Absence of Variable Fluorescence from Guard Cell Chloroplasts of Stenotaphrum secundatum

The lack of detectable variable fluorescence from guard cell chloroplasts in both the albino and green portions of variegated leaves of St. Augustine grass (Stenotaphrum secundatum var variegatum A.S. Hitchc.) is reported. Fluorescence was measured either with a highly sensitive, modified fluorescence microscope which was capable of recording fluorescence induction curves from single chloroplasts, or with a spectrofluorometer. Both fast and slow fluorescence transients from S. secundatum guard cells showed a rapid rise and then remained at a steady level. Neither variable fluorescence increase (induction) nor decrease (quenching), properties normally associated with photosystem II, was observed from these chloroplasts. These fluorescence kinetics did not change either with alterations of the specimen preparation procedure or with alterations of the excitation light intensities and wavelengths. These results indicate that guard cell chloroplasts in this variety of S. secundatum do not conduct normal photosystem II electron transport. Light regulation of stomatal conductance in intact leaves of this plant did occur, however, and was similar to light regulation observed in other species. The conductance of the green portion of the leaves was much greater in the light than in the dark, and was much greater than the conductance of the albino portion of the leaves. Stomata in the green portion of the leaves also showed greater opening in blue light than in red light. These results provide evidence that stomatal regulation in this variety of S. secundatum does not rely on photosystem II electron transport in guard cell chloroplasts.     

Syringomycin, a bacterial phytotoxin, closes stomata

The effects of the bacterial phytotoxin, syringomycin, on stomata were investigated using detached leaves of Xanthium strumarium and isolated epidermes of Vicia faba. Syringomycin is known to cause K⁺ efflux in fungal and higher plant cells. Doses of syringomycin as low as 0.3 unit per square centimeter (about 0.88 pmole per square centimeter) resulted in measurable stomatal closure when applied through the transpiration stream of detached leaves; higher doses produced larger reductions in stomatal conductance. Stomatal apertures of isolated epidermes were also reduced by low concentrations (3.2 units per milliliter; 10⁻⁸ molar) of syringomycin. The effects of syringomycin were similar to those of ABA. Both compounds closed stomata at a similar rate and at similar concentrations. In addition, neither compound significantly affected the relationship between photosynthesis and intercellular CO₂ based on data taken after stomatal conductance had stabilized following the treatment. It is possible that syringomycin and ABA activate the same K⁺ export system in guard cells, and syringomycin may be a valuable tool for studying the molecular basis of ABA effects on guard cells.     

Changes in photosynthesis caused by adaptation of maize seedlings to short-term drought

Detached leaf is in the state of increasing water deficit; it is a good experimental model for looking into the hardening effect of adaptation of eight-day-old maize (Zea mays L.) seedlings to short-term drought (five days without watering). The light stage of photosynthesis and photosynthetic CO₂/H₂O exchange in detached leaves were studied. Specific surface density of leaf tissue (SSDL), the content of chlorophylls a and b, proline, MDA as well as photosynthetic parameters: quantum yield of photosystem II fluorescence, assimilation of CO₂, and transpiration at room temperature and light saturation (density of PAR quantum flux of 2000 μmol/(m² s)) at normal and half atmospheric CO₂ concentration were determined. The leaves of seedlings exposed to short-term drought differed from control material by a greater SSDL and higher content of proline. The hardening effect of the stress agent on the dark stage of photosynthesis was detected; it was expressed in the maintenance of the higher photosynthetic CO₂ assimilation against control material due to the elevation of stomatal conductance for CO₂ diffusing into the leaf. Judging from the lack of differences in the MDA content, short-term drought did not injure photosynthetic membranes. In detached leaves of experimental maize seedlings, photosynthesis was maintained on a higher level than in control material.     

