Classical phenotyping and deep learning concur on genetic control of stomatal density and area in sorghum

Stomatal density (SD) and stomatal complex area (SCA) are important traits that regulate gas exchange and abiotic stress response in plants. Despite sorghum (Sorghum bicolor) adaptation to arid conditions, the genetic potential of stomata-related traits remains unexplored due to challenges in available phenotyping methods. Hence, identifying loci that control stomatal traits is fundamental to designing strategies to breed sorghum with optimized stomatal regulation. We implemented both classical and deep learning methods to characterize genetic diversity in 311 grain sorghum accessions for stomatal traits at two different field environments. Nearly 12,000 images collected from abaxial (Ab) and adaxial (Ad) leaf surfaces revealed substantial variation in stomatal traits. Our study demonstrated significant accuracy between manual and deep learning methods in predicting SD and SCA. In sorghum, SD was 32%–39% greater on the Ab versus the Ad surface, while SCA on the Ab surface was 2%–5% smaller than on the Ad surface. Genome-Wide Association Study identified 71 genetic loci (38 were environment-specific) with significant genotype to phenotype associations for stomatal traits. Putative causal genes underlying the phenotypic variation were identified. Accessions with similar SCA but carrying contrasting haplotypes for SD were tested for stomatal conductance and carbon assimilation under field conditions. Our findings provide a foundation for further studies on the genetic and molecular mechanisms controlling stomata patterning and regulation in sorghum. An integrated physiological, deep learning, and genomic approach allowed us to unravel the genetic control of natural variation in stomata traits in sorghum, which can be applied to other plants.

High-throughput phenotyping using deep learning tools integrated with genome-wide association studies revealed genes that control SD and area in grain sorghum.     

The physiological basis for genetic variation in water use efficiency and carbon isotope composition in Arabidopsis thaliana

Ecologists and physiologists have documented extensive variation in water use efficiency (WUE) in Arabidopsis thaliana, as well as association of WUE with climatic variation. Here, we demonstrate correlations of whole-plant transpiration efficiency and carbon isotope composition (δ¹³C) among life history classes of A. thaliana. We also use a whole-plant cuvette to examine patterns of co-variation in component traits of WUE and δ¹³C. We find that stomatal conductance (g _s) explains more variation in WUE than does A. Overall, there was a strong genetic correlation between A and g _s, consistent with selection acting on the ratio of these traits. At a more detailed level, genetic variation in A was due to underlying variation in both maximal rate of carboxylation (V _cmax) and maximum electron transport rate (Jmax). We also found strong effects of leaf anatomy, where lines with lower WUE had higher leaf water content (LWC) and specific leaf area (SLA), suggesting a role for mesophyll conductance (g _m) in variation of WUE. We hypothesize that this is due to an effect through g _m, and test this hypothesis using the abi4 mutant. We show that mutants of ABI4 have higher SLA, LWC, and g _m than wild-type, consistent with variation in leaf anatomy causing variation in g _m and δ¹³C. These functional data also add further support to the central, integrative role of ABI4 in simultaneously altering ABA sensitivity, sugar signaling, and CO₂ assimilation. Together our results highlight the need for a more holistic approach in functional studies, both for more accurate annotation of gene function and to understand co-limitations to plant growth and productivity.     

Comparison of arabidopsis stomatal density mutants indicates variation in water stress responses and potential epistatic effects

Recent physiological analysis of Arabidopsis stomatal density (SD) mutants indicated that SD was not the major factor controlling aboveground biomass accumulation. Despite the general theory that plants with fewer stomata have limited biomass acquisition capabilities, epf1 and several other Arabidopsis mutants varied significantly in leaf fresh weight despite having similar stomatal numbers. The indepth mechanisms controlling increased or decreased leaf area biomass remain undetermined. This work used calculations of SD, overnight water-loss, and LI6400XT measurements to reject the premise that SD is a primary factor controlling leaf biomass accumulation in Arabidopsis. With respect to our data, SD is not the primary factor influencing biomass accumulation in Arabidopsis epf1 mutants as it did not positively correlate to any of the physiological parameters examined. Further observation of morphological differences between the mutants hinted that additional pathways were interrupted when these mutants were generated. Each mutant examined showed a variation in physiological measurements despite SD. Many SD mutants also showed morphological abnormalities in addition to altered stomatal numbers. These phenotypes may indicate epistatic effects related to the mutation of SD genes in the studied mutants.     

