Metabolic Background, Not Photosynthetic Physiology, Determines Drought and Drought Recovery Responses in C3 and C2 Moricandias.

Distinct photosynthetic physiologies are found within the Moricandia genus, both C3-type and C2-type representatives being known. As C2-physiology is an adaptation to drier environments, a study of physiology, biochemistry and transcriptomics was conducted to investigate whether plants with C2-physiology are more tolerant of low water availability and recover better from drought. Our data on Moricandia moricandioides (Mmo, C3), M. arvensis (Mav, C2) and M. suffruticosa (Msu, C2) show that C3 and C2-type Moricandias are metabolically distinct under all conditions tested (well-watered, severe drought, early drought recovery). Photosynthetic activity was found to be largely dependent upon the stomatal opening. The C2-type M. arvensis was able to secure 25-50% of photosynthesis under severe drought as compared to the C3-type M. moricandioides. Nevertheless, the C2-physiology does not seem to play a central role in M. arvensis drought responses and drought recovery. Instead, our biochemical data indicated metabolic differences in carbon and redox-related metabolism under the examined conditions. The cell wall dynamics and glucosinolate metabolism regulations were found to be major discriminators between M. arvensis and M. moricandioides at the transcription level.

CO2 -induced biochemical changes in leaf volatiles decreased fire-intensity in the run-up to the Triassic-Jurassic boundary.

The Triassic-Jurassic boundary marks the third largest mass extinction event in the Phanerozoic, characterized by a rise in CO2 -concentrations from c. 600 ppm to c. 2100-2400 ppm, coupled with a c. 3.0-4.0°C temperature rise. This is hypothesized to have induced major floral turnover, altering vegetation structure, composition and leaf morphology, which in turn are hypothesized to have driven changes in wildfire. However, the effects of elevated CO2 on fuel properties, such as chemical composition of leaves, are also important in influencing fire behaviour, but yet have not been considered. We test this by selecting three Triassic analogue species grown experimentally in different atmospheric compositions, and analyse variations in leaf chemistry, and leaf level flammability. These data were used to inform a fire behaviour model. We find that all three species tested showed a reduction in their volatile component, leading to lower flammability. Accounting for these variations in a model, our results suggest that leaf intrinsic flammability has a measurable impact on modelled fire behaviour. If scaled up to ecosystem level, periods of elevated CO2 may therefore be capable of inducing both biochemical and morphological changes in fuel properties, and thus may be capable of influencing fire behaviour.

Species-specific variation of photosynthesis and mesophyll conductance to ozone and drought in three Mediterranean oaks.

Mesophyll conductance (gmCO2 ) is one of the most important components in plant photosynthesis. Tropospheric ozone (O3 ) and drought impair physiological processes, causing damage to photosynthetic systems. However, the combined effects of O3 and drought on gmCO2 are still largely unclear. We investigated leaf gas exchange during mid-summer in three Mediterranean oaks exposed to O3 (ambient [35.2 nmol mol-1 as daily mean]; 1.4 × ambient) and water treatments (WW [well-watered] and WD [water-deficit]). We also examined if leaf traits (leaf mass per area [LMA], foliar abscisic acid concentration [ABA]) could influence the diffusion of CO2 inside a leaf. The combination of O3 and WD significantly decreased net photosynthetic rate (PN ) regardless of the species. The reduction of photosynthesis was associated with a decrease in gmCO2 and stomatal conductance (gsCO2 ) in evergreen Quercus ilex, while the two deciduous oaks (Q. pubescens, Q. robur) also showed a reduction of the maximum rate of carboxylation (Vcmax ) and maximum electron transport rate (Jmax ) with decreased diffusive conductance parameters. The reduction of gmCO2 was correlated with increased [ABA] in the three oaks, whereas there was a negative correlation between gmCO2 with LMA in Q. pubescens. Interestingly, two deciduous oaks showed a weak or no significant correlation between gsCO2 and ABA under high O3 and WD due to impaired stomatal physiological behaviour, indicating that the reduction of PN was related to gmCO2 rather than gsCO2 . The results suggest that gmCO2 plays an important role in plant carbon gain under concurrent increases in the severity of drought and O3 pollution.

Integrating stomatal physiology and morphology: evolution of stomatal control and development of future crops.

