Correlation between the inhibition of photosynthesis and the decrease in area of detached leaf discs or volume/absorbance of protoplasts under osmotic stress in pea (Pisum sativum)

Exposure to osmotic stress reduces leaf area and protoplast volume while decreasing photosynthesis. But the measurement of protoplast volume is tedious, while rapid determinations of leaf area in the field are difficult. We evaluated the quantitative relationship between the extent of decrease in area of detached leaf discs or the volume of protoplast of pea ( Pisum sativum ) and reduction in their photosynthetic capacity under osmotic stress. Osmotic stress was induced by increasing sorbitol concentration in the surrounding medium of the leaf discs from zero to 1.0 M (-3.1 MPa), and in case of protoplasts from 0.4 M (-1.3 MPa, isotonicity) to 1.0 M (-3.1 MPa, hypertonicity). There was a high degree of positive correlation between the extent of reduction in the area of detached leaf discs or the volume of protoplasts (indicated by diameter or absorption at 440 nm) and the decrease in photosynthesis. The correlation coefficients between inhibition of photosynthesis and the decrease in leaf disc area or protoplast volume were 0.96 and 0.99, respectively. We therefore suggest that the decrease in absorbance at 440 nm (corrected for turbidity at 750 nm) can be used as a simple measure to predict the inhibition due to osmotic stress of photosynthesis in mesophyll protoplasts. Similarly, the reduction in area of detached leaf discs could also be a very simple and useful criterion to assess osmotic tolerance of photosynthesis.     

Importance of AOX pathway in optimizing photosynthesis under high light stress: role of pyruvate and malate in activating AOX

The present study shows the importance of alternative oxidase (AOX) pathway in optimizing photosynthesis under high light (HL). The responses of photosynthesis and respiration were monitored as O₂ evolution and O₂ uptake in mesophyll protoplasts of pea pre‐incubated under different light intensities. Under HL (3000 µmol m^?2 s^?1), mesophyll protoplasts showed remarkable decrease in the rates of NaHCO₃‐dependent O₂ evolution (indicator of photosynthetic carbon assimilation), while decrease in the rates of respiratory O₂ uptake were marginal. While the capacity of AOX pathway increased significantly by two fold under HL, the capacity of cytochrome oxidase (COX) pathway decreased by >50% compared with capacities under darkness and normal light (NL). Further, the total cellular levels of pyruvate and malate, which are assimilatory products of active photosynthesis and stimulators of AOX activity, were increased remarkably parallel to the increase in AOX protein under HL. Upon restriction of AOX pathway using salicylhydroxamic acid (SHAM), the observed decrease in NaHCO₃‐dependent O₂ evolution or p‐benzoquinone (BQ)‐dependent O₂ evolution [indicator of photosystem II (PSII) activity] and the increase in total cellular levels of pyruvate and malate were further aggravated/promoted under HL. The significance of raised malate and pyruvate levels in activation of AOX protein/AOX pathway, which in turn play an important role in dissipating excess chloroplastic reducing equivalents and sustenance of photosynthetic carbon assimilation to balance the effects of HL stress on photosynthesis, was depicted as a model.     

Beneficial interactions of mitochondrial metabolism with photosynthetic carbon assimilation

Chloroplasts and mitochondria are traditionally considered to be autonomous organelles but they are not as independent as they were once thought to be. Mitochondrial metabolism, particularly the bioenergetic reactions of oxidative electron transport and phosphorylation, continue to be active in the light and are essential for sustaining photosynthetic carbon assimilation. The marked and mutually beneficial interaction between mitochondria and chloroplasts is intriguing. The key compartments within plant cells, including not only mitochondria and chloroplasts but also the peroxisomes and cytosol, appear to be in a delicate metabolic equilibrium. Disturbance of any of these compartments perturbs the metabolism of whole cell. Nevertheless, mitochondria appear to be the key players because they function during both photorespiration and dark respiration.     

Importance of oxidative electron transport over oxidative phosphorylation in optimizing photosynthesis in mesophyll protoplasts of pea (Pisum sativum L.)

The role of mitochondrial respiration in optimizing photosynthesis was assessed in mesophyll protoplasts of pea ( Pisum sativum L., cv. Arkel) by using low concentrations of oligomycin (an inhibitor of oxidative phosphorylation), antimycin A (inhibits cytochrome pathway of electron transport) and salicylhydroxamic acid (SHAM, an inhibitor of alternative oxidase). All three compounds decreased the rate of photosynthetic O₂ evolution in mesophyll protoplasts, but did not affect chloroplast photosynthesis. The inhibition of photosynthesis by these mitochondrial inhibitors was stronger at optimal CO₂ (1.0 m M NaHCO₃) than that at limiting CO₂ (0.1 m M NaHCO₃). We conclude that mitochondrial metabolism through both cytochrome and alternative pathways is essential for optimizing photosynthesis at limiting as well as at optimal CO₂. The ratios of ATP to ADP in whole protoplast extracts were hardly affected, despite the marked decrease in their photosynthetic rates by SHAM. Similarly, the decrease in the ATP/ADP ratio by oligomycin or antimycin A was more pronounced at limiting CO₂ than at optimal CO₂. The mitochondrial oxidative electron transport, through both cytochrome and alternative pathways, therefore akppears to be more important than oxidative phosphorylation in optimizing photosynthesis, particularly at limiting CO₂ (when ATP demand is expected to be low). Our results also confirm that the alternative pathway has a significant role in contributing to the cellular ATP, when the cytochrome pathway is limited.     

