Effect of nitrogen on carbonic anhydrase activity, stomatal conductance, net photosynthetic rate and yield of mustard

Mustard (Brassica juncea L.) cv. Rohini was grown under three levels of urea nitrogen fertilization [0, 2, and 4 g(N) pot^-1]. Carbonic anhydrase activity and net photosynthetic rate in leaves of 50 d-old plants as well as yield attributes at harvest increased with increasing levels of nitrogen. Stomatal conductance was not affected, and oil content decreased. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of nitrogen on carbonic anhydrase activity,stomatal conductance,net photosynthetic rate and yield of mustard

Mustard (Brassica juncea L.) cv. Rohini was grown under three levels of urea nitrogen fertilization [0, 2, and 4 g(N) pot^-1]. Carbonic anhydrase activity and net photosynthetic rate in leaves of 50 d-old plants as well as yield attributes at harvest increased with increasing levels of nitrogen. Stomatal conductance was not affected, and oil content decreased.     

Emerging roles for carbonic anhydrase in mesophyll conductance and photosynthesis

Carbonic anhydrase (CA) is an abundant protein in most photosynthesizing organisms and higher plants. This review paper considers the physiological importance of the more abundant CA isoforms in photosynthesis, through their effects on CO₂ diffusion and other processes in photosynthetic organisms. In plants, CA has multiple isoforms in three different families (α, β and γ) and is mainly known to catalyze the CO₂ equilibrium. This reversible conversion has a clear role in photosynthesis, primarily through sustaining the CO₂ concentration at the site of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco). Despite showing the same major reaction mechanism, the three main CA families are evolutionarily distinct. For different CA isoforms, cellular localization and total gene expression as a function of developmental stage are predicted to determine the role of each family in relation to the net assimilation rate. Reaction–diffusion modeling and observational evidence support a role for CA activity in reducing resistance to CO₂ diffusion inside mesophyll cells by facilitating CO₂ transfer in both gas and liquid phases. In addition, physical and/or biochemical interactions between CAs and other membrane‐bound compartments, for example aquaporins, are suggested to trigger a CO₂‐sensing response by stomatal movement. In response to environmental stresses, changes in the expression level of CAs and/or stimulated deactivation of CAs may correspond with lower photosynthetic capacity. We suggest that further studies should focus on the dynamics of the relationship between the activity of CAs (with different subcellular localization, abundance and gene expression) and limitations due to CO₂ diffusivity through the mesophyll and supply of CO₂ to photosynthetic reactions.     

