Analysis of Guard Cell Viability and Action in Senescing Leaves of Nicotiana glauca (Graham), Tree Tobacco

In an attempt to determine whether low epidermal conductances to water vapor diffusion of senescing leaves were caused by internal changes in guard cells or by factors external to guard cells, stomatal behavior was examined in intact senescing and nonsenescing leaves of Nicotiana glauca (Graham), tree tobacco, grown in the field or in an environmental chamber. Conductances of senescing leaves were 5 to 10% of the maximum conductances of nonsenescing leaves of the same plant, yet guard cell duplexes isolated from epidermal peels of senescing leaves developed full turgor in the light in solutions containing KCl, and sodium cobaltinitrite staining showed that K⁺ accumulated as turgor developed. Ninety-five per cent of the guard cells isolated from senescing leaves concentrated neutral red and excluded trypan blue. Intercellular leaf CO₂ concentrations of senescing and nonsenescing leaves of chamber-grown plants were not significantly different (about 240 microliters per liter), but the potassium contents of adaxial and abaxial epidermes of senescing leaves taken from plants grown in the field were less than half those of nonsenescing leaves. We conclude that guard cells do not undergo the orderly senescence process that characteristically takes place in mesophyll tissue during whole-leaf senescence and that the reduced conductances of senescing leaves are produced by factors external to guard cells.     

Distinct light responses of the adaxial and abaxial stomata in intact leaves of Helianthus annuus L

Using a laboratory-constructed system that can measure the gas exchange rates of two leaf surfaces separately, the light responses of the adaxial and abaxial stomata in intact leaves of sunflower ( Helianthus annuus L.) were investigated, keeping the intercellular CO₂ concentration ( C _i) at 300  µ L L⁻¹. When evenly illuminating both sides of the leaf, the stomatal conductance ( g _s) of the abaxial surface was higher than that of the adaxial surface at any light intensity. When each surface of the leaf was illuminated separately, both the adaxial and abaxial stomata were more sensitive to the light transmitted through the leaf (self-transmitted light) than to direct illumination. Relationships between the whole leaf photosynthetic rate ( A _n) and the g _s for each side highlighted a strong dependence of stomatal opening on mesophyll photosynthesis. Light transmitted through another leaf was more effective than the direct white light for the abaxial stomata, but not for the adaxial stomata. Moreover, green monochromatic light induced an opening of the abaxial stomata, but not of the adaxial stomata. As the proportion of blue light in the transmitted light is less than that in the white light, there may be some uncharacterized light responses, which are responsible for the opening of the abaxial stomata by the transmitted, green light.     

Physiological effects of long-term exposure to low and moderate concentrations of atmospheric NH3 on poplar leaves

Abstract. Poplar shoots ( Populus euramericana L.) obtained from cuttings were exposed for 6 or 8 weeks to NH₃ concentrations of 50 and 100 μgm⁻³ or filtered air in fumigation chambers. After this exposure the rates of NH₃ uptake, transpiration, CO₂ assimilation and respiration of leaves were measured using a leaf chamber. During the long-term exposure also modulated chlorophyll fluorescence measurements were carried out to obtain information about the photosynthetic performance of individual leaves. Both fluorescence and leaf chamber measurements showed a higher photosynthetic activity of leaves exposed to 100 μg NH₃ m⁻³. These leaves showed also a larger leaf conductance and a larger uptake rate of NH₃ than leaves exposed to 50 μg m⁻³ NH₃ or filtered air. The long-term NH₃ exposure did not induce an internal resistance against NH₃ transport in the leaf, nor did it affect the leaf cuticle. So, not only at a short time exposure, but also at a long-term exposure NH₃ uptake into leaves can be calculated from data on the boundary layer and stomatal resistance for H₂O and ambient NH₃-concentration. Furthermore, the NH₃ exposure had no effect on the relation between CO₂-assimilation and stomatal conductance, indicating that NH₃ in concentrations up to 100 μg m⁻³ has no direct effect on stomatal behaviour; for example, by affecting the guard or contiguous cells of the stomata.     

