Reponses of morphological and biochemical traits of bamboo trees under elevated atmospheric O3 enrichment.

Dwarf bamboo (Indocalamus decorus) is an O3-tolerant plant species. To identify the possible mechanism and response of leaf morphological, antioxidant, and anatomical characteristics to elevated atmospheric O3 (EO3) concentrations, we exposed three-year-old I. decorus seedlings to three O3 levels (low O3-LO: ambient air; medium O3-MO: Ambient air+70 ppb high O3-HO: Ambient air+140 ppb O3) over a growing season using open-top chambers. Leaf shape and stomatal characteristics, and leaf microscopic structure of I. decorus were examined. The results indicated that 1) the stomata O3 flux (Fst) of HO decreased more rapidly under EO3 as the exposure time increased. The foliar O3 injury of HO and MO occurred when AOT40 was 26.62 ppm h and 33.20 ppm h, respectively, 2) under EO3, leaf number, leaf mass per area, leaf area, and stomata length/width all decreased, while leaf thickness, stomatal density, width, and area increased compared to the control, 3) MDA and total soluble protein contents all showed significantly increase under HO (36.57% and 32.77%) and MO(31.91% and 19.52%) while proline contents only increased under HO(33.27%). 4) MO and HO increased bulliform cells numbers in the leaves by 6.28% and 23.01%, respectively. HO reduced the transverse area of bulliform cells by 13.73%, while MO treatments had no effect, and 5) the number of fusoid cells interspace, the transverse area of fusoid cells interspace, and mesophyll thickness of HO significantly increased by 11.16%, 28.58%, and 13.42%, respectively. In conclusion, I. decorus exhibits strong O3 tolerance characteristics, which stem from adaptions in the leaf's morphological, structural, antioxidant, and anatomical features. One critical attribute was the enlargement of the bulliform cell transverse area and the transverse area of fusoid cells interspace that drove this resistance to O3. Local bamboo species with high resistance to O3 pollution thus need to be promoted for sustained productivity and ecosystem services in areas with high O3 pollution.

How Important Are Functional and Developmental Constraints on Phenotypic Evolution? An Empirical Test with the Stomatal Anatomy of Flowering Plants.

AbstractQuantifying the relative contribution of functional and developmental constraints on phenotypic variation is a long-standing goal of macroevolution, but it is often difficult to distinguish different types of constraints. Alternatively, selection can limit phenotypic (co)variation if some trait combinations are generally maladaptive. The anatomy of leaves with stomata on both surfaces (amphistomatous) present a unique opportunity to test the importance of functional and developmental constraints on phenotypic evolution. The key insight is that stomata on each leaf surface encounter the same functional and developmental constraints but potentially different selective pressures because of leaf asymmetry in light capture, gas exchange, and other features. Independent evolution of stomatal traits on each surface imply that functional and developmental constraints alone likely do not explain trait covariance. Packing limits on how many stomata can fit into a finite epidermis and cell size-mediated developmental integration are hypothesized to constrain variation in stomatal anatomy. The simple geometry of the planar leaf surface and knowledge of stomatal development make it possible to derive equations for phenotypic (co)variance caused by these constraints and compare them with data. We analyzed evolutionary covariance between stomatal density and length in amphistomatous leaves from 236 phylogenetically independent contrasts using a robust Bayesian model. Stomatal anatomy on each surface diverges partially independently, meaning that packing limits and developmental integration are not sufficient to explain phenotypic (co)variation. Hence, (co)variation in ecologically important traits like stomata arises in part because there is a limited range of evolutionary optima. We show how it is possible to evaluate the contribution of different constraints by deriving expected patterns of (co)variance and testing them using similar but separate tissues, organs, or sexes.

Anatomical drivers of stomatal conductance in sorghum lines with different leaf widths grown under different temperatures.

