Kollipara Padmasree

Essentiality of mitochondrial oxidative metabolism for photosynthesis: optimization of carbon assimilation and protection against photoinhibition.

Referee: Christine Foyer, Dept. of Biochemistry and Physiology, IACR - Rothamsted, Harpemden, Herts AL5 2JQ, England, United Kingdom The review emphasizes the essentiality of mitochondrial oxidative metabolism for photosynthetic carbon assimilation. Photosynthetic activity in chloroplasts and oxidative metabolism in mitochondria interact with each other and stimulate their activities. During light, the partially modified TCA cycle supplies oxoglutarate to cytosol and chloroplasts. The marked stimulation of O2 uptake after few minutes of photosynthetic activity, termed as light enhanced dark respiration (LEDR), is now a well-known phenomenon. Both the cytochrome and alternative pathways of mitochondrial electron transport are important in such interactions. The function of chloroplast is optimized by the complementary nature of mitochondrial metabolism in multiple ways: facilitation of export of excess reduced equivalents from chloroplasts, shortening of photosynthetic induction, maintenance of photorespiratory activity, and supply of ATP for sucrose biosynthesis as well as other cytosolic needs. Further, the mitochondrial oxidative electron transport and phosphorylation also protects chloroplasts against photoinhibition. Besides mitochondrial respiration, reducing equivalents (and ATP) are used for other metabolic phenomena, such as sulfur or nitrogen metabolism and photorespiration. These reactions often involve peroxisomes and cytosol. The beneficial interaction between chloroplasts and mitochondria therefore extends invariably to also peroxisomes and cytosol. While the interorganelle exchange of metabolites is the known basis of such interaction, further experiments are warranted to identify other biochemical signals between them. The uses of techniques such as on-line mass spectrometric measurement, novel mutants/transgenics, and variability in metabolism by growth conditions hold a high promise to help the plant biologist to understand this interesting topic.

Induction of the AOX1D isoform of alternative oxidase in A. thaliana T-DNA insertion lines lacking isoform AOX1A is insufficient to optimize photosynthesis when treated with antimycin A.

Plant respiration is characterized by two pathways for electron transfer to O(2), namely the cytochrome pathway (CP) that is linked to ATP production, and the alternative pathway (AP), where electrons from ubiquinol are directly transferred to O(2) via an alternative oxidase (AOX) without concomitant ATP production. This latter pathway is well suited to dispose of excess electrons in the light, leading to optimized photosynthetic performance. We have characterized T-DNA-insertion mutant lines of Arabidopsis thaliana that do not express the major isoform, AOX1A. In standard growth conditions, these plants did not show any phenotype, but restriction of electron flow through CP by antimycin A, which induces AOX1A expression in the wild-type, led to an increased expression of AOX1D in leaves of the aox1a-knockout mutant. Despite the increased presence of the AOX1D isoform in the mutant, antimycin A caused inhibition of photosynthesis, increased ROS, and ultimately resulted in amplified membrane leakage and necrosis when compared to the wild-type, which was only marginally affected by the inhibitor. It thus appears that AOX1D was unable to fully compensate for the loss of AOX1A when electron flow via the CP is restricted. A combination of inhibition studies, coupled to metabolite profiling and targeted expression analysis of the P-protein of glycine decarboxylase complex (GDC), suggests that the aox1a mutants attempt to increase their capacity for photorespiration. However, given their deficiency, it is intriguing that increase in expression neither of AOX1D nor of GDC could fully compensate for the lack of AOX1A to optimize photosynthesis when treated with antimycin A. We suggest that the aox1a mutants can further be used to substantiate the current models concerning the influence of mitochondrial redox on photosynthetic performance and gene expression.

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.

