Leaf surface wax is a source of plant methane formation under UV radiation and in the presence of oxygen

The terrestrial vegetation is a source of UV radiation‐induced aerobic methane (CH₄) release to the atmosphere. Hitherto pectin, a plant structural component, has been considered as the most likely precursor for this CH₄ release. However, most of the leaf pectin is situated below the surface wax layer, and UV transmittance of the cuticle differs among plant species. In some species, the cuticle effectively absorbs and/or reflects UV radiation. Thus, pectin may not necessarily contribute substantially to the UV radiation‐induced CH₄ emission measured at surface level in all species. Here, we investigated the potential of the leaf surface wax itself as a source of UV radiation‐induced leaf aerobic CH₄ formation. Isolated leaf surface wax emitted CH₄ at substantial rates in response to UV radiation. This discovery has implications for how the phenomenon should be scaled to global levels. In relation to this, we demonstrated that the UV radiation‐induced CH₄ emission is independent of leaf area index above unity. Further, we observed that the presence of O₂ in the atmosphere was necessary for achieving the highest rates of CH₄ emission. Methane formation from leaf surface wax is supposedly a two‐step process initiated by a photolytic rearrangement reaction of the major component followed by an α‐cleavage of the generated ketone.     

Interacting elevated CO2 and tropospheric O3 predisposes aspen (Populus tremuloides Michx.) to infection by rust (Melampsora medusae f. sp. tremuloidae)

We investigated the interaction of elevated CO₂ and/or (Ozone) O₃ on the occurrence and severity of aspen leaf rust (Melampsora medusae Thuem. f. sp. tremuloidae) on trembling aspen (Populus tremuloides Michx.). Furthermore, we examined the role of changes in leaf surface properties induced by elevated CO₂ and/or O₃ in this host–pathogen interaction. Three‐ to five‐fold increases in levels of rust infection index were found in 2 consecutive years following growing‐season‐long exposures with either O₃ alone or CO₂ + O₃ depending on aspen clone. Examination of leaf surface properties (wax appearance, wax amount, wax chemical composition, leaf surface and wettability) suggested significant effects by O₃ and CO₂ + O₃. We conclude that elevated O₃ is altering aspen leaf surfaces in such a way that it is likely predisposing the plants to increased infection by aspen leaf rust.     

The Inhibitor of wax 1 locus (Iw1) prevents formation of β‐ and OH‐β‐diketones in wheat cuticular waxes and maps to a sub‐cM interval on chromosome arm 2BS

Glaucousness is described as the scattering effect of visible light from wax deposited on the cuticle of plant aerial organs. In wheat, two dominant genes lead to non‐glaucous phenotypes: Inhibitor of wax 1 (Iw1) and Iw2. The molecular mechanisms and the exact extent (beyond visual assessment) by which these genes affect the composition and quantity of cuticular wax is unclear. To describe the Iw1 locus we used a genetic approach with detailed biochemical characterization of wax compounds. Using synteny and a large number of F₂ gametes, Iw1 was fine‐mapped to a sub‐cM genetic interval on wheat chromosome arm 2BS, which includes a single collinear gene from the corresponding Brachypodium and rice physical maps. The major components of flag leaf and peduncle cuticular waxes included primary alcohols, β‐diketones and n‐alkanes. Small amounts of C19–C27 alkyl and methylalkylresorcinols that have not previously been described in wheat waxes were identified. Using six pairs of BC₂F₃ near‐isogenic lines, we show that Iw1 inhibits the formation of β‐ and hydroxy‐β‐diketones in the peduncle and flag leaf blade cuticles. This inhibitory effect is independent of genetic background or tissue, and is accompanied by minor but consistent increases in n‐alkanes and C₂₄ primary alcohols. No differences were found in cuticle thickness and carbon isotope discrimination in near‐isogenic lines differing at Iw1.     

Changes in the composition of coffee leaf wax with development

Coffee leaf wax contains alkanes, free primary alcohols and free acids, together with unidentified substances. The relative amounts of each fraction varied with age: alkanes from 22–35% alcohols from 28-25%, and free acids from 22-14%. The major homologues of the alkane fraction were C₂₉ and C₃₁, of the alcohol fraction C₃₀ and C₃₂ and of the acid fraction C₂₈, C₃₀ and C₃₂. The ratio of both C₂₉:C₃₁ alkanes and C_3O:C₃₂ alcohols changed from 1:1 to 1:2 during development, although their combined sum in each case remained constant at 90% (for alkanes) and 73% (for alcohols) of the weight of the respective fractions.     

