Phytochelatins are the principal heavy metal-detoxifying components in plants. To investigate phytochelatin (PHC) production and the importance of these compounds for heavy metal tolerance, sunflower (Helianthus annuus) was exposed to cadmium. The leaves and roots of sunflower plants cultivated in the presence of 15, 25 and 50  M Cd(NO3)2 for 3
and 9 days showed increased tolerance to cadmium and contained higher concentrations of phytochelatins. The phytochelatin level was assayed by using HPLC and the Cd level was determined by atomic spectrum analysis

F. Yurekli

Z. Kucukbay

Hrčak - Portal of scientific journals of Croatia

Acta Bot. Croat. 62 (1), 21–25, 2003 CODEN: ABCRA25ISSN 0365–0588Synthesis of phytochelatins in Helianthus annuusis enhanced by cadmium nitrateFUSUN YUREKLI1*, ZEHRA KUCUKBAY21 Inonu University, Arts and Science Faculty, Department of Biology, Malatya, Turkey2 Inonu University, Arts and Science Faculty, Department of Chemistry, Malatya, TurkeyPhytochelatins are the principal heavy metal-detoxifying components in plants. To inves-tigate phytochelatin (PHC) production and the importance of these compounds for heavymetal tolerance, sunflower (Helianthus annuus) was exposed to cadmium. The leaves androots of sunflower plants cultivated in the presence of 15, 25 and 50  M Cd(NO3)2 for 3and 9 days showed increased tolerance to cadmium and contained higher concentrationsof phytochelatins. The phytochelatin level was assayed by using HPLC and the Cd levelwas determined by atomic spectrum analysis.Key words: Phytochelatin, Helianthus annuus, cadmium nitrate, phytoremediationIntroductionHeavy metals such as Cd, Pb, Hg, As, and Se are environmental pollutants of increasingimportance in the world (ZHU et al. 1999). Heavy metal toxicity poses major environmentaland health problems. Cadmium, for example, is a non-essential heavy metal which is toxicto living cells at very low concentrations. Cd ions displace Ca or Zn in proteins and cancause oxidative stress (CLEMENS et al. 1999). Plants under heavy metal stress synthesizephytochelatin by a  -glutamyl-cysteinyl-dipeptidyl trans-peptidase using glutathione as asubstrate (GRILL et al. 1985). Metal-binding phytochelatins may be universal in the plantkingdom (GRILL et.al. 1985). Plants can be used to remove heavy metals from the soil byaccumulating, stabilizing, or biochemically transforming them. This cost-effective and en-vironment-friendly technology has been called phytoremediation (ZHU et al. 1999). Heavymetals, unlike organic pollutants, cannot be chemically degraded or biodegraded by micro-organisms. An alternative biological approach is to use plants to clean up polluted watersand soils.The detoxification mechanisms of plants in response to heavy metals have been widelystudied, especially where heavy metals in contaminated soil could be removed by plantsthrough bioremediation processes. Plants produce metal-binding peptides under heavy--metal stress (YAN et al. 2000). ( -Glu-Cys)nGly peptides (n = 2–11) were found in a Cd-,ACTA BOT. CROAT. 62 (1), 2003 21* Corresponding author: e-mail: fyurekli@inonu.edu.trAg-, Bi-, Pb-, Hg-binding complex produced by higher plants and named phytochelatins(PHCs) (LOEFFLER et al.1989, KLAPHECK et al.1995, INOUHE et al.2000). PHCs can beviewed as linear polymers of the  -glutamylcysteine ( -Glu-Cys) portion of glutathione.These peptides could be enzymatically produced by stepwise condensation of  -Glu-Cysmoieties to glutathione itself and the growing phytochelatin chain (GRILL et al.1989).In the present study, we examined the effect of Cd(NO3)2 on PHC synthesis and deter-mined Cd levels in the leaves and roots of sunflower plants. The aim of the study was toevaluate whether sunflower plants can be used in bioremediation.Materials and MethodsPlant material: Sunflower (Helianthus annuus L. cv. B 950) seeds were sterilized in2% hypochlorite solution for 15 min. After being thoroughly washed, the seeds were keptbetween two pads of moistened cotton, for 3 days, and transferred to Hoagland’s solutionwith/without 15, 25 50  M Cd(NO3)2 for 15 days in a growth chamber with 250  mol m–2 s–1light intensity and a 16 h/8h light/dark light cycle. Both control and Cd treated leaves androots were frozen in liquid nitrogen immediately after harvesting and stored at –20 °C.Cd Analysis: Total Cd in tissues was determined using the method of HAN and LEE(1996) employing atomic absorption spectroscopy. Frozen tissues (1.0 g) were ground in amill and then stirred in a solution containing 5 mL sulfuric acid at 4 °C for 10 min beforecentrifugation at 13 000 g for 15 min. A one-mL aliquot of the reaction mixture was sub-jected to atomic absorption spectroscopy analysis.HPLC Analysis: Analysis of the PHCs was performed according to INOUHE et al.