Plasticity in stomatal size and density of potato leaves under different irrigation and phosphorus regimes

The morphological features of stomata including their size and density could be modulated by environmental cues; however, the underlying mechanisms remain largely elusive. Here, the effect of different irrigation and phosphorus (P) regimes on stomatal size (SS) and stomatal density (SD) of potato leaves was investigated. The plants were grown in split-root pots under two P fertilization rates (viz., 0 and 100 mg kg⁻¹ soil, denoted as P0 and P1, respectively) and subjected to full (FI), deficit (DI), and partial root-zone drying (PRD) irrigation regimes. Results showed that SS and SD were unresponsive to P but significantly affected by the irrigation treatment. FI plants had the largest SS, followed by DI, and PRD the smallest; and the reverse was the case for SD. Compared to FI and DI, PRD plants had significantly lower values of specific leaf area (SLA) and leaf carbon isotope discrimination (Δ¹³C) under P0. Midday leaf water potential (Ψ_leaf) and stomatal conductance (g_s) was similar for DI and PRD, which was significantly lower than that of FI. Leaf contents of C, N, K, Ca and Mg were higher in PRD than in DI plants, particularly under P0. When analyzed across the three irrigation regimes, it was found that the P1 plants had significantly higher leaf contents of P and Mg, but significantly lower leaf K content compared to the P0 plants. Linear correlation analyses revealed that SS was positively correlated with Ψ_leaf and Δ¹³C; whereas SD was negatively correlated with Ψ_leaf, Δ¹³C and SLA, and positively correlated with leaf C, N and Ca contents. And g_s was positively correlated with SS but negatively correlated with SD. Collectively, under low P level, the smaller and denser stomata in PRD plants may bring about a more efficient stomatal control over gas exchange, hereby potentially enhance water-use efficiency as exemplified by the lowered leaf Δ¹³C under fluctuating soil moisture conditions.     

Transpiration and stomatal control: a cross-species study of leaf traits in 39 evergreen and deciduous broadleaved subtropical tree species

Key message

Using an extensive dataset for 39 subtropical broadleaved tree species, we found traits of the leaf economics spectrum to be linked to mean stomatal conductance but not to stomatal regulation.

Abstract

The aim of our study was to establish links between stomatal control and functional leaf traits. We hypothesized that mean and maximum stomatal conductance (g _s) varies with the traits described by the leaf economics spectrum, such as specific leaf area and leaf dry matter content, and that high g _s values correspond to species with tender leaves and high photosynthetic capacity. In addition, we hypothesized that species with leaves of low stomata density have more limited stomatal closure than those with high stomata density. In order to account for confounding site condition effects, we made use of a common garden situation in which 39 deciduous and evergreen species of the same age were grown in a biodiversity ecosystem functioning experiment in Jiangxi (China). Daily courses of g _s were measured with porometry, and the species-specific g _s~vpd relationships were modeled. Our results show that mean stomatal conductance can be predicted from leaf traits that represent the leaf economics spectrum, with a positive relationship being related to leaf nitrogen content and a negative relationship with the leaf carbon: nitrogen ratio. In contrast, parameters of stomatal control were related to traits unassociated with the leaf economics spectrum. The maximum of the conductance~vpd curve was positively related to leaf carbon content and vein length. The vpd at the point of inflection of the conductance~vpd curve was lower for species with higher stomata density and higher for species with a high leaf carbon content. Overall, stomata size and density as well as vein length were more effective at explaining stomatal regulation than traits used in the leaf economics spectrum.     

