Stomatal action directly feeds back on leaf turgor: new insights into the regulation of the plant water status from non‐invasive pressure probe measurements

Uptake of CO₂ by the leaf is associated with loss of water. Control of stomatal aperture by volume changes of guard cell pairs optimizes the efficiency of water use. Under water stress, the protein kinase OPEN STOMATA 1 (OST1) activates the guard‐cell anion release channel SLOW ANION CHANNEL‐ASSOCIATED 1 (SLAC1), and thereby triggers stomatal closure. Plants with mutated OST1 and SLAC1 are defective in guard‐cell turgor regulation. To study the effect of stomatal movement on leaf turgor using intact leaves of Arabidopsis, we used a new pressure probe to monitor transpiration and turgor pressure simultaneously and non‐invasively. This probe permits routine easy access to parameters related to water status and stomatal conductance under physiological conditions using the model plant Arabidopsis thaliana. Long‐term leaf turgor pressure recordings over several weeks showed a drop in turgor during the day and recovery at night. Thus pressure changes directly correlated with the degree of plant transpiration. Leaf turgor of wild‐type plants responded to CO₂, light, humidity, ozone and abscisic acid (ABA) in a guard cell‐specific manner. Pressure probe measurements of mutants lacking OST1 and SLAC1 function indicated impairment in stomatal responses to light and humidity. In contrast to wild‐type plants, leaves from well‐watered ost1 plants exposed to a dry atmosphere wilted after light‐induced stomatal opening. Experiments with open stomata mutants indicated that the hydraulic conductance of leaf stomata is higher than that of the root–shoot continuum. Thus leaf turgor appears to rely to a large extent on the anion channel activity of autonomously regulated stomatal guard cells.     

Interpretation of an empirical model for stomatal conductance in terms of guard cell function

An empirical model for stomatal conductance (g), proposed by Leuning (1995, this issue) as a modification of Ball, Woodrow & Berry's (1987) model, is interpreted in terms of a simple, steady-state model of guard cell function. In this model, stomatal aperture is a function of the relative turgor between guard cells and epidermal cells. The correlation between g and leaf surface vapour pressure deficit in Leuning's model is interpreted in terms of stomatal sensing of the transpiration rate, via changes in the gradient of total water potential between guard cells and epidermal cells. The correlation between g, CO₂ assimilation rate and leaf surface CO₂ concentration in Leuning's model is interpreted as a relationship between the corresponding osmotic gradient, irradiance, temperature, intercellular CO₂ concentration and stomatal aperture itself. The explicit relationship between osmotic gradient and stomatal aperture (possibly describing the effect of changes in guard cell volume on the membrane permeability for ion transport) results in a decrease in the transpiration rate in sufficiently dry air. Possible extension of the guard cell model to include stomatal responses to soil water status is discussed.     

Guard cell photosynthesis is critical for stomatal turgor production,yet does not directly mediate CO2‐ and ABA‐induced stomatal closing

Stomata mediate gas exchange between the inter‐cellular spaces of leaves and the atmosphere. CO₂ levels in leaves (Ci) are determined by respiration, photosynthesis, stomatal conductance and atmospheric [CO₂]. [CO₂] in leaves mediates stomatal movements. The role of guard cell photosynthesis in stomatal conductance responses is a matter of debate, and genetic approaches are needed. We have generated transgenic Arabidopsis plants that are chlorophyll‐deficient in guard cells only, expressing a constitutively active chlorophyllase in a guard cell specific enhancer trap line. Our data show that more than 90% of guard cells were chlorophyll‐deficient. Interestingly, approximately 45% of stomata had an unusual, previously not‐described, morphology of thin‐shaped chlorophyll‐less stomata. Nevertheless, stomatal size, stomatal index, plant morphology, and whole‐leaf photosynthetic parameters (PSII, qP, qN, F_V′/F_M′) were comparable with wild‐type plants. Time‐resolved intact leaf gas‐exchange analyses showed a reduction in stomatal conductance and CO₂‐assimilation rates of the transgenic plants. Normalization of CO₂ responses showed that stomata of transgenic plants respond to [CO₂] shifts. Detailed stomatal aperture measurements of normal kidney‐shaped stomata, which lack chlorophyll, showed stomatal closing responses to [CO₂] elevation and abscisic acid (ABA), while thin‐shaped stomata were continuously closed. Our present findings show that stomatal movement responses to [CO₂] and ABA are functional in guard cells that lack chlorophyll. These data suggest that guard cell CO₂ and ABA signal transduction are not directly modulated by guard cell photosynthesis/electron transport. Moreover, the finding that chlorophyll‐less stomata cause a ‘deflated’ thin‐shaped phenotype, suggests that photosynthesis in guard cells is critical for energization and guard cell turgor production.     

