Changes in cuticular transpiration rate and cuticular lipids of oat (Avena sativa) seedlings induced by water stress

Two cultivars of oat ( Avena sativa L. cvs Pendek and Stormogul II) were exposed to short periods of water-deficit stress on five consecutive days. The plants responded to the stress by decreasing their cuticular transpiration rate. After two stress periods the cuticular transpiration rate was reduced by 30% for Pendek and by 47% for Stormogul II, and after another three stress periods by 30% and 20%, respectively. These reductions were correlated neither to changes in the total amount of what is generally called epicuticular lipids, nor to changes in any of the major individual constituents of the epicuticular lipids (alkanes, free and esterified fatty acids or free primary alcohols). After removal of the epicuticular lipids the long chain free primary alcohols of the leaves were extracted and determined. The amount of these presumably intracuticular alcohols increased after stress and changed to shorter chain length. From these results it is concluded that the intra- as well as the epicuticular lipids must be taken into consideration when discussing leaf surface lipids as protecting agents against water loss.     

Cuticular transpiration and epicuticular lipids of primary leaves of barley (Hordeum vulgare)

Twenty cultivars of barley and 15 eceriferum mutants from one of the cultivars have been analysed for cuticular transpiration and epicuticular lipids of their primary leaves. The relative cuticular transpiration rates of the cultivars ranged from 0.61 to 1.98. In spite of this variation in transpiration most of the cultivars had almost the same amount of epicuticular lipids per leaf area, about 16 μg cm⁻². The eceriferum mutants showed a wider range in amount of epicuticular lipids, from 5.0 to 15.5 μg cm⁻². Nevertheless, most of the mutants transpired almost at the same rate. Only a weak correlation was found between cuticular transpiration and total amount of epicuticular lipids. None of the analysed lipid components (alkanes, aldehydes, primary alcohols, esters or fatty acids) was better correlated to the cuticular transpiration than the total amount of lipids. When the cultivars were exposed to a mild water stress their cuticular transpiration rates decreased by about 11%. This reduction was not accompanied by any corresponding increase in total amount of epicuticular lipids. The most pronounced effect of the water stress treatment was a stimulation in the ester formation and a reduced formation of primary alcohols. This shift in lipid composition could not be correlated to the decreased cuticular transpiration rates of the individual cultivars. From this investigation it is concluded that the cuticular transpiration is poorly correlated to the amount or composition of the epicuticular lipids in this barley material. As a consequence it was not possible to use any characteristic of the epicuticular lipids as a selection criterion in breeding for drought resistance.     

空气湿度与土壤水分胁迫对紫花苜蓿叶表皮蜡质特性的影响

选用2个抗旱性不同的紫花苜蓿品种,敖汉(强抗旱)和三得利(弱抗旱),设置空气湿度(45%-55%和75%-85%)和土壤水分胁迫(75%和35%田间持水量)处理,分析紫花苜蓿叶表皮蜡质含量、组分及晶体结构、气体交换参数、水势及脯氨酸含量的变化规律。结果表明,单独土壤水分胁迫时,紫花苜蓿叶表皮蜡质晶体结构及蜡质总量无显著变化;敖汉蜡质组分中烷类、酯类含量增加,醇类含量下降;三得利醇类含量下降,烷类、酯类含量变化不显著。低空气湿度胁迫时,两品种蜡质总量无显著变化,烷类和酯类含量显著增加,醇类含量显著下降,叶表皮片状蜡质晶体结构熔融呈弥漫性,扩大了对叶表面积的覆盖,其蒸腾速率显著低于正常湿度。复合胁迫处理时,叶表皮片状蜡质晶体结构继续呈弥漫性,烷类、酯类、未知蜡质组分含量均高于单独胁迫处理,醇类含量最低,而蜡质总量除三得利显著高于对照外,其余均无显著差异。紫花苜蓿叶表皮蜡质各组分含量(除醇类)及蜡质总量与光合速率呈显著负相关,与蒸腾速率无显著相关关系。蜡质总量与叶水势呈显著正相关。总体上,敖汉蜡质总量显著高于三得利,蜡质组分中烷类物质的增加有助于提高植株的抗旱性。在复合胁迫下,强抗旱品种主要通过气孔因素控制水分散失,而弱抗旱品种通过气孔和非气孔因素共同控制植物水分散失。     

