The presence of cutan limits the interpretation of cuticular chemistry and structure: Ficus elastica leaf as an example

Plant cuticles have been traditionally classified on the basis of their ultrastructure, with certain chemical composition assumptions. However, the nature of the plant cuticle may be misinterpreted in the prevailing model, which was established more than 150 years ago. Using the adaxial leaf cuticle of Ficus elastica, a study was conducted with the aim of analyzing cuticular ultrastructure, chemical composition and the potential relationship between structure and chemistry. Gradual chemical extractions and diverse analytical and microscopic techniques were performed on isolated leaf cuticles of two different stages of development (i.e. young and mature leaves). Evidence for the presence of cutan in F. elastica leaf cuticles has been gained after chemical treatments and tissue analysis by infrared spectroscopy and electron microscopy. Significant calcium, boron and silicon concentrations were also measured in the cuticle of this species. Such mineral elements which are often found in plant cell walls may play a structural role and their presence in isolated cuticles further supports the interpretation of the cuticle as the most external region of the epidermal cell wall. The complex and heterogeneous nature of the cuticle, and constraints associated with current analytical procedures may limit the chance for establishing a relationship between cuticle chemical composition and structure also in relation to organ ontogeny.     

Mid-infrared spectroscopy is a fast screening method for selecting Arabidopsis genotypes with altered leaf cuticular wax

Arabidopsis eceriferum (cer) mutants with unique alterations in their rosette leaf cuticular wax accumulation and composition established by gas chromatography have been investigated using attenuated total reflection (ATR)-Fourier transform infrared (FTIR) spectroscopy in combination with univariate and multivariate analysis. Objectives of this study were to evaluate the utility of ATR-FTIR for detection of chemical diversity in leaf cuticles, obtain spectral profiles of cer mutants in comparison with the wild type, and identify changes in leaf cuticles caused by drought stress. FTIR spectra revealed both genotype- and treatment-dependent differences in the chemical make-up of Arabidopsis leaf cuticles. Drought stress caused specific changes in the integrated area of the CH₃ peak, asymmetrical and symmetrical CH₂ peaks, ester carbonyl peak and the peak area ratio of ester CO to CH₂ asymmetrical vibration. CH₃ peak positively correlated with the total wax accumulation. Thus, ATR-FTIR spectroscopy is a valuable tool that can advance our understanding of the role of cuticle chemistry in plant response to drought and allow selection of superior drought-tolerant varieties from large genetic resources.     

Vapour pressure deficit during growth has little impact on genotypic differences of transpiration efficiency at leaf and whole‐plant level: an example from Populus nigra L.

Poplar genotypes differ in transpiration efficiency (TE) at leaf and whole‐plant level under similar conditions. We tested whether atmospheric vapour pressure deficit (VPD) affected TE to the same extent across genotypes. Six Populus nigra genotypes were grown under two VPD. We recorded (1) ¹³C content in soluble sugars; (2) ¹⁸O enrichment in leaf water; (3) leaf‐level gas exchange; and (4) whole‐plant biomass accumulation and water use. Whole‐plant and intrinsic leaf TE and ¹³C content in soluble sugars differed significantly among genotypes. Stomatal conductance contributed more to these differences than net CO₂ assimilation rate. VPD increased water use and reduced whole‐plant TE. It increased intrinsic leaf‐level TE due to a decline in stomatal conductance. It also promoted higher ¹⁸O enrichment in leaf water. VPD had no genotype‐specific effect. We detected a deviation in the relationship between ¹³C in leaf sugars and ¹³C predicted from gas exchange and the standard discrimination model. This may be partly due to genotypic differences in mesophyll conductance, and to its lack of sensitivity to VPD. Leaf‐level ¹³C discrimination was a powerful predictor of the genetic variability of whole‐plant TE irrespective of VPD during growth.     

PENETRATION OF 2,4-D IN RELATION TO CUTICLE THICKNESS

Cuticle thickness was measured, either by direct microscopic examination or as weight per unit area, for the astomatous cuticle from the upper leaf surface of nine species and from tomato fruit. Thickness ranged from 1.4 μm for peach leaf cuticle to 10.8 μm for oleander leaf cuticle, and the weight from 0.19 mg/cm² to 1.26 mg/cm², respectively. Cuticles were isolated by the pectinase method and permeability to 2,4-D was determined. There was no correlation between cuticle thickness and penetration of 2,4-D, either for non-dewaxed cuticles or after chloroform extraction of waxes. Penetration of 2,4-D was increased following wax removal, but there was no correlation between wax content and the magnitude of the increase. It is suggested that cutin and wax qualitative composition are probably more important than thickness in determining relative permeability of cuticle from different plant species to 2,4-D.     

