Cuticular Wax Deposition Research Articles

A Non-specific Setaria italica Lipid Transfer Protein Gene Plays a Critical Role under Abiotic Stress.

Lipid transfer proteins (LTPs) are a class of cysteine-rich soluble proteins having small molecular weights. LTPs participate in flower and seed development, cuticular wax deposition, also play important roles in pathogen and abiotic stress responses. A non-specific LTP gene (SiLTP) was isolated from a foxtail millet (Setaria italica) suppression subtractive hybridization library enriched for differentially expressed genes after abiotic stress treatments. A semi-quantitative reverse transcriptase PCR analysis showed that SiLTP was expressed in all foxtail millet tissues. Additionally, the SiLTP promoter drove GUS expression in root tips, stems, leaves, flowers, and siliques of transgenic Arabidopsis. Quantitative real-time PCR indicated that the SiLTP expression was induced by NaCl, polyethylene glycol, and abscisic acid (ABA). SiLTP was localized in the cytoplasm of tobacco leaf epidermal cells and maize protoplasts. The ectopic expression of SiLTP in tobacco resulted in higher levels of salt and drought tolerance than in the wild type (WT). To further assess the function of SiLTP, SiLTP overexpression (OE) and RNA interference (RNAi)-based transgenic foxtail millet were obtained. SiLTP-OE lines performed better under salt and drought stresses compared with WT plants. In contrast, the RNAi lines were much more sensitive to salt and drought compared than WT. Electrophoretic mobility shift assays and yeast one-hybrids indicated that the transcription factor ABA-responsive DRE-binding protein (SiARDP) could bind to the dehydration-responsive element of SiLTP promoter in vitro and in vivo, respectively. Moreover, the SiLTP expression levels were higher in SiARDP-OE plants compared than the WT. These results confirmed that SiLTP plays important roles in improving salt and drought stress tolerance of foxtail millet, and may partly be upregulated by SiARDP. SiLTP may provide an effective genetic resource for molecular breeding in crops to enhance salt and drought tolerance levels.

Variations of Leaf Cuticular Waxes Among C3 and C4 Gramineae Herbs

Modern C4 plants are commonly distributed in hot and dry environments whereas C3 plants predominate in cool and shade areas. At the outmost of plant surface, the deposition and chemical composition of cuticular waxes vary under different environmental conditions. However, whether such variation of cuticular wax is related to the distribution of C3 and C4 under different environmental conditions is still not clear. In this study, leaves of six C3 Gramineae herbs distributed in spring, Roegneriakamoji, Polypogonfugax, Poaannua, Avenafatua, Alopecurusaequalis, and Oplismenusundulatifolius, and four C4 and one C3 Gramineae herbs distributed in summer, Digitariasanguinalis, Eleusineindica, Setariaviridis, S. plicata, and O. undulatifolius, were sampled and analyzed for cuticular wax. Plates were the main epicuticular wax morphology in both C3 and C4 plants except S.plicata. The plates melted in C4 plants but not in C3 plants. The total cuticular wax amounts in C4 plants were significantly lower than those in C3 plants, except for O.undulatifolius. Primary alcohols were the most abundant compounds in C3 plants, whereas n-alkanes were relatively the most abundant compounds in C4 plants. C29 was the most abundant n-alkane in C3 plants except for O.undulatifolius, whereas the most abundant n-alkane was C31 or C33 in C4 plants. The average chain length (ACL) of n-alkanes was higher in C4 than in C3 plants, whereas the ACL of n-alkanoic acids was higher in C3 than C4 plants. The cluster analysis based on the distribution of n-alkanes clearly distinguished C3 and C4 plants into two groups, except for O.undulatifolius which was grouped with C4 plants. These results suggest that the variations of cuticular waxes among C3 and C4 Gramineae herbs are related to the distribution of C3 and C4 plants under different environmental conditions.

