VLCFAs. These results indicate that the apparent stimulatory role of GLOSSY2 or GLOSSY2-LIKE to enable the synthesis of longer chain VLCFAs in diploid yeast cells may be associated with mixing plant enzyme components with the endogenous FAE complex.

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.

A long non-coding RNA functions as a competitive endogenous RNA to modulate TaNAC018 by acting as a decoy for tae-miR6206.

Increasing evidence indicates a strong correlation between the deposition of cuticular waxes and drought tolerance. However, the precise regulatory mechanism remains elusive. Here, we conducted a comprehensive transcriptome analysis of two wheat (Triticum aestivum) near-isogenic lines, the glaucous line G-JM38 rich in cuticular waxes and the non-glaucous line NG-JM31. We identified 85,143 protein-coding mRNAs, 4,485 lncRNAs, and 1,130 miRNAs. Using the lncRNA-miRNA-mRNA network and endogenous target mimic (eTM) prediction, we discovered that lncRNA35557 acted as an eTM for the miRNA tae-miR6206, effectively preventing tae-miR6206 from cleaving the NAC transcription factor gene TaNAC018. This lncRNA-miRNA interaction led to higher transcript abundance for TaNAC018 and enhanced drought-stress tolerance. Additionally, treatment with mannitol and abscisic acid (ABA) each influenced the levels of tae-miR6206, lncRNA35557, and TaNAC018 transcript. The ectopic expression of TaNAC018 in Arabidopsis also improved tolerance toward mannitol and ABA treatment, whereas knocking down TaNAC018 transcript levels via virus-induced gene silencing in wheat rendered seedlings more sensitive to mannitol stress. Our results indicate that lncRNA35557 functions as a competing endogenous RNA to modulate TaNAC018 expression by acting as a decoy target for tae-miR6206 in glaucous wheat, suggesting that non-coding RNA has important roles in the regulatory mechanisms responsible for wheat stress tolerance.

Dynamic changes to the plant cuticle include the production of volatile cuticular wax-derived compounds.

The cuticle is a hydrophobic structure that seals plant aerial surfaces from the surrounding environment. To better understand how cuticular wax composition changes over development, we conducted an untargeted screen of leaf surface lipids from black cottonwood (Populus trichocarpa). We observed major shifts to the lipid profile across development, from a phenolic and terpene-dominated profile in young leaves to an aliphatic wax-dominated profile in mature leaves. Contrary to the general pattern, levels of aliphatic cis-9-alkenes decreased in older leaves following their accumulation. A thorough examination revealed that the decrease in cis-9-alkenes was accompanied by a concomitant increase in aldehydes, one of them being the volatile compound nonanal. By applying exogenous alkenes to P. trichocarpa leaves, we show that unsaturated waxes in the cuticle undergo spontaneous oxidative cleavage to generate aldehydes and that this process occurs similarly in other alkene-accumulating systems such as balsam poplar (Populus balsamifera) leaves and corn (Zea mays) silk. Moreover, we show that the production of cuticular wax-derived compounds can be extended to other wax components. In bread wheat (Triticum aestivum), 9-hydroxy-14,16-hentriacontanedione likely decomposes to generate 2-heptadecanone and 7-octyloxepan-2-one (a caprolactone). These findings highlight an unusual route to the production of plant volatiles that are structurally encoded within cuticular wax precursors. These processes could play a role in modulating ecological interactions and open the possibility for engineering bioactive volatile compounds into plant waxes.

Reveal the relationship between the quality and the cuticle composition of Satsuma mandarin (Citrus unshiu) by postharvest heat treatment.

