The impact of drought on wheat leaf cuticle properties.
ABSTRACT: The plant cuticle is the outermost layer covering aerial tissues and is composed of cutin and waxes. The cuticle plays an important role in protection from environmental stresses and glaucousness, the bluish-white colouration of plant surfaces associated with cuticular waxes, has been suggested as a contributing factor in crop drought tolerance. However, the cuticle structure and composition is complex and it is not clear which aspects are important in determining a role in drought tolerance. Therefore, we analysed residual transpiration rates, cuticle structure and epicuticular wax composition under well-watered conditions and drought in five Australian bread wheat genotypes, Kukri, Excalibur, Drysdale, RAC875 and Gladius, with contrasting glaucousness and drought tolerance.Significant differences were detected in residual transpiration rates between non-glaucous and drought-sensitive Kukri and four glaucous and drought-tolerant lines. No simple correlation was found between residual transpiration rates and the level of glaucousness among glaucous lines. Modest differences in the thickness of cuticle existed between the examined genotypes, while drought significantly increased thickness in Drysdale and RAC875. Wax composition analyses showed various amounts of C31 ?-diketone among genotypes and increases in the content of alkanes under drought in all examined wheat lines.The results provide new insights into the relationship between drought stress and the properties and structure of the wheat leaf cuticle. In particular, the data highlight the importance of the cuticle's biochemical makeup, rather than a simple correlation with glaucousness or stomatal density, for water loss under limited water conditions.
Project description:A plant cuticle forms a hydrophobic layer covering plant organs, and plays an important role in plant development and protection from environmental stresses. We examined epicuticular structure, composition, and a MYB-based regulatory network in two Australian wheat cultivars, RAC875 and Kukri, with contrasting cuticle appearance (glaucousness) and drought tolerance. Metabolomics and microscopic analyses of epicuticular waxes revealed that the content of ?-diketones was the major compositional and structural difference between RAC875 and Kukri. The content of ?-diketones remained the same while those of alkanes and primary alcohols were increased by drought in both cultivars, suggesting that the interplay of all components rather than a single one defines the difference in drought tolerance between cultivars. Six wheat genes encoding MYB transcription factors (TFs) were cloned; four of them were regulated in flag leaves of both cultivars by rapid dehydration and/or slowly developing cyclic drought. The involvement of selected MYB TFs in the regulation of cuticle biosynthesis was confirmed by a transient expression assay in wheat cell culture, using the promoters of wheat genes encoding cuticle biosynthesis-related enzymes and the SHINE1 (SHN1) TF. Two functional MYB-responsive elements, specifically recognized by TaMYB74 but not by other MYB TFs, were localized in the TdSHN1 promoter. Protein structural determinants underlying the binding specificity of TaMYB74 for functional DNA cis-elements were defined, using 3D protein molecular modelling. A scheme, linking drought-induced expression of the investigated TFs with downstream genes that participate in the synthesis of cuticle components, is proposed.
Project description:The cuticle of terrestrial plants functions as a protective barrier against many biotic and abiotic stresses. In wheat and other Triticeae, ?-diketone waxes are major components of the epicuticular layer leading to the bluish-white glaucous trait in reproductive-age plants. Glaucousness in durum wheat is controlled by a metabolic gene cluster at the WAX1 (W1) locus and a dominant suppressor INHIBITOR of WAX1 (Iw1) on chromosome 2B. The wheat D subgenome from progenitor Aegilops tauschii contains W2 and Iw2 paralogs on chromosome 2D. Here we identify the Iw1 gene from durum wheat and demonstrate the unique regulatory mechanism by which Iw1 acts to suppress a carboxylesterase-like protein gene, W1-COE, within the W1 multigene locus. Iw1 is a long noncoding RNA (lncRNA) containing an inverted repeat (IR) with >80% identity to W1-COE The Iw1 transcript forms a miRNA precursor-like long hairpin producing a 21-nt predominant miRNA, miRW1, and smaller numbers of related sRNAs associated with the nonglaucous phenotype. When Iw1 was introduced into glaucous bread wheat, miRW1 accumulated, W1-COE and its paralog W2-COE were down-regulated, and the phenotype was nonglaucous and ?-diketone-depleted. The IR region of Iw1 has >94% identity to an IR region on chromosome 2 in Ae. tauschii that also produces miRW1 and lies within the marker-based location of Iw2 We propose the Iw loci arose from an inverted duplication of W1-COE and/or W2-COE in ancestral wheat to form evolutionarily young miRNA genes that act to repress the glaucous trait.
