Stomatal regulation in Douglas fir following a fungal-mediated chronic reduction in leaf area

Pathogens can cause chronic premature needle abscission in coniferous species. To assess the potential impacts on tree productivity, stomatal regulation was investigated in Douglas fir with chronic stomatal occlusion and defoliation from varying levels of the Swiss needle cast (SNC) fungus, Phaeocryptopus gaeumannii. Levels of SNC disease and subsequent defoliation were manipulated by choosing six sites with varying levels of disease and by foliar applications of fungicides on six trees per site. Diurnal measurements of leaf water potential (Ψ_leaf), stomatal conductance (g _s) and vapor pressure deficit (D) were made on six fungicide treated and six control trees per site. In addition, leaf specific hydraulic conductance was calculated on a single branch (K _{L_B}) from three trees per treatment per site. Stomatal conductance at D=1 kPa (g _sref) was negatively correlated with fungal colonization (number of fruiting bodies present in needle stomata) and positively correlated with K _{L_B}. Despite reduced needle retention in diseased trees, K _L declined due to a reduction in sapwood area and permeability (i.e., increasing presence of latewood in functional sapwood). In general, stomatal sensitivity to D for all foliage was consistent with stomatal regulation based on a simple hydraulic model [g _s=K _L(Ψ_soil?Ψ_leaf)/ D], which assumes strict stomatal regulation of Ψ_leaf. However, when fungal presence reduced maximum g _s below the potential maximum supported by hydraulic architecture, stomatal sensitivity was lower than expected based on the theoretical relationship: dg _s/dlnD=0.6·g _sref. The results indicate that losses in productivity associated with physical blockage of stomata and defoliation are compounded by additional losses in K _L and a reduction in g _s in remaining functional stomata.     

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     

Stomatal conductance in mature deciduous forest trees exposed to elevated CO<Subscript>2</Subscript>

Stomatal conductance (g _s) of mature trees exposed to elevated CO₂ concentrations was examined in a diverse deciduous forest stand in NW Switzerland. Measurements of g _s were carried out on upper canopy foliage before noon, over four growing seasons, including an exceptionally dry summer (2003). Across all species reductions in stomatal conductance were smaller than 25% most likely around 10%, with much variation among species and trees. Given the large heterogeneity in light conditions within a tree crown, this signal was not statistically significant, but the responses within species were surprisingly consistent throughout the study period. Except during a severe drought, stomatal conductance was always lower in trees of Carpinus betulus exposed to elevated CO₂ compared to Carpinus trees in ambient air, but the difference was only statistically significant on 2 out of 15 days. In contrast, stomatal responses in Fagus sylvatica and Quercus petraea varied around zero with no consistent trend in relation to CO₂ treatment. During the 2003 drought in the third treatment year, the CO₂ effect became reversed in Carpinus, resulting in higher g _s in trees exposed to elevated CO₂ compared to control trees, most likely due to better water supply because of the previous soil water savings. This was supported by less negative predawn leaf water potential in CO₂ enriched Carpinus trees, indicating an improved water status. These findings illustrate (1) smaller than expected CO₂-effects on stomata of mature deciduous forest trees, and (2) the possibility of soil moisture feedback on canopy water relations under elevated CO₂.     

Water loss regulation to soil drought associated with xylem vulnerability to cavitation in temperate ring-porous and diffuse-porous tree seedlings

Key message

Sustainable stomatal opening despite xylem cavitation occurs in ring-porous species and stomatal closure prior to cavitation in diffuse-porous species during soil drought.

Abstract

To elucidate the relationship between water loss regulation and vulnerability to cavitation associated with xylem structure, stomatal conductance (g _s), defoliation, vulnerability curves, and vessel features were measured on seedlings of ring-porous Zelkova serrata and Melia azedarach, and diffuse-porous Betula platyphylla var. japonica, Cerasus jamasakura and Carpinus tschonoskii. Under prolonged drought conditions, the percentage loss of hydraulic conductivity (PLC) increased and g _s decreased gradually with decreasing predawn (Ψ_pd) or xylem water potential (Ψ_xylem) in Z. serrata. During the gentle increase of PLC in M. azedarach, g _s increased in the early stages of dehydration while leaves were partly shed. A sharp reduction in g _s was observed before the onset of an increase in the PLC for drying plants of the three diffuse-porous species, suggesting cavitation avoidance by stomatal regulation. In the ring-porous species, xylem-specific hydraulic conductivity (K _s) was higher, whereas the vessel multiple fractions, the ratio of the number of grouped vessels to total vessels, was lower than that in the diffuse-porous species, suggesting that many were distributed as solitary vessels. This may explain the gradual increase in the PLC with decreasing Ψ_xylem because isolated vessels provide less opportunity for air seeding. Different water loss regulation to soil drought was identified among the species, with potential mechanisms being sustainable gas exchange at the expense of xylem dysfunction or partial leaf shedding, and the avoidance of xylem cavitation by strict stomatal regulation. These were linked to vulnerability to cavitation that appears to be governed by xylem structural properties.     

Evolution of protein transport to the chloroplast envelope membranes

Comparing with other angiosperms, most members within the family Orchidaceae have lower photosynthetic capacities. However, the underlying mechanisms remain unclear. Cypripedium and Paphiopedilum are closely related phylogenetically in Orchidaceae, but their photosynthetic performances are different. We explored the roles of internal anatomy and diffusional conductance in determining photosynthesis in three Cypripedium and three Paphiopedilum species, and quantitatively analyzed their diffusional and biochemical limitations to photosynthesis. Paphiopedilum species showed lower light-saturated photosynthetic rate (A _N), stomatal conductance (g _s), and mesophyll conductance (g _m) than Cypripedium species. A _N was positively correlated with g _s and g _m. And yet, in both species A _N was more strongly limited by g _m than by biochemical factors or g _s. The greater g _s of Cypripedium was mainly affected by larger stomatal apparatus area and smaller pore depth, while the less g _m of Paphiopedilum was determined by the reduced surface area of mesophyll cells and chloroplasts exposed to intercellular airspace per unit of leaf area, and much thicker cell wall thickness. These results suggest that leaf anatomical structure is the key factor affecting g _m, which is largely responsible for the difference in photosynthetic capacity between those two genera. Our findings provide new insight into the photosynthetic physiology and functional diversification of orchids.     

