Wax Load Research Articles

Eco-physiological responses of Aeluropus lagopoides (grass halophyte) and Suaeda nudiflora (non-grass halophyte) under individual and interactive sodic and salt stress

Aeluropus lagopoides (grass halophyte) and Suaeda nudiflora (non-grass halophyte) collected from extreme saline sodic Kachchh plains, Bhuj (Gujrat), were established in micro-plot research facility of CSSRI, Karnal, to evaluate their eco-physiological responses under different salt stresses. The experiment was designed in split plot design (two factorial randomized complete block design) having 2 halophytes and 9 different treatments of salinity/sodicity, i.e., control (pH2 7.1; ECe: 0.56), sodic (pH2 9.5 and 10.0), saline (ECe: 15, 25, 35 dS m−1), and saline sodic (pH2 9.0 with ECe: 10, 15, 20 dS m−1). Eco-physiological responses were measured in terms of gas exchange attributes, chlorophyll fluorescence, ionic relations (Na+, K+, and Cl− content) and biochemicals (total soluble sugars, total soluble protein, proline content, epicuticular wax load, and peroxidase activity). Both these halophytes showed maximum gas exchange properties under unstressed control conditions. The highest photosynthetic rate (34.5 and 33.5μmol CO2 m−2s−1) was recorded in control treatment, which was decreased with the intensified stress and reduced to minimum under stress condition of pH2 9.0+ECe 20 dS m−1 (18.1 and 16.9μmol CO2 m−2s−1) in A.lagopoides and S.nudiflora, respectively. Reductions were also noticed in the rates of stomatal conductance and transpiration rate under different saline/sodic levels. As the stress conditions prevailed, these grass and non-grass halophytes accumulated higher amount of Na+ and Cl− in their leaves. Aeluropus accumulated 10.23% Na+ at ECe 35 dS m−1, which was approximately 6 times higher than control (1.65%) and 2 times than pH2 9.0+ECe 20 dS m−1 (5.8%) stress level, whereas S. nudiflora accumulated 2.75% Na+ in control, which increased to 17.33% at ECe 35 dS m−1 and 22.25% at pH2 9.0+ECe 20 dS m−1 treatment. Chloride content showed similar trend of increase. Increased accumulation of K+, i.e., 103.3% and 39.5%, was observed at ECe 35dS m−1 in A. lagopoides and S. nudiflora, respectively, with respect to control treatment. TSS content was decreased under sodic environment, while increasing pattern was observed under salinity and combined stress. Maximum protein accumulation of 23.24mg/gF.W. was observed at ECe 35 dS m−1 in A. lagopoides while in S. nudiflora (19.18mg/gF.W.) at pH2 9.0+ECe 20 dS m−1. Approximately 10 times higher proline accumulation was observed in both halophytes with increasing stress conditions, which showed higher osmotic adaptations. This study confers the eco-physiological potential of salt tolerance in both the halophytes and these could be used as good material for forages under salt-affected environments.

Variability in Epicuticular Wax in 35 Woody Plants in Linares, Northeast Mexico

A study has been undertaken on epicuticular wax on the leaves of 35 woody species in Linares, Northeast Mexico at the experimental station of Facultad de Ciencias Forestales, Universidad Autonoma de Nuevo Leon, located in the municipality of Linares. Considerable variation in wax accumulation was found among species showing prominent interspecific variation. Wax load varied from 11.18 to 702.04 μg/cm2 among species studied during summer. Few species selected with high epicuticular wax viz, Forestiera angustifolia (702.04 μg/cm2), Diospyros texana (607.65 μg/cm2), Bernardia myricifolia (437.53 μg/cm2), Leucophylum leucocephala (388.50 μg/cm2), during summer which could well be adapted under semi-arid environments for their efficiency in the reflection of radiation load, reduced transpiration, gas exchange and probably impart drought resistance. The large variations in epicuticular wax could be related to their physiological functions such as transpiration, gas exchange, water relations etc.