Stomata are central players in the hydrological and carbon cycles, regulating the uptake of carbon dioxide (CO2) for photosynthesis and transpirative loss of water (H2O) between plants and the atmosphere. The necessity to balance water-loss and CO2-uptake has played a key role in the evolution of plants, and is increasingly important in a hotter and drier world. The conductance of CO2 and water vapour across the leaf surface is determined by epidermal and stomatal morphology (the number, size, and spacing of stomatal pores) and stomatal physiology (the regulation of stomatal pore aperture in response to environmental conditions). The proportion of the epidermis allocated to stomata and the evolution of amphistomaty are linked to the physiological function of stomata. Moreover, the relationship between stomatal density and [CO2] is mediated by physiological stomatal behaviour; species with less responsive stomata to light and [CO2] are most likely to adjust stomatal initiation. These differences in the sensitivity of the stomatal density-[CO2] relationship between species influence the efficacy of the 'stomatal method' that is widely used to infer the palaeo-atmospheric [CO2] in which fossil leaves developed. Many studies have investigated stomatal physiology or morphology in isolation, which may result in the loss of the 'overall picture' as these traits operate in a coordinated manner to produce distinct mechanisms for stomatal control. Consideration of the interaction between stomatal morphology and physiology is critical to our understanding of plant evolutionary history, plant responses to on-going climate change and the production of more efficient and climate-resilient food and bio-fuel crops.

Interactive effect of leaf age and ozone on mesophyll conductance in Siebold's beech.

Mesophyll conductance (Gm ) is one of the most important factors determining photosynthesis. Tropospheric ozone (O3 ) is known to accelerate leaf senescence and causes a decline of photosynthetic activity in leaves. However, the effects of age-related variation of O3 on Gm have not been well investigated, and we, therefore, analysed leaf gas exchange data in a free-air O3 exposure experiment on Siebold's beech with two levels (ambient and elevated O3 : 28 and 62 nmol mol-1 as daylight average, respectively). In addition, we examined whether O3 -induced changes on leaf morphology (leaf mass per area, leaf density and leaf thickness) may affect CO2 diffusion inside leaves. We found that O3 damaged the photosynthetic biochemistry progressively during the growing season. The Gm was associated with a reduced photosynthesis in O3 -fumigated Siebold's beech in August. The O3 -induced reduction of Gm was negatively correlated with leaf density, which was increased by elevated O3 , suggesting that the reduction of Gm was accompanied by changes in the physical structure of mesophyll cells. On the other hand, in October, the O3 -induced decrease of Gm was diminished because Gm decreased due to leaf senescence regardless of O3 treatment. The reduction of photosynthesis in senescent leaves after O3 exposure was mainly due to a decrease of maximum carboxylation rate (Vcmax ) and/or maximum electron transport rate (Jmax ) rather than diffusive limitations to CO2 transport such as Gm . A leaf age×O3 interaction of photosynthetic response will be a key for modelling photosynthesis in O3 -polluted environments.

Ambulances are for emergencies: shifting behaviour through a research-informed behaviour change campaign.

BACKGROUND: A major review of Victoria's ambulance services identified the need to improve public awareness of the role of ambulances as an emergency service. A communications campaign was developed to address this challenge. This research paper expands on an initial evaluation of the campaign by focusing on the long-term behavioural outcomes. METHODS: The behavioural evaluation involved two types of data collection - administrative data (routine collection from various health services) and survey data (cross-sectional community-wide surveys to measure behavioural intentions). RESULTS: Behavioural intentions for accessing two of the targeted non-emergency services increased after the second phase of the campaign commenced. There was also a significant change in the slope of call trends for emergency ambulances. This decrease is also likely attributed to the second phase of the campaign as significant level effects were identified 3 and 9 months after it commenced. CONCLUSIONS: A long-term campaign developed through evidence review, stakeholder consultation and behavioural theory was successful in reducing the number of daily calls requesting an emergency ambulance in Victoria and in increasing intentions to use alternative services. This research highlights the importance of collaborative intervention design along with the importance of implementing a robust monitoring and evaluation framework.

A Comparison of the Variable J and Carbon-Isotopic Composition of Sugars Methods to Assess Mesophyll Conductance from the Leaf to the Canopy Scale in Drought-Stressed Cherry.