The effects of soil and atmospheric drought on photosynthesis and stomatal control of gas exchange in three coniferous species

The responses of steady-state CO₂ assimilation rate (A), transpiration rate (E), and stomatal conductance (g_s) to changes in leaf-to-air vapour pressure difference (δW) on one hand and to increasing soil drought on the other hand were examined in 2-year-old seedlings of Pseudotsuga menziesii, Pseudotsuga macrocarpa and Cedrus atlantica. Analysing the data through A vs intercellular CO₂ molar fraction (c_i) graphs, we could determine stomatal and mesophyll contributions to changes in A as δW or soil drought were increased. Increasing soil drought affected g_s and mesophyll photosynthesis independently, since clearly distinct predawn leaf water potential (ψ_p) regions appeared in which either stomatal or mesophyll effects prevailed for explaining the changes in A. The two Pseudotsuga species exhibited a large ψ_P range (between ca -0.8 and -1.5 to -1.9 MPa) in which only stomata were responsible for the decrease in A. A dramatic decline in mesophyll photosynthesis was noticed starting from values as high as -1.2 MPa ( C. atlantica ), -1.5 MPa ( P. macrocarpa ) and -1.9 MPa ( P. menziesii ). Increasing ΔW at high soil water content led to a sharp decline in A primarily due to an alteration of mesophyll photosynthesis. Stomatal conductance for CO₂ diffusion was affected in a lesser extent and in close correlation with the changes in mesophyll photosynthesis, which could suggest the existence of a functional linkage between mesophyll photosynthesis and stomata. Surprisingly, the drought resistant P. macrocarpa exhibited the least conservative water use efficiency in response to the two types of drought. In this species drought adaptation seems to be mainly due to its high root growth and soil prospection ability.     

Stomatal Responses and the Senescence of Leaves

Guard cell responses were examined in green and senescing leavesof Victa faba using detached epidermal strips to eliminate influencesfrom the mesophyll. Stomatal opening was greater in epidermalstrips from mature leaves than from senescing leaves althoughthe latter still retained the ability to respond to CO₂ andto kinetin. It was concluded that the decline in stomatal activityduring senescence is an independent but parallel process tochanges occurring in the mesophyll. Vicia faba, leaf senescence, stomata, kinetin     

The influence of leaf thickness on the CO2 transfer conductance and leaf stable carbon isotope ratio for some evergreen tree species in Japanese warm-temperate forests

1. The influence of leaf thickness on internal conductance for CO₂ transfer from substomatal cavity to chloroplast stroma ( g _i) and carbon isotope ratio (δ¹³C) of leaf dry matter was investigated for some evergreen tree species from Japanese temperate forests. g _i was estimated based on the combined measurements of gas exchange and concurrent carbon isotope discrimination.
2. Leaves with thicker mesophyll tended to have larger leaf dry mass per area (LMA), larger surface area of mesophyll cells exposed to intercellular air spaces per unit leaf area ( S _mes) and smaller volume ratio of intercellular spaces to the whole mesophyll (mesophyll porosity).
3. g _i of these leaves was correlated positively to S _mes but negatively to mesophyll porosity. The variation in g _i among these species would be therefore primarily determined by variation of the conductance in liquid phase rather than that in gas phase.
4. δ¹³C was positively correlated to mesophyll thickness and leaf nitrogen content on an area basis. However, g _i values did not correlate to δ¹³C. These results suggest that difference in δ¹³C among the species was not caused by the variation in g _i, but mainly by the difference in long-term photosynthetic capacity.
5. Comparison of our results with those of previous studies showed that the correlation between leaf thickness and g _i differed depending on leaf functional types (evergreen, deciduous or annual). Differences in leaf properties among these functional types were discussed.     

Opinion: Stomatal responses to light and CO2 depend on the mesophyll

The mechanisms by which stomata respond to red light and CO₂ are unknown, but much of the current literature assumes that these mechanisms reside wholly within the guard cells. However, responses of guard cells in isolated epidermes are typically much smaller than those in leaves, and there are several lines of evidence in the literature suggesting that the mesophyll is necessary for these responses in leaves. This paper advances the opinion that although guard cells may have small direct responses to red light and CO₂, most of the stomatal response to these factors in leaves is caused by an unknown signal that originates in the mesophyll.     