Biodiversity in Leaf Chemistry (Pigments, Epicuticular Wax and Leaf Nutrients) in Woody Plant Species in North-eastern Mexico, a Synthesis

The leaves of trees and shrubs possess various chemical components such as leaf pigments, epicuticular wax and various macro and micronutrients. These components influence the growth, development and productivity and adaptation of the species to environments. The results of studies undertaken on the more than 30 woody species in North-eastern Mexico results reveal that there exist large variations in pigments (chlorophyll, carotenoids), epicuticular wax and macro and micro nutrients Several species have been selected with high values of the pigments, epicuticular wax and various macro and micronutrients. For example, species with high Chlorophyll a were: Ebenopsis ebano (1.755), Cercidium suveoleon (0.589), Amyrys texana (1.66), and those with high chlorophyll b were Ebenopsis ebano (0.398), Amyrys texana (1.66) and species with high Chlorophyll total (Chl a + b) were: Ebenopsis ebano (2.253), Leucaena leucocephala (1.687). Species with high carptenoids were: Berberis chococo (0.585), Diospyros palmeri (0.433. The species showing high epicuticular wax load are Forestiera angustifolia (702.04 μg/cm2), Diospyros texana (607.65 μg/cm2), Bernardia myricifolia (437.53 μg/cm2). There is a here is a need to conform the efficiency of these selected species for productivity and adaptation of the species to environment.

Barrier against water loss: relationship between epicuticular wax composition, gene expression and leaf water retention capacity in banana.

In the present study we examined 13 banana (Musa spp.) genotypes belonging to different genomic groups with respect to total leaf cuticular wax concentration, chemical composition, carbon chain length and their relationship with leaf water retention capacity (LWRC). A positive correlation between epicuticular wax content and LWRC clearly indicated that the cuticular wax plays an important role in maintaining banana leaf water content. The classification of hexane soluble cuticular wax components into different classes based on functional group and their association with LWRC showed that alcohol and ester compounds have a positive correlation. Further, the compounds with >C28 carbon chain length had a positive correlation with LWRC, indicating the role of longer carbon chain length in maintaining the water status of banana leaves. Also, the gene expression analysis showed higher expression of the wax biosynthetic genes FATB and KCS11 in higher wax load genotypes whereas lower expression was seen in low wax banana genotypes. Here, we report for the first time the compositional variations of cuticular wax in different banana genotypes, followed by their association with leaf water retention capacity. The results were also supported by variation in gene expression analysis of cuticular wax biosynthetic genes - FATB and KCS11.

Shape stabilised phase change materials based on a high melt viscosity HDPE and paraffin waxes

Shape stabilised phase change materials (SSPCMs) based on a high density poly(ethylene)(hv-HDPE) with high (H-PW, Tm=56–58°C) and low (L-PW, Tm=18–23°C) melting point paraffin waxes were readily prepared using twin-screw extrusion. The thermo-physical properties of these materials were assessed using a combination of techniques and their suitability for latent heat thermal energy storage (LHTES) assessed. The melt processing temperature (160°C) of the HDPE used was well below the onset of thermal decomposition of H-PW (220°C), but above that for L-PW (130°C), although the decomposition process extended over a range of 120°C and the residence time of L-PW in the extruder was <30s. The SSPCMs prepared had latent heats up to 89J/g and the enthalpy values for H-PW in the respective blends decreased with increasing H-PW loading, as a consequence of co-crystallisation of H-PW and hv-HDPE. Static and dynamic mechanical analysis confirmed both waxes have a plasticisation effect on this HDPE. Irrespective of the mode of deformation (tension, flexural, compression) modulus and stress decreased with increased wax loading in the blend, but the H-PW blends were mechanically superior to those with L-PW.

Developmental Changes in Composition and Morphology of Cuticular Waxes on Leaves and Spikes of Glossy and Glaucous Wheat (Triticum aestivum L.).