Project description:Using a series of multiplexed experiments we studied the quantitative changes in protein abundance of three Australian bread wheat cultivars (Triticum aestivum L.) in response to a drought stress. Three cultivars differing in their ability to maintain grain yield during drought, Kukri (intolerant), Excalibur (tolerant), and RAC875 (tolerant), were grown in the glasshouse with cyclic drought treatment that mimicked conditions in the field. Proteins were isolated from leaves of mature plants and isobaric tags were used to follow changes in the relative protein abundance of 159 proteins. This is the first shotgun proteomics study in wheat, providing important insights into protein responses to drought as well as identifying the largest number of wheat proteins (1,299) in a single study. The changes in the three cultivars at the different time points reflected their differing physiological responses to drought, with the two drought tolerant varieties (Excalibur and RAC875) differing in their protein responses. Excalibur lacked significant changes in proteins during the initial onset of the water deficit in contrast to RAC875 that had a large number of significant changes. All three cultivars had changes consistent with an increase in oxidative stress metabolism and reactive O(2) species (ROS) scavenging capacity seen through increases in superoxide dismutases and catalases as well as ROS avoidance through the decreases in proteins involved in photosynthesis and the Calvin cycle.
Project description:The cuticle is regarded as a non-living tissue; it remains unknown whether the cuticle could be reversibly modified and what are the potential mechanisms. In this study, three tea germplasms (<i>Wuniuzao</i>, <i>0202-10</i>, and <i>0306A</i>) were subjected to water deprivation followed by rehydration. The epicuticular waxes and intracuticular waxes from both leaf surfaces were quantified from the mature 5th leaf. Cuticular transpiration rates were then measured from leaf drying curves, and the correlations between cuticular transpiration rates and cuticular wax coverage were analyzed. We found that the cuticular transpiration barriers were reinforced by drought and reversed by rehydration treatment; the initial weak cuticular transpiration barriers were preferentially reinforced by drought stress, while the original major cuticular transpiration barriers were either strengthened or unaltered. Correlation analysis suggests that cuticle modifications could be realized by selective deposition of specific wax compounds into individual cuticular compartments through multiple mechanisms, including <i>in vivo</i> wax synthesis or transport, dynamic phase separation between epicuticular waxes and the intracuticular waxes, <i>in vitro</i> polymerization, and retro transportation into epidermal cell wall or protoplast for further transformation. Our data suggest that modifications of a limited set of specific wax components from individual cuticular compartments are sufficient to alter cuticular transpiration barrier properties.
Project description:Cuticle is the major transpiration barrier that restricts non-stomatal water loss and is closely associated with plant drought tolerance. Although multiple efforts have been made, it remains controversial what factors shape up the cuticular transpiration barrier. Previously, we found that the cuticle from the tender tea leaf was mainly constituted by very-long-chain-fatty-acids and their derivatives while alicyclic compounds dominate the mature tea leaf cuticle. The presence of two contrasting cuticle within same branch offered a unique system to investigate this question. In this study, tea seedlings were subjected to water deprivation treatment, cuticle structures and wax compositions from the tender leaf and the mature leaf were extensively measured and compared. We found that cuticle wax coverage, thickness, and osmiophilicity were commonly increased from both leaves. New waxes species were specifically induced by drought; the composition of existing waxes was remodeled; the chain length distributions of alkanes, esters, glycols, and terpenoids were altered in complex manners. Drought treatment significantly reduced leaf water loss rates. Wax biosynthesis-related gene expression analysis revealed dynamic expression patterns dependent on leaf maturity and the severity of drought. These data suggested that drought stress-induced structural and compositional cuticular modifications improve cuticle water barrier property. In addition, we demonstrated that cuticle from the tender leaf and the mature leaf were modified through both common and distinct modes.
Project description:<h4>Key message</h4>The cuticle is the plant's barrier against abiotic and biotic stresses, and the deposition of epicuticular wax crystals results in the scattering of light, an effect termed glaucousness. Here, we dissect the genetic architecture of flag leaf glaucousness in wheat toward a future targeted design of the cuticle. The cuticle serves as a barrier that protects plants against abiotic and biotic stresses. Differences in cuticle composition can be detected by the scattering of light on epicuticular wax crystals, which causes a phenotype termed glaucousness. In this study, we dissected the genetic architecture of flag leaf glaucousness in a panel of 1106 wheat cultivars of global origin. We observed a large genotypic variation, but the geographic pattern suggests that other wax layer characteristics besides glaucousness may be important in conferring tolerance to abiotic stresses such as heat and drought. Genome-wide association mapping identified two major quantitative trait loci (QTL) on chromosomes 3A and 2B. The latter corresponds to the W1 locus, but further characterization revealed that it is likely to contain additional QTL. The same holds true for the major QTL on 3A, which was also found to show an epistatic interaction with another locus located a few centiMorgan distal to it. Genome-wide prediction and the identification of a few additional putative QTL revealed that small-effect QTL also contribute to the trait. Collectively, our results illustrate the complexity of the genetic control of flag leaf glaucousness, with additive effects and epistasis, and lay the foundation for the cloning of the underlying genes toward a more targeted design of the cuticle by plant breeding.