Fluorescence Properties of Guard Cell Chloroplasts: EVIDENCE FOR LINEAR ELECTRON TRANSPORT AND LIGHT-HARVESTING PIGMENTS OF PHOTOSYSTEMS I AND II

The presence of chloroplasts in guard cells from leaf epidermis, coleoptile, flowers, and albino portions of variegated leaves was established by incident fluorescence microscopy, thus confirming the notion that guard cell chloroplasts are remarkably conserved. Room temperature emission spectra from a few chloroplasts in a single guard cell of Vicia faba showed one major peak at around 683 nanometers. Low-temperature (77 K) emission spectra from peels of albino portions of Chlorophytum comosum leaves and from mesophyll chloroplasts of green parts of the same leaves showed major peaks at around 687 and 733 nanometers, peaks usually attributed to photosystem II and photosystem I pigment systems, respectively. Spectra of peels of V. faba leaves showed similar peaks. However, fluorescence microscopy revealed that the Vicia peels, as well as those from Allium cepa and Tulipa sp., were contaminated with non-guard cell chloroplasts which were practically undetectable under bright field illumination. These observations pose restrictions on the use of epidermal peels as a source of isolated guard cell chloroplasts. Studies on the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-sensitive variable fluorescence kinetics of uncontaminated epidermal peels of C. comosum indicated that guard cell chloroplasts operate a normal, photosystem II-dependent, linear electron transport. The above properties in combination with their reported inability to fix CO₂ photosynthetically may render the guard cell chloroplasts optimally suited to supply the reducing and high-energy phosphate equivalents needed to sustain active ion transport during stomatal opening in daylight.     

Guard Cell Chloroplasts Are Essential for Blue Light-Dependent Stomatal Opening in Arabidopsis

Blue light (BL) induces stomatal opening through the activation of H⁺-ATPases with subsequent ion accumulation in guard cells. In most plant species, red light (RL) enhances BL-dependent stomatal opening. This RL effect is attributable to the chloroplasts of guard cell, the only cells in the epidermis possessing this organelle. To clarify the role of chloroplasts in stomatal regulation, we investigated the effects of RL on BL-dependent stomatal opening in isolated epidermis, guard cell protoplasts, and intact leaves of Arabidopsis thaliana. In isolated epidermal tissues and intact leaves, weak BL superimposed on RL enhanced stomatal opening while BL alone was less effective. In guard cell protoplasts, RL enhanced BL-dependent H⁺-pumping and DCMU, a photosynthetic electron transport inhibitor, eliminated this effect. RL enhanced phosphorylation levels of the H⁺-ATPase in response to BL, but this RL effect was not suppressed by DCMU. Furthermore, DCMU inhibited both RL-induced and BL-dependent stomatal opening in intact leaves. The photosynthetic rate in leaves correlated positively with BL-dependent stomatal opening in the presence of DCMU. We conclude that guard cell chloroplasts provide ATP and/or reducing equivalents that fuel BL-dependent stomatal opening, and that they indirectly monitor photosynthetic CO₂ fixation in mesophyll chloroplasts by absorbing PAR in the epidermis.     

The effects of enhanced UV-B radiation on photosynthetic and biochemical activities in super-high-yield hybrid rice Liangyoupeijiu at the reproductive stage

We investigated the light reactions, CO₂ assimilation, but also the chloroplast ultrastructure in the upper three functional leaves (flag, 2^nd, and 3^rd leaves) of the Chinese super-high-yield hybrid rice (Oryza sativa L.) Liangyoupeijiu (LYPJ) with ultraviolet-B (UV-B) treatment during reproductive development. Photosynthetic parameters showed that the upper 3 functional leaves of LYPJ entered into senescence approximately 15 days after flag leaf emergence (DAE). Leaves in UV-B treatment exhibited greater efficiency in absorbing and utilizing light energy of photosystem II (PSII), characterized by higher chlorophyll (Chl) content and the whole chain electron transport rate (ETR). However, UV-B radiation reduced activities of Ca²⁺-ATPase and photophosphorylation. The significantly decreased activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was greatly associated with the decline in photosynthetic efficiency. The net photosynthetic rate (P _N) and stomatal conductance (g _s) suffered strong reductions before 25 DAE, and afterwards showed no significant difference between control and treatment. UV-B treatment delayed chloroplasts development of flag leaves. Chloroplast membranes later swelled and disintegrated, and more stromal thylakoids were parallel to each other and were arranged in neat rows, which might be responsible for better performance of the primary light reaction. It is likely that accumulation of starch and an increase in the number of lipid droplet and translucent plastoglobuli were results of an inhibition of carbohydrate transport. Our results suggest that long-term exposure to enhanced UV-B radiation was unlikely to have detrimental effects on the absorption flux of photons and the transport of electrons, but it resulted in the decrease of photophosphorylation and Rubisco activation of LYPJ. The extent of the damage to the chloroplast ultrastructure was consistent with the degree of the inhibition of photosynthesis.     