Cuticular wax biosynthesis is positively regulated by WRINKLED4, an AP2/ERF‐type transcription factor, in Arabidopsis stems

The aerial surfaces of terrestrial plants are covered by a cuticular wax layer, which protects the plants from environmental stresses such as desiccation, high irradiance, and UV radiation. Cuticular wax deposition is regulated in an organ-specific manner; Arabidopsis stems have more than 10-fold higher wax loads than leaves. In this study, we found that WRINKLED4 (WRI4), encoding an AP2/ERF (ethylene-responsive factor) transcription factor (TF), is predominantly expressed in stem epidermis, is upregulated by salt stress, and is involved in activating cuticular wax biosynthesis in Arabidopsis stems. WRI4 harbors a transcriptional activation domain at its N-terminus, and fluorescent signals from a WRI4:eYFP construct were localized to the nuclei of tobacco leaf protoplasts. Deposition of epicuticular wax crystals on stems was reduced in wri4-1 and wri4-3 knockout mutants. Total wax loads were reduced by ~28% in wri4 stems but were not altered in wri4 siliques or leaves compared to the wild type. The levels of 29-carbon long alkanes, ketones, and secondary alcohols, which are the most abundant components of stem waxes, were significantly reduced in wri4 stems relative to the wild type. A transactivation assay in tobacco protoplasts and a chromatin immunoprecipitation (ChIP) assay showed that the expression of long-chain acyl-CoA synthetase1 (LACS1), β-ketoacyl CoA reductase1 (KCR1), PASTICCINO2 (PAS2), trans-2,3-enoyl-CoA reductase (ECR), and bifunctional wax synthase/acyl-CoA: diacylglycerol acyltransferase (WSD1) is positively regulated by direct binding of WRI4 to their promoters. Taken together, these results suggest that WRI4 is a transcriptional activator that specifically controls cuticular wax biosynthesis in Arabidopsis stems.

A Metabolic Gene Cluster in the Wheat W1 and the Barley Cer-cqu Loci Determines β-Diketone Biosynthesis and Glaucousness.

The glaucous appearance of wheat (Triticum aestivum) and barley (Hordeum vulgare) plants, that is the light bluish-gray look of flag leaf, stem, and spike surfaces, results from deposition of cuticular β-diketone wax on their surfaces; this phenotype is associated with high yield, especially under drought conditions. Despite extensive genetic and biochemical characterization, the molecular genetic basis underlying the biosynthesis of β-diketones remains unclear. Here, we discovered that the wheat W1 locus contains a metabolic gene cluster mediating β-diketone biosynthesis. The cluster comprises genes encoding proteins of several families including type-III polyketide synthases, hydrolases, and cytochrome P450s related to known fatty acid hydroxylases. The cluster region was identified in both genetic and physical maps of glaucous and glossy tetraploid wheat, demonstrating entirely different haplotypes in these accessions. Complementary evidence obtained through gene silencing in planta and heterologous expression in bacteria supports a model for a β-diketone biosynthesis pathway involving members of these three protein families. Mutations in homologous genes were identified in the barley eceriferum mutants defective in β-diketone biosynthesis, demonstrating a gene cluster also in the β-diketone biosynthesis Cer-cqu locus in barley. Hence, our findings open new opportunities to breed major cereal crops for surface features that impact yield and stress response.

Identification of candidate genes involved in wax deposition in Poa pratensis by RNA-seq.

BackgroundThe cuticular wax plays important roles in plant resistance to various biotic and abiotic stresses. Understanding the synthesis and secretion of cuticular waxes is necessary in utilizing cuticular waxes to improve crop productivity and plant ecological adaptation. Due to the lack of genomic resources, little genetic research on cuticular wax deposition has been focused on Poa pratensis, a perennial forage and turf grass species that is widely distributed under various habitats. In this study, we performed de novo transcriptome sequencing to explore differentially expressed genes between the leaf non-elongation zone (NEZm) and the emerged blade zone (EBZ) and to identify genes related to cuticular wax deposition.ResultsA total of 77,707,414 high quality reads were obtained from llumina HiSeq 2500 platform, which were then assembled into 106,766 unigenes. Among them, 6019 unigenes showed significant differences in expression between NEZm and EBZ. In our assembled sequences, 3087 SSRs molecular markers were discovered. All the unigenes were searched against the NR, Swissprot, GO, COG, and KEGG databases using BLAST program for functional annotation. From 3156 unigenes with more expression in NEZm compared to EBZ, a number of unigenes involved in very long chain fatty acids (VLCFAs) and cuticular wax biosynthesis, transportation and regulation were identified. Several unigenes related to defense response and epidermal patterning were also found. Twelve putative genes involved in VLCFAs and cuticular wax biosynthesis were further analyzed for their expressions using qRT-PCR.ConclusionsThe transcriptome of P. pratensis leaf was deep sequenced, de novo assembled and annotated, and the candidate genes potentially involved in VLCFAs and cuticular wax biosynthesis, secretion and regulation in P. pratensis were identified. This provides fundamental genetic resources in improving plant adaptation to abiotic and biotic stresses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2641-2) contains supplementary material, which is available to authorized users.