To investigate the influence of heat treatment (HT) on Satsuma mandarin fruit's postharvest quality and cuticle composition, we immersed the fruit for 3 min in hot water at 52°C and subsequently stored them at room temperature (25°C) for 28 days, and fruit quality parameters, such as good fruit rate, weight loss rate, firmness, total soluble solids, total titratable acidity, and ascorbic acid content, were monitored. Additionally, changes in the peel's cuticle composition were analyzed, and wax crystal morphologies on the fruit surface were examined using scanning electron microscopy (SEM). The findings revealed that appropriate HT effectively preserved fruit quality. The main compositions of wax and cutin on the fruit's surface remained consistent between the HT and the CK during storage. The total content of wax and cutin initially increased, peaking on the 14th day of storage, and then decreased, falling below the levels observed on day 0. Notably, the total amount of cutin in the HT group exceeded that of the control group. Specifically, ω-hydroxy fatty acids with mid-chain oxo groups and mid-oh-ω-hydroxy fatty acids constituted approximately 90% of the total cutin content. Moreover, the HT group exhibited higher (p < 0.05) total wax content in relation to the control. Fatty acids and alkanes were the predominant components, accounting for approximately 87.5% of the total wax. SEM analysis demonstrated that HT caused wax crystals to melt and redistribute, effectively filling wax gaps. It suggests that HT holds promising potential as a green, safe, and eco-friendly commercial treatment for preserving the postharvest quality of Satsuma mandarin. PRACTICAL APPLICATION: In this study, Satsuma citrus (Citrus unshiu) underwent heat treatment (HT) and was subsequently preserved at room temperature (25°C) for 28 days. The findings revealed that HT significantly improved fruit quality compared to the control group. These findings provide valuable insights into the advancement of eco-friendly and pollution-free citrus preservation methods, offering essential strategies and process parameters for their practical application.

Long-term rain exclusion in a Mediterranean forest: response of physiological and physico-chemical traits of Quercus pubescens across seasons.

With climate change, an aggravation in summer drought is expected in the Mediterranean region. To assess the impact of such a future scenario, we compared the response of Quercus pubescens, a drought-resistant deciduous oak species, to long-term amplified drought (AD) (partial rain exclusion in natura for 10 years) and natural drought (ND). We studied leaf physiological and physico-chemical trait responses to ND and AD over the seasonal cycle, with a focus on chemical traits including major groups of central (photosynthetic pigments and plastoquinones) and specialized (tocochromanols, phenolic compounds, and cuticular waxes) metabolites. Seasonality was the main driver of all leaf traits, including cuticular triterpenoids, which were highly concentrated in summer, suggesting their importance to cope with drought and thermal stress periods. Under AD, trees not only reduced CO2 assimilation (-42%) in summer and leaf concentrations of some phenolic compounds and photosynthetic pigments (carotenoids from the xanthophyll cycle) but also enhanced the levels of other photosynthetic pigments (chlorophylls, lutein, and neoxanthin) and plastochromanol-8, an antioxidant located in chloroplasts. Overall, the metabolomic adjustments across seasons and drought conditions reinforce the idea that Q. pubescens is highly resistant to drought although significant losses of antioxidant defenses and photoprotection were identified under AD.

Cuticular Wax Variability of Abies alba, A.×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.×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 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.×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.

Alteration of surface morphologies and chemical composition of cuticle in response to chilling injury in papaya (Carica papaya L.) after harvest.

The alteration of surface microstructures and chemical composition in cuticle of papaya fruit in response to chilling stress were comparatively studied between cultivars of 'Risheng' and 'Suihuang' after harvest. Fruit surface was covered by fissured wax layers in both cultivars. The presence of granule crystalloids was cultivar dependent, with higher abundance in 'Risheng' and lower in 'Suihuang'. Various typical very-long-chain aliphatics i.e., fatty acids, aldehydes, n-alkanes, primary alcohols, and n-alkenes dominated waxes; and cutin monomers were prominently 9/10,16-dihydroxyhexadecanoic acid in papaya fruit cuticle. Chilling pitting symptom was accompanied by modification of granule crystalloids into flat appearance and decreased primary alcohols, fatty acids, and aldehydes in 'Risheng', but no evident changes in 'Suihuang'. The response of cuticle to chilling injury in papaya fruit might be not directly related to the overall amount of waxes and cutin monomers, but more likely to the alteration of appearance morphologies and chemical composition in cuticle.

Triterpenoids of Three Apple Cultivars-Biosynthesis, Antioxidative and Anti-Inflammatory Properties, and Fate during Processing.

Triterpenoids are a group of secondary plant metabolites, with a remarkable pharmacological potential, occurring in the cuticular waxes of the aerial parts of plants. The aim of this study was to analyze triterpenoid variability in the fruits and leaves of three apple cultivars during the growing season and gain new insights into their health-promoting properties and fate during juice and purée production. The identification and quantification of the compounds of interest were conducted using gas chromatography coupled with mass spectrometry. The waxes of both matrices contained similar analytes; however, their quantitative patterns varied: triterpenic acids prevailed in the fruits, while higher contents of steroids and esterified forms were observed in the leaves. The total triterpenoid content per unit area was stable during the growing season; the percentage of esters increased in the later phases of growth. Antioxidative and anti-inflammatory properties were evaluated with a series of in vitro assays. Triterpenoids were found to be the main anti-inflammatory compounds in the apples, while their impact on antioxidant capacity was minor. The apples were processed on a lab scale to obtain juices and purées. The apple purée and cloudy juice contained only some of the triterpenoids present in the raw fruit, while the clear juices were virtually free of those lipophilic compounds.

