Monitoring Methods of Plant Viability in Genetic Conservation

Transcription

1 Monitoring Methods of Plant Viability in Genetic Conservation OLGA PINTILIE 1, LAURA ION 2, ANDRIANA SURLEVA 3, MARIUS ZAHARIA 2 *, ELENA TODIRASCU CIORNEA 3, ELENA CIUBOTARIU 2, ADRIANA BALAN 5, GABI DROCHIOIU 2, ION SANDU 6,7 1 Alexandru Ioan Cuza University of Iasi, Department of Geography and Geology, 11 Carol I, , Iasi, Romania 2 Alexandru Ioan Cuza University of Iasi, Department of Chemistry, 11 Carol I, , Iasi, Romania, 3 University of Chemical Technology and Metallurgy, Analytical Chemistry Department, 8 St. Kl. Ohridski av., 1756 Sofia, Bulgaria 4 Alexandru Ioan Cuza University of Iasi, Department of Biology, 11 Carol I, , Iasi, Romania, 5 Grigore T. Popa University of Medicine and Pharmacy of Iasi, 16 Universitatii Str., , Iasi, Romania 6 Alexandru Ioan Cuza University of Iasi, ARHEOINVEST Interdisciplinary Platform, 22 Carol I,700506, Iasi, Romania 7 Romanian Inventors Forum, 3 Sf. Petru Movila St., Bl. L11, III/3, , Iasi, Romania One of the most severe consequences of unwise human activities is extinction of numerous species of plants: today 90% of food production is provided by 120 different species of crop plants. Industrialized agriculture and improper use of chemical compounds are responsible for the enhanced genetic erosion, affected being the local varieties of crops and old varieties as well. Losing of local varieties is more than their total disappearance. Therefore, this study investigates the effect of nitrophenolic pesticides on the plants viability at molecular level and trying to draw attention on the danger of the widespread use of toxic compounds. Keywords: nitrophenolic pesticides, genetic conservation, enzymatic determination, biodiversity, germination assays Biotic Earth s shell contains over 500,000 plants species. Different biological communities, developed in millions of years, began to be destroyed by human activities [1-3]. A large number of species are suffering a rapid decline as a result of: excessive hunting, habitat destruction, attack of wild predators, etc. [4]. The main enemy of biodiversity is poverty and by improving of humanity wellbeing and by struggle against underdevelopment we can help protect the environment [5, 6]. Demonstrating the value of biodiversity and natural resources is a very complex issue, it is determined by a variety of economic and ethic factors [7]. A major objective of ecological economy is developing of evaluation methods for biological diversity. There were developed different methods for assigning economic value of genetic variability, biological communities and ecosystems [8, 9]. Conservation of genetic resources in gene banks is considered the most advanced form in doing this. Gene banks are special institutions which propose to collect, evaluate and conserve the plant genetic resources. Gene Bank has the responsibility to conserve plant germplasm in Romania and, through international cooperation, contribute to ensuring global plant genetic resources. Plant genetic resources contribute to food security being the most precious gift given to us by nature [10]. Currently, agricultural production growth is based primarily on the massive use of chemicals for fertilization and pest control, the use of fossil fuels, irrigation, modern varieties of high productivity [11]. Expanding of these agricultural technologies is explained by the fact that they are much simpler and more manageable [12, 13]. Long term, industrial agriculture mean environmental pollution, weakening the health of human society and irreversible loss of agricultural biodiversity. Therefore the need to preserve and revitalize traditional seeds, which are in danger not only because of unfavorable legislation but also due to rural population aging, migration of young people towards urban areas and low interest in agriculture, expansion of industrial agro fuels agriculture pollutants and genetically modified organisms [14]. In this work we studied the plant viability which may be partially degraded and reversibly under the action of dinitrophenol pesticides. In our researchers, were used plants in the presence of pesticides nitrophenolic (phenolic compounds). The viability of the plant were monitoring by studying the enzymatic changes occurring due to the toxic action of phenolic compounds. Experimental part Materials and methods Reagents The reagents used were analytical grade and the aqueous solutions were prepared in distilled water of high purity (R = 18.2 Ω). Dinitrophenols were purchased from Sigma Aldrich (USA). Bradford reagent 10 mg Coomassie Brilliant Blue G-250 was dissolve in 10 ml ethanol (96%), then 9.8 ml of phosphoric acid (89%) was added. The solution was filtered and stored at room temperature in amber glass. This reagent is stable over a period of 4 weeks. Reagents used for catalase activity determination: - potassium dichromate (K 2 Cr 2 O 7 ), 5%. 5 g of potassium dichromate was dissolved in 100 ml of hydrogen peroxide. - potassium dichromate glacial acetic acid solution. 20 ml of 5% potassium dichromate was mixed with 60 ml of glacial acetic acid. - potassium phosphate buffer 0.01 M, ph = 7.0. Dissolve g KH 2 PO 4 and g K 2 HPO 4 in 300 ml distilled * zaharia.marius2011@yahoo.com REV.CHIM.(Bucharest) 67 No