Automatic measurement of stomatal density from microphotographs

Key message

An automated process using a cascade classifier allowed the rapid assessment of the density and distribution of stomata on microphotographs from leaves of two oak species.

Abstract

Stomatal density is the number of stomata per unit area, an intensively studied trait, involved in the control of CO₂ and H₂O exchange between leaf and atmosphere. This trait is usually estimated by counting manually each stoma on a given surface (e.g., a microphotograph), usually repeating the procedure with images from different parts of the leaf. To improve this procedure, we tested the performance of a cascade classifier to automatically detect stomata on microphotographs from two oak species: Quercus afares Pomel and Quercus suber L. The two species are phylogenetically close with similar stomatal morphology, which allowed testing the reuse of the cascade classifier on stomata with similar shape. The results showed that a cascade classifier trained on only 100 sample views of stomata from Q. afares was able to rapidly detect stomata in Q. afares as well as in Q. suber with a very low number of false positives (5 %/1.9 %) and a small number of undetected stomata (14.8 %/0.74 %), when partial stomata near the edge of the microphotographs were ignored. The remaining undetected stomata were due to obstacles such as trichomes. As an example of further applications, we used the positions detected by the cascade classifier to assess the spatial distribution of stomata and group them on the leaf surface. To our knowledge this is the first time that a cascade classifier has been applied to plant microphotographs, and we were able to show that it can dramatically decrease the time needed to estimate stomatal density and spatial distribution.     

Adaptive temperature regulation in the little bird in winter: predictions from a stochastic dynamic programming model

Stomatal CO₂ responsiveness and photosynthetic capacity vary greatly among plant species, but the factors controlling these physiological leaf traits are often poorly understood. To explore if these traits are linked to taxonomic group identity and/or to other plant functional traits, we investigated the short-term stomatal CO₂ responses and the maximum rates of photosynthetic carboxylation (V _cmax) and electron transport (J _max) in an evolutionary broad range of tropical woody plant species. The study included 21 species representing four major seed plant taxa: gymnosperms, monocots, rosids and asterids. We found that stomatal closure responses to increased CO₂ were stronger in angiosperms than in gymnosperms, and in monocots compared to dicots. Stomatal CO₂ responsiveness was not significantly related to any of the other functional traits investigated, while a parameter describing the relationship between photosynthesis and stomatal conductance in combined leaf gas exchange models (g ₁) was related to leaf area-specific plant hydraulic conductance. For photosynthesis, we found that the interspecific variation in V _cmax and J _max was related to within leaf nitrogen (N) allocation rather than to area-based total leaf N content. Within-leaf N allocation and water use were strongly co-ordinated (r ² = 0.67), such that species with high fractional N investments into compounds maximizing photosynthetic capacity also had high stomatal conductance. We conclude that while stomatal CO₂ responsiveness of tropical woody species seems poorly related to other plant functional traits, photosynthetic capacity is linked to fractional within-leaf N allocation rather than total leaf N content and is closely co-ordinated with leaf water use.     

Chemical and hydraulic signals regulate stomatal behavior and photosynthetic activity in maize during progressive drought

The objectives of this study were to investigate stomatal regulation in maize seedlings during progressive soil drying and to determine the impact of stomatal movement on photosynthetic activity. In well-watered and drought-stressed plants, leaf water potential (Ψ _leaf), relative water content (RWC), stomatal conductance (g _s), photosynthesis, chlorophyll fluorescence, leaf instantaneous water use efficiency (iWUE_leaf), and abscisic acid (ABA) and zeatin-riboside (ZR) accumulation were measured. Results showed that g _s decreased significantly with progressive drought and stomatal limitations were responsible for inhibiting photosynthesis in the initial stages of short-term drought. However, after 5 days of withholding water, non-stomatal limitations, such as damage to the PSII reaction center, became the main limiting factor. Stomatal behavior was correlated with changes in both hydraulic and chemical signals; however, changes in ABA and ZR occurred prior to any change in leaf water status. ABA in leaf and root tissue increased progressively during soil drying, and further analysis found that leaf ABA was negatively correlated with g _s (R ² = 0.907, p < 0.05). In contrast, leaf and root ZR decreased gradually. ZR in leaf tissue was positively correlated with g _s (R ² = 0.859, p < 0.05). These results indicate that ABA could induce stomatal closure, and ZR works antagonistically against ABA in stomatal behavior. In addition, the ABA/ZR ratio also had a strong correlation with g _s, suggesting that the combined chemical signal (the interaction between ABA and cytokinin) plays a role in coordinating stomatal behavior. In addition, Ψ _leaf and RWC decreased significantly after only 3 days of drought stress, also affecting stomatal behavior.     

