Chilling syndrome in light-exposed maize leaves and its easing by low doses of DCMU

When maize plants ( Zea mays L. line ps-lye) were subjected to chilling (at 8 ± 2°C, 80% relative humidity for 24 h under illumination by 80 W m² between 400–700 nm), the leaves were wilted and photosynthetic membranes were permanently damaged. This was shown by the swelling of grana thylakoids and a deerease in the charging capacity of the electron transport chain. Water loss and photosynthetic dysfunction were connected in the process of a chilling-induced increase of stomatal aperture. Chilling injury could be eased to a considerable extent by a mild treatment with DCMU preventing stomatal opening, wilting, and the irreversible loss of CO₂ fixation capacity.     

Oxygen-dependent stomatal opening in Zea mays leaves. Effect of Light and carbon dioxide

The oxygen requirement for stomatal opening in maize plants ( Zea mays L. hybrid INRA 508) was studied at different CO₂ concentrations and light intensities. In the absence of CO₂, stomatal opening always required O₂, but this requirement decreased with increasing light intensity. In darkness, the lowest O₂ partial pressure needed to obtain a weak stomatal movement was about 50 Pa. This value was lowered to ca 10 Pa in light (320 μmol m⁻² s⁻¹).
On the other hand. in the absence of O₂, CO₂enabled stomatal opening to occur in the light, presumably due to the evolved photosynthetic O₂. Thus, CO₂, which generally reduced stomatal aperture, could induce stomatal movement in anoxia and light. The effect of CO₂ on stomatal opening was closely dependent on O₂ concentration and light intensity. Stomatal aperture appeared CO₂-independent at an O₂ partial pressure which was dependent on light intensity and was about 25 Pa at 320 umol m⁻² s⁻¹.
The presence of a plasmalemma oxidase, in addition to mitochondrial oxidase, might explain the differences in the O² requirement at various light intensities. The possible involvement of such a system in relation to the effect of CO₂ is discussed.     

Possible involvement of phospholipase A2 in light signal transduction of guard cells of Commelina communis

Polyunsaturated fatty acids induce stomatal opening (Y. Lee, H. Lee, R. C. Crain, A. Lee and S. J. Korn. 1994. Cell Signal. 6: 181–186), but it is not known whether they function as second messengers in guard cells exposed to signals that open stomata. To test the hypothesis that phospholipase A₂ (PLA₂), which produces fatty acids and lysophospholipids, is involved in light signal transduction in guard cells, we treated epidermal peels of Commelina communis L. with PLA₂ inhibitors and followed the changes in stomatal apertures in response to light. Stomatal opening by white, blue, or red light was inhibited by 2–3 different PLA₂ inhibitors in concentration ranges that have been reported to inhibit PLA₂ activity. However, the PLA₂ inhibitors could not block stomatal opening induced by a polyunsaturated fatty acid. These results suggest that PLA₂ functions as a signal transducer for both blue and red light in guard cells.     

Photosynthesis affects following night leaf conductance in Vicia faba

Night-time stomatal opening in C₃ plants may result in significant water loss when no carbon gain is possible. The objective of this study was to determine if endogenous patterns of night-time stomatal opening, as reflected in leaf conductance, in Vicia faba are affected by photosynthetic conditions the previous day. Reducing photosynthesis with low light or low CO₂ resulted in reduced night-time stomatal opening the following night, irrespective of the effects on daytime stomatal conductance. Likewise, increasing photosynthesis with enriched CO₂ levels resulted in increased night-time stomatal opening the following night. Reduced night-time stomatal opening was not the result of an inability to regulate stomatal aperture as leaves with reduced night-time stomatal opening were capable of greater night-time opening when exposed to low CO₂. After acclimating plants to long or short days, it was found that night-time leaf conductance was greater in plants acclimated to short days, and associated with greater leaf starch and nitrate accumulation, both of which may affect night-time guard cell osmotic potential. Direct measurement of guard cell contents during endogenous night-time stomatal opening will help identify the mechanism of the effect of daytime photosynthesis on subsequent night-time stomatal regulation.     

