Department of Biotechnology and Bioinformatics

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    Interdependence of photosynthesis and respiration in plant cells: interactions between chloroplasts and mitochondria
    ( 1994-01-01) Raghavendra, A. S. ; Padmasree, K. ; Saradadevi, K.
    Photosynthesis and respiration in an illuminated plant cell are not only interdependent but also mutually beneficial. Respiratory rates increase after hours of illumination due to carbohydrate (substrate) accumulation. Besides such long-term effects, photosynthesis and respiration interact even during short illumination periods of a few minutes. The rate of respiration in isolated leaf protoplasts increases severalfold after 10-15 min of illumination. Such light-enhanced dark respiration (LEDR) has been demonstrated in protoplasts as well as in leaves. The stimulation of LEDR by bicarbonate and its sensitivity to inhibitors of photosynthesis (DCMU) or the Calvin cycle (d,l-glyceraldehyde point out the importance of photosynthetic carbon metabolism for respiration. From metabolite analyses of protoplasts, the majority of LEDR is due to mitochondrial oxidation of malate produced by chloroplasts. Simultaneous measurements of photosynthesis and respiration, using mass spectrometry, demonstrate that mitochondrial TCA cycle-based CO2 evolution is inhibited by illumination while O2 uptake is either unaffected or stimulated. The marked sensitivity of photosynthesis in leaves or protoplasts to classic mitochondrial inhibitors such as oligomycin, sodium azide or antimycin A implies that mitochondrial metabolism is essential for photosynthesis. Respiration not only benefits photosynthesis but also protects illuminated leaf protoplasts against photoinhibition. Oxidative electron transport and phosphorylation play a much more important role than the reactions of glycolysis and the TCA cycle in this beneficial interaction. The metabolite shuttles involving PGA-DHAP and/or OAA-malate across the chloroplast and mitochondrial membranes could form the biochemical basis of the interaction between photosynthesis and respiration. Alternatively, cytosolic NAD(P)H, derived from photosynthetic products, can be directly acted upon by the mitochondrial external NAD(P)H dehydrogenase and oxidised through the mitochondrial electron transport system. Mitochondrial oxidation of NAD(P)H (even if indirect) helps to prevent the over-reduction of the cytosol and, consequently, the chloroplast in illuminated leaf cells. Besides the direct interaction with chloroplasts, mitochondria can supply reducing equivalents through malate to peroxisomes during photorespiration and provide citrate as the precursor of oxoglutarate, necessary for glutamine and glutamate formation. These two phenomena further complement the strong interdependence of photosynthesis and mitochondrial metabolism. © 1994.
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    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)
    ( 1996-01-01) Saradadevi, K. ; Padmasree, K. ; Raghavendra, A. S.
    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.
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    Bacterial ice nucleation: Prospects and perspectives
    ( 1997-03-10) Pattnaik, P. ; Batish, V. K. ; Graver, Sunita ; Ahmed, Niyaz
    In this new emerging era of biotechnology, ice nucleating proteins find varied applications in different fields like artificial snow making, freeze texturization of foods, freeze drying, freeze concentration, detection of bacterial pathogens and also in the control of frost damage to plants. However, the commercial exploitation of this novel biobased and energy saving technology is still under consideration.
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    PCR-based rapid detection of Mycobacterium tuberculosis in blood from immunocompetent patients with pulmonary tuberculosis
    ( 1998-01-01) Ahmed, Niyaz ; Mohanty, Ashok Kumar ; Mukhopadhyay, Utpal ; Batish, Virender Kumar ; Grover, Sunita
    A PCR test based on insertion sequence IS1081 was developed to detect Mycobacterium tuberculosis complex organisms in the peripheral blood. The method was applied to blood samples from immunocompetent individuals with localized pulmonary tuberculosis. Seven of 16 (43.75%) blood samples were found to be positive for the circulating DNA copies of M. tuberculosis complex.
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    Alterations in electron transport characteristics during senescence of Cucumis cotyledonary leaves. Analysis of the effects of inhibitors
    ( 1998-12-01) Prakash, J. S.S. ; Baig, M. A. ; Mohanty, Prasanna
    Cotyledonary leaves of Cucumis sativus cv. Poinsette exhibited senescence-induced losses in chlorophyll (Chl) and protein contents within three weeks since germination. Chl and protein concentrations in cotyledonary leaves approached maximum on 6(th) d after germination and they declined to 50 and 41%, respectively, by the 20(th) day of growth. Activities of both photosystem (PS) 2 and PS1 decreased by 33 and 31%, respectively, on the 20(th) day, compared to the control 6(th) day. Changes in sensitivity of PS2 to inhibitors like atrazine and dibromothymoquinone and sensitivity of PS1 to KCN accompanied the changes in PS2 and PS1 activities. Hence both the acceptor side of PS2 and the donor side of PS1 are affected by senescence-induced changes in cucumber cotyledonary leaves.
