Cloning and expression analysis of candidate genes involved in wax deposition along the growing barley (Hordeum vulgare) leaf.

The aim of the present study was to isolate clones of genes which are likely to be involved in wax deposition on barley leaves. Of particular interest were those genes which encode proteins that take part in the synthesis and further modification of very long chain fatty acids (VLCFAs), the precursors of waxes. Previously, it had been shown that wax deposition commences within a spatially well-defined developmental zone along the growing barley leaf (Richardson et al. in Planta 222:472-483, 2005). In the present study, a barley microarray approach was used to screen for candidate contig-sequences (www.barleybase.org) that are expressed particularly in those leaf zones where wax deposition occurs and which are expressed specifically within the epidermis, the site of wax synthesis. Candidate contigs were used to screen an established in-house cDNA library of barley. Six full-length coding sequences clones were isolated. Based on sequence homologies, three clones were related to Arabidopsis CER6/CUT1, and these clones were termed HvCUT1;1, HvCUT1;2 and HvCUT1;3. A fourth clone, which was related to Arabidopsis Fiddlehead (FDH), was termed HvFDH1;1. These clones are likely to be involved in synthesis of VLCFAs. A fifth and sixth clone were related to Arabidopsis CER1, and were termed HvCER1;1 and HvCER1;2. These clones are likely to be involved in the decarbonylation pathway of VLCFAs. Semi-quantitative RT-PCR confirmed microarray expression data. In addition, expression analyses at 10-mm resolution along the blade suggest that HvCUT1;1 (and possibly HvCUT1;2) and HvCER1;1 are involved in commencement of wax deposition during barley leaf epidermal cell development.

Cuticular permeance in relation to wax and cutin development along the growing barley (Hordeum vulgare) leaf.

The developing leaf three of barley provides an excellent model system for the direct determination of relationships between amounts of waxes and cutin and cuticular permeance. Permeance of the cuticle was assessed via the time-course of uptake of either toluidine blue or (14)C-labelled benzoic acid ([(14)C] BA) along the length of the developing leaf. Toluidine blue uptake only occurred within the region 0-25 mm from the point of leaf insertion (POLI). Resistance--the inverse of permeance--to uptake of [(14)C] BA was determined for four leaf regions and was lowest in the region 10-20 mm above POLI. At 20-30 and 50-60 mm above POLI, it increased by factors of 6 and a further 32, respectively. Above the point of emergence of leaf three from the sheath of leaf two, which was 76-80 mm above POLI, resistance was as high as at 50-60 mm above POLI. GC-FID/MS analyses of wax and cutin showed that: (1) the initial seven fold increase in cuticular resistance coincided with increase in cutin coverage and appearance of waxes; (2) the second, larger and final increase in cuticle resistance was accompanied by an increase in wax coverage, whereas cutin coverage remained unchanged; (3) cutin deposition in barley leaf epidermis occurred in parallel with cell elongation, whereas deposition of significant amounts of wax commenced as cells ceased to elongate.

Water transport in plant cuticles: an update.

The scale, mechanism, and physiological importance of cuticular transpiration were last reviewed in this journal 5 and 10 years ago. Progress in our basic understanding of the underlying processes and their physiological and structural determinants has remained frustratingly slow ever since. There have been major advances in the quantification of cuticular water permeability of stomata-bearing leaf and fruit surfaces and its dependence on leaf temperature in astomatous surfaces, as well as in our understanding of the respective roles of epicuticular and intracuticular waxes and molecular-scale aqueous pores in its physical control. However, understanding the properties that determine the thousand-fold differences between permeabilities of different cuticles remains a huge challenge. Molecular biology offers unique opportunities to elucidate the relationships between cuticular permeability and structure and chemical composition of cuticles, provided care is taken to quantify the effects of genetic manipulation on cuticular permeability by reliable experimental approaches.

Quantification of cuticular permeability in genetically modified plants.

More and more studies on genetically modified plants are identifying parts of the genetic code with putative involvement in creating the cuticular barrier. Unfortunately, many of these studies suffer from the inadequacy of the chosen methods to quantify, in a reasonably unambiguous way, if and how the efficacy of the cuticular barrier is affected by the genetic change. A short overview of relevant findings is given and a more stringent experimental approach to quantifying effects on cuticular permeability in genetically modified plants proposed.