The maize fused leaves1 (fdl1) gene controls organ separation in the embryo and seedling shoot and promotes coleoptile opening.

The fdl1-1 mutation, caused by an Enhancer/Suppressor mutator (En/Spm) element insertion located in the third exon of the gene, identifies a novel gene encoding ZmMYB94, a transcription factor of the R2R3-MYB subfamily. The fdl1 gene was isolated through co-segregation analysis, whereas proof of gene identity was obtained using an RNAi strategy that conferred less severe, but clearly recognizable specific mutant traits on seedlings. Fdl1 is involved in the regulation of cuticle deposition in young seedlings as well as in the establishment of a regular pattern of epicuticular wax deposition on the epidermis of young leaves. Lack of Fdl1 action also correlates with developmental defects, such as delayed germination and seedling growth, abnormal coleoptile opening and presence of curly leaves showing areas of fusion between the coleoptile and the first leaf or between the first and the second leaf. The expression profile of ZmMYB94 mRNA-determined by quantitative RT-PCR-overlaps the pattern of mutant phenotypic expression and is confined to a narrow developmental window. High expression was observed in the embryo, in the seedling coleoptile and in the first two leaves, whereas RNA level, as well as phenotypic defects, decreases at the third leaf stage. Interestingly several of the Arabidopsis MYB genes most closely related to ZmMYB94 are also involved in the activation of cuticular wax biosynthesis, suggesting deep conservation of regulatory processes related to cuticular wax deposition between monocots and dicots.

Differentiated cuticular wax content and expression patterns of cuticular wax biosynthetic genes in bloomed and bloomless broccoli (Brassica oleracea var. italica)

The aerial surfaces of plants are covered with a wax layer that serves the essential functions of limiting non-stomatal water loss and acting as protective barrier against environmental stresses. We selected two broccoli lines, bloomed (MC91) and bloomless (MC117), and analyzed their phenotypes related to cuticular wax accumulation. The total wax amount was 1.93-fold higher in MC91 leaves compared to MC117 leaves. All of the studied cuticular wax compounds were 1.07–3.79-fold higher in MC91 plants compared to MC117 plants except for the C31 alkane. The wax compositions did not essentially different between the two broccoli lines, but some compounds were found at significantly higher levels in MC91 plants compared to MC117 plants, mainly reflecting differences in C29 alkanes, C29 secondary alcohols and C29 ketones. To investigate gene regulation by bloom phenotype, we analyzed the mRNA expression patterns of various cuticular wax biosynthetic genes. Our results revealed that LACS1, KCS1, KCR1, ECR, CER3 and MAH1 were expressed more in MC91 plants compared to MC117 plants at both 3 and 10 weeks. The expression levels of the studied cuticular wax biosynthetic genes were significantly induced by drought stress, which is known to induce cuticular wax deposition. Together, these results show that the cuticular wax accumulation of broccoli is regulated by cuticular wax biosynthetic gene expression and can be affected by environmental signals.

Over-expression of SlSHN1 gene improves drought tolerance by increasing cuticular wax accumulation in tomato.

