O2 reduction was investigated in photosystem I (PSI) complexes isolated from cyanobacteria Synech... more O2 reduction was investigated in photosystem I (PSI) complexes isolated from cyanobacteria Synechocystis sp. PCC 6803 wild type (WT) and menB mutant strain, which is unable to synthesize phylloquinone and contains plastoquinone at the quinone-binding site A1. PSI complexes from WT and menB mutant exhibited different dependencies of O2 reduction on light intensity, namely, the values of O2 reduction rate in WT did not reach saturation at high intensities, in contrast to the values in menB mutant. The obtained results suggest the immediate phylloquinone involvement in the light-induced O2 reduction by PSI.
Light-dependent oxygen reduction in the photosynthetic electron transfer chain, i.e. the Mehler r... more Light-dependent oxygen reduction in the photosynthetic electron transfer chain, i.e. the Mehler reaction, has been studied using isolated pea thylakoids. The role of the plastoquinone pool in the Mehler reaction was investigated in the presence of dinitrophenyl ether of 2-iodo-4-nitrothymol (DNP-INT), the inhibitor of plastohydroquinone oxidation by cytochrome b6/f complex. Oxygen reduction rate in the presence of DNP-INT was higher than in the absence of the inhibitor in low light at pH 6.5 and 7.6, showing that the capacity of the plastoquinone pool to reduce molecular oxygen in this case exceeded that of the entire electron transfer chain. In the presence of DNP-INT, appearance of superoxide anion radicals outside thylakoid membrane represented approximately 60% of the total superoxide anion radicals produced. The remaining 40% of the produced superoxide anion radicals was suggested to be trapped by plastohydroquinone molecules within thylakoid membrane, leading to the formation of hydrogen peroxide (HO). To validate the reaction of superoxide anion radical with plastohydroquinone, xanthine/xanthine oxidase system was integrated with thylakoid membrane in order to generate superoxide anion radical in close vicinity of plastohydroquinone. Addition of xanthine/xanthine oxidase to the thylakoid suspension resulted in a decrease in the reduction level of the plastoquinone pool in the light. The obtained data provide additional clarification of the aspects that the plastoquinone pool is involved in both reduction of oxygen to superoxide anion radicals and reduction of superoxide anion radicals to HO. Significance of the plastoquinone pool involvement in the Mehler reaction for the acclimation of plants to light conditions is discussed.
Effect of knockout of the At4g20990 gene encoding α-carbonic anhydrase 4 (α-CA4) in Arabidopsis t... more Effect of knockout of the At4g20990 gene encoding α-carbonic anhydrase 4 (α-CA4) in Arabidopsis thaliana in plants grown in low light (LL, 80 μmol photons m(-2) s(-1)) or in high light (HL, 400 μmol photons m(-2) s(-1)) under long (LD, 16 h) or short (SD, 8 h) day length was studied. In α-CA4 knockout plants, under all studied conditions, the non-photochemical quenching was lower; the decrease was more pronounced under HL. This pointed to α-CA4 implication in the processes leading to energy dissipation in PSII antenna. In this context the content of major antenna proteins Lhcb1 and Lhcb2 was lower in α-CA4 knockouts than in wild-type (WT) plants under all growth conditions. The expression level of lhcb2 gene was also lower in mutants grown under LD, LL and HL in comparison to WT. At the same time, this level was higher in mutants grown under SD, LL and it was the same under SD, HL. Overall, the data showed that the knockout of the At4g20990 gene affected both the contents of protein...
ABSTRACT Carbonic anhydrase (CA) activities of the mesophyll protoplasts from Pisum sativum L. le... more ABSTRACT Carbonic anhydrase (CA) activities of the mesophyll protoplasts from Pisum sativum L. leaves (plasmalemma CA), the supernatant after thylakoid precipitation (soluble CA), and the washed thylakoids (thylakoid CA) were studied. It was found that the Km (CO2) were 104 mM for the plasmalemma CA, 20 mM for the soluble CA, and 9 mM for the thylakoid CA. The activity of the last differed from the first two in response to the inhibitor acetazolamide by increasing at submicromolar concentrations of inhibitor. pH-dependencies of Km (HCO3-) for soluble CA and thylakoid CA differed in that the former increased as pH increasd from 7.0 to 8.0, while the latter slightly decreased over this pH change. A comparison of the pH-dependencies of the soluble CA and thylakoid CA dehydrase activities expressed in Wilbur-Andersen units demonstrated that, while the activity of soluble CA was unchanged over pH range 6–8, the activity of thylakoid CA had a distinct pH optimum at pH 6.8–7.0. The possible functions of the three forms of CA in the leaf cell of C3 -plants are briefly discussed.