Effects of turgor potentials of epidermal cells neighbouring guard cells on stomatal opening in detached leaf epidermis and intact leaflets of Vicia faba L. (faba bean)

Leaflets of Vicia faba L. (faba bean) were used to determine whether the mechanical forces resulting from the turgor potentials (Φ_p) of the larger epidermal cells neighbouring guard cells play a significant role in regulating stomatal aperture. When Φ_p, of epidermis and Φ_p of bulk leaflet tissue were compared at midday, Φ_p of epidermis were only 15–25% those of bulk leaflet tissue at all but the most negative leaflet water potentials (Φ). When plants were bagged to increase Φ by reducing vapour pressure differences between leaflets and air, Φ_p of bulk leaflet tissue increased to predawn values, but Φ_p, of epidermis increased to only = 20% of predawn values and stomata opened to their widest apertures. Stomatal apertures were positively correlated with Φ_p of bulk leaflet tissue but they were not correlated with Φ_p of epidermis. Reductions in epidermal Φ_p, began predawn, before stomata were open, and reached minimum values at midday, when stomata were open. We conclude that, in Vicia faba, (1) reduction of Φ_p of epidermal cells begins predawn, reducing the counterforce to stomatal opening that would exist if full epidermal turgor were maintained throughout the day, and (2) changes in Φ_p, of leaf epidermal cells do not play a significant role in regulating stomatal aperture.     

Jasmonate‐mediated stomatal closure under elevated CO2 revealed by time‐resolved metabolomics

Foliar stomatal movements are critical for regulating plant water loss and gas exchange. Elevated carbon dioxide (CO₂) levels are known to induce stomatal closure. However, the current knowledge on CO₂ signal transduction in stomatal guard cells is limited. Here we report metabolomic responses of Brassica napus guard cells to elevated CO₂ using three hyphenated metabolomics platforms: gas chromatography‐mass spectrometry (MS); liquid chromatography (LC)‐multiple reaction monitoring‐MS; and ultra‐high‐performance LC‐quadrupole time‐of‐flight‐MS. A total of 358 metabolites from guard cells were quantified in a time‐course response to elevated CO₂ level. Most metabolites increased under elevated CO₂, showing the most significant differences at 10 min. In addition, reactive oxygen species production increased and stomatal aperture decreased with time. Major alterations in flavonoid, organic acid, sugar, fatty acid, phenylpropanoid and amino acid metabolic pathways indicated changes in both primary and specialized metabolic pathways in guard cells. Most interestingly, the jasmonic acid (JA) biosynthesis pathway was significantly altered in the course of elevated CO₂ treatment. Together with results obtained from JA biosynthesis and signaling mutants as well as CO₂ signaling mutants, we discovered that CO₂‐induced stomatal closure is mediated by JA signaling.     