Sustaining crop productivity and resilience in water-limited environments and under rising temperatures are matters of concern worldwide. We investigated the leaf anatomical traits that underpin our recently identified link between leaf width (LW) and intrinsic water use efficiency (iWUE), as traits of interest in plant breeding. Ten sorghum lines with varying LW were grown under three temperatures to expand the range of variation of both LW and gas exchange rates. Leaf gas exchange, surface morphology and cross-sectional anatomy were measured and analysed using structural equations modelling. Narrower leaves had lower stomatal conductance (gs ) and higher iWUE across growth temperatures. They also had smaller intercellular airspaces, stomatal size, percentage of open stomatal aperture relative to maximum, hydraulic pathway, mesophyll thickness, and leaf mass per area. Structural modelling revealed a developmental association among leaf anatomical traits that underpinned gs variation in sorghum. Growing temperature and LW both impacted leaf gas exchange rates, but only LW directly impacted leaf anatomy. Wider leaves may be more productive under well-watered conditions, but consume more water for growth and development, which is detrimental under water stress.

Stomatal morphology and physiology explain varied sensitivity to abscisic acid across vascular plant lineages.

Abscisic acid (ABA) can induce rapid stomatal closure in seed plants, but the action of this hormone on the stomata of fern and lycophyte species remains equivocal. Here, ABA-induced stomatal closure, signaling components, guard cell K+ and Ca2+ fluxes, vacuolar and actin cytoskeleton dynamics, and the permeability coefficient of guard cell protoplasts (Pf) were analyzed in species spanning the diversity of vascular land plants including 11 seed plants, 6 ferns, and 1 lycophyte. We found that all 11 seed plants exhibited ABA-induced stomatal closure, but the fern and lycophyte species did not. ABA-induced hydrogen peroxide elevation was observed in all species, but the signaling pathway downstream of nitric oxide production, including ion channel activation, was only observed in seed plants. In the angiosperm faba bean (Vicia faba), ABA application caused large vacuolar compartments to disaggregate, actin filaments to disintegrate into short fragments and Pf to increase. None of these changes was observed in the guard cells of the fern Matteuccia struthiopteris and lycophyte Selaginella moellendorffii treated with ABA, but a hypertonic osmotic solution did induce stomatal closure in fern and the lycophyte. Our results suggest that there is a major difference in the regulation of stomata between the fern and lycophyte plants and the seed plants. Importantly, these findings have uncovered the physiological and biophysical mechanisms that may have been responsible for the evolution of a stomatal response to ABA in the earliest seed plants.

Stomatal density in Pinus sylvestris as an indicator of temperature rather than CO2 : Evidence from a pan-European transect.

The commonly observed negative relationship between stomatal density (SD) and atmospheric CO2 has led to SD being proposed as an indicator of atmospheric CO2 concentration. The use of SD as a proxy for CO2 , however, has been hampered by an insufficient understanding of the intraspecific variation of this trait. We hypothesized that SD in Pinus sylvestris, a widely distributed conifer, varies geographically and that this variation is determined by major climatic variables. By sampling needles from naturally growing trees along a latitudinal range of 32.25°, equivalent to 13.7°C gradient of mean annual temperature (MAT) across Europe, we found that SD decreased from the warmest southern sites to the coldest sites in the north at a rate of 4 stomata per mm2 for each 1°C, with MAT explaining 44% of the variation. Additionally, samples from a provenance trial exhibited a positive relationship between SD and the MAT of the original localities, suggesting that high SD is an adaptation to warm temperature. Our study revealed one of the strongest intraspecific relationships between SD and climate in any woody species, supporting the utility of SD as a temperature, rather than direct CO2 , proxy. In addition, our results predict the response of SD to climate warming.

The influence of stomatal morphology and distribution on photosynthetic gas exchange.

