Stomatal density and aperture length in four plant species grown across a subambient CO2 gradient

Stomatal density, stomatal aperture length, area/leaf, and number of stomata/leaf were measured after the annual C₃ agronomic grasses oats (Avena sativa) and wheat (Triticum aestivum), the C, woody legume honey mesquite (Prosopis glandulosa), and the perennial C₄ grass little bluestem (Schizachyrium scoparium) were grown across a subambient carbon dioxide concentration ([CO₂]) gradient from near 200 to 350 μmol/mol in a growth chamber. The purpose was to determine if the size and density of stomata vary in response to atmospheric [CO₂] during growth, across a subambient [CO₂] range representative of the doubling that has occurred since the last ice age. Changes in stomatal density and aperture length with increasing [CO₂] were small when detected. Stomatal density decreased on adaxial flag leaf surfaces of wheat, and aperture length increased slightly with [CO₂], Leaf area and number of stomata/flag leaf increased by similar proportions with [CO₂] in two wheat cultivars. No consistent relationship between [CO₂] and stomatal density or size was detected in mesquite, oats, or little bluestem. We conclude that individual plants of these species lack the plasticity to significantly alter stomatal density and aperture length in response to increasing atmospheric [CO₂] in a single generation (annuals) or growing season (perennials).     

Guard cell photosynthesis is critical for stomatal turgor production,yet does not directly mediate CO2‐ and ABA‐induced stomatal closing

Stomata mediate gas exchange between the inter‐cellular spaces of leaves and the atmosphere. CO₂ levels in leaves (Ci) are determined by respiration, photosynthesis, stomatal conductance and atmospheric [CO₂]. [CO₂] in leaves mediates stomatal movements. The role of guard cell photosynthesis in stomatal conductance responses is a matter of debate, and genetic approaches are needed. We have generated transgenic Arabidopsis plants that are chlorophyll‐deficient in guard cells only, expressing a constitutively active chlorophyllase in a guard cell specific enhancer trap line. Our data show that more than 90% of guard cells were chlorophyll‐deficient. Interestingly, approximately 45% of stomata had an unusual, previously not‐described, morphology of thin‐shaped chlorophyll‐less stomata. Nevertheless, stomatal size, stomatal index, plant morphology, and whole‐leaf photosynthetic parameters (PSII, qP, qN, F_V′/F_M′) were comparable with wild‐type plants. Time‐resolved intact leaf gas‐exchange analyses showed a reduction in stomatal conductance and CO₂‐assimilation rates of the transgenic plants. Normalization of CO₂ responses showed that stomata of transgenic plants respond to [CO₂] shifts. Detailed stomatal aperture measurements of normal kidney‐shaped stomata, which lack chlorophyll, showed stomatal closing responses to [CO₂] elevation and abscisic acid (ABA), while thin‐shaped stomata were continuously closed. Our present findings show that stomatal movement responses to [CO₂] and ABA are functional in guard cells that lack chlorophyll. These data suggest that guard cell CO₂ and ABA signal transduction are not directly modulated by guard cell photosynthesis/electron transport. Moreover, the finding that chlorophyll‐less stomata cause a ‘deflated’ thin‐shaped phenotype, suggests that photosynthesis in guard cells is critical for energization and guard cell turgor production.     

Similarity of stomatal index in the C4 plant Salsola kali L. in material Collected in 1843 and in 1987: relevance to changes in atmospheric CO2 content

Summary

Earlier work (by F.I. Woodward) demonstrated that those plants with the C₃ pathway of photosynthesis which were examined, i.e. present day plants and dated herbarium material showed a decreased stomatal index (stomata as a percentage of stomata plus epidermal cells) over the last 200 years. Growth of some of these, and of some other, C₃ plants at different CO₂ partial pressures shows that the fall in stomatal index over the last 200 years is probably a consequence of the increased atmospheric CO₂ partial pressure resulting from the industrial revolution. The work reported here shows that Salsola kali (one of the few plants with the C₄ pathway which are native to Scotland) showed the same stomatal index 144 years ago as it does today. Properties of photosynthetic gas exchange in C₃ and C₄ plants are reviewed. It is concluded that responses to decreased CO₂ partial pressure for growth which involve increased stomatal index in C₃ plants are achieved in C₄ plants without an increase in stomatal index. While C₃ plants growing at low CO₂ appear to sacrifice water use efficiency in partially maintaining net photosynthetic rate, the C₄ plants may emphasize more the maintenance of their already high water use efficiency.     