Hydrogen sulfide effects on stomatal apertures

Hydrogen sulfide (H₂S) has recently been reported to be a signaling molecule in plants. It has been well established that is has such roles in animals and it has been suggested that it is included into the group of gasotransmitters. We have recently shown that hydrogen sulfide causes stomatal opening in the model plant Arabidopsis thaliana. H₂S can be supplied to the plant tissues from donors such as sodium hydrosulfide (NaSH) or more recently from slow release H₂S donor molecules such as GYY4137. Both give similar effects, that is, they cause stomatal opening. Furthermore both H₂S donors reduced the accumulation of nitric oxide (NO) induced by abscisic acid (ABA) treatment of leaf tissues. Here similar work has been repeated in a crop plant, Capsicum anuum, and similar data has been obtained, suggesting that such effects of hydrogen sulfide on plants is not confined to model species.Key words: abscisic acid, GYY4137, hydrogen sulfide, nitric oxide, stomatal apertureThe effects of hydrogen sulfide on plants have been studied for many years, but it is only recently that it has been suggested that this gas is acting as a signaling molecule. In animals this has been well established^1,2 and it has been suggested that H₂S be grouped together with other gasotransmitters.^2,3 This group will also contain nitric oxide (NO) which as well as having established roles in animals is also known to cause stomatal closure in plants.^4,5 With this in mind, we previously investigated whether H₂S may also have an effect on stomatal closure, using a model organism Arabidopsis thaliana.⁶ The study used two different H₂S donors, sodium hydrosulfide (NaSH) and morpholin-4-ium 4 methoxyphenyl(morpholino) phosphinodithionate (GYY4137). The former will release H₂S in an instant burst which soon dissipates, which questions the wisdom of its use. GYY4137 on the other hand will release H₂S much more slowly and in a manner which is more likely to reflect physiological generation of H₂S.^7,8 Both donors caused stomatal that had previously been exposed to light to open even further. If leaf tissues were not light treated H₂S compounds once again caused stomata to open. Furthermore, H₂S treatment prevented stomatal closure caused by dark treatment. To investigate the possible mechanism of this effect, tissues were treated with the plant hormone abscisic acid (ABA) to initiate NO generation and then NO accumulation was measured in the absence and presence of H₂S donors using fluorescent probes and confocal microscopy.⁹ Both NaSH and GYY4137 caused a reduction in the accumulation of NO. This suggests that H₂S may be acting by a disruption of NO signaling, which results in the alteration of guard cell physiology.Others have reported different effects of H₂S on stomatal movements. Garcia-Mata and Lamattina¹⁰ found that both H₂S donors NaSH and GY4137 caused stomatal closure in different plant species including Vicia faba, Arabidopsis thaliana and Impatiens walleriana. Use of glibenclamide, which is an ABC transport inhibitor, reduced the effect. Cystathione γ lyase and L-Cys desulfhydrase are enzymes which may be responsible for H₂S synthesis and stomatal movements were also reduced by propargylglycine, an inhibitor of these enzymes. It was suggested therefore that H₂S helps to mediate ABA signaling pathway in guard cells. This paper was further discussed following its publication by Desikan.¹¹ However, this seems to be in conflict with the work we reported. This would not be the first time that there has been contradictory data when it comes to reporting stomatal movements, as ethylene has been shown to mediate auxin-induced opening¹² and to cause stomatal closure.¹³More recently it has been reported that stomatal conductance was increased by carbonyl sulfide (COS).¹⁴ The authors went on to suggest that this effect was mediated by H₂S which was produced from COS hydrolysis. This seems to support our original data. Therefore, here we report on the effects of both NaSH and GYY4137 on a different plant species and one which has relevance as an important crop, that is Capsicum anuum. GYY4137 was supplied as in our previous paper in reference ⁶ and ⁷. As can be seen in Figure 1A NaSH caused stomata to open further, even though the leaf tissue had been exposed to the light. Stomata were able to close, as ABA treatment demonstrated, therefore showing that the stomata were not defective. When the experiments were repeated with GYY4137 (Fig. 1B) and smaller but similar effect of the addition of the H₂S donor was seen. This would be expected as the release of H₂S from GYY4137 would be slower and more prolonged than from NaSH.^7,8 To investigate if NO accumulation is also effected in Capsicum when treated with H₂S donors, leaf tissue was treated with ABA to initiate NO generation and NO measured by the use of DAF2-DA as previously reported in references ⁶ and ⁹. Once again the presence of H₂S donors dramatically reduced the amount of NO that was measured following ABA treatment (Fig. 2). This once again suggests that H₂S is having an effect on NO metabolism which may account for the stomata aperture measurements. It has been suggested in animal systems that H₂S and NO react, resulting in the formation of nitrosothiols/nitrothiol-like species¹⁵ which could have signaling effects in their own right. NO in plants has been reported to lead to increased cGMP and/or increased nitrosylation of proteins,⁵ but if H₂S was removing the bioavailability of NO both mechanisms are likely to be reduced.Open in a separate windowFigure 1H₂S donors cause stomatal opening in Capsicum anuum. The leaves of analyzed from Capsicum anuum plants which were between 6 and 7 weeks old. Stomatal bioassays were performed as described previously by Desikan et al.⁹ Epidermal peels were incubated in MES-KCl buffer [10 mM 2-morpholino ethane sulfonic acid (MES), 5 mM KCl, 50 µM CaCl₂, pH 6.15] for 2.5 h exposed to the direct lightning (in 60–100 IE m⁻² s⁻¹) before the addition of various compounds. (A) Samples were sheltered from direct lighting and treated with ABA or NaHS for 2.5 h and left under the day light conditions before stomata apertures were analyzed. (B) Samples were sheltered from direct lighting and treated with ABA or GYY 4137 for next 2 h and left under the day light conditions before stomata apertures were analyzed. Apertures were measured using a light microscope and imaging camera with LEICA QWIN image processing and analysis software (Leica Microsystems and Imaging Solutions, Cambridge, UK). n = 40 stomatal apertures, ±SE. GYY4137 was synthesis as previously described in reference ⁷.Open in a separate windowFigure 2H₂S donors reduce NO accumulation in Capsicum anuum. Nitric oxide accumulation was estimated using the specific NO dye DAF2-DA (Calbiochem, Nottingham, UK), using the method described previously by Desikan et al.⁹ Epidermal fragments in MES-KCl buffer (10 mM MES, 5 mM KCl, 50 µM CaCl₂, pH 6.15) were exposed to the direct lightning for 2 h. After 2 h samples were loaded with 30 µM DAF2-DA for 15 min before washing with MES-KCl buffer; three times for 10 min. Fragments were subsequently incubated for a further 30 min in the presence of various compounds (as indicated below) before images were visualized using CLSM (excitation 488 nm, emission 515 nm; Nikon PCM2000, Kingston-upon-Thames, UK). Images acquired were analyzed using SCION IMAGE software (Scion, Frederick, MD, USA). (A) Control with no treatment; (B) ABA (50) treatment; (C) NaHS (100 µm) treatment alone; (D) ABA treatment in the presence of NaHS; (E) GYY4137 (100 µm) treatment alone; (F) ABA treatment in the presence of NaHS.NO metabolism is involved in a wide range of plant functions, including seed germination,¹⁶ floral development,¹⁷ root gravitropism¹⁸ and gene expression¹⁹ as well as controlling stomatal function.⁴ H₂S on the other hand may be present in or around plants for a variety to reasons. H₂S can be produced endogenously by for example by plastid located cysteine desulfhydrases,²⁰ or H₂S may come from the environment,²¹ including the soil and waters.²² This is further discussed in a recent review in reference ²³. Therefore future work should be focused on the interplay between H₂S from a variety of sources on the NO metabolism of a range of plant tissues. Not all affects of H₂S will be mediated by NO, with alterations of glutathione on H₂S treatment being reported for example.²⁴ But the full extent of the modulation of NO accumulation and signal by both exogenous and endogenous H₂S needs to be explored so the role of these gasotransmitters^2,3 in mediating hormone and stress responses in plants can be fully understood.     

