Changes in the amount of metal complexing organic acids were investigated in barley leaves during 0,1, 0,5, 1mM Ni treatment parallel with the measurement of Ni accumulation. The content of five organic acids (oxaloacetate, citrate, malate, succinate and fumarate) was measured in the leaves of barley seedlings hoping to identify them as parts of nickel detoxification mechanisms. Besides a 3 fold increase of citrate and malate, the amount of oxalate and fumarate increased by 5-6 fold. It was found that oxalate and fumarate are also take part in nickel complexation mechanisms

Droppa, Magdolna

Jócsák, Ildikó

Végvári, György

English

Jócsák Ildikó

Végvári György

Droppa Magdolna

University of Szeged

Volume 49(1-2):99-101, 2005Acta Biologica Szegediensishttp://www.sci.u-szeged.hu/ABS1Department of Plant Anatomy and Plant Physiology, Szent István University, Gödöllô, Hungary, 2Department of Plant Physiology and Plant Biochemistry, Corvinus University of Budapest, Budapest, Hungary, 3Department of Fruit Science, Corvinus University of Budapest, Budapest, HungaryIldikó Jócsák1,2, György Végvári3, Magdolna Droppa2*ABSTRACT                        Changes in the amount of metal complexing organic acids were investigated in barley leaves during 0,1, 0,5, 1mM Ni treatment parallel with the measurement of Ni accumula-tion. The content of five organic acids (oxaloacetate, citrate, malate, succinate and fumarate) was measured in the leaves of barley seedlings hoping to identify them as parts of nickel de-toxification mechanisms. Besides a 3 fold increase of citrate and malate, the amount of oxalate and fumarate increased by 5-6 fold. It was found that oxalate and fumarate are also take part in nickel complexation mechanisms. Acta Biol Szeged 49(1-2):99-101 (2005)KEY WORDSnickelorganic acidsHPLC*Corresponding author. E-mail: droppa@uni-corvinus.hu99Heavy metal detoxification by organic acids in barley seedlingsProceedings of the 8th Hungarian Congress on Plant Physiologyand the 6th Hungarian Conference on Photosynthesis, 2005Once an essential element is absorbed by root cells, mecha-nisms must be present to maintain the solubility and further availability for use in mineral nutrition. Similar processes affect polluting elements as well. Microelements, such as nickel can get into plants easily, since their uptake channels are developed through the evolution. Nickel, is an essential heavy metal in the forming of urease enzyme (Klucas et al. 1983). When in excess amount, nickel tolerance mostly achieved through organic acids. Organic acids, such as citrate, fumarate, malate, succinate and oxaloacetate play an impor-tant role in several major metabolic pathways in plants such as in fatty acid biosynthesis and oxidation, glyoxalate acid cycle, carbohydrate biosynthesis and especially have significance in the citrate cycle of the respiratory processes (Popova and Pinheiro de Cavalho 1998). Citrate and malate are supposed to be responsible for chelation and transportation of nickel into the vacuoles (Brooks et al. 1981), however citrate seems to be most likely the nickel chelator (Tatár et al. 2000). On the other hand we did not find information whether other or-ganic acids of citratre cycle also take part in the detoxification mechanisms or not. That is why we investigated the amount of five organic acids (oxaloacetate, citrate, malate, succinate and fumarate) in barley seedlings hoping to identify more that play role in nickel detoxification mechanisms.Materials and MethodsGrowth conditionsBarley (Hordeum vulgare L. „Triangel”) seeds germinated for three days then grown on modified Hoagland solution (Hegedûs et al. 2001) for seven days. 0,1; 0,5 and 1mM NiSO4 was added to the solution of ten days old seedlings. The growth conditions were: 20°C; 90 µE m-2·s-1 light intensity; 12-12 h light/dark period. Seedlings were sampled for organic acid, and Ni content measurement 1, 4 and 7 days after treat-ment. The experiment was repeated three times.Organic acid content measurementShoots (0.8 g) of barley seedlings were ground in liquid nitrogen and quartz sand then were centrifuged then filtered through 45 µm nylon membrane of which 20 µl and the mea-surement of organic acids with RP HPLC (Waters). Ni content measurement 0.2 g of powdered dry material was dissolved in 2 cm3 of HNO3:H2O2 mixture (1:1 v/v). The nickel content of the filtered solution was determined by an ICAP 61E Plasma Spectrometer (Horváth et al. 1996).  