The intricate and interconnecting reactions of C3 photosynthesis are often limited by one of two fundamental processes: the conversion of solar energy into chemical energy, or the diffusion of CO2 from the atmosphere through the stomata, and ultimately into the chloroplast. In this review, we explore how the contributions of stomatal morphology and distribution can affect photosynthesis, through changes in gaseous exchange. The factors driving this relationship are considered, and recent results from studies investigating the effects of stomatal shape, size, density and patterning on photosynthesis are discussed. We suggest that the interplay between stomatal gaseous exchange and photosynthesis is complex, and that a disconnect often exists between the rates of CO2 diffusion and photosynthetic carbon fixation. The mechanisms that allow for substantial reductions in maximum stomatal conductance without affecting photosynthesis are highly dependent on environmental factors, such as light intensity, and could be exploited to improve crop performance.

Form, development and function of grass stomata.

Stomata are cellular breathing pores on leaves that open and close to absorb photosynthetic carbon dioxide and to restrict water loss through transpiration, respectively. Grasses (Poaceae) form morphologically innovative stomata, which consist of two dumbbell-shaped guard cells flanked by two lateral subsidiary cells (SCs). This 'graminoid' morphology is associated with faster stomatal movements leading to more water-efficient gas exchange in changing environments. Here, we offer a genetic and mechanistic perspective on the unique graminoid form of grass stomata and the developmental innovations during stomatal cell lineage initiation, recruitment of SCs and stomatal morphogenesis. Furthermore, the functional consequences of the four-celled, graminoid stomatal morphology are summarized. We compile the identified players relevant for stomatal opening and closing in grasses, and discuss possible mechanisms leading to cell-type-specific regulation of osmotic potential and turgor. In conclusion, we propose that the investigation of functionally superior grass stomata might reveal routes to improve water-stress resilience of agriculturally relevant plants in a changing climate.

Leaf water relations in epiphytic ferns are driven by drought avoidance rather than tolerance mechanisms.

Opportunistic diversification has allowed ferns to radiate into epiphytic niches in angiosperm dominated landscapes. However, our understanding of how ecophysiological function allowed establishment in the canopy and the potential transitionary role of the hemi-epiphytic life form remain unclear. Here, we surveyed 39 fern species in Costa Rican tropical forests to explore epiphytic trait divergence in a phylogenetic context. We examined leaf responses to water deficits in terrestrial, hemi-epiphytic and epiphytic ferns and related these findings to functional traits that regulate leaf water status. Epiphytic ferns had reduced xylem area (-63%), shorter stipe lengths (-56%), thicker laminae (+41%) and reduced stomatal density (-46%) compared to terrestrial ferns. Epiphytic ferns exhibited similar turgor loss points, higher osmotic potential at saturation and lower tissue capacitance after turgor loss than terrestrial ferns. Overall, hemi-epiphytic ferns exhibited traits that share characteristics of both terrestrial and epiphytic species. Our findings clearly demonstrate the prevalence of water conservatism in both epiphytic and hemi-epiphytic ferns, via selection for anatomical and structural traits that avoid leaf water stress. Even with likely evolutionarily constrained physiological function, adaptations for drought avoidance have allowed epiphytic ferns to successfully endure the stresses of the canopy habitat.

Understanding airspace in leaves: 3D anatomy and directional tortuosity.

The leaf intercellular airspace is a tortuous environment consisting of cells of different shapes, packing densities, and orientation, all of which have an effect on the travelling distance of molecules from the stomata to the mesophyll cell surfaces. Tortuosity, the increase in displacement over the actual distance between two points, is typically defined as encompassing the whole leaf airspace, but heterogeneity in pore dimensions and orientation between the spongy and palisade mesophyll likely result in heterogeneity in tortuosity along different axes and would predict longer traveling distance along the path of least tortuosity, such as vertically within the columnar cell matrix of the palisade layer. Here, we compare a previously established geometric method to a random walk approach, novel for this analysis in plant leaves, in four different Eucalyptus species. The random walk method allowed us to quantify directional tortuosity across the whole leaf profile, and separately for the spongy and palisade mesophyll. For all species tortuosity was higher in the palisade mesophyll than the spongy mesophyll and horizontal (parallel to the epidermis) tortuosity was consistently higher than vertical (from epidermis to epidermis) tortuosity. We demonstrate that a random walk approach improves on previous geometric approaches and is valuable for investigating CO2 and H2 O transport within leaves.