2 Fig. 1. The effect of the aromatic derivatives on the wheat seeds during 7 days of germination water. - hydrogen peroxide solution, 0.16 M in potassium phosphate buffer 0.01 M, ph = standard solution of hydrogen peroxide 0.08 M in potassium phosphate buffer 0.01 M ph = disodium phosphate 0.1 M g sodium hydrogen phosphate (NaH 2 PO 4 H 2 O) was dissolved in 500 ml distilled water. Reagents used for quantitative determination of peroxidase activity: - phosphate buffer 0.1 M, ph = ortho-dianisidine solution, 0.03 M. 95 mg orthodianisidine was dissolved in 10 ml of 96% ethyl alcohol). - hdrogen peroxide (10 mm). 50 ml of phosphate buffer, 0.1 M was mixed with 0.06 ml of concentrated hydrogen peroxide (30%). - substrate buffer. 9.6 ml phosphate buffer (0.1 M, ph = 6) was mixed with 0.1 ml of 0.03 M alcoholic solution of ortho-dianisidine. Biological materials Wheat seed, Triticum aestivum, variety Gasprom, were purchased from Agricultural Research Station, Suceava, Romania. Instruments. UV-Vis Spectrophotometer Libbra S35 model PC UV / VIS (Biochrom, UK) with quartz cuvettes with 1 cm in length. Microprocessor Cole Parmer Ultrasonic (USA, Illinois) used for enzymatic extraction. Mikro22R centrifuge (Hettich) used for the separation of soluble protein mixture containing insoluble components (cell membrane). HANNA ph meter PH 211 used for determining and adjusting the ph buffers. Procedure. Germination tests. Was studied the wheat seeds assortment Gasparom, from Bank of genetic resources, Suceava which have been undergone to germination in Petri dishes in laboratory conditions, after pretreatment for one hour with prepared solution 2x10-3 M of 2,4-dinitrophenol (2,4-DNP), 2,5-dinitrophenol (2,5- DNP), 2,6-dinitrophenol (2,6-DNP) solutions, picric acid, m-nitrophenol and p-nitroaniline. Also it has been put to germinate in the same time, three Petri dishes with seeds soaked in distilled water, the samples constituting the control. Samples were daily wetted with constant volume of distilled water for seven days and afterword the metric, gravimetric and biochemical analysis were performed (by determining the activity of oxidative stress enzymes. Quantitative determination of soluble vegetable proteins by Bradford method. The method is based on the reaction of Bradford reagent, under acidic conditions, with arginine, histidine, lysine, tyrosine, tryptophan and phenylalanine from protein structure and the complex formed can be measured at 595 nm [15]. Spectrophotometric determination of catalase activity (Sinha method). The catalase is left to interact with hydrogen peroxide for a certain amount of time, and then the enzyme is inactivated by adding a mixture consisting of potassium dichromate and acetic acid. The amount of hydrogen peroxide that wasn t decomposed by catalase reduces the potassium dichromate (Cr 6+ ) to chromic acetate (Cr 3+ ) in acidic medium. Chromic acetate can be determined by spectrophotometry at 570 nm. Because potassium dichromate doesn t absorb at this wavelength, its presence doesn t interfere with the spectrophotometric determination of chromic acetate. The amount of water decomposed by catalase can be determined by calculating the difference between the initial and final amount of hydrogen peroxide [16]. Determination of peroxidase activity. Peroxidase activity is determined by measuring the optical density of the oxidation product resulted from the reaction of ortodianisidine with hydrogen peroxide in the presence of peroxidase [17]. Results and discussions The wheat seedlings germinated differently as a result of the treatment of batches composed of 50 seeds with 5 ml of different toxic agents (fig.1). It can be noticed the the picric acid inhibits the most the development of wheat seeds. Considering the number of grown seedlings, the toxicity of the analyzed compounds grows in the following order: p-nitroaniline < 2,4-dinitrophenol (2,4-DNP) < 2,5- dinitrophenol (2,5-DNP) < m-nitrophenol < 2,6- dinitrophenol (2,6-DNP) < picric acid (fig.2). Fig. 2. The effect of different nitro-derivatives on the development of wheat seeds Quantative determination of soluble vegetable proteins by Bradford method Enzymatic extract After the development period (7 days of germination), the seedlings were cut in pieces of approximately 1 cm each and were detached from roots in order to study the enzymatic activity in both parts of plant. A quantity of 5 g of seedlings, and 5 g of roots respectively were suspended in 10 ml phosphate buffer and sonicated three times for 30 s, with 5 min break and then the extract was centrifuged and kept at 4 o C for 4-5 days [18]. Results calculations The proteins concentration is determined from a calibration curve, that is made by consequent dilutions of REV.CHIM.(Bucharest) 67 No