Fractionation and Characterization of Triterpenoids from Vaccinium vitis-idaea L. Cuticular Waxes and Their Potential as Anticancer Agents.

Fruit and leaf cuticular waxes are valuable source materials for the isolation of triterpenoids that can be applied as natural antioxidants and anticancer agents. The present study aimed at the semi-preparative fractionation of triterpenoids from cuticular wax extracts of Vaccinium vitis-idaea L. (lingonberry) leaves and fruits and the evaluation of their cytotoxic potential. Qualitative and quantitative characterization of obtained extracts and triterpenoid fractions was performed using HPLC-PDA method, followed by complementary analysis by GC-MS. For each fraction, cytotoxic activities towards the human colon adenocarcinoma cell line (HT-29), malignant melanoma cell line (IGR39), clear renal carcinoma cell line (CaKi-1), and normal endothelial cells (EC) were determined using MTT assay. Furthermore, the effect of the most promising samples on cancer spheroid growth and viability was examined. This study allowed us to confirm that particular triterpenoid mixtures from lingonberry waxes may possess stronger cytotoxic activities than crude unpurified extracts. Fractions containing triterpenoid acids plus fernenol, complexes of oleanolic:ursolic acids, and erythrodiol:uvaol were found to be the most potent therapeutic candidates in the management of cancer diseases. The specificity of cuticular wax extracts of lingonberry leaves and fruits, leading to different purity and anticancer potential of obtained counterpart fractions, was also enclosed. These findings contribute to the profitable utilization of lingonberry cuticular waxes and provide considerable insights into the anticancer effects of particular triterpenoids and pharmacological interactions.

Sugars and cuticular waxes impact sugarcane aphid (Melanaphis sacchari) colonization on different developmental stages of sorghum.

Sugarcane aphid (SCA; Melanaphis sacchari) is a devastating pest of sorghum (Sorghum bicolor) that colonizes sorghum plants at different growth stages. Leaf surface characteristics and sugars often influence aphid settling and feeding on host plants. However, how changes in cuticular waxes and sugar levels affect SCA establishment and feeding at different development stages of sorghum have not been explored. In this study, two- and six-week-old BTx623 plants, a reference line of sorghum, was used to evaluate plant-aphid interactions. Monitoring aphid feeding behavior using Electrical Penetration Graph (EPG) technique revealed that aphids spent more time in the sieve element phase of six-week-old plants compared to two-week-old plants. Significant differences were found in the time spent to reach the first sieve element and pathway phases between the two- and six-week-old plants. However, no-choice aphid bioassays displayed that SCA population numbers were higher in two-week-old plants compared to six-week-old plants. Differences in the abundance of wax and sugar contents were analyzed to determine how these plant components influenced aphid feeding and proliferation. Among the cuticular wax compounds analyzed, α-amyrin and isoarborinone increased after 10 days of aphid infestation only in six-week-old plants. Trehalose content was significantly increased by SCA feeding on two- and six-week-old plants. Furthermore, SCA feeding depressed sucrose content and increased levels of glucose and fructose in two-week-old but not in six-week-old plants. Overall, our study indicates that plant age is a determinant for SCA feeding, and subtle changes in triterpenoids and available sugars influence SCA establishment on sorghum plants.

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.

Chemical Composition of Cuticle and Barrier Properties to Transpiration in the Fruit of Clausena lansium (Lour.) Skeels.