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    A novel method of measuring volume changes of mesophyll cell protoplasts and the effect of mercuric chloride on their osmotically-induced swelling
    ( 1999-01-01) Willmer, C. M. ; Padmasree, K. ; Raghavendra, A. S.
    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 μM irreversibly damaged protoplasts, 5-10 μM had a negligible inhibitory effect on swelling while 100 μM 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.
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    Response of photosynthetic carbon assimilation in mesophyll protoplasts to restriction on mitochondrial oxidative metabolism: Metabolites related to the redox status and sucrose biosynthesis
    ( 1999-01-01) Padmasree, K. ; Raghavendra, A. S.
    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.
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    Age dependent alterations in photosystem II acceptor side in Cucumis sativus cotyledonary leaf thylakoids: Analysis of binding characteristics of herbicide [ < sup > 14 < /sup > C]-atrazine
    ( 1999-02-01) Prakash, J. S.S. ; Baig, M. A. ; Mohanty, Prasanna
    Senescence induced temporal changes in photosystems can be conveniently studied in cotyledonary leaves. We monitored the protein, chlorophyll and electron transport activities in Cucumis sativus cv Poinsette cotyledonary leaves and observed that by 20th day, there was a 50%, 41% and 30-33% decline in the chlorophyll, protein and photosystem II activity respectively when compared to 6th day cotyledonary leaves taken as control. We investigated the changes in photosystem II activity (O2 evolution) as a function of light intensity. The photosystem II functional antenna decreased by 27% and the functional photosystem II units decreased by 30% in 20-day old cotyledonary leaf thylakoids. The herbicide [14C]-atrazine binding assay to monitor specific binding of the herbicide to the acceptor side of photosystem II reaction centre protein, D1, showed an increase in the affinity for atrazine towards D1 protein and decrease in the QB binding sites in 20th day leaf thylakoids when compared to 6th day leaf thylakoids. The western blot analysis also suggested a decrease in steady state levels of D1 protein in 20th day cotyledonary leaf thylakoids as compared to 6th day sample which is in agreement with [14C]-atrazine binding assay and light saturation kinetics.
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    Importance of oxidative electron transport over oxidative phosphorylation in optimizing photosynthesis in mesophyll protoplasts of pea (Pisum sativum L.)
    ( 1999-03-01) Padmasree, K. ; Raghavendra, A. S.
    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 mM NaHCO3) than that at limiting CO2 (0.1 mM 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 appears 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.
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    Prolongation of photosynthetic induction as a consequence of interference with mitochondrial oxidative metabolism in mesophyll protoplasts of the pea (Pisum sativum L.)
    ( 1999-03-02) Padmasree, K. ; Raghavendra, A. S.
    The restriction of mitochondrial oxidative metabolism by metabolic inhibitors caused a prolongation of induction of photosynthetic O2 evolution in mesophyll protoplasts of the pea (Pisum sativum L.) at both optimal and limiting CO2. There was a significant increase in the lag period of photosynthesis in the presence of oligomycin (an inhibitor of oxidative phosphorylation) or antimycin A (an inhibitor of cytochrome pathway), while the increase in lag was marginal in presence of salicylhydroxamic acid (SHAM) or propyl gallate (inhibitors of alternative oxidase (AOX) pathway). There was a pronounced decrease in steady-state levels of ribulose-1,5-bisphosphate (RuBP) in the presence of oligomycin or antimycin A, but not in the presence of SHAM or propyl gallate. Our results suggest that the restriction of the cytochrome pathway of mitochondrial electron transport or oxidative phosphorylation, leads to a prolongation of the photosynthetic induction period and a marked decrease in the steady state levels of RuBP.
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    Inhibition of topoisomerase II catalytic activity by two ruthenium compounds: A ligand-dependent mode of action
    ( 1999-04-06) Gopal, Y. N.Vashisht ; Jayaraju, D. ; Kondapi, Anand K.