(2000) and modified by KLAPHECK et al. (1995) as follows. Frozen tissues were extractedwith (1 mL g–1 FW) 10% (w/v) 5-sulfosalicylic acid at 0 °C. Extracts were centrifuged at8.000 g for 5 min and supernatants were kept at 0 °C for 30 min immediately before HPLC.20  L samples were injected to a reversed-phase LC18 column (250  4.6 mm, 5  m)(Supelco) connected to a HPLC pump (Cecil 1100, Cambridge, UK) and the column waseluted with linear gradient of acetonitrile in 0.1% (w/v) aqueous trifluoroacetic acid at aflow rate of 1 mL min–1. The column eluent was derivatized with 75  M 5,5’-dithio--bis-(2-nitrobenzoic acid) (NBA) in 50 mM potassium phosphate (pH 7.6) at a flow rate of1 mL min–1 and monitored 410 nm. PHC concentrations were expressed as  mol–1 g FW,based on peak areas of the glutathion (GSH) standard.ResultsThe levels of phytochelatins and Cd assayed in roots and leaves of sunflower were de-termined to be time course and Cd(NO3)2 concentration. Phytochelatin and Cd levels arepresented in Table 1–2. Addition of 25  M and 50  m Cd(NO3)2 to the nutrient solutionleads to a significant enhancement of PHCs in the leaves and roots of sunflower plants. ThePHC content changes during the growth of the seedlings, reaching different concentrationsafter 9 d (nearly 45  M g–1 FW and 30  M g–1 FW) in roots and leaves respectively (Tab. 1).The highest phytochelatin level (49.84  mol g–1 FW) was observed in the roots by day 9 ofincubation with 50  M Cd(NO3)2 (Fig. 1). The level of phytochelatin was determined to behigher (1.3–1.8 fold) in the roots than the leaves at different Cd(NO3)2 concentrations. In-cubation with Cd(NO3)2 leads to synthesis of PHCs in roots and leaves.22 ACTA BOT. CROAT. 62 (1), 2003YUREKLI F., KUCUKBAY Z.To investigate distribution of Cd in roots and leaves we conducted a study on thetime-dependent accumulation of Cd. Remarkable amounts of Cd had accumulated in theroots of the sunflower (Tab. 2). Also as shown in Table 2, more Cd accumulated in the rootthan in the leaves.In the present study, we found that sunflower (Helianthus annuus Lc 950) leaves androot tissues showed increasing PHC levels after exposure to Cd; here we examine the ef-fects of Cd on PHC synthesis with regards to Cd(NO3)2 concentration and time course.ACTA BOT. CROAT. 62 (1), 2003 23PHYTOCHELATINS IN HELIANTHUS ANNUUSTab. 1. Phytochelatin levels in the roots and leaves of sunflower plants after exposure to Cd(NO3)2Phytochelatin Levels ( M g–1 FW)Cd(NO3)2 Roots (3d) Roots (9d) Leaves (3d) Leaves (9d)15  M 24.60  1.30 43.14  2.28 13.10  0.78 17.35  1.8025  M 27.20  1.84 47.16  2.08 17.22  0.88 31.42  2.4050  M 32.40  3.62 49.84  3.14 21.42  1.66 37.10  1.78Fig. 1. HPLC analysis of reduced glutathion (GSH) (a) and phytochelatins (b). Peaks 1–5 representphytochelatins in the extract of roots of sunflower plants exposed to Cd(NO3)2.DiscussionHeavy metals, unlike organic pollutants, cannot be chemically degraded or biode-graded by microorganisms. An alternative biological approach to deal with this problem isphytoremediation, i.e. the use of plants to clean up polluted waters and soils. Heavy metalscan be removed from polluted sites by phytoreaction (ZHU et al. 1999). PHCs can be de-tected in plant tissues in liquid cultures exposed to heavy metals and the level of PHCs ob-served in plant tissues correlates with the depletion of metal ions from the medium. PHCswere induced to varying levels by a wide range of metal ion tested. The most effective ap-peared to be Cd, Ag, arsenate, Cu, Hg, and Pb ions. PHCs appeared to play an importantrole in Cd and arsenate detoxification in Arabidopsis and yeast (COBBET et al. 2000). Previ-ous studies have demonstrated a phytochelatin