Response of photosynthetic carbon assimilation in mesophyll protoplasts to restriction on mitochondrial oxidative metabolism: Metabolites related to the redox status and sucrose biosynthesis

The patterns of cellular metabolites related to redox status and sucrose biosynthesis in mesophyll protoplasts of pea (Pisum sativum L.) were examined 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 increase on illumination in the rate of photosynthesis or cellular metabolites was more at optimal CO2 (1.0 mM NaHCO3) compared to that at limiting CO2 (0.1 mM NaHCO3). Furthermore, the inhibition of photosynthesis in presence of mitochondrial inhibitors was more pronounced at optimal CO2 than that at limiting CO2. There was a marked increase in steady-state levels of triose-P/PGA (phosphoglyceric acid) and glucose-6-phosphate (Glc-6-P) in the presence of oligomycin and antimycin A. In contrast, SHAM caused a marked increase in malate/OAA (oxaloacetate). We suggest that dissipation of excess redox equivalents generated in photosynthesis occurs through both cytochrome and alternative pathways, while sucrose biosynthesis is backed up by cytochrome pathway alone. Thus, mitochondrial respiration (through both cytochrome and alternative pathways of mitochondrial electron transport) optimizes chloroplast photosynthesis by modulating cellular metabolites related to both intracellular redox state and sucrose biosynthesis.

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 O(2) evolution and O(2) uptake in mesophyll protoplasts of pea pre-incubated under different light intensities. Under HL (3000 micromol m(-2) s(-1)), mesophyll protoplasts showed remarkable decrease in the rates of NaHCO(3)-dependent O(2) evolution (indicator of photosynthetic carbon assimilation), while decrease in the rates of respiratory O(2) 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 NaHCO(3)-dependent O(2) evolution or p-benzoquinone (BQ)-dependent O(2) 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.

Importance of the alternative oxidase (AOX) pathway in regulating cellular redox and ROS homeostasis to optimize photosynthesis during restriction of the cytochrome oxidase pathway in Arabidopsis thaliana

BACKGROUND AND AIMS The importance of the alternative oxidase (AOX) pathway, particularly AOX1A, in optimizing photosynthesis during de-etiolation, under elevated CO2, low temperature, high light or combined light and drought stress is well documented. In the present study, the role of AOX1A in optimizing photosynthesis was investigated when electron transport through the cytochrome c oxidase (COX) pathway was restricted at complex III. METHODS Leaf discs of wild-type (WT) and aox1a knock-out mutants of Arabidopsis thaliana were treated with antimycin A (AA) under growth-light conditions. To identify the impact of AOX1A deficiency in optimizing photosynthesis, respiratory O2 uptake and photosynthesis-related parameters were measured along with changes in redox couples, reactive oxygen species (ROS), lipid peroxidation and expression levels of genes related to respiration, the malate valve and the antioxidative system. KEY RESULTS In the absence of AA, aox1a knock-out mutants did not show any difference in physiological, biochemical or molecular parameters compared with WT. However, after AA treatment, aox1a plants showed a significant reduction in both respiratory O2 uptake and NaHCO3-dependent O2 evolution. Chlorophyll fluorescence and P700 studies revealed that in contrast to WT, aox1a knock-out plants were incapable of maintaining electron flow in the chloroplastic electron transport chain, and thereby inefficient heat dissipation (low non-photochemical quenching) was observed. Furthermore, aox1a mutants exhibited significant disturbances in cellular redox couples of NAD(P)H and ascorbate (Asc) and consequently accumulation of ROS and malondialdehyde (MDA) content. By contrast, WT plants showed a significant increase in transcript levels of CSD1, CAT1, sAPX, COX15 and AOX1A in contrast to aox1a mutants. CONCLUSIONS These results suggest that AOX1A plays a significant role in sustaining the chloroplastic redox state and energization to optimize photosynthesis by regulating cellular redox homeostasis and ROS generation when electron transport through the COX pathway is disturbed at complex III.