Stomatal density and aperture in non-vascular land plants are non-responsive to above-ambient atmospheric CO2 concentrations

Background and Aims Following the consensus view for unitary origin and conserved function of stomata across over 400 million years of land plant evolution, stomatal abundance has been widely used to reconstruct palaeo-atmospheric environments. However, the responsiveness of stomata in mosses and hornworts, the most basal stomate lineages of extant land plants, has received relatively little attention. This study aimed to redress this imbalance and provide the first direct evidence of bryophyte stomatal responsiveness to atmospheric CO₂.Methods A selection of hornwort (Anthoceros punctatus, Phaeoceros laevis) and moss (Polytrichum juniperinum, Mnium hornum, Funaria hygrometrica) sporophytes with contrasting stomatal morphologies were grown under different atmospheric CO₂ concentrations ([CO₂]) representing both modern (440 p.p.m. CO₂) and ancient (1500 p.p.m. CO₂) atmospheres. Upon sporophyte maturation, stomata from each bryophyte species were imaged, measured and quantified.Key Results Densities and dimensions were unaffected by changes in [CO₂], other than a slight increase in stomatal density in Funaria and abnormalities in Polytrichum stomata under elevated [CO₂].Conclusions The changes to stomata in Funaria and Polytrichum are attributed to differential growth of the sporophytes rather than stomata-specific responses. The absence of responses to changes in [CO₂] in bryophytes is in line with findings previously reported in other early lineages of vascular plants. These findings strengthen the hypothesis of an incremental acquisition of stomatal regulatory processes through land plant evolution and urge considerable caution in using stomatal densities as proxies for paleo-atmospheric CO₂ concentrations.     

No heat-stimulated germination found in herbaceous species from burned subtropical grassland

The inverse relationship between numbers of stomata (stomatal frequency) on tree leaves and ambient CO₂ concentration is increasingly applied for reconstructing past atmospheric CO₂ levels. The abundance of leaf remains of Quercus robur in Holocene peat and lake deposits in Europe makes this species potentially suitable for high-resolution stomatal frequency analysis. In order to quantify the CO₂ responsiveness of the species, the behavior of the stomatal index for Q. robur during the current anthropogenic CO₂ increase is determined on the basis of buried, herbarium and modern leaf material from the Netherlands. The stomatal index (SI), expressing the ratio of the number of stomata in a given area divided by the total number of stomata and other epidermal cells in that same area, is used in order to minimize influences on stomatal frequency of environmental conditions other than CO₂. The sigmoid SI response pattern recorded for Q. robur resembles that of the closely related species Q. petraea, although there is a difference in the timing of the response limitation of the two species to increasing atmospheric CO₂. For calibration purposes only the linear phase of the sigmoidal response curve is taken into consideration in the presented CO₂ response model, which allows confident combination of Q. robur and Q. petraea over the interval from 290 to 325 ppmv CO₂. The model is conservative in reconstructing past CO₂ mixing ratios outside the range of monitored response. As a result of the observed SI response limit, the model predicts CO₂ levels below 325 ppmv with a mean error of 10.2 ppmv, whereas higher CO₂ levels are underestimated.     

Stomatal function,density and pattern,and CO2 assimilation in Arabidopsis thaliana tmm1 and sdd1‐1 mutants

• Stomata modulate the exchange of water and CO₂ between plant and atmosphere. Although stomatal density is known to affect CO₂ diffusion into the leaf and thus photosynthetic rate, the effect of stomatal density and patterning on CO₂ assimilation is not fully understood.
• We used wild types Col‐0 and C24 and stomatal mutants sdd1‐1 and tmm1 of Arabidopsis thaliana, differing in stomatal density and pattern, to study the effects of these variations on both stomatal and mesophyll conductance and CO₂ assimilation rate. Anatomical parameters of stomata, leaf temperature and carbon isotope discrimination were also assessed.
• Our results indicate that increased stomatal density enhanced stomatal conductance in sdd1‐1 plants, with no effect on photosynthesis, due to both unchanged photosynthetic capacity and decreased mesophyll conductance. Clustering (abnormal patterning formed by clusters of two or more stomata) and a highly unequal distribution of stomata between the adaxial and abaxial leaf sides in tmm1 mutants also had no effect on photosynthesis.
• Except at very high stomatal densities, stomatal conductance and water loss were proportional to stomatal density. Stomatal formation in clusters reduced stomatal dynamics and their operational range as well as the efficiency of CO₂ transport.