Association genetics,geography and ecophysiology link stomatal patterning in Populus trichocarpa with carbon gain and disease resistance trade‐offs

Stomata are essential for diffusive entry of gases to support photosynthesis, but may also expose internal leaf tissues to pathogens. To uncover trade‐offs in range‐wide adaptation relating to stomata, we investigated the underlying genetics of stomatal traits and linked variability in these traits with geoclimate, ecophysiology, condensed foliar tannins and pathogen susceptibility in black cottonwood (Populus trichocarpa). Upper (adaxial) and lower (abaxial) leaf stomatal traits were measured from 454 accessions collected throughout much of the species range. We calculated broad‐sense heritability (H²) of stomatal traits and, using SNP data from a 34K Populus SNP array, performed a genome‐wide association studies (GWAS) to uncover genes underlying stomatal trait variation. H² values for stomatal traits were moderate (average H² = 0.33). GWAS identified genes associated primarily with adaxial stomata, including polarity genes (PHABULOSA), stomatal development genes (BRASSINOSTEROID‐INSENSITIVE 2) and disease/wound‐response genes (GLUTAMATE‐CYSTEINE LIGASE). Stomatal traits correlated with latitude, gas exchange, condensed tannins and leaf rust (Melampsora) infection. Latitudinal trends of greater adaxial stomata numbers and guard cell pore size corresponded with higher stomatal conductance (g_s) and photosynthesis (A_max), faster shoot elongation, lower foliar tannins and greater Melampsora susceptibility. This suggests an evolutionary trade‐off related to differing selection pressures across the species range. In northern environments, more adaxial stomata and larger pore sizes reflect selection for rapid carbon gain and growth. By contrast, southern genotypes have fewer adaxial stomata, smaller pore sizes and higher levels of condensed tannins, possibly linked to greater pressure from natural leaf pathogens, which are less significant in northern ecosystems.     

Do litter manipulations affect leaf functional traits of savanna woody plants?

Plant litter is the layer composed of dead plant material that covers soil surfaces in terrestrial ecosystems. It is an important pool of essential nutrients for soil and plants, serving also as a protective layer on the soil surface. In this study, we investigated the effects of litter addition and removal on leaf functional traits of woody Neotropical savanna trees. We measured maximum photosynthesis (A _max), stomatal conductance (g _s), leaf transpiration (E), intrinsic water use efficiency (IWUE), specific leaf area (SLA), and chlorophyll content (CCI) in 15 species belonging to three different phenological groups (evergreen, briefly deciduous, and deciduous species) that were subjected to three distinct litter availability treatments (addition, removal, and control plots) in a Neotropical Savanna site in Brazil. Although SLA and CCI differed among phenology groups, they were not affected by the litter treatments. In contrast, when considered at the community level, we found that the availability of litter affected the leaf traits linked with the water status of the plants (E, g _s and IWUE). Plants in the litter removal plots exhibited lower g _s and E (25 % of reduction in comparison with control group) but higher IWUE, while plants in the litter addition plots had a 10 % decrease in IWUE but a 12 % increase in g _s and E compared with plants in control plots. Savanna woody plants responded promptly to litter manipulation by adjusting leaf water loss, which suggests that in the short term, changes in the amount of litter in Cerrado ecosystems can affect the soil water availability to the plant community.     

Diurnal and seasonal variations in photosynthetic and morphological traits of the tree ferns Dicksonia antarctica (Dicksoniaceae) and Cyathea australis (Cyatheaceae) in wet sclerophyll forests of Australia

Steady state and dynamic responses of two tree fern species of contrasting origins, Dicksonia antarctica (of Gondwanan origin) and Cyathea australis (Pan-tropical), were studied over two consecutive years under field conditions in a wet sclerophyll forest of south-east Australia. Irrespective of their different origins, there were no significant differences in photosynthetic performance between the two species. Growth irradiance and leaf temperature, but not plant water status, was significantly related to photosynthetic and morphological traits. At a common leaf temperature, maximum light-use efficiency of photosystem II (F_v/F_m) was significantly lower in winter than in summer, suggesting some limitation to PSII efficiency potentially associated with cold winter mornings. Both species displayed seasonal acclimation in a number of measured photosynthetic parameters and frond traits (i.e. F_v/F_m, A_sat, g_s, N_A, total chlorophyll, SLA). Acclimation of stomatal density to spatial variation in growth irradiance seemed limited in both species, although stomatal pattern differed between species. Because there were no significant differences between the two species in photosynthetic parameters, both species can be described by common carbon gain and water use models at the leaf scale.     