Macroscopic variation in Arabidopsis mutants despite stomatal uniformity across soil nutrient environments.

Stomata are essential pores flanked by guard cells that control gas exchange in plants. We can utilize stomatal size and density measurements as a proxy for a plant's capacity for gas exchange. While stomatal responses to stressful environments are well studied; data are lacking in the responses across mutant genotypes of the same species in these trait and treatment interactions or genetic variation in phenotypic plasticity. We evaluated the effects of soil nutrient variation on macroscopic and stomatal traits of Arabidopsis thaliana T-DNA insertion mutants for which prior performance in a single benign growing condition were available. Nutrient-induced stress significantly impacted traits including plant biomass, height, fruit number, and leaf number which we denote as macroscopic traits. We found evidence that genotype by environment effects exist for macroscopic traits, yet total stomatal area variation, or "microscopic variation" across environments was modest. Divergence from the wildtype line varied by mutant background and these responses were variable among traits. These findings suggest that Arabidopsis employs a strategy of physiological compensation, sacrificing morphological traits to maintain stomatal production.

Exogenous 1',4'-trans-Diol-ABA Induces Stress Tolerance by Affecting the Level of Gene Expression in Tobacco (Nicotiana tabacum L.).

1',4'-trans-diol-ABA is a key precursor of the biosynthesis of abscisic acid (ABA) biosynthesis in fungi. We successfully obtained the pure compound from a mutant of Botrytis cinerea and explored its function and possible mechanism on plants by spraying 2 mg/L 1',4'-trans-diol-ABA on tobacco leaves. Our results showed that this compound enhanced the drought tolerance of tobacco seedlings. A comparative transcriptome analysis showed that a large number of genes responded to the compound, exhibiting 1523 genes that were differentially expressed at 12 h, which increased to 1993 at 24 h and 3074 at 48 h, respectively. The enrichment analysis demonstrated that the differentially expressed genes (DEGs) were primarily enriched in pathways related to hormones and resistance. The DEGs of transcription factors were generally up-regulated and included the bHLH, bZIP, ERF, MYB, NAC, WRKY and HSF families. Moreover, the levels of expression of PYL/PYR, PP2C, SnRK2, and ABF at the ABA signaling pathway responded positively to exogenous 1',4'-trans-diol-ABA. Among them, seven ABF transcripts that were detected were significantly up-regulated. In addition, the genes involved in salicylic acid, ethylene and jasmonic acid pathways, reactive oxygen species scavenging system, and other resistance related genes were primarily induced by 1',4'-trans-diol-ABA. These findings indicated that treatment with 1',4'-trans-diol-ABA could improve tolerance to plant abiotic stress and potential biotic resistance by regulating gene expression, similar to the effects of exogenous ABA.

Rafflesia patma Blume flower organs: histology of the epidermis and vascular structures, and a search for stomata.