Conductance of CO2 across the mesophyll (Gm) frequently constrains photosynthesis (PN) but cannot be measured directly. We examined Gm of cherry (Prunus avium L.) subjected to severe drought using the variable J method and carbon-isotopic composition (δ13C) of sugars from the centre of the leaf, the leaf petiole sap, and sap from the largest branch. Depending upon the location of the plant from which sugars are sampled, Gm may be estimated over scales ranging from a portion of the leaf to a canopy of leaves. Both the variable J and δ13C of sugars methods showed a reduction in Gm as soil water availability declined. The δ13C of sugars further from the source of their synthesis within the leaf did not correspond as closely to the diffusive and C-isotopic discrimination conditions reflected in the instantaneous measurement of gas exchange and chlorophyll-fluorescence utilised by the variable J approach. Post-photosynthetic fractionation processes and/or the release of sugars from stored carbohydrates (previously fixed under different environmental and C-isotopic discrimination conditions) may reduce the efficacy of the δ13C of sugars from leaf petiole and branch sap in estimating Gm in a short-term study. Consideration should be given to the spatial and temporal scales at which Gm is under observation in any experimental analysis.

Dynamic changes in ABA content in water-stressed Populus nigra: effects on carbon fixation and soluble carbohydrates.

BACKGROUND AND AIMS: Hydraulic and chemical signals operate in tandem to regulate systemic plant responses to drought. Transport of abscisic acid (ABA) through the xylem and phloem from the root to shoot has been suggested to serve as the main signal of water deficit. There is evidence that ABA and its ABA-glycosyl-ester (ABA-GE) are also formed in leaves and stems through the chloroplastic 2-C-methylerythritol-5-phosphate (MEP) pathway. This study aimed to evaluate how hormonal and hydraulic signals contribute to optimize stomatal (gs), mesophyll (gm) and leaf hydraulic (Kleaf) conductance under well-watered and water-stressed conditions in Populus nigra (black poplar) plants. In addition, we assessed possible relationships between ABA and soluble carbohydrates within the leaf and stem. METHODS: Plants were subjected to three water treatments: well-watered (WW), moderate stress (WS1) and severe stress (WS2). This experimental set-up enabled a time-course analysis of the response to water deficit at the physiological [leaf gas exchange, plant water relations, (Kleaf)], biochemical (ABA and its metabolite/catabolite quantification in xylem sap, leaves, wood, bark and roots) and molecular (gene expression of ABA biosynthesis) levels. KEY RESULTS: Our results showed strong coordination between gs, gm and Kleaf under water stress, which reduced transpiration and increased intrinsic water use efficiency (WUEint). Analysis of gene expression of 9-cis-epoxycarotenoid dioxygenase (NCED) and ABA content in different tissues showed a general up-regulation of the biosynthesis of this hormone and its finely-tuned catabolism in response to water stress. Significant linear relationships were found between soluble carbohydrates and ABA contents in both leaves and stems, suggesting a putative function for this hormone in carbohydrate mobilization under severe water stress. CONCLUSIONS: This study demonstrates the tight regulation of the photosynthetic machinery by levels of ABA in different plants organs on a daily basis in both well-watered and water stress conditions to optimize WUEint and coordinate whole plant acclimation responses to drought.

The effect of summer drought on the yield of Arundo donax is reduced by the retention of photosynthetic capacity and leaf growth later in the growing season.