Responses to ultraviolet-B radiation (280–315 nm) of pea (Pisum sativum) lines differing in leaf surface wax

To test the hypothesis that leaf surface wax influences plant responses to UV-B, 6 lines of cultivated pea (Pisum sativum L.), selected as having more or less wax, were grown at 0 or 6.5 kJ m^-2 day^-1 plant-weighted UV-B against a background of 850–950 μmol m^-2 s^-1 photosynthetically active radiation. In the 4 lines with least leaf surface wax the amount of wax on adaxial and abaxial leaf surfaces was increased following exposure to 6.5 kJ m^-2 day^-1 UV-B, but UV-B decreased surface wax in Scout, which had the greatest wax deposits. On the adaxial leaf surface, UV-B radiation caused a shift in wax composition from alcohols to esters and hydrocarbons and the ratio of short to long chain length alkyl ester homologues was increased. There was no evidence of a shortening in carbon chain length of hydrocarbons, primary alcohols or fatty acids due to UV-B and no significant correlation between wax amount and UV reflectance from leaves. UV-B induced significant increases in UV-absorbing compounds in the expanded leaves and buds of most lines. UV-B reduced the growth of all lines. Foliage area (leaves plus stipules) declined by 5–30%, plant dry weight by 12–30%, and plant height by 24–38%. Reductions in growth occurred in the absence of any changes in chlorophyll fluorescence or photosynthetic rate. UV-B also had no major effect on carbon allocation patterns. The effects of UV-B on growth appeared to be due to changes in tissue extension and expansion. Indeed, many of the responses to UV-B observed in this study of pea appear more consistent with indirect effects being expressed in developing tissues rather than through the direct action of UV-B on mature tissues. There was no evidence that wax amount or biochemistry was associated with the sensitivity of the lines to UV-B radiation. Furthermore, induction of pigments was not correlated with changes in growth. However, lines with the greatest constitutive amounts of pigments in unexpanded bud tissues were most tolerant of elevated UV-B.     

Wax synthesis in Brassica oleracea as modified by trichloroacetic acid and glossy mutations

Two different mutations in Brassica oleracea, gl₅ and gl₄ have been re-investigated using acetate-1-¹⁴C labelling in an attempt to define more closely the nature of the genetic blocks to wax synthesis. It has been found that gl₅ is a mutation which blocks elongation in the Step C₂₈–C₃₀. The mutation gl₄ exhibits no elongation block and could be blocked in the decarboxylation Step C₃₀–C₂₉. 0·1 mM TCA supplied in the culture solution of cauliflower seedlings affected the leaf surface by producing a glossy appearance similar to that induced by gl₃ and gl₄. At this concentration growth was not inhibited and the appearance of the plants was normal except for the surface wax. The amount of surface wax produced was about 40% of that in untreated seedlings on a leaf area basis. Slight, but significant changes in wax composition were noted, mainly involving a reduction in C₃₀ acids and aldehydes, a slight reduction (33–29%) in alkane content, and a marked difference in chain length composition of the alkanes with C₂₇ increased relative to C₂₉. Over a range of concentrations from 0·1–1 mM, TCA inhibited incorporation of label from acetate-1-¹⁴C into C₃₀ acids and aldehydes more than into C₂₈ at concentrations 0·4–0·8 mM while label tended to accumulate in C₂₄ and C₂₆ acids; thus elongation C₂₈–C₃₀ was especially sensitive to TCA. TCA also inhibited incorporation into primary alcohols and esters almost as much as into C₂₉ compounds. In spite of relatively specific effects on incorporation of label into longer chain lengths, the resulting block to C₃₀ synthesis is not sufficient to make much difference to the overall rate of C₂₉ synthesis. Both results of analysis of wax from whole plants and experiments with tissue slices in vitro indicated that the effect of TCA in reducing the glaucousness of the leaf surface is a combination of overall reduction of wax synthesis together with slight but significant changes in wax composition.     