Enhanced Stomatal Conductance by a Spontaneous Arabidopsis Tetraploid,Me-0, Results from Increased Stomatal Size and Greater Stomatal Aperture

The rate of gas exchange in plants is regulated mainly by stomatal size and density. Generally, higher densities of smaller stomata are advantageous for gas exchange; however, it is unclear what the effect of an extraordinary change in stomatal size might have on a plant’s gas-exchange capacity. We investigated the stomatal responses to CO₂ concentration changes among 374 Arabidopsis (Arabidopsis thaliana) ecotypes and discovered that Mechtshausen (Me-0), a natural tetraploid ecotype, has significantly larger stomata and can achieve a high stomatal conductance. We surmised that the cause of the increased stomatal conductance is tetraploidization; however, the stomatal conductance of another tetraploid accession, tetraploid Columbia (Col), was not as high as that in Me-0. One difference between these two accessions was the size of their stomatal apertures. Analyses of abscisic acid sensitivity, ion balance, and gene expression profiles suggested that physiological or genetic factors restrict the stomatal opening in tetraploid Col but not in Me-0. Our results show that Me-0 overcomes the handicap of stomatal opening that is typical for tetraploids and achieves higher stomatal conductance compared with the closely related tetraploid Col on account of larger stomatal apertures. This study provides evidence for whether larger stomatal size in tetraploids of higher plants can improve stomatal conductance.Gas exchange is a vital activity for higher plants that take up atmospheric CO₂ and release oxygen and water vapor through epidermal stomatal pores. Gas exchange affects CO₂ uptake, photosynthesis, and biomass production (Horie et al., 2006; Evans et al., 2009; Tanaka et al., 2014). Stomatal conductance (gs) is used as an indicator of gas-exchange capacity (Franks and Farquhar, 2007). Maximum stomatal conductance (gsmax) is controlled mainly by stomatal size and density, two parameters that change with environmental conditions and are negatively correlated with each other (Franks et al., 2009).Given a constant total stomatal pore area, large stomata are generally disadvantageous for gas exchange compared with smaller stomata, because the greater pore depth in larger stomata increases the distance that gas molecules diffuse through. This increased distance is inversely proportional to g_smax (Franks and Beerling, 2009). The fossil record indicates that ancient plants had small numbers of large stomata when atmospheric CO₂ levels were high, and falling atmospheric [CO₂] induced a decrease in stomatal size and an increase in stomatal density to increase g_s for maximum carbon gain (Franks and Beerling, 2009). The positive relationship between a high g_s and numerous small stomata also holds true among plants living today under various environmental conditions (Woodward et al., 2002; Galmés et al., 2007; Franks et al., 2009). Additionally, the large stomata of several plant species (e.g. Vicia faba and Arabidopsis [Arabidopsis thaliana]) are often not effective for achieving rapid changes in g_s, due to slower solute transport to drive movement caused by their lower membrane surface area-to-volume ratios (Lawson and Blatt, 2014).Stomatal size is strongly and positively correlated with genome size (Beaulieu et al., 2008; Franks et al., 2012; Lomax et al., 2014). Notably, polyploidization causes dramatic increases in nucleus size and stomatal size (Masterson, 1994; Kondorosi et al., 2000). In addition to the negative effects of large stomata on gas exchange (Franks et al., 2009), polyploids may have another disadvantage; del Pozo and Ramirez-Parra (2014) showed that artificially induced tetraploids of Arabidopsis have a reduced stomatal density (stomatal number per unit of leaf area) and a lower stomatal index (stomatal number per epidermal cell number). Moreover, tetraploids of Rangpur lime (Citrus limonia) and Arabidopsis have lower transpiration rates and changes in the expression of genes involved in abscisic acid (ABA), a phytohormone that induces stomatal closure (Allario et al., 2011; del Pozo and Ramirez-Parra, 2014). On the other hand, an increase in the ploidy level of Festuca arundinacea results in an increase in the CO₂-exchange rate (Byrne et al., 1981); hence, polyploids may not necessarily have a reduced gas-exchange capacity.Natural accessions provide a wide range of information about mechanisms for adaptation, regulation, and responses to various environmental conditions (Bouchabke et al., 2008; Brosché et al., 2010). Arabidopsis, which is distributed widely throughout the Northern Hemisphere, has great natural variation in stomatal anatomy (Woodward et al., 2002; Delgado et al., 2011). Recently, we investigated leaf temperature changes in response to [CO₂] in a large number of Arabidopsis ecotypes (374 ecotypes; Takahashi et al., 2015) and identified the Mechtshausen (Me-0) ecotype among ecotypes with low CO₂ responsiveness; Me-0 had a comparatively low leaf temperature, implying a high transpiration rate. In this study, we revealed that Me-0 had a higher g_s than the standard ecotype Columbia (Col), despite having tetraploid-dependent larger stomata. Notably, the g_s of Me-0 was also higher than that of tetraploid Col, which has stomata as large as those of Me-0. This finding resulted from Me-0 having a higher g_s-to-g_smax ratio due to more opened stomata than tetraploid Col. In addition, there were differences in ABA responsiveness, ion homeostasis, and gene expression profiles in guard cells between Me-0 and tetraploid Col, which may influence their stomatal opening. Despite the common trend of smaller stomata with higher gas-exchange capacity, the results with Me-0 confirm the theoretical possibility that larger stomata can also achieve higher stomatal conductance if pore area increases sufficiently.     

Gas exchanges of an endangered species <Emphasis Type="Italic">Syringa pinnatifolia</Emphasis> and a widespread congener <Emphasis Type="Italic">S. oblata</Emphasis>

Net photosynthetic rate (P_N), transpiration rate (E), water use efficiency (WUE), stomatal conductance (g_s), and stomatal limitation (L_s) were investigated in two Syringa species. The saturation irradiance (SI) was 400 µmol m^-2s^-1 for S. pinnatifolia and 1 700 µmol m^-2s^-1 for S. oblata. Compared with S. oblata, S. pinnatifolia had extremely low g _s . Unlike S. oblata, the maximal photosynthetic rate (P_max) in S. pinnatifoliaoccurred around 08:00 and then fell down, indicating this species was sensitive to higher temperature and high photosynthetic photon flux density. However, such phenomenon was interrupted by the leaf development rhythms before summer. A relatively lower P_N together with a lower leaf area and shoot growth showed the capacity for carbon assimilation was poorer in S. pinnatifolia.     