BACKGROUND AND AIMS: The development of Arundo donax as a biomass crop for use on drought-prone marginal lands in areas with warm to hot climates is constrained by the lack of variation within this species. We investigated the effect of morphological and physiological variation on growth and tolerance to drought under field conditions in three ecotypes of A. donax collected from habitats representing a climate gradient: a pre-desert in Morocco, a semi-arid Mediterranean climate in southern Italy and a warm sub-humid region of central Italy. METHODS: The three A. donax ecotypes were grown under irrigated and rain-fed conditions in a common garden field trial in a region with a semi-arid Mediterranean climate. Physiological and morphological characteristics, and carbohydrate metabolism of the ecotypes were recorded to establish which traits were associated with yield and/or drought tolerance. KEY RESULTS: Variation was observed between the A. donax ecotypes. The ecotype from the most arid habitat produced the highest biomass yield. Stem height and the retention of photosynthetic capacity later in the year were key traits associated with differences in biomass yield. The downregulation of photosynthetic capacity was not associated with changes in foliar concentrations of sugars or starch. Rain-fed plants maintained photosynthesis and growth later in the year compared with irrigated plants that began to senescence earlier, thus minimizing the difference in yield. Effective stomatal control prevented excessive water loss, and the emission of isoprene stabilized photosynthetic membranes under drought and heat stress in A. donax plants grown under rain-fed conditions without supplementary irrigation. CONCLUSIONS: Arundo donax is well adapted to cultivation in drought-prone areas with warm to hot climates. None of the A. donax ecotypes exhibited all of the desired traits consistent with an 'ideotype'. Breeding or genetic (identification of quantitative trait loci) improvement of A. donax should select ecotypes on the basis of stem morphology and the retention of photosynthetic capacity.

Leaf and canopy photosynthesis of a chlorophyll deficient soybean mutant.

The photosynthetic, optical, and morphological characteristics of a chlorophyll-deficient (Chl-deficient) "yellow" soybean mutant (MinnGold) were examined in comparison with 2 green varieties (MN0095 and Eiko). Despite the large difference in Chl content, similar leaf photosynthesis rates were maintained in the Chl-deficient mutant by offsetting the reduced absorption of red photons by a small increase in photochemical efficiency and lower non-photochemical quenching. When grown in the field, at full canopy cover, the mutants reflected a significantly larger proportion of incoming shortwave radiation, but the total canopy light absorption was only slightly reduced, most likely due to a deeper penetration of light into the canopy space. As a consequence, canopy-scale gross primary production and ecosystem respiration were comparable between the Chl-deficient mutant and the green variety. However, total biomass production was lower in the mutant, which indicates that processes other than steady state photosynthesis caused a reduction in biomass accumulation over time. Analysis of non-photochemical quenching relaxation and gas exchange in Chl-deficient and green leaves after transitions from high to low light conditions suggested that dynamic photosynthesis might be responsible for the reduced biomass production in the Chl-deficient mutant under field conditions.

Adaptation to high temperature mitigates the impact of water deficit during combined heat and drought stress in C3 sunflower and C4 maize varieties with contrasting drought tolerance.

Heat and drought stress frequently occur together, however, their impact on plant growth and photosynthesis (PN ) is unclear. The frequency, duration and severity of heat and drought stress events are predicted to increase in the future, having severe implications for agricultural productivity and food security. To assess the impact on plant gas exchange, physiology and morphology we grew drought tolerant and sensitive varieties of C3 sunflower (Helianthus annuus) and C4 maize (Zea mays) under conditions of elevated temperature for 4 weeks prior to the imposition of water deficit. The negative impact of temperature on PN was most apparent in sunflower. The drought tolerant sunflower retained ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) activity under heat stress to a greater extent than its drought sensitive counterpart. Maize exhibited no varietal difference in response to increased temperature. In contrast to previous studies, where a sudden rise in temperature induced an increase in stomatal conductance (Gs ), we observed no change or a reduction in Gs with elevated temperature, which alongside lower leaf area mitigated the impact of drought at the higher temperature. The drought tolerant sunflower and maize varieties exhibited greater investment in root-systems, allowing greater uptake of the available soil water. Elevated temperatures associated with heat-waves will have profound negative impacts on crop growth in both sunflower and maize, but the deleterious effect on PN was less apparent in the drought tolerant sunflower and both maize varieties. As C4 plants generally exhibit water use efficiency (WUE) and resistance to heat stress, selection on the basis of tolerance to heat and drought stress would be more beneficial to the yields of C3 crops cultivated in drought prone semi-arid regions.

Coordination of stomatal physiological behavior and morphology with carbon dioxide determines stomatal control.