    The ability of two structurally different ruthenium complexes to interfere with the catalytic activity of topoisomerase II was studied to elucidate their molecular mechanism of action and relative antineoplastic activity. The first complex, [RuCl2(C6H6)(dmso)], could completely inhibit DNA relaxation activity of topoisomerase II and form a drag-induced cleavage complex. This strongly suggests that the drug interferes with topoisomerase II activity by cleavage complex formation. The bi-directional binding of [RuCl2(C6H6)(dmso)] to DNA and topoisomerase II was verified by immunoprecipitation experiments which confirmed the presence of DNA and ruthenium in the cleavage complex. The second complex, Ruthenium Salicylaldoxime, could not inhibit topoisomerase II relaxation activity appreciably and also could not induce cleavage complex formation, though its DNA-binding characteristics and antiproliferation activity were almost comparable to those of [RuCl2(C6H6)(dmso)]. The results suggest that the difference in ligands and their orientation around a metal atom may be responsible for topoisomerase II poisoning by the first complex and not by the second. A probable mechanism is proposed for [RuCl2(C6H6)(dmso)], where the ruthenium atom interacts with DNA and ligands of the metal atom form cross-links with topoisomerase II. This may facilitate the formation of a drug-induced cleavage complex.
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    Topoisomerase II is a cellular target for antiproliferative cobalt salicylaldoxime complex
    ( 1999-09-01) Jayaraju, D. ; Gopal, Y. N.Vashisht ; Kondapi, Anand K.
    Topoisomerase II is a cellular target for a number of clinically relevant antitumor drugs. To elucidate the possible cellular target for the antiproliferation activity of cobalt salicylaldoxime (CoSAL), which inhibits 50% of leukemic cell proliferation at a concentration of 60 μM, DNA binding studies and studies of the action of this complex on topoisomerase H catalytic activities were carried out. The results from DNA binding studies show that CoSAL binds DNA strongly with a stoichiometric ratio of two drug molecules for five nucleotide bases and shows a mode of interaction similar to that of DNA groove binding agents. The results from topoisomerase II inhibition studies show that the complex inhibits the relaxation activity of topoisomerase II in a dose-dependent manner and poisons its activity through cleavage complex formation. To see if the hydroxyl group present on imine nitrogen is involved in topoisomerase II poisoning, we synthesized an analogue of CoSAL in which the hydroxyl group was replaced with semicarbazone. This complex too binds DNA with an affinity similar to that of CoSAL, but with a small difference in the mode of interaction; however, it marginally inhibits leukemic cell proliferation and does not inhibit topoisomerase II activity, which suggests the involvement of a hydroxyl group. An immunoprecipitation assay was conducted which showed that the cleavage complex formed in the presence of CoSAL contained 75% of the complex, while the other complex shows only 7.65%. Cyclic voltametric spectra of the complexes in the presence of DNA show that they do not oxidize DNA. These results suggest that CoSAL shows a bidirectional mode of interaction with enzyme and DNA and inhibits topoisomerase II activity by forming a drug- mediated cleavage complex. Our data strongly suggest that topoisomerase II may be one of the cellular targets for antiproliferation activity of CoSAL.
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    Topoisomerase II poisoning and antineoplastic action by DNA-nonbinding diacetyl and dicarboxaldoxime derivatives of ferrocene
    ( 2000-04-01) Vashisht Gopal, Y. N. ; Jayaraju, D. ; Kondapi, Anand K.
    Topoisomerase II is a major molecular target for a number of DNA-binding anticancer drugs. In the present study, we report topoisomerase II inhibition and anticancer activity by four substituted ferrocene derivatives which do not bind to DNA. The first derivative, acetyl-substituted ferrocene (monoacetylferrocene), showed a minor inhibition of topoisomerase II activity along with a consequent inhibition of cancer cell proliferation. The second derivative (diacetylferrocene) showed a higher potency of action compared to the monosubstituted derivative. The third and fourth derivatives, with mono- and disubstituted carboxaldoxime groups (ferrocenecarboxaldoxime and ferrocenedicarboxaldoxime), showed a higher anticancer action and stronger topoisomerase II inhibition. To understand their molecular mechanism of action, cleavage assays were carried out to monitor the drug-induced, topoisomerase II mediated DNA cleavage. The results show that diacetylferrocene and ferrocenedicarboxaldoxime could form an enzyme-drug-DNA ternary complex, called a 'cleavage complex,' resulting in DNA cleavage. These results along with those of an immunoprecipitation assay indicate that the two compounds interact with topoisomerase II alone and poison its activity by trapping the enzyme and enzyme-cleaved DNA in the covalently closed cleavage complex. The formation of such a complex has numerous genetic implications, which ultimately results in neoplastic cell death. (C) 2000 Academic Press.
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    Topoisomerase II poisoning by indazole and imidazole complexes of ruthenium
    ( 2001-01-01) Vashisht Gopal, Y. N. ; Kondapi, A. K.