Pea thylakoids with high carbonic anhydrase (CA) activity (average rates of 5000 micromol H(+) (m... more Pea thylakoids with high carbonic anhydrase (CA) activity (average rates of 5000 micromol H(+) (mg Chl)(-1) h(-1) at pH 7.0) were prepared. Western blot analysis using antibodies raised against the soluble stromal beta-CA from spinach clearly showed that this activity is not a result of contamination of the thylakoids with the stromal CA but is derived from a thylakoid membrane-associated CA. Increase of the CA activity after partial membrane disintegration by detergent treatment, freezing or sonication implies the location of the CA in the thylakoid interior. Salt treatment of thylakoids demonstrated that while one part of the initial enzyme activity is easily soluble, the rest of it appears to be tightly associated with the membrane. CA activity being measured as HCO(3) (-) dehydration (dehydrase activity) in Photosystem II particles (BBY) was variable and usually low. The highest and most reproducible activities (approximately 2000 micromol H(+) (mg Chl)(-1) h(-1)) were observed in the presence of detergents (Triton X-100 or n-octyl-beta-D-glucopyranoside) in low concentrations. The dehydrase CA activity of BBY particles was more sensitive to the lipophilic CA inhibitor, ethoxyzolamide, than to the hydrophilic CA inhibitor, acetazolamide. CA activity was detected in PS II core complexes with average rate of 13,000 micromol H(+) (mg Chl)(-1) h(-1) which was comparable to CA activity in BBY particles normalized on a PS II reaction center basis.
O2 reduction was investigated in photosystem I (PSI) complexes isolated from cyanobacteria Synech... more O2 reduction was investigated in photosystem I (PSI) complexes isolated from cyanobacteria Synechocystis sp. PCC 6803 wild type (WT) and menB mutant strain, which is unable to synthesize phylloquinone and contains plastoquinone at the quinone-binding site A1. PSI complexes from WT and menB mutant exhibited different dependencies of O2 reduction on light intensity, namely, the values of O2 reduction rate in WT did not reach saturation at high intensities, in contrast to the values in menB mutant. The obtained results suggest the immediate phylloquinone involvement in the light-induced O2 reduction by PSI.
Light-dependent oxygen reduction in the photosynthetic electron transfer chain, i.e. the Mehler r... more Light-dependent oxygen reduction in the photosynthetic electron transfer chain, i.e. the Mehler reaction, has been studied using isolated pea thylakoids. The role of the plastoquinone pool in the Mehler reaction was investigated in the presence of dinitrophenyl ether of 2-iodo-4-nitrothymol (DNP-INT), the inhibitor of plastohydroquinone oxidation by cytochrome b6/f complex. Oxygen reduction rate in the presence of DNP-INT was higher than in the absence of the inhibitor in low light at pH 6.5 and 7.6, showing that the capacity of the plastoquinone pool to reduce molecular oxygen in this case exceeded that of the entire electron transfer chain. In the presence of DNP-INT, appearance of superoxide anion radicals outside thylakoid membrane represented approximately 60% of the total superoxide anion radicals produced. The remaining 40% of the produced superoxide anion radicals was suggested to be trapped by plastohydroquinone molecules within thylakoid membrane, leading to the formation of hydrogen peroxide (HO). To validate the reaction of superoxide anion radical with plastohydroquinone, xanthine/xanthine oxidase system was integrated with thylakoid membrane in order to generate superoxide anion radical in close vicinity of plastohydroquinone. Addition of xanthine/xanthine oxidase to the thylakoid suspension resulted in a decrease in the reduction level of the plastoquinone pool in the light. The obtained data provide additional clarification of the aspects that the plastoquinone pool is involved in both reduction of oxygen to superoxide anion radicals and reduction of superoxide anion radicals to HO. Significance of the plastoquinone pool involvement in the Mehler reaction for the acclimation of plants to light conditions is discussed.