Dynamics of stomatal water relations during the humidity response: implications of two hypothetical mechanisms

The feasibility of two hypothetical mechanisms for the stomatal response to humidity was evaluated by identifying theoretical constraints on these mechanisms and by analysing timecourses of stomatal aperture following a step change in humidity. The two hypothetical mechanisms, which allow guard cell turgor pressure to overcome the epidermal mechanical advantage, are: (1) active regulation of guard cell osmotic pressure, requiring no hydraulic disequilibrium between guard and epidermal cells, and (2) a substantial hydraulic resistance between guard and epidermal cells, resulting in hydraulic disequilibrium between them. Numerical simulations of the system are made possible by recently published empirical relationships between guard cell pressure and volume and between stomatal aperture, guard cell turgor pressure, and epidermal cell turgor pressure; these data allow the hypothetical control variables to be inferred from stomatal aperture and evaporative demand, given physical assumptions that characterize either hypothesis. We show that hypothesis (1) predicts that steady‐state π_g is monotonically related to transpiration rate, whereas hypothesis (2) suggests that the relationship between transpiration rate and the steady‐state guard to epidermal cell hydraulic resistance may be either positive or negative, and that this resistance must change substantially during the transient phase of the stomatal response to humidity.     

Critical Water Potential for Stomatal Closure in Sitka Spruce

Steady state rates of net photosynthesis and stomatal conductance at high water potentials were measured under controlled conditions in a leaf chamber on Sitka spruce [Picea sitchensis (Bong.) Carr.] shoots detached from the forest canopy or on seedlings. The water supply to the seedlings was terminated by excision and the shoot water potential (or critical water potential) and osmotic potential at the onset of stomatal closure measured. The turgor potential was calculated. The initial osmotic potential before insertion of the shoot into the chamber was also measured. Shoot water potential and osmotic potential at stomatal closure, and initial osmotic potential were significantly higher (less negative) in foliage from the lowest level in the canopy compared with foliage in the upper canopy, and higher in shoots of seedlings transferred to low light than in those at high light. Critical water potential also varied with season, being higher in July than in October and November. In all except one instance, turgor potential at the onset of stomatal closure was negative, possibly because of dilution of the cell sap by the extracellular water during the estimate of osmotic potential. Over all the experiments variation in critical water potential was correlated with variation in critical osmotic potential and, to a lesser extent, the initial osmotic potential. However, turgor potential at the critical potential varied from +0.6 to -4.6 bar. This suggests that difference in turgor between the guard cells and subsidiary cells, which controls stomatal aperture, is only loosely coupled with the bulk leaf turgor and hence that bulk leaf turgor is not a good index of the turbor relations of the guard cells.     

The Effect of Epidermal Cell Water Potential on Stomatal Response to Illumination of Leaf Discs of Vicia faba

Illuminated leaf discs of Vicia faba were brought into equilibrium with a series of mannitol solutions. The width of stomatal aperture and the osmotic potential of guard cells and epidermal cells were determined. It was found that the maximal aperture was obtained when epidermal cells were at about incipient plasmolysis and that any increase in their turgor pressure brought about a decrease in stomatal aperture. These findings emphasize the importance of epidermal cells in determining the width of the stomatal pore.     

Insights into the Molecular Mechanisms of CO2-Mediated Regulation of Stomatal Movements

Plants must continually balance the influx of CO₂ for photosynthesis against the loss of water vapor through stomatal pores in their leaves. This balance can be achieved by controlling the aperture of the stomatal pores in response to several environmental stimuli. Elevation in atmospheric [CO₂] induces stomatal closure and further impacts leaf temperatures, plant growth and water-use efficiency, and global crop productivity. Here, we review recent advances in understanding CO₂-perception mechanisms and CO₂-mediated signal transduction in the regulation of stomatal movements, and we explore how these mechanisms are integrated with other signaling pathways in guard cells.     

