Simulations and observations of patchy stomatal behavior in leaves of <Emphasis Type="Italic">Quercus crispula</Emphasis>, a cool-temperate deciduous broad-leaved tree species

We investigated the occurrence of patchy stomatal behavior in leaves of saplings and a forest canopy tree of Quercus crispula Blume. Through a combination of leaf gas-exchange measurements and numerical simulation, we detected patterns of stomatal closure (either uniform or patchy bimodal) coupled with depression of net assimilation rate (A). There was a clear inhibition of A associated with stomatal closure in leaves of Q. crispula during the day, but the magnitude of inhibition varied among days and growing conditions. Comparisons of observed and simulated A values for both saplings and the canopy tree identified patterns of stomatal behavior that shifted flexibly between uniform and patchy frequency distributions depending on environmental conditions. Bimodal stomatal closure explained severe depression of A in saplings under conditions of relatively high leaf temperature and vapor pressure deficit. Model simulations of A depression through bimodal stomatal closure were corroborated by direct observations of stomatal aperture distribution using Suzuki’s Micro-Printing method; these demonstrated that there was a real bimodal frequency distribution of stomatal apertures. Although there was a heterogeneous distribution of stomatal apertures both within and among patches, induction of heterogeneity in intercellular CO₂ concentration among patches, and hence severe depression of A, resulted only from bimodal stomatal closure among patches (rather than within patches).     

Short communication. Abscisic acid is not necessarily required for the induction of patchy stomatal closure

Patchy stomatal closure was observed in leaves of transgenic plants of Nicotiana plumbaginifolia producing antibodies that block the action of abscisic acid. Stomatal patchiness was induced by leaf detachment and subsequent water loss. Stomatal closure was followed by an irreversible reduction of maximal chlorophyll fluorescence. The degree of deviation from the A/ci-curve is correlated with steady-state diffusion conductance before leaf detachment. It is concluded that a heterogeneous sensitivity of stomata to abscisic acid is not directly involved in the induction of patchy stomatal closure.Keywords: Abscisic acid, chlorophyll fluorescence imaging, patchy stomatal closure, Nicotiana plumbaginifolia.      

Stomatal patchiness in Mediterranean evergreen sclerophylls

Midday depression of net photosynthesis and transpiration in the Mediterranean sclerophylls Arbutus unedo L. and Quercus suber L. occurs with a depression of mesophyll photosynthetic activity as indicated by calculated carboxylation efficiency (CE) and constant diurnal calculated leaf intercellular partial pressure of CO₂ (C_i). This work examines the hypothesis that this midday depression can be explained by the distribution of patches of either wide-open or closed stomata on the leaf surface, independent of a coupling mechanism between stomata and mesophyll that results in a midday depression of photosynthetic activity of the mesophyll. Pressure infiltration of four liquids differing in their surface tension was used as a method to show the occurrence of stomatal patchiness and to determine the status of stomatal aperture within the patches. Liquids were selected such that the threshold leaf conductance necessary for infiltration through the stomatal pores covered the expected diurnal range of calculated leaf conductance (g) for these species. Infiltration experiments were carried out with leaves of potted plants under simulated Mediterranean summer conditions in a growth chamber. For all four liquids, leaves of both species were found to be fully infiltratable in the morning and in the late afternoon while during the periods leading up to and away from midday the leaves showed a pronounced patchy distribution of infiltratable and non-infiltratable areas. Similar linear relationships between the amount of liquid infiltrated and g (measured by porometry prior to detachment and infiltration) for all liquids clearly revealed the existence of pneumatically isolated patches containing only wide-open or closed stomata. The good correspondence between the midday depression of CE, calculated under the assumption of no stomatal patchiness, and the diurnal changes in non-infiltratable leaf area strongly indicates that the apparent reduction in mesophyll activity results from assuming no stomatal patchiness. It is suggested that simultaneous responses of stomata and mesophyll activity reported for other species may also be attributed to the occurrence of stomatal patchiness. In Quercus coccifera L., where the lack of constant diurnal calculated C_i and major depression of measured CE at noontime indicates different stomatal behavior, non-linear and dissimilar relationships between g and the infiltratable quantities of the four liquids were found. This indicates a wide distribution of stomatal aperture on the leaf surface rather than only wide-open or closed stomata.Dedicated to Professor Otto L. Lange on the occasion of his 65th birthday     