The response of stomata to CO2 relates to its effect on respiration and ATP level

External ATP enhanced stomatal opening of Commelina communis L. differently from EDTA. ATP was more effective in opening stomata than EDTA, when both were applied in amounts yielding equivalent free Ca²⁺ concentration. The stimulation by ATP depended upon its de-phosphorylation and was not due to the P₁ released. Hence an energetical contribution of external ATP appears possible. Increase in CO₂ concentration increased the stimulation of stomatal opening by ATP and diminished the internal ATP level, ATP/(ADP+AMP) ratio and respiration rate.     

Further support for the involvement of phytoehrome in stomatal movement

The involvement of phytochrome in stomatal movement in Commelina communis L. is indicated by the following observations: 1) Short irradiation with red or blue light causes opening, of isolated stomata and swelling of guard cell protoplasts. This is reversed by subsequent far red irradiation. 2) In a similar way, stomatal response to prolonged irradiation with red or blue light is decreased by concomitant far red irradiation. 3) Pretreatment with filipin, which interferes with phytochrome binding to membranes, decreases stomatal opening in red and blue light. The stomatal responses to blue and red light are modified by DCMU, N₂, CO_2-enriched atmosphere, and CO_2-free air, which are known to affect, among other processes, chlorophyll fluorescence. Increased chlorophyll fluorescence by DCMU, N₂ and CO₂-enriched atmosphere enhanced stomatal opening in blue light and inhibited it in red light. CO₂-free air, which decreases chlorophyll fluorescence, had the opposite effect.     

Silencing of NtMPK4 impairs CO-induced stomatal closure, activation of anion channels and cytosolic Casignals in Nicotiana tabacum guard cells

Light-induced stomatal opening in C3 and C4 plants is mediated by two signalling pathways. One pathway is specific for blue light and involves phototropins, while the second pathway depends on photosyntheticaly active radiation (PAR). Here, the role of Nt MPK4 in light-induced stomatal opening was studied, as silencing of this MAP kinase stimulates stomatal opening. Stomata of Nt MPK4-silenced plants do not close in elevated atmospheric CO₂, and show a reduced response to PAR. However, stomatal closure can still be induced by abscisic acid. Measurements using multi-barrelled intracellular micro-electrodes showed that CO₂ activates plasma membrane anion channels in wild-type Nicotiana tabacum guard cells, but not in Nt MPK4-silenced cells. Anion channels were also activated in wild-type guard cells after switching off PAR. In approximately half of these cells, activation of anion channels was accompanied by an increase in the cytosolic free Ca²⁺ concentration. The activity of anion channels was higher in cells showing a parallel increase in cytosolic Ca²⁺ than in those with steady Ca²⁺ levels. Both the darkness-induced anion channel activation and Ca²⁺ signals were repressed in Nt MPK4-silenced guard cells. These data show that CO₂ and darkness can activate anion channels in a Ca²⁺-independent manner, but the anion channel activity is enhanced by parallel increases in the cytosolic Ca²⁺ concentration. Nt MPK4 plays an essential role in CO₂- and darkness-induced activation of guard-cell anion channels, through Ca²⁺-independent as well as Ca²⁺-dependent signalling pathways.     

Photosynthesis of Coffea arabica after chilling

Net photosynthetic CO₂ exchange of 1-year-old plants of Coffea arabica L. was studied after the above-ground parts had been exposed once or repeatedly to night temperatures in the chilling range. Chill-reduced rates of CO₂ uptake (measured at 24°C and at natural CO, level) were observed after a 12 h night exposure to about 6°C. After exposure to 4°C, activity was reduced to less than half of that of the controls, and after exposure to 0.5°C the leaves suffered visible necrotic injury and were no longer able to take up Co₂ If the leaves were not lethally injured, net photosynthesis recovered completely within 2 to 6 days. About 25% of chill-induced reduction of CO₂ uptake was due to reduced stomatal aperture and 75% to impairment of carboxylation efficiency.
Chilling on successive nights at 4–6°C reduced CO, uptake progressively on each day following treatment. After 10 nights, activity was decreased to less than 10% of initial performance. Conditioning at temperatures slightly above the chilling level (e.g. 15/I2°C) for 2 weeks led to almost complete impairment of photosynthetic activity without additional chilling stress instead of improving chilling tolerance.     