Parameterization, comparison, and validation of models quantifying relative change of cuticular permeability with physicochemical properties of diffusants.

Predictions from two previously published models and a new model for the relative change in cuticular permeability with boiling point, octanol/air partition coefficient, and/or molar volume of a wide range of diffusants (not including ions and large hydrophilic compounds) are compared with each other and to experimental data sets not used for model parameterization. While the models work in a similar way for all cuticles for which data are available, it is not yet possible to predict in absolute terms the permeability of any cuticles for which no data are available-that is, while the slope of a plot representing the change in permeability with diffusant properties is predictable, the position of the linear relationship along the ordinate needs to be determined experimentally for each type of cuticle at or near the relevant temperature(s).

Rising atmospheric CO2 reduces sequestration of root-derived soil carbon.

Forests have a key role as carbon sinks, which could potentially mitigate the continuing increase in atmospheric carbon dioxide concentration and associated climate change. We show that carbon dioxide enrichment, although causing short-term growth stimulation in a range of European tree species, also leads to an increase in soil microbial respiration and a marked decline in sequestration of root-derived carbon in the soil. These findings indicate that, should similar processes operate in forest ecosystems, the size of the annual terrestrial carbon sink may be substantially reduced, resulting in a positive feedback on the rate of increase in atmospheric carbon dioxide concentration.

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

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

Exchange of polychlorinated biphenyls (PCBs) and polychlorinated naphthalenes (PCNs) between air and a mixed pasture sward.

To improve understanding of air-to-vegetation transfer of persistent organic pollutants (POPs), uptake and depuration of polychlorinated biphenyls (PCBs) and polychlorinated naphthalenes (PCNs) between grass sward and air was investigated. Pasture swards were placed in fanned (2 m s(-1) wind speed) and unfanned conditions for a period of 20 days and sampled at intervals. Depuration was carried out after a short (4 days) and a long (14 days) exposure period. Prior to contamination, a mixed pasture sward at a semi-rural location contained sigmaPCN concentrations 15-20% of the sigmaPCB concentration. Uptake of both PCBs and PCNs was broadly linear in fanned and unfanned conditions over the 20-day period, i.e., the pasture did not reach equilibrium with the air. Uptake rates (fluxes) were greater under the fanned conditions. The difference in uptake rates between fanned and unfanned conditions increased with degree of chlorination for both PCBs and PCNs, ranging between a factor of 2 for tri-chlorinated PCBs and PCNs and a factor 5 for octa-chlorinated PCBs. Depuration results over the first hours were very scattered, showing an initial period of loss, followed by an increase in concentrations, possibly as a result of re-volatilization of PCBs from the soil in the trays, with consequent recapture by the overlying sward. Rapid clearance was observed over the following days, but depuration of PCBs and PCNs was still incomplete after 14 days, with 20% of the initial concentration of the sigmaPCBs and 10% of the sigmaPCNs retained by the sward. There was no difference in the proportion of POPs retained in the sward between the 4- and 14-day contamination treatments. POP-specific differences in the amount of compound "trapped" in leaves after contamination were observed. The results show that, although changes in the rate of air movement around a pasture have an effect on the uptake rate of POPs into the vegetation, plant-side resistance controls both the air-to-pasture and pasture-to-air exchange of gas-phase PCBs and PCNs; i.e., differences between plant species in cuticle composition and/or structure affecting the permeability of the cuticle are of greater importance than differences in leaf morphology affecting aerodynamic roughness.

Current issues and uncertainties in the measurement and modelling of air-vegetation exchange and within-plant processing of POPs.