Whole-plant gas exchange responses of Spartina alterniflora (Poaceae) to a range of constant and transient salinities

A two-chamber-system was used to study whole-plant gas exchange responses of Spartina alterniflora to long-term and transient salinity treatments over the range of 5 to 40 ppt NaCl. Lower photosynthetic rates, leaf water vapor conductances, belowground respiration rates, and higher aboveground respiration rates in plants adapted to 40 ppt NaCl were observed. Area-specific leaf weight increased with salinity, although the salt content of leaf tissues did not. A reduced rate of gross photosynthesis and higher aboveground respiration rate in 40-ppt NaCl plants significantly lowered the net whole-plant CO₂ gain below that of 5-ppt NaCl plants, while the net CO₂ gain of 25-ppt NaCl plants was intermediate. Within 6 hr of increasing the salinity of 5- and 25-ppt NaCl plants by 20 and 15 ppt NaCl, S. alterniflora responded by reducing leaf water vapor conductance, which in turn reduced the photosynthetic rate. This response was reversed by returning the plants to their original salinity, which indicates that S. alterniflora adjusts water loss and gas exchange in response to transient salinity stress by regulating stomatal aperture. On the other hand, decreasing salinity of the growth media of plants cultured at 25 and 40 ppt NaCl had little or no effect on gas exchange characteristics. This suggests that S. alterniflora adapts to constant salinity through fixed, salinity-dependent structural modifications, such as stomatal density.     

Malate-sensitive anion channels enable guard cells to sense changes in the ambient CO2 concentration

Malate is a characteristic metabolite in the photosynthesis of C4 and CAM plants. Furthermore, changes in the intracellular concentration of this organic acid provide part of the osmotic motor for guard cells. Since alterations in the malate concentration influence the photosynthetic capacity on one side and stomatal action on the other, it was studied whether the extracellular malate level represents an indicator of changes in the ambient CO₂ concentration and a key regulator of ion transport in guard cells. Here it is demonstrated that alterations in the ambient CO₂ level modify the extracellular malate concentration of Vicia faba leaves. Elevated external malate caused stomatal closure in a concentration-dependent manner (K_m^mal = 0.3 mM). Slight variations in the external malate concentration strongly regulate the voltage-dependent properties of GCAC1, an anion-release channel in the plasma membrane of guard cells. Superfusion of guard cell protoplasts with malate levels in the physiological range (K_m^mal = 0.4 mM) caused the voltage gate to shift towards the resting potential of the cell-activating GCAC1. Single-channel conductance was dependent on the extracellular chloride concentration (K_m^Cl = 3 mM). In the absence of extracellular chloride the plasma membrane lacked anion conductance until the addition of malate induced channel opening. Isophthalate was a powerful agonist in both malate-induced processes, channel regulation and stomatal closure, indicating that modulation of GCAC1 is a key step in stomatal action. It was thus concluded that feedback regulation of volume and turgor with respect to the ambient CO₂ concentration via malate-sensitive anion channels may provide a CO₂ sensor to guard cells.     

The relationship between guard cell water potential and the aperture of stomata in Populus

Abstract Previous work with clones of Populus trichocarpa demonstrated that the water vapour conductance of leaves from well-watered cuttings of this species does not decline with loss of turgor from the bulk leaf. In the present study, stomatal responses to water potential in Populus were examined with detached epidermal strips. Stomata in epidermal strips from well-watered plants of P. trichocarpa did not close at low water potentials which led to plasmolysis of the guard cells. In contrast, stomata of P. deltoides and a P. trichocarpa×deltoides hybrid closed when the guard cells lost turgor. A period of water stress preconditioning resulted in modified stomatal responses in P. trichocarpa such that stomata of stressed and re-watered plants nearly closed when guard cell turgor was lost.     

