Analele tiinifice ale Universitii Alexandru Ioan Cuza, Seciunea Genetic i Biologie Molecular, TOM XIII, 2012

INVESTIGATIONS REGARDING THE ANTHROPIC IMPACT ON THE KREBS CYCLE DEHYDROGENASES SYSTEM ON CERTAIN WOODSPECIES IN MINING AREAS, SUCEAVA COUNTY
ELENA TUTU1, MARIUS VIOREL ONICIUC1, ELENA CIORNEA1*
Keywords: dehydrogenases, Krebs cycle, pollution, copper, barite, sulphur. Abstract: The Krebs cycle, a second stage of cellular respiration that occurs in the mitochondrion of the leaf cell and consist in a multistep processes plays a central role in catabolism of organic fuel molecules. The mining extraction technologies for both underground and surface, the preparation of copper ore and barite applied in Tarnia, respectively to the sulphur in Calimani Mountain and the excess of these elements in natural environment may cause malfunction of ecosystem. The dehydrogenases of Krebs cycle can give information on the type and the duration of the effects of pollutants on the metabolic activity in leaves, to subsequent area pollution, therefore, the aim of the present study has been to determine these effects on this enzymatic system activity. For this reason, the isocitrate dehydrogenase, the -ketoglutate dehydrogenase, the succinate dehydrogenase and the malate dehydrogenase activity was determined using the spectrophotometric method with triphenyl-tetrazolium and the analysis of experimental results shows the differences from one sample to another sample of closely related species specificity, but also the effect of environmental factors.

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INTRODUCTION MATERIALS AND METHOD RESULTS AND DISCUSION
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The Krebs cycle, the second stage of cellular breathing which takes place in the mitochondria of the vegetal cells, a multi-staged process in its turn, has a central place in the catabolism of the organic molecules that furnishes the necessary fuel for cell activity. The technologies used in underground and surface mining, the preparation of the copper and barite ores in Tarnia and of sulphur in the Climani Mountains, as well as the excess of these elements in the natural environment can produce malfunctions in the ecosystem. In this regard, the Krebs cycle dehydrogenases can offer information regarding the length and type of the effects on the metabolic activities in leaves that appear due to the contamination of the abovementioned areas. The purpose of this paper was to evaluate these effects on the activity of the enzymatic system. For this reason, the activity of the isocitrate-dehydrogenase, -ketoglutarate-dehydrogenase, succinate-dehydrogenase and malate-dehydrogenase was determined trough the method using triphenyl-tetrazolium and the analysis of the experimental results highlights differences from one sample of probes to other, in close connection with the species specificity but also with environmental factors.

Work had been done on foliar tissue sampled from specimens of Picea abies, Populus tremula, Salix alba, Larix decidua, Betula verrucosa and Fagus sylvatica from polluted (Climani sulphur exploitation and Tarnia nonferrous ore flotation station) and non-polluted (Rdui) areas of Suceava county, determining the activity of the Krebs cycle dehydrogenases (isocitrate-dehydrogenase, -ketoglutarate dehydrogenase, succinate-dehydrogenase and malatedehydrogenase) using the method with triphenyl-tetrazolium (Cojocaru, 2009).

The paper systematizes the results of the research regarding the determination of the activity of the main enzymes of the Krebs cycle, considering the fact that the acclimatization of plants at stress is associated with profound changes in the composition of the cellular proteome, the proteins being directly involved in the resistance of the plants to stress induced by metallic excess. From the data shown in figure 1, at Picea abies, we can see a balanced evolution of the Krebs cycle, the enzymes catalyzing the different stages in well-defined proportions. Hence, the activity of the isocitrate-dehydrogenase, (8.9444 g formazan/g. vegetal mat.) is followed by a