PREMISE OF THE STUDY: Stomatal control is determined by the ability to alter stomatal aperture and/or the number of stomata on the surface of new leaves in response to growth conditions. The development of stomatal control mechanisms to the concentration of CO2within the atmosphere ([CO2]) is fundamental to our understanding of plant evolutionary history and the prediction of gas exchange responses to future [CO2]. METHODS: In a controlled environment, fern and angiosperm species were grown in atmospheres of ambient (400 ppm) and elevated (2000 ppm) [CO2]. Physiological stomatal behavior was compared with the stomatal morphological response to [CO2]. KEY RESULTS: An increase in [CO2] or darkness induced physiological stomatal responses ranging from reductions (active) to no change (passive) in stomatal conductance. Those species with passive stomatal behavior exhibited pronounced reductions of stomatal density in new foliage when grown in elevated [CO2], whereas species with active stomata showed little morphological response to [CO2]. Analysis of the physiological and morphological stomatal responses of a wider range of species suggests that patterns of stomatal control to [CO2] do not follow a phylogenetic pattern associated with plant evolution. CONCLUSIONS: Selective pressures may have driven the development of divergent stomatal control strategies to increased [CO2]. Those species that are able to actively regulate guard cell turgor are more likely to respond to [CO2] through a change in stomatal aperture than stomatal number. We propose a model of stomatal control strategies in response to [CO2] characterized by a trade-off between short-term physiological behavior and longer-term morphological response.

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 ([CO(2)]). 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 [CO(2)] (C(a)) in an evolutionary cross-section of vascular plants grown in atmospheres of elevated [CO(2)] (1,500 ppm) and sub-ambient [O(2)] (13.0 %) compared to control conditions (380 ppm CO(2), 20.9 % O(2)). We found that active control of stomatal aperture to [CO(2)] 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 [CO(2)]. 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 [CO(2)] 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.

Ethylenediurea protects against ozone phytotoxicity not by adding nitrogen or controlling stomata in a stomata-unresponsive hybrid poplar.

Ozone (O3) pollution is a persistent environmental issue worldwide, which causes widespread damage to vegetation, deteriorating plant health and reducing plant productivity. Ethylenediurea (EDU) is a synthetic chemical that has been widely applied in scientific studies as a protectant against O3 phytotoxicities. Despite four decades of active research, the exact mechanisms to explain its mode of action remain unclear. Here, we aimed to reveal whether EDU's phytoprotective property is due to its control over stomatal regulation and/or its action as a nitrogen (N) fertilizer, utilizing stomatal-unresponsive plants of a hybrid poplar (Populus koreana × trichocarpa cv. Peace) grown in a free-air O3-concenctration enrichment (FACE) facility. Plants were treated with water (WAT), EDU (400 mg L-1), or EDU's constitutive amount of N every nine days, and exposed to ambient (AOZ) or elevated (EOZ) O3 during a growing season (June-September). EOZ led to extensive foliar injuries (but protected against rust disease), lower photosynthetic rate (A), impaired dynamics of responses of A to changes in light intensity, and smaller total plant leaf area. EDU protected against common phytotoxicities caused by EOZ without inducing stomatal closure, since stomatal conductance (gs) was generally unresponsive to the experimental treatments. EDU also modulated the dynamic response of A to light fluctuations under O3 stress. N addition acted as a fertilizer but did not satisfactorily protect plants against O3 phytotoxicities. The results suggest that EDU protects against O3 phytotoxicity not by adding N or controlling stomata, which provides a new insight into our understanding of the mode of action of EDU as a protectant against O3 phytotoxicity.

The functional significance of the stomatal size to density relationship: Interaction with atmospheric [CO2] and role in plant physiological behaviour.

The limits for stomatal conductance are set by stomatal size (SS) and density (SD). An inverse relationship between SS and SD has been observed in fossil and living plants. This has led to hypotheses proposing that the ratio of SS to SD influences the diffusion pathway for CO2 and degree of physiological stomatal control. However, conclusive evidence supportive of a functional role of the SS-SD relationship is not evident, and patterns in SS-SD may simply reflect geometric constraints in stomatal spacing over a leaf surface. We examine published and new data to investigate the potential functional significance of the relationship between SS and SD to atmospheric [CO2] in multiple generation adaptive responses and short-term acclamatory adjustment of stomatal morphology. Consistent patterns in SS and SD were not evident in fossil and living plants adapted to high [CO2] over many generations. However, evolutionary adaptation to [CO2] strongly affected SS and SD responses to elevated [CO2], with plants adapted to the 'low' [CO2] of the past 10 million years (Myr) showing adjustment of SS-SD, while members of the same species adapted to 'high' [CO2] showed no response. This may suggest that SS and SD responses to future [CO2] will likely constrain the stimulatory effect of 'CO2-fertilisation' on photosynthesis. Angiosperms generally possessed higher densities of smaller stomata that corresponded to a greater degree of physiological stomatal control consistent with selective pressures induced by declining [CO2] over the past 90 Myr. Atmospheric [CO2] has likely shaped stomatal size and density relationships alongside the interaction with stomatal physiological behaviour. The rate and predicted extent of future increases in [CO2] will have profound impacts on the selective pressures shaping SS and SD. Understanding the trade-offs involved in SS-SD and the interaction with [CO2], will be central to the development of more productive climate resilient crops.