Development of plant cuticles: fine structure and cutin composition of Clivia miniata Reg. leaves

The fine structure and monomeric composition of the ester-cutin fraction (susceptible to BF₃/CH₃OH transesterification) of the adaxial leaf cuticle of Clivia miniata Reg. were studied in relation to leaf and cuticle development. Clivia leaves grow at their base such that cuticle and tissues increase in age from the base to the tip. The zone of maximum growth (cell expansion) was located between 1 and 4 cm from the base. During cell expansion, the projected surface area of the upper epidermal cells increased by a factor of nine. In the growth region the cuticle consists mainly of a polylamellate cuticle proper of 100–250 nm thickness. After cell expansion has ceased both the outer epidermal wall and the cuticle increase in thickness. Thickening of the cuticle is accomplished by interposition of a cuticular layer between the cuticle proper and the cell wall. The cuticular layer exhibits a reticulate fine structure and contributes most of the total mass of the cuticle at positions above 6 cm from the leaf base. The composition of ester cutin changed with the age of cuticles. In depolymerisates from young cuticles, 26 different monomers could be detected whereas in older ones their number decreased to 13. At all developmental stages, 9,16-/10,16-dihydroxyhexadecanoic acid (positional isomers not separated), 18-hydroxy-9-octadecenoic acid, 9,10,18-trihydroxyoctadecanoic acid and 9,10-epoxy-18-hydroxyoctadecanoic acid were most frequent with the epoxy alkanoic acid clearly predominating (47% at 16 cm). The results are discussed as to (i) the age dependence of cutin composition, (ii) the relationship between fine structure and composition, (iii) the composition of the cuticle proper, the cuticular layer and the non-depolymerizable cutin fraction, and (iv) the polymeric structure of cutin.Abbreviations CL cuticular layer - CP cuticle proper - MX cutin polymer matrix     

The 18O-signal transfer from water vapour to leaf water and assimilates varies among plant species and growth forms

The ¹⁸O signature of atmospheric water vapour (δ¹⁸O_V) is known to be transferred via leaf water to assimilates. It remains, however, unclear how the ¹⁸O-signal transfer differs among plant species and growth forms. We performed a 9-hr greenhouse fog experiment (relative humidity ≥ 98%) with ¹⁸O-depleted water vapour (−106.7‰) on 140 plant species of eight different growth forms during daytime. We quantified the ¹⁸O-signal transfer by calculating the mean residence time of O in leaf water (MRT_LW) and sugars (MRT_Sugars) and related it to leaf traits and physiological drivers. MRT_LW increased with leaf succulence and thickness, varying between 1.4 and 10.8 hr. MRT_Sugars was shorter in C₃ and C₄ plants than in crassulacean acid metabolism (CAM) plants and highly variable among species and growth forms; MRT_Sugars was shortest for grasses and aquatic plants, intermediate for broadleaf trees, shrubs, and herbs, and longest for conifers, epiphytes, and succulents. Sucrose was more sensitive to δ¹⁸O_V variations than other assimilates. Our comprehensive study shows that plant species and growth forms vary strongly in their sensitivity to δ¹⁸O_V variations, which is important for the interpretation of δ¹⁸O values in plant organic material and compounds and thus for the reconstruction of climatic conditions and plant functional responses.     

Changes in photosynthesis,xanthophyll cycle,and sugar accumulation in two North Australia tropical species differing in leaf angles

Two tropical species of North Australia, Acacia crassicarpa and Eucalyptus pellita, have similar leaf size and leaf structure but different leaf angles. A. crassicarpa with near vertical leaf angle directly reduced photon absorption and leaf temperature (T _l) and had relatively high photosynthetic activity (P _max) and low xanthophyll cycle activity. In contrast, E. pellita with a small leaf angle exhibited high T _l, low P _max, and high activity of xanthophyll cycle which was useful for the dissipation of excessive energy and reduction of photoinhibition. In the dry season, contents of soluble sugars including pinitol, sucrose, fructose, and glucose in A. crassicarpa increased whereas larger amounts of only fructose and glucose were accumulated in E. pellita. Different sugar accumulation may be involved in osmotic adjustment of leaves during water stress that makes photosynthesis more efficient. The leaf angle may be critical for developing different protective mechanisms in these two tropical tree species that ensure optimal growth in the high irradiance and drought stress environment in North Australia.     