Project description:The cuticle plays a major role in restricting nonstomatal water transpiration in plants. There is therefore a long-standing interest to understand the structure and function of the plant cuticle. Although many efforts have been devoted, it remains controversial to what degree the various cuticular parameters contribute to the water transpiration barrier. In this study, eight tea germplasms were grown under normal conditions; cuticle thickness, wax coverage, and compositions were analyzed from the epicuticular waxes and the intracuticular waxes of both leaf surfaces. The cuticular transpiration rates were measured from the individual leaf surface as well as the intracuticular wax layer. Epicuticular wax resistances were also calculated from both leaf surfaces. The correlation analysis between the cuticular transpiration rates (or resistances) and various cuticle parameters was conducted. We found that the abaxial cuticular transpiration rates accounted for 64-78% of total cuticular transpiration and were the dominant factor in the variations for the total cuticular transpiration. On the adaxial surface, the major cuticular transpiration barrier was located on the intracuticular waxes; however, on the abaxial surface, the major cuticular transpiration barrier was located on the epicuticular waxes. Cuticle thickness was not a factor affecting cuticular transpiration. However, the abaxial epicuticular wax coverage was found to be significantly and positively correlated with the abaxial epicuticular resistance. Correlation analysis suggested that the very-long-chain aliphatic compounds and glycol esters play major roles in the cuticular transpiration barrier in tea trees grown under normal conditions. Our results provided novel insights about the complex structure-functional relationships in the tea cuticle.
Project description:The plant cuticle is the major barrier that limits unrestricted water loss and hence plays a critical role in plant drought tolerance. Due to the presence of stomata on the leaf abaxial surface, it is technically challenging to measure abaxial cuticular transpiration. Most of the existing reports were only focused on leaf astomatous adaxial surface, and few data are available regarding abaxial cuticular transpiration. Developing a method that can measure cuticular transpiration from both leaf surfaces simultaneously will improve our understanding about leaf transpiration barrier organization. Here, we developed a new method that enabled the simultaneous measurement of cuticular transpiration rates from the adaxial and abaxial surfaces. The proposed method combined multi-step leaf pretreatments including water equilibration under dark and ABA treatment to close stomata, as well as gum arabic or vaseline application to remove or seal the epicuticular wax layer. Mathematical formulas were established and used to calculate the transpiration rates of individual leaf surfaces from observed experimental data. This method facilitates the simultaneous quantification of cuticular transpiration from adaxial and abaxial leaf surfaces. By applying this method, we demonstrated that the adaxial intracuticular waxes and the abaxial epicuticular waxes constitute the major transpiration barriers in Camellia sinensis. Wax analysis indicated that adaxial intracuticular waxes had higher coverage of very long chain fatty acids, 1-alkanol esters, and glycols, which may be attributed to its higher transpiration barrier than that of the abaxial intracuticular waxes.
Project description: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, ?- and OH-?-diketones were first detected in flag leaves from 200-day-old plants, and the amounts of ?- and OH-?-diketones were significantly higher in glaucous varieties compared with glossy varieties. In spike waxes, ?-diketone existed in all varieties, but OH-?-diketone was detectable only in the glaucous varieties. Unexpectedly, the glaucous variety Fanmai 5 yielded large amounts of OH-?-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.
Project description:Previous studies have shown that wheat grain yield is seriously affected by drought stress, and leaf cuticular wax is reportedly associated with drought tolerance. However, most studies have focused on cuticular wax biosynthesis and model species. The effects of cuticular wax on wheat drought tolerance have rarely been studied. The aims of the current study were to study the effects of leaf cuticular wax on wheat grain yield under drought stress using the above-mentioned wheat NILs and to discuss the possible physiological mechanism of cuticular wax on high grain yield under drought stress. Compared to water-irrigated (WI) conditions, the cuticular wax content (CWC) in glaucous and non-glaucous NILs under drought-stress (DS) conditions both increased; mean increase values were 151.1 and 114.4%, respectively, which was corroborated by scanning electronic microscopy images of large wax particles loaded on the surfaces of flag leaves. The average yield of glaucous NILs was higher than that of non-glaucous NILs under DS conditions in 2014 and 2015; mean values were 7368.37 kg·ha<sup>-1</sup> and 7103.51 kg·ha<sup>-1</sup>. This suggested that glaucous NILs were more drought-tolerant than non-glaucous NILs (<i>P</i> = 0.05), which was supported by the findings of drought tolerance indices TOL and SSI in both years, the relatively high water potential and relative water content, and the low ELWL. Furthermore, the photosynthesis rate (<i>P<sub>n</sub></i> ) of glaucous and non-glaucous wheat NILs under DS conditions decreased by 7.5 and 9.8%, respectively; however, glaucous NILs still had higher mean values of <i>P<sub>n</sub></i> than those of non-glaucous NILs, which perhaps resulted in the higher yield of glaucous NILs. This could be explained by the fact that glaucous NILs had a smaller <i>F<sub>v</sub>/F<sub>m</sub></i> reduction, a smaller <i>PI</i> reduction and a greater <i>ABS/RC</i> increase than non-glaucous NILs under DS conditions. This is the first report to show that wheat cuticular wax accumulation is associated with drought tolerance. Moreover, the leaf CWC can be an effective selection criterion in the development of drought-tolerant wheat cultivars.