Preparation and characterisation of lignin nanoparticles: evaluation of their potential as antioxidants and UV protectants

Lignin, an abundant plant biopolymer, is known to possess antioxidant and UV protectant properties in its native state. Nanoparticles exhibit either improved or different properties corresponding to their bulk materials or parent polymers. In the present study, using nanoprecipitation method, dioxane lignin nanoparticles (DLNP) and alkali lignin nanoparticles (ALNP) are fabricated from two different sources of lignin, i.e., hardwood dioxane lignin (DL) extracted from subabul stems and softwood alkali lignin (AL) which is available as a commercial source. Both DLNP and ALNP were fabricated in spherical shape with mean size of 80–104 nm. Analysis of radical scavenging activity revealed that both DLNP and ALNP possess higher antioxidant activity when compared with their parent polymers DL and AL, respectively. UV protectant potential of DLNP and ALNP was validated by monitoring the survival rates of Escherichia coli upon UV-induced mortality. DLNP and ALNP were more efficient than DL and AL in protecting E. coli against UV-irradiation-induced mortality. However, upon irradiation for different time periods, DLNP offered pronounced protection for E. coli against UV when compared with ALNP. Thus, the present study demonstrates that the antioxidant and UV protection properties of DLNP can be exploited further in food, pharmaceutical and cosmetic industries.

Gum resin of Boswellia serrata inhibited human monocytic (THP-1) cell activation and platelet aggregation.

ETHNOPHARMACOLOGICAL RELEVANCE Stem bark gum resin extract of Boswellia serrata is traditionally used in India for its hemostatic, antiinflammatory and cardiovascular health effects and it is named as Śallakī in Ayurvedic medicine. AIM OF THE STUDY This study was conducted to evaluate the antioxidative and antithrombotic properties of stem bark gum resin extracts of Boswellia serrata (BS). MATERIALS AND METHODS The inhibitory activity of the BSWE and BSAE on FeCl(3) induced lipid peroxidation (in vitro) in rat liver and heart homogenates was measured spectrophotometrically. Their effect on H(2)O(2) induced reactive oxygen species (ROS) generation in human monocytic (THP-1) cells was investigated by tracking intensity of a cell permeable fluorescent dye, H(2)DCFDA and subjecting the cell samples to confocal microscopy. Further, the effect of BSAE and BSWE on ADP-induced platelet aggregation was assessed using a multimode detection plate reader, plasma coagulation times using an automated blood coagulation analyzer and on human blood clotting factors Xa and XIa using chromogenic substrate. Phytomarker analysis of the water (BSWE) and hydroalcoholic (BSAE) extracts of BS-gum resin was done through HPLC using a standard compound AKβBA. RESULTS BSAE and BSWE inhibited, to varied extents, the lipid peroxidation in liver (80%) and heart (50%) tissue homogenates of male Wistar rats. Further, BSAE (30 μg dwt/mL) and BSWE (300 μg dwt/mL) attenuated ≥ 60% of H(2)O(2) mediated ROS generation in THP-1 cells. In case of standard compounds, ascorbate (20 μg dwt/mL) and butylated hydroxytoluene (BHT) (10 μg dwt/mL) completely scavenged ROS in the cells. BSAE and BSWE at 3 mg dwt/mL completely inhibited ADP induced platelet aggregation and activities were comparable to 20 μg/mL of heparin. The extracts also showed very high activity in prolonging coagulation time periods. Both types of extracts extended prothrombin time (PT) from ∼13 to >60s and activated partial thromboplastin time (APTT) from ∼32s to >90s. BSAE inhibited clotting factors Xa and XIa remarkably at 6 μg of dwt where as BSWE did not show much effect on FXa and showed 30% inhibition on FXIa at 120 μg. 10 μg of heparin was required to inhibit about 30% activity of the above factors. HPLC analyses suggested that BSAE and BSWE had AKβBA of 9% (w/w) and 7.8% (w/w) respectively. CONCLUSION Present study demonstrated antioxidant and antithrombotic anticoagulant activities of water and hydroalcoholic extracts of Boswellia serratas gum resin. We suggest that BS-gum resin as a good source for lead/therapeutic compounds possessing antioxidant, antiplatelet and anticoagulant activities.