Ni c o n t e n t  05 01 0 01 5 02 0 02 5 00 2 4 6 8T im e  (d a y )c o n tr o l0 ,1  mM Ni0 ,5  mM Ni1  mM Ni Ni content (�g/g fr. wt.)Figure 1. Ni content of leaves of barley seedlings.100ResultsThe nickel accumulation increased significantly, although the slope of the curves was not proportional to the applied concentration. After 7 days of treatment, the nickel content of the 1 mM NiSO4 treated seedlings was 250 times than that of the controls, but at 0,1 mM treatment it was only six times higher (Fig. 1).The organic acid content of nickel treated seedlings in-creased during the experiment in a concentration dependent way (Fig. 2), although not in a similar rate. In the 1 mM nickel treated seedlings the oxalate and fumarate contents were approximately six times higher, but the rest of the five identified organic acid (malate, citrate and succinate) contents were only three times higher than that of the control. Furthermore, the content of oxalate- and fumarate increased during the first day of nickel treatment, whereas the others began to increase later, between the first and fourth day. Citr ic  a c id01 0 02 0 03 0 04 0 05 0 06 0 07 0 00 2 4 6 8T ime  ( d a y )c o n tro l0 ,1  mM N i0 ,5  mM N i1  mM Ni Citrc acid in the % of controlMa lic  a c id01 0 02 0 03 0 04 0 05 0 06 0 07 0 00 2 4 6 8T im e  (d a y )c o n tr o l0 ,1  mM Ni0 ,5  mM Ni1  mM Ni Malic acid in the % of controlS u c c in ic  a c id01 0 02 0 03 0 04 0 05 0 06 0 07 0 00 2 4 6 8T ime  ( d a y )c o n tro l0 ,1  mM Ni0 ,5  mM Ni1  mM Ni Succinic acid in the % of controlO x a lo a c e t ic  a c id  01 0 02 0 03 0 04 0 05 0 06 0 07 0 00 2 4 6 8Time  (d a y )c o n tro l0 ,1  mM Ni0 ,5  mM Ni1  mM Ni Oxalic acid in the % of controlFu ma r ic  a c id01 0 02 0 03 0 04 0 05 0 06 0 07 0 00 2 4 6 8Time  (d a y )c o n tr o l0 ,1  mM Ni0 ,5  mM Ni1  mM NiFumaric acid in the % of controlFigure 2. Changes of organic acid contents of in the percentage of control.101Treatments with 0,5 mM nickel also increased all organic acid contents, but only approximately to their double except for fumarate (four times higher). The lowest nickel concentration caused the most pronounced change in the level of fumarate. It has to be noted that the absolute values of fumarate and oxaloacetate were approximately one and two orders of mag-nitude lower than the values of malate, citrate and succinate (data not shown). Discussion Barley leaves are able to accumulate considerable amount of nickel in a short time, but the highest concentration of nickel in the mineral solution (1mM) causes more rapid accumula-tion than the lowest. (0,1mM). As consequence of nickel ac-cumulation, the organic acid content increased, which shows an enhanced need for these compounds in order to maintain homeostatic conditions. Tatár et al. found citrate, malate and fumarate in the xylem sap of cucumber, indicating that these organic acids take part in the transport mechanism of nickel. According to our findings, the amount of oxaloacetate and succinate also increased. Consequently, they also play role in the detoxification mechanisms of nickel poisoning, not in the transportation, but in the chelation processes, since the majority of the accumulated organic acids are stored in the vacuole to where the transportation of nickel directs (Farré et al. 2001). Citrate and malate supposed to chelate nickel however there is no direct evidence for this (Brooks et al. 1998; Tatár et al. 2000).Although the absolute value of oxaloacetate and fumarate was much lower than in case of malate, citrate or succinate, they seemed to react the most rapidly to increased nickel availability. In order to achieve the increased amount of ci-trate and malate, which are the most abundant organic acids in barley leaves; first, other intermediates of the Krebs cycle are synthesized in an enhanced level. Furthermore, some of the components of mitochondrial electron transport chain of Krebs cycle leak out a few electrons and produce free oxygen radicals. It is believed that heavy metals enhance these leakages. The probable sites seem to be complex I and ubiquinone-cytochrome b region (Lund et al. 1991). The rapid rise of fumarate and oxaloacetate level may be related to this change of the electron transport route.AcknowledgementsThis work was supported by OTKA grants T 037748 and M 036652. ReferencesBrooks RR, Shaw S, Asensi Marfil A (1981) The chemical form and physiological function of nickel in some Iberian Alyssum species. Physiol Plant 51:167-170.Farré EM, Tiessen A, Roessner U, Geigenberger P, Trethewey RN, Willmitzer L (2001) Analysis of the Compartmentation of Glycolytic Intermediates, Nucleotides, Sugars, Organic Acids, Amino Acids, and Sugar Alcohols in Potato Tubers Using a Nonaqueous Fractionation Method. Plant Physiol 127:685-700.Hegedûs A, Erdei S, Horváth G (2001) Comparative studies of H2O2 detoxifying enzymes in green and greening barley seedlings under cadmium stress. Plant Sci 160:1085-109. Horváth G, Droppa M, Orazeez A, RaskinVI, Marder JB (1996) Formation of photosynthetic apparatus during greening of cadmim- poisoned barley leaves. Planta 199:238-243.Klucas RV, Hanus FJ, Russell SA, Evans H (1983) Nickel: A micronutrient element for hydrogen-dependent growth of rhizobium japonicum and for expression of urease activity in soybean leaves. PNAS 80:2253-2257.Lund BO, Miller DM, Woods JS (1991) Mercury – induced H2O2 production an dlipid peroxidation in vitro in rat kidney mitochondria. Biochem Pharmacol 42:S181-S187.Popova TN, Pinheiro de Cavalho MAA (1998) Citrate and isocitrate in plant metabolism. Biochim Biophys Acta 1364:307-325.Tatár E, Mihucz VG, Kmethy B, Záray Gy, Fodor F (2000) Determination of organic acids and their role in nickel transport within cucumber plants. Microchem J 67:73-81.

Heavy metal detoxification by organic acids in barley seedlings

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Heavy metal detoxification by organic acids in barley seedlings

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