3 Fig. 3. Calibration curve using standard BSA solution Fig. 4. The evolution of the specific proteic activity from wheat roots after the treatment with different nitroderivatives Table 1 VALUES FOR SPECIFIC ACTIVITY AND PROTEINS CONCENTRATION IN THE PROTEIC EXTRACT OBTAINED FROM ROOTS a standard BSA solution (bovine serum albumin) to a final concentration µg/ml). The calibration curve for this method is linear in the concentration domain of µg/mL of bovine serum albumin. If the extinction of the unknown sample is outside this domain, the determination error will be very high. Another consequence of the linearity domain is that the samples must be within the linearity range, and if not a supplementary dilution is applied (fig. 3). Results obtained from the calibration curve and calculated specific activities are presented in table 1 and table 2. It can be noticed that the samples extracted from roots and treated with nitrophenols present a different specific activity compare to the activity value of the witness solution that didn t contain nitrophenolic derivative. Thus, the specific activities vary depending on the toxic compound used in the treatment of wheat seeds and the highest proteic activity was identified at the treatment with picric acid and 2,6-dinitrophenol, while the treatment with p- nitroaniline, 2,4-dinitrophenol and m-nitrophenol determines the enzymatic inhibition (fig. 4). It can be noticed from figure 5 that the extracts from wheat seedlings present different values for specific activity as a result of the treatment with different toxic compounds. Thus, after the treatment with 2,5-dinitrophenol and m- nitrophenol can be identified a smaller activity; while after the treatment with 2,6-dinitrophenol, 2,4-dinitrophenol, picric acid and p-nitroaniline the activities are higher. Determination of catalase activity Catalase activity was expressed in enzyme units (EU), a unit representing the amount of the enzyme catalase which dissolve one µm of hydrogen peroxide (0.034 mg) in one minute at 20 C and ph = 7. In practice, is monitoring the consumption of hydrogen peroxide at 570 nm for 60 seconds in 3 ml potassium phosphate buffer 0.01 M (ph = 7.0), 0.08 M H 2 O 2 and 50 µl extract. Determination of hydrogen peroxide micromoles is Table 2 VALUES OF SPECIFIC ACTIVITIES AS FUNCTION OF ABSORBANCE, PROTEIN AMOUNT AND PROTEIN CONCENTRATION FROM THE ENZYMATIC EXTRACT OBTAINED FROM SEEDLINGS REV.CHIM.(Bucharest) 67 No

4 Fig. 5. The evolution of specific activity og the proteic extract from wheat seedlings after the treatment with different nitroderivatives Fig, 6. Calibration curve for catalase activity based on a standard curve using standard solution of hydrogen peroxide (80 µm/ml) (fig. 6). After determining concentration of hydrogen peroxide for controls and samples, the catalase activity is calculated in micromoles of hydrogen peroxide / min / gram of analyzed tissue. If one EU is 1 µm of hydrogen peroxide, then the difference between the number of micromoles of hydrogen peroxide in controls (n 1 = 80 micromoles of hydrogen peroxide corresponding to the 0.5 ml solution of hydrogen peroxide 0.16 M) and the number of micromoles of hydrogen peroxide remaining decomposed in the sample (n 2 ) after inactivation of catalase with potassium dichromate acetic acid solution will give us X enzymatic units (EU). Value of n 2 can be calculated from the standard curve or from the extinction ratio between control and sample: Fig.7. Catalase activity in wheat plants after 7 days treatment The release activity of H 2 O 2 as a result of reactions catalyzed by superoxide dismutase, the catalase activity follows the schedule graph of SOD activity and, therefore, as can be easily seen, the high quantities of hydrogen peroxide are decomposed under the action of the second enzymes (fig. 7). Activity intensification of both enzymes show that under nitroderivatives treatment, shows that these compounds are able to induce a significant uncoupling of the electron transport and oxidative phosphorylation, as well [19]. Therefore dissipation of membrane gradient and increased of reactive oxygen species production levels can be occurs. This facts help as to understand and better understand the increased catalase activity in plants which has been treated with picric acid ( o