Is stomatal conductance optimized over both time and space in plant crowns? A field test in grapevine (Vitis vinifera)

Crown carbon gain is maximized for a given total water loss if stomatal conductance (g_s) varies such that the marginal carbon product of water (?A/?E) remains invariant both over time and among leaves in a plant crown, provided the curvature of assimilation rate (A) versus transpiration rate (E) is negative. We tested this prediction across distinct crown positions in situ for the first time by parameterizing a biophysical model across 14 positions in four grapevine crowns (Vitis vinifera), computing optimal patterns of g_s and E over a day and comparing these to the observed patterns. Observed water use was higher than optimal for leaves in the crown interior, but lower than optimal in most other positions. Crown carbon gain was 18% lower under measured g_s than under optimal g_s. Positive curvature occurred in 39.6% of cases due to low boundary layer conductance (g_bw), and optimal g_s was zero in 11% of cases because ?A/?E was below the target value at all g_s. Some conclusions changed if we assumed infinite g_bw, but optimal and measured E still diverged systematically in time and space. We conclude that the theory's spatial dimension and assumption of positive curvature require further experimental testing.     

Qualitative effects of patchy stomatal conductance distribution features on gas-exchange calculations

The qualitative influence of patchy stomatal conductance distributions on the values of photosynthesis (A) and intercellular CO₂ concentration (c_i) as determined by gas-exchange measurements were investigated using computer modelling. Gas-exchange measurements were simulated for different conductance distributions by modelling photosynthesis explicitly for each patch, summing these rates, and inferring c_i from a diffusion equation. Qualitative relationships are presented between conductance distribution features and the difference between assimilation rates measured for patchy and homogeneous leaves at the same c_i (A_p and A_h, respectively). These data show that, although most conductance distributions have little effect on the value of A measured for a given c_i, some distribution features (which we have termed ‘bimodality’, ‘position’, ‘skewness’ and ‘range’) play a key role in controlling the magnitude of these effects. Distributions that are more nearly bimodal, span regions of lower conductance, are right-skewed, or have broader conductance ranges are associated with larger effects on the A(c_i) relationship. To clarify our mathematical analysis and illustrate some of the trends it predicts, we present conductance distributions and gas-exchange data from leaves of Malus dolgo var. Spring Snow Dial were treated with ABA. The results are discussed in the light of recent controversy over the effect of patchy stomatal conductance on gas-exchange data.     

Stomatal response to environment and a possible interrelation between stomatal effects on transpiration and CO2 assimilation

Abstract It had been hypothesized that if daily CO₂ assimilation is to be maximized at a given level of daily transpiration, stomatal apertures should change during the day so that the gain ratio (?A/?g)/(?E/?g) remains constant. These partial differentials describe the sensitivity of assimilation rate (A) and transpiration rate (E) to changes in stomatal conductance (g). Experiments were conducted to determine whether stomata respond to environment in a manner which results in constant gain ratios. Gas–exchange measurements were made of the stomatal and photosynthetic responses of Vigna unguiculata L. Walp. in controlled environments. Leaf conductance to water vapour responded to step changes in temperature and humidity so that for different steady-state conditions the gain ratio remained constant on all but one day. Depletion of water in the root zone resulted in day-to-day increases in gain ratio which were correlated with decreases in maximum leaf conductance to water vapour. The significance of the results for plant adaptation and stomatal mechanisms, and methods for measuring the gain ratio, are discussed.     

Stomatal responses to non-local changes in PFD: evidence for long-distance hydraulic interactions

Interactions among stomata within a single areole have recently been reported, and evidence suggests that hydraulic mechanisms may be responsible for these interactions. Such interactions may play a role in patchy stomatal behaviour by coordinating stomatal behaviour within areoles. However, models suggest that longer‐distance interactions may be required to produce the large‐scale discoordination that is characteristic of stomatal patchiness. This study was undertaken to characterize long‐distance interactions between ‘artificial patches’ of stomata under varying conditions of evaporative demand and soil water stress. Gas‐exchange was monitored in two adjacent regions (‘patches’) of a wheat leaf by two independent gas mixing and analysis systems. When photon flux density (PFD) was changed in only one of these patches, stomatal conductance responded in both patches in a manner consistent with hydraulic interactions propagated by changes in xylem water potential. These data are discussed in the context of mechanisms for patchy stomatal conductance and implications for the design and analysis of gas‐exchange experiments.     