    Trans-imidazolium (bis imidazole) tetrachloro ruthenate (RuIm) and trans-indazolium (bis indazole) tetrachloro ruthenate (RuInd) are ruthenium coordination complexes, which were first synthesized and exploited for their anticancer activity. These molecules constitute two of the few most effective anticancer ruthenium compounds. The clinical use of these compounds however was hindered due to toxic side effects on the human body. Our present study on topoisomerase II poisoning by these compounds shows that they effectively poison the activity of topoisomerase II by forming a ternary cleavage complex of DNA, drug and topoisomerase II. The thymidine incorporation assays show that the inhibition of cancer cell proliferation correlates with topoisomerase II poisoning. The present study on topoisomerase II poisoning by these two compounds opens a new avenue for renewing further research on these compounds. This is because they could be effective lead candidates for the development of more potent and less toxic ruthenium containing topoisomerase II poisons. Specificity of action on this molecular target may reduce the toxic effects of these ruthenium-containing molecules and thus improve their therapeutic index.
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    Differential changes in the steady state levels of thylakoid membrane proteins during senescence in Cucumis sativus cotyledons
    ( 2001-01-01) Prakash, J. S.S. ; Baig, M. A. ; Mohanty, P.
    Chloroplast structure and function is known to alter during foliar senescence. Besides, the alterations in the structural organisation of thylakoid membranes changes in the steady state levels of thylakoid membrane proteins occur due to leaf ageing. We monitored temporal changes in some of the specific proteins of thylakoid membrane protein complexes by western blotting in the Cucumis sativus cotyledons as a function of the cotyledon age. We observed that the levels of D1 and D2 proteins of photosystem II started declining at the early stages of senescence of Cucumis cotyledons and continued to decline with the progress of cotyledon age. Similarly the level of Cyt f of Cyt b6/f complex declined rapidly with progress of senescence in these cotyledons. The reaction centre proteins of photosystem I were relatively found to be more stable than that of photosystem II reaction centre proteins reflecting possibly the disorganisation of photosystem II prior to photosystem I. The 33 kDa extrinsic protein (MSP) of oxygen evolving complex, the LHCII apoprotein and the β-subunit of ATPsynthase showed the declined levels with the progress of cotyledon age. However, the extents of loss of these proteins were not as high as the reaction centre proteins of photosystem II and the Cyt f. These results provide that during senescence, proteins of thylakoid membranes degrade in a specific temporal sequence and thereby affect the temporal photochemical functions in Cucumis sativus cotyledons.
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    Synthesis of novel non-cross-linking pyrrolobenzodiazepines with remarkable DNA binding affinity and potent antitumour activity
    ( 2001-03-07) Kamal, A. ; Laxman, N. ; Ramesh, G. ; Neelima, K. ; Kondapi, A. K.
    Mixed imine-amide pyrrolobenzodiazepine dimers have been prepared which exhibit potent antitumour activity and have significant DNA binding affinity; one of them, 1c, has been shown to cause a remarkable rise in the melting temperature of calf thymus DNA.
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    Consequence of restricted mitochondrial oxidative metabolism on photosynthetic carbon assimilation in mesophyll protoplasts: Decrease in light activation of four chloroplastic enzymes
    ( 2001-08-06) Padmasree, K. ; Raghavendra, A. S.
    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 mM NaHCO3) than that at optimal CO2 (1.0 mM 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.
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    Restriction of mitochondrial oxidative metabolism leads to suppression of photosynthetic carbon assimilation but not of photochemical electron transport in pea mesophyll protoplasts
    ( 2001-09-25) Padmasree, K. ; Raghavendra, A. S.
    The present study examines and establishes the primary effect of mitochondrial inhibitors, oligomycin, antimycin A and salicylhydroxamic acid (SHAM), on the photosynthetic carbon assimilation and photochemical electron transport activities, monitored in intact mesophyll protoplasts. These inhibitors caused a marked restriction of malate (+ glutamate)-dependent O2 uptake in mitochondria (53-73%) isolated from pea leaves. When mesophyll protoplasts were illuminated in the presence of mitochondrial inhibitors, there was a significant decrease ( > 45%) in HCO3--dependent O2 evolution, while the decrease in O2 evolution was marginal ( < 10%) in the presence of benzoquinone (BQ) (photosystem PSII-mediated) and NO2--(dependent on PSII + PSI) as electron acceptors. 3-(3.4-dichlorophenyl)-1,1-dimethylurea (DCMU), a typical photosynthetic inhibitor decreased drastically all the three reactions: HCO3- or BQ or NO2--dependent O2 evolution in mesophyll protoplasts. Our results indicate that mitochondrial oxidative metabolism (through both cytochrome and alternative pathways) is essential for maintenance of photosynthetic carbon assimilation, but not for PSI or PSII-dependent photochemical electron transport activities in mesophyll protoplasts of pea.