The Anisotropy of the Mesophyll and CO2 Capture Sites in Vicia faba L. Leaves at Low Light Intensities

Experiments are reported on the spatial distributions of isotopiccarbon within the mesophyll of detached leaves of the C₃ plantVicia faba L. fed ¹⁴CO₂ at different light intensities. Eachleaf was isolated in a cuvette and ten artificial stomata providedspatial continuity between the ambient atmosphere (0.03–0.05%v/v CO₂) and the mesophyll from the abaxial leaf side. Paradermalleaf layers exhibited spatial profiles of radioactivity whichvaried with the intensity of incident light in 2 min exposures.At low light, when biochemical kinetics should limit CO₂ uptake,sections through palisade cells contained most radioactivity.As the light intensity was increased to approximately 20% offull sunlight, peak radioactivity was observed in the spongycells near the geometric mid-plane of the mesophyll. The resultsindicate that diffusion of carbon dioxide within the mesophyllregulated the relative photosynthetic activity of the palisadeand spongy cells at incident photosynthetically active lightintensities as little as 110 µE m^–2 s^–1 whenCO₂ entered only through the lower leaf surface. Key words: CO₂ capture sites, Vicia faba L., Artificial stomata     

Stomatal sensing of the environment

The effects of environmental factors on stomatal behaviour are reviewed and the questions of whether photosynthesis and transpiration eontrol stomata or whether stomata themselves control the rates of these processes is addressed. Light affects stomata directly and indirectly. Light can act directly as an energy source resulting in ATP formation within guard cells via photophosphorylation, or as a stimulus as in the case of the blue light effects which cause guard cell H⁺ extrusion. Light also acts indirectly on stomata by affecting photosynthesis which influences the intercellular leaf CO₂ concentration ( C _i). Carbon dioxide concentrations in contact with the plasma membrane of the guard cell or within the guard cell acts directly on cell processes responsible for stomatal movements. The mechanism by which CO₂ exerts its effect is not fully understood but, at least in part, it is concerned with changing the properties of guard cell plasma membranes which influence ion transport processes. The C _i may remain fairly constant for much of the day for many species which is the result of parallel responses of stomata and photosynthesis to light. Leaf water potential also influences stomatal behaviour. Since leaf water potential is a resultant of water uptake and storage by the plant and transpirational water loss, any factor which affects these processes, such as soil water availability, temperature, atmospheric humidity and air movement, may indirectly affect stomata. Some of these factors, such as temperature and possibly humidity, may affect stomata directly. These direct and indirect effects of environmental factors interact to give a net opening response upon which is superimposed a direct effect of stomatal circadian rhythmic activity.     

Protective cytokinin action switches to damaging during senescence of detached wheat leaves in continuous light

The effect of exogenous application of the cytokinin meta -topolin [mT; N⁶-( meta -hydroxybenzyl)adenine] on artificial senescence of detached wheat leaves ( Triticum aestivum L. cv. Hereward) was studied and compared in leaves senescing under continuous light (100 µmol photons m⁻² s⁻¹) and darkness. Senescence-induced deterioration in structure and function of the photosynthetic apparatus was characterized by reduction in chlorophyll content, maximal efficiency of photosystem (PS) II photochemistry ( F _v/ F _m) and the rate of CO₂ assimilation, by increase in the excitation pressure on PSII (1 −  q _P) and a level of lipid peroxidation and by modifications in chloroplast ultrastructure. While in darkened leaf segments mT effectively slowed senescence-induced changes in all measured parameters, in light-senescing segments the effect of mT changed into opposite a few days after detachment. We observed an overexcitation of photosynthetic apparatus, as indicated by pronounced increases in the excitation pressure on PSII and in a deepoxidation state of xanthophyll cycle pigments, marked starch grain accumulation in chloroplasts and stimulation of lipid peroxidation in light-senescing leaf segments in mT. Possible mechanisms of acceleration of senescence-accompanying decrease in photosynthetic function and increase in lipid peroxidation during mT influence are discussed. We propose that protective mT action in darkness becomes damaging during artificial senescence in continuous light due to overexcitation of photosynthetic apparatus resulting in oxidative damage.     