Stomatal patchiness in conifers: experiments with Picea abies (L.) Karst. and Abies alba Mill.

Summary Strong evidence for the occurrence of pronounced stomatal patchiness in needles of Picea abies (L.) Karst. and Abies alba Mill. was found using various indirect methods. Anatomical investigations revealed a septate leaf anatomy for both species, a phenomenon expected if a patchy distribution of stomatal aperture is present. Calculation of some photosynthetic characteristics (e.g. carboxylation efficiency) from gas exchange measurements is shown to be markedly affected by the patchy distribution of stomatal apertures on the needles. The importance of stomatal patchiness in connection with air pollution related forest decline symptoms as well as an hypothesis suggesting a possible role of the phenomenon as a protective mechanism against photoinhibition are discussed.     

Stomatal patchiness

Different behaviour of small groups of stomata on a single leaf blade (stomatal patchiness) is reviewed. The occurrence of stomatal patchiness depends on plant species, age, leaf position, environmental conditions,etc. The possibility of errors in conventional evaluation of stomatal and non-stomatal (biochemical) limitations of photosynthesis resulting from patchy stomatal closure is analysed. The consequences of stomatal patchiness for leaf and plant photosynthesis and water economy are discussed. A brief survey of the techniques currently used for detection and quantification of stomatal patchiness is presented.     

Changes in photon flux can induce stomatal patchiness

Images of chlorophyll fluorescence were used to detect the occurrence of stomatal patchiness in leaves from eight species under variable photon flux conditions. Pronounced stomatal patchiness was induced within 5–10 min after PFD was changed from intermediate (～450 μmol quanta m^?2 s^?1) to low (～150 μmol quanta m^?2 s^?1) levels. This effect was completely reversible by returning PFD to intermediate levels. The pattern of heterogeneous fluorescence for each leaf was usually similar during repeated applications of medium and low PFD. In three species, stomatal patchiness could only be induced in slightly water-stressed plants. Leaves of more severely water-stressed Xanthium strumarium plants in low air humidity exhibited oscillations in fluorescence that corresponded with oscillatory changes in leaf diffusion conductance for water vapour. Stomatal patchiness was also induced by illuminating dark-adapted leaves with low PFD (below 200–300 μmol quanta m^?2 s^?1). Infiltration of leaves with distilled water showed that heterogeneous chlorophyll fluorescence was caused by changes in stomatal apertures.     

Effects of patchy stomatal closure on gas exchange measurements following abscisic acid treatment

Gas exchange data and images of leaf fluorescence were collected concurrently as stomata responded to abscisic acid (ABA) application. When 10^?5kmolm^?3 ABA was applied to the transpiration stream in a short pulse, stomatal conductance (g_s), photosynthesis (A) and intercellular CO2 concentration (C_i) decreased rapidly after a short lag period and became approximately constant after 2h. There was an apparent reduction in the A versus c1 relationship as stomata closed, but the data returned to the A versus C1 curve while stomatal conductance was constant or slowly rising during the second hour after ABA treatment. Larger amounts of ABA administered during the pulse caused larger deviations from the A versus c1 relationship. When 10^?7kmolm^?3 ABA was applied continuously through the transpiration stream, gs, A and Cⁱ decreased, but there was no substantial deviation from the A versus c{ curve. Fluorescence images were patchy as stomata closed for all experiments, but became slowly more uniform during the time that gas exchange was returning to the A versus Cj curve. The distribution of con-ductance among patches was not bimodal, and larger devi-ations from the A versus ct curve had greater ranges of pixel values and more pixel values representing low values of Cj during stomatal closure than did experiments show-ing small or no deviation. Estimates of A and gs from fluo-rescence images compared favourably with measured val-ues in most cases, suggesting that the patchy distributions of fluorescence were caused by patchy distributions of stomatal conductance and that apparent reductions in the A versus ct relationship were the result of these patchy stomatai distributions and not direct effects of ABA on mesophyll functioning. The data show that stomatal patches can be temporary and that patchiness may not be reflected in gas exchange data if the range of stomatal con-ductances is not large. These observations may explain some of the discrepancies among previous studies concerning the effect of ABA on the A versus Cⁱ relationship.     