Plant Physiological Analysis to Overcome Limitations to Plant Phenotyping.

Plant physiological status is the interaction between the plant genome and the prevailing growth conditions. Accurate characterization of plant physiology is, therefore, fundamental to effective plant phenotyping studies; particularly those focused on identifying traits associated with improved yield, lower input requirements, and climate resilience. Here, we outline the approaches used to assess plant physiology and how these techniques of direct empirical observations of processes such as photosynthetic CO2 assimilation, stomatal conductance, photosystem II electron transport, or the effectiveness of protective energy dissipation mechanisms are unsuited to high-throughput phenotyping applications. Novel optical sensors, remote/proximal sensing (multi- and hyperspectral reflectance, infrared thermography, sun-induced fluorescence), LiDAR, and automated analyses of below-ground development offer the possibility to infer plant physiological status and growth. However, there are limitations to such 'indirect' approaches to gauging plant physiology. These methodologies that are appropriate for the rapid high temporal screening of a number of crop varieties over a wide spatial scale do still require 'calibration' or 'validation' with direct empirical measurement of plant physiological status. The use of deep-learning and artificial intelligence approaches may enable the effective synthesis of large multivariate datasets to more accurately quantify physiological characters rapidly in high numbers of replicate plants. Advances in automated data collection and subsequent data processing represent an opportunity for plant phenotyping efforts to fully integrate fundamental physiological data into vital efforts to ensure food and agro-economic sustainability.

The 'microbiome counterattack': Insights on the soil and root-associated microbiome in diverse chickpea and lentil genotypes after an erratic rainfall event.

Legumes maintain soil fertility thanks to their associated microbiota but are threatened by climate change that causes soil microbial community structural and functional modifications. The core microbiome associated with different chickpea and lentil genotypes was described after an unexpected climatic event. Results showed that chickpea and lentil bulk soil microbiomes varied significantly between two sampling time points, the first immediately after the rainfall and the second 2 weeks later. Rhizobia were associated with the soil of the more productive chickpea genotypes in terms of flower and fruit number. The root-associated bacteria and fungi were surveyed in lentil genotypes, considering that several parcels showed disease symptoms. The metabarcoding analysis revealed that reads related to fungal pathogens were significantly associated with one lentil genotype. A lentil core prokaryotic community common to all genotypes was identified as well as a genotype-specific one. A higher number of specific bacterial taxa and an enhanced tolerance to fungal diseases characterized a lentil landrace compared to the commercial varieties. This outcome supported the hypothesis that locally adapted landraces might have a high recruiting efficiency of beneficial soil microbes.

The evolution of diffusive and biochemical capacities for photosynthesis was predominantly shaped by [CO2] with a smaller contribution from [O2].