Air-vegetation exchange of POPs is an important process controlling the entry of POPs into terrestrial food chains, and may also have a significant effect on the global movement of these compounds. Many factors affect the air-vegetation transfer including: the physicochemical properties of the compounds of interest; environmental factors such as temperature, wind speed, humidity and light conditions; and plant characteristics such as functional type, leaf surface area, cuticular structure, and leaf longevity. The purpose of this review is to quantify the effects these differences might have on air/plant exchange of POPs, and to point out the major gaps in the knowledge of this subject that require further research. Uptake mechanisms are complicated, with the role of each factor in controlling partitioning, fate and behaviour process still not fully understood. Consequently, current models of air-vegetation exchange do not incorporate variability in these factors, with the exception of temperature. These models instead rely on using average values for a number of environmental factors (e.g. plant lipid content, surface area), ignoring the large variations in these values. The available models suggest that boundary layer conductance is of key importance in the uptake of POPs, although large uncertainties in the cuticular pathway prevents confirmation of this with any degree of certainty, and experimental data seems to show plant-side resistance to be important. Models are usually based on the assumption that POP uptake occurs through the lipophilic cuticle which covers aerial surfaces of plants. However, some authors have recently attached greater importance to the stomatal route of entry into the leaf for gas phase compounds. There is a need for greater mechanistic understanding of air-plant exchange and the 'scaling' of factors affecting it. The review also suggests a number of key variables that researchers should measure in their experiments to allow comparisons to be made between studies in order to improve our understanding of what causes any differences in measured data between sites.

Study of plant-air transfer of PCBs from an evergreen shrub: implications for mechanisms and modeling.

The depuration of gas-phase polychlorinated biphenyls (PCBs) from a slow-growing evergreen shrub, Skimmia japonica Thunb., was studied to investigate the reversibility of uptake and the compartmentalization of PCB congeners within leaves with respect to air-plant exchange processes. Depuration of PCBs was monitored over periods of hours, days, and weeks. Equilibrium had not been attained between air and leaves during the uptake phase after many weeks. Depuration followed two-phase clearance kinetics, with phase 1 occurring over the order of hours and phase 2 continuing slowly over weeks. In phase 1, a substantial part (ca. 40%) of the PCB burden that the plants had accumulated over weeks was lost in 2-3 h. This observation is further evidence for the close dynamic coupling of air and vegetation compartments. In the second phase, very slow depuration over 28 d only removed a further approximately 25% of the accumulated PCB burden. Depuration rates in phase 2 varied between compounds and were not influenced by growth dilution. Depuration rates for both phases were not correlated with KOA, indicating that plant-air mass transfer coefficients were proportional to plant-air partition coefficients and, therefore, probably dominated by the plant-side resistance to diffusion. Photolysis and metabolism are unlikely to have influenced the rates of congener disappearance. Pathways into the leaf and possible storage locations within the plant are discussed with respect to the observed differences between uptake and clearance rates. Uptake and depuration are not mirror image processes, with a fraction of accumulated PCBs effectively stored in the leaves. This has important implications for terrestrial food chain transfer and global cycling with leaf concentrations remaining elevated long after a contamination event.

Investigation into the importance of the stomatal pathway in the exchange of PCBs between air and plants.

The transfer of persistent organic pollutants (POPs) from air to vegetation is an important air-surface exchange process that affects global cycling and can result in human and wildlife exposure via the terrestrial food chain. To improve understanding of this process, the role of stomata in uptake of gas-phase polychlorinated biphenyls (PCBs) was investigated using Hemerocallis x hybrida "Black Eyed Stella", a plant with a high stomatal density. Uptake of PCBs was monitored over a 72-h period in the presence and absence of light. Uptake rates were significantly greater in illuminated (stomata open) plants than unilluminated (stomata closed) plants for 18 of the 28 measured PCB congeners (p < 0.05). Depuration of PCBs was monitored in a subsequent experiment over a period of 3 weeks. Levels after 3 weeks of depuration time were still much higher than the concentration prior to contamination. Tri- and tetrachlorinated PCBs showed the greatest depuration, with less than 20% and 50% of accumulated PCBs respectively remaining, while approximately 70% of higher chlorinated PCB congeners remained in the plants at the end of the experiment. Treatments with/without light (to control stomatal opening during uptake) and with/without abscisic acid (ABA) application (to control stomatal opening during depuration) were compared. After contamination indoors for 3 days, there was a significantly higher concentration of PCBs (p < 0.05) in the light contaminated plants than the dark-contaminated plants for 13 of the 28 measured PCB congeners. The ABA treatment affected depuration of PCB-18 only. "Light/ABA-treated" plants had a significantly slower depuration rate for PCB-18 than "light/untreated", "dark/ABA-treated", and "dark/untreated" plants (p < 0.05). The results of the study indicate that there is a stomatal effect on the rate of exchange of PCBs between Hemerocallis leaves and air.

Air-side and plant-side resistances influence the uptake of airborne PCBs by evergreen plants.