The cellular basis of guard cell sensing of rising CO2

Numerous studies conducted on both whole plants and isolated epidermes have documented stomatal sensitivity to CO₂. In general, CO₂ concentrations below ambient stimulate stomatal opening, or an inhibition of stomatal closure, while CO₂ concentrations above ambient have the opposite effect. The rise in atmospheric CO₂ concentrations which has occurred since the industrial revolution, and which is predicted to continue, will therefore alter rates of transpirational water loss and CO₂ uptake in terrestrial plants. An understanding of the cellular basis for guard cell CO₂ sensing could allow us to better predict, and perhaps ultimately to manipulate, such vegetation responses to climate change. However, the mechanisms by which guard cells sense and respond to the CO₂ signal remain unknown. It has been hypothesized that cytosolic pH and malate levels, cytosolic Ca²⁺ levels, chloroplastic zeaxanthin levels, or plasma-membrane anion channel regulation by apoplastic malate are involved in guard cell perception and response to CO₂. In this review, these hypotheses are discussed, and the evidence for guard cell acclimation to prevailing CO₂ concentrations is also considered.     

Nitrate reductase activity, nitrogenase activity and photosynthesis of black alder exposed to chilling temperatures

Actinorhizal ( Frankia -nodulated) black alder [ Alnus glutinosa (L.) Gaertn.] seedlings fertilized with 0.36 m M nitrate (low nitrate fertilizer treatment) or 7.14 m M nitrate (high nitrate fertilizer treatment) and acclimated in a growth chamber for 2 weeks were exposed to 2.5 h of night-time chilling temperatures of −1 to 4°C. Cold treatment decreased nitrogenase activity (acetylene reduction activity) 33% for low nitrate fertilized plants and 41% for high nitrate fertilized plants. Recovery of nitrogenase activity occurred within 7 days after chilling treatment. In contrast, in vivo nitrate reductase (NR) activities of leaves and fine roots increased immediately after chilling then decreased as nitrogenase activities recovered. Fine roots of alder seedlings exhibited NR activities proportional to the amounts of nitrate in the rooting medium. In contrast, the NR activities of leaves were independent of substrate and tissue nitrate levels and corresponded to nitrogenase activity in the root nodules. In a separate experiment, net photosynthesis (PS) of similarly treated black alder seedlings was measured before and after chilling treatments. Net PS declined in response to chilling by 17% for plants receiving low nitrate fertilizer and 19% for plants receiving high nitrate fertilizer. After chilling, stomatal conductance (g_s) decreased by 39% and internal CO₂ concentration (c_i) decreased by 5% in plants receiving the high nitrate fertilizer, whereas plants receiving the low nitrate fertilizer showed no change in g_s and a 13% increase in c_i. Results indicate that chilling stimulates stomatal closure only at the high nitrate level and that interference with biochemical functions is probably the major impact of chilling on PS.     

C4 photosynthesis at low temperatures

Abstract. C₄ plants grown in optimum conditions are, by comparison to C₃, capable of higher maximum dry-matter yields and greater efficiencies of water and nitrogen use, yet they are rare outside the subtropics. Both latitudinal and altitudinal limits of C₄ distributions correlate most closely with a mean minimum temperature of 8-10°C during the period of active growth. The possibility that the C₄ process is inherently incapable of functioning at low temperatures is examined. The reversible effects of chilling on the quantum efficiency of C₄ photosynthesis and the functioning of the individual steps in the C₄ cycle are examined. Chilling also produces an irreversible loss of capacity to assimilate CO₂ which is directly proportional to the light received during chilling. It is suggested that the reversible reduction in capacity to assimilate CO₂ and the lack of an alternative pathway for the utilization of lightgenerated reducing power may make C₄ species more prone to chilling-dependent photoinhibition. Laboratory studies and limited field observations suggest that this damage would be most likely to occur during photosynthetic induction at the temperatures and light levels encountered on clear, cool mornings during the spring and early summer in cool climates. Even those C₄ species occurring naturally in cool climates do not appear fully capable of tolerating these conditions; indeed their growth patterns suggest that they may be adapted by avoiding 'rather than enduring' such conditions.     