Growth and photosynthetic responses in Jatropha curcas L. seedlings of different provenances to watering regimes

Seedlings from four provenances of Jatropha curcas were subjected to 80, 50, and 30% of soil field capacity in potted experiments in order to study their responses to water availability. Our results showed that with the decline of soil water availability, plant growth, biomass accumulation, net photosynthetic rate, stomatal conductance (g_s), and transpiration rate (E) decreased, whereas leaf carbon isotope composition (δ¹³C), leaf pigment contents, and stomatal limitation value increased, while maximal quantum yield of PSII photochemistry was not affected. Our findings proved that stomatal limitation to photosynthesis dominated in J. curcas under low water availability. The increase of δ¹³C should be attributed to the decrease in g_s and E under the lowest water supply. J. curcas could adapt to low water availability by adjusting its plant size, stomata closure, reduction of E, increasing δ¹³C, and leaf pigment contents. Moreover, effects of provenance and the interaction with the watering regime were detected in growth and many physiological parameters. The provenance from xeric habitats showed stronger plasticity in the plant size than that from other provenances under drought. The variations may be used as criteria for variety/provenance selection and improvement of J. curcas performance.     

The Developmental Basis of Stomatal Density and Flux

Equations for stomatal density and maximum theoretical stomatal conductance as functions of stomatal initiation rate, epidermal cell size, and stomatal size enable scaling from development to flux.Since the first published measurements of stomatal density by Johann Hedwig (1793) and Alexander von Humboldt (1798), the counting and measuring of stomata has been one of the most typical botanical activities, with an important role across fields of plant biology (Willmer and Fricker, 1996). Stomatal density (d) and size (s) are indicators of acclimation and adaptation to contrasting environments, and permit estimation of the theoretical anatomical maximum stomatal conductance (g_max; units: mol m⁻² s⁻¹; Brown and Escombe, 1900; Lawson et al., 1998; Franks and Beerling, 2009; Franks et al., 2009), which represents a first quantitative estimate of the anatomical constraint on maximum stomatal gas exchange. While decades of theory have focused on d and g_max, their basis in traits with a transparent relationship to epidermal development has not been expressed. We derived exact mathematical equations for d and g_max as functions of stomatal differentiation rate, also known as stomatal index (i, no. of stomata per no. of epidermal cells plus stomata), s, and epidermal cell size (e). These equations unify the quantitative understanding of epidermal development and maximum flux, revealing the developmental bases for d and g_max across genotypes or species, and enabling targeting of specific epidermal development traits in plant breeding for productivity.The genetic and developmental basis for high stomatal density and stomatal conductance is a research priority in plant physiology, agriculture, and paleobiology (Asl et al., 2011; Doheny-Adams et al., 2012; Dow et al., 2014; Franks et al., 2015; Roche, 2015; Wang et al., 2015b). Indeed, a higher g_max should benefit species under low CO₂, higher irradiance or nutrient supply, or under selection for high productivity or competition (Franks and Beerling, 2009; Taylor et al., 2012; Jones, 2014). Under the opposite conditions, a lower g_max would provide the potential benefits of reduced water loss and/or increased CO₂ gain relative to water loss (Franks and Beerling, 2009; Taylor et al., 2012; Jones, 2014; Franks et al., 2015). Decades of theory have focused on the basis of g_max in stomatal anatomy (Fig. 1, A–C). According to a classic formulation of g_max (Brown and Escombe, 1900) in a recently updated form,(1)where D (m² s⁻¹) represents the diffusivity in air of water or CO₂ (which differ by a factor of 1.6); ν the molar volume of air (m³ mol⁻¹); and d, a_max, and l, respectively, the stomatal density (pores m⁻²), the mean maximum area of a single stomatal pore (m²), and stomatal pore depth (m; Franks and Beerling, 2009; Franks et al., 2009). The most recent extensions of this equation incorporated basic assumptions about allometries among guard cell dimensions, which have become standard in the stomatal literature (e.g. Franks and Beerling, 2009; Franks et al., 2009; Taylor et al., 2012; Dow et al., 2014; McElwain et al., 2016) and enable the estimation of g_max as a function of d and s. In its simplest form:(2)where and such that b is a biophysical constant and m a morphological constant based on scaling factors representing the proportionality of stomatal length (L) and width (W), and pore length (p) and depth (l), with c = p/L, j = W/L, and h = l/W all treated as constant for the estimation of g_max (c, h, and j = 0.5 for nongrasses with kidney bean-shaped guard cells, or c = 0.5, h = 0.5, and j = 0.125 for grasses with their dumbbell-shaped guard cells; Franks and Beerling, 2009; McElwain et al., 2016), though these ratios can be allowed to vary for individual species or genotypes when more detailed information is available on stomatal dimensions (Franks and Farquhar, 2007; Franks et al., 2014).Open in a separate windowFigure 1.Anatomical variables determining maximum stomatal conductance (g_max). A to C, Stomatal dimensions (guard cell length, L; stomatal pore length, p; guard cell width, W; stomatal area, s; stomatal maximum pore area, a_max; stomatal depth, l) and epidermal development traits (epidermal cell area, e; stomatal index, i). D to F, The influence on stomatal density (d) and g_max of e and i: increasing i as from D to E would lead to higher d and g_max; reducing e as from E to F would lead to higher d and g_max. Larger s would also lead to lower d and g_max, though with a much smaller effect. Stomatal images after Beaulieu et al. (2008).The g_max estimated this way strongly predicted the operating stomatal conductance measured with leaf gas exchange systems (g_op) across Arabidopsis (Arabidopsis thaliana) genotypes under low CO₂, high humidity, and high red and blue light (Dow et al., 2014). However, across diverse species, the g_max values estimated by Equation 2 tend to be much higher than g_op (Feild et al., 2011; McElwain et al., 2016) for several reasons. First, for typical leaves transpiring even under the best conditions, the effective area of the stomatal pore (a’) is smaller than the anatomical maximum a_max, by an amount that varies across