Changes of epicuticular wax induced by enhanced UV-B radiation impact on gas exchange in Brassica napus

The epicuticular wax covering on plant surface plays important roles in protecting plants against UV radiation. However, the role of epicuticular wax in affecting leaf gas exchange under enhanced ultraviolet-B (UV-B) radiation remains obscure. In the present study, different aged leaves of Brassica napus were used to analyze the responses of crystal structure and chemical constituents of epicuticular wax to UV-B radiation and the effects of such responses on gas exchange indices. Enhanced UV-B radiation significantly decreased the amount of esters in all leaves except the first leaf, amount of secondary alcohols in the second, third and fourth leaves, and amount of primary alcohols in the second and third leaves, while increased the amounts of ketones and aldehydes in the first leaf. Enhanced UV-B level had no significant effect on the amounts of alkanes and total wax in all leaves. Exposure to UV-B radiation resulted in wax fusion on adaxial leaf and stomata opening on abaxial leaf. Fusions of plates and rods on adaxial leaf surface covered most of the stomata, thereby influencing the photosynthesis in the upper mesophyll of leaves. Enhanced UV-B level significantly reduced the net photosynthesis rate (P _N) but increased the stomata conductance (g _s), concentrations of intercellular CO₂ (C _i ), and transpiration rate (E) in all leaves. Both UV-B radiation and the wax fusion induced by enhanced UV-B radiation resulted in different stomata status on abaxial and adaxial leaf surface, causing decrease of P _N, and increase of g _s, C _i and E in leaves.     

Epicuticular waxes of Sorghum and some compositional changes with plant age

Epicuticular wax from mature plants of Sorghum bicolor SD-102 was compared with that from panicles and seedlings of the same variety at the fourth-fifth leaf stage of growth. The composition of wax from SD-102 panicles was quite different from that of mature leaf blades and sheaths. Free fatty alcohols were the dominant class of wax from SD-102 seedlings and C₃₂ was the major homologue of alcohols and aldehydes. For comparison purposes, the epicuticular waxes from whole plants of two other S. bicolor varieties, Alliance A and Martin A, grown up to the tasseling stage, have been analysed. The major wax components were free fatty acids. The typical chain lengths of aldehydes, free alcohols and free fatty acids were C₂₈ and C_30.p-Hydroxybenzaldehyde was not a wax component of the studied varieties of sorghum.     

Will the climate of plant origins influence the chemical profiles of cuticular waxes on leaves of Leymus chinensis in a common garden experiment?

Cuticular wax covering the leaf surface plays important roles in protecting plants from biotic and abiotic stresses. Understanding the way in which plant leaf cuticles reflect their growing environment could give an insight into plant resilience to future climate change. Here, we analyzed the variations of cuticular waxes among 59 populations of Leymus chinensis in a common garden experiment, aiming to verify how environmental conditions influence the chemical profiles of cuticular waxes. In total, eight cuticular wax classes were identified, including fatty acids, aldehydes, primary alcohols, alkanes, secondary alcohols, ketones, β‐diketones, and alkylresorcinols, with β‐diketones the predominant compounds in all populations (averaged 67.36% across all populations). Great intraspecific trait variations (ITV) were observed for total wax coverage, wax compositions, and the relative abundance of homologues within each wax class. Cluster analysis based on wax characteristics could separate 59 populations into different clades. However, the populations could not be separated according to their original longitudes, latitudes, annual temperature, or annual precipitation. Redundancy analysis showed that latitude, arid index, and the precipitation from June to August were the most important parameters contributing to the variations of the amount of total wax coverage and wax composition and the relative abundance of wax classes. Pearson's correlation analysis further indicated that the relative abundance of wax classes, homologues in each wax class, and even isomers of certain compound differed in their responses to environmental factors. These results suggested that wax deposition patterns of L. chinensis populations formed during adaptations to their long‐term growing environments could inherit in their progenies and exhibit such inheritance even these progenies were exported to new environments.     

Composition of the epicuticular and intracuticular wax layers on Kalanchoe daigremontiana (Hamet et Perr. de la Bathie) leaves

Epicuticular and intracuticular waxes from both adaxial and abaxial surfaces of the leaves of Kalanchoe daigremontiana were analyzed. All wax mixtures were found to contain approximately equal amounts of triterpenoids and very long chain fatty acid (VLCFA) derivatives. The triterpenoid fraction consisted of glutinol (8-19% of the total wax) and friedelin (4-9%), together with smaller amounts of glutanol, glutinol acetate, epifriedelanol, germanicol and β-amyrin. The VLCFA derivatives comprised C₂₇-C₃₅ alkanes (19-37% of the total wax), C₃₂-C₃₄ aldehydes (3-7%), C₃₂ and C₃₄ fatty acids (0.2-3%), C₂₆-C₃₆ primary alcohols (4-8%), and C₄₂-C₅₂ alkyl esters (2-9%). The wax layers were found to differ in triterpenoid amounts, with the intracuticular wax containing higher percentages of most triterpenoids than the epicuticular wax. Friedelin, the only triterpenoid ketone present, showed the opposite distribution with higher proportions in the epicuticular wax. VLCFA derivatives also accumulated to higher percentages in the epicuticular than in the intracuticular wax layer. Epicuticular wax crystals were observed on both the adaxial and abaxial leaf surfaces.     