Stem growth habit affects leaf morphology and gas exchange traits in soybean

Backgrounds and Aims

The stem growth habit, determinate or indeterminate, of soybean, Glycine max, varieties affects various plant morphological and developmental traits. The objective of this study is to identify the effect of stem growth habit in soybean on the stomatal conductance of single leaves in relation to their leaf morphology in order to better understand the ecological and agronomic significance of this plant trait.

Methods

The stomatal conductance of leaves on the main stem was measured periodically under favourable field conditions to evaluate g_max, defined as the maximum stomatal conductance at full leaf expansion, for four varieties of soybean and their respective determinate or indeterminate near isogenic lines (NILs). Leaf morphological traits including stomatal density, guard cell length and vein density were also measured.

Key Results

The value of g_max ranged from 0·383 to 0·754 mol H₂O m⁻² s⁻¹ across all the genotypes for both years. For the four pairs of varieties, the indeterminate lines exhibited significantly greater g_max, stomatal density, numbers of epidermal cells per unit area and total vein length per unit area than their respective determinate NILs in both years. The guard cell length, leaf mass per area and single leaf size all tended to be greater in the determinate types. The variation of g_max across genotypes and years was well explained by the product of stomatal density and guard cell length (r = 0·86, P < 0·01).

Conclusions

The indeterminate stem growth habit resulted in a greater maximum stomatal conductance for soybean than the determinate habit, and this was attributed to the differences in leaf structure. This raises the further hypothesis that the difference in stem growth habit results in different water use characteristics of soybean plants in the field. Stomatal conductance under favourable conditions can be modified by leaf morphological traits.Key words: Soybean, Glycine max, stem growth habit, stomatal conductance, stomatal density, guard cell length, near isogenic lines     

Leaf Photosynthetic Rate of Tropical Ferns Is Evolutionarily Linked to Water Transport Capacity

Ferns usually have relatively lower photosynthetic potential than angiosperms. However, it is unclear whether low photosynthetic potential of ferns is linked to leaf water supply. We hypothesized that there is an evolutionary association of leaf water transport capacity with photosynthesis and stomatal density in ferns. In the present study, a series of functional traits relating to leaf anatomy, hydraulics and physiology were assessed in 19 terrestrial and 11 epiphytic ferns in a common garden, and analyzed by a comparative phylogenetics method. Compared with epiphytic ferns, terrestrial ferns had higher vein density (D_vein), stomatal density (SD), stomatal conductance (g_s), and photosynthetic capacity (A_max), but lower values for lower epidermal thickness (LET) and leaf thickness (LT). Across species, all traits varied significantly, but only stomatal length (SL) showed strong phylogenetic conservatism. A_max was positively correlated with D_vein and g_s with and without phylogenetic corrections. SD correlated positively with A_max, D_vein and g_s, with the correlation between SD and D_vein being significant after phylogenetic correction. Leaf water content showed significant correlations with LET, LT, and mesophyll thickness. Our results provide evidence that A_max of the studied ferns is linked to leaf water transport capacity, and there was an evolutionary association between water supply and demand in ferns. These findings add new insights into the evolutionary correlations among traits involving carbon and water economy in ferns.     

Optical topometry and machine learning to rapidly phenotype stomatal patterning traits for maize QTL mapping

Stomata are adjustable pores on leaf surfaces that regulate the tradeoff of CO₂ uptake with water vapor loss, thus having critical roles in controlling photosynthetic carbon gain and plant water use. The lack of easy, rapid methods for phenotyping epidermal cell traits have limited discoveries about the genetic basis of stomatal patterning. A high-throughput epidermal cell phenotyping pipeline is presented here and used for quantitative trait loci (QTL) mapping in field-grown maize (Zea mays). The locations and sizes of stomatal complexes and pavement cells on images acquired by an optical topometer from mature leaves were automatically determined. Computer estimated stomatal complex density (SCD; R² = 0.97) and stomatal complex area (SCA; R² = 0.71) were strongly correlated with human measurements. Leaf gas exchange traits were genetically correlated with the dimensions and proportions of stomatal complexes (r_g = 0.39–0.71) but did not correlate with SCD. Heritability of epidermal traits was moderate to high (h² = 0.42–0.82) across two field seasons. Thirty-six QTL were consistently identified for a given trait in both years. Twenty-four clusters of overlapping QTL for multiple traits were identified, with univariate versus multivariate single marker analysis providing evidence consistent with pleiotropy in multiple cases. Putative orthologs of genes known to regulate stomatal patterning in Arabidopsis (Arabidopsis thaliana) were located within some, but not all, of these regions. This study demonstrates how discovery of the genetic basis for stomatal patterning can be accelerated in maize, a C₄ model species where these processes are poorly understood.