Involvement of Singlet Oxygen in 5-Aminolevulinic Acid-Induced Photodynamic Damage of Cucumber (Cucumis sativus L.) Chloroplasts

Cucumber (Cucumis sativus L., cv Poinsette) plants were sprayed with 20 millimolar 5-aminolevulinic acid and then incubated in the dark for 14 hours. The intact chloroplasts were isolated from the above plants in the dark and were exposed to weak light (250 micromoles per square meter per second). Within 30 minutes, photosystem II activity was reduced by 50%. The singlet oxygen (¹O₂) scavengers, histidine and sodium azide (NaN₃) significantly protected against the damage caused to photosystem II. The hydroxyl radical scavenger formate failed to protect the thylakoid membranes. The production of ¹O₂ monitored as N,N-dimethyl p-nitrosoaniline bleaching increased as a function of light exposure time of treated chloroplasts and was abolished by the ¹O₂ quencher, NaN₃. Membrane lipid peroxidation monitored as malondialdehyde production was also significantly reduced when chloroplasts were illuminated in the presence of NaN₃ and histidine. Protochlorophyllide was the most abundant pigment accumulated in intact chloroplasts isolated from 5-aminolevulinic acid-treated plants and was probably acting as type II photosensitizer.     

Structural and functional aspects of the Nuphar lutea (L.) Smith heterophylly: Ultrastructure and photosynthesis

The ultrastructure and functional characteristics of the photosynthetic apparatus of floating and submersed leaves of the heterophyllous plant Nuphar lutea (L.) Smith have been examined. Differences have been revealed in mesophyll cell chloroplasts, content of pigments, and chlorophyll fluorescence parameters between floating and submersed leaves and submersed leaves at different depths. A sharp decline in the PSII (photosystem II) efficiency of submersed leaves when exposed to an actinic light intensity of more than 60 ??mol m^?2 s^?1 has been noted. The described differences may be considered as an adaptation mechanism of submersed leaves to life in an aquatic environment with a reduced light intensity and changed light spectral composition.     

Physiological characterization of photosynthesis,chloroplast ultrastructure,and nutrient content in bracts and rosette leaves from <Emphasis Type="Italic">Glaucium flavum</Emphasis>

Glaucium flavum is a biennial plant that bears a rosette of leaves, producing a flower stalk, bracteate monochasium, in its second year. The aims of this work were both to investigate the contribution of bracts to gas-exchange activities in this species and to compare this contribution to that of rosette leaves. In addition, we investigated the extent to which its responses can be explained by chloroplast ultrastructure, as well as the possible role of nutrient concentrations in the physiological responses of both leaf types. Gas exchange and plant characteristics regarding chlorophyll fluorescence were examined in a field experiment; we also determined leaf relative water content, tissue concentrations of photosynthetic pigments, chloroplast ultrastructure and nutrient contents. Although bracts indeed contributed to gas-exchange activities of G. flavum, rosette leaves showed higher values of net photosynthetic rate and stomatal conductance to CO₂ for photosynthetic photon flux density above 200 μmol m⁻² s⁻¹. The incongruities in photosynthetic rates between bracts and leaves may be explained by the bigger chloroplasts of rosette leaves, which results in a larger membrane surface area. This agrees with the higher pigment concentrations and quantum efficiency of photosystem II values recorded as well for rosette leaves. On the other hand, bracts showed higher sodium concentrations, which could be a mechanism for salt tolerance of G. flavum.     