Attenuation of hydrogen peroxide-mediated oxidative stress by Brassica juncea annexin-3 counteracts thiol-specific antioxidant (TSA1) deficiency in Saccharomyces cerevisiae

Brassica juncea annexin-3 (BjAnn3) was functionally characterized for its ability to modulate H₂O₂-mediated oxidative stress in Saccharomyces cerevisiae. BjAnn3 showed a significant protective role in cellular-defense against oxidative stress and partially alleviated inhibition of mitochondrial respiration in presence of exogenously applied H₂O₂. Heterologous expression of BjAnn3 protected membranes from oxidative stress-mediated damage and positively regulated antioxidant gene expression for ROS detoxification. We conclude that, BjAnn3 partially counteracts the effects of thioredoxin peroxidase 1 (TSA1) deficiency and aids in cellular-protection across kingdoms. Despite partial compensation of TSA1 by BjAnn3 in cell-viability tests, the over-complementation in ROS-related features suggests the existence of both redundant (e.g. ROS detoxification) and distinct features (e.g. membrane protection versus proximity-based redox regulator) of both proteins.     

Purification and characterization of a Bowman-Birk proteinase inhibitor from the seeds of black gram (Vigna mungo)

A proteinase inhibitor (BgPI) was purified from black gram, Vigna mungo (cv. TAU-1) seeds by using ammonium sulfate fractionation, followed by ion-exchange, affinity and gel-filtration chromatography. BgPI showed a single band in SDS-PAGE under non-reducing condition with an apparent molecular mass of ∼8 kDa correlating to the peak 8041.5 Da in matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrum. BgPI existed in different isoinhibitor forms with pI values ranging from 4.3 to 6.0. The internal sequence “SIPPQCHCADIR” of a peak 1453.7 m/z, obtained from MALDI-TOF-TOF showed 100% similarity with Bowman-Birk inhibitor (BBI) family. BgPI exhibited non-competitive-type inhibitory activity against both bovine pancreatic trypsin (K_i of 309.8 nM) and chymotrypsin (K_i of 10.7 μM), however, with a molar ratio of 1:2 with trypsin. BgPI was stable up to a temperature of 80 °C and active over a wide pH range between 2 and 12. The temperature-induced conformational changes in secondary structure are reversed when BgPI was cooled from 90 to 25 °C. Further, upon reduction with dithiothreitol, BgPI lost both its inhibitory activity as well as secondary structural conformation. Lysine residue(s) present in the reactive site of BgPI play an important role in inhibiting the bovine trypsin activity. The present study provides detailed biochemical characteristic features of a BBI type serine proteinase inhibitor isolated from V. mungo.     

Importance of ROS and antioxidant system during the beneficial interactions of mitochondrial metabolism with photosynthetic carbon assimilation

The present study suggests the importance of reactive oxygen species (ROS) and antioxidant metabolites as biochemical signals during the beneficial interactions of mitochondrial metabolism with photosynthetic carbon assimilation at saturating light and optimal CO₂. Changes in steady-state photosynthesis of pea mesophyll protoplasts monitored in the presence of antimycin A [AA, inhibitor of cytochrome oxidase (COX) pathway] and salicylhydroxamic acid [SHAM, inhibitor of alternative oxidase (AOX) pathway] were correlated with total cellular ROS and its scavenging system. Along with superoxide dismutase (SOD) and catalase (CAT), responses of enzymatic components—ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), glutathione reductase (GR) and non-enzymatic redox components of ascorbate–glutathione (Asc–GSH) cycle, which play a significant role in scavenging cellular ROS, were examined in the presence of mitochondrial inhibitors. Both AA and SHAM caused marked reduction in photosynthetic carbon assimilation with concomitant rise in total cellular ROS. Restriction of electron transport through COX or AOX pathway had differential effect on ROS generating (SOD), ROS scavenging (CAT and APX) and antioxidant (Asc and GSH) regenerating (MDAR and GR) enzymes. Further, restriction of mitochondrial electron transport decreased redox ratios of both Asc and GSH. However, while decrease in redox ratio of Asc was more prominent in the presence of SHAM in light compared with dark, decrease in redox ratio of GSH was similar in both dark and light. These results suggest that the maintenance of cellular ROS at optimal levels is a prerequisite to sustain high photosynthetic rates which in turn is regulated by respiratory capacities of COX and AOX pathways.     