C/mg protein), compared to the control group ( o C/mg protein). In catalase activity evaluation should be considered that the intensity is much higher than superoxide dismutase intensity. As in the cellular oxidative balance are involved other enzymatic or non-enzymatic systems which are able to reduce the production of reactive oxygen species (e.g. Thiamine) [20, 21], it can be said that this increased activity of oxidoreductases is due to the influence of picric acid. Moreover, it is known that both germination and wheat fermentation increase iron availability which is known as catalase central component. Determination of peroxidase activity A volume of 0.94 ml and 50 ml buffer-substrate solution are mixed. The enzymatic reaction is achieved by adding 10 ml of hydrogen peroxide (10 mm) (fig. 8). The absorbance is measured at 436 nm, after 3 min incubation time. where: The extinction coefficient for control (40 mm -1 cm -1 ). The number of micromoles, X, of hydrogen peroxide decomposed for one minute in one gram of animal or plant tissue, can be calculated using the following expression: where: t = incubation time (min) p = weight of biological material (g) The specific activity of the catalase is obtained by dividing the number of micromoles of hydrogen peroxide/ min to the mg of protein from the enzyme extract. Fig. 8. Determination of peroxidase activity in wheat Determination of extracts specific activity For determination of extracts specific activity follow the ecquations: REV.CHIM.(Bucharest) 67 No

5 the applied treatment or sources. For example, in case of nitrophenol treatment, the leaves SOD activity is not influenced as in the case of roots, when the influence on the enzyme activity is important. In conclusion the enzyme activity is higher in roots case than in the leaves case and the phenolic extract type used affects the enzyme activity. Acknowledgments Financial support within project from the Romanian Government (PN-II-PT-PCCA , Contract 107/2014). Ion Laura s work was supported by the strategic POSDRU/159/1.5/S/137750, co-financed by the European Social Fund within the Sectorial Operational Program Human Resources Development Fig.9. Peroxidase activity in wheat plants after 7 days treatment with nitrophenols derivatives Peroxidase shows to be an antioxidant defense parameter which can be used as a biomarker to indicate picric acid toxicity on wheats, because the enzyme activity, as shown in the results below, proved to be five times higher in the treated wheat ( UP/mg protein) than in control case (2.515 UP/mg protein) (fig. 9). We can draw the conclusion that the intense peroxidase activity in experimental sample can be due to the picric acid, which can be explain by the fact that the picric acid can influence the aromatic monomers polymerization and influence the germination and growth processes of wheat plants as well. It is known that the seeds deterioration process begins after harvest, under different intrinsic and extrinsic factors [22] and, therefore, to prevent the rapid loss of viability, vigor and productivity is necessary a controlled approach for their storage, especially in environments where the rate of deterioration is accelerated by an hostile environment (high heat and humidity). Implementation of various treatments can rapidly slowing down deterioration rate and increase seed viability. Conclusions In the present work was determined the enzymatic activity of plant species. Wheat extracts were subjected to further steps of concentration and dialysis. The enzyme extracts stability were relatively highly for short periods of time (one week, at 4 C), but changes can occurs when there are stored for a long period of time (the enzymatic activity decreases at 30-40% after 3 weeks). The enzymes involved in respiratory processes are a good example for understanding the evolution of life, from eukaryotic stage to the prokaryotic one, and pathological changes that occur in malign processes, where the reverse occurs from normal cell breathing to lactic fermentation, (body fluids changes their ph, degradation of essential amino acid, etc.). Enzymatic determination method can be successfully applied, in particularly, in plant roots case due to more pronounced inhibition. The extraction is influenced by the size of plant parts used for extraction. The enzymatic activity in the various parts of the plant, differ significantly due to References 1.POPESCU, I., NIMIRCIAG, R., VINA, G., MARSAN, A.F., Rev. Chim. (Bucharest), 64, no.5, 2013, p ROMANESCU, G., CRETU, M. A., SANDU, I. G., PAUN, E., SANDU, I., Rev. Chim. (Bucharest), 64, no. 12, 2013, p CHITU, E., LACATUS, V., GAIDAU, C., IONITA, A. D., FILIPESCU, L., Rev. Chim. (Bucharest), 61, no.11, 2010, p ENJALBERT, J. 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