Specific inhibition of alkane synthesis with accumulation of very long chain compounds by dithioerythritol, dithiothreitol, and mercaptoethanol in Pisum sativum

Sodium [1-¹⁴C]acetate and [1-¹⁴C]stearic acid were readily incorporated into hydrocarbons, secondary alcohols, wax esters, aldehydes, primary alcohols, and fatty acids in young pea leaves (Pisum sativum). Dithioerythritol, dithiothreitol, and mercaptoethanol (but not glutathione and cysteine) severely inhibited the incorporation of labeled acetate into alkanes and secondary alcohols with accumulation of label in wax ester and aldehyde fractions. Detailed radio gas-chromatographic analyses of the fatty acids of both the surface lipid components and internal lipids showed that dithioerythritol and mercaptoethanol specifically inhibited n-hentriacontane (C₃₁) synthesis and caused accumulation of C₃₂ aldehyde, suggesting that the inhibition was at or near the terminal step in alkane biosynthesis, presumably decarboxylation. Trichloroacetate, at a concentration that inhibited C₃₁ alkane synthesis but not the synthesis of alcohols (C₂₆ and C₂₈) specifically inhibited the formation of C₃₂ aldehyde but not that of the C₂₆ or C₂₈ aldehyde. From these results, it is concluded that the C₃₂ aldehyde is derived from the C₃₂ acyl derivative which is the precursor of C₃₁ alkane.     

Epicuticular waxes of Secale cereale and Triticale hexaploide leaves

Wax on leaves of rye and of hexaploid Triticale (60–70-day-old plants) contains hydrocarbons (6–8%), esters (10%), free alcohols (14-8%), free acids (3%), hentriacontane-14,16-dione (39–45%), 25 (S)-hydroxyhentriacontane-14,16-dione (13–11%) and unidentified (14–15%). Diesters (1–3%) are also present in rye wax. Compositions of hydrocarbons (C₂₇-C₃₃) and esters (C₂₈,C₅₈) are similar for both waxes. Free and combined alcohols of rye wax are mainly hexacosanol but alcohols of Triticale wax are mainly octacosanol. The composition of Triticale wax is close to that of its wheat parent Triticum durum (cv. Stewart 63). Esters of wax from ripe rye contain 58% of trans 2,3-unsaturated esters. *NRCC No. 14033.     

Quantification of plant surface metabolites by matrix‐assisted laser desorption–ionization mass spectrometry imaging: glucosinolates on Arabidopsis thaliana leaves

The localization of metabolites on plant surfaces has been problematic because of the limitations of current methodologies. Attempts to localize glucosinolates, the sulfur‐rich defense compounds of the order Brassicales, on leaf surfaces have given many contradictory results depending on the method employed. Here we developed a matrix‐assisted laser desorption–ionization (MALDI) mass spectrometry protocol to detect surface glucosinolates on Arabidopsis thaliana leaves by applying the MALDI matrix through sublimation. Quantification was accomplished by spotting glucosinolate standards directly on the leaf surface. The A. thaliana leaf surface was found to contain approximately 15 nmol of total glucosinolate per leaf with about 50 pmol mm^?2 on abaxial (bottom) surfaces and 15–30 times less on adaxial (top) surfaces. Of the major compounds detected, 4‐methylsulfinylbutylglucosinolate, indol‐3‐ylmethylglucosinolate, and 8‐methylsulfinyloctylglucosinolate were also major components of the leaf interior, but the second most abundant glucosinolate on the surface, 4‐methylthiobutylglucosinolate, was only a trace component of the interior. Distribution on the surface was relatively uniform in contrast to the interior, where glucosinolates were distributed more abundantly in the midrib and periphery than the rest of the leaf. These results were confirmed by two other mass spectrometry‐based techniques, laser ablation electrospray ionization and liquid extraction surface analysis. The concentrations of glucosinolates on A. thaliana leaf surfaces were found to be sufficient to attract the specialist feeding lepidopterans Plutella xylostella and Pieris rapae for oviposition. The methods employed here should be easily applied to other plant species and metabolites.     