Alternative Oxidase Pathway Optimizes Photosynthesis During Osmotic and Temperature Stress by Regulating Cellular ROS, Malate Valve and Antioxidative Systems.

The present study reveals the importance of alternative oxidase (AOX) pathway in optimizing photosynthesis under osmotic and temperature stress conditions in the mesophyll protoplasts of Pisum sativum. The responses of photosynthesis and respiration were monitored at saturating light intensity of 1000 μmoles m–2 s–1 at 25°C under a range of sorbitol concentrations from 0.4 to 1.0 M to induce hyper-osmotic stress and by varying the temperature of the thermo-jacketed pre-incubation chamber from 25 to 10°C to impose sub-optimal temperature stress. Compared to controls (0.4 M sorbitol and 25°C), the mesophyll protoplasts showed remarkable decrease in NaHCO3-dependent O2 evolution (indicator of photosynthetic carbon assimilation), under both hyper-osmotic (1.0 M sorbitol) and sub-optimal temperature stress conditions (10°C), while the decrease in rates of respiratory O2 uptake were marginal. The capacity of AOX pathway increased significantly in parallel to increase in intracellular pyruvate and reactive oxygen species (ROS) levels under both hyper-osmotic stress and sub-optimal temperature stress under the background of saturating light. The ratio of redox couple (Malate/OAA) related to malate valve increased in contrast to the ratio of redox couple (GSH/GSSG) related to antioxidative system during hyper-osmotic stress. Further, the ratio of GSH/GSSG decreased in the presence of sub-optimal temperature, while the ratio of Malate/OAA showed no visible changes. Also, the redox ratios of pyridine nucleotides increased under hyper-osmotic (NADH/NAD) and sub-optimal temperature (NADPH/NADP) stresses, respectively. However, upon restriction of AOX pathway by using salicylhydroxamic acid (SHAM), the observed changes in NaHCO3-dependent O2 evolution, cellular ROS, redox ratios of Malate/OAA, NAD(P)H/NAD(P) and GSH/GSSG were further aggravated under stress conditions with concomitant modulations in NADP-MDH and antioxidant enzymes. Taken together, the results indicated the importance of AOX pathway in optimizing photosynthesis under both hyper-osmotic stress and sub-optimal temperatures. Regulation of ROS through redox couples related to malate valve and antioxidant system by AOX pathway to optimize photosynthesis under these stresses are discussed.

Inhibitors from Pigeonpea Active Against Lepidopteran Gut Proteinases

ABSTRACT The proteinase inhibitors (PIs) active against bovine pancreatic trypsin, chymotrypsin, and insect midgut trypsin-like proteinases were found in the seeds of 14 cultivars and eight wild types of pigeonpea, Cajanus cajan L.. The inhibitory activity of PIs against trypsin and chymotrypsin, as well as their activity profile on gelatin-polyacrylamide gel electrophoresis (PAGE) were identical among the various cultivars. In contrast to cultivars, the wild types showed differences in inhibitory activity of PIs and their activity profile on gelatin-PAGE. The PIs from all cultivars and few wild types showed 10- to 50-fold higher activity against midgut trypsin-like proteinases of Achaea Janata (L.) (Lepidoptera: Noctuidae), compared with bovine pancreatic trypsin. However, the PIs from both cultivars and wild types showed three- to nine-fold less activity against Spodoptera litura (F.) (Lepidoptera: Noctuidae) midgut trypsin-like proteinases, compared with bovine pancreatic trypsin. This inhibitory potential of PIs from cultivars and wild types, toward midgut trypsin-like proteinases from A. Janata was further evident by the strong activity profile visualized on gelatin-PAGE. These results further suggest that the inhibitory potential of PIs from pigeonpea cultivars and wild types could be exploited in management of nonhost insects.