Decomposition and sorption characterization of plant cuticles in soil

The sorption of organic compounds by plant cuticular matter has been extensively investigated; however, little has been studied regarding the effect of plant cuticle degradation on their role in the sorption of organic compounds in soils. The sorption of phenanthrene was studied in soil samples which had been incubated for up to 9 months with three different types of plant cuticle isolated from tomato fruits, pepper fruits and citrus leaves. The main change in the diffuse reflectance Fourier-transform infrared (DRIFT) spectra during incubation of the cuticles was related to cutin decomposition. The peaks assigned to methyl and ethyl vibration and C=O vibration in ester links decreased with decomposition. In general, with all samples, the phenanthrene sorption coefficients calculated for the whole incubated soils (K _d) decreased with incubation time. In contrast, the carbon-normalized K _d values (K _oc) did not exhibit a similar trend for the different cuticles during incubation. The origin of the cuticle also affected the linearity of the sorption isotherms. With the tomato and citrus cuticle samples, the Freundlich N values were close to unity and were stable throughout incubation. However with the green pepper cuticle, the N values exhibited a significant decrease (from 0.98 to 0.70). This study demonstrates that the structural composition of the plant cuticle affects its biodegradability and therefore its ability to sorb organic compounds in soils. Of the residues originating from plant cuticular matter in soils, the cutan biopolymer and lignin-derived structures appear to play a dominant role in sorption as decomposition progresses. Responsible Editor: Alfonso Escudero.     

Development of plant cuticles: occurrence and role of non-ester bonds in cutin of Clivia miniata Reg. leaves

The effect of BF₃-methanol treatment on the mass and fine structure of isolated Clivia leaf cuticles at different stages of development has been investigated. BF₃-methanol cleaves ester linkages in cutin; however, the cuticles are not completely depolymerized. With increasing age, the residue left after BF₃-methanol treatment increases in mass. In very young cuticles, 10% of the total cutin resisted BF₃-methanol and the fraction of nonester cutin increased up to 62% in mature leaves. Transmission electron microscopy shows that fine structure of the cuticle proper is severely distorted but not destroyed. The internal cuticular layer, which exhibits a heavy contrast when fixed with KMnO₄, is completely depolymerized, while the external cuticular layer is hardly affected. The results are discussed in relation to cuticle development and to the function of cuticles as transpiration resistances.Abbreviation CP cuticle proper - ECL external cuticular layer - E cutin ester bonded cutin - ICL internal cuticular layer - MX-membrane polymer matrix membrane - NE-cutin non-ester bonded cutin - TEM transmission electron microscopy     

Bennettitalean leaf cuticle fragments (here Anomozamites and Pterophyllum) can be used interchangeably in stomatal frequency‐based palaeo‐CO2 reconstructions

Abstract: Bennettites are an abundant and frequently well‐preserved component of many Mesozoic fossil floras, often playing an important ecological role in flood plain vegetation communities. During a recent study focusing on stomatal indices of Triassic–Jurassic fossil plants, it became evident that the leaf fragments of two bennettite genera Anomozamites Schimper (1870) emend. Harris (1969) and Pterophyllum Brongniart (1825) display a significant overlap of leaf shape as well as cuticular characters. Owing to the preference of recognition of single taxa (ideally species) for the stomatal method, we use a database of 70 leaf fragments of Anomozamites and Pterophyllum compressions from five isotaphonomic Late Triassic sedimentary beds of Astartekløft in East Greenland to test whether leaf and cuticle fragments of the two genera can be separated using a range of quantitative and qualitative morphological and statistical analyses. None of the observed characters – including stomatal frequencies – could be applied to separate the fragments of the two genera into well‐defined groups. Our results therefore indicate that fragmented material and dispersed cuticles cannot be utilized to distinguish between Anomozamites or Pterophyllum at the genus level, but that instead these cuticle fragments may be used interchangeably as stomatal proxies. Classification of fossil leaves into either of these genera is thus only possible given adequate preservation of macro‐morphology and is not possible based solely on cuticle morphology. We suggest that this large inter‐ and intra‐generic morphological variation in both leaf and cuticle traits within Anomozamites and Pterophyllum may be related to the bennettites’ role as understory plants, experiencing a range of micro‐environmental conditions, perhaps depending mainly on sun exposure. Based on the results obtained in this study, we conclude that Anomozamites and Pterophyllum cuticle fragments can be employed interchangeably in palaeo [CO₂] reconstructions based on the stomatal method, thus potentially annexing a plethora of bennettitalean fossil plant material as CO₂ proxies, including dispersed cuticles.     