Increasing cuticular wax accumulation in plants has been associated with improving drought tolerance in plants. In this study, a cDNA clone encoding the SlSHN1 transcription factor, the closest ortholog to WIN/SHN1 gene in Arabidopsis, was isolated from tomato plant. Expression analysis of SlSHN1 indicated that it is induced in response to drought conditions. The over-expression of SlSHN1 in tomato under the control of the constitutive CaMV 35S promoter produced plants that showed mild growth retardation phenotype with shiny and dark green leaves. Scanning electron microscopy showed that the over-expression of SlSHN1 in tomato resulted in higher cuticular wax deposition on leaf epidermial tissue when compared to non-transformed plants. Expression analysis in transgenic lines over-expressing SlSHN1 indicated that several wax-related synthesis genes were induced. Transgenic tomato plants over-expressing SlSHN1 showed higher drought tolerance when compared with wild type plants; this was reflected in delayed wilting of transgenic lines, improved water status and reduced water loss rate when compared with wild type plants. In conclusion, the SlSHN1 gene can modulate wax accumulation and could be utilized to enhance drought tolerance in tomato plant.

The TsnsLTP4, a Nonspecific Lipid Transfer Protein Involved in Wax Deposition and Stress Tolerance

Nonspecific lipid transfer proteins (nsLTPs), a group of small, basic proteins that are ubiquitously distributed throughout the plant kingdom, are thought to participate in cutin formation as well as in defense reactions against abiotic and biotic stresses. However, whether nsLTPs are involved in cuticular wax deposition remains unknown. We identified a salt-induced gene, TsnsLTP4, encoding an nsLTP from Thellungiella salsuginea. TsnsLTP4 expression was significantly induced by salt, polyethylene glycol (PEG), abscisic acid (ABA), and high (37 °C) and low (4 °C) temperatures. Transgenic onion epidermal cells transiently expressing a TsnsLTP4-DsRed fusion protein demonstrated that TsnsLTP4 was targeted to the cell wall. Overexpression of TsnsLTP4 in transgenic Arabidopsis plants resulted in increased epicuticular wax deposition, particularly of wax components with a carbon chain length of more than C27. Moreover, the amount of wax deposited was strongly reduced in RNA interference (RNAi)-knockdown TsnsLTP4 transgenic T. salsuginea lines. Cuticle permeability was inversely related to the expression level of TsnsLTP4. Further analyses indicated that overexpression of TsnsLTP4 resulted in significantly enhanced drought and salt tolerance in transgenic Arabidopsis. In summary, our studies suggested that TsnsLTP4 may play a role in wax deposition and in plant tolerance against abiotic stresses.

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.

Cell geometry guides the dynamic targeting of apoplastic GPI-linked lipid transfer protein to cell wall elements and cell borders in Arabidopsis thaliana.

During cellular morphogenesis, changes in cell shape and cell junction topology are fundamental to normal tissue and organ development. Here we show that apoplastic Glycophosphatidylinositol (GPI)-anchored Lipid Transfer Protein (LTPG) is excluded from cell junctions and flat wall regions, and passively accumulates around their borders in the epidermal cells of Arabidopsis thaliana. Beginning with intense accumulation beneath highly curved cell junction borders, this enrichment is gradually lost as cells become more bulbous during their differentiation. In fully mature epidermal cells, YFP-LTPG often shows a fibrous cellulose microfibril-like pattern within the bulging outer faces. Physical contact between a flat glass surface and bulbous cell surface induces rapid and reversible evacuation from contact sites and accumulation to the curved wall regions surrounding the contact borders. Thus, LTPG distribution is dynamic, responding to changes in cell shape and wall curvature during cell growth and differentiation. We hypothesize that this geometry-based mechanism guides wax-carrying LTPG to functional sites, where it may act to “seal” the vulnerable border surrounding cell-cell junctions and assist in cell wall fortification and cuticular wax deposition.

Coexpression patterns indicate that GPI-anchored non-specific lipid transfer proteins are involved in accumulation of cuticular wax, suberin and sporopollenin