The atmospheric concentration of carbon dioxide ([CO2]) and oxygen ([O2]) directly influence rates of photosynthesis (PN) and photorespiration (RPR) through the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). Levels of [CO2] and [O2] have varied over Earth history affecting rates of both CO2 uptake and loss, alongside associated transpirative water-loss. The availability of CO2 has likely acted as a stronger selective pressure than [O2] due to the greater specificity of RubisCO for CO2. The role of [O2], and the interaction of [O2] and [CO2], in plant evolutionary history is less understood. We exposed twelve phylogenetically diverse species to combinations of sub-ambient, ambient and super-ambient [O2] and [CO2] to examine the biochemical and diffusive components of PN and the possible role of [O2] as a selective pressure. Photosynthesis, photosynthetic capacity and stomatal, mesophyll and total conductance to CO2 were higher in the derived eudicot and monocot angiosperms than the more basal ferns, gymnosperms and basal angiosperms which originated in atmospheres characterised by higher CO2:O2 ratios. The ratio of RPR:PN was lower in the monocots, consistent with greater carboxylation capacity and higher stomatal and mesophyll conductance making easier CO2 delivery to chloroplasts. The effect of [O2] and [CO2] on PN/RPR was less evident in more derived species with a higher conductance to CO2. The effect of [O2] was less apparent at high [CO2], suggesting that atmospheric [O2] may only have exerted a strong selective pressure on plant photosynthetic processes during periods characterised by low atmospheric CO2:O2 ratios. Current rising [CO2] will predominantly enhance PN rates in species with low diffusive conductance to CO2.

The stomatal CO2 proxy does not saturate at high atmospheric CO2 concentrations: evidence from stomatal index responses of Araucariaceae conifers.

The inverse relationship between the number of stomata on a leaf surface and the atmospheric carbon dioxide concentration ([CO(2)]) in which the leaf developed allows plants to optimise water-use efficiency (WUE), but it also permits the use of fossil plants as proxies of palaeoatmospheric [CO(2)]. The ancient conifer family Araucariaceae is often represented in fossil floras and may act as a suitable proxy of palaeo-[CO(2)], yet little is known regarding the stomatal index (SI) responses of extant Araucariaceae to [CO(2)]. Four Araucaria species (Araucaria columnaris, A. heterophylla, A. angustifolia and A. bidwillii) and Agathis australis displayed no significant relationship in SI to [CO(2)] below current ambient levels (~380 ppm). However, representatives of the three extant genera within the Araucariaceae (A. bidwillii, A. australis and Wollemia nobilis) all exhibited significant reductions in SI when grown in atmospheres of elevated [CO(2)] (1,500 ppm). Stomatal conductance was reduced and WUE increased when grown under elevated [CO(2)]. Stomatal pore length did not increase alongside reduced stomatal density (SD) and SI in the three araucariacean conifers when grown at elevated [CO(2)]. These pronounced SD and SI reductions occur at higher [CO(2)] levels than in other species with more recent evolutionary origins, and may reflect an evolutionary legacy of the Araucariaceae in the high [CO(2)] world of the Mesozoic Era. Araucariacean conifers may therefore be suitable stomatal proxies of palaeo-[CO(2)] during periods of "greenhouse" climates and high [CO(2)] in the Earth's history.

Stomatal index responses of Agrostis canina to CO2 and sulphur dioxide: implications for palaeo-[CO2] using the stomatal proxy.

• Stomatal index values of fossil plants are widely used in reconstructing palaeo-[CO(2)]. This depends upon the assumption that the stomatal index is determined by the atmospheric concentration of CO(2) ([CO(2)]). This study investigates whether fumigation with, and resistance to, sulphur dioxide (SO(2)) induces a reduction in the stomatal index that may affect stomatal reconstructions of palaeo-[CO(2)] coinciding with episodes of global-scale volcanism. • Agrostis canina from Mefite di Ansanto, Italy, grow in atmospheres of elevated-[CO(2)], SO(2) and hydrogen sulphide (H(2)S). Mefite A. canina were compared with a control population in a 'common-garden' experiment and a controlled-environment study under elevated-[CO(2)] and SO(2) fumigation. • In A. canina, resistance to toxic volcanic gases is not associated with reduced stomatal index, and fumigation with SO(2) does not cause a decrease in stomatal initiation. The two populations of A. canina analyzed in this study exhibit different stomatal index-[CO(2)] 'responses', with control plants showing a reduction in stomatal index and Mefite plants showing no response. • Stomatal reconstructions of palaeo-[CO(2)] during past episodes of global-scale volcanism probably reflect atmospheric [CO(2)] and not [SO(2)]. The lack of a reduction in the stomatal index in response to elevated [CO(2)] in the Mefite plants, suggests that resistance to toxic gases and/or long-term growth at high [CO(2)] reduces, or negates, sensitivity of the stomatal index-[CO(2)] relationship, or that stomatal index-[CO(2)] in the Mefite plants is attuned to [CO(2)] fluctuations at much higher concentrations.