species, particularly as the actual pore geometry usually deviates from simplified cylindrical geometry (Franks and Farquhar, 2007). Second, as guard cells close under adverse conditions, a’ declines (Fanourakis et al., 2015). Third, there may be a substantial contribution of diffusion resistances in the intercellular airspaces, especially in the case of a partly cutinized substomatal chamber (Roth-Nebelsick, 2007; Feild et al., 2011). Fourth, leaf surface features such as hairs or papillae surrounding the stomata, or encryption of stomata, may affect the diffusion through stomata, and especially will influence the boundary layer, which in addition to stomatal conductance determines overall diffusional conductance and therefore gas exchange (Kenzo et al., 2008; Hassiotou et al., 2009; Maricle et al., 2009). Clearly, much more research is needed to establish models that include all the factors that determine the anatomical influence of stomata on gas exchange rates and to validate these against a wide diversity of plants, yet the anatomical maximum defined as in Equations 1 and 2 is a strong constraint: g_max correlates across diverse species with g_op and light-saturated photosynthetic rate (McElwain et al., 2016), and scales up, in combination with leaf area allocation, to the determination of ecosystem net primary productivity (Wang et al., 2015a). The anatomical g_max is therefore a theoretical value estimating the maximum stomatal diffusion capacity, and like other theoretical physiological variables, such as photosynthetic parameters including the maximum carboxylation rate (V_cmax), it cannot be reached in practice, but is useful for generating hypotheses regarding the capacity for stomatal diffusion in various domains, such as comparisons of genotypes or species, functional types, or trends in evolutionary time (Franks and Beerling, 2009; Doheny-Adams et al., 2012; Taylor et al., 2012; McElwain et al., 2016; de Boer et al., 2016).Despite the well-recognized importance of both d and g_max, there has been limited understanding of their genetic and developmental basis and their relationships to other epidermal traits. Ever since the seminal work of E.J. Salisbury early last century, d has been known to be positively associated with stomatal initiation rate, also known as stomatal index (i = no. of stomata per no. of epidermal cells plus stomata; Salisbury, 1927; Wengier and Bergmann, 2012), and negatively with mean epidermal cell area (e), as increases in e would space stomata apart (Fig. 1, D–F). Studies of plants of different species (Beaulieu et al., 2008; Brodribb et al., 2013) or of given species grown in different irradiance and vapor pressure deficit treatments (Carins Murphy et al., 2012, 2014) found that d related negatively to e. Further, a negative relationship of d with s within plant canopies or across species has been found numerous times and sometimes attributed to a “general association” or “trade-off” (e.g. Weiss, 1865; Grubb et al., 1975; Tichá, 1982; Hetherington and Woodward, 2003; Sack et al., 2003; Franks and Beerling, 2009; Brodribb et al., 2013; Wang et al., 2015a; de Boer et al., 2016). Yet, while numerous correlational studies within and across species have confirmed these relationships, their formal mathematical basis has remained unclear.To directly link g_max, and thus stomatal flux, to underlying epidermal development traits, we derived new equations for d and g_max as functions of e, i, and s, where e and s are projected cell areas (units: m²).As defined by Salisbury (1927), i is the number of stomata (n_s) divided by the sum of n_s and the number of epidermal cells (n_e):(3)Stomatal density (d) is related to n_s, n_e, s, and e as:(4)where area is that of the whole leaf (units: m²). Equation 4 can be rearranged as(4a)The ratio n_e/n_s can be expressed in terms of i by rearranging Equation 3:(5)Applying Equation 5 to Equation 4a gives(6)This equation gives d as a function of e, i, and s—traits with a transparent relationship to development, all being related to epidermal cell differentiation and expansion. Equation 6 can be applied to Equation 2 to give g_max as a function of e, i, and s:(7)These expressions rely on mean values for e, s, and i, so their accuracy may be affected by variation of these variables within leaves, or by variation in sampling methods as there exists no standard measurement protocol (see “Materials and Methods”). We tested the correctness of the derivation of Equation 6 and its applicability to real measurements of d, e, i, and s for abaxial leaf surfaces compiled from the published literature for 141 values from 81 species from 28 angiosperm families (“Supplemental Data”). We further checked for quantitative consistency between g_max as estimated from e, s, and i (Eq. 7) and the literature standard estimate of g_max from d and s (Eq. 2) using the same dataset. In both cases we found extremely tight correspondence (Fig. 2). Considering relationships within individual plant families for which ≥ 6 points were available showed similarly tight correspondence (R² = 0.96–1.0, P < 0.001, n = 6–30 for Betulaceae, Ericaceae, Fabaceae, Fagaceae, Orchidaceae, Rosaceae, and Sapindaceae; slopes and intercepts did not differ at P < 0.05 among families or from 1.0 and 0, respectively).Open in a separate windowFigure 2.Developmental basis for maximum stomatal flux variables: the estimation of abaxial leaf stomatal density (d; A) and theoretical maximum stomatal conductance (g_max; B) as functions of epidermal cell area (e), stomatal index (i), and stomatal area (s). A, Values estimated using Equation 6 plotted against reported values of d; B, values estimated using Equation 7 plotted against values estimated using Equation 2 with inputs of s and d as standard in the current literature. Data were compiled from single or mean values for leaves from published papers for seedlings (circles) and adults (squares) of 54 European woody species; four species of annual herbs of genus Gomphrena (Amaranthaceae; triangles); 22 species of genus Stanhopea, Orchidaceae (diamonds); and one grass species (Paspalum dilatatum, Poaceae; hexagon). The lines are ordinary least squares regressions fitted to the data with fixed zero intercept. The high R² values indicate the correctness of the derivation, its applicability to real measurements, and the quality of the measurements.These equations clarify precise geometric linkages among stomatal flux, anatomy, and development. We propose five examples of potentially powerful applications of these relationships to inform fundamental research across plant development, physiology, paleobiology, and crop science.