Epi- and intracuticular lipids and cuticular transpiration rates of primary leaves of eight barley (Hordeum vulgare) cultivars

The major constituents of the epi- and intracuticular lipids of primary leaves of 8 cultivars of barley ( Hordeum vulgare L.) have been studied together with cuticular transpiration rates. The total amount of analysed cuticular lipids ranged from 9.6 to 13.4 μg cm⁻² and was dominated by the epicuticular fraction, which made up 73–84% of the total. There were variations in the percentages of the analysed lipid classes, alkanes, esters, aldehydes, β-diketones and alcohols, between epi- and intracuticular lipids among individual cultivars, but no clear tendency in these variations, except for the aldehydes, was found. The epicuticular lipids were richer in aldehydes than the intracuticular lipids. The cuticular transpiration rates were poorly correlated with the levels or composition of epi-, intra- or total cuticular lipids. The cuticular transpiration rates were considerably altered as a response to a water stress treatment, but these changes could not be correlated with any changes in amount or composition of the cuticular lipids. From these results it is concluded that some property other than amount or composition of cuticular lipids is the most important in regulation of water diffusion through the cuticle.     

Leaf cuticular waxes and physiological parameters in alfalfa leaves as influenced by drought

Drought significantly constrains higher yield of alfalfa (Medicago sativa L.) in arid and semiarid areas all over the world. This study evaluated the responses of leaf cuticular wax constituents to drought treatment and their relations to gas-exchange indexes across six alfalfa cultivars widely grown in China. Water deficit was imposed by withholding water for 12 d during branching stage. Cuticular waxes on alfalfa leaves were dominated by primary alcohols (41.7?C54.2%), alkanes (13.2?C26.9%) and terpenes (17.5?C28.9%), with small amount of aldehydes (1.4?C3.4%) and unknown constituents (4.5?C18.4%). Compared to total wax contents, the wax constituents were more sensitive to drought treatment. Drought decreased the contents of primary alcohol and increased alkanes in all cultivars. Alkane homologs, C25, C27, and C29, were all negatively correlated with photosynthetic rate, transpiration rate, stomatal conductance, and leaf water potential. Under drought conditions, both stomatal and nonstomatal factors were involved in controlling water loss from alfalfa leaves. No direct relationship was observed between wax contents and drought resistance among alfalfa cultivars. An increase in alkane content might be more important in improving drought tolerance of alfalfa under water deficit, which might be used as an index for selecting and breeding drought resistant cultivars of alfalfa.     

高羊茅叶片表皮蜡质含量与其抗旱性的关系

以14个高羊茅品种为试验材料,在田间试验中对干旱高温胁迫下的叶片表皮蜡质含量、净光合速率、蒸腾速率、气孔导度、胞间CO2浓度等生理指标测定分析。结果表明,干热胁迫下高羊茅品种间的叶片表皮蜡质含量和水分利用效率均存在极显著差异(P<0.01);叶片蜡质含量与综合抗旱性和水分利用效率的等级相关系数分别为0.78(P<0.01)和0.68(P<0.01);蜡质含量越高的品种,其叶片气孔导度和胞间CO2浓度越低,水分利用效率越高,但所有品种的水分利用效率绝对值都较低。研究发现,在干热胁迫时,高羊茅叶片表皮蜡质可通过对气孔导度的调节来减少气孔蒸腾,提高水分利用效率,最终提高其抗旱性;表皮蜡质含量可以作为高羊茅品种抗旱性鉴定的一个新指标。     

A morpho-physiological approach differentiates bread wheat cultivars of contrasting tolerance under cyclic water stress