Optical topometry and machine learning tools allow assessment of epidermal cell patterning and analysis of its genetic architecture alongside leaf photosynthetic gas exchange in maize.     

Variability in water use efficiency at the leaf level among Mediterranean plants with different growth forms

Assessing natural variability of leaf water use efficiency in plants adapted to extreme conditions of the Mediterranean climate represents an important step in the evaluation of the usefulness of some plant ecophysiological traits under water stress. Eleven Mediterranean species naturally inhabiting the Balearic Islands and corresponding to different growth forms (herbs, semi-deciduous shrubs, woody evergreen shrubs and woody evergreen semi-shrubs) were subject to progressive soil water depletion. Leaf intrinsic water use efficiency was measured by gas exchange at four different degrees of water stress. Under well watered conditions, differences in leaf intrinsic water use efficiency (A _N/g _s) among growth forms were limited to woody evergreen semi-shrubs, which presented the highest values. Under water stress conditions, differences became more evident, with a trend for an increase in A _N/g _s from woody evergreen shrubs, through semi-deciduous shrubs and herbaceous to woody evergreen semi-shrubs. The observed variation in A _N/g _s correlated with several physiological (leaf water potential, soil to leaf hydraulic conductance and stomatal conductance) and morphological (stomatal density) parameters, displaying a general relationship for all growth forms. This suggests that the capacity for withstanding water limitation is adaptive for all Mediterranean species. However, when A _N/g _s was related to leaf mass area, this relationship was not generally applicable, and depended on growth forms, suggesting that different growth forms display specific morphological adjustments in response to water shortage.     

Association mapping of maturity and plant height using SNP markers with the sorghum mini core collection

Plant height and maturity are two critical traits in sorghum breeding. To develop molecular tools and to identify genes underlying the traits for molecular breeding, we developed 14,739 SNP markers used to genotype the complete sorghum [Sorghum bicolor (L.) Moench] mini core collection. The collection was evaluated in four rainy and three post-rainy season environments for plant height and maturity. Association analysis identified six marker loci linked to height and ten to maturity in at least two environments with at least two SNPs in each locus. Of these, 14 were in close proximity to previously mapped height/maturity QTL in sorghum. Candidate genes for maturity or plant height close to the marker loci include a sugar transporter (SbSUC9), an auxin response factor (SbARF3), an FLC and FT regulator (SbMED12), and a photoperiod response gene (SbPPR1) for maturity and peroxidase 53, and an auxin transporter (SbLAX4) for plant height. Linkage disequilibrium analysis showed that SbPPR1 and SbARF3 were in regions with reduced sequence variation among early-maturing accessions, suggestive of past purifying selection. We also found a linkage disequilibrium block that existed only among the accessions with short plant height in rainy season environments. The block contains a gene homologous to the Arabidopsis flowering time gene, LUMINIDEPENDENS (LD). Functional LD promotes early maturity while mutation delays maturity, affecting plant height. Previous studies also found reduced sequence variations within this gene. These newly-mapped SNP markers will facilitate further efforts to identify plant height or maturity genes in sorghum.     

Most stomatal closure in woody species under moderate drought can be explained by stomatal responses to leaf turgor

Reduced stomatal conductance (g_s) during soil drought in angiosperms may result from effects of leaf turgor on stomata and/or factors that do not directly depend on leaf turgor, including root‐derived abscisic acid (ABA) signals. To quantify the roles of leaf turgor‐mediated and leaf turgor‐independent mechanisms in g_s decline during drought, we measured drought responses of g_s and water relations in three woody species (almond, grapevine and olive) under a range of conditions designed to generate independent variation in leaf and root turgor, including diurnal variation in evaporative demand and changes in plant hydraulic conductance and leaf osmotic pressure. We then applied these data to a process‐based g_s model and used a novel method to partition observed declines in g_s during drought into contributions from each parameter in the model. Soil drought reduced g_s by 63–84% across species, and the model reproduced these changes well (r² = 0.91, P < 0.0001, n = 44) despite having only a single fitted parameter. Our analysis concluded that responses mediated by leaf turgor could explain over 87% of the observed decline in g_s across species, adding to a growing body of evidence that challenges the root ABA‐centric model of stomatal responses to drought.     

