Characterization of the effects of metabolic inhibitors, ATPase inhibitors and a potassium-channel blocker on stomatal opening and closing in isolated epidermis of Commelina communis L.

Abstract. The specific effects of hypoxia and various inhibitors on stomatal opening in the light and closing in the dark were characterized in isolated epidermis from Commelina communis L. Reducing the guard cell metabolism with hypoxia and the uncoupler carbonyl cyanide-m-chloro-phenyl-hydrazone, CCCP, respectively, inhibited both stomatal opening and closing. Stomatal closing was very efficiently blocked by CCCP and this effect could be readily reversed by washing out the inhibitor. The authors were unable to inhibit stomatal opening with ATPase-inhibitors, without also affecting closing. Orthovanadate, up to 2 mol m⁻³, affected neither opening nor closing. Dicyclohexylcarbodiimide, DCCD, and diethylstilbestrol, DES, inhibited opening as well as closing to about 50%. The K⁺ -channel blocker tetraethylammonium chloride, TEA-Cl, inhibited both stomatal opening and closing, as did phenyl acetic acid, PAA, a compound considered to interfere with blue light induced stomatal opening. The results are discussed in the view that the uncontrolled K⁺ leakage from the guard cells is low, that K⁺ efflux during stomatal closing, as well as K⁺ influx during opening, occurs through specific K⁺-channels and that ATP and/or a membrane potential seems to be needed to keep these channels open.     

Putative involvement of cytosolic Ca2+ and GTP-binding proteins in cyclic-GMP-mediated induction of stomatal opening by auxin in Commelina communis L.

To investigate whether cyclic GMP (cGMP) would mediate, in an intracellular Ca²⁺ -dependent manner, coupling of auxin to stomatal opening, the stomatal opening responses to the auxin indolyl-3-butyric acid (IBA) and to the cGMP membrane-permeable derivative 8-bromoguanosine 3^′,5^′-cyclic monophosphate (8-Br-cGMP) were compared in epidermal strips of Commelina communis. In this comparison were studied possible effects of intracellular Ca²⁺ modulators, GTP-binding protein (G-protein) modulators and selective inhibitors of enzymatic reactions which use or generate cGMP. The stomatal response to IBA was almost similarly reversed by the Ca²⁺ buffer 1,2-bis(o-aminophenoxy)ethane-N,N,N^′,N^′-tetraacetic acid (BAPTA), the intracellular Ca²⁺-release inhibitors ruthenium red and procaine, the inactive cGMP analog Rp-8-bromoguanosine 3^′,5^′-cyclic monophosphorothioate (Rp-8-Br-cGMPS), the inhibitor of cGMP-producing guanylyl cyclase LY 83583, the G-protein inhibitor mas17 and the G-protein antagonist pGlu-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH₂. Comparison with stomatal opening in response to 8-Br-cGMP, which was almost completely suppressed by either BAPTA, ruthenium red, procaine or Rp-8-Br-cGMPS, strongly suggests that cGMP acts downstream of G-protein activation as a second messenger for IBA signal transduction and that the cGMP pathway likely depends on cytosolic Ca²⁺signaling. Received: 8 November 1997 / Accepted: 6 March 1998     

Free calcium ion concentration in the sieve-tube sap of Ricinus communis L. seedlings

Changes in free Ca²⁺ in sieve-tube sap have been proposed to be important in the regulation of phloem transport, and Ca²⁺-activated protein kinase activity has been described in phloem exudate (S.A. Avdiushko et al. 1997 J Plant Physiol 150: 552–559). Using atomic absorption spectrometry, we have determined that the total Ca²⁺ concentration in sieve-tube sap from Ricinus seedlings containing the endosperm is about 100 μM (range 80–150 μM). We used three independent methods to determine the free calcium ion concentration in the phloem sap ([Ca²⁺]_p). The first method was to calculate [Ca²⁺]_p from the total Ca²⁺ concentration, in combination with the binding constants and concentrations of the ionic solutes in phloem sap. The resultant estimate of [Ca²⁺]_p was 63 μM. The second method used the Ca-specific fluorescent dye 2-[2-(5-carboxy)oxazole]-5-hydroxy-6-aminobenzofuran-N,N,O-triacetic-acid (FURAPTRA) on exuded sieve-tube sap. Although the sap interfered severely with the fluorescence properties of the dye, Ca²⁺ titrations enabled a value of [Ca²⁺]_p = 20 μM to be deduced. The third method used Ca²⁺-selective microelectrodes on exuded sap samples, which gave an average value for [Ca²⁺]_p = 13 μM. No significant change in this value was observed during the sap exudation period. The Ca²⁺ buffer capacity was determined and the result of about 0.6 mmol · l⁻¹ · pCa⁻¹ displayed excellent agreement with the measured values of free and total Ca²⁺ concentration in sieve-tube sap. Since the measured values for free Ca²⁺ are 20- to 100-fold higher than those usually reported for the cytosol of a range of plant cells in resting conditions, it is concluded that either regulation of [Ca²⁺]_p is of limited physiological importance, or that the Ca²⁺-dependent proteins respond only to relatively high [Ca²⁺]_p. The implications for regulation of cytosolic free Ca²⁺ in symplastically connected companion cells is discussed. Received: 15 February 1998 / Accepted: 14 March 1998     

Photosynthetic and stomatal responses of the halophyte,Plantago maritima L. to fluctuations in salinity