Optimal water-use efficiency in a California shrub

Abstract. The stomatal behaviour of a California chaparral shrub was compared with that predicted by a model of optimal water-use efficiency (Cowan & Farquhar, 1977). The daily courses of stomatal conductance, evaporation, and assimilation were calculated as was the derivative of evaporation with respect to assimilation (∂ E /∂ A ). The derivative ∂ E /∂ A was not constant, and the measured courses of conductance were not optimal, but daily courses of evaporation and assimilation were usually very close to what would have been predicted had ∂ E /∂ A been constant. This discrepancy arises because evaporation and assimilation are sometimes so severely constrained by the microenvironment that stomatal conductance has almost no effect on them.     

A fast method to detect the occurrence of nonhomogeneous distribution of stomatal aperture in heterobaric plant leaves

Summary Pressure infiltration of water into a leaf via the stomatal pores can be used to quickly determine whether all stomata are open, or as recently described for several mesophytic and xerophytic species, whether there is a non-homogeneous distribution of stomatal opening (stomatal patchiness) on the leaf surface. Information about this phenomenon is important since the commonly used algorithms for calculation of leaf conductance from water vapor exchange measurements imply homogeneously open stomata, which in the occurrence of stomatal patchiness will lead to erroneous results. Infiltration experiments in a growth chamber with leaves of the Mediterranean evergreen shrub Arbutus unedo, carried out under simulated Mediterranean summer day conditions, where the species typically exhibits a strong midday stomatal closure, revealed a temporary occurrence of stomatal patchiness during the phase of stomatal closure in the late morning and during the stomatal reopening in the afternoon. Leaves were, however, found to be fully (i.e. homogeneously) infiltratable in the morning and in the evening. At midday during maximum stomatal closure, leaves were almost non-infiltratable. During the day, the infiltrated amount of water was found to be linearly correlated with porometer measurements of leaf conductance of the same leaves, carried out with the attached leaves immediately before infiltration.     

Dynamics of stomatal patches for a single surface of Xanthium strumarium L. leaves observed with fluorescence and thermal images

Fluorescence and thermal imaging were used to examine the dynamics of stomatal patches for a single surface of Xanthium strumarium L. leaves following a decrease in ambient humidity. Patches were not observed in all experiments, and in many experiments the patches were short-lived. In some experiments, however, patches persisted for many hours and showed complex temporal and spatial patterns. Rapidly sampled fluorescence images showed that the measurable variations of these patches were sufficiently slow to be captured by fluorescence images taken at 3-min intervals using a saturating flash of light. Stomatal patchiness with saturating flashes of light was not demonstrably different from that without saturating flashes of light, suggesting that the regular flashes of light did not directly cause the phenomenon. Comparison of simultaneous fluorescence and thermal images showed that the fluorescence patterns were largely the result of stomatal conductance patterns, and both thermal and fluorescence images showed patches of stomatal conductance that propagated coherently across the leaf surface. These nondispersing patches often crossed a given region of the leaf repeatedly at regular intervals, resulting in oscillations in stomatal conductance for that region. The existence of these coherently propagating structures has implications for the mechanisms that cause patchy stomatal behaviour as well as for the physiological ramifications of this phenomenon.     