Differences in leaf gas exchange strategies explain Quercus rubra and Liriodendron tulipifera intrinsic water use efficiency responses to air pollution and climate change

Trees continuously regulate leaf physiology to acquire CO₂ while simultaneously avoiding excessive water loss. The balance between these two processes, or water use efficiency (WUE), is fundamentally important to understanding changes in carbon uptake and transpiration from the leaf to the globe under environmental change. While increasing atmospheric CO₂ (iCO₂) is known to increase tree intrinsic water use efficiency (iWUE), less clear are the additional impacts of climate and acidic air pollution and how they vary by tree species. Here, we couple annually resolved long-term records of tree-ring carbon isotope signatures with leaf physiological measurements of Quercus rubra (Quru) and Liriodendron tulipifera (Litu) at four study locations spanning nearly 100 km in the eastern United States to reconstruct historical iWUE, net photosynthesis (A_net), and stomatal conductance to water (g_s) since 1940. We first show 16%–25% increases in tree iWUE since the mid-20th century, primarily driven by iCO₂, but also document the individual and interactive effects of nitrogen (NO_x) and sulfur (SO₂) air pollution overwhelming climate. We find evidence for Quru leaf gas exchange being less tightly regulated than Litu through an analysis of isotope-derived leaf internal CO₂ (C_i), particularly in wetter, recent years. Modeled estimates of seasonally integrated A_net and g_s revealed a 43%–50% stimulation of A_net was responsible for increasing iWUE in both tree species throughout 79%–86% of the chronologies with reductions in g_s attributable to the remaining 14%–21%, building upon a growing body of literature documenting stimulated A_net overwhelming reductions in g_s as a primary mechanism of increasing iWUE of trees. Finally, our results underscore the importance of considering air pollution, which remains a major environmental issue in many areas of the world, alongside climate in the interpretation of leaf physiology derived from tree rings.     

Leaf gas exchange of trees in old-growth and young secondary forest stands in Sulawesi, Indonesia

In the tropics, old-growth forests are converted to other land cover types at a high rate and young secondary forest may gain in importance. Information on associated changes in leaf gas exchange and other leaf traits can be valuable for modelling biogeochemical fluxes under altered land-use patterns. We studied in situ photosynthetic parameters and stomatal conductance for water vapour in eight abundant tree species of young secondary forest and eight tree species of natural old-growth forest in Central Sulawesi, Indonesia. In sun leaves, the average maximal stomatal conductance (g _smax) in the secondary forest (SF) species was 2.1 times higher than in the old-growth forest (OGF) species. Species with a high g _smax reduced g _s sharply when vapour pressure deficit of the air increased, whereas species with a low g _smax were much less sensitive to air humidity. For area-based photosynthetic capacity (A _max-area), the SF species had a 2.3 times higher average than the OGF species. For both, g _smax and A _max-area the variation among species was higher in the OGF than in the SF. When all tree species (n=16) are considered, species means of specific leaf area (SLA), leaf N concentration and leaf P concentration were significantly correlated with g _smax and A _max-area. The strong correlation between A _max-area and foliar P (r ²=0.8) is remarkable as the alluvial soils in the study region are rich in nutrients. If the eight OGF species are analysed separately, the only significant correlation was observed between SLA and mass-based A _max; in the SF species strong correlations were found between leaf size and A _max-area and g _smax. These results show that the conversion of old-growth forest to young secondary forest in Sulawesi significantly alters tree leaf gas exchange characteristics and that chemical and structural leaf traits can be used for the prediction of these changes. The best correlations between leaf gas exchange parameters and leaf traits were obtained by different traits in the SF species, the OGF species and the entire pool of studied species.     