The non-specific lipid transfer proteins (nsLTP) are unique to land plants. The nsLTPs are characterized by a compact structure with a central hydrophobic cavity and can be classified to different types based on sequence similarity, intron position or spacing between the cysteine residues. The type G nsLTPs (LTPGs) have a GPI-anchor in the C-terminal region which attaches the protein to the exterior side of the plasma membrane. The function of these proteins, which are encoded by large gene families, has not been systematically investigated so far. In this study we have explored microarray data to investigate the expression pattern of the LTPGs in Arabidopsis and rice. We identified that the LTPG genes in each plant can be arranged in three expression modules with significant coexpression within the modules. According to expression patterns and module sizes, the Arabidopsis module AtI is functionally equivalent to the rice module OsI, AtII corresponds to OsII and AtIII is functionally comparable to OsIII. Starting from modules AtI, AtII and AtIII we generated extended networks with Arabidopsis genes coexpressed with the modules. Gene ontology analyses of the obtained networks suggest roles for LTPGs in the synthesis or deposition of cuticular waxes, suberin and sporopollenin. The AtI-module is primarily involved with cuticular wax, the AtII-module with suberin and the AtIII-module with sporopollenin. Further transcript analysis revealed that several transcript forms exist for several of the LTPG genes in both Arabidopsis and rice. The data suggests that the GPI-anchor attachment and localization of LTPGs may be controlled to some extent by alternative splicing.

Rice OsGL1-6 is involved in leaf cuticular wax accumulation and drought resistance.

Cuticular wax is a class of organic compounds that comprises the outermost layer of plant surfaces. Plant cuticular wax, the last barrier of self-defense, plays an important role in plant growth and development. The OsGL1-6 gene, a member of the fatty aldehyde decarbonylase gene family, is highly homologous to Arabidopsis CER1, which is involved in cuticular wax biosynthesis. However, whether OsGL1-6 participates in cuticular wax biosynthesis remains unknown. In this study, an OsGL1-6 antisense-RNA vector driven by its own promoter was constructed and introduced into the rice variety Zhonghua11 by Agrobacterium-mediated transformation to obtain several independent transgenic plants with decreased OsGL1-6 expression. These OsGL1-6 antisense-RNA transgenic plants showed droopy leaves at the booting stage, significantly decreased leaf cuticular wax deposition, thinner cuticle membrane, increased chlorophyll leaching and water loss rates, and enhanced drought sensitivity. The OsGL1-6 gene was constitutively expressed in all examined organs and was very highly expressed in leaf epidermal cells and vascular bundles. The transient expression of OsGL1-6-GFP fusion indicated that OsGL1-6 is localized in the endoplasmic reticulum. Qualitative and quantitative analysis of the wax composition using gas chromatography-mass spectrometry revealed a significantly reduced total cuticular wax load on the leaf blades of the OsGL1-6 antisense-RNA transgenic plants as well as markedly decreased alkane and aldehyde contents. Their primary alcohol contents increased significantly compared with those in the wild type plants, suggesting that OsGL1-6 is associated with the decarbonylation pathways in wax biosynthesis. We propose that OsGL1-6 is involved in the accumulation of leaf cuticular wax and directly impacts drought resistance in rice.

RDR1 and SGS3, components of RNA-mediated gene silencing, are required for the regulation of cuticular wax biosynthesis in developing inflorescence stems of Arabidopsis.

The cuticle is a protective layer that coats the primary aerial surfaces of land plants and mediates plant interactions with the environment. It is synthesized by epidermal cells and is composed of a cutin polyester matrix that is embedded and covered with cuticular waxes. Recently, we have discovered a novel regulatory mechanism of cuticular wax biosynthesis that involves the ECERIFERUM7 (CER7) ribonuclease, a core subunit of the exosome. We hypothesized that at the onset of wax production, the CER7 ribonuclease degrades an mRNA specifying a repressor of CER3, a wax biosynthetic gene whose protein product is required for wax formation via the decarbonylation pathway. In the absence of this repressor, CER3 is expressed, leading to wax production. To identify the putative repressor of CER3 and to unravel the mechanism of CER7-mediated regulation of wax production, we performed a screen for suppressors of the cer7 mutant. Our screen resulted in the isolation of components of the RNA-silencing machinery, RNA-DEPENDENT RNA POLYMERASE1 and SUPPRESSOR OF GENE SILENCING3, implicating RNA silencing in the control of cuticular wax deposition during inflorescence stem development in Arabidopsis (Arabidopsis thaliana).