Leaf micromorphological traits and some physiological parameters with potential relevance to drought tolerance mechanisms were investigated in four selected winter wheat varieties. Plants were subjected to two cycles of drought treatment at anthesis. Yield components confirmed contrasting drought-sensitive and -tolerant behavior of the genotypes. Drought tolerance was associated with small flag leaf surfaces and less frequent occurrence of stomata. Substantial variation of leaf cuticular thickness was found among the cultivars. Thin cuticle coincided with drought sensitivity and correlated with a high rate of dark-adapted water loss from leaves. Unlike in Arabidopsis, thickening of the cuticular matrix in response to water deprivation did not occur. Water stress induced epicuticular wax crystal depositions preferentially on the abaxial leaf surfaces. According to microscopy and electrolyte leakage measurements from leaf tissues, membrane integrity was lost earlier or to a higher extent in sensitive than in tolerant genotypes. Cellular damage and a decline of relative water content of leaves in sensitive cultivars became distinctive during the second cycle of water deprivation. Our results indicate strong variation of traits with potential contribution to the complex phenotype of drought tolerance in wheat genotypes. The maintained membrane integrity and relative water content values during repeated water limited periods were found to correlate with drought tolerance in the selection of cultivars investigated.     

The After-Effect of Water Stress on Transpiration Rate and Changes in Abscisic Acid Content of Young Wheat Plants

Young wheat plants (Triticum aestivum L. cv. Weibulls Starke II) were exposed to water stress for 1, 2 or 3 hours by cooling the roots. The plants were subjected to a constant water stress during the stress periods. By this treatment the leaf water potential was lowered from ?6.5 to ?11.5 bars. Leaf water potential, transpiration rate and abscisic acid content were determined during the stress periods and during the recovery. The water stressed plants showed an after-effect on transpiration rate lasting for between 10 and 24 hours depending on the duration of the stress. The amount of water stress in the stressed plants compared with the controls is defined as the difference in leaf water potential between the controls and the stressed plants during the stress period integrated over time. The amount of after-affect on transpiration is analogously defined as the difference in transpiration rate between the controls and the stressed plants during the recovery period integrated over time. There was a linear relationship between the amount of water stress and the amount of after-effect on transpiration of the leaves. The abscisic acid content of the leaves increased between 3.0 and 4.5 times the original content depending on the duration of the stress. However, during the recovery the abscisic acid content reattained the pre-stress level within 3 hours for all three stress periods. There was thus no direct relationship between the after-effect and the abscisic acid content of the leaf.     

Ontogenetic variation in chemical and physical characteristics of adaxial apple leaf surfaces

The reaction of plants to environmental factors often varies with developmental stage. It was hypothesized, that also the cuticle, the outer surface layer of plants is modified during ontogenesis. Apple plantlets, cv. Golden Delicious, were grown under controlled conditions avoiding biotic and abiotic stress factors. The cuticular wax surface of adaxial apple leaves was analyzed for its chemical composition as well as for its micromorphology and hydrophobicity just after unfolding of leaves ending in the seventh leaf insertion. The outer surface of apple leaves was formed by a thin amorphous layer of epicuticular waxes. Epidermal cells of young leaves exhibited a distinctive curvature of the periclinal cell walls resulting in an undulated surface of the cuticle including pronounced lamellae, with the highest density at the centre of cells. As epidermal cells expanded during ontogenesis, the upper surface showed only minor surface sculpturing and a decrease in lamellae. With increasing leaf age the hydrophobicity of adaxial leaf side decreased significantly indicated by a decrease in contact angle. Extracted from plants, the amount of apolar cuticular wax per area unit ranged from only 0.9 microgcm(-2) for the oldest studied leaf to 1.5 microgcm(-2) for the youngest studied leaf. Differences in the total amount of cuticular waxes per leaf were not significant for older leaves. For young leaves, triterpenes (ursolic acid and oleanolic acid), esters and alcohols were the main wax components. During ontogenesis, the proportion of triterpenes in total mass of apolar waxes decreased from 32% (leaf 1) to 13% (leaf 7); absolute amounts decreased by more than 50%. The proportion of wax alcohols and esters, and alkanes to a lesser degree, increased with leaf age, whereas the proportion of acids decreased. The epicuticular wax layer also contained alpha-tocopherol described for the first time to be present also in the epicuticular wax. The modifications in the chemical composition of cuticular waxes are discussed in relation to the varying physical characteristics of the cuticle during ontogenesis of apple leaves.     