Human tear film and meibum. Very long chain wax esters and (O-acyl)-omega-hydroxy fatty acids of meibum

Human meibum was targetly analyzed for the presence of intact wax esters (WEs) and related compounds by means of reverse-phase HPLC in combination with ion trap mass spectrometry. The major detected WEs were based on C_18:n (n = 1–4) unsaturated FAs ranking in the following order of abundance: C_18:1>C_18:2>C_18:3>C_18:4. The major fatty alcohols (FAls) found in WE were of saturated nature and varied from C_18:0 to C_28:0. The three most abundant species were C_18:1-FA esters of C_24:0, C_25:0, and C_26:0-FAl. Typically, a major compound based on C_18:1-FA and a saturated FAl was accompanied by a few related compounds based on a C_18:2, C_18:3, and C_18:4-FA. Contrary to previous reports, no epoxy-WEs or epoxy-FAs were detected in fresh and 1-year-old meibum samples. More than 20 (O-acyl)-ω-hydroxy-FAs (OAHFAs) were observed. The main detected OAHFAs were based on very long-chain ω-hydroxy-FA (C_30:1, C_32:1, and C_34:1) acylated through their ω-hydroxyls by a C_18:1-FA. Due to their amphiphilic anionogenic nature, OAHFAs may be responsible for stabilization of the tear film lipid layer by creating an interface between the vast pool of strictly nonpolar lipids of meibum (WEs, cholesteryl esters, etc.) and the aqueous subphase beneath it, a role previously attributed to phospholipids.     

The composition of external lipids from adult whiteflies,Bemisia tabaci and Trialeurodes vaporariorum

The total surface lipids, including the wax particles, of the adult whiteflies of Bemisia tabaci and Trialeurodes vaporariorum were characterized. At eclosion, there were similar amounts of long-chain hydrocarbons, aldehydes, alcohols and wax esters. Within a few hours post-eclosion, long-chain aldehydes and long-chain alcohols were the dominant surface lipid components, C₃₄ on B. tabaci and C₃₂ on T. vaporariorum. Hydrocarbons, mainly n-alkanes, were minor components of the surface lipids. The major wax esters were C₄₆ on B. tabaci and C₄₂ on T. vaporariorum. The major acid and alcohol moieties in the wax esters of B. tabaci were C₂₀ and C₂₆, respectively, and of T. vaporariorum were C₂₀ and C₂₂, respectively. Both B. tabaci and T. vaporariorum had a minor wax ester composed of the fatty acid C_18:1 esterified to the major alcohols, C₃₄ and C₃₂, respectively. Bemisia were readily distinguished from Trialeurodes based on the composition of their wax particles and/or their wax esters; however, no differentiating surface lipid components were detected between biotypes A and B of B. tabaci.     

Surface wax of Andreaea and Pogonatum species

The mosses Andreaea rupestris, Pogonatum aloides and P. urnigerum contain surface waxes in amounts of 0.05–0.12% dry wt. The waxes consisted of esters (C₃₈-C₅₄), primary alcohols (C₂₀-C₃₂), free fatty acids (C₁₆-C₃₀), and alkanes (C₂₁-C₃₁). Additionally, aldehydes (C₂₂-C₃₀) were major constituents in the wax of P. urnigerum. The classes and their chain length distributions in the surface waxes of these mosses are comparable to those of epicuticular waxes of higher plants.     

A genetic link between leaf carbon isotope composition and whole‐plant water use efficiency in the C4 grass Setaria