The glossy varieties (A14 and Jing 2001) and glaucous varieties (Fanmai 5 and Shanken 99) of wheat (Triticum aestivum L.) were selected for evaluation of developmental changes in the composition and morphology of cuticular waxes on leaves and spikes. The results provide us with two different wax development patterns between leaf and spike. The general accumulation trend of the total wax load on leaf and spike surfaces is first to increase and then decrease during the development growth period, but these changes were caused by different compound classes between leaf and spike. Developmental changes of leaf waxes were mainly the result of variations in composition of alcohols and alkanes. In addition, diketones were the third important contributor to the leaf wax changes in the glaucous varieties. Alkanes and diketones were the two major compound classes that caused the developmental changes of spike waxes. For leaf waxes, β- and OH-β-diketones were first detected in flag leaves from 200-day-old plants, and the amounts of β- and OH-β-diketones were significantly higher in glaucous varieties compared with glossy varieties. In spike waxes, β-diketone existed in all varieties, but OH-β-diketone was detectable only in the glaucous varieties. Unexpectedly, the glaucous variety Fanmai 5 yielded large amounts of OH-β-diketone. There was a significant shift in the chain length distribution of alkanes between early stage leaf and flag leaf. Unlike C28 alcohol being the dominant chain length in leaf waxes, the dominant alcohol chain length of spikes was C24 or C26 depending on varieties. Epicuticular wax crystals on wheat leaf and glume were comprised of platelets and tubules, and the crystal morphology changed constantly throughout plant growth, especially the abaxial leaf crystals. Moreover, our results suggested that platelets and tubules on glume surfaces could be formed rapidly within a few days.

Cuticular wax variation in the tomato (Solanum lycopersicum L.), related wild species and their interspecific hybrids

The tomato (Solanum lycopersicum L.) is one of the world's most important vegetable crop species. Among the many tomato accessions available, only a few are tolerant to abiotic stresses, which are responsible for the majority of the crop losses worldwide. Wild tomato species are then secondary gene pool in the breeding of more resistant tomato cultivars. In the current study, the composition of leaf cuticular waxes from fourteen tomato accessions, including S. lycopersicum, Solanum pennellii, Solanum pimpinellifolium, and their interspecific hybrids was studied in order to select the most adequate chemotaxonomic markers. Total cuticular wax load of S. pennellii plants was much higher than in the other plant species. Hydrocarbons were usually the most abundant wax components, followed by minor quantities of triterpenes and other compounds. Interspecific hybrids showed intermediate wax characteristics. The amount and composition of surface waxes were not correlated with the abiotic stress tolerance in S. lycopersicum. The composition of the hydrocarbon fraction was the least variable both within a single accession and between all the plants studied. Based on the results, cuticular hydrocarbons are proposed as potential chemotaxonomic markers in the classification of tomato and related species.

Leaf water status, osmoregulation and secondary metabolism as a model for depicting drought tolerance in Argania spinosa

The present investigation was undertaken to characterise and to distinguish four contrasting Argania spinosa ecotypes in terms of drought tolerance by exploring the changes of leaf water status, osmoregulation and secondary metabolism. A. spinosa plants corresponding to four contrasting ecotypes (Lks, Alz, Rab and Adm) were subjected to drought stress. The results exhibited that there was a significant decrease in predawn leaf water potential (Ψpd), stomatal conductance (g s) and leaf relative water content under the influence of the intensity and duration of drought stress. Negative and significant correlations were recorded between epicuticular wax load (EWL) and residual transpiration rate. Electrolyte leakage (EL) increased significantly in leaves of plants under drought stress treatment compared to control plants. Furthermore, our data revealed that drought stress can induce shikimate and phenylpropanoid pathways in A. spinosa. A significant induction of phenylalanine ammonium lyase (PAL), shikimate dehydrogenase (SKDH) and cinnamate 4-hydroxylase (C4H) enzymes and an increase in polyphenol content were recorded, of which Lks showed the highest induction and accumulation among ecotypes. Accumulation of polyphenols was positively correlated with the SKDH, PAL and C4H activities. The strong induction of secondary metabolism in Lks might be linked to its better ability of drought tolerance. The proline and soluble sugar content in leaves of all ecotypes increased substantially in parallel with the severity of stress-induced. According to canonical discriminant analysis of our data, the four ecotypes were separated by the following physiological and biochemical parameters: EL, g s, EWL, soluble sugars and polyphenols.

OsGL1-3 is involved in cuticular wax biosynthesis and tolerance to water deficit in rice.