Tomato fruit cuticular waxes and their effects on transpiration barrier properties: functional characterization of a mutant deficient in a very-long-chain fatty acid beta-ketoacyl-CoA synthase

Cuticular waxes play a pivotal role in limiting transpirational water loss across the plant surface. The correlation between the chemical composition of the cuticular waxes and their function as a transpiration barrier is still unclear. In the present study, intact tomato fruits (Lycopersicon esculentum) are used, due to their astomatous surface, as a novel integrative approach to investigate this composition- function relationship: wax amounts and compositions of tomato were manipulated before measuring unbiased cuticular transpiration. First, successive mechanical and extractive wax-removal steps allowed the selective modification of epi- and intracuticular wax layers. The epicuticular film consisted exclusively of very-long-chain aliphatics, while the intracuticular compartment contained large quantities of pentacyclic triterpenoids as well. Second, applying reverse genetic techniques, a loss-of-function mutation with a transposon insertion in a very-long-chain fatty acid elongase beta-ketoacyl-CoA synthase was isolated and characterized. Mutant leaf and fruit waxes were deficient in n-alkanes and aldehydes with chain lengths beyond C30, while shorter chains and branched hydrocarbons were not affected. The mutant fruit wax also showed a significant increase in intracuticular triterpenoids. Removal of the epicuticular wax layer, accounting for one-third of the total wax coverage on wild-type fruits, had only moderate effects on transpiration. By contrast, reduction of the intracuticular aliphatics in the mutant to approximately 50% caused a 4-fold increase in permeability. Hence, the main portion of the transpiration barrier is located in the intracuticular wax layer, largely determined by the aliphatic constituents, but modified by the presence of triterpenoids, whereas epicuticular aliphatics play a minor role.     

The impact of epicuticular wax on gas-exchange and photoinhibition in Leucadendron lanigerum (Proteaceae)

This study investigated the seasonal modification of wax deposition, and the impact of epicuticular wax on gas-exchange as well as photoinhibition in Leucadendron lanigerum, a species from the Proteaceae family with wax-covered leaf surfaces and the stomata also partially occluded by wax. The results of this study demonstrated that the deposition of epicuticular wax in L. lanigerum is dependent on the age of the leaf as well as the season, and generation and regeneration of wax occur mostly in spring while transformation and also degeneration of wax crystals occur in winter. Epicuticular waxes decreased cuticular water loss, but had little impact on leaf reflectance. The temperature of leaves without wax was lower than that of wax-covered leaves, indicating that the rate of transpiration impacted more on leaf temperature than reflectance of light in the PAR range in L. lanigerum. The wax coverage at the entrance of stomata in L. lanigerum increased resistance to gas diffusion and as a consequence decreased stomatal conductance, transpiration and photosynthesis. Also, the results indicated that epicuticular waxes do help prevent photodamage in L. lanigerum, and so this property could benefit plants living in arid environments with high solar radiation.     

Plant response to drought stress simulated by ABA application: Changes in chemical composition of cuticular waxes

Plant cuticles form the interface between epidermal plant cells and the atmosphere. The cuticle creates an effective barrier against water loss, bacterial and fungal infection and also protects plant tissue from UV radiation. It is composed of the cutin matrix and embedded soluble lipids also called waxes. Chemical composition of cuticular waxes and physiological properties of cuticles are affected by internal regulatory mechanisms and environmental conditions (e.g. drought, light, and humidity). Here, we tested the effect of drought stress simulation by the exogenous application of abscisic acid (ABA) on cuticular wax amount and composition. ABA-treated plants and control plants differed in total aboveground biomass, leaf area, stomatal density and aperture, and carbon isotope composition. They did not differ in total wax amount per area but there were peculiar differences in the abundance of particular components. ABA-treated plants contained significantly higher proportions of aliphatic components characterized by chain length larger than C₂₆, compared to control plants. This trend was consistent both between and within different functional groups of wax components. This can lead to a higher hydrophobicity of the cuticular transpiration barrier and thus decrease cuticular water loss in ABA-treated plants. At both ABA-treated and control plants alcohols with chain length C₂₄ and C₂₆ were predominant. Such a shift towards wax compounds having a higher average chain length under drought conditions can be interpreted as an adaptive response of plants towards drought stress.     