response to heavy metal exposure. Evi-dence suggests that during exposure, phytochelatins accumulate in root tissues and leaves.The present study established the formation of PHCs in the root tissues and leaves of sun-flower plants. Several analyses in PHC occurrence studies have documented a ligation ofCd+2, Ag+2, Hg+2 and Pb+2 by thiolate coordination, as is known for investigated hyper-accumulator species. Evidence for the hyperaccumulating species derives from PHC anal-ysis studies. GALLEGO et al. (1996) have reported that exposure to Cd+2 increased PHCs insunflower and noted that PHCs protected the plant against its harmful effects.The documented study of the binding of the metalloid to PHCs presented here indicatesa detoxifying role for PHCs in an economically important species. In the light of this evi-dence we emphasize the concept of metal complexation and detoxification by the PHCpeptides during the Cd exposure.The results presented suggest that Cd(NO3)2 exposure induces the formation of phyto-chelatins in the root tissues and leaves. As seen in Table 1 and Table 2 Cd and PHC levelsincreased in the roots and leaves. Although a high level phytochelatin exists in the root tis-sue, the leaves accumulate a remarkable quantity of Cd and synthesis phytochelatins. Froma comparison of Table 1 and Table 2, it can be seen that cadmium probably stimulates verysignificant phytochelatin synthesis. It would seem that there is a positive feedback in thesynthesis of phytochelatins after exposure to Cd(NO3)2. It is expected that phytochelatinsmay be found to play an important role in removing heavy metals from polluted environ-ments and in bioremediation.24 ACTA BOT. CROAT. 62 (1), 2003YUREKLI F., KUCUKBAY Z.Tab. 2. Cadmium levels in roots and leaves of sunflower plants after exposure to Cd(NO3)2.Cadmium levels ( M g–1 FW)Cd(NO3)2 Roots (3d) Roots (9d) Leaves (3d) Leaves (9d)15  M 0.08  0.01 0.11  0.005 0.02  0.00 0.04  0.0025  M 0.17  0.02 0.29  0.08 0.07  0.00 0.11  0.00150  M 0.24  ?.02 0.36  0.01 0.17  0.001 0.20  0.00ReferencesCLEMENS, S., KIME, EJ., NEUMANN, D., SCHRODER, J. I., 1999: Tolerance to toxic metals bya gene family of phytochelatin synthases from plants and yeast. EMBO 18, 3325–3333.COBBET, C. S., 2000: Phytochelatins and their roles in heavy metal detoxification. PlantPhysiol. 123, 825–832.GALLEGO, SM., BENAVÝDES, MP., TOMARO, ML., 1996: Oxidative damage caused by cad-mium chloride in sunflower (Helianthus annuus L) plants. Int. Exptl. Bot. 58, 51–52.GRILL, E., WINNACKER E. L., ZENK, M. H., 1985: Phytochelatins: The principal heavy--metal complexing peptides of higher plants. Science. 230, 674–676.GRILL, E., LÖFFLER, S., WINNACKER, E. L., ZENK, M. H., 1989: Phytochelatins, the heavy--metal-binding-peptides of plants, are synthesized from glutathione by a specific -glutamylcysteine dipeptidyl transpeptidase (phytochelatin synthase). Proc. Natl. Acad.86, 6838–6842.HAN, D., LEE, J. H., 1996: Effects of liming on uptake of lead and cadmium by Raphanussativa. Arch. Environ. Contam. Toxicol. 31, 488–493.INOUHE, M., ITO, R., ITO, S., SASADA, N., TOHOYAMA, H., JOHO, M., 2000: Azuki bean cellsare hypersensitive to cadmium and do not synthesize phytochelatins. Plant Physiol.123, 1029–1036.KLAPHECK, S., SCHLUNZ, S., BERGMANN, L., 1995: Synthesis of phytochelatins and homo--phytochelatins in Pisum sativum L. Plant Physiol. 107, 515–521.LÖEFFLER, S., HOCHBERGER, A., GRILL, E., WINNACKER, E. L., ZENK, M. H., 1989: Termina-tion of phytochelatin synthase reaction through sequestration of heavy metals by the re-action product. FEBS Letters. 258, 42–46.YAN, S-H., TSAY, C-C., CHEN, Y-R., 2000: Isolation and characterization of phytochelatinsynthase in rice seedlings. Proc. Natl. Counc. ROC (B) 24, 202–207.ZHU,Y. L., PILON-SMITS, E. A. H., TARUN, A. S., WEBER, S. U., JOUANIN, L., TERRY, N.,1999: Cadmium tolerance and accumulation in Indian mustard is enhanced by over-expressing  -glutamylcysteine synthase. Plant Physiol. 121, 1169–1177.ACTA BOT. CROAT. 62 (1), 2003 25PHYTOCHELATINS IN HELIANTHUS ANNUUS

Synthesis of phytochelatins in Helianthus annuus is enhanced by cadmium nitrate

Abstract

Similar works

Full text

Available Versions

Hrčak - Portal of scientific journals of Croatia