     

A stomatal optimization theory to describe the effects of atmospheric CO2 on leaf photosynthesis and transpiration

Background and Aims

Global climate models predict decreases in leaf stomatal conductance and transpiration due to increases in atmospheric CO₂. The consequences of these reductions are increases in soil moisture availability and continental scale run-off at decadal time-scales. Thus, a theory explaining the differential sensitivity of stomata to changing atmospheric CO₂ and other environmental conditions must be identified. Here, these responses are investigated using optimality theory applied to stomatal conductance.

Methods

An analytical model for stomatal conductance is proposed based on: (a) Fickian mass transfer of CO₂ and H₂O through stomata; (b) a biochemical photosynthesis model that relates intercellular CO₂ to net photosynthesis; and (c) a stomatal model based on optimization for maximizing carbon gains when water losses represent a cost. Comparisons between the optimization-based model and empirical relationships widely used in climate models were made using an extensive gas exchange dataset collected in a maturing pine (Pinus taeda) forest under ambient and enriched atmospheric CO₂.

Key Results and Conclusion

In this interpretation, it is proposed that an individual leaf optimally and autonomously regulates stomatal opening on short-term (approx. 10-min time-scale) rather than on daily or longer time-scales. The derived equations are analytical with explicit expressions for conductance, photosynthesis and intercellular CO₂, thereby making the approach useful for climate models. Using a gas exchange dataset collected in a pine forest, it is shown that (a) the cost of unit water loss λ (a measure of marginal water-use efficiency) increases with atmospheric CO₂; (b) the new formulation correctly predicts the condition under which CO₂-enriched atmosphere will cause increasing assimilation and decreasing stomatal conductance.     

Water Relations,CO2 Exchange,Water-use Efficiency and Growth of Welwitschia mirabilis Hook. fil. in three Contrasting Habitats of the Namib Desert1

CO₂ exchange, transpiration and leaf water potential of Welwitschia mirabilis were measured in three contrasting habitats of the Namib desert. From these measurements stomatal conductance, internal CO₂concentration and WUE were calculated. In two of the three habitats photosynthetic CO₂ uptake decreased and transpiration increased with increasing leaf age while in the third habitat CO₂ uptake increased and transpiration decreased with leaf age. Except for the stomata of young leaf sections in this habitat, stomata closed with increasing δw leading to a pronounced midday depression of CO₂ uptake. The high stomatal limitation of photosynthetic CO₂ uptake of glasshouse-grown plants was verified in the natural habitat. Photosynthetic CO₂ uptake saturated between 800 and 1300 μmol photons m^?2 s^?1depending on leaf age and habitat. CO₂ uptake had a broad temperature optimum declining significantly beyond 32 °C. Predawn leaf water potential reflected water availability and atmospheric conditions in the three habitats and ranged from ? 2.5 to ? 6.2 MPa. There was a pronounced diurnal course of leaf water potential in all habitats. During the day a gradient in water potential developed along the leaf axis with the lowest potential at the leaf's tip. With respect to whole plant balances of CO₂ exchange and transpiration, there were marked differences between Welwitschias in the three habitats. Despite a negative CO₂ balance over a period of five months, leaves in the driest habitat grew constantly at the expense of carbon reserves in the plant. Only at the wettest site did carbon gain exceed carbon demand for growth. The WUE of whole plants was insignificant in all habitats. The results were as contrasting as the habitats and plants and did not allow generalisations about adaptational features of Welwitschia mirabilis.     

