EFFECTS OF REDOX  REGULATORS ON THE HYDROLYTIC ACTIVITY OF PROTON PUMPS IN THE TONOPLAST OF RED BEET AT DIFFERENT STAGES OF PLANT DEVELOPMENT

        

Natalia V. Ozolina, Еlena V. Pradedova, Yulia G. Sapega, Ryrik K. Salyaev

Siberian Institute of Plant Physiology and Biochemistry Siberian Division of the

Russian Academy of Sciences, P.O.Box 317, 664033 Irkutsk, Russia,

Fax(3952)510754; e-mail ozol@sifibr.irk.ru

 

Abstract

The effects of physiological and chemical redox-regulators (oxidized and reduced glutation, sulphite) on the hydrolytic activity of vacuolar Н⁺-АТPase and Н⁺-pyrophosphatase (Н⁺-PPase) were investigated on vacuoles  isolated from red beet  (Beta vulgaris L. cv. Bordo) taproots at different stages of  plant development.  All redox-regulators  influenced on the hydrolytic activity both  Н⁺-translocating enzymes, although sensitivity of proton pumps to redox-regulators was found to depend from stage of  plant development. In the stage of plant development during intensive growth and accumulation of metabolites (first growing season)  Н⁺-pyrophosphatase proved to be more sensitive to redox-regulators, and in the period of dormancy (storage) Н⁺-АТPase was more sensitive. If changes in redox-conditions affected the activity of tonoplast proton pumps, it may be did not depend on the direction of the shift (oxidation or reduction), that allows to presume regulatory effect of redox-homeostasis change. 

Key words: redox-regulators, redox-homeostasis, proton pumps, Н⁺-АТPase,  Н⁺-pyrophosphatase, vacuole, tonoplast, ontogenesis.

 

Introduction

The  proton pumps of the tonoplast Н⁺-pyrophosphotase (ЕС 3.6.1.1.) and Н⁺-АТPase (ЕС 3.6.1.3) play a key role in transport processes since they generate the transmembrane proton gradient, which is required to accumulate reserve substances in vacuoles. An elucidation of the mechanism regulating the activity of these pumps is of great practical interest from the viewpoint of the possibility of controlling the transport of essential metabolites (carbohydrates, amino acids, and other compounds) into plant vacuoles [1]. It is assumed that plant vacuolar proton pumps may be reversibly regulated by the change of redox-conditions in vivo [2]. At vacuolar Н⁺-АТPases this type of regulation was studied on the yeast, Neurospora crassa [3, 4]. Redox-regulation was recently shown to act as one of the mechanisms controlling  the activity of Н⁺-АТPase and Н⁺-pyrophosphatase of beetroot vacuolar membrane [5].  The present study was aimed at the investigation of redox-conditions influence on the activity of tonoplast proton pumps at different stages of plant development, when membrane functions significantly differed.

     

Materials and Methods

Vacuoles were isolated from red beet (Beta vulgaris L., cv.Bordo) taproots  harvested at two stages of plant development. There stages are characterized by qualitative changes in the biochemical properties and physiological functions of the plants. We performed experiments at two stages of plant development: during intensive growth and accumulation of metabolites (first growing season) and period of dormancy (storage).

      The root tissue was sliced in a medium containing 800 mM KCI, 20 mM EDTA, 50 mM NaH₂PO₄, pH 8.0.  The solution containing the tissue was sieved and centrifuged for 10 min at 250 g [6]. The vacuolar preparations obtained were used in the experiments. The hydrolytic activity of the Н⁺-АТPase and Н⁺-pyrophosphatase in the tonoplast was determined by incubating the isolated vacuoles 30 minutes at 37^оС  in a stabilizing solution containing 70 mM KCl, 3 mM Mg₂SO₄, 0.5 mM of ammonium molybdate, 30 mM Tris/MES (рН 7.4). The activities of the enzymes were determined by the amount of phosphate cleaved from the substrate during hydrolysis.  Mg-АТP or potassium pyrophosphate at the concentration of 3 mM (Sigma) was used as a substrate. The phosphate was incorporated into a phosphorus molybdenum complex and reduced with ascorbic acid to form a blue colour and absorbance was determined at 600 nm. For colour formation to occur, the solution analyzed was supplemented with 5 mL of 0.1 M acetate buffer, pH 4, 0.5 mL of 1% ammonium molybdate diluted with 0.05 N H₂SO₄ and 0.5 mL of 1% ascorbic acid diluted with 0.01 M CuSO₄ [7].  Oxidized or reduced glutathione (10, 20 мМ), sodium sulphite (5 and 25 mM) and potassium nitrate (50 mM) were added to the incubation media of test variants. In case of sodium sulfite, incubation solutions were equalized by the content of sodium ions via adding NaCI in relevant concentration.  The activities of the enzymes were expressed in arbitrary units as the ratio between  inorganic phosphate (Pi) and protein determined by the method of Bradford [8] during time of incubation. Enzyme activity in vacuoles exposed to redox-regulators was expressed as a percentage of the activity in control samples without added redox-regulators.