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.     

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 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.     

Direct and indirect effects of light on stomata. I. In Scots pine and Sitka spruce

Abstract. The response of stomatal conductance to broadband blue and red light was measured in whole shoots of Scots pine and Sitka spruce, two species which have low stomatal sensitivity to CO². In Scots pine, blue light was more than three times more effective than red light (on an incident quantum basis) in opening stomata, particularly at low quantum flux densities (<100μmiol m⁻² s⁻¹). However, the apparent quantum yield of net CO² assimilation rate in blue light was only half that in red light. The contrasting effects of red and blue light on conductance and assimilation led to higher intercellular CO² concentrations (C_i) in blue light (up to 100 μmol mol⁻¹ higher) than in red light. Similar results were obtained with Sitka spruce shoots, though differences in the effectiveness of red and blue light were less marked. In both species, both red and blue light increased conductance in normal and CO²-free air, indicating that neither red nor blue light exert effects through changes in C_i or mesophyll assimilation. However, decreases in C_i caused increases in conductance in both red and blue light, suggesting that these direct effects of light are not wholly independent of CO².     

Physiological response of Pinus halepensis needles under ozone and water stress conditions

The aim of this study was to evaluate how physiological processes of potted Pinus halepensis plants, grown under controlled conditions, were affected by ozone (O₃) and/or water stress, integrating the gas exchange and biochemical data with fluorescence OJIP polyphasic transient data. Plants submitted to only water stress (T₁) and with ozone (T₃) showed a strong decrease in stomatal conductance and gas exchange, coinciding with a reduction of maximum yield of photochemistry ( φ _po) and very negative values of leaf water potential. Simultaneously, a great increase of both PSII antenna size, indicated by absorption per reaction centre, and electron transport per reaction centre were found. The reduction of photosynthesis in the O₃-treated plants (T₂) by a slowing down of the Calvin cycle was supported by the increase of related fluorescence parameters such as relative variable fluorescence, heat de-excitation constant, energy de-excitation by spillover, and the decrease of φ _po. We suggest an antagonistic effect between the two stresses to explain the delayed ozone-induced decrease of stomatal conductance values for T₃ with respect to T₁ plants, by an alteration of the physiological mechanisms of stomatal opening, which involve the increase of intra-cellular free-calcium induced by ABA under co-occurring water shortage. We emphasise the importance of considering the intensity of the individual stress factor in studies concerning the interaction of stresses.     

Stomatal frequency of Betula pubescens and Pinus sylvestris shows no proportional relationship with atmospheric CO2 concentration

We investigated the relationship between stomatal frequency and a range of atmospheric CO₂ concentrations ([CO₂]_atm) in Betula pubescens and Pinus sylvestris , two important boreal trees in Scandinavia. If strong relationships exist, they can be used to reconstruct past [CO₂]_atm from stomatal frequency of fossil Betula and Pinus leaves. Responses of epidermal characters (stomatal density (SD), epidermal cell density (ED), stomatal index (SI)) to different CO₂ concentrations were investigated utilising (1) the lower partial pressure of CO₂ at increasing altitudes for B. pubescens , and in herbarium specimens of B. pubescens and P. sylvestris collected during the post-industrial rise of [CO₂]_atm from c. 280 ppmv to c. 360 ppmv in 1997 and (2) concentrations (560 ppmv) and temperatures (3° summer) above present day in the CLIMEX greenhouse experiment. All the results show no clear relationship between SD or SI and [CO₂] _atm for either B. pubescens or P. sylvestris. Most likely there are stronger genetically and environmentally induced factors that affect the development of the leaves. Problems with collecting representative samples from herbarium specimens are discussed. Since the effects of changes in [CO₂]_atm cannot be statistically modelled, B. pubescens and P. sylvestris are not suitable for reconstructing past atmospheric CO₂ concentrations from fossil leaves using stomatal density or stomatal index     