Genetic selection for whole‐plant water use efficiency (yield per transpiration; WUE_plant) in any crop‐breeding programme requires high‐throughput phenotyping of component traits of WUE_plant such as intrinsic water use efficiency (WUE_i; CO₂ assimilation rate per stomatal conductance). Measuring WUE_i by gas exchange measurements is laborious and time consuming and may not reflect an integrated WUE_i over the life of the leaf. Alternatively, leaf carbon stable isotope composition (δ¹³C_leaf) has been suggested as a potential time‐integrated proxy for WUE_i that may provide a tool to screen for WUE_plant. However, a genetic link between δ¹³C_leaf and WUE_plant in a C₄ species has not been well established. Therefore, to determine if there is a genetic relationship in a C₄ plant between δ¹³C_leaf and WUE_plant under well watered and water‐limited growth conditions, a high‐throughput phenotyping facility was used to measure WUE_plant in a recombinant inbred line (RIL) population created between the C₄ grasses Setaria viridis and S. italica. Three quantitative trait loci (QTL) for δ¹³C_leaf were found and co‐localized with transpiration, biomass accumulation, and WUE_plant. Additionally, WUE_plant for each of the δ¹³C_leaf QTL allele classes was negatively correlated with δ¹³C_leaf, as would be predicted when WUE_i influences WUE_plant. These results demonstrate that δ¹³C_leaf is genetically linked to WUE_plant, likely to be through their relationship with WUE_i, and can be used as a high‐throughput proxy to screen for WUE_plant in these C₄ species.     

Elevated CO2 affects photosynthetic responses in canopy pine and subcanopy deciduous trees over 10 years: a synthesis from Duke FACE

Leaf responses to elevated atmospheric CO₂ concentration (C_a) are central to models of forest CO₂ exchange with the atmosphere and constrain the magnitude of the future carbon sink. Estimating the magnitude of primary productivity enhancement of forests in elevated C_a requires an understanding of how photosynthesis is regulated by diffusional and biochemical components and up‐scaled to entire canopies. To test the sensitivity of leaf photosynthesis and stomatal conductance to elevated C_a in time and space, we compiled a comprehensive dataset measured over 10 years for a temperate pine forest of Pinus taeda, but also including deciduous species, primarily Liquidambar styraciflua. We combined over one thousand controlled‐response curves of photosynthesis as a function of environmental drivers (light, air C_a and temperature) measured at canopy heights up to 20 m over 11 years (1996–2006) to generate parameterizations for leaf‐scale models for the Duke free‐air CO₂ enrichment (FACE) experiment. The enhancement of leaf net photosynthesis (A_net) in P. taeda by elevated C_a of +200 μmol mol^?1 was 67% for current‐year needles in the upper crown in summer conditions over 10 years. Photosynthetic enhancement of P. taeda at the leaf‐scale increased by two‐fold from the driest to wettest growing seasons. Current‐year pine foliage A_net was sensitive to temporal variation, whereas previous‐year foliage A_net was less responsive and overall showed less enhancement (+30%). Photosynthetic downregulation in overwintering upper canopy pine needles was small at average leaf N (N_area), but statistically significant. In contrast, co‐dominant and subcanopy L. styraciflua trees showed A_net enhancement of 62% and no A_net–N_area adjustments. Various understory deciduous tree species showed an average A_net enhancement of 42%. Differences in photosynthetic responses between overwintering pine needles and subcanopy deciduous leaves suggest that increased C_a has the potential to enhance the mixed‐species composition of planted pine stands and, by extension, naturally regenerating pine‐dominated stands.     

Genotype differences in 13C discrimination between atmosphere and leaf matter match differences in transpiration efficiency at leaf and whole‐plant levels in hybrid Populus deltoides ×nigra

¹³C discrimination between atmosphere and bulk leaf matter (Δ¹³C_lb) is frequently used as a proxy for transpiration efficiency (TE). Nevertheless, its relevance is challenged due to: (1) potential deviations from the theoretical discrimination model, and (2) complex time integration and upscaling from leaf to whole plant. Six hybrid genotypes of Populus deltoides×nigra genotypes were grown in climate chambers and tested for whole‐plant TE (i.e. accumulated biomass/water transpired). Net CO₂ assimilation rates (A) and stomatal conductance (g_s) were recorded in parallel to: (1) ¹³C in leaf bulk material (δ¹³C_lb) and in soluble sugars (δ¹³C_ss) and (2) ¹⁸O in leaf water and bulk leaf material. Genotypic means of δ¹³C_lb and δ¹³C_ss were tightly correlated. Discrimination between atmosphere and soluble sugars was correlated with daily intrinsic TE at leaf level (daily mean A/g_s), and with whole‐plant TE. Finally, g_s was positively correlated to ¹⁸O enrichment of bulk matter or water of leaves at individual level, but not at genotype level. We conclude that Δ¹³C_lb captures efficiently the genetic variability of whole‐plant TE in poplar. Nevertheless, scaling from leaf level to whole‐plant TE requires to take into account water losses and respiration independent of photosynthesis, which remain poorly documented.