• (1) An expansion of available data on stomatal differentiation. Measurements of i can be technically challenging given the need to resolve all epidermal pavement cells in an image, but Equation 6 can be rearranged to allow estimation of i from measurements of d, e, and s, greatly expanding the data availability for this important developmental trait:(8)
• (2) Analysis of the developmental and genetic drivers of d and g_max across genotypes of a given species or across phylogenetically diverse species; i.e. quantifying how much of the variation in d and g_max arises due to differences in i, e, or s. Thus, the developmental basis for observed shifts in g_max in response to climate, CO₂, and lifeform evolution can be inferred using Equations 6 and 7.
• (3) Clarifying the quantitative role of shifts in genome and cell sizes (i.e. e and s) on g_max. The question of the role and impact of cell size is especially important given the strong developmental plasticity and evolutionary lability of cell size, and its relationship to other traits. For example, within some lineages, epidermal cell size correlates positively with genome size and leaf size and/or negatively with venation density (Beaulieu et al., 2008; Brodribb et al., 2013).
• (4) Resolving the coordinated shifts of stomatal traits in fossils and experimental plants, thereby improving inferences concerning shifts in response to global temperature and atmospheric CO₂. Previous studies of adaptation and acclimation in response to CO₂ have tended to quantify d and/or i (e.g. Beerling et al., 1998; Royer, 2001) and/or more rarely s and e (e.g. Ogaya et al., 2011; Haworth et al., 2014), and assumed that a shift in any one of these traits was an important marker of adaptation. Equations 6 and 7 allow estimation of the quantitative dependency of shifts in d and g_max on other variables.
• (5) Prediction of how each trait should be adjusted, through breeding or genetic manipulation, to optimize productivity through changes in g_max. Equations 6 and 7 clarify the separate roles of e, i, and s in determining higher g_max. Given the increasing resolution of the genetic basis for these traits in model species (e.g. Ferris et al., 2002; Delgado et al., 2011), these traits can be made specific targets for breeding for higher g_max and thereby for productivity. Other traits would also need to be targeted (e.g. hydraulic and photosynthetic traits) to enable higher productivity above and beyond the potential cost of constructing, maintaining, and operating additional stomatal apparatus (Assmann and Zeiger, 1987).

By linking leaf epidermal anatomy and development with physiological flux, these equations allow scaling from the differentiation and expansion of epidermal cells and stomata to plant productivity. Given ongoing improvement of models for the influence of the anatomy and dynamic behavior of stomata and of internal and external leaf tissues on gas exchange, consideration of these important traits in terms of their development will have potential applications across the widest range of fields in plant biology and earth system science.     

Changes in stomatal frequency, stomatal conductance and cuticle thickness during leaf expansion in the broad-leaved evergreen species, Eucalyptus regnans

Aspects of leaf anatomical and physiological development were investigated in the broad-leaved evergreen species, Eucalyptus regnans F.Muell. Newly emergent leaves were tagged in the field and measured for stomatal conductance while a subset was collected every 14 days for the measurement of stomata and cuticle over a 113-day period. Cuticle thickness increased during leaf expansion, the increase following a sigmoid curve. Stomatal frequency (no. mm⁻²) decreased from 56 to 113 days after leaf emergence. The frequency of both immature and intermediate developmental stages of stomata also decreased over this time, but the total number of stomata per leaf remained relatively constant. Stomatal conductance (g _s) of young expanding leaves increased during expansion, and was significantly linearly correlated with stomatal frequency (excluding immature stomata), and with cuticle thickness. The progressive increase in g _s in young developing leaves was contrary to the observed changes in structural characteristics (increased cuticle thickness and decreased stomatal frequency). This increase in g _s with development may be related to the progressive increase in number of mature stomata with larger apertures and, therefore, a higher total pore area in fully expanded leaves.     

Variability of stomatal conductance, leaf anatomy, and seasonal leaf wettability of young and adult European beech leaves along a vertical canopy gradient

This study assessed the variation of leaf anatomy, chlorophyll content index (CCI), maximal stomatal conductance (g _s ^max ) and leaf wettability within the canopy of an adult European beech tree (Fagus sylvatica L.) and for beech saplings placed along the vertical gradient in the canopy. At the top canopy level (CL_28m) of the adult beech, CCI and leaf anatomy reflected higher light stress, while g _s ^max increased with height, reflecting the importance of gas exchange in the upper canopy layer. Leaf wettability, measured as drop contact angle, decreased from 85.5°?±?1.6° (summer) to 57.5°?±?2.8° (autumn) at CL_28m of the adult tree. At CL_22m, adult beech leaves seemed to be better optimized for photosynthesis than the CL_28m leaves because of a large leaf thickness with less protective and impregnated substances, and a higher CCI. The beech saplings, in contrast, did not adapt their stomatal characteristics and leaf anatomy according to the same strategy as the adult beech leaves. Consequently, care is needed when scaling up experimental results from seedlings to adult trees.     