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.     

Gas permeability of plant cuticles

Cuticles from the adaxial surface of Citrus aurantium L. leaves and from the pericarp of Lycopersicon esculentum L. and Capsicum annuum L. were isolated enzymatically and their oxygen permeability was determined. Isolated cuticles were mounted between a gaseous and an aqueous compartment with the physiological outer side of the membrane facing the gaseous compartment. Permeability for oxygen was characterized by permeability (P) and diffusion (D) coefficients. P and D were independent of the driving force (gradient of oxygen concentration) across the cuticle, thus, Henry's law was obeyed. P values for the diffusion of oxygen varied between 3·10^-7 (Citrus), 1.4·10^-6 (Capsicum), and 1.1·10^-6 (Lycopersicon) m·s^-1. Extraction of soluble lipids from the cuticles increased the permeability. By treating the cutin matrix and the soluble lipids as resistances in series, it could be demonstrated that the soluble lipids were the main resistance for oxygen permeability in Citrus cuticles. However, in Lycopersicon and Capsicum, both the cutin matrix and the soluble lipids determined the total resistance. P values were not affected by either the proton concentration (pH 3–9) or the cations (Na⁺, Ca²⁺) present at the morphological inner side of the cuticles. It is concluded that the water content of cuticles does not affect the permeability properties for oxygen. Partition coefficients indicated a high solubility of oxygen in the cuticle of Citrus. The data suggest a solubility process in the cuticle of Citrus with respect to oxygen permeation.Abbreviations CM cuticular membrane - MX cutin polymer matrix - SCL soluble cuticular lipids     

Protecting against water loss: analysis of the barrier properties of plant cuticles

The cuticle is the major barrier against uncontrolled water loss from leaves, fruits and other primary parts of higher plants. More than 100 mean values for water permeabilities determined with isolated leaf and fruit cuticles from 61 plant species are compiled and discussed in relation to plant organ, natural habitat and morphology. The maximum barrier properties of plant cuticles exceed that of synthetic polymeric films of equal thickness. Cuticular water permeability is not correlated to the thickness of the cuticle or to wax coverage. Relationships between cuticular permeability, wax composition and physical properties of the cuticle are evaluated. Cuticular permeability to water increases on the average by a factor of 2 when leaf surface temperature is raised from 15 degrees C to 35 degrees C. Organic compounds of anthropogenic and biogenic origin may enhance cuticular permeability. The pathway taken by water across the cuticular transport barrier is reviewed. The conclusion from this discussion is that the bulk of water diffuses as single molecules across a lipophilic barrier while a minor fraction travels along polar pores. Open questions concerning the mechanistic understanding of the plant cuticular transport barrier and the role the plant cuticle plays in ensuring the survival and reproductive success of an individual plant are indicated.     

Cuticular penetration of abscisic acid

Summary Penetration of 2-¹⁴C abscisic acid (ABA) through enzymatically isolated cuticles from tomato fruit and from the upper epidermis of apricot, pear and orange leaves was assessed. Penetration was linear with time, greater as the undissociated than the dissociated ion, and greater through dewaxed than non-dewaxed cuticles. Significantly less (3–6 times) (2-¹⁴C)ABA penetrated the tomato fruit cuticle than NAA or 2,4-D. The leaf cuticles were less permeable than the tomato fruit cuticle. There was no evidence that the ABA was altered during transfer across the cuticle.     