Effect of knockout of the At4g20990 gene encoding α-carbonic anhydrase 4 (α-CA4) in Arabidopsis t... more Effect of knockout of the At4g20990 gene encoding α-carbonic anhydrase 4 (α-CA4) in Arabidopsis thaliana in plants grown in low light (LL, 80 μmol photons m(-2) s(-1)) or in high light (HL, 400 μmol photons m(-2) s(-1)) under long (LD, 16 h) or short (SD, 8 h) day length was studied. In α-CA4 knockout plants, under all studied conditions, the non-photochemical quenching was lower; the decrease was more pronounced under HL. This pointed to α-CA4 implication in the processes leading to energy dissipation in PSII antenna. In this context the content of major antenna proteins Lhcb1 and Lhcb2 was lower in α-CA4 knockouts than in wild-type (WT) plants under all growth conditions. The expression level of lhcb2 gene was also lower in mutants grown under LD, LL and HL in comparison to WT. At the same time, this level was higher in mutants grown under SD, LL and it was the same under SD, HL. Overall, the data showed that the knockout of the At4g20990 gene affected both the contents of protein...
ABSTRACT Carbonic anhydrase (CA) activities of the mesophyll protoplasts from Pisum sativum L. le... more ABSTRACT Carbonic anhydrase (CA) activities of the mesophyll protoplasts from Pisum sativum L. leaves (plasmalemma CA), the supernatant after thylakoid precipitation (soluble CA), and the washed thylakoids (thylakoid CA) were studied. It was found that the Km (CO2) were 104 mM for the plasmalemma CA, 20 mM for the soluble CA, and 9 mM for the thylakoid CA. The activity of the last differed from the first two in response to the inhibitor acetazolamide by increasing at submicromolar concentrations of inhibitor. pH-dependencies of Km (HCO3-) for soluble CA and thylakoid CA differed in that the former increased as pH increasd from 7.0 to 8.0, while the latter slightly decreased over this pH change. A comparison of the pH-dependencies of the soluble CA and thylakoid CA dehydrase activities expressed in Wilbur-Andersen units demonstrated that, while the activity of soluble CA was unchanged over pH range 6–8, the activity of thylakoid CA had a distinct pH optimum at pH 6.8–7.0. The possible functions of the three forms of CA in the leaf cell of C3 -plants are briefly discussed.
Pea thylakoids with high carbonic anhydrase (CA) activity (average rates of 5000 micromol H(+) (m... more Pea thylakoids with high carbonic anhydrase (CA) activity (average rates of 5000 micromol H(+) (mg Chl)(-1) h(-1) at pH 7.0) were prepared. Western blot analysis using antibodies raised against the soluble stromal beta-CA from spinach clearly showed that this activity is not a result of contamination of the thylakoids with the stromal CA but is derived from a thylakoid membrane-associated CA. Increase of the CA activity after partial membrane disintegration by detergent treatment, freezing or sonication implies the location of the CA in the thylakoid interior. Salt treatment of thylakoids demonstrated that while one part of the initial enzyme activity is easily soluble, the rest of it appears to be tightly associated with the membrane. CA activity being measured as HCO(3) (-) dehydration (dehydrase activity) in Photosystem II particles (BBY) was variable and usually low. The highest and most reproducible activities (approximately 2000 micromol H(+) (mg Chl)(-1) h(-1)) were observed in the presence of detergents (Triton X-100 or n-octyl-beta-D-glucopyranoside) in low concentrations. The dehydrase CA activity of BBY particles was more sensitive to the lipophilic CA inhibitor, ethoxyzolamide, than to the hydrophilic CA inhibitor, acetazolamide. CA activity was detected in PS II core complexes with average rate of 13,000 micromol H(+) (mg Chl)(-1) h(-1) which was comparable to CA activity in BBY particles normalized on a PS II reaction center basis.
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