Stomatal function,density and pattern,and CO2 assimilation in Arabidopsis thaliana tmm1 and sdd1‐1 mutants

• Stomata modulate the exchange of water and CO₂ between plant and atmosphere. Although stomatal density is known to affect CO₂ diffusion into the leaf and thus photosynthetic rate, the effect of stomatal density and patterning on CO₂ assimilation is not fully understood.
• We used wild types Col‐0 and C24 and stomatal mutants sdd1‐1 and tmm1 of Arabidopsis thaliana, differing in stomatal density and pattern, to study the effects of these variations on both stomatal and mesophyll conductance and CO₂ assimilation rate. Anatomical parameters of stomata, leaf temperature and carbon isotope discrimination were also assessed.
• Our results indicate that increased stomatal density enhanced stomatal conductance in sdd1‐1 plants, with no effect on photosynthesis, due to both unchanged photosynthetic capacity and decreased mesophyll conductance. Clustering (abnormal patterning formed by clusters of two or more stomata) and a highly unequal distribution of stomata between the adaxial and abaxial leaf sides in tmm1 mutants also had no effect on photosynthesis.
• Except at very high stomatal densities, stomatal conductance and water loss were proportional to stomatal density. Stomatal formation in clusters reduced stomatal dynamics and their operational range as well as the efficiency of CO₂ transport.

     

Stomatal Opening in Isolated Epidermal Strips of Vicia faba. I. Response to Light and to CO(2)-free Air

This paper reports a consistent and large opening response to light + CO₂-free air in living stomata of isolated epidermal strips of Vicia faba. The response was compared to that of non-isolated stomata in leaf discs floating on water; stomatal apertures, guard cell solute potentials and starch contents were similar in the 2 situations. To obtain such stomatal behavior, it was necessary to float epidermal strips on dilute KCl solutions. This suggests that solute uptake is necessary for stomatal opening.

The demonstration of normal stomatal behavior in isolated epidermal strips provides a very useful system in which to investigate the mechanism of stomatal opening. It was possible to show independent responses in stomatal aperture to light and to CO₂-free air.

     

Control of Volume and Turgor in Stomatal Guard Cells

Water loss from plants is determined by the aperture of stomatal pores in the leaf epidermis, set by the level of vacuolar accumulation of potassium salt, and hence volume and turgor, of a pair of guard cells. Regulation of ion fluxes across the tonoplast, the key to regulation of stomatal aperture, can only be studied by tracer flux measurements. There are two transport systems in the tonoplast. The first is a Ca²⁺-activated channel, inhibited by phenylarsine oxide (PAO), responsible for the release of vacuolar K⁺(Rb⁺) in response to the “drought” hormone, abscisic acid (ABA). This channel is sensitive to pressure, down-regulated at low turgor and up-regulated at high turgor, providing a system for turgor regulation. ABA induces a transient stimulation of vacuolar ion efflux, during which the flux tracks the ion content (volume, turgor), suggesting ABA reduces the set-point of a control system. The second system, which is PAO-insensitive, is responsible for an ion flux from vacuole to cytoplasm associated with inward water flow following a hypo-osmotic transfer. It is suggested that this involves an aquaporin as sensor, and perhaps also as responder; deformation of the aquaporin may render it ion-permeable, or, alternatively, the deformed aquaporin may signal to an associated ion channel, activating it. Treatment with inhibitors of aquaporins, HgCl₂ or silver sulfadiazine, produces a large transient increase in ion release from the vacuole, also PAO-insensitive. It is suggested that this involves the same aquaporin, either rendered directly ion-permeable, or signalling to activate an associated ion channel.     

Asymmetric responses of adaxial and abaxial stomata to elevated CO2: impacts on the control of gas exchange by leaves