Hydraulic responses to height growth in maritime pine trees

As trees grow taller, decreased xylem path conductance imposes a major constraint on plant water and carbon balance, and is thus a key factor underlying forest productivity decline with age. The responses of stomatal conductance, leaf area: sapwood area ratio (A_L : A_S) and soil–leaf water potential gradient (ΔΨ_S–L) to height growth were investigated in maritime pine trees. Extensive measurements of in situ sap flow, stomatal conductance and (non‐gravitational) needle water potential (_L = Ψ_L ? ρ_wgh) were made during 2 years in a chronosequence of four even‐aged stands, under both wet and dry soil conditions. Under wet soil conditions, _L was systematically lower in taller trees on account of differences in gravitational potential. In contrast, under dry soil conditions, our measurements clearly showed that _L was maintained above a minimum threshold value of ?2.0 MPa independently of tree height, thus limiting the range of compensatory change in ΔΨ_S–L. Although a decrease in the A_L : A_S ratio occurred with tree height, this compensation was not sufficient to prevent a decline in leaf‐specific hydraulic conductance, K_L (50% lower in 30 m trees than in 10 m trees). An associated decline in stomatal conductance with tree height thus occurred to maintain a balance between water supply and demand. Both the increased investment in non‐productive versus productive tissues (A_S : A_L) and stomatal closure may have contributed to the observed decrease in tree growth efficiency with increasing tree height (by a factor of three from smallest to tallest trees), although other growth‐limiting responses (e.g. soil nutrient sequestration, increased respiratory costs) cannot be excluded.     

Dynamics of patchy stomatal movements, and their contribution to steady-state and oscillating stomatal conductance calculated using gas-exchange techniques

Patchy stomatal movements were induced in leaves of Helianthus annuus L. and Xanthium strumarium L. by increasing Δw and decreasing light in a gas-exchange cuvette. The dynamics of the patchy movements were recorded and analysed using images of chlorophyll fluorescence, and the influence of heterogeneous stomatal activity on gas-exchange measurements of whole-leaf stomatal conductance was explored. Image series and gas-exchange measurements from two contrasting 100 min experiments are presented. One series of images, taken using Helianthus annuus, was characterized by strongly oscillating stomatal conductance induced by a decrease in light at high Δw. Fluorescence analysis revealed that individual patches of the leaf displayed a variety of behaviours (from static to strongly oscillating fluorescence), which, when averaged, matched the time dependence of the oscillating stomatal conductance measured by gas-exchange techniques. During the second series of images, taken using Xanthium strumarium, stomatal conductance (measured with gas exchange) declined slightly after an increase in Δw, and then maintained a steady state. Again, some patches in this leaf showed highly dynamic q_NP, although on the whole q_NP varied without any obvious pattern or frequency. When all patch activity in this series was averaged, it paralleled the steady whole-leaf stomatal conductance determined by gas-exchange measurements. It is clear from this work that coordinated patchy stomatal movements can contribute significantly to the dynamics of whole-leaf stomatal conductance, and, in contrast, that dynamic but uncoordinated patchy movements can average to produce a steady gas-exchange trace.     

Asymmetric patchy stomatal closure for the two surfaces of Xanthium strumarium L. leaves at low humidity

Images of chlorophyll fluorescence were used to demonstrate patchy stomatal closure at low humidities in leaves of well-watered Xanthium strumarium plants. The pattern and extent of patchy stomatal closure were shown to be different for the two surfaces of amphistomatous leaves by taking images of leaves with CO₂ available to only one leaf was exposed to low humidity, patchiness was more extensive on that surface. Gas-exchange experiments were also conducted to determine the apparent photosynthetic capacity of the mesophyll (photosynthesis rate at constant c_i when it was supplied with CO₂ through both surfaces or through each surface alone. These experiments showed declines in the apparent photosynthetic capacity of the mesophyll at low humidities that were consistent with patchy stomatal closure on one or both surfaces. The results suggest that patchy stomatal closure can be a factor in the steady-state reponses of stomata to humidity. In amphistomatous leaves this is further complicated by the fact that patches on one epidermis may not coincide with those of the other surface.     

Rapid and Specific Modulation of Stomatal Conductance by Blue Light in Ivy (Hedera helix) : An Approach to Assess the Stomatal Limitation of Carbon Assimilation

Low intensity (0.015 millimole per square meter per second) blue light applied to leaves of Hedera helix under a high intensity red light background (0.50 millimole per square meter per second red light) induced a specific stomatal opening response, with rapid kinetics comparable to those previously reported for stomata with `grass type' morphology. The response of stomatal conductance to blue light showed a transient `overshoot' behavior at high vapor pressure difference (2.25 ± 0.15 kiloPascals), but not at low vapor pressure difference (VPD) (0.90 ± 0.10 kilo-Pascal). The blue light-induced conductance increase was accompanied by an increase in net photosynthetic carbon assimilation, mediated by an increase in the intercellular concentration of carbon dioxide. Values of assimilation once the blue light-stimulated conductance increase reached steady state were less than those at the peak of the overshoot, but the ratios of assimilation to transpiration (A/E) and blue light-stimulated ΔA/ΔE were greater during the steady-state response than during the overshoot. These results indicate that significant stomatal limitation of assimilation can occur, but that this limitation may improve water use efficiency under high VPD conditions. Under high intensity red light, the decline in A/E associated with an increase in VPD was minimized when conductance was stimulated by additional low intensity blue light. This effect indicates that the blue light response of stomata may be important in H. helix for the optimization of water use efficiency under natural conditions of high irradiance and VPD.     