Effect of salt stress on physiological response of tomato fruit grown in hydroponic culture system

The effect of salt stress on physiological response of hydroponically grown tomato fruit was investigated. Fruit growth rate, water status, cuticle permeability and induction of blossom-end rot (BER) of tomato fruit were considered for this study. Salt stress was applied by using Ca salt treatment and it plays an important role on all parameters studied in this experiment. Fruit growth rate, predawn water potential, osmotic potential and cuticle permeability were significantly lower in treated plants than in control plants. On the other hand, tissue turgor of control and treated fruit showed almost similar values 12 days after flowering (DAF). This result indicated that turgor was osmotically regulated in fruit under stress condition. Fruit growth rate was found to decline from 12 DAF and eventually ceased when BER externally appeared on fruit surface at the age of 19 DAF in this experiment. The reduction of growth rate coincided with the reduction of water potential in fruit tissue due to salt stress. Although BER externally appeared at 19 DAF anatomical investigation showed that intercellular air space becomes discoloured at least one week before external symptoms appeared on fruit tip. Different levels of cuticular permeability indicated that the deposition of cuticular wax on fruit surface was enhanced by the salt stress condition in tomato fruit. Since, BER was found to appear on fruit tip under high calcium concentration in solution it can be concluded that calcium deficiency was not the only the cause of BER in tomato, rather salt stress might alter metabolic activity in developing tomato fruit.

Rice OsGL1-1 Is Involved in Leaf Cuticular Wax and Cuticle Membrane

Cuticular wax forms a hydrophobic barrier on aerial plant organs; it plays an important role in protecting a plant from damage caused by many forms of environmental stress. In the present study, we characterized a rice leaf wax-deficient mutant osgl1-1 derived from a spontaneous mutation, which exhibited a wax-deficient and highly hydrophilic leaf phenotype. We cloned the OsGL1-1 gene by the map-based cloning method and performed a complementation test to confirm the function of the candidate gene. Molecular studies revealed that OsGL1-1 was a member of the OsGL1 family, and contained regions that were homologous to some regions in sterol desaturases and short-chain dehydrogenases/reductases. Compared to the wild-type, the osgl1-1 mutant showed decreased cuticular wax deposition, thinner cuticular membrane, decreased chlorophyll leaching, increased rate of water loss, and enhanced sensitivity to drought. OsGL1-1 is expressed ubiquitously in rice. The transient expression of OsGL1-1-green fluorescent protein fusion protein indicated that OsGL1-1 is localized in the cytoplasm, plasma membrane, and nucleus.

The MYB96 transcription factor regulates cuticular wax biosynthesis under drought conditions in Arabidopsis.

Drought stress activates several defense responses in plants, such as stomatal closure, maintenance of root water uptake, and synthesis of osmoprotectants. Accumulating evidence suggests that deposition of cuticular waxes is also associated with plant responses to cellular dehydration. Yet, how cuticular wax biosynthesis is regulated in response to drought is unknown. We have recently reported that an Arabidopsis thaliana abscisic acid (ABA)-responsive R2R3-type MYB transcription factor, MYB96, promotes drought resistance. Here, we show that transcriptional activation of cuticular wax biosynthesis by MYB96 contributes to drought resistance. Microarray assays showed that a group of wax biosynthetic genes is upregulated in the activation-tagged myb96-1D mutant but downregulated in the MYB96-deficient myb96-1 mutant. Cuticular wax accumulation was altered accordingly in the mutants. In addition, activation of cuticular wax biosynthesis by drought and ABA requires MYB96. By contrast, biosynthesis of cutin monomers was only marginally affected in the mutants. Notably, the MYB96 protein acts as a transcriptional activator of genes encoding very-long-chain fatty acid-condensing enzymes involved in cuticular wax biosynthesis by directly binding to conserved sequence motifs present in the gene promoters. These results demonstrate that ABA-mediated MYB96 activation of cuticular wax biosynthesis serves as a drought resistance mechanism.