Fluorescence and Delayed Light Emission from Mesophyll and Bundle Sheath Cells in Leaves of Normal and Salt-Treated Panicum miliaceum

A modified fluorescence microscope system was used to measure chlorophyll fluorescence and delayed light emission from mesophyll and bundle sheath cells in situ in fresh-cut sections from leaves of Panicum miliaceum L. The fluorescence rise in 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU)-treated leaves and the slow fluorescence kinetics in untreated leaves show that mesophyll chloroplasts have larger photosystem II unit sizes than do bundle sheath chloroplasts. The larger photosystem II units imply more efficient noncyclic electron transport in mesophyll chloroplasts. Quenching of slow fluorescence also differs between the cell types with mesophyll chloroplasts showing complex kinetics and bundle sheath chloroplasts showing a relatively simple decline. Properties of the photosynthetic system were also investigated in leaves from plants grown in soil containing elevated NaCl levels. As judged by changes in both fluorescence kinetics in DCMU-treated leaves and delayed light emission in leaves not exposed to DCMU, salinity altered photosystem II in bundle sheath cells but not in mesophyll cells. This result may indicate different ionic distributions in the two cell types or, alternatively, different responses of the two chloroplast types to environmental change.     

Gas Exchange, Stomatal Behavior, and deltaC Values of the flacca Tomato Mutant in Relation to Abscisic Acid

The relationship between stomatal conductance and capacity for assimilation was investigated in flacca, a mutant of tomato (Lycopersicon esculentum Mill.) that has abnormal stomatal behavior and low abscisic acid (ABA) content. The assimilation capacity, determined by measuring assimilation rate as a function of intercellular CO₂ pressure, did not differ in leaves of flacca and its parent variety, Rheinlands Ruhm (RR). On the other hand, stomatal conductance of flacca leaves was greater than that of RR, and could be phenotypically reverted by spraying with 30 micromolar ABA. Stomatal conductance of flacca leaves was also reduced by increasing CO₂ pressure, increasing leaf to air vapor pressure difference, and decreasing quantum flux, irrespective of ABA treatment.

The high conductance of flacca leaves resulted in a high intercellular CO₂ pressure. This allowed greater discrimination against ¹³CO₂, as evidenced by more negative δ ¹³C values for flacca as compared to RR. The δ ¹³C values of both flacca and RR plants as influenced by ABA treatment were consistent with predictions based on gas exchange measurements, using a recent model of discrimination.

     

Effect of Leaf Detachment on Chlorophyll Fluorescence during Chilling Experiments

The effect of leaf detachment on chlorophyll fluorescence was analyzed for Zea mays, Cucumis sativus, Phaseolus vulgaris, and Echinochloa crus-galli. Results clearly indicate that detachment hastens the decrease in chlorophyll fluorescence during the course of chilling experiments. For maize and bean, the activity of photosystem II of chloroplasts isolated from detached leaves is lower than that of chloroplasts isolated from attached leaves. There are also large differences in ionic loss between detached and attached leaves of barnyard grass which could correlate with changes in leaf water status. The detached leaves lost some 50% of their total ionic content. Finally, detachment alters the ranking of the species with regard to their chilling tolerance.     

Nutrient Influences on Leaf Photosynthesis: EFFECTS OF NITROGEN, PHOSPHORUS, AND POTASSIUM FOR GOSSYPIUM HIRSUTUM L

The net rate of CO₂ uptake for leaves of Gossypium hirsutum L. was reduced when the plants were grown at low concentrations of NO₃^-, PO₄^2-, or K⁺. The water vapor conductance was relatively constant for all nutrient levels, indicating little effect on stomatal response. Although leaves under nutrient stress tended to be lower in chlorophyll and thinner, the ratio of mesophyll surface area to leaf area did not change appreciably. Thus, the reduction in CO₂ uptake rate at low nutrient levels was due to a decrease in the CO₂ conductance expressed per unit mesophyll cell wall area (g^cell_CO2). The use of g^cell_CO2 and nutrient levels expressed per unit of mesophyll cell wall provides a new means of assessing nutrient effects on CO₂ uptake of leaves.     

Effect of temperature on senescing detached rice leaves: I. Photoelectron transport activity of chloroplasts

Detached rice(Oryza sativaL cv Mousouri)leaves were induced to senesce in darkness at 0°C(cold), 27°C(room temperature) and 40°C(heat). 2,6-dichlorophenol indophenol (DCPIP) Hill reaction activity of chloroplasts isolated from senescing leaves under all experimental temperatures with H₂O, Mn²⁺ or diphenyl carbazide (DPC) as electron donor declined during the period of incubation. Since DPC and Mn²⁺ augmented 2,6-dichlorophenol indophenol photoreduction by chloroplasts from senescing leaves, damage and/or change in the conformation of a site between H₂O and DCPIP in photosystem II (PS II) is suggested. Heat caused a faster decline of the Hill activity compared to cold or room temperature. However, cold treatment showed no significant effect on the photoelectron transport from H₂O to DCPIP compared with room temperature.     