MAIN CONCLUSION: A histological study of Rafflesia patma revealed the simplicity of a flower's vascular tissue and epidermal features of flower organs, including their structures and pigmentation. Rafflesia is an endophytic holoparasitic plant that infects Tetrastigma. In a previous study, we characterized the shape of the strands of an endophyte (Rafflesia patma Blume) and hypothesized their distribution. In this study, we deepened our analysis by assessing parts of flower tissue sampled during anthesis, performed surface casting of the abaxial and adaxial sides of the perigone lobe to profile their surface features, and histologically characterized the perigone lobe, perigone tube, and central column base, including the anther and cupula region. The objective of these observations was to compare tissues from different organs and the distribution of cells staining positive for tannin, suberin, and lignin. Observable features in this study were vascular and epidermal tissue. We also observed reduced vascular tissue with xylem and vascular parenchyma in multiple organs. The adaxial epidermis found in the perigone lobes and tube had papillate cells, and their function might be to assist with the emission of odor through chemical evaporation. The abaxial epidermis, also found in perigone lobes and tube, had flattened cells. These, combined with the nearby flattened parenchyma cells, especially in the outermost, early perigone lobe, might provide a tougher (stiffer) outer protective barrier for the flower. The accumulation of tannin in perigone lobes might offer protection to the flower from herbivores prior to anthesis. Although a previous observation indicated the possibility of stomata on the surface of Rafflesia flowers, no stomata were found in this study.

Increased adaxial stomatal density is associated with greater mesophyll surface area exposed to intercellular air spaces and mesophyll conductance in diverse C4 grasses.

Mesophyll conductance (gm ) is the diffusion of CO2 from intercellular air spaces (IAS) to the first site of carboxylation in the mesophyll cells. In C3 species, gm is influenced by diverse leaf structural and anatomical traits; however, little is known about traits affecting gm in C4 species. To address this knowledge gap, we used online oxygen isotope discrimination measurements to estimate gm and microscopy techniques to measure leaf structural and anatomical traits potentially related to gm in 18 C4 grasses. In this study, gm scaled positively with photosynthesis and intrinsic water-use efficiency (TEi ), but not with stomatal conductance. Also, gm was not determined by a single trait but was positively correlated with adaxial stomatal densities (SDada ), stomatal ratio (SR), mesophyll surface area exposed to IAS (Smes ) and leaf thickness. However, gm was not related to abaxial stomatal densities (SDaba ) and mesophyll cell wall thickness (TCW ). Our study suggests that greater SDada and SR increased gm by increasing Smes and creating additional parallel pathways for CO2 diffusion inside mesophyll cells. Thus, SDada , SR and Smes are important determinants of C4 -gm and could be the target traits selected or modified for achieving greater gm and TEi in C4 species.

Are stomata in ferns and allies sluggish? Stomatal responses to CO2 , humidity and light and their scaling with size and density.

Fast stomatal reactions enable plants to successfully cope with a constantly changing environment yet there is an ongoing debate on the stomatal regulation mechanisms in basal plant groups. We measured stomatal morphological parameters in 29 fern and allied species from temperate to tropical biomes and two outgroup angiosperm species. Stomatal dynamic responses to environmental drivers were measured in 16 ferns and the two angiosperms using a gas-exchange system. Principal components analyses were used to further reveal the structure-function relationships in stomata. We show a > 10-fold variation for stomatal opening delays and 20-fold variation for stomatal closing delays in ferns. Across species, stomatal responses to vapor pressure deficit (VPD) were the fastest, while light and [CO2 ] responses were slower. In most cases the outgroup species' reaction speeds to changes in environmental variables were similar to those of ferns. Correlations between stomatal response rate and size were apparent for stomatal opening in light and low [CO2 ] while not evident for closing reactions and changes in VPD. No correlations between stomatal density and response speed were observed. Together, this study demonstrates different mechanisms controlling stomatal reactions in ferns at different environmental stimuli, which should be considered in future studies relating stomatal morphology and function.

Allometric co-variation of xylem and stomata across diverse woody seedlings.