Effects of dextran-bound inhibitors on carbonic anhydrase activity in isolated rat lungs

Effects of macromolecular Prontosil-dextran inhibitors (PD) on carbonic anhydrase (CA) activity in isolated rat lungs were studied. Isolated lungs were perfused with Krebs-Ringer bicarbonate (KRB) solutions containing no inhibitor, PD 100,000 (mol wt 100,000), PD 5,000 (mol wt 5,000), or low-molecular-weight inhibitors (Prontosil or acetazolamide). The time course of effluent perfusate pH equilibration was measured in a stop-flow pH electrode apparatus. Pulmonary CO2 excretion (Vco2) was monitored by continuously recording expired CO2 concentration. The lungs were ventilated with room air and perfused at 37 degrees C with KRB prebubbled with 5% CO2- 20% O2- 75% N2. The results obtained show that both the low-molecular-weight inhibitors and PD's caused postcapillary pH disequilibria (delta pH) in effluent perfusate. However, only acetazolamide and Prontosil caused a reduction in Vco2. These results suggest that there is an intravascular CA, presumably associated with endothelial cell membranes, that is accessible to all inhibitors used and is responsible in part for equilibration of the CO2- HCO3- -H+ reactions in the perfusate but, under the conditions used, does not affect CO2 excretion; and there is an extravascular (possibly intracellular) CA that can be inhibited by low-molecular-weight inhibitors, is primarily responsible for enhanced CO2 transfer across the alveolar-capillary barrier (perhaps via facilitation of CO2 diffusion), and is in part responsible for pH equilibration.     