Cuticular wax covers aerial organs of plants and functions as the outermost barrier against non-stomatal water loss. We reported here the functional characterization of the Glossy1(GL1)-homologous gene OsGL1-3 in rice using overexpression and RNAi transgenic rice plants. OsGL1-3 gene was ubiquitously expressed at different level in rice plants except root and its expression was up-regulated under ABA and PEG treatments. The transient expression of OsGL1-3–GFP fusion protein indicated that OsGL1-3 is mainly localized in the plasma membrane. Compared to the wild type, overexpression rice plants exhibited stunted growth, more wax crystallization on leaf surface, and significantly increased total cuticular wax load due to the prominent changes of C30–C32 aldehydes and C30 primary alcohols. While the RNAi knockdown mutant of OsGL1-3 exhibited no significant difference in plant height, but less wax crystallization and decreased total cuticular wax accumulation on leaf surface. All these evidences, together with the effects of OsGL1-3 on the expression of some wax synthesis related genes, suggest that OsGL1-3 is involved in cuticular wax biosynthesis. Overexpression of OsGL1-3 decreased chlorophyll leaching and water loss rate whereas increased tolerance to water deficit at both seedling and late-tillering stages, suggesting an important role of OsGL1-3 in drought tolerance.

Effect of drought stress on epicuticular wax load in peanut genotypes

The changes in epicuticular wax load (EWL) and the leaf temperature in two peanut genotypes such as JL-24 (drought susceptible) and K-9 (drought tolerant) was studied under drought stress and its recovery. The drought stress was induced at different soil moisture level (100%, 60%, 40% and 20% FC) for 7 days and subsequent recovery for 7 days. In both JL-24 and K-9 genotypes, the EWL was found to be increase with the increase in the intensity of drought stress. However, the increase in EWL in K-9 genotype was found to be significantly higher than JL-24 genotype. Moreover, the leaf temperature was also found to be increased with the increase in the intensity of drought stress and the degree of increase in leaf temperature was more pronounced in JL-24 compared to K-9 genotype. Further, after the recovery of drought stress, the EWL and leaf temperature decreased consistently in both the genotypes. The results are discussed in terms of the differences in EWL and temperature of leaf in two genotypes studied under drought stress.

Effects of Acute Moisture Stress on Creeping Bentgrass Cuticle Morphology and Associated Effects on Foliar Nitrogen Uptake

Foliar fertilization is a common practice to deliver nitrogen (N) to turfgrasses. The mechanisms of foliar applied nutrient uptake, particularly the effects of the leaf cuticle layer, have not been clearly characterized in turfgrasses. The objectives of this study were to determine the effect of acute moisture stress on the morphological and compositional components of the cuticle and the resulting effect on foliar-applied N absorption. Creeping bentgrass (Agrostis stolonifera L.) was irrigated to return 100% or 50% evapotranspiration rate (ET) for 10 days to examine cuticular modifications resulting from acute moisture stress and foliar N uptake with and without a surfactant. Acute water stress increased the total cuticle wax by 11%, mostly as a result of the compound 1-hexacosanol, and increased crystalloid density creating a rougher leaf surface. The 50% ET treatment significantly reduced recovery of 15N-labeled urea by 14%, which was attributed to the increased total cuticle wax and crystalloid density making the surface less receptive to foliar applications. The surfactant addition to the urea solution increased 15N-labeled urea recovery by 21% and absorption of 15N in 50% ET plants to levels consistent with the 100% ET plants. These results suggest that acute moisture stress modifies the cuticle wax load and morphology, thereby hindering foliar absorption; however, a surfactant addition can help to mitigate this effect and increase absorption of N.

Cuticular wax biosynthesis is up-regulated by the MYB94 transcription factor in Arabidopsis.

The aerial parts of all land plants are covered with hydrophobic cuticular wax layers that act as the first barrier against the environment. The MYB94 transcription factor gene is expressed in abundance in aerial organs and shows a higher expression in the stem epidermis than within the stem. When seedlings were subjected to various treatments, the expression of the MYB94 transcription factor gene was observed to increase approximately 9-fold under drought, 8-fold for ABA treatment and 4-fold for separate NaCl and mannitol treatments. MYB94 harbors the transcriptional activation domain at its C-terminus, and fluorescent signals from MYB94:enhanced yellow fluorescent protein (eYFP) were observed in the nucleus of tobacco epidermis and in transgenic Arabidopsis roots. The total wax loads increased by approximately 2-fold in the leaves of the MYB94-overexpressing (MYB94 OX) lines, as compared with those of the wild type (WT). MYB94 activates the expression of WSD1, KCS2/DAISY, CER2, FAR3 and ECR genes by binding directly to their gene promoters. An increase in the accumulation of cuticular wax was observed to reduce the rate of cuticular transpiration in the leaves of MYB94 OX lines, under drought stress conditions. Taken together, a R2R3-type MYB94 transcription factor activates Arabidopsis cuticular wax biosynthesis and might be important in plant response to environmental stress, including drought.