抗旱性不同的小麦幼苗对水分和NaCl胁迫的反应

分别测定抗旱小麦的８１３９（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ Ｌ．ｃｖ．８１３９）和干旱敏感品种甘麦８号（Ｔ．ａｅｓｔｉｖｕｍ Ｌ．ｃｖ．Ｇａｎｍａｉ Ｎｏ．８）在２０％ＰＥＧ６０００和１．２％ＮａＣｌ胁迫下的生长、光合作用、蒸腾作用及抗氧化保护系统的变化。结果表明,抗旱小麦８１３９对ＰＥＧ６０００有较强的抗性,但对ＮａＣｌ胁迫的抗性较差。ＮａＣｌ胁迫下,两种小麦根的生长均受到严重抑制,而在ＰＥ     

Contribution of arbuscular mycorrhizal fungi (AMF) to the adaptations exhibited by the deciduous shrub Anthyllis cytisoides L. under water deficit

Anthyllis cytisoides L. is highly colonized by arbuscular mycorrhizal fungi (AMF) and behaves as a drought-avoider species in the field. Our objectives were: (1) to study the response of A. cytisoides when exposed to moderate (acclimation) or severe (peak) drought and subsequent rewatering under nursery conditions; and (2) to verify if AMF improved the adaptation of A. cytisoides to stress. The soil compactness in drought-acclimated treatments increased four times compared with that of well-watered controls, which could reinforce the effects of water deficit on plant physiology. Photosynthetic rates decreased by around 50% and 70% and leaf conductance decreased by 40% and 50% in drought-acclimated non-mycorrhizal and mycorrhizal plants, respectively. Peak drought limited plant growth, accelerated leaf senescence and induced the conversion of starch into soluble sugars in the leaves of stressed plants. The accumulation of sugars could contribute to a decrease in water potential in order to achieve the required tension to let water move from soil to shoot. Mycorrhizal plants showed a two-fold higher chlorotic leaf biomass than non-mycorrhizal plants under severe drought. Moreover, mycorrhizal A. cytisoides showed enhanced epicuticular waxes on the surfaces of the remaining green leaves. Increased leaf senescence, together with wax deposition, could reduce whole plant transpiration, thus allowing mycorrhizal plants to maintain a higher leaf relative water content (50%) than non-mycorrhizal plants (35%). After drought recovery, leaf abscission in stressed mycorrhizal plants was 10 times greater than that in non-mycorrhizal plants. The results suggest that AMF conferred greater responsiveness of A. cytisoides to drought. Enhanced wax deposition and leaf senescence could be an ecological adaptation to cope with severe water deficit.     

Leaf cuticular waxes of potted rose cultivars as affected by plant development, drought and paclobutrazol treatments

The present work was carried out to evaluate how plant growth and cultural practices influence the amount and composition of cuticular waxes on leaves of rose cultivars. The total amount of cuticular wax per leaf area was higher for rose cultivar Apollo Parade than for Charming Parade. Both cultivars had waxes dominated by alkanes, with the major alkanes being the C₃₁ and C₃₃ homologues. Primary alcohols were the next most abundant constituent class, with C₂₆ as the dominant homologue. Compared with Charming Parade, Apollo Parade had higher proportions of its total wax load as primary alcohols but lower acids and aldehydes. The proportion of alkanes in the total load on these cultivars was similar. Commercially produced roses are routinely treated with paclobutrazol (PBZ) to retard growth. PBZ treatments caused a 10% increase in total wax load and changes in the proportions of certain wax constituents within 11 days of application. Notable was an increase in the total proportion of acids in the total load 25 days after PBZ application, primarily because of increased C₂₈ acids. An alternative method of retarding plant growth is production of roses under limited water availability. When Apollo Parade roses experienced periods of moderate drought stress during production, the wax load per leaf area increased 14 and 8% above control levels at 24 and 38 days after imposition of drought, respectively. Drought caused similar changes in the proportions of individual wax constituents as did PBZ application.     