Focus on Water: Stomatal Size,Speed, and Responsiveness Impact on Photosynthesis and Water Use Efficiency

The control of gaseous exchange between the leaf and bulk atmosphere by stomata governs CO₂ uptake for photosynthesis and transpiration, determining plant productivity and water use efficiency. The balance between these two processes depends on stomatal responses to environmental and internal cues and the synchrony of stomatal behavior relative to mesophyll demands for CO₂. Here we examine the rapidity of stomatal responses with attention to their relationship to photosynthetic CO₂ uptake and the consequences for water use. We discuss the influence of anatomical characteristics on the velocity of changes in stomatal conductance and explore the potential for manipulating the physical as well as physiological characteristics of stomatal guard cells in order to accelerate stomatal movements in synchrony with mesophyll CO₂ demand and to improve water use efficiency without substantial cost to photosynthetic carbon fixation. We conclude that manipulating guard cell transport and metabolism is just as, if not more likely to yield useful benefits as manipulations of their physical and anatomical characteristics. Achieving these benefits should be greatly facilitated by quantitative systems analysis that connects directly the molecular properties of the guard cells to their function in the field.In order for plants to function efficiently, they must balance gaseous exchange between inside and outside the leaf to maximize CO₂ uptake for photosynthetic carbon assimilation (A) and to minimize water loss through transpiration. Stomata are the “gatekeepers” responsible for all gaseous diffusion, and they adjust to both internal and external environmental stimuli governing CO₂ uptake and water loss. The pathway for CO₂ uptake from the bulk atmosphere to the site of fixation is determined by a series of diffusional resistances, which start with the layer of air immediately surrounding the leaf (the boundary layer). Stomatal pores provide a major resistance to flux from the atmosphere to the substomatal cavity within the leaf. Further resistance is encountered by CO₂ across the aqueous and lipid boundaries into the mesophyll cell and chloroplasts (mesophyll resistance). Water leaving the leaf largely follows the same pathway in reverse, but without the mesophyll resistance component. Guard cells surround the stomatal pore. They increase or decrease in volume in response to external and internal stimuli, and the resulting changes in guard cell shape adjust stomatal aperture and thereby affect the flux of gases between the leaf internal environment and the bulk atmosphere. Stomatal behavior, therefore, controls the volume of CO₂ entering the intercellular air spaces of the leaf for photosynthesis. It also plays a key role in minimizing the amount of water lost. Transpiration, by virtue of the concentration differences, is an order of magnitude greater than CO₂ uptake, which is an inevitable consequence of free diffusion across this pathway. Although the cumulative area of stomatal pores only represents a small fraction of the leaf surface, typically less than 3%, some 98% of all CO₂ taken up and water lost passes through these pores. When fully open, they can mediate a rate of evaporation equivalent to one-half that of a wet surface of the same area (Willmer and Fricker, 1996).Early experiments illustrated that photosynthetic rates were correlated with stomatal conductance (g_s) when other factors were not limiting (Wong et al., 1979). Low g_s limits assimilation rate by restricting CO₂ diffusion into the leaf, which, when integrated over the growing season, will influence the carbohydrate status of the leaf with consequences for crop yield. Stomata of well-watered plants are thought to reduce photosynthetic rates by about 20% in most C3 species and by less in C4 plants in the field (Farquhar and Sharkey, 1982; Jones, 1987). However, even this restriction has been shown to impact substantially on yield. For example, Fischer et al. (1998) demonstrated a close correlation between g_s and yield in eight different wheat (Triticum aestivum) cultivars. Those studies highlighted the effects g_s can have on crop yield, not only through reduced CO₂ diffusion but also through the impact on water loss and evaporative cooling of the leaf. Indeed, enhancing photosynthesis yields by only 2% to 3% is sufficient to substantially increase plant growth and biomass over the course of a growing season (Lefebvre et al., 2005; Zhu et al., 2007).Stomata and their behavior profoundly affect the global fluxes of CO₂ and water, with an estimated 300 × 10¹⁵ g of CO₂ and 35 × 10¹⁸ g of water vapor passing through stomata of leaves every year (Hetherington and Woodward, 2003). Changes in stomatal behavior in response to changing climatic conditions are thought to impact on water levels and fluxes. For example, it is estimated that partial stomatal closure driven by increasing CO₂ concentration over the past two decades has led to increased CO₂ uptake and reduced evapotranspiration in temperate and boreal northern hemisphere forests (Keenan et al., 2013), with implications for continental runoff and freshwater availability associated with the global rise in CO₂ (Gedney et al., 2006). Concurrently, the increase in global water usage over the past 100 years and the expectation that this is set to double before 2030 (UNESCO, 2009) has put pressure on breeders and scientists to find new crop varieties, breeding traits, or potential targets for manipulation that would result in crop plants that are able to sustain yield with less water input. The fact that stomata are major players in plant water use and the entire global water cycle makes the functional and physical attributes of stomata potential targets for manipulation to improve carbon gain and plant productivity as well as global water fluxes.There are several approaches for improving carbon gain and plant water use efficiency (WUE) that focus on stomata. It is possible to increase or decrease the gaseous conductance of the ensemble of stomata per unit of leaf area (g_s) through the manipulation of stomatal densities (Büssis et al., 2006). In addition, there is potential to alter the stomatal response or sensitivity to environmental signals through the manipulation of guard cell characteristics that affect stomatal mechanics (e.g. OPEN STOMATA [ost] mutants; Merlot et al., 2002). Such approaches have produced an array of mutant plants with altered characteristics and varying impacts on CO₂ uptake and transpiration, several of which we discuss in greater detail below. An intuitive measure of the efficacy of such manipulations is the WUE, commonly defined as the amount of carbon fixed in photosynthesis per unit of water transpired. In general, higher WUE values have been observed in plants with lower g_s, but these gains are usually achieved together with a reduction in A and slower plant growth. Plants with higher g_s have greater assimilation rates and grow faster under optimal conditions, but they generally exhibit lower WUE. An approach that has not been fully explored or considered in any depth is to select plants for differences in the kinetics of stomatal response or to manipulate stomatal kinetics in ways that improve the synchrony with mesophyll CO₂ demand (Lawson et al., 2010, 2012). To date, the majority of studies assessing the impact of stomatal behavior on photosynthetic carbon gain have focused on steady-state measurements of g_s in relation to photosynthesis. These studies do not take account of the dynamic situation in the field. As we discuss below, a cursory analysis of stomatal synchrony with mesophyll CO₂ demand suggests that gains of 20% to 30% are theoretically possible.Here, we address the question of the kinetics of the stomatal response to the naturally fluctuating environment, notably to fluctuations in light that are typical of the conditions experienced in the field. We focus on vascular seed plants, to which crop plants belong, and do not address seedless vascular plants, such as ferns. The characteristics of the latter, and hence the issues and challenges they present, are very different. In our minds, of paramount importance is whether there is potential for engineering guard cells of crop plants to manipulate the dynamic behavior of stomata so as to improve WUE without substantial cost in assimilation. Of course, in many circumstances, stomata are not the only factor to limit water flux through the plant (see other articles in this issue), but they are one of the most important “gatekeepers” and therefore, serve as a good starting point for such considerations. Thus, we explore the physical and functional attributes of stomata, their signaling, and the solute transport mechanisms that determine pore aperture as targets for potential manipulation of stomatal responses to changing environmental cues.     