Leaf stomatal density is known to co-vary with leaf vein density. However, the functional underpinning of this relation, and how it scales to whole-plant water transport anatomy, is still unresolved. We hypothesized that the balance of water exchange between the vapour phase (in stomata) and liquid phase (in vessels) depends on the consistent scaling between the summed stomatal areas and xylem cross-sectional areas, both at the whole-plant and single-leaf level. This predicted size co-variation should be driven by the co-variation of numbers of stomata and terminal vessels. We examined the relationships of stomatal traits and xylem anatomical traits from the entire plant to individual leaves across seedlings of 53 European woody angiosperm species. There was strong and convergent scaling between total stomatal area and stem xylem area per plant and between leaf total stomatal area and midvein xylem area per leaf across all the species, irrespective of variation in leaf habit, growth-form or relative growth rate. Moreover, strong scaling was found between stomatal number and terminal vessel number, whereas not in their respective average areas. Our findings have broad implications for integrating xylem architecture and stomatal distribution and deepen our understanding of the design rules of plants' water transport network.

SlTLFP8 reduces water loss to improve water-use efficiency by modulating cell size and stomatal density via endoreduplication.

Improving plant water-use efficiency (WUE) is important to plant survival and crop yield in the context of water limitation. In this study, SlTLFP8 (Tubby-like F-box protein 8) was identified as an osmotic-induced gene in tomato. Transgenic tomato with up-regulated expression of SlTLFP8 showed enhanced water-deficient resistance, whereas knockout mutants generated by CRISPR/Cas9 were more sensitive to water deficit. SlTLFP8 overexpression significantly enhanced WUE by suppressing transpiration under both water-sufficient and water-deficient conditions. Further study showed that overexpressing SlTLFP8 significantly increased leaf epidermal cell size and thereby decreased stomatal density 10-20%, conversely SlTLFP8 knockout resulted in decreased cell size and thereby increased stomatal density 20-50%. SlTLFP8 overexpression and knockout modulated ploidy levels in leaf cells. Changes in expression of cell cycle related genes also indicated that SlTLFP8 affected cell size and stomatal density through endocycle transition. Despite changes in stomata density and transpiration, altering the expression of SlTLFP8 did not change photosynthesis. Additionally, biomass was not altered and there was little difference in fruit yield for transgenic and wild type lines under water-sufficient and water-deficient conditions. Our results demonstrate the effect of SlTLFP8 on endoreduplication and the potential of SlTLFP8 for improvement of WUE. BRIEF SUMMERY: This work found a new mechanism of TLP (Tubby like protein) response to water-deficient stress. SlTLFP8, a member of TLP family, regulates water-deficient resistance by modulating water loss via affecting stomatal density. Expression of SlTLFP8 was induced by osmotic stress. Transgenic tomato lines with SlTLFP8 overexpression or SlTLFP8 knockout showed significantly differences in water-use efficiency (WUE) and water-deficient resistance. The difference of leaf water loss caused by transpiration is the main explanation of the difference in WUE and water-deficient resistance. Additionally, overexpressing SlTLFP8 significantly decreased stomatal density, while SlTLFP8 knockout resulted in increased stomatal density, and SlTLFP8 affected stomatal density through endoreduplication and altered epidermal cell size. Despite changes in stomata density, altering the expression of SlTLFP8 did not result in distinct changes in photosynthesis, biomass and yield of tomato.

The ratio of trichomes to stomata is associated with water use efficiency in Solanum lycopersicum (tomato).

Trichomes are specialised structures that originate from the aerial epidermis of plants, and play key roles in the interaction between the plant and the environment. In this study we investigated the trichome phenotypes of four lines selected from the Solanum lycopersicum × Solanum pennellii introgression line (IL) population for differences in trichome density, and their impact on plant performance under water-deficit conditions. We performed comparative analyses at morphological and photosynthetic levels of plants grown under well-watered (WW) and also under water-deficit (WD) conditions in the field. Under WD conditions, we observed higher trichome density in ILs 11-3 and 4-1, and lower stomatal size in IL 4-1 compared with plants grown under WW conditions. The intrinsic water use efficiency (WUEi ) was higher under WD conditions in IL 11-3, and the plant-level water use efficiency (WUEb ) was also higher in IL 11-3 and in M82 for WD plants. The ratio of trichomes to stomata (T/S) was positively correlated with WUEi and WUEb , indicating an important role for both trichomes and stomata in drought tolerance in tomato, and offering a promising way to select for improved water use efficiency of major crops.