Elena Tutu et al Investigations regarding the anthropic impact on the Krebs cycle dehydrogenases system on certain wood-species in mining areas, Suceava county

much smaller activity of -ketoglutarate dehydrogenase, 4.950 g formazan/g. vegetal mat., a situation that can be explained by the fact that possibly, a large portion of the substrate could have entered in the anabolic pathways connected in this point (the biosynthesis of glutamate family amino acids, for example), depending on the necessities of the vegetal cell. Moreover, it is known that the substrate represented by -ketoglutarate is not only a target for species of reactive oxygen generated at mitochondrial level, but also a generator. It is very likely that trough endogenous mechanisms, the vegetal cell might have adopted a strategy to counter the oxidative stress, in order to maintain the integrity of the cellular mitochondrion, for a good evolution of the intermediary metabolism and for the prolonging of the cellular viability. The succinate comes out of the Krebs cycle under the form of malate, the enzyme conditioning this transformation reaching a level 6.395 g formazan/g. vegetal mat. It can be seen, at a careful analysis, that the succinate and the malate are in higher quantities when, catalyzed by the nearby dehydrogenases, they enter in the stages of the Krebs cycle, which leads us towards two possible explanations: the first one would be that it is possible that the extra quantity might have entered in the path of the tricarboxylic acids from other metabolic pathways; the second is that, at certain chronic concentration of sulphur, the activity of malate-dehidrogenase is stimulated. In the foliar materials sampled from Populus tremula, the isocitrate-dehydrogenase reached the highest level of all species taken into account 30.043 g formazan/g. vegetal mat. A convincing explanation would be that the sulphur had produced at this species an excess opening of the stomatas, considering its well-known capacity to do this, which provoked a hydric stress at the level of the spruce needles, and in concordance with the data obtained by Pierre, M. and Queiroz, O. (1988), in these conditions the isocitrate-dehydrogenase had a escalation of enzymatic activity. However, the oxidative decarboxylation had progressed at much smaller levels (4.950 g formazan/g. vegetal mat.). It is established that a known mechanism used by plants at stress is to produce and accumulate osmoprotectans such as amino acids. Some of these synthesize at different amplitudes, starting from ketoglutarate, on anabolic pathways connected to the Krebs cycle. The succinate-dehydrogenase had close values (13.759 g formazan/g. vegetal mat.), not higher than those of malate-dehidrogenase at this species: 12.598 g formazan/g. vegetal mat. Ziegler, I. (1974) had shown that malate-dehidrogenase dependent on NAD+ and NADP+, at treatments higher than 0,05 mM with sulfites on the vegetal cell, is strongly inhibed, probably due to the decomposition of the disulphide proteins trough the sulfitolysis of the S S bridges in the polypeptides that where followed in this experiment. In contrast, Rao M. I. et al. (1983) had found that at other species of angiosperms, that the foliar epidermal tissues had presented, at short chronic exposures to sulphur, a reduced activity of the malate-dehidrogenase, while in the entire leaf, exposed to concentrations higher than 0.05 mM NAD+ and NADP+, the malate-dehidrogenase showed a higher activity. The different answer given by the epidermis and the rest of the leaf was caused, in the opinion of the above-mentioned authors, by the mezophyll. In its turn, Salix alba, had shown a balanced evolution of the Krebs cycle, the isocitratedehydrogenase having one of the highest values of activity from all species taken into account (22.679 g formazan/g. vegetal mat.). This can be explained trough the fact that this enzyme is capable of furnishing enough NAD(P)H to maintain the inter-cellular detoxification. In its turn, the behaviour of the -ketoglutarate dehydrogenase was similar to that of the other species, the enzyme having the maximum of its activity at 11.129 g formazan/g.vegetal mat., the succinatedehydrogenase having in its turn the same behaviour: 13.629 g formazan/g. vegetal mat. The higher level of malate-dehidrogenase certifies a higher toxicity of sulfur on the foliar material