DEWAX-mediated transcriptional repression of cuticular wax biosynthesis in Arabidopsis thaliana

The aerial parts of plants are covered with a cuticular wax layer, which is the first barrier between a plant and its environment. Although cuticular wax deposition increases more in the light than in the dark, little is known about the molecular mechanisms underlying the regulation of cuticular wax biosynthesis. Recently DEWAX (Decrease Wax Biosynthesis) encoding an AP2/ERF transcription factor was found to be preferentially expressed in the epidermis and induced by darkness. Wax analysis of the dewax knockout mutant, wild type, and DEWAX overexpression lines (OX) indicates that DEWAX is a negative regulator of cuticular wax biosynthesis. DEWAX represses the expression of wax biosynthetic genes CER1, LACS2, ACLA2, and ECR via direct interaction with their promoters. Cuticular wax biosynthesis is negatively regulated twice a day by the expression of DEWAX; throughout the night and another for stomata closing. Taken together, it is evident that DEWAX-mediated negative regulation of the wax biosynthetic genes plays role in determining the total wax loads produced in Arabidopsis during daily dark and light cycles. In addition, significantly higher levels of DEWAX transcripts in leaves than stems suggest that DEWAX-mediated transcriptional repression might be involved in the organ-specific regulation of total wax amounts on plant surfaces.

Overexpression of Arabidopsis MYB96 confers drought resistance in Camelina sativa via cuticular wax accumulation.

Camelina has been highlighted as an emerging oilseed crop. Transgenic Camelina plants overexpressing Arabidopsis MYB96 exhibited drought resistance by activating expression of Camelina wax biosynthetic genes and accumulating wax load. Camelina (Camelina sativa L.) is an oilseed crop in the Brassicaeae family with potential to expand biofuel production to marginal land. The aerial portion of all land plants is covered with cuticular wax to protect them from desiccation. In this study, the Arabidopsis MYB96 gene was overexpressed in Camelina under the control of the CaMV35S promoter. Transgenic Camelina plants overexpressing Arabidopsis MYB96 exhibited normal growth and development and enhanced tolerance to drought. Deposition of epicuticular wax crystals and total wax loads increased significantly on the surfaces of transgenic leaves compared with that of non-transgenic plants. The levels of alkanes and primary alcohols prominently increased in transgenic Camelina plants relative to non-transgenic plants. Cuticular transpiration occurred more slowly in transgenic leaves than that in non-transgenic plants. Genome-wide identification of Camelina wax biosynthetic genes enabled us to determine that the expression levels of CsKCS2, CsKCS6, CsKCR1-1, CsKCR1-2, CsECR, and CsMAH1 were approximately two to sevenfold higher in transgenic Camelina leaves than those in non-transgenic leaves. These results indicate that MYB96-mediated transcriptional regulation of wax biosynthetic genes is an approach applicable to generating drought-resistant transgenic crops. Transgenic Camelina plants with enhanced drought tolerance could be cultivated on marginal land to produce renewable biofuels and biomaterials.

Thiabendazole residue loading in dip, drench and wax coating applications to control green mould and chilling injury on citrus fruit