Elevated CO2 concentration induces photosynthetic down-regulation with changes in leaf structure, non-structural carbohydrates and nitrogen content of soybean.

BACKGROUND: Understanding the mechanisms of crops in response to elevated CO2 concentrations is pivotal to estimating the impacts of climate change on the global agricultural production. Based on earlier results of the "doubling-CO2 concentration" experiments, many current climate models may overestimate the CO2 fertilization effect on crops, and meanwhile, underestimate the potential impacts of future climate change on global agriculture ecosystem when the atmospheric CO2 concentration goes beyond the optimal levels for crop growth. RESULTS: This study examined the photosynthetic response of soybean (Glycine max (L.) Merr.) to elevated CO2 concentration associated with changes in leaf structure, non-structural carbohydrates and nitrogen content with environmental growth chambers where the CO2 concentration was controlled at 400, 600, 800, 1000, 1200, 1400, 1600 ppm. We found CO2-induced down-regulation of leaf photosynthesis as evidenced by the consistently declined leaf net photosynthetic rate (An) with elevated CO2 concentrations. This down-regulation of leaf photosynthesis was evident in biochemical and photochemical processes since the maximum carboxylation rate (Vcmax) and the maximum electron transport rate (Jmax) were dramatically decreased at higher CO2 concentrations exceeding their optimal values of about 600 ppm and 400 ppm, respectively. Moreover, the down-regulation of leaf photosynthesis at high CO2 concentration was partially attributed to the reduced stomatal conductance (Gs) as demonstrated by the declines in stomatal density and stomatal area as well as the changes in the spatial distribution pattern of stomata. In addition, the smaller total mesophyll size (palisade and spongy tissues) and the lower nitrogen availability may also contribute to the down-regulation of leaf photosynthesis when soybean subjected to high CO2 concentration environment. CONCLUSIONS: Down-regulation of leaf photosynthesis associated with the changes in stomatal traits, mesophyll tissue size, non-structural carbohydrates, and nitrogen availability of soybean in response to future high atmospheric CO2 concentration and climate change.

Speedy stomata, photosynthesis and plant water use efficiency.

Contents Summary 93 I. Introduction 93 II. Influence of the speed of gs responses on A and Wi 93 III. Determinants of the rapidity of gs responses 95 IV. Conclusion 97 Acknowledgements 97 References 97 SUMMARY: Stomatal movements control CO2 uptake for photosynthesis and water loss through transpiration, and therefore play a key role in plant productivity and water use efficiency. The predicted doubling of global water usage by 2030 mean that stomatal behaviour is central to current efforts to increase photosynthesis and crop yields, particularly under conditions of reduced water availability. In the field, slow stomatal responses to dynamic environmental conditions add a temporal dimension to gaseous fluxes between the leaf and atmosphere. Here, we review recent work on the rapidity of stomatal responses and present some of the possible anatomical and biochemical mechanisms that influence the rapidity of stomatal movements.

StomataCounter: a neural network for automatic stomata identification and counting.

Stomata regulate important physiological processes in plants and are often phenotyped by researchers in diverse fields of plant biology. Currently, there are no user-friendly, fully automated methods to perform the task of identifying and counting stomata, and stomata density is generally estimated by manually counting stomata. We introduce StomataCounter, an automated stomata counting system using a deep convolutional neural network to identify stomata in a variety of different microscopic images. We use a human-in-the-loop approach to train and refine a neural network on a taxonomically diverse collection of microscopic images. Our network achieves 98.1% identification accuracy on Ginkgo scanning electron microscropy micrographs, and 94.2% transfer accuracy when tested on untrained species. To facilitate adoption of the method, we provide the method in a publicly available website at http://www.stomata.science/.