Effects of elevated ozone on CO2 uptake and leaf structure in sugar maple under two light environments

The interactive effects of ozone and light on leaf structure, carbon dioxide uptake and short-term carbon allocation of sugar maple ( Acer saccharum Marsh.) seedlings were examined using gas exchange measurements and ¹⁴C-macroautoradiographic techniques. Two-year-old sugar maple seedlings were fumigated from budbreak for 5 months with ambient or 3 × ambient ozone in open-top chambers, receiving either 35% (high light) or 15% (low light) of full sunlight. Ozone accelerated leaf senescence, and reduced net photosynthesis, ¹⁴CO₂ uptake and stomatal conductance, with the effects being most pronounced under low light. The proportion of intercellular space increased in leaves of seedlings grown under elevated ozone and low light, possibly enhancing the susceptibility of mesophyll cells to ozone by increasing the cumulative dose per mesophyll cell. Indeed, damage to spongy mesophyll cells in the elevated ozone × low light treatment was especially frequent. ¹⁴C macroautoradioraphy revealed heterogeneous uptake of ¹⁴CO₂ in well defined areole regions, suggesting patchy stomatal behaviour in all treatments. However, in seedlings grown under elevated ozone and low light, the highest ¹⁴CO₂ uptake occurred along larger veins, while interveinal regions exhibited little or no uptake. Although visible symptoms of ozone injury were not apparent in these seedlings, the cellular damage, reduced photosynthetic rates and reduced whole-leaf chlorophyll levels corroborate the visual scaling of whole-plant senescence, suggesting that the ozone × low light treatment accelerated senescence or senescence-like injury in sugar maple.     

Division of guard cell protoplasts of Nicotiana glauca (Graham) in liquid cultures

Abstract. Guard cells are uniquely differentiated to transduce signals into the metabolic and ion transport processes that result in turgor-driven stomatal movements. We tested the hypothesis that these highly specialized cells are terminally differentiated. Guard cell protoplasts were isolated from abaxial epidermal tissue of leaves of Nicotiana glauca (Graham) and cultured in a medium designed for culturing mesophyll protoplasts of Nicotiana tabacum. Protoplasts were incubated at densities of 2–5 × 10¹¹ cells m⁻³ in eight-well microchamber slides under 50μmol m⁻² s⁻¹ of photons of continuous fluorescent light at 25°C. When the medium was modified by the addition of 100mol m⁻³ of sucrose and by buffering with 10mol m⁻³ of MES buffer at pH 6.1, cell division began within 96h of the time the culture was initiated. After 9d of culture, 80% of surviving cells had synthesized new cell walls, had dedifferentiated, and were dividing to form small colonies. Callus tissue was visible after 4–5 weeks. We conclude that guard cells of Nicotiana glauca are not terminally differentiated, and that guard cell protoplasts of this species have the capacity to grow, synthesize cell walls and divide.     

The stomatal response to CO2 is linked to changes in guard cell zeaxanthin*

The mechanisms mediating CO₂ sensing and light–CO₂ interactions in guard cells are unknown. In growth chamber-grown Vicia faba leaves kept under constant light (500 μ mol m^–2 s^–1) and temperature, guard cell zeaxanthin content tracked ambient [CO₂] and stomatal apertures. Increases in [CO₂] from 400 to 1200 cm³ m^–3 decreased zeaxanthin content from 180 to 80 mmol mol^–1 Chl and decreased stomatal apertures by 7·0 μ m. Changes in zeaxanthin and aperture were reversed when [CO₂] was lowered. Guard cell zeaxanthin content was linearly correlated with stomatal apertures. In the dark, the CO₂-induced changes in stomatal aperture were much smaller, and guard cell zeaxanthin content did not change with chamber [CO₂]. Guard cell zeaxanthin also tracked [CO₂] and stomatal aperture in illuminated stomata from epidermal peels. Dithiothreitol (DTT), an inhibitor of zeaxanthin formation, eliminated CO₂-induced zeaxanthin changes in guard cells from illuminated epidermal peels and reduced the stomatal CO₂ response to the level observed in the dark. These data suggest that CO₂-dependent changes in the zeaxanthin content of guard cells could modulate CO₂-dependent changes of stomatal apertures in the light while a zeaxanthin-independent CO₂ sensing mechanism would modulate the CO₂ response in the dark.     