Stomatal conductance and not stomatal density determines the long-term reduction in leaf transpiration of poplar in elevated CO<Subscript>2</Subscript>

Using a free-air CO₂ enrichment (FACE) experiment, poplar trees (Populus × euramericana clone I214) were exposed to either ambient or elevated [CO₂] from planting, for a 5-year period during canopy development, closure, coppice and re-growth. In each year, measurements were taken of stomatal density (SD, number mm⁻²) and stomatal index (SI, the proportion of epidermal cells forming stomata). In year 5, measurements were also taken of leaf stomatal conductance (g _s, μmol m⁻² s⁻¹), photosynthetic CO₂ fixation (A, mmol m⁻² s⁻¹), instantaneous water-use efficiency (A/E) and the ratio of intercellular to atmospheric CO₂ (C_i:C_a). Elevated [CO₂] caused reductions in SI in the first year, and in SD in the first 2 years, when the canopy was largely open. In following years, when the canopy had closed, elevated [CO₂] had no detectable effects on stomatal numbers or index. In contrast, even after 5 years of exposure to elevated [CO₂], g _s was reduced, A/E was stimulated, and C_i:C_a was reduced relative to ambient [CO₂]. These outcomes from the long-term realistic field conditions of this forest FACE experiment suggest that stomatal numbers (SD and SI) had no role in determining the improved instantaneous leaf-level efficiency of water use under elevated [CO₂]. We propose that altered cuticular development during canopy closure may partially explain the changing response of stomata to elevated [CO₂], although the mechanism for this remains obscure.     

Photosynthesis in the water-stressed C4 grass Setaria sphacelata is mainly limited by stomata with both rapidly and slowly imposed water deficits

A comparison of the effects of a rapid and a slowly imposed water deficit on photosynthesis was performed in Setaria sphacelata var. splendida (Stapf) Clayton, a C₄ NADP‐ME grass. Gas exchange was measured in rapidly and slowly dehydrated adult leaves either under atmospheric CO₂ partial pressure with an infrared gas analyser or under saturating CO₂ partial pressure with a leaf disc oxygen electrode. These measurements were used to calculate stomatal and non‐stomatal limitations to photosynthesis. These were further investigated using modulated chlorophyll a fluorescence measurements and photosynthetic pigment quantification. The decrease of net photosynthesis, leaf conductance and water use efficiency was more pronounced under rapid stress than in slow stress. However, photosynthesis is always mainly limited by stomata in both types of stress, albeit the contribution of non‐stomatal limitations increases at severe water deficits in slow stress experiments. The substomatal CO₂ partial pressure significantly increased in both types of stress, suggesting an increased resistance due to an internal barrier to CO₂ diffusion. Physical alterations in the structure of the intercellular spaces due to leaf shrinkage may account for these results. The maximal photochemical efficiency of photosystem II (PSII) was remarkably resistant to stress, as the F_v/F_m ratio decreased only at severe water deficit. On the contrary, the effective photochemical efficiency of PSII (ΔF/F′_m) measured under high actinic light decreased linearly in both types of stress, although in a more pronounced way under rapid stress. A similar variation in photochemical quenching suggests that the decrease of ΔF/F′_m is mainly due to the closure of PSII reaction centres. The non‐photochemical quenching did not change significantly except under severe dehydration indicating that the energization state of thylakoids remained stable under stress. The decrease observed in photosynthetic pigments may be an adaptation to stress rather than a limiting factor to photosynthesis. Results suggests that, although intrinsic mesophyll metabolic inhibitions occur, stomatal limitation to CO₂ diffusion is the main reason for the decrease in photosynthesis.     

CO2-Gaswechsel amphistomatischer Blätter

Summary The time-response of the CO₂-exchange of both leaf surfaces was measured separately. Leaves of Primula palinuri and Zea mays were used for the study. After short dark-periods (3 min) the stomata are not closed. Consequently CO₂-uptake starts quickly after re-illumination and reaches the steady-state value very rapidly. The time-response of stripped leaves of Primula and of normal leaves after short dark-periods is identical. Accordingly, the conclusion seems to be evident that in both cases we are measuring the time-response of photosynthesis, which is not influenced by stomatal reactions. After long dark-periods (60 min) the stomata are closed. After re-illumination the CO₂ released by respiration is immediately reassimilated. There is a distinct lag-phase in time-response which is more or less located in the CO₂-compensation point. This lag-phase is of different length for both leaf surfaces, and is interpreted as being the lag-phase of stomatal opening reactions. The consequence of the observed different time response of photosynthesis and stomatal reactions is discussed: under non-steady-state conditions photosynthesis is limited by slow stomatal opening reactions.     