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Analele tiinifice ale Universitii Alexandru Ioan Cuza, Seciunea Genetic i Biologie Molecular, TOM XIII, 2012

sampled from Salix alba (like at Fagus sylvatica). It is a known fact that a higher degree of malate is strongly related to a higher activity of phosphoenolpyruvate carboxylase, an enzyme involved in the guard cell metabolism during the stomata opening and in the furnishing of the malate as a breathing substrate in the foliar materials subjected to different amounts of stress (Dizengremel, P. et al., 2009; OLeary, B. et al., 2011). Moreover, the malate, together with other organic acids has an important role in the redox cell equilibrium of NAD(P)H / NAD(P)+ (Sriram, G. et al., 2007). Betula verrucosa is a species known to be tolerant to contamination, due to its extremely evolved radicular ectomycorrhizant system, capable of accumulating the contaminators at this level, offering the leaves the capability to have a normal metabolic activity, with little to no influence from pollution. The Krebs cycle, in this series of determination, was found to have a balanced evolution of its stages, the highest activity having been noted in the case of the isocitrate-dehydrogenase15.641 g formazan/g. vegetal mat. However, the activity of the -ketoglutarate dehydrogenase was found to be the smallest from all the studied species 7.149 g formazan/g. vegetal mat., only half of the activity of the isocitrate-dehydrogenase at birch. The activity of the succinatedehydrogenase is at a similar level - 7.3456 g formazan/g. vegetal mat., while the activity of the malate-dehidrogenase is closer to that of the isocitrate-dehydrogenase - 11.981 g formazan/g. vegetal mat., as a metabolic reaction of the plant to the stress factors, both enzymes acting as regulator arms of the intermediary metabolism and of the breathing in order to accommodate the plant to contamination. It cannot be ruled out, knowing the fact that the breathing at the foliar system is directly correlated to the season in which the determinations where made, that this factor, besides the genetic background, the pedoclimatic factors and the species phenotype, might had in its turn, a decisive role in the activity of this enzymes. However, in the case of the larch, the activity of the isocitrate-dehydrogenase was the lowest of all the studied species 6.450 g formazan/g. vegetal mat., a similar level being shown by the oxoglutarate-dehydrogenase 5.7592 g formazan/g. vegetal mat., while the activity of the succinate-dehydrogenase had proven to be much smaller, the level attained by this enzyme was only 3.820 g formazan/g. vegetal mat., followed by a slight increase of malatedehidrogenase -5.179 g formazan/g. vegetal mat.
35 30 25 20 15 10

g formazan/g biological matter

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5 0
la ua ies osa alba a tica e mu a ab rruc decid ss p s sylv lus tr Pic e la v e Larix S alix Fagu P opu Be tu

Isocitrate-DH

Malate - DH

Succinate - DH

-Keto-glutarate - DH

Fig. 1. The Krebs cycle dehydrogenases activity in leaves of some Angiosperms and Gymnosperms species of Climani Massive

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Elena Tutu et al Investigations regarding the anthropic impact on the Krebs cycle dehydrogenases system on certain wood-species in mining areas, Suceava county