Responses of Photosynthetic Electron Transport in Stomatal Guard Cells and Mesophyll Cells in Intact Leaves to Light, CO2, and Humidity

High-resolution images of the chlorophyll fluorescence parameter Fq'/Fm' from attached leaves of commelina (Commelina communis) and tradescantia (Tradescantia albiflora) were used to compare the responses of photosynthetic electron transport in stomatal guard cell chloroplasts and underlying mesophyll cells to key environmental variables. Fq'/Fm' estimates the quantum efficiency of photosystem II photochemistry and provides a relative measure of the quantum efficiency of non-cyclic photosynthetic electron transport. Over a range of light intensities, values of Fq'/Fm' were 20% to 30% lower in guard cell chloroplasts than in mesophyll cells, and there was a close linear relationship between the values for the two cell types. The responses of Fq'/Fm' of guard and mesophyll cells to changes of CO2 and O2 concentration were very similar. There were similar reductions of Fq'/Fm' of guard and mesophyll cells over a wide range of CO2 concentrations when the ambient oxygen concentration was decreased from 21% to 2%, suggesting that both cell types have similar proportions of photosynthetic electron transport used by Rubisco activity. When stomata closed after a pulse of dry air, Fq'/Fm' of both guard cell and mesophyll showed the same response; with a marked decline when ambient CO2 was low, but no change when ambient CO2 was high. This indicates that photosynthetic electron transport in guard cell chloroplasts responds to internal, not ambient, CO2 concentration.     

The role of the mesophyll in stomatal responses to light and CO2

Stomatal responses to light and CO₂ were investigated using isolated epidermes of Tradescantia pallida , Vicia faba and Pisum sativum . Stomata in leaves of T. pallida and P. sativum responded to light and CO₂, but those from V. faba did not. Stomata in isolated epidermes of all three species could be opened on KCl solutions, but they showed no response to light or CO₂. However, when isolated epidermes of T. pallida and P. sativum were placed on an exposed mesophyll from a leaf of the same species or a different species, they regained responsiveness to light and CO₂. Stomatal responses in these epidermes were similar to those in leaves in that they responded rapidly and reversibly to changes in light and CO₂. Epidermes from V. faba did not respond to light or CO₂ when placed on mesophyll from any of the three species. Experiments with single optic fibres suggest that stomata were being regulated via signals from the mesophyll produced in response to light and CO₂ rather than being sensitized to light and CO₂ by the mesophyll. The data suggest that most of the stomatal response to CO₂ and light occurs in response to a signal generated by the mesophyll.     

The responses of guard and mesophyll cell photosynthesis to CO2, O2, light,and water stress in a range of species are similar

High resolution chlorophyll a fluorescence imaging was used to compare the photosynthetic efficiency of PSII electron transport (estimated by Fq'/Fm') in guard cell chloroplasts and the underlying mesophyll in intact leaves of six different species: Commelina communis, Vicia faba, Amaranthus caudatus, Polypodium vulgare, Nicotiana tabacum, and Tradescantia albifora. While photosynthetic efficiency varied between the species, the efficiencies of guard cells and mesophyll cells were always closely matched. As measurement light intensity was increased, guard cells from the lower leaf surfaces of C. communis and V. faba showed larger reductions in photosynthetic efficiency than those from the upper surfaces. In these two species, guard cell photosynthetic efficiency responded similarly to that of the mesophyll when either light intensity or CO2 concentration during either measurement or growth was changed. In all six species, reducing the O2 concentration from 21% to 2% reduced guard cell photosynthetic efficiency, even for the C4 species A. caudatus, although the mesophyll of the C4 species did not show any O2 modulation of photosynthetic efficiency. This suggests that Rubisco activity is significant in the guard cells of these six species. When C. communis plants were water-stressed, the guard cell photosynthetic efficiency declined in parallel with that of the mesophyll. It was concluded that the photosynthetic efficiency in guard cells is determined by the same factors that determine it in the mesophyll.     