Green mould (caused by Penicillium digitatum) is a major cause of postharvest losses in citrus. Residue loading of thiabendazole (TBZ) with application methods typically used in South African packhouses and green mould control was studied. TBZ was applied curatively and protectively in dip, drench and wax coating treatments and fruit were inoculated with a TBZ-sensitive or a TBZ-resistant isolate of P. digitatum. The dip treatments consisted of TBZ concentrations of 0–2000μgmL−1; fruit were dipped for 60s at 22°C at a pH of 7. Residues differed between fruit batches and ranged from 0.5 to 1.7μgg−1 at 1000μgmL−1 TBZ. Curative dip treatments almost completely controlled green mould (>96% at 1000μgmL−1 TBZ). The residue level needed for 75% curative control ranged from 0.06 to 0.22μgg−1, depending on citrus type. Protective treatments were unreliable and control varied from 17% to 97.9% at 1000μgmL−1 TBZ between fruit batches. Drench treatments consisted of exposure times of 30, 60 and 90s with 1000 or 2000μgmL−1 TBZ. Average TBZ residues were 2.14μgg−1 for Clementine mandarin fruit and 3.50μgg−1 for navel orange fruit. Green mould control on navel orange fruit resulted in 66–92%, 34–90% and 9–38% control for curative treatments after 6 and 24h and protective treatments, respectively, depending on fruit batch. Wax with 4000μgmL−1 TBZ was applied at 0.6, 1.2 and 1.8Lwaxton−1 fruit. Chilling injury was evaluated after fruit storage at −0.5°C for 40 days. Average TBZ residues loaded was 1.3, 1.3 and 2.7μgg−1 at the recommended 1.2Lton−1 for Satsuma mandarin, Clementine mandarin and Valencia orange fruit, respectively. Protective treatments showed lower infection levels (14–20%) than curative treatments (27–40%) for Valencia orange fruit. The same trend was observed with Satsuma (92–95% curative; 87–90% protective) and Clementine mandarin fruit (82–90% curative; 59–88% protective), but control was relatively poor. TBZ application in wax exceeded 5μgg−1 at higher wax loads (1.2 and 1.8Lton−1). Wax treatments showed a significant reduction in chilling injury; TBZ had an additive effect. TBZ resistant isolates could not be controlled.

ArabidopsisCuticular Wax Biosynthesis Is Negatively Regulated by theDEWAXGene Encoding an AP2/ERF-Type Transcription Factor

The aerial parts of plants are protected from desiccation and other stress by surface cuticular waxes. The total cuticular wax loads and the expression of wax biosynthetic genes are significantly downregulated in Arabidopsis thaliana under dark conditions. We isolated Decrease Wax Biosynthesis (DEWAX), which encodes an AP2/ERF-type transcription factor that is preferentially expressed in the epidermis and induced by darkness. Disruption of DEWAX leads to an increase in total leaf and stem wax loads, and the excess wax phenotype of dewax was restored to wild type levels in complementation lines. Moreover, overexpression of DEWAX resulted in a reduction in total wax loads in leaves and stems compared with the wild type and altered the ultrastructure of cuticular layers. DEWAX negatively regulates the expression of alkane-forming enzyme, long-chain acyl-CoA synthetase, ATP citrate lyase A subunit, enoyl-CoA reductase, and fatty acyl-CoA reductase, and chromatin immunoprecipitation analysis suggested that DEWAX directly interacts with the promoters of wax biosynthesis genes. Cuticular wax biosynthesis is negatively regulated twice a day by the expression of DEWAX, throughout the night and at stomata closing. Significantly higher levels (10- to 100-fold) of DEWAX transcripts were found in leaves than in stems, suggesting that DEWAX-mediated transcriptional repression may be an additional mechanism contributing to the different total wax loads in leaves and stems.

Fine mapping and metabolic and physiological characterization of the glume glaucousness inhibitor locus Iw3 derived from wild wheat

This research provided the first view of metabolic and physiological effect of a tissue-specific glaucousness inhibitor in wheat and laid foundation for map-based cloning of the Iw3 locus. Cuticular wax constitutes the outermost layer of plant skin, and its composition greatly impacts plant appearance and plant-environment interaction. Epicuticular wax in the upper part of adult wheat plants can form the glaucousness, which is associated with drought tolerance. In this research, we characterized a glume-specific glaucousness inhibitor, Iw3, by fine mapping, physiological, and molecular approaches. Iw3 inhibits glaucousness formation by altering wax composition. Compared to the wild type, Iw3 eliminated β-diketone, reduced 47 % primary alcohols, but increased aldehyde 400-fold and alkanes fivefold, which led to 30 % reduction of total glume wax load. Loss of the glaucousness increased cuticle permeability, suggesting an important role in drought sensitivity. Genetically, the glaucousness-inhibiting effect by Iw3 is partially dominant in a dosage-dependent manner. We localized the Iw3 locus within a 0.13-cM interval delimited by marker loci Xpsp3000 and XWL3096. Of the 53 wax genes assayed, we detected transcription changes in nine genes by Iw3, downregulation of Cer4-1 and upregulation of other five Cer4 and three KCS homologs. All these results provided initial insights into Iw3-mediated regulation of wax metabolism and paved way for in-depth characterization of the Iw3 locus and the glaucousness-related β-diketone pathway.