Utility of the modified ATP III defined metabolic syndrome and severe obesity as predictors of insulin resistance in overweight children and adolescents: a cross-sectional study

Background

Inhibition of vascular smooth muscle cell (vSMC) proliferation by oral anti-hyperglycemic agents may have a role to play in the amelioration of vascular disease in diabetes. Thiazolidinediones (TZDs) inhibit vSMC proliferation but it has been reported that they anomalously stimulate [³H]-thymidine incorporation. We investigated three TZDs, two biguanides and two sulfonylureas for their ability of inhibit vSMC proliferation. People with diabetes obviously have fluctuating blood glucose levels thus we determined the effect of media glucose concentration on the inhibitory activity of TZDs in a vSMC preparation that grew considerably more rapidly under high glucose conditions. We further explored the mechanisms by which TZDs increase [³H]-thymidine incorporation.

Methods

VSMC proliferation was investigated by [³H]-thymidine incorporation into DNA and cell counting. Activation and inhibition of thymidine kinase utilized short term [³H]-thymidine uptake. Cell cycle events were analyzed by FACS.

Results

VSMC cells grown for 3 days in DMEM with 5% fetal calf serum under low (5 mM glucose) and high (25 mM glucose) increased in number by 2.5 and 4.7 fold, respectively. Rosiglitazone and pioglitazone showed modest but statistically significantly greater inhibitory activity under high versus low glucose conditions (P < 0.05 and P < 0.001, respectively). We confirmed an earlier report that troglitazone (at low concentrations) causes enhanced incorporation of [³H]-thymidine into DNA but did not increase cell numbers. Troglitazone inhibited serum mediated thymidine kinase induction in a concentration dependent manner. FACS analysis showed that troglitazone and rosiglitazone but not pioglitazone placed a slightly higher percentage of cells in the S phase of a growing culture. Of the biguanides, metformin had no effect on proliferation assessed as [³H]-thymidine incorporation or cell numbers whereas phenformin was inhibitory in both assays albeit at high concentrations. The sulfonylureas chlorpropamide and gliclazide had no inhibitory effect on vSMC proliferation assessed by either [³H]-thymidine incorporation or cell numbers.

Conclusion

TZDs but not sulfonylureas nor biguanides (except phenformin at high concentrations) show favorable vascular actions assessed as inhibition of vSMC proliferation. The activity of rosiglitazone and pioglitazone is enhanced under high glucose conditions. These data provide further in vitro evidence for the potential efficacy of TZDs in preventing multiple cardiovascular diseases. However, the plethora of potentially beneficial actions of TZDs in cell and animal models have not been reflected in the results of major clinical trials and a greater understanding of these complex drugs is required to delineate their ultimate clinical utility in preventing macrovascular disease in diabetes.     

Consequence of restricted mitochondrial oxidative metabolism on photosynthetic carbon assimilation in mesophyll protoplasts: Decrease in light activation of four chloroplastic enzymes

The patterns of light activation of 4 chloroplastic enzymes were examined in mesophyll protoplasts of pea ( Pisum sativum ) in the absence or presence of oligomycin (inhibitor of oxidative phosphorylation) or antimycin A (inhibitor of cytochrome pathway) or salicylhydroxamic acid (SHAM, inhibitor of alternative pathway). The results were compared with those of DCMU (inhibitor of photosynthetic electron transport). The light activation of NADP glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH), fructose-1,6-bisphosphatase (FBPase), phosphoribulokinase (PRK) (enzymes of the Calvin cycle) and NADP malate dehydrogenase (NADP-MDH) (reflects chloroplast redox state) was more pronounced at limiting CO₂ (0.1 m M NaHCO₃) than that at optimal CO₂ (1.0 m M NaHCO₃). SHAM decreased markedly (up to 33%) the light activation of all 4 enzymes, while antimycin A or oligomycin exerted only a limited effect (<10% decrease). Antimycin A or oligomycin or SHAM had no significant effect on light activation of these 4 enzymes in isolated chloroplasts. However, DCMU caused a remarkable decrease in light activation of enzymes in both protoplasts (up to 78%) and chloroplasts (up to 69%). These results suggest that the restriction of alternative pathway of mitochondrial metabolism results in a marked decrease in the light activation of key chloroplastic enzymes in mesophyll protoplasts but not in isolated chloroplasts. Such a decrease in the light activation of enzymes could be also a secondary feedback effect because of the restriction on carbon assimilation.     

Short communication. A novel method of measuring volume changes of mesophyll cell protoplasts and the effect of mercuric chloride on their osmotically-induced swelling

A quick and accurate method of monitoring changes of volume of mesophyll cell protoplasts (MCP) of pea was devised using the difference in absorbance of the protoplasts at 440 nm and 750 nm (OD 440-750); when protoplasts expanded in response to changing the external medium from 0.4 M sorbitol to 0.3 M sorbitol OD 440-750 values increased and, conversely, when protoplasts were transferred from 0.4 to 0.5 M sorbitol, protoplasts contracted. The kinetics of expansion or contraction of the protoplasts could also be monitored using this method and the half-time for water exchange for expanding protoplasts (about 10 s) was slightly higher than that for contracting protoplasts. A study of the effects of the water channel blocker, mercuric chloride, on swelling protoplasts showed that 500 M irreversibly damaged protoplasts, 5-10 M had a negligible inhibitor effect on swelling while 100 M had a large inhibitory effect often completely inhibiting swelling. A preliminary study indicated that mercapto-ethanol reversed the inhibitory effect of mercuric chloride on protoplast swelling.