Use of lysophosphatidylethanolamine, a natural lipid, to retard tomato leaf and fruit senescence

We studied the influence of lysophosphatidylethanolamine (LPE) on the pattern and rate of ethylene production and respiration of tomato ( Lycopersicon esculentum cv. H7155) leaflets and fruit. Leaflets that had been senescing on the plant showed a climacteric-like rise in ethylene production but not in respiration rate which decreased continuously with leaf age. Detached leaflets had a climacteric-like pattern in respiration whether they were incubated in complete darkness or in light. Detached leaflets incubated in the dark had higher rates of ethylene production and CO₂ evolution than did light-incubated leaves. There was no change in the pattern of ethylene production or CO₂ evolution as a result of LPE treatment. However, LPE-treated attached and detached leaflets had consistently lower rates of CO₂ evolution. The reduction in CO₂ evolution by LPE was most pronounced at the climacteric-like peak of the detached leaves. LPE-treated leaflets had a higher chlorophyll content and fresh weight and lower electrolyte leakage than the control. LPE-treated fruits had lower rates of ethylene and CO₂ production than did the control. LPE-treated fruits also had higher pericarp firmness and lower electrolyte leakage than the control. The results of the present study provide evidence that LPE is able to retard senescence of attached leaves and detached leaves and tomato fruits. Several recent studies suggest that lysolipids can act in a specific manner as metabolic regulators. Our results suggest a specific role of lysolipid LPE in aging and senescence     

Modelling environmental controls on ecosystem photosynthesis and the carbon isotope composition of ecosystem-respired CO2 in a coastal Douglas-fir forest

We developed and applied an ecosystem-scale model that calculated leaf CO₂ assimilation, stomatal conductance, chloroplast CO₂ concentration and the carbon isotope composition of carbohydrate formed during photosynthesis separately for sunlit and shaded leaves within multiple canopy layers. The ecosystem photosynthesis model was validated by comparison to leaf-level gas exchange measurements and estimates of ecosystem-scale photosynthesis from eddy covariance measurements made in a coastal Douglas-fir forest on Vancouver Island. A good agreement was also observed between modelled and measured δ ¹³C values of ecosystem-respired CO₂ ( δ _R). The modelled δ _R values showed strong responses to variation in photosynthetic photon flux density (PPFD), air temperature, vapour pressure deficit (VPD) and available soil moisture in a manner consistent with leaf-level studies of photosynthetic ¹³C discrimination. Sensitivity tests were conducted to evaluate the effect of (1) changes in the lag between the time of CO₂ fixation and the conversion of organic matter back to CO₂; (2) shifts in the proportion of autotrophic and heterotrophic respiration; (3) isotope fractionation during respiration; and (4) environmentally induced changes in mesophyll conductance, on modelled δ _R values. Our results indicated that δ _R is a good proxy for canopy-level C _c/ C _a and ¹³C discrimination during photosynthetic gas exchange, and therefore has several applications in ecosystem physiology.     

Effect of elevated CO2 on the stomatal distribution and leaf physiology of Alnus glutinosa

Variation in stomatal development and physiology of mature leaves from Alnus glutinosa plants grown under reference (current ambient, 360 μmol mol⁻¹ CO₂) and double ambient (720 μmol mol⁻¹ CO₂) carbon dioxide (CO₂) mole fractions is assessed in terms of relative plant growth, stomatal characters (i.e. stomatal index and density) and leaf photosynthetic characters. This is the first study to consider the effects of elevated CO₂ concentration on the distribution of stomata and epidermal cells across the whole leaf and to try to ascertain the cause of intraleaf variation. In general, a doubling of the atmospheric CO₂ concentration enhanced plant growth and significantly increased stomatal index. However, there was no significant change in relative stomatal density. Under elevated CO₂ concentration there was a significant decrease in stomatal conductance and an increase in assimilation rate. However, no significant differences were found for the maximum rate of carboxylation ( V _cmax) and the light saturated rate of electron transport ( J _max) between the control and elevated CO₂ treatment.     

The diurnal time course of net photosynthesis of soybean leaves: Analysis with a physiologically based steady-state photosynthesis model

Summary A physiologically based steady-state model of whole leaf photosynthesis (WHOLEPHOT) is used to analyze observed net photosynthesis daily time courses of soybean, Glycine max (L.) Merr., leaves. Observations during two time periods of the 1978 growing season are analyzed and compared. After adjustment of the model for soybean, net photosynthesis rates are calculated with the model in response to measured incident light intensity, leaf temperature, air carbon dioxide concentration, and leaf diffusion resistance. The steady-state calculations closely approximate observed net photosynthesis. Results of the comparison reveal a decrease in photosynthetic capacity in leaves sampled during the second time period, which is associated with decreasing ability of leaves to respond to light intensity and internal air space carbon dioxide concentration, increasing mesophyll resistance, and increasing stomatal resistance.