Enzymes activity in control variants was accepted as 100% and amounted on average for Н⁺-pyrophosphatase 2.05 ± 0.08 µM Pi/mg of protein in the first growing season  and 1.48 ± 0.09 µM Pi/mg of protein in the period of dormancy. Н⁺-АТPase activity in the first growing season amounted to 0.98 ± 0.07 and 0.45 ± 0.08 µM Pi/mg of protein in the period of dormancy.

            The data in figures represent the means of 5-7 replicates and their standart errors.

            The following chemical reagents produced by the company «Sigma» (USA) were used for the study: Tris, 2-N-morpholino-ethane-sulphonic acid (МES), ATP (magnesium salt), pyrophosphate, ammonium molybdate, glutathione. Other reagents used were produced in Russia.

 

            Results and discussion

 

            Glutathione physiological redox-pair known to participate in numerous physiological processes within the cell in vivo is currently most frequently used for the investigation of redox-conditions influence on enzymes activity [9].  In the first series of experiments we compared the impact of oxidized glutathione (GSSG) and reduced glutathione (GSH) on hydrolytic activity of tonoplast proton pumps. The results acquired are presented in figures  1 and 2.  Sensitivity towards redox-regulators for both enzymes depended from  stage of plant development during which the test was conducted. This is particularly well illustrated by the tests with Н⁺-pyrophosphatase (Fig.1). Vacuoles extracted from the roots in period of dormancy (storage)  (Fig.1B) demonstrated no reliable influence of the redox-agents under study, whereas in the period of the first growing season (Fig.1А) hydrolytic activity of Н⁺-pyrophosphatase reduced by 60-70% with the change of redox-environment caused by both forms of glutathione Oxidized glutathione manifested a more pronounced influence.

       These physiological regulators demonstrated a different influence on Н⁺-АТPase (Fig.2) at different stages of plant development.  In the period of dormancy (Fig.2B) hydrolytic activity of Н⁺-АТPase reduced in the presence of both forms of glutathione by 20%, whereas in the period of  the first growing season its activity increased by 15-20% due to the same regulators. This proton pump was slightly more affected by reduced glutathione. Tests with different glutathione concentrations (10 and 20 mM) did not show significant differences. The results acquired allow presuming that hydrolytic activity of vacuolar membrane proton pumps is affected not by an individual reduced or oxidizes form of glutathione, but the change of homeostasis regardless of its direction.

            In the second series of experiments we studied the influence of sulfite chemical regulator on Н⁺-АТPase at different stages of plant development. The studies of fungi  (Saccharomyces cerevisiae, Neurospora crassa) [3, 9] demonstrated that sulfite may be regulated the activity of vacuolar membrane Н⁺-АТPase in vivo by oxidation/reduction of sulfhydryl groups. At the same time sulfite ability to prevent Н⁺-АТPase activity inhibition by nitrite was revealed. In our experiments we studied sulfite influence on Н⁺-АТPase of beetroot tonoplast at different stages of plant development.  These experiments also showed the change in sensitivity of the enzyme studied depended from stages of plant development. Reliable increase of activity by 40-50% under the influence of sulfite was noted only in the period of dormancy (Fig.3). In the same period addition on sulfite eliminated inhibiting effect of nitrate on the Н⁺-АТPase activity. Sulfite is a reducer and the results acquired allow presume that the inhibiting influence of nitrate ions is conditioned by its oxidizing properties, which neutralize sulfite in the experiments conducted. During the period of active growth processes in first growing season Н⁺-АТPase showed no sensitivity towards this chemical redox-agent.  Stimulating influence of sulfite did not vary significantly with the use of different concentrations.

            The results acquired showed that enzymes sensitivity towards change in redox environment depended from the stages  of plant development. During active growth processes in the first growing season Н⁺-pyrophosphatase was more sensitive to the change of redox-homeostasis in any direction; its activity decreased considerably, whereas the same conditions caused slight increase in the activity of Н⁺-АТPase. During the period of dormancy, redox-homeostasis change produced and inhibiting impact only Н⁺-АТPase. Changes of functions performed by the membrane at various stages of ontogenesis may be presumed to affect micro-environment of the enzymes and cause changes in its conformation, when the redox-agent sensitive part of molecule became inaccessible. Or we might be dealing with different iso-forms of enzymes at different stages of ontogenesis.