The response of adaxial and abaxial stomatal conductance in Rumex obtusifolius to growth at elevated atmospheric concentrations of CO₂ (250 μmol mol^?1 above ambient) was investigated over two growing seasons. The conductance of both the adaxial and abaxial leaf surfaces was found to be reduced by elevated concentrations of CO₂. Elevated CO₂ caused a much greater reduction in conductance for the adaxial surface than for the abaxial surface. The absence of effects upon stomatal density indicated that the reductions were probably the result of changes in stomatal aperture. Partitioning of gas exchange between the leaf surfaces revealed that increased concentrations of CO₂ caused increased rates of photosynthesis only via the abaxial surface. Additionally, leaf thickness was found to increase during growth at elevated concentrations of CO₂. The tendency for these amphistomatous leaves to develop a distribution of conductance approaching that of hypostomatous leaves clearly reduced their maximum photosynthetic potential. This conclusion was supported by measurements of stomatal limitation, which showed greater values for the adaxial surfaces, and greater values at elevated CO₂. This reduction in photosynthesis may in part be caused by higher diffusive limitations imposed because of increased leaf thickness. In an uncoupled canopy, asymmetrical stomatal responses of the kind identified here may appreciably reduce transpiration. Species which show symmetrical responses are less likely to show reduced transpirational rates, and a redistribution of water loss between species may occur. The implications of asymmetrical stomatal responses for photosynthesis and canopy transpiration are discussed.     

Drought-induced H2O2 accumulation in subsidiary cells is involved in regulatory signaling of stomatal closure in maize leaves

Increasing H₂O₂ levels in guard cells in response to environmental stimuli are recently considered a general messenger involved in the signaling cascade for the induction of stomatal closure. But little is known as to whether subsidiary cells participate in the H₂O₂-mediated stomatal closure of grass plants. In the present study, 2-week-old seedlings of maize (Zea mays) were exposed to different degrees of soil water deficit for 3 weeks. The effects of soil water contents on leaf ABA and H₂O₂ levels and stomatal aperture were investigated using physiological, biochemical, and histochemical approaches. The results showed that even under well-watered conditions, significant amounts of H₂O₂ were observed in guard cells, whereas H₂O₂ concentrations in the subsidiary cells were negligible. Decreasing soil water contents led to a significant increase in leaf ABA levels associated with significantly enhanced O₂ ^? and H₂O₂ contents, consistent with reduced degrees of stomatal conductance and aperture. The significant increase in H₂O₂ appeared in both guard cells and subsidiary cells of the stomatal complex, and H₂O₂ levels increased with decreasing soil water contents. Drought-induced increase in the activity of antioxidative enzymes could not counteract the significant increase in H₂O₂ levels in guard cells and subsidiary cells. These results indicate that subsidiary cells participate in H₂O₂-mediated stomatal closure, and drought-induced H₂O₂ accumulation in subsidiary cells is involved in the signaling cascade regulating stomatal aperture of grass plants such as maize.     

Slow photosynthetic induction and low photosynthesis in Paphiopedilum armeniacum are related to its lack of guard cell chloroplast and peculiar stomatal anatomy

Paphiopedilum and Cypripedium are close relatives in the subfamily Cypripedioideae. Cypripedium leaves contain guard cell chloroplasts, whereas Paphiopedilum do not. It is unclear whether the lack of guard cell chloroplasts affects photosynthetic induction, which is important for understory plants to utilize sunflecks. To understand the role of guard cell chloroplasts in photosynthetic induction of Paphiopedilum and Cypripedium, the stomatal anatomy and photosynthetic induction of Paphiopedilum armeniacum and Cypripedium flavum were investigated at different ratios of red to blue light. The highest stomatal opening and photosynthesis of intact leaves in P. armeniacum were induced by irradiance enriched with blue light. Its stomatal opening could be induced by red light 250 µmol m^?2 s^?1, but the magnitude of stomatal opening was lower than those at the other light qualities. However, the stomatal opening and photosynthesis of C. flavum were highly induced by mixed blue and red light rather than pure blue or red light. The two orchid species did not differ in stomatal density, but P. armeniacum had smaller stomatal size than C. flavum. The stomata of P. armeniacum were slightly sunken into the leaf epidermis, while C. flavum protruded above the leaf surface. The slower photosynthetic induction and lower photosynthetic rate of P. armeniacum than C. flavum were linked to the lack of guard cell chloroplasts and specific stomatal structure, which reflected an adaptation of Paphiopedilum to periodic water deficiency in limestone habitats. These results provide evidence for the morphological and physiological evolution of stomata relation for water conservation under natural selection.     