Abstract Measurements of photosynthesis as a function of intercellular CO₂ (A-C₁ curve) were made on single. attached leaves of Plantago maritima L. while plants were exposed to changes in salinity. Salinity was increased in steps from 50 to 500 mol m^-3 NaCl and then returned to 50 mol m^-3 NaCl at two rates, 75 mol m^-3 (NaCl) day^-1 (experiment 1) and 150 mol m^-3 (NaCl) day^-1 (experiment 2). In experiment one, the CO₂ assimilation rate declined at high CO₂ concentrations, but the initial slope of the A-C₁ curve was unaffected in young leaves after salinity was increased to 500 mol m^-3 NaCl. The insensitivity of photosynthesis to increases in CO₂ concentration above air levels was not associated with insensitivity to a reduction in oxygen concentration. In experiment two increasing the rate at which salinity was changed resulted in larger declines in photosynthesis and leaf conductance than were observed in experiment one. Both the initial slope and the CO₂ saturated region of the A-C₁ curve were substantially reduced at high salinity suggesting that mesophyll biochemical capacity had been inhibited. However, concurrent measurements of photosynthesis as oxygen evolution under 5% CO₂ indicated no effect of increased salinity on photosynthetic capacity. This suggests that the apparent non-stomatal limitations indicated by A-C₁ measurements were artifacts caused by strong, nonuniform stomatal closure.     

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.     

Effects of long-term exposure to elevated CO2 and N fertilization on the development of photosynthetic capacity and biomass accumulation in Quercus suber L.

The effects of long‐term (4 year) CO₂ enrichment (70 Pa versus 35 Pa) and nitrogen nutrition (8 mm versus 1 mm NO₃^–) on biomass accumulation and the development of photosynthetic capacity in leaves of cork oak (Quercus suber L., a Mediterranean evergreen tree) were studied. The evolution of photosynthetic parameters with leaf development was estimated by fitting the biochemical model of Farquhar et al. (Planta 149, 78–90, 1980) with modifications by Sharkey (Botanical Review 78, 71–75, 1985) to A–C_i response curves. CO₂ enrichment had a small reduction effect on the development of the maximum CO₂ fixation capacity by Rubisco (V_Cmax), and no effect over maximum electron transport capacity (J_max), day‐time respiration (R_d) and Triose‐P utilization (TPU). However, there was a statistically significant effect of N fertilization and the interaction CO₂ × N over the evolution of V_Cmax, J_max and TPU. Relative stomatal limitation (estimated from A–C_i curves) was higher (+20%) for plants grown under ambient CO₂ than for plants grown under elevated CO₂. There was a significant effect of CO₂ and N fertilization over total biomass accumulation as well as leaf area. Plants grown at elevated CO₂ had 27% more biomass than plants grown at ambient CO₂ when given high N. However, for plants grown under low N there was no significant effect of CO₂ enrichment on biomass accumulation. Plants grown under low N also had significantly higher root : shoot ratios whereas there were no differences between CO₂ treatments. The larger biomass accumulation of Q. suber under elevated CO₂ is attributable to a higher availability of CO₂ coupled to a larger leaf area, with no significant decrease in photosynthetic capacity under CO₂ enrichment and elevated N fertilization. For low N fertilization, the effects of CO₂ enrichment over leaf area and biomass accumulation are lost, suggesting that in native ecosystems with low N availability, the effects of CO₂ enrichment may be insignificant.     

Cytochrome bd-I in Escherichia coli is less sensitive than cytochromes bd-II or bo′' to inhibition by the carbon monoxide-releasing molecule,CORM-3: N-acetylcysteine reduces CO-RM uptake and inhibition of respiration

Background: CO-releasing molecules (CO-RMs) are potential therapeutic agents, able to deliver CO – a critical gasotransmitter – in biological environments. CO-RMs are also effective antimicrobial agents; although the mechanisms of action are poorly defined, haem-containing terminal oxidases are primary targets. Nevertheless, it is clear from several studies that the effects of CO-RMs on biological systems are frequently not adequately explained by the release of CO: CO-RMs are generally more potent inhibitors than is CO gas and other effects of the molecules are evident. Methods: Because sensitivity to CO-RMs cannot be predicted by sensitivity to CO gas, we assess the differential susceptibilities of strains, each expressing only one of the three terminal oxidases of E. coli — cytochrome bd-I, cytochrome bd-II and cytochrome bo′, to inhibition by CORM-3. We present the first sensitive measurement of the oxygen affinity of cytochrome bd-II (K_m 0.24 μM) employing globin deoxygenation. Finally, we investigate the way(s) in which thiol compounds abolish the inhibitory effects of CORM-2 and CORM-3 on respiration, growth and viability, a phenomenon that is well documented, but poorly understood. Results: We show that a strain expressing cytochrome bd-I as the sole oxidase is least susceptible to inhibition by CORM-3 in its growth and respiration of both intact cells and membranes. Growth studies show that cytochrome bd-II has similar CORM-3 sensitivity to cytochrome bo′. Cytochromes bo′ and bd-II also have considerably lower affinities for oxygen than bd-I. We show that the ability of N-acetylcysteine to abrogate the toxic effects of CO-RMs is not attributable to its antioxidant effects, or prevention of CO targeting to the oxidases, but may be largely due to the inhibition of CO-RM uptake by bacterial cells. Conclusions: A strain expressing cytochrome bd-I as the sole terminal oxidase is least susceptible to inhibition by CORM-3. N-acetylcysteine is a potent inhibitor of CO-RM uptake by E. coli. General significance: Rational design and exploitation of CO-RMs require a fundamental understanding of their activity. CO and CO-RMs have multifaceted effects on mammalian and microbial cells; here we show that the quinol oxidases of E. coli are differentially sensitive to CORM-3. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.