Variation in climatic tolerance,but not stomatal traits,partially explains Pooideae grass species distributions

Background and AimsGrasses in subfamily Pooideae live in some of the world’s harshest terrestrial environments, from frigid boreal zones to the arid windswept steppe. It is hypothesized that the climate distribution of species within this group is driven by differences in climatic tolerance, and that tolerance can be partially explained by variation in stomatal traits.MethodsWe determined the aridity index (AI) and minimum temperature of the coldest month (MTCM) for 22 diverse Pooideae accessions and one outgroup, and used comparative methods to assess predicted relationships for climate traits versus fitness traits, stomatal diffusive conductance to water (g_w) and speed of stomatal closure following drought and/or cold.Key ResultsResults demonstrate that AI and MTCM predict variation in survival/regreening following drought/cold, and g_w under drought/cold is positively correlated with δ ¹³C-measured water use efficiency (WUE). However, the relationship between climate traits and fitness under drought/cold was not explained by g_w or speed of stomatal closure.ConclusionsThese findings suggest that Pooideae distributions are at least partly determined by tolerance to aridity and above-freezing cold, but that variation in tolerance is not uniformly explained by variation in stomatal traits.     

Limited evidence for CO2‐related growth enhancement in northern Rocky Mountain lodgepole pine populations across climate gradients

Forests sequester large amounts of carbon annually and are integral in buffering against effects of global change. Increasing atmospheric CO₂ may enhance photosynthesis and/or decrease stomatal conductance (g_s) thereby enhancing intrinsic water‐use efficiency (iWUE), having potential indirect and direct benefits to tree growth. While increasing iWUE has been observed in most trees globally, enhanced growth is not ubiquitous, possibly due to concurrent climatic constraints on growth. To investigate our incomplete understanding of interactions between climate and CO₂ and their impacts on tree physiology and growth, we used an environmental gradient approach. We combined dendrochronology with carbon isotope analysis (δ¹³C) to assess the covariation of basal area increment (BAI) and iWUE over time in lodgepole pine. Trees were sampled at 18 sites spanning two climatically distinct elevation transects on the lee and windward sides of the Continental Divide, encompassing the majority of lodgepole pine's northern Rocky Mountain elevational range. We analyzed BAI and iWUE from 1950 to 2015, and explored correlations with monthly climate variables. As expected, iWUE increased at all sites. However, concurrent growth trends depended on site climatic water deficit (CWD). Significant growth increases occurred only at the driest sites, where increases in iWUE were strongest, while growth decreases were greatest at sites where CWD has been historically lowest. Late summer drought of the previous year negatively affected growth across sites. These results suggest that increasing iWUE, if strong enough, may indirectly benefit growth at drier sites by effectively extending the growing season via reductions in g_s. Strong growth decreases at high elevation windward sites may reflect increasing water stress as a result of decreasing snowpack, which was not offset by greater iWUE. Our results imply that increasing iWUE driven by decreasing g_s may benefit tree growth in limited scenarios, having implications for future carbon uptake potential of semiarid ecosystems.     

Differential coordination of stomatal conductance,mesophyll conductance,and leaf hydraulic conductance in response to changing light across species

Stomatal conductance (g_s) and mesophyll conductance (g_m) represent major constraints to photosynthetic rate (A), and these traits are expected to coordinate with leaf hydraulic conductance (K_leaf) across species, under both steady‐state and dynamic conditions. However, empirical information about their coordination is scarce. In this study, K_leaf, gas exchange, stomatal kinetics, and leaf anatomy in 10 species including ferns, gymnosperms, and angiosperms were investigated to elucidate the correlation of H₂O and CO₂ diffusion inside leaves under varying light conditions. Gas exchange, K_leaf, and anatomical traits varied widely across species. Under light‐saturated conditions, the A, g_s, g_m, and K_leaf were strongly correlated across species. However, the response patterns of A, g_s, g_m, and K_leaf to varying light intensities were highly species dependent. Moreover, stomatal opening upon light exposure of dark‐adapted leaves in the studied ferns and gymnosperms was generally faster than in the angiosperms; however, stomatal closing in light‐adapted leaves after darkening was faster in angiosperms. The present results show that there is a large variability in the coordination of leaf hydraulic and gas exchange parameters across terrestrial plant species, as well as in their responses to changing light.