The transfer of persistent organic pollutants (POPs) from airto vegetation is an important air-surface exchange process that affects global cycling and can result in human and wildlife exposure via the terrestrial food chain. To improve understanding of this process, the uptake of gas-phase polychlorinated biphenyls (PCBs) by two slow-growing evergreen shrubs, Skimmia japonicaThunb. and Hebe"Great Orme", was studied to investigate the influence of air-side and plant-side resistances. Uptake of PCBs was monitored over periods of hours, days, and weeks. Uptake rates were higher in the smaller Hebe leaves than the Skimmia leaves. Equilibrium was not attained between air and plants in the duration of the experiments; uptake curves were indicative of a two-phase uptake-step 1 over the order of hours and step 2 continuing steadily over days to weeks. Uptake rates (h(-1)) were greater in conditions simulating typical ambient wind speeds (2 m s(-1)) than under still air, indicating a significant impact of air-side resistance relative to plant-side resistance in still air. Wind speed is an important variable that has not been previously considered in studies of the air-planttransfer of persistent organic pollutants (POPs). Uptake rate constants increased with increasing level of chlorination (and hence K(OA)) both in still air and under turbulent conditions. This was inconsistent with the idea of air-side resistance dominating uptake, since diffusion rates in air decrease with molecular weight (and hence KOA). Greater uptake of particle-bound PCBs may have contributed to this finding, but the most likely explanation is the previously established relationship that the permeability of cuticles increases with increasing KOA of the diffusing chemical. The findings indicate that plant-side resistance can have an important effect on uptake rates of different PCB congeners in the field, even when air-side resistance is high.

Sodium-related partial stomatal closure and salt tolerance of Aster tripolium.

• When Aster tripolium is grown at high salinity, stomatal closure is induced by the presence of sodium ions in the apoplast surrounding the guard cells. The occurrence of this system in Aster tripolium and not in the closely related glycophyte Aster amellus suggests that it could be an important factor in the network of physiological attributes required for salt tolerance. • Gas exchange and growth parameters were measured in Aster tripolium plants grown at different levels of salinity. A simple mechanistic model was constructed to test whether the Na-sensing feature of the guard cells was a realistic component of salinity tolerance. • The model captured very well the behaviour of plants in terms of salt uptake and reduction of growth with increasing salinity. There was moderate variance between measured and modelled rates of decrease of conductance with increasing levels of salinity. • No evidence was found to refute our hypothesis that stomatal closure in response to sodium plays an important role in salt tolerance of Aster tripolium.

In vivo manipulation of cuticular water permeance and its effect on stomatal response to air humidity.

Cuticular water permeance was manipulated in Corylus avellana L., Hypericum androsaemum L. and Populus tremula L. by (1) long-term application of low doses of various systemic herbicides inhibiting biosynthesis of cuticular waxes, (2) very short-term application of organic solvents to the leaf surface, and (3) exposure to natural strong winds. Treatment effects were very variable, but increased the natural range of permeances by a factor of 10 or so in undamaged leaves. All species had hypostomatous leaves. Relative change of leaf conductance (g) in response to stepwise increases of leaf-to-air water vapour pressure difference (VPD) was measured for individual leaves (Corylus) or groups of leaves at the shoot or branch tip. Adaxial cuticular water permeance (P) was determined for the same leaves after measurement of the VPD-response. A proportional measure of relative change of g with VPD, d(log e .g)dVPD, was then plotted against P. No increase in the strength of the closing response to increasing VPD was found with increasing P, as would have been expected if water loss through the cuticle was involved in stomatal response to changes in VPD via a direct effect on guard cell turgor. By contrast, high P coincided, most clearly in Corylus, with a reduced strength of the stomatal closing response to increasing VPD, i.e. less negative d(loge g)dVPD. As the responses were non-linear, the value of d(loge g)dVPD changed with VPD. With rising VPD, all three species and a fourth one previously studied showed a decline in the value of [d(loge g)dVPD]/d(log P), reaching negative values in one species. This is interpreted in terms of two independent and antagonistic effects of increased cuticular water permeance on guard cell response to VPD, one acting by reducing the backpressure exerted on guard cells by the epidermis, and the other one possibly causing greater depression of guard cell turgor through delivery of more chemical messengers (such as abscisic acid) to the guard cells with the cuticular transpiration stream.