The intensity of the intermediary metabolism had been found the strongest in the foliar samples of Fagus sylvatica where, despite the fact that the intensity of the isocitratedehydrogenase was not as high as in Populus tremula 24.370 g formazan/g.vegetal mat., the other enzymes of the Krebs cycle had higher intensities than in other species (succinatedehydrogenase 16.345 g formazan/g. vegetal mat., malate-dehidrogenase 22.524 g formazan/g. vegetal mat.) in contrast with the -ketoglutarate dehydrogenase that attained a medium level in comparison with other species, 7.141 g formazan/g. vegetal mat.). At a careful analysis of the main enzymes of the Krebs cycle, it can be observed that at all species grown on soils contaminated with heavy metals, as it is shown in the fig. 2, the oxidative decarboxylation of the isocitrate, mediated by the isocitrate-dehydrogenase at 2oxoglutarate, had reached the highest level in the foliar samples taken from Salix alba (21.438 g formazan/g. vegetal mat.), all the other species having in their own turn, a high amplitude of the same enzyme (20.858 438 g formazan/g. vegetal mat. at Fagus sylvatica, 18.389 g formazan/g. vegetal mat. at Larix decidua, 17.746 g formazan/g. vegetal mat. at Betula verrucosa, 11.962 g formazan/g. vegetal mat. at Picea abies and 9.962 g formazan/g. vegetal mat. at Populus tremula). Despite the fact that in previous studies, (Kasim, W.A., 2007) excess zinc had been found capable of inhibiting the activity of isocitrate-dehydrogenase, in the interpretation of our results we must take into account other data from the literature that signal the fact that the activity of the enzyme in plants exposed to large quantities of heavy metals is higher, together with the activity of other enzymes such as malic enzyme, glucose - 6 - phosphate - dehidrogenase and glutamate dehidrogenase. Ernst (1980) quoted by Lagriffoul, A. et al. (1998) explains this fact that trough the activity of these enzymes, directly or indirectly involved into the Krebs cycle, the pentosophosphate pathway might be stimulated as a compensation for the decrease of the level of ATP and NADPH furnished in normal conditions by photosynthetic reactions with the metals. Considering the presence of pyrite in the environment, but also of Cu, Mn, Pb and other metals resulted from the ore-processing activities, the highest activity of -ketoglutarate dehydrogenase was found in the foliar samples taken from Salix alba (9.1666 g formazan/g. vegetal mat.), followed by that from Fagus sylvatica (9.067 g formazan/g. vegetal mat.), in the leafs belonging to Populus tremula (7.228 g formazan/g. vegetal mat.), those of larch (6.679 g formazan/g. vegetal mat.), Betula verrucosa (5.506 g formazan/g. vegetal mat.), the lowest intensity having been found at Picea abies (4.141 g formazan/g. vegetal mat.). According to previous studies, the high concentration of metals in the environment is capable of inhibiting with 50% the activity of malate-dehidrogenase (Mansfield, T.A., 1976), together with that of isocitrate-dehydrogenase, glucose-6-phosphate-dehydrogenase and peroxidasis. At Picea abies the enzyme has a low level (4.259 /g g formazan/ vegetal mat.), but also at Salix alba (12.86 g formazan/g vegetal mat.), but its activity is only slightly lower than that of succinate-dehydrogenase (5.197 g formazan/ vegetal mat. at Picea abies and 15.358 g formazan/g. vegetal mat. at Salix alba) which indicates the fact that in the condition of excess copper, pyrite and barite ore in the environment, the hydratation of the fumarate resulted from the oxidation of the succinate to L-malate, is the result of a balanced reaction of the Krebs cycle and not of the inhibited activity of succinate - dehydrogenase. However, the other species showed in the presence of contaminating ores in the environment activities even higher than that of the oxoglutarate-dehydrogenase and succinate-dehydrogenase (Fagus sylvatica - 19.290 g formazan/g. vegetal mat. and 15.228 g formazan/g. vegetal mat., Larix decidua - 12.388 g

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Analele tiinifice ale Universitii Alexandru Ioan Cuza, Seciunea Genetic i Biologie Molecular, TOM XIII, 2012

formazan/g. vegetal mat. and 10.475 g formazan/g. vegetal mat. Betula verrucosa (10.845 g formazan/g. vegetal mat. and 9.598 g formazan/g. vegetal mat., Populus tremula - 8.555 g formazan/g. vegetal mat. and 7.635 g formazan/ vegetal mat.), while at Picea abies i Salix alba, the activity of the malate - dehydrogenase was lower than that of succinate-dehydrogenase (4.425 g formazan/g. vegetal mat. and 5.197 g formazan/g. vegetal mat. at spruce, respectively 12.864 g formazan/g. vegetal mat. and 15.358 g formazan/g. vegetal mat. at willow). At Salix alba - the activity of the succinate - dehydrogenase is specifically high, comparative with that of the -ketoglutarate dehydrogenase (15.358 g formazan/g. vegetal mat.). Although we could adopt the idea that the extra succinate had entered the Krebs cycle on other metabolic pathways, we cannot ignore the fact that succinate, together with citrate, ftalate, tartrate, salicilate and acetate had proven to be, according to certain experiments (Vara Prasad, M.N. and De Oliveira Freitas, H.M., 2003) excellent chellators due to their quick mobility and assimilation of metals by plants in contaminated areas. It is very likely that because of the usage of metals in different cell processes, the used substrate might enter into the Krebs cycle to be transformed in malate, who is in its turn, an excellent chellator.
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g formazan/g biological matter 20 15 10 5 0
a ab Pic e ies P op ulus u tre m la ss Salix a ua osa a tica p alb rruc decid sylv la v e arix gus a L tu F e B