The plant cuticle, as a lipid membrane covering aerial plant surfaces, functions primarily against uncontrolled water loss. Herein, the cuticle chemical composition and the transpiration of wampee fruit (Clausena lansium (Lour.) Skeels) at the green, turning, and yellow stages in cultivars of "Jixin" and "Tianhuangpi" were comprehensively studied. The coverage of wax and cutin monomers per unit of fruit surface area at the green stage was lower in "Jixin" than in "Tianhuangpi" and increased gradually during development. Cutin monomers accumulated ranging from 22.5 µg cm-2 (green) to 52.5 µg cm-2 (turning) in "Jixin" and from 36.5 µg cm-2 (green) to 81.7 µg cm-2 (yellow) in "Tianhuangpi." The total composition of waxes ranged between 6.0 µg cm-2 (green) and 11.1 µg cm-2 (turning) in "Jixin," while they increased from 7.4 µg cm-2 (green) to 16.7 µg cm-2 (yellow) in "Tianhuangpi." Cutin monomers were dominated by ω-, mid-dihydroxy fatty acids (over 40%), followed by multiple monomers of α,ω-dicarboxylic acids with or without added groups, α-monocarboxylic acids with or without ω- or mid-chain hydroxy or mid-epoxy groups, primary alcohols, and phenolics. The very-long-chain (VLC) aliphatic pattern of cuticular waxes was prominently composed of n-alkanes (ranging from 21.4% to 39.3% of total wax content), fatty acids, primary alcohols, and aldehydes. The cyclic waxes were dominated by triterpenoids (between 23.9 and 51.2%), sterols, and phenolics. Water loss in wampee fruit exhibited linear changes over time, indicating an overall monofunctional barrier to transpiration. Permeance for water in wampee fruit was higher at the green stage than at the yellow stage in both "Jixin" and "Tianhuangpi," which showed a negative correlation with the changes of VLC n-alkanes. The results showed the cuticular chemicals, including cutin monomers and waxes, in wampee fruit and further indicated the potential contributions of the cuticular chemical composition to the physiological functions in fruits.

Synthesis of C20-38 Fatty Acids in Plant Tissues.

Very-long-chain fatty acids (VLCFA) are involved in a number of important plant physiological functions. Disorders in the expression of genes involved in the synthesis of VLCFA lead to a number of phenotypic consequences, ranging from growth retardation to the death of embryos. The elongation of VLCFA in the endoplasmic reticulum (ER) is carried out by multiple elongase complexes with different substrate specificities and adapted to the synthesis of a number of products required for a number of metabolic pathways. The information about the enzymes involved in the synthesis of VLCFA with more than 26 atoms of Carbon is rather poor. Recently, genes encoding enzymes involved in the synthesis of both regular-length fatty acids and VLCFA have been discovered and investigated. Polyunsaturated VLCFA in plants are formed mainly by 20:1 elongation into new monounsaturated acids, which are then imported into chloroplasts, where they are further desaturated. The formation of saturated VLCFA and their further transformation into a number of aliphatic compounds included in cuticular waxes and suberin require the coordinated activity of a large number of different enzymes.

Transcriptome and Physiological Analyses of a Navel Orange Mutant with Improved Drought Tolerance and Water Use Efficiency Caused by Increases of Cuticular Wax Accumulation and ROS Scavenging Capacity.

Drought is one of the main abiotic stresses limiting the quality and yield of citrus. Cuticular waxes play an important role in regulating plant drought tolerance and water use efficiency (WUE). However, the contribution of cuticular waxes to drought tolerance, WUE and the underlying molecular mechanism is still largely unknown in citrus. 'Longhuihong' (MT) is a bud mutant of 'Newhall' navel orange with curly and bright leaves. In this study, significant increases in the amounts of total waxes and aliphatic wax compounds, including n-alkanes, n-primary alcohols and n-aldehydes, were overserved in MT leaves, which led to the decrease in cuticular permeability and finally resulted in the improvements in drought tolerance and WUE. Compared to WT leaves, MT leaves possessed much lower contents of malondialdehyde (MDA) and hydrogen peroxide (H2O2), significantly higher levels of proline and soluble sugar, and enhanced superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activities under drought stress, which might reduce reactive oxygen species (ROS) damage, improve osmotic regulation and cell membrane stability, and finally, enhance MT tolerance to drought stress. Transcriptome sequencing results showed that seven structural genes were involved in wax biosynthesis and export, MAPK cascade, and ROS scavenging, and seven genes encoding transcription factors might play an important role in promoting cuticular wax accumulation, improving drought tolerance and WUE in MT plants. Our results not only confirmed the important role of cuticular waxes in regulating citrus drought resistance and WUE but also provided various candidate genes for improving citrus drought tolerance and WUE.

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

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

Increased Cuticle Waxes by Overexpression of WSD1 Improves Osmotic Stress Tolerance in Arabidopsis thaliana and Camelina sativa.