Analyses of Tomato Fruit Brightness Mutants Uncover Both Cutin-Deficient and Cutin-Abundant Mutants and a New Hypomorphic Allele of GDSL Lipase

The cuticle is a protective layer synthesized by epidermal cells of the plants and consisting of cutin covered and filled by waxes. In tomato (Solanum lycopersicum) fruit, the thick cuticle embedding epidermal cells has crucial roles in the control of pathogens, water loss, cracking, postharvest shelf-life, and brightness. To identify tomato mutants with modified cuticle composition and architecture and to further decipher the relationships between fruit brightness and cuticle in tomato, we screened an ethyl methanesulfonate mutant collection in the miniature tomato cultivar Micro-Tom for mutants with altered fruit brightness. Our screen resulted in the isolation of 16 glossy and 8 dull mutants displaying changes in the amount and/or composition of wax and cutin, cuticle thickness, and surface aspect of the fruit as characterized by optical and environmental scanning electron microscopy. The main conclusions on the relationships between fruit brightness and cuticle features were as follows: (1) screening for fruit brightness is an effective way to identify tomato cuticle mutants; (2) fruit brightness is independent from wax load variations; (3) glossy mutants show either reduced or increased cutin load; and (4) dull mutants display alterations in epidermal cell number and shape. Cuticle composition analyses further allowed the identification of groups of mutants displaying remarkable cuticle changes, such as mutants with increased dicarboxylic acids in cutin. Using genetic mapping of a strong cutin-deficient mutation, we discovered a novel hypomorphic allele of GDSL lipase carrying a splice junction mutation, thus highlighting the potential of tomato brightness mutants for advancing our understanding of cuticle formation in plants.

Putative megaenzyme DWA1 plays essential roles in drought resistance by regulating stress-induced wax deposition in rice

Drought stress is a major limiting factor for crop production. Cuticular wax plays an important role in preventing water loss from drought stress. However, the genetic control of cuticular wax deposition under drought stress conditions has not been characterized. Here, we identified a rice gene Drought-Induced Wax Accumulation 1 (DWA1) encoding a very large protein (2,391 aa in length) containing multiple enzymatic structures, including an oxidoreductase-like domain; a prokaryotic nonribosomal peptide synthetase-like module, including an AMP-binding domain; and an allene oxide synthase-like domain. This previously unreported putative megaenzyme is conserved in vascular plants. A dwa1 KO mutant was highly sensitive to drought stress relative to the WT. DWA1 was preferentially expressed in vascular tissues and epidermal layers and strongly induced by drought stress. The dwa1 mutant was impaired in cuticular wax accumulation under drought stress, which significantly altered the cuticular wax composition of the plant, resulting in increased drought sensitivity. The mutant had reduced levels of very-long-chain fatty acids, and plants overexpressing DWA1 showed elevated levels of very-long-chain fatty acids relative to the WT. The expression of many wax-related genes was significantly suppressed in dwa1 under drought conditions. The AMP-binding domain exhibited in vitro enzymatic activity in activating long-chain fatty acids to form acyl-CoA. Our results suggest that DWA1 controls drought resistance by regulating drought-induced cuticular wax deposition in rice. This finding may have significant implications for improving the drought resistance of crop varieties.

Ectopic Expression of a WRKY Homolog from Glycine soja Alters Flowering Time in Arabidopsis

Flowering is a critical event in the life cycle of plants; the WRKY-type transcription factors are reported to be involved in many developmental processes sunch as trichome development and epicuticular wax loading, but whether they are involved in flowering time regulation is still unknown. Within this study, we provide clear evidence that GsWRKY20, a member of WRKY gene family from wild soybean, is involved in controlling plant flowering time. Expression of GsWRKY20 was abundant in the shoot tips and inflorescence meristems of wild soybean. Phenotypic analysis showed that GsWRKY20 over-expression lines flowered earlier than the wild-type plants under all conditions: long-day and short-day photoperiods, vernalization, or exogenous GA3 application, indicating that GsWRKY20 may mainly be involved in an autonomous flowering pathway. Further analyses by qRT-PCR and microarray suggests that GsWRKY20 accelerating plant flowering might primarily be through the regulation of flowering-related genes (i.e., FLC, FT, SOC1 and CO) and floral meristem identity genes (i.e., AP1, SEP3, AP3, PI and AG). Our results provide the evidence demonstrating the effectiveness of manipulating GsWRKY20 for altering plant flowering time.