Multilevel regulation and signalling processes associated with adaptation to terminal drought in wild emmer wheat

Low water availability is the major environmental factor limiting crop productivity. Transcriptome analysis was used to study terminal drought response in wild emmer wheat, Triticum dicoccoides, genotypes contrasting in their productivity and yield stability under drought stress. A total of 5,892 differentially regulated transcripts were identified between drought and well-watered control and/or between drought resistant (R) and drought susceptible (S) genotypes. Functional enrichment analyses revealed that multilevel regulatory and signalling processes were significantly enriched among the drought-induced transcripts, in particular in the R genotype. Therefore, further analyses were focused on selected 221 uniquely expressed or highly abundant transcripts in the R genotype, as potential candidates for drought resistance genes. Annotation of the 221 genes revealed that 26% of them are involved in multilevel regulation, including: transcriptional regulation, RNA binding, kinase activity and calcium and abscisic acid signalling implicated in stomatal closure. Differential expression patterns were also identified in genes known to be involved in drought adaptation pathways, such as: cell wall adjustment, cuticular wax deposition, lignification, osmoregulation, redox homeostasis, dehydration protection and drought-induced senescence. These results demonstrate the potential of wild emmer wheat as a source for candidate genes for improving drought resistance.

Heterologous expression of two Medicago truncatula putative ERF transcription factor genes, WXP1 and WXP2, in Arabidopsis led to increased leaf wax accumulation and improved drought tolerance, but differential response in freezing tolerance

Cuticular waxes are the major components of plant cuticle and play an important role in protecting aerial organs from damage caused by biotic and abiotic stresses. Here we report the functional characterization of two putative ERF transcription factor genes WXP1 and its paralog WXP2 from Medicago truncatula. Transgenic expression of WXP1 and WXP2 in Arabidopsis (ecotype Columbia) led to significantly increased cuticular wax deposition on leaves of 4-week-old and 6-week-old transgenic plants, assessed based on fresh weight or based on surface area. Differences in the accumulation of various wax components as well as their chain length distributions were found in the WXP1 and WXP2 plants. The major wax component in Arabidopsis, n-alkanes, increased substantially in both WXP1 and WXP2 transgenics, however, another wax component, primary alcohols, increased in WXP1 plants but decreased in WXP2 plants. Cuticle properties of the transgenic leaves were analyzed by chlorophyll leaching assay; while the WXP1 plants had no change, the WXP2 plants showed more chlorophyll leaching. Analysis of fresh weight loss from detached leaves revealed that the transgenic leaves tend to retain more water than the control. Both WXP1 and WXP2 transgenic plants showed significantly enhanced whole plant drought tolerance. Analysis of freezing tolerance at the whole plant level and measurement of electrolyte leakage from detached leaves revealed that the WXP1 plants had increased freezing tolerance while the WXP2 plants were more sensitive to low temperature when compared to the control. Transgenic expression of WXP1 had no obvious effects on plant growth and development, however, the expression of WXP2 led to slower plant growth. These results indicate that WXP1 is a useful candidate gene for improving plant drought and freezing tolerance by genetic transformation.

Cuticular wax deposition in growing barley (Hordeum vulgare) leaves commences in relation to the point of emergence of epidermal cells from the sheaths of older leaves

In grasses, leaf cells divide and expand within the sheaths of older leaves, where the micro-environment differs from the open atmosphere. By the time epidermal cells are displaced into the atmosphere, they must have a functional cuticle to minimize uncontrolled water loss. In the present study, gas chromatography and scanning electron microscopy were used to follow cuticular wax deposition along the growing leaf three of barley (Hordeum vulgare L.). 1-Hexacosanol (C(26) alcohol) comprised more than 75% of extractable cuticular wax and was used as a marker for wax deposition. There was no detectable wax along the first 20 mm from the point of leaf insertion. Deposition started within the distal portion of the elongation zone (23-45 mm) and continued beyond the point of leaf emergence from the sheath of leaf two. The region where wax deposition commenced shifted towards more proximal (basal) positions when the point of leaf emergence was lowered by stripping back part of the sheath. When relative humidity in the shoot environment was elevated from 70% (standard growth conditions) to 92-96% for up to 4 days prior to analysis, wax deposition did not change significantly. The results show that cuticular waxes are deposited along the growing grass leaf independent of cell age or developmental stage. Instead, the reference point for wax deposition appears to be the point of emergence of cells into the atmosphere. The possibility of changes in relative humidity between enclosed and emerged leaf regions triggering wax deposition is discussed.