Water use strategy affects avoidance of ozone stress by stomatal closure in Mediterranean trees—A modelling analysis

Both ozone (O₃) and drought can limit carbon fixation by forest trees. To cope with drought stress, plants have isohydric or anisohydric water use strategies. Ozone enters plant tissues through stomata. Therefore, stomatal closure can be interpreted as avoidance to O₃ stress. Here, we applied an optimization model of stomata involving water, CO₂, and O₃ flux to test whether isohydric and anisohydric strategies may affect avoidance of O₃ stress by stomatal closure in four Mediterranean tree species during drought. The data suggest that stomatal closure represents a response to avoid damage to the photosynthetic mechanisms under elevated O₃ depending on plant water use strategy. Under high-O₃ and well-watered conditions, isohydric species limited O₃ fluxes by stomatal closure, whereas anisohydric species activated a tolerance response and did not actively close stomata. Under both O₃ and drought stress, however, anisohydric species enhanced the capacity of avoidance by closing stomata to cope with the severe oxidative stress. In the late growing season, regardless of the water use strategy, the efficiency of O₃ stress avoidance decreased with leaf ageing. As a result, carbon assimilation rate was decreased by O₃ while stomata did not close enough to limit transpirational water losses.     

Responses of Two Olive Tree (Olea Europaea L.) Cultivars to Elevated CO2 Concentration in the Field

Five-year-old plants of two olive cultivars (Frantoio and Moraiolo) grown in large pots were exposed for 7 to 8 months to ambient (AC) or elevated (EC) CO₂ concentration in a free-air CO₂ enrichment (FACE) facility. Exposure to EC enhanced net photosynthetic rate (P _N) and decreased stomatal conductance, leading to greater instantaneous transpiration efficiency. Stomata density also decreased under EC, while the ratio of intercellular (C _i) to atmospheric CO₂ concentration and chlorophyll content did not differ, except for the cv. Moraiolo after seven months of exposure to EC. Analysis of the relationship between photosynthesis and C _i indicated no significant change in carboxylation efficiency of ribulose-1,5-bisphosphate carboxylase/oxygenase after five months of exposure to EC. Based on estimates derived from the P _N-C _i relationship, there were no apparent treatment differences in daytime respiration, CO₂ compensation concentration, CO₂-saturated photosynthetic rate, or photosynthetic rate at the mean C _i, but there was a reduction in stomata limitation to P _N at EC. Thus 5-year-old olive trees did not exhibit down regulation of leaf-level photosynthesis in their response to EC, though some indication of adjustment was evident for the cv. Frantoio with respect to the cv. Moraiolo.     

Increasing canopy photosynthesis in rice can be achieved without a large increase in water use—A model based on free‐air CO2 enrichment

Achieving higher canopy photosynthesis rates is one of the keys to increasing future crop production; however, this typically requires additional water inputs because of increased water loss through the stomata. Lowland rice canopies presently consume a large amount of water, and any further increase in water usage may significantly impact local water resources. This situation is further complicated by changing the environmental conditions such as rising atmospheric CO₂ concentration ([CO₂]). Here, we modeled and compared evapotranspiration of fully developed rice canopies of a high‐yielding rice cultivar (Oryza sativa L. cv. Takanari) with a common cultivar (cv. Koshihikari) under ambient and elevated [CO₂] (A‐CO₂ and E‐CO₂, respectively) via leaf ecophysiological parameters derived from a free‐air CO₂ enrichment (FACE) experiment. Takanari had 4%–5% higher evapotranspiration than Koshihikari under both A‐CO₂ and E‐CO₂, and E‐CO₂ decreased evapotranspiration of both varieties by 4%–6%. Therefore, if Takanari was cultivated under future [CO₂] conditions, the cost for water could be maintained at the same level as for cultivating Koshihikari at current [CO₂] with an increase in canopy photosynthesis by 36%. Sensitivity analyses determined that stomatal conductance was a significant physiological factor responsible for the greater canopy photosynthesis in Takanari over Koshihikari. Takanari had 30%–40% higher stomatal conductance than Koshihikari; however, the presence of high aerodynamic resistance in the natural field and lower canopy temperature of Takanari than Koshihikari resulted in the small difference in evapotranspiration. Despite the small difference in evapotranspiration between varieties, the model simulations showed that Takanari clearly decreased canopy and air temperatures within the planetary boundary layer compared to Koshihikari. Our results indicate that lowland rice varieties characterized by high‐stomatal conductance can play a key role in enhancing productivity and moderating heat‐induced damage to grain quality in the coming decades, without significantly increasing crop water use.     