Isocitrate-DH

Malate - DH

Succinate - DH

-Keto-glutarate - DH

Fig. 2. The Krebs cycle dehydrogenases activity in leaves of some Angiosperms and Gymnosperms species of Tarnita, Suceava County

Regarding the activity of the main dehydrogenases of the cycle of tricarboxylic acids, at the studied species in the area of Rdui, considered to be non-contaminated, as it can be seen in figure 4, the intensity of the intermediary metabolism and of the breathing processes is much lower comparing to those found in the contaminated areas, with a few exceptions. So, the maximum level of activity of isocitrate-dehydrogenase found at Picea abies (5.907 g formazan/g. vegetal mat.) was seconded by that found in the leaves of Betula verrucosa (5.1234 g formazan/g. vegetal mat.) and followed, equally by Populus tremula and Fagus sylvatica (4.006 g formazan/g. vegetal mat.), Larix decidua and Salix alba having the lowest activity of this enzyme (3.006 g formazan/g. vegetal mat. and 2.382 g formazan/g. vegetal mat.). Generally, had been found that the activity of -ketoglutarate-dehydrogenase was smaller than that of the isocitrate-dehydrogenase, but in the case of Larix decidua (5.086 g

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Elena Tutu et al Investigations regarding the anthropic impact on the Krebs cycle dehydrogenases system on certain wood-species in mining areas, Suceava county

formazan/g. vegetal mat.) and Salix alba (2.561 g formazan/g. vegetal mat.) the intensity of it was higher than that of other enzymes of the cycle. In descending order, the activity of the enzyme had been noted: Betula verrucosa 4.765 g formazan/g. vegetal mat., Fagus sylvatica 2.6111 g formazan/g. vegetal mat. and Picea abies 2.209 g formazan/g. vegetal mat. and the succinate - dehydrogenase had registered a downward array of values in the foliar materials as it follows: Betula verrucosa (6.018 g formazan/g. vegetal mat.) > Picea abies (4.623 g formazan/g. vegetal mat.) > Populus tremula (3.691 g formazan/g. vegetal mat.) > Fagus sylvatica (3.185 g formazan/g. vegetal mat.) > Larix decidua (2.709 g formazan/g. vegetal mat.) > Salix alba (2.061 g formazan/g. vegetal mat.). At Betula verrucosa, in the case of malate dehydrogenase had been found a higher level of enzyme comparing with the isocitrate dehydrogenase and lower than that of succinate dehydrogenase 5.432 g formazan/g. vegetal mat., situation that had been found also in the samples taken from Larix decidua 3.6111 g formazan/g. vegetal mat., a fact that can be explained either trough the fact that the substrate, metabolized by the enzyme had entered into the Krebs cycle trough its junctions with certain catabolic pathways of some products, either through a possible link with the openings of the stomata, as it is well known its connection with the activity of the enzyme and its direct relation with the intensity of the light. At other species had been found in a descending order: Picea abies -5.401 g formazan/g. vegetal mat., Fagus sylvatica -3.481 g formazan/g. vegetal mat., Populus tremula 3.450 g formazan/g. vegetal mat., Salix alba 2.709 Fagus sylvatica -3.481 g formazan/g. vegetal mat.