Oscillations in stomatal conductance and plant functioning associated with stomatal conductance: Observations and a model

Summary Measurements of transpiration, leaf water content, and flux of water in a cotton plant exhibiting sustained oscillations, in stomatal conductance are presented, and a model of the mechanism causing this behaviour is developed. The dynamic elements, of the model are capacitors—representing the change of water content with water potential in mesophyll, subsidiary and guard cells—interconnected by resistances representing flow paths in the plant. Increase of water potential in guard cells causes an increase in stomatal conductance. Increase of water potential in the subsidiary cells has the opposite effect and provides the positive feed-back which can cause stomatal conductance to oscillate. The oscillations are shown to have many of the characteristics of free-running oscillations in real plants. The behaviour of the model has been examined, using an analogue computer, with constraints and perturbations representing some of those which could be applied to real plants in physiological experiments. Aspects of behaviour which have been simulated are (a) opening and closing of stomata under the influence of changes in illumination, (b) transient responses due to step changes in potential transpiration, root permeability and potential of water surrounding the roots, (c) the influence of these factors on the occurrence and shape of spontaneous oscillations, and (d) modulation of sustained oscillations due to a circadian rhythm in the permeability of roots.     

Affinity chromatography of carbonic anhydrase

An insoluble support for affinity chromatography of carbonic anhydrase has been prepared by coupling Sulfamylon (p-aminomethylbenzene sulfonamide) to Sepharose 4B. Carbonic anhydrase binds to Sulfamylon-Sepharose very strongly and can be eluted under mild conditions by the addition of enzyme inhibitors. The gel was used to purify carbonic anhydrase from human erythrocytes and to separate isozymes B and C. It was also employed to separate native enzyme from modified carbonic anhydrases. The apoenzyme and the carboxymethyl enzyme of human carbonic anhydrase B were both isolated by this method.     

Immunohistochemical demonstration of carbonic anhydrase

Summary Rabbits were immunized using human erythroxyte carbonic anhydrase B (HCA B) purified. by the modified methods of Armstrong et al. (1966) and Bernstein and Schraer (1972). The globulin fraction was isolated by ammonium sulphate precipitation. The anti-HCA B globulin was specific, when judged using the double diffusion technique of Ouchterlony and immunoelectrophoresis. No cross reaction with human erythrocyte carbonic anhydrase C was found, but cross reactions with erythrocyte carbonic anhydrase from rat, mouse and guinea pig were observed. Fluorescein isothiocyanate conjugated goat anti-rabbit globulin was used for the localization of HCA B in tissue sections and erythrocytes on slides.     

Carbon-water balance and patchy stomatal conductance

Stomata govern carbon-water balance by simultaneously controlling photosynthesis (A) and transpiration (E). It is unclear how patchy stomatal conductance influences this control. Cowan and Farquhar showed that for a given water supply available during a fixed time interval, carbon gain is maximized by a pattern of stomatal behavior that keeps the partial derivative of A with respect to E constant. This result implies that spatially uniform stomatal conductance is optimal (provided photosynthetic performance and environmental conditions are spatially uniform), so patchy stomatal conductance should be detrimental to carbon-water balance. However, these results required that the curvature of A versus E be uniformly negative. Using mathematical arguments and computer modeling, we show that (1) this caveat is violated under some environmental conditions, (2) water-use efficiency (A/E) is nearly unaffected, and can actually be improved, by patchiness under these conditions, and (3) patchiness has most often been observed under conditions similar to these. These results imply that under many conditions, patchiness may not significantly influence carbon-water balance, consistent with recent work suggesting patchiness may be common but unobserved. Additionally, we discuss implications of these results that muddle the definition of `optimal' in the context of plant gas exchange in some situations, and extend the work of Cowan and Farquhar under conditions causing positive curvature in A versus E. Received: 15 May 1998 / Accepted: 14 October 1998     