Water permeability of isolated cuticular membranes: The effect of cuticular waxes on diffusion of water

Summary The water permeability of astomatous cuticular membranes isolated from Citrus aurantium L. leaves, pear (Pyrus communis L.) leaves and onion (Allium cepa L.) bulb scales was determined before and after extraction of cuticular waxes with lipid solvents. In pear, the permeability coefficients for diffusion of tritiated water across cuticular membranes (CM) prior to extraction [P _d(CM)] decreased by a factor of four during leaf expansion. In all three species investigated P _d(CM) values of cuticular membranes from fully expanded leaves varied between 1 to 2×10^-7 cm^-3 s^-1·P _d(CM) values were not affected by pH. Extraction of cuticular waxes from the membranes increased their water permeability by a factor of 300 to 500. Permeability coefficients for diffusion of THO across the cutin matrix (MX) after extraction [P _d(MX)] increased with increasing pH. P _dvalues were not inversely proportional to the thickness of cuticular membranes. By treating the cutin matrix and cuticular waxes as two resistances acting in series it was shown that the water permeability of cuticles is completely determined by the waxes. The lack of the P _d(CM) values to respond to pH appeared to be due to structural effects of waxes in the cutin matrix. Cuticular membranes from the submerse leaves of the aquatic plant Potamogeton lucens L. were three orders of magnitude more permeable to water than the cuticular membranes of the terrestrial species investigated.Abbreviations CM cuticular membrane - MX cutin matrix - WAX waxes This study was supported by a grant from the Deutsche Forschungsgemeinschaft.     

AgCl precipitates in isolated cuticular membranes reduce rates of cuticular transpiration

Counter diffusion of chloride, applied as NaCl at the inner side of isolated cuticles, and silver, applied as AgNO₃ at the outer side, lead to the formation of insoluble AgCl precipitates in isolated cuticles. AgCl precipitates could be visualized by light and scanning electron microscopy. The presence of AgCl precipitates in isolated cuticles was verified by energy dispersive X-ray analysis. It is argued that insoluble AgCl precipitates formed in polar pores of cuticles and as a consequence, cuticular transpiration of 13 out of 15 investigated species was significantly reduced up to three-fold. Water as a small and uncharged but polar molecule penetrates cuticles via two parallel paths: a lipophilic path, formed by lipophilic cutin and wax domains, and a aqueous pathe, formed by polar pores. Thus, permeances P (m s⁻¹) of water, which is composed of the two quantities P _Lipid and P _Pore, decreased, since water transport across polar pores was affected by AgCl precipitates. Cuticles with initially high rates of cuticular transpiration were generally more sensitive towards AgCl precipitates compared to cuticles with initially low rates of transpiration. Results presented here, significantly improves the current model of the structure of the cuticular transpiration barrier, since the pronounced heterogeneity of the cuticular transport barrier, composed of lipophilic as well as polar paths of diffusion, has to be taken into account in future.     

Influence of gibberellic acid on <Superscript>14</Superscript>CO<Subscript>2</Subscript> metabolism,growth, and production of alkaloids in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

Changes in growth parameters, carbon assimilation efficiency, and utilization of ¹⁴CO₂ assimilate into alkaloids in plant parts were investigated at whole plant level by treatment of Catharanthus roseus with gibberellic acid (GA). Application of GA (1 000 g m⁻³) resulted in changes in leaf morphology, increase in stem elongation, leaf and internode length, plant height, and decrease in biomass content. Phenotypic changes were accompanied by decrease in contents of chlorophylls and in photosynthetic capacity. GA application resulted in higher % of total alkaloids accumulated in leaf, stem, and root. GA treatment produced negative phenotypic response in total biomass production but positive response in content of total alkaloids in leaf, stem, and roots. ¹⁴C assimilate partitioning revealed that ¹⁴C distribution in leaf, stem, and root of treated plants was higher than in untreated and variations were observed in contents of metabolites as sugars, amino acids, and organic acids. Capacity to utilize current fixed ¹⁴C derived assimilates for alkaloid production was high in leaves but low in roots of treated plants despite higher content of ¹⁴C metabolites such as sugars, amino acids, and organic acids. In spite of higher availability of metabolites, their utilization into alkaloid production is low in GA-treated roots.     