Photosynthesis Inhibition During Gas Exchange Oscillations in ABA-Treated Helianthus annuus: Relative Role of Stomatal Patchiness and Leaf Carboxylation Capacity

Environmental factors that induce spatial heterogeneity of stomatal conductance, g _s, called stomatal patchiness, also reduce the photochemical capacity of CO₂ fixation, yet current methods cannot distinguish between the relative effect of stomatal patchiness and biochemical limitations on photosynthetic capacity. We evaluate effects of stomatal patchiness and the biochemical capacity of CO₂ fixation on the sensitivity of net photosynthetic rate (P _N) to stomatal conductance (g _s), θ (θ = δP _N/g _s). A qualitative model shows that stomatal patchiness increases the sensitivity θ while reduced biochemical capacity of CO₂ fixation lowers θ. We used this feature to distinguish between stomatal patchiness and mesophyll impairments in the photochemistry of CO₂ fixation. We compared gas exchange of sunflower (Helianthus annuus L.) plants grown in a growth chamber and fed abscisic acid, ABA (10⁻⁵ M), for 10 d with control plants (-ABA). P _N and g _s oscillated more frequently in ABA-treated than in control plants when the leaves were placed into the leaf chamber and exposed to a dry atmosphere. When compared with the initial CO₂ response measured at the beginning of the treatment (day zero), both ABA and control leaves showed reduced P _N at particular sub-stomatal CO₂ concentration (c _i) during the oscillations. A lower reduction of P _N at particular g _s indicated overestimation of c _i due to stomatal patchiness and/or omitted cuticular conductance, g _c. The initial period of damp oscillation was characterised by inhibition of chloroplast processes while stomatal patchiness prevailed at the steady state of gas exchange. The sensitivity θ remained at the original pre-treatment values at high g _s in both ABA and control plants. At low g _s, θ decreased in ABA-treated plants indicating an ABA-induced impairment of chloroplast processes. In control plants, g _c neglected in the calculation of g _s was the likely reason for apparent depression of photosynthesis at low g _s. This revised version was published online in August 2006 with corrections to the Cover Date.     

Photosynthesis Decrease and Stomatal Control of Gas Exchange in Abies alba Mill. in Response to Vapor Pressure Difference

The responses of steady state CO₂ assimilation rate (A), transpiration rate (E), and stomatal conductance (g_s) to changes in leaf-to-air vapor pressure difference (ΔW) were examined on different dates in shoots from Abies alba trees growing outside. In Ecouves, a provenance representative of wet oceanic conditions in Northern France, both A and g_s decreased when ΔW was increased from 4.6 to 14.5 Pa KPa⁻¹. In Nebias, which represented the dry end of the natural range of A. alba in southern France, A and g_s decreased only after reaching peak levels at 9.0 and 7.0 Pa KPa⁻¹, respectively. The representation of the data in assimilation rate (A) versus intercellular CO₂ partial pressure (C_i) graphs allowed us to determine how stomata and mesophyll photosynthesis interacted when ΔW was increased. Changes in A were primarily due to alterations in mesophyll photosynthesis. At high ΔW, and especially in Ecouves when soil water deficit prevailed, A declined, while C_i remained approximately constant, which may be interpreted as an adjustment of g_s to changes in mesophyll photosynthesis. Such a stomatal control of gas exchange appeared as an alternative to the classical feedforward interpretation of E versus ΔW responses with a peak rate of E. The gas exchange response to ΔW was also characterized by considerable deviations from the optimization theory of IR Cowan and GD Farquhar (1977 Symp Soc Exp Biol 31: 471-505).