Effects of Growth Temperature on the Thermal Stability of the Photosynthetic Apparatus of Atriplex lentiformis (Torr.) Wats

High growth temperatures induced a substantial increase in the thermal stability of the photosynthetic apparatus of Atriplex lentiformis. This was manifested as a much reduced inhibition of light-saturated photosynthesis and the initial slope of the light-dependence curves by exposure to high temperatures in high as compared to moderate temperature-grown plants. Heat treatment at 46 C of leaves from moderate temperature-grown plants resulted in a marked reduction in photosystem II activities of chloroplasts isolated from them. In contrast, heat treatment of leaves from high temperature-grown plants resulted in no reduction of photosystem II activities. In vivo estimates of photosystem II functioning, the 515 nm light-induced absorbance change, and the ratio initial to maximum fluorescence (F₀/F_max) indicated a similar increase in the thermal stability of photosystem II in high temperature-grown plants.     

Salt tolerance in crop plants monitored by chlorophyll fluorescence in vivo

The potential of measurements of chlorophyll fluorescence in vivo to detect cellular responses to salinity and degrees of salt stress in leaves was investigated for three crop plants. Sugar beet (Beta vulgaris L.) (salt tolerant), sunflower (Helianthus annuus L.) (moderately salt tolerant), and bean (Phaseolus Vulgaris L. cv Canadian Wonder) (salt intolerant) were grown in pots and watered with mineral nutrient solution containing 100 millimolar NaCl. The fast rise in variable chlorophyll fluorescence yield that is correlated with photoreduction of photosystem II acceptors increased in leaves of sugar beet plants treated with salt suggesting stimulation of photosystem II activity relative to photosystem I. In sunflower, this fast rise was depressed by approximately 25% and the subsequent slow rate of quenching of the chlorophyll fluorescence was stimulated. These differences were more marked in the older mature leaves indicating an increasing gradient of salt response down the plant. The salt effect in vivo was reversible since chloroplasts isolated from mature leaves of salt-treated and control sunflower plants gave similar photosystem II activities. Unlike in sugar beet and sunflower, leaves of salt-treated bean progressively lost chlorophyll. The rate of slow quenching of chlorophyll fluorescence decreased indicating development of a partial block after photosystem II and possible initial stimulation of photosystem II activity. With further loss of chlorophyll photosystem II activity declined. It was concluded that measurements of chlorophyll fluorescence in vivo can provide a rapid means of detecting salt stress in leaves, including instances where photosynthesis is reduced in the absence of visible symptoms. The possible application to screening for salt tolerance is discussed.     

Stress Tolerance of Photosystem II in Vivo: Antagonistic Effects of Water, Heat, and Photoinhibition Stresses

The in vivo photochemical activity of photosystem II was inferred from modulated chlorophyll fluorescence and photoacoustic measurements in intact leaves of several plant species (Lycopersicon esculentum Mill., Solanum tuberosum L., Solanum nigrum L.) exposed to various environmental stresses (drought, heat, strong light) applied separately or in combination. Photosystem II was shown to be highly drought-resistant: even a drastic desiccation in air of detached leaf samples only marginally affected the quantum yield for photochemistry in photosystem II. However, water stress markedly modified the responses of photosystem II to superimposed constraints. The stability of photosystem II to heat was observed to increase strongly in leaves exposed to water stress conditions: heat treatments (e.g. 42°C in the dark), which caused a complete and irreversible inhibition of photosystem II in well-watered (tomato) leaves, resulted in a small and fully reversible reduction of the photochemical efficiency of photosystem II in drought-stressed leaves. In vivo photoacoustic data indicated that photosystem I was highly resistant to both heat and water stresses. When leaves were illuminated with intense white light at 25°C, photoinhibition damage of photosystem II was more pronounced in water-stressed leaves than in undesiccated controls. However, in nondehydrated leaves, photoinhibition of photosystem II was strongly temperature dependent, being drastically stimulated at high temperatures above 38 to 40°C. As a consequence, when exposed to strong light at high temperature, photosystem II photochemistry was significantly less inhibited in dehydrated leaves than in control well-hydrated leaves. Our results demonstrate the existence of a marked antagonism between physicochemical stresses, with water stress enhancing the resistance of photosystem II to constraints (heat, strong light at high temperature) that are usually associated with drought in the field.     