Do Stomata Respond to CO(2) Concentrations Other than Intercellular?

Most studies on stomatal responses to CO₂ assume that guard cells respond only to intercellular CO₂ concentration and are insensitive to the CO₂ concentrations in the pore and outside the leaf. If stomata are sensitive to the CO₂ concentration at the surface of the leaf or in the stomatal pore, the stomatal response to intercellular CO₂ concentration will be incorrect for a `normally' operating leaf (where ambient CO₂ concentration is a constant). In this study asymmetric CO₂ concentrations for the two surfaces of amphistomatous leaves were used to vary intercellular and leaf surface CO₂ concentrations independently in Xanthium strumarium L. and Helianthus annuus L. The response of stomata to intercellular CO₂ concentration when the concentration at the leaf surface was held constant was found to be the same as the response when the surface concentration was varied. In addition, stomata did not respond to changes in leaf surface CO₂ concentration when the intercellular concentration for that surface was held constant. It is concluded that stomata respond to intercellular CO₂ concentration and are insensitive to the CO₂ concentration at the surface of the leaf and in the stomatal pore.     

Changes in graminoid stomatal morphology over the last glacial–interglacial transition: evidence from Mount Kenya,East Africa

Stomatal size and density were measured from graminoid cuticular fragments extracted from dated sediments in two tropical-montane crater lakes on Mount Kenya. The sediments had been dated in other studies and spanned 1500–37 000 calibrated years BP. Changes in the mean size and density of the graminoid stomata were found. Using a coarse signal analysis the two lakes gave fairly similar results, although there was some divergence at the start and end of the time period analyzed. There is some correspondence between the atmospheric CO₂ concentration and graminoid stomatal density during the transition from the LGM to the start of the Holocene, where stomatal density decreased while CO₂ concentrations increased. All the changes observed may have been plastic responses within existing species at the site or competitive replacements of grass floras. We argue that higher stomatal density may have been a response to falling CO₂ levels during the last glaciation, accompanying the replacement of a C₃ flora by C₄ species. The stomatal size changes exhibited over this time period may have adapted plants to changes in soil water availability. That stomatal morphology changes in a sample flora (not a single taxon) over millennia is a novel finding, and one that may have implications for paleoecological interpretation and the prediction of grass behavior in the future.     

Stomatal sensitivity to carbon dioxide and humidity: a comparison of two c(3) and two c(4) grass species

The sensitivity of stomatal conductance to changes of CO₂ concentration and leaf-air vapor pressure difference (VPD) was compared between two C₃ and two C₄ grass species. There was no evidence that stomata of the C₄ species were more sensitive to CO₂ than stomata of the C₃ species. The sensitivity of stomatal conductance to CO₂ change was linearly proportional to the magnitude of stomatal conductance, as determined by the VPD, the same slope fitting the data for all four species. Similarly, the sensitivity of stomatal conductance to VPD was linearly proportional to the magnitude of stomatal conductance. At small VPD, the ratio of intercellular to ambient CO₂ concentration, C_i/C_a, was similar in all species (0.8-0.9) but declined with increasing VPD, so that, at large VPD, C_i/C_a was 0.7 and 0.5 (approximately) in C₃ and C₄ species, respectively. Transpiration efficiency (net CO₂ assimilation rate/transpiration rate) was larger in the C₄ species than in the C₃ species at current atmospheric CO₂ concentrations, but the relative increase due to high CO₂ was larger in the C₃ than in the C₄ species.     