Ultrastructure and Radiolabelling of Leaf Cuticles from Ivy (Hedera helixL.) Plantsin vitroand duringex vitroAcclimatization

Cuticle ultrastructure and radiolabelling of isolated cuticlesafter incorporation of [¹⁴C] acetate in foliar discs were investigatedwith ivy plants grownin vitrothenex vitro. Results show an increasein thickness, mass and wax content, between young and expandedleaves, for bothin vitroandex vitrocuticles. The cuticle ofinvitrounexpanded leaves was very thin and only constituted alamellate zone. The ultrastructure ofin vitroyoung and expandedleaf cuticles showed characteristics similar toin situcuticles.The thickness of the lamellate zone remained fairly constantand represented 33% of the cuticle thickness in young leaves,but only 11.4% in expanded leaves. The number of lamellar unitsdecreased from 14 to nine between these two growth stages. Themain difference between young leaves developedin vitroorex vitrowasa thinner lamellate zone forex vitrocuticles. However, theselatter cuticles had an intermediary zone between the lamellateand reticulate zones. The cuticle thickness of expanded leaveswas greater forin vitrocuticles suggesting a temporary decreasein cuticle biosynthesis after transfer of the plant fromin vitrotoexvitro.Results from cuticle radiolabelling show higher radioactivityincorporation in cuticles isolated from leaves developedex vitrocomparedtoin vitro. This radiolabelling was particularly marked forexvitroyoung leaf cuticles and depended on the duration of theexvitrogrowth period revealing a progressive activation of cuticlebiosynthesis in response to new environmental conditions. Hedera helix; ivy leaf cuticle; in vitroplants; electron microscopy; radiolabelling; isolated cuticles     

Photosynthetic and gas exchange characteristics of dominant woody plants on a moisture gradient in an African savanna

We determined key photosynthetic gas exchange parameters, and their temperature dependence, in dominant woody plants at four savanna sites on a moisture gradient in Botswana, southern Africa. Leaf stable carbon and nitrogen (N) isotope and morphological measures were made concurrently. Sampling of these predominantly non‐N‐fixing species took place during an exceptional rainfall season, representing near‐optimum conditions for primary production at these sites. The mean specific leaf area and leaf size were positively related to mean annual rainfall (MAR); species with larger leaves of lower density were more abundant in wetter sites. Almost all species at all sites showed high net light‐saturated photosynthetic rates (A_max?10 μmol CO₂ m^?2 s^?1) due both to high CO₂ carboxylation (V_c,max) and RubP‐regeneration capacity (J_max). These high rates were associated with high values of leaf [N]. Across all sites, the temperature response of A_max showed no clear optimum, and a gradual drop from 25°C to 35°C, without notable temperature limitation at leaf temperatures in excess of 35°C. Dark respiration rate (R_day) across all species and sites increased exponentially with increasing leaf temperature. Species sampled at selected sites revealed a negative relationship between leaf δ¹³C (stable carbon isotope ratio) and MAR, suggesting higher leaf‐level water‐use efficiency at drier sites when integrated over the life of the leaf. At wetter sites, specific leaf [N] was lower and photosynthetic nitrogen‐use efficiency increased, a pattern reflected at the ecosystem level by less ¹⁵N enrichment of leaves at these sites. Taken together, the results suggest a switch from water‐use to nitrogen‐use efficiency constraints with increasing moisture availability. These constraints impact leaf form and function significantly, and may emerge at the ecosystem level in aspects of water and N cycling.     

Function of leaf hamamelitol as a compatible solute during water stress treatment of Hedera helix L.

Hamamelitol is an unusual branched-chain sugar alcohol previously suggested to function as a leaf compatible solute. In this study, we have examined the leaf metabolism and intracelluiar compartmentalization of hamamelitol and other soluble sugars during long-term water stress treatment of Hedera helix (English ivy). Total leaf hamamelitol content was relatively low in greenhouse control plants, but increased 2-fold during water stress treatment to levels approaching those observed in field-grown plants (6–7 μmol g^?1 fresh weight). Using density gradient fractionation with non-aqueous solvents, we showed that hamamelitol occurs primarily in the cytoplasm and vacuoles of leaf mesophyll cells. During water stress treatment most of the increase in leaf hamamelitol occurred in the mesophyll cytoplasm, compensating osmotically for a decrease in cytoplasmic sucrose concentration. The maximum concentration of cytoplasmic hamamelitol was 155 mol m^?3 and occurred in field-grown plants. Labelling experiments showed that hamamelitol is slowly synthesized from ¹⁴CO₂ in leaves of H. helix, but is very long-lived (estimated t_1/2 of 4 years). Together, these data indicate that hamamelitol probably functions during long-term stress conditions as an osmotically active, compatible solute in plant leaves. We suggest that the signal for enhanced accumulation of hamamelitol during the water stress treatment was initiated by decreased plant growth and increased leaf sucrose hydrolysis.