Kinetic pathways and carbonic anhydrase mechanisms

Carbonic anhydrase (CA) exists in three forms: the low-pH form (L); the high-pH form (H); and the anion-inhibited from (A). The latter includes the bicarbonate complex. All three forms have been demonstrated in CA I and, when sulfate is removed, in CA II. The L-form of CA III has not yet been seen, even at pH 5. Equilibrium among the three forms in a sample of CA can be established, in principle, by kinetic pathways connecting any two forms; which pathway dominates is as yet an open question. By invoking the usual ping-pong mechanism of CA, during which hydration of CO₂ causes the enzyme to go from H to L, the kinetic pathway connecting A and H is ignored, essentially by definition. Rarely has the AH pathway been considered (cf. Koenig et al., 1980). Though there are few data to demonstrate the relative kinetics of the AL and AH pathways, it can be argued that the latter is buffer-mediated, which could distinguish the two. In this case, the lifetime of a bound anion would be buffer-dependent. We have investigated this point by measuring the nuclear relaxation rates of fluorine of trifluoroacetate in Co²⁺CA II solutions. The fluorine linewidth, and thus the anion exchange rate, is independent of buffer concentration up to ～50 mM, which argues for the AL pathway predominating.     

Effects of dopaminergic compounds on carbonic anhydrase isozymes I, II, and VI

Studies on carbonic anhydrase (CA, EC 4.2.1.1) inhibitors have increased due to several therapeutic applications while there are few investigations on activators. Here we investigated CA inhibitory and activatory capacities of a series of dopaminergic compounds on human carbonic anhydrase (hCA) isozymes I, II, and VI. 2-Amino-1,2,3,4-tetrahydronaphthalene-6,7-diol hydrobromide and 2-amino-1,2,3,4-tetrahydronaphthalene-5,6-diol hydrobromide were found to show effective inhibitory action on hCA I and II whereas 2-amino-5,6-dibromoindan hydrobromide and 2-amino-5-bromoindan hydrobromide exhibited only moderate inhibition against both isoforms, being more effective inhibitors of hCA VI. K(i) values of the molecules 3-6 were in the range of 41.12-363 μM against hCA I, of 0.381-470 μM against hCA II and of 0.578-1.152 μM against hCA VI, respectively. Compound 7 behaved as a CA activator with K(A) values of 27.3 μM against hCA I, of 18.4 μM against hCA II and of 8.73 μM against hCA VI, respectively.     

Effects of dopaminergic compounds on carbonic anhydrase isozymes I,II, and VI

Studies on carbonic anhydrase (CA, EC 4.2.1.1) inhibitors have increased due to several therapeutic applications while there are few investigations on activators. Here we investigated CA inhibitory and activatory capacities of a series of dopaminergic compounds on human carbonic anhydrase (hCA) isozymes I, II, and VI. 2-Amino-1,2,3,4-tetrahydronaphthalene-6,7-diol hydrobromide and 2-amino-1,2,3,4-tetrahydronaphthalene-5,6-diol hydrobromide were found to show effective inhibitory action on hCA I and II whereas 2-amino-5,6-dibromoindan hydrobromide and 2-amino-5-bromoindan hydrobromide exhibited only moderate inhibition against both isoforms, being more effective inhibitors of hCA VI. K_i values of the molecules 3–6 were in the range of 41.12–363 μM against hCA I, of 0.381–470 μM against hCA II and of 0.578–1.152 μM against hCA VI, respectively. Compound 7 behaved as a CA activator with K_A values of 27.3 μM against hCA I, of 18.4 μM against hCA II and of 8.73 μM against hCA VI, respectively.     

Purification and localization of human carbonic anhydrase

Summary Three different isoenzymes of human carbonic anhydrase are now well characterized. Carbonic anhydrase I and II have been known for several years and are located in high amounts in red blood cells as well as in many other tissues.Carbonic anhydrase III, a protein showing CO₂ hydratase and p-nitrophenylphosphatase activity was isolated from skeletal muscle some years ago. Earlier observations based on enzyme activity and radioimmunoassay studies have suggested that this protein is present in greater quantities in red skeletal muscles than in white ones. We have purified CA III from human soleus muscle and using obtained monospecific polyclonal antibody localized this protein in the same muscle fibers which show acid resistant ATPase activity. Using this protein as a marker for type I muscle fibers, fiber classification into type I and II could now be done also from paraffin embedded sections.This study is supported by the Research Council of Physical Education and Sport, Ministry of Education, Finland