Stomatal sensitivity to vapour pressure difference over a subambient to elevated CO2 gradient in a C3/C4 grassland

In the present study the response of stomatal conductance (g_s) to increasing leaf‐to‐air vapour pressure difference (D) in early season C₃ (Bromus japonicus) and late season C₄ (Bothriochloa ischaemum) grasses grown in the field across a range of CO₂ (200–550 µmol mol^?1) was examined. Stomatal sensitivity to D was calculated as the slope of the response of g_s to the natural log of externally manipulated D (dg_s/dlnD). Increasing D and CO₂ significantly reduced g_s in both species. Increasing CO₂ caused a significant decrease in stomatal sensitivity to D in Br. japonicus, but not in Bo. ischaemum. The decrease in stomatal sensitivity to D at high CO₂ for Br. japonicus fit theoretical expectations of a hydraulic model of stomatal regulation, in which g_s varies to maintain constant transpiration and leaf water potential. The weaker stomatal sensitivity to D in Bo. ischaemum suggested that stomatal regulation of leaf water potential was poor in this species, or that non‐hydraulic signals influenced guard cell behaviour. Photosynthesis (A) declined with increasing D in both species, but analyses of the ratio of intercellular to atmospheric CO₂ (C_i/C_a) suggested that stomatal limitation of A occurred only in Br. japonicus. Rising CO₂ had the greatest effect on g_s and A in Br. japonicus at low D. In contrast, the strength of stomatal and photosynthetic responses to CO₂ were not affected by D in Bo. ischaemum. Carbon and water dynamics in this grassland are dominated by a seasonal transition from C₃ to C₄ photosynthesis. Interspecific variation in the response of g_s to D therefore has implications for predicting seasonal ecosystem responses to CO₂.     

Photosynthetic Responses of a Temperate Liana to Xylella fastidiosa Infection and Water Stress

Xylella fastidiosa is a xylem‐limited bacterial plant pathogen that causes bacterial leaf scorch in its hosts. Our previous work showed that water stress enhances leaf scorch symptom severity and progression along the stem of a liana, Parthenocissus quinquefolia, infected by X. fastidiosa. This paper explores the photosynthetic gas exchange responses of P. quinquefolia, with the aim to elucidate mechanisms behind disease expression and its interaction with water stress. We used a 2 × 2‐complete factorial design, repeated over two growing seasons, with high and low soil moisture levels and infected and non‐infected plants. In both years, low soil moisture levels reduced leaf water potentials, net photosynthesis and stomatal conductance at all leaf positions, while X. fastidiosa‐infection reduced these parameters at basally located leaves only. Intercellular CO₂ concentrations were reduced in apical leaves, but increased at the most basal leaf location, implicating a non‐stomatal reduction of photosynthesis in leaves showing the greatest disease development. This result was supported by measured reductions in photosynthetic rates of basal leaves at high CO₂ concentrations, where stomatal limitation was eliminated. Repeated measurements over the summer of 2000 showed that the effects of water stress and infection were progressive over time, reaching their greatest extent in September. By reducing stomatal conductances at moderate levels of water stress, P. quinquefolia maintained relatively high leaf water potentials and delayed the onset of photosynthetic damage due to pathogen and drought‐induced water stress. In addition, chlorophyll fluorescence measurements showed that P. quinquefolia has an efficient means of dissipating excess light energy that protects the photosynthetic machinery of leaves from irreversible photoinhibitory damage that may occur during stress‐induced stomatal limitation of photosynthesis. However, severe stress induced by disease and drought eventually led to non‐stomatal decreases in photosynthesis associated with leaf senescence.