Responses of individual stomata in Ipomoea pes-caprae to various CO2 concentrations

The responses of individual stomata to CO₂ concentrations ranging from 0 to 900 μmol mol⁻¹ air were analysed in Ipomoea pes-caprae L. Sweet (Convolvulaceae). The stomata were directly observed using a measurement system that permitted continuous observation of stomatal movement under controlled light and CO₂ conditions. A CO₂ concentration of 350 μmol mol⁻¹ or higher induced stomatal closure, whereas concentrations below 350 μmol mol⁻¹ did not. The time lag before stomatal closure decreased with increasing CO₂ concentration, as did the steady-state aperture of the stomata after a change in CO₂ concentration. However, the rate of stomatal closure increased with increasing CO₂ concentration. Therefore, not only the stomatal closure rate but also the time from the CO₂ concentration change to the beginning of stomatal closure changed with increasing CO₂ concentration. These results suggest that atmospheric CO₂ may be the stimulus for the closure of guard cells. No significant differences were observed between adaxial and abaxial stomata in terms of their responses to CO₂. However, heterogeneous responses were detected between neighbouring stomata on each leaf surface.     

Photosynthetic acclimation to elevated CO2 is modified by source:sink balance in three component species of chalk grassland swards grown in a free air carbon dioxide enrichment (FACE) experiment

Artificial chalk grassland swards were exposed to either ambient air or air enriched to 600 μ mol mol^–1 CO₂, using free-air CO₂ enrichment technology, and subjected to an 8 week simulated grazing regime. After 14 months of treatment, ribulose-1,5-bisphosphate carboxylase (Rubisco) activity ( V _c,max) and electron transport mediated ribulose-1,5-bisphosphate (RuBP) regeneration capacity ( J _max), estimated from leaf gas exchange, were significantly lower in fully expanded leaves of Anthyllis vulneraria L. (a legume) and Sanguisorba minor Scop. grown in elevated CO₂. After a change in source:sink balance brought about by defoliation, photosynthetic capacity was fully restored in A. vulneraria and S. minor, but acclimation continued in the grass Bromopsis erecta (Hudson) Fourr. Changes in net photosynthesis ( P _n) with growth at elevated CO₂ ranged from a 1·6% reduction in precut leaves of A. vulneraria to a 47·1% stimulation in postcut leaves of S. minor . Stomatal acclimation was observed in leaves of A. vulneraria (reduced stomatal density) and B. erecta (reduced stomatal conductance). The results are discussed in terms of whole-plant resource-use optimization and chalk grassland community competitive interactions at elevated CO₂.     

An heuristic hypothesis of chilling injury in plants: a role for calcium as the primary physiological transducer of injury

Abstract. It is suggested that increased levels of free cytosolic calcium ([Ca²⁺]_cyt) may serve as the primary physiological transducer of chilling injury in plants. Numerous similarities between the effects of [Ca²⁺]_cyt-raising treatments on plants and the effects of chilling temperatures on chilling-sensitive (CS) plants are noted. It is proposed that chilling temperatures may lead to increases in [Ca²⁺]_cyt in CS plant cells by reducing the rate at which they exclude Ca²⁺ from their cytosol and that rapid cooling (coldshock) may cause rapid increases in [Ca²⁺]_cyt due to the activation of voltage-dependent cation channels. Chill-induced increases in [Ca²⁺]_cyt in the cells of CS plants may reflect either an inherent inability of such plants to maintain homeostatic levels of Ca²⁺ at low temperatures or a stress-induced reaction which has evolved to enable such cells to cope more effectively with the short-term hardships imposed by cold. Previous proposals concerning the physiological transduction of chilling injury are also discussed. It is argued that there is little evidence to suggest that the immediate effects of low temperatures on CS cells include either decreases in ATP levels, general increases in the passive permeability of membranes, or increased rates of fermentation.