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.     

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.     

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.     

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     

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.     

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.     

Ultrastructural immunocytochemical localization of carbonic anhydrase

Summary Specific antibodies against human erythrocyte carbonic anhydrase isozyme C were used to determine the ultrastructural localization of this enzyme in the collecting ducts of rat kidney. Using a pre-embedding labeling technique, carbonic anhydrase C was found in the cytoplasm of intercalated cells, whereas the principal cells were negative.     

Metal poison inhibition of carbonic anhydrase

Carbonic anhydrase is inhibited by the “metal poison” cyanide. Several spectroscopic investigations of carbonic anhydrase where the natural zinc ion has been replaced by cobalt have further strengthened the view that cyanide and cyanate bind directly to the metal. We have determined the structure of human carbonic anhydrase II inhibited by cyanide and cyanate, respectively, by X-ray crystallography. It is shown that the inhibitors replace a molecule of water, which forms a hydrogen bond to the peptide nitrogen of Thr-199 in the native structure. The coordination of the zinc ion is hereby left unaltered compared to the native crystal structure, so that the zinc coordinates three histidines and one molecule of water or hydroxyl ion in a tetrahedral fashion. The binding site of the two inhibitors is identical to what earlier has been suggested to be the position of the substrate (CO₂) when attacked by the zinc bound hydroxyl ion. The peptide chain undergoes no significant alterations upon binding of either inhibitor. © 1993 Wiley-Liss, Inc.     

Thermal denaturation of erythrocyte carbonic anhydrase

An experimental study on the thermal behaviour of erythrocyte carbonic anhydrase was carried out with the main aim to estimate the thermodynamic parameters that control the stability of the enzyme. The effects of thermal denaturation on the catalytic properties of the enzyme were also investigated. Below 60 degrees C the enzyme was found to be very stable, whereas between 60 and 65 degrees C a drastic decrease in the biological activity was observed. From the obtained results some considerations were made about the stabilization of the active form of the protein.