PHYSIOLOGICAL RESPONSES TO RAINFALL IN OPUNTIA BASILARIS (CACTACEAE)

An immediate, marked response to small amounts of rainfall occurs in Opuntia basilaris, despite previous drought conditions. The effect of rainfall is upon plant water potential, which is the single most important parameter influencing stomatal opening, CO₂ assimilation, and organic acid synthesis. Nocturnal stomatal opening is initiated following rainfall, and stomata remain open during the daytime. Decreasing stomatal and mesophyll resistances correlate with increasing rates of nocturnal assimilation of ¹⁴CO₂. Photosynthetic rates of ¹⁴CO₂ assimilation are low, despite high plant water potentials and low stomatal diffusion resistances. The decreased mesophyll resistances and increased rates of nocturnal ¹⁴CO₂ assimilation correlate with the increases of nocturnal efficiency of water use and CO₂ assimilation. The diurnal efficiency of water use and CO₂ assimilation is lower than the nocturnal gas exchange efficiency values.     

Carbon isotope discrimination and stomatal responses of mature Pinus sylvestris L. trees exposed in situ for three years to elevated CO2 and temperature

The Climate Change Experiment (CLIMEX) is a unique large scale facility in which an entire undisturbed catchment of boreal vegetation has been exposed to elevated CO₂ (560 ppm) and temperature (+3°C summer, +5°C winter) for the past three years with all the soil-plant-atmosphere linkages intact. Here, carbon isotope composition and stomatal density have been analysed from sequential year classes of needles of mature Scots pine trees (Pinus sylvestris L.) to investigate the response of time-integrated water-use efficiency (UWE) and stomatal density to CO₂ enrichment and climate change. Carbon isotope discrimination decreased and WUE increased in cohorts of needles developing under increased CO₂ and temperature, compared to needles on the same trees developing in pretreatment years. Mid-season instantaneous gas exchange, measured on the same trees for the past four years, indicated that these responses resulted from higher needle photosynthetic rates and reduced stomatal conductance. Needles of P. sylvestris developing under increased CO₂ and temperature had consistently lower stomatal densities than their ambient grown counterparts on the same trees. The stomatal density of P. sylvestris needles was inversely correlated with δ¹³C-derived WUE, implying some effect of this morphological response on leaf gas exchange. Future atmospheric CO₂ and temperature increases are therefore likely to improve the water economy of P. sylvestris, at least at the scale of individual needles, by affecting stomatal density and gas exchange processes.     

Co-ordination of physiological and morphological responses of stomata to elevated [CO2] in vascular plants

Plant stomata display a wide range of short-term behavioural and long-term morphological responses to atmospheric carbon dioxide concentration ([CO₂]). The diversity of responses suggests that plants may have different strategies for controlling gas exchange, yet it is not known whether these strategies are co-ordinated in some way. Here, we test the hypothesis that there is co-ordination of physiological (via aperture change) and morphological (via stomatal density change) control of gas exchange by plants. We examined the response of stomatal conductance (G _s) to instantaneous changes in external [CO₂] (C _a) in an evolutionary cross-section of vascular plants grown in atmospheres of elevated [CO₂] (1,500 ppm) and sub-ambient [O₂] (13.0 %) compared to control conditions (380 ppm CO₂, 20.9 % O₂). We found that active control of stomatal aperture to [CO₂] above current ambient levels was not restricted to angiosperms, occurring in the gymnosperms Lepidozamia peroffskyana and Nageia nagi. The angiosperm species analysed appeared to possess a greater respiratory demand for stomatal movement than gymnosperm species displaying active stomatal control. Those species with little or no control of stomatal aperture (termed passive) to C _a were more likely to exhibit a reduction in stomatal density than species with active stomatal control when grown in atmospheres of elevated [CO₂]. The relationship between the degree of stomatal aperture control to C _a above ambient and the extent of any reduction in stomatal density may suggest the co-ordination of physiological and morphological responses of stomata to [CO₂] in the optimisation of water use efficiency. This trade-off between stomatal control strategies may have developed due to selective pressures exerted by the costs associated with passive and active stomatal control.