Leaf chlorosis in oilseed rape plants (Brassica napus) grown on cadmium-polluted soil: causes and consequences for photosynthesis and growth

Brassica napus L. (oilseed rape) was grown from seeds on a reconstituted soil contaminated with cadmium (100 mg Cd kg⁻¹ dry soil), resulting in a marked chlorosis of the leaves which was investigated using a combination of biochemical, biophysical and physiological methods. Spectroscopic and chromatographic analyses of the photosynthetic pigments indicated that chlorosis was not due to a direct interaction of Cd with the chlorophyll biosynthesis pathway. In addition, mineral deficiency and oxidative stress were apparently not involved in the pigment loss. Leaf chlorosis was attributable to a marked decrease in the chloroplast density caused by a reduction in the number of chloroplasts per cell and a change in cell size, suggesting that Cd interfered with chloroplast replication and cell division. Relatively little Cd was found in the chloroplasts and the properties of the photosynthetic apparatus (electron transport, protein composition, chlorophyll antenna size, chloroplast ultrastructure) were not affected appreciably in plants grown on Cd-polluted soil. Depth profiling of photosynthetic pigments by phase-resolved photoacoustic spectroscopy revealed that the Cd-induced decrease in pigment content was very pronounced at the leaf surface (stomatal guard cells) compared to the leaf interior (mesophyll). This observation was consistent with light transmission and fluorescence microscopy analyses, which revealed that stomata density in the epidermis was noticeably reduced in Cd-exposed leaves. Concomitantly, the stomatal conductance estimated from gas-exchange measurements was strongly reduced with Cd. When plants were grown in a high-CO₂ atmosphere (4,000 μl CO₂ l⁻¹), the inhibitory effect of Cd on growth was not cancelled, suggesting that the reduced availability of CO₂ at the chloroplast level associated with the low stomatal conductance was not the main component of Cd toxicity in oilseed rape. Received: 14 July 2000 / Accepted: 27 August 2000     

Interspecific Variation in SO(2) Flux : Leaf Surface versus Internal Flux, and Components of Leaf Conductance

The objective of this study was to clarify the relationships among stomatal, residual, and epidermal conductances in determining the flux of SO₂ air pollution to leaves. Variations in leaf SO₂ and H₂O vapor fluxes were determined using four plant species: Pisum sativum L. (garden pea), Lycopersicon esculentum Mill. flacca (mutant of tomato), Geranium carolinianum L. (wild geranium), and Diplacus aurantiacus (Curtis) Jeps. (a native California shrub). Fluxes were measured using the mass-balance approach during exposure to 4.56 micromoles per cubic meter (0.11 microliters per liter) SO₂ for 2 hours in a controlled environmental chamber. Flux through adaxial and abaxial leaf surfaces with closed stomata ranged from 1.9 to 9.4 nanomoles per square meter per second for SO₂, and 0.3 to 1.3 millimoles per square meter per second for H₂O vapor. Flux of SO₂ into leaves through stomata ranged from ~0 to 8.5 (dark) and 3.8 to 16.0 (light) millimoles per square meter per second. Flux of H₂O vapor from leaves through stomata ranged from ~0 to 0.6 (dark) to 0.4 to 0.9 (light) millimole per square meter per second. Lycopersicon had internal flux rates for both SO₂ and H₂O vapor over twice as high as for the other species. Stomatal conductance based on H₂O vapor flux averaged from 0.07 to 0.13 mole per square meter per second among the four species. Internal conductance of SO₂ as calculated from SO₂ flux was from 0.04 mole per square meter per second lower to 0.06 mole per square meter per second higher than stomatal conductance. For Pisum, Geranium, and Diplacus stomatal conductance was the same or slightly higher than internal conductance, indicating that, in general, SO₂ flux could be predicted from stomatal conductance for H₂O vapor. However, for the Lycopersicon mutant, internal leaf conductance was much higher than stomatal conductance, indicating that factors inside leaves can play a significant role in determining SO₂ flux.