Considering the samples studied, the Krebs cycle dehidrogenases activity in Picea abies, Populus tremula, Salix alba, Betula verrucosa, Larix decidua and Fagus sylvatica leaves from polluted areas with sulphur (Climani), copper ores and barite (Tarnita) and unpolluted areas (Rduti) is influenced by their presence in the environment, but also, by the wood species.

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7 6 5 4 3 2 1 0 g formazan/g biological matter
a ab Pic e ies P op ulus u tre m la Salix a ca osa alba cidu lva ti rruc ss p x de s sy la v e Lari Fagu Be tu

Isocitrate-DH

Malate - DH

Succinate - DH

-Keto-glutarate - DH

Fig. 3. The Krebs cycle dehydrogenases activity in leaves of some Angiosperms and Gymnosperms species of Rduti, Suceava County

CONCLUSIONS

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Analele tiinifice ale Universitii Alexandru Ioan Cuza, Seciunea Genetic i Biologie Molecular, TOM XIII, 2012

After analyzing the features of this study, we conclude that the seasonal active biomonitoring of dehydrogenases of tricarboxilic acid cycle activity in leaves of these wood species is required, but, on a longer time. In generally, it can not established a correlation between the activity of this enzymes and the degree of resistance and accommodation of the wood species to anthropic pollution. REFERENCES
1. Cojocaru, D., (2009): Enzimologie practica, Ed. Tehnopress, Iasi. 2. Dizengremel, P., Le Thiec, D., Hasenfratz-Sauder, M.P., Vaultier,M.N., Bagard, M. and Jolivet, Y., (2009): Metabolic-dependent changes in plant cell redox power after ozone exposure, Plant Biology, Special Issue: Plant Functioning in a Changing Global Environment, Volume 11, Issue Supplement s1, 3542 3. Kasim, W.A., (2007): Physiological consequences of structural and ultra-structural changes induced by Zn stress in Phaseolus vulgaris. II. enzymes, amino acids and protein profile. Int. J. Bot., 3: 33-39. 4. Lagriffoul, A., Mocquot, B., Mench, M. and Vangronsveld, J., (1998): Cadmium toxicity effects on growth, mineral and chlorophyll contents and activities of stress related enzymes in young maize plants (Zea mays L.), Plant and Soil, vol. 200: 241-250. 5. Mansfield, T.A., (1976): Effects of air pollutants on plants, CUP Archive, page 121. 6. OLeary, B., Park, J. and Plaxton, W.C., (2011): Review Article. The remarkable diversity of plant PEPC (phosphoenolpyruvate carboxylase): recent insights into the physiological functions and post-translational controls of non-photosynthetic PEPCs, Biochem. J., 436: 1534 7. Pierre, M. and Queiroz, O., (1988): Air pollution by SO2 amplifies the effects of water stress on enzymes and total soluble proteins of spruce needles, Physiologia Plantarum, vol. 73, issue 3: 412417. 8. Rao, M.I., Amundson, R.G., Alscher-Herman, R. and Anderson, L.E., (1983): Effects of SO2 on Stomatal Metabolism in Pisum sativum L.1, Plant Physiol., vol. 72: 573-577. 9. Sriram, G., Fulton, D. B. and Shanks, J. V., (2007): Flux quantication in central carbon metabolism of Catharanthus roseus hairy roots by C-13 labeling and comprehensive bondomer balancing. Phytochemistry, vol. 68: 22432257. 10. Vara Prasad, M.N. and De Oliveira Freitas, H.M., (2003): Metal hyperaccumulation in plants - Biodiversity prospecting for phytoremediation technology, Electronic Journal of Biotechnology, 6 (3): 285-305. 11. Ziegler, I., (1974): Action of sulfite on plant malate dehydrogenase. Phytochemistry, vol. 13: 2411-2416

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1) Alexandru Ioan Cuza University of Jassy, Romania * ciornea@uaic.ro

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Analele tiinifice ale Universitii Alexandru Ioan Cuza, Seciunea Genetic i Biologie Molecular, TOM XIII, 2012

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