Epicuticular Wax and Cuticular Resistance in Rice

High leaf cuticular resistance has been reported as a component adaptation of plants to drought prone regions, Experiments were conducted to evaluate and characterize the role of epicuticular wax as a component of cuticular resistance to water vapor loss from rice (Oryza sativa L.) leaves. This information is necessary to determine the applicability of including higher cuticular resistance in an upland rice breeding program and to evaluate potential selection methods. Diffusion porometry, electron microscopy, and gas liquid chromatography were employed. Measurement of cuticular resistance by leaf diffusive resistance porometry after stomatal closure by exposure of rice leaves to pure CO₂ for 15 min was found sufficient to induce complete stomatal closure regardless of light level, and was superior to dark acclimation for this purpose. Removal of epicuticular wax from rice leaves by chloroform dip significantly reduced the cuticular resistance. Stressed plants were observed to increase cuticular resistance, illustrating the responsive nature of this characteristic. Gas liquid chromatography (GLC) of the chloroform leaf dip proved to be an expedient method of characterizing both quantitative and qualitative differences in the epicuticular wax of rice cullivars. The porometry and GLC techniques may be useful in selecting parents, spot checking in a pedigree program, or checking lines reaching the yield testing stage, but ate not well suited lor mass screening early generation progeny. Significant differences were found in the two rice cultivurs, ‘63–83′, an upland adapted rice from West Africa, and “IR20′, bred and selected in submerged paddy culture in ihc Philippines, by tlie above methods of characterizing cuticular resistance and epicuticular wax. These results are of ecological significance to plant breeders.     

Changes in leaf cuticular waxes of sesame (Sesamum indicum L.) plants exposed to water deficit

Sesame (Sesamum indicum L.) is one of the most important oilseed crops, having seeds and oil that are highly valued as a traditional health food. The objective of this study was to evaluate leaf cuticular wax constituents across a diverse selection of sesame cultivars, and the responses of these waxes to drought-induced wilting. Water-deficit was imposed on 18 sesame cultivars by withholding irrigation for 15d during the post-flowering stage, and the effect on seed yield and leaf waxes compared with a well-watered control. Leaf cuticular waxes were dominated by alkanes (59% of total wax), with aldehydes being the next-most abundant class. Compared to well-irrigated plants, drought treatment caused an increase in wax amount on most cultivars, with only three cultivars having a notable reduction. When expressed as an average across all cultivars, drought treatment caused a 30% increase in total wax amount, with a 34% increase in total alkanes, a 13% increase in aldehydes, and a 28% increase in the total of unknowns. In all cultivars, the major alkane constituents were the C27, C29, C31, C33, and C35 homologs, whereas the major aldehydes were the C30, C32, and C34 homologs, and drought exposure had only minor effects on the chain length distribution within these and other wax classes. Drought treatments caused a large decrease in seed yield per plant, but did not affect the mean weight of individual seeds, showing that sesame responds to post-flowering drought by reducing seed numbers, but not seed size. Seed yield was inversely correlated with the total wax amount (-0.466*), indicating that drought induction of leaf wax deposition does not contribute directly to seed set. Further studies are needed to elucidate the ecological role for induction of the alkane metabolic pathway by drought in regulating sesame plant survival and seed development in water-limiting environments.     

Chemical composition of the Prunus laurocerasus leaf surface. Dynamic changes of the epicuticular wax film during leaf development

The seasonal development of adaxial Prunus laurocerasus leaf surfaces was studied using newly developed methods for the mechanical removal of epicuticular waxes. During epidermal cell expansion, more than 50 microg leaf(-1) of alkyl acetates accumulated within 10 d, forming an epicuticular wax film approximately 30 nm thick. Then, alcohols dominated for 18 d of leaf development, before alkanes accumulated in an epicuticular wax film with steadily increasing thickness (approximately 60 nm after 60 d), accompanied by small amounts of fatty acids, aldehydes, and alkyl esters. In contrast, the intracuticular waxes stayed fairly constant during development, being dominated by triterpenoids that could not be detected in the epicuticular waxes. The accumulation rates of all cuticular components are indicative for spontaneous segregation of intra- and epicuticular fractions during diffusional transport within the cuticle. This is the first report quantifying the loss of individual compound classes (acetates and alcohols) from the epicuticular wax mixture. Experiments with isolated epicuticular films showed that neither chemical conversion within the epicuticular film nor erosion/evaporation of wax constituents could account for this effect. Instead, transport of epicuticular compounds back into the tissue seems likely. Possible ecological and physiological functions of the coordinate changes in the composition of the plant surface layers are discussed.