Shoot hydraulic characteristics, plant water status and stomatal response in olive trees under different soil water conditions

Aims

To evaluate the impact of the amount and distribution of soil water on xylem anatomy and xylem hydraulics of current-year shoots, plant water status and stomatal conductance of mature ‘Manzanilla’ olive trees.

Methods

Measurements of water potential, stomatal conductance, hydraulic conductivity, vulnerability to embolism, vessel diameter distribution and vessel density were made in trees under full irrigation with non-limiting soil water conditions, localized irrigation, and rain-fed conditions.

Results

All trees showed lower stomatal conductance values in the afternoon than in the morning. The irrigated trees showed water potential values around ?1.4 and ?1.6 MPa whereas the rain-fed trees reached lower values. All trees showed similar specific hydraulic conductivity (K _s) and loss of conductivity values during the morning. In the afternoon, K _s of rain-fed trees tended to be lower than of irrigated trees. No differences in vulnerability to embolism, vessel-diameter distribution and vessel density were observed between treatments.

Conclusions

A tight control of stomatal conductance was observed in olive which allowed irrigated trees to avoid critical water potential values and keep them in a safe range to avoid embolism. The applied water treatments did not influence the xylem anatomy and vulnerability to embolism of current-year shoots of mature olive trees.     

Leaf responses to iron nutrition and low cadmium in peanut: anatomical properties in relation to gas exchange

Aims

This study evaluated how iron nutrition affect leaf anatomical and photosynthetic responses to low cadmium and its accumulation in peanut plants.

Methods

Seedlings were treated with Cd (0 and 0.2 μM CdCl₂) and Fe (0, 10, 25, 50 or 100 μM EDTA-Na₂Fe) in hydroponic culture.

Results

Cadmium accumulation is highest in Fe-deficient plants, and dramatically decreased with increasing Fe supply. The biomass, gas exchange, and reflectance indices were highest at 25 μM Fe²⁺ treatments, indicating the concentration is favorable for the growth of peanut plants. Both Fe deficiency and Cd exposure impair photosynthesis and reduce reflectance indices. However, they show different effects on leaf anatomical traits. Fe deficiency induces more and smaller stomata in the leaf surface, but does not affect the inner structure. Low Cd results in a thicker lamina with smaller stomata, thicker palisade and spongy tissues, and lower palisade to spongy thickness ratio. The stomatal length and length/width ratio in the upper epidermis, spongy tissue thickness, and palisade to spongy thickness ratio were closely correlated with net photosynthetic rate, stomatal conductance, and transpiration rate.

Conclusions

Cd accumulation rather than Fe deficiency alters leaf anatomy that may increase water use efficiency but inhibit photosynthesis.     

Environmental conditions modulate plasticity in the physiological responses of three plant species of the Neotropical savannah

Plasticity in plants could be changed due to abiotic factors, tending to increase fitness across environments. In the Neotropical savannah, a strong water deficit during the dry season is one of the main factors limiting the plasticity in physiological responses of plants. The present study aims to assess the plasticity in physiological responses and vegetative phenology of three plant species of the Neotropical savannah (Cerrado in Brazil) during the dry and the rainy seasons. The three species, Byrsonima verbascifolia, Roupala montana, and Solanum lycocarpum, occur in Serra do Cipó in the state of Minas Gerais, Brazil. The development and vegetative phenology of individuals of these three species were evaluated over the course of 1 year. In February 2012 (rainy season) and August 2012 (dry season), stomatal conductance (g _s), water potential (Ψ), photosynthetic quantum yield, and concentration of leaf photosynthetic pigments were measured. The relative distance among the physiological parameters of all individuals within each season was measured using the relative distance plasticity index. B. verbascifolia has pronounced senescence in July and lost leaves completely by the early September, while R. montana and S. lycocarpum have green leaves throughout the year. The three studied species had greater control of stomatal opening during the dry season. S. lycocarpum and R. montana had negative water potential values in the dry season and in the middle of the day in both seasons. In the dry season, the three species exhibited a decrease in F _v/F _m, with values between 0.7 and 0.75. The relative distance plasticity index varied from 0 to 1. R. montana demonstrated the greatest plasticity and S. lycocarpum had lower plasticity. Then, a seasonal effect on physiological response was observed in all three model-species, with lower values for leaf water potential and stomatal conductance, and increased photoinhibition, in the dry season. Ecophysiological traits, such as stomatal conductance and leaf water potential, exhibited the greatest plasticity. In addition, there was a seasonal effect on the plasticity in physiological responses of the three plants species of the Neotropical savannah. The results are contradicting the idea that water restriction in the dry season would reduce the plasticity in most species of the Neotropical savannah.