To ensure global food security under the changing climate, there is a strong need for developing 'climate resilient crops' that can thrive and produce better yields under extreme environmental conditions such as drought, salinity, and high temperature. To enhance plant productivity under the adverse conditions, we constitutively overexpressed a bifunctional wax synthase/acyl-CoA:diacylglycerol acyltransferase (WSD1) gene, which plays a critical role in wax ester synthesis in Arabidopsis stem and leaf tissues. The qRT-PCR analysis showed a strong upregulation of WSD1 transcripts by mannitol, NaCl, and abscisic acid (ABA) treatments, particularly in Arabidopsis thaliana shoots. Gas chromatography and electron microscopy analyses of Arabidopsis seedlings overexpressing WSD1 showed higher deposition of epicuticular wax crystals and increased leaf and stem wax loading in WSD1 transgenics compared to wildtype (WT) plants. WSD1 transgenics exhibited enhanced tolerance to ABA, mannitol, drought and salinity, which suggested new physiological roles for WSD1 in stress response aside from its wax synthase activity. Transgenic plants were able to recover from drought and salinity better than the WT plants. Furthermore, transgenics showed reduced cuticular transpirational rates and cuticle permeability, as well as less chlorophyll leaching than the WT. The knowledge from Arabidopsis was translated to the oilseed crop Camelina sativa (L.) Crantz. Similar to Arabidopsis, transgenic Camelina lines overexpressing WSD1 also showed enhanced tolerance to drought stress. Our results clearly show that the manipulation of cuticular waxes will be advantageous for enhancing plant productivity under a changing climate.

Origin and diversification of ECERIFERUM1 (CER1) and ECERIFERUM3 (CER3) genes in land plants and phylogenetic evidence that the ancestral CER1/3 gene resulted from the fusion of pre-existing domains.

ECERIFERUM1 (CER1) and ECERIFERUM3 (CER3) are key genes in synthesis of alkanes, a major component of cuticular waxes in land plants. The genes share extensive similarity, including the N-terminal (ERG3/FAH) and C-terminal (WAX2) domains. This study, traces the origin, evolutionary history, phylogenetic relationships and variation in copy number of the two genes within and beyond the Viridiplantae (green plants). Protein homologs of both CER1 and CER3 were identified across most Embryophyta (land plants), a single homolog (CER1/3) in charophytes and prasinophytes, and none in the other green, red or brown algae. Ancestral state reconstructions in 100 sequenced Archaeplastida using presence/absence of CER1/3 family genes revealed that the CER1/3 gene probably originated in the common ancestor of Viridiplantae. Phylogenetic analysis of CER1 and CER3 protein sequences from 146 plant species strongly suggests that the two genes originated by duplication of CER1/3 in the ancestral embryophyte. The evolution of CER1 and CER3 genes involved differential divergence of the two domains. Outside Embryophyta, CER1/3 similar sequences identified in diatoms and a cryptophyte, were the closest relatives of the CER1/3 family proteins. Proteins harbouring WAX2-wxAR (WAX2 associated region) similar regions were identified in proteins of bacteria, Archaea, cryptophytes, dinoflagellates and Stramenopiles. The independent existence of both ERG3/FAH and WAX2-wxAR domains in diverse lineages strongly points to the origin of CER1/3 gene in green plants by the fusion of pre-existing domains.

Minimum Leaf Conductance (g min) Is Higher in the Treeline of Pinus uncinata Ram. in the Pyrenees: Michaelis' Hypothesis Revisited.

The search for a universal explanation of the altitudinal limit determined by the alpine treeline has given rise to different hypotheses. In this study, we revisited Michaelis' hypothesis which proposed that an inadequate "ripening" of the cuticle caused a greater transpiration rate during winter in the treeline. However, few studies with different explanations have investigated the role of passive mechanisms of needles for protecting against water loss during winter in conifers at the treeline. To shed light on this, the cuticular transpiration barrier was studied in the transition from subalpine Pinus uncinata forests to alpine tundra at the upper limit of the species in the Pyrenees. This upper limit of P. uncinata was selected here as an example of the ecotones formed by conifers in the temperate mountains of the northern hemisphere. Our study showed that minimum leaf conductance in needles from upper limit specimens was higher than those measured in specimens living in the lower levels of the sub-alpine forest and also displayed lower cuticle thickness values, which should reinforce the seminal hypothesis by Michaelis. Our study showed clear evidence that supports the inadequate development of needle cuticles as one of the factors that lead to increased transpirational water losses during winter and, consequently, a higher risk of suffering frost drought.