 

            Acknowledgments

 

The work was accomplished with the support of Russian Foundation fo Basic research (project № 05-04-48351-а).

                                                 

            Literature

 

1. Maeshima M. // Annu. Rew. Plant Physoil. Plant. Mol. Biol., 2001, v. 52, p. 469-497.

2. Tavakoli N., Kluge C., Golldack D., Mimura T., Dietz K. // Plant J. 2001, v. 28, p. 28-51.

3. Feng Y., Forgac M. // J. Biol. Chem. 1992, v. 267, p. 5817-5822.

4. Forgas M. // J. Exp. Bot., 2000, v.  203, p. 71-80.

 5.  Pradedova E.V.,  Sapega  Yu.G.,  Zheleznykh A.O., Ozolina N.V.,  Salyaev R.K. // Biological membranes., v. 23, № 4, p. 269-301.

6. Salyaev R.K., Kuzevanov V.Ya., Khaptagayev V.Ya., Kopytchuk V.N. // Plant physiology, 1981, v.  28, p. 1295-1305.

7. Nikulina G.N. Review of methods of quantitative determination of phosphorus by formation of molybdenum blue//  L.: Nauka, 1965. p.45.

8. Bradford M. //  Anal. Biochem.  72: 248-254, 1976.

9. Dschida W., Bowman B. // J. Biol. Chem., 1995, v. 270, p. 1557-1563.

     

 

 

 

 

 

 

                                                       Figure legends

 

Figure 1.    Effect of reduced glutathione (GSH) and oxidized glutathione                 (GSSG) in various concentrations on  Н⁺-pyrophosphatase activity at different stages of plant development: А -  period of  growth (first growing season); B – period of dormancy (storage).

 

Figure 2.    Effect of reduced glutathione (GSH) and oxidized glutathione                (GSSG) in various concentrations on  Н⁺-ATPase activity at different stages of ontogenesis: А -  period of  growth (first growing season); B – period of dormancy (storage).

 

Figure 3.       Effect of Na₂SO₃ in various concentrations  and 50 mM KNO₃ on  Н⁺-АТPase activity at different stages of plant development:  А -  period of  growth (first growing season); B – period of dormancy (storage).

 

 

 

Figure 1.    Effect of reduced glutathione (GSH) and oxidized glutathione                 (GSSG) in various concentrations on  Н⁺-pyrophosphatase activity at different stages of plant development: А -  period of  growth (first growing season); B – period of dormancy (storage).

 

 

 

 

 

 

 

Figure 2.    Effect of reduced glutathione (GSH) and oxidized glutathione(GSSG) in various concentrations on  Н⁺-ATPase activity at different stages of ontogenesis: А -  period of  growth (first growing season); B – period of dormancy (storage).

 

 

Figure 3.         Effect of Na₂SO₃ in various concentrations  and 50 mM KNO₃ on  Н⁺-АТPase activity at different stages of plant development:  А -  period of  growth (first growing season); B – period of dormancy (storage).

 

 

 

OZOLINA Natalia Vladimirovna, D.Sc., senior scientific researcher. Home address: 664056 Irkutsk, Primorskyi str.,4, ap.21. Home telephone: (3952) 427295, office telephone: (3952) 425878. Fax: (3952) 510754. E-mail: Ozol@sifibr.irk.ru.

PRADEDOVA Elena Vladimirovna, Ph.D Biology, senior scientific researcher. Home address: 664053 Irkutsk, Pomjalovski str., 28, ap.30. Home telephone: (3952) 420311, office telephone: (3952) 425878. Fax: (3952) 510754. E-mail: praded@sifibr.irk.ru.

SAPEGA Yulia Gennadievna, post-gradute student. Home address: 664012 Irkutsk, 25 October str., 25, ap.2. Office telephone: (3952) 425878. Fax: (3952) 510754. E-mail: sapegal@sifibr.irk.ru.

SALYAEV Ryrik Konstantinovich D.Sc., Prof. Home address: 664000 Irkutsk, Rossiiskaya str., 8, ap.18. Home telephone: (3952) 341018, office telephone: (3952) 420721. Fax: (3952) 510754. E-mail: salyaev@sifibr.irk.ru.

         Correspondence is to be conducted with N.V. Ozolina.

 

 

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