PLANT TOLERANCE TO HEAVY METALS,

A RISK FOR FOOD TOXICITY OR A MEANS

FOR FOOD FORTIFICATION WITH ESSENTIAL

METALS: THE ALLIUM SCHOENOPRASUM MODEL

Avi Golan-Goldhirsh Ben Gurion University of the Negev, Jacob Blausetin Institutes for Desert Research, Albert

Katz Department of Dryland Biotechnologies, Sede Boqer Campus, Israel

Abstract: There is a duality in plant tolerance and response to heavy metal stress. On one hand tolerant plants having heavy metal hyper accumulation potential could be beneficial in phytoremediation for clean-up of soil and water; on the other hand tolerant food crops, if exposed to heavy metals in their growth medium, may be dangerous as carriers of toxic metals in the food chain leading to food toxicity. Tolerant food crops could also be used for enrichment of the diet with essential metals. Heavy metal stress causes oxidative damage, but some reactive oxygen species (ROS) can participate in signal transduction pathways. A new hypothetical mechanism for the interaction of cupric ions and ascorbic acid with proteins in response to metal stress is presented. The dualities mentioned above are discussed. Allium schoenoprasum (Chives) is the model plant to be presented. Like other members of the Allium genus it has a wide spectrum of sulfur-containing compounds in high concentrations, this may render it more tolerant to heavy metals. Some of these compounds may function as reductants and/or chelators to prevent the toxic effect of the metal. The results presented, point to chives phytoremediation potential, but also to the potential risk in accumulation of heavy metals in a commonly edible plant.

Key words: phytoremediation; tolerance; cadmium; iron fortification; ascorbic acid-copper

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and Water Pollution Monitoring, Protection and Remediation, 3–23.

© 2006 Springer.

Soil

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1. INTRODUCTION

This paper focuses on dualities which stem from plant tolerance to heavy metals (Figure 1). On one hand tolerant plants having heavy metals hyperaccumulation potential could be beneficial in phytoremediation for clean-up of soil and water. On the other hand tolerant food crops, if exposed to heavy metals in their growth medium, may be dangerous as carriers of toxic metals in the food chain leading to food toxicity.

DamageOxidative

Competition in transport

Competition as cofactors

Defense

Anti-oxidative systems

Chelators

Metal

Benefit

ROS as signals

Nutritional

Phytoremediation

CellularCompartmentation

DamageOxidative

Competition in transport

Competition as cofactors

Defense

Anti-oxidative systems

Chelators

Metal

Benefit

ROS as signals

Nutritional

Phytoremediation

ROS as signals

Nutritional

Phytoremediation

CellularCompartmentation

Figure 1. Schematic view of plant response to metal stress and some biotechnological aspects.

Still another aspect of this duality lies in the potential use of tolerant crops for fortification of food with essential metals (Guerinot and Salt, 2001). There is also duality in plant tolerance to heavy metals at the biochemical level, on one hand metals cause oxidative damage, on the other hand reactive oxygen species (ROS) can enter signal transduction pathways (Davletova et al., 2005). In this paper I shall discuss these dualities using Allium schoenoprasum (Chives) as the model system. Phytoremediation, iron fortification and a new hypothetical mechanism for the interaction of Cu and ascorbic acid with proteins in response to metal stress is presented.

2. METHODS AND MATERIALS

2.1 The Allium Schoenoprasum Model

Allium schoenoprasum (Chives) plants were grown hydroponically in 'aerated half strength Hoagland medium, with metals added as indicated in the tables (for experimental details see Barazani et al., 2004). The plants were grown in bunches of 25 seedlings each and were reharvested every 2

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weeks by cutting the shoot approximately 3 cm above the bulb. This approach enabled uptake of metals from the medium over extended periods on the same root system (Figure 2).

Figure 2. Experimental system - hydroponically grown Chives.

3. RESULTS

3.1 Metal Accumulation in Chives

Treatment of chives with Ni, Co and Cd at 0.05 and 0.25 mM, showed that the plant is tolerant and hyperaccumulates Ni and Cd (Table 1).

Table 1. Metal accumulation and stress symptoms in chives. Contents, (mg/kg, d.w.)1Treatment

(mM)Time (days)

Harvest(number)

Leaf Root

Stress symptoms

Ni 0.05 96 6 1110 ± 66 789 ±73 No

Ni 0.25 16 1 503 ± 35 1453 ±72 Wilting, GI2

Co 0.05 96 6 1002 ±59 726 ±125 Yellowing

Co 0.25 48 3 2643 ± 625 3000 ± 0 Yellowing, GI2

Cd 0.05 96 6 1166 ± 121 1314 ±123 No

Cd 0.25 64 4 2719 ± 723 2933 ± 116 GI2

1 d.w. - dry weight; 2 GI - growth inhibition

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482 Avi Golan-Goldhirsh

There were no obvious stress symptoms over a period of 96 days (6 reharvests) at a concentration of 0.05 mM metal in the medium.

Comparison of Cd accumulation in chives to several other species reported in the literature (Table 2) showed that chives accumulated Cd to higher concentrations than Brassica juncea, Allium sativum, Pisum sativum,Arabidopsis halleri, Lactuca sativa or Phragmites australis (Table 2). The accumulation in Allium schoenoprasum leaves was at the level of accumulation in Medicago sativa. At the 0.25 mM Cd treatment, the metal concentration in Allium schoenoprasum leaves was higher than in the other species (Table 2).

Table 2. Cadmium accumulation in several plant species reported in the literature. Contents

(mg/kg, d.w.)1Plant species Cd

treatment µM

Medium Timedays

Leaf Root

References

Silene

vulgaris

20.0 Liquid 21 13002 >13000 Verkleij and Prast (1989)

Brassica

juncea

17.8 Soil 14 104 - Kumar et al. (1995)

Thlaspi

caerulescens

63.2 Liquid 28 1290 3200 Brown et al. (1995)

Allium

sativum

10000 Liquid 9 62.9 10100 Jiang et al. (2001)

Pisum

sativum

40.0 Liquid 7 89 640 Dixit et al. (2001)

Medicago

sativa

440.0 Soil 15 20022 - Peralta-Vedia et al. (2002)

Lactuca

sativa

8.9 Liquid 16 162 139 Ramos et al. (2002)

Phragmites

australis

50.0 Liquid 21 77 3690 Iannelli et al. (2002)

Arabidopsis

halleri

10.0 Liquid 7 1572 660 Bert et al. (2003)

Allium

schoenop-

rasum

(chives)

50.0 Liquid 14 199 (1166 )3

1713 Khadka (2003)

Allium

schoeno-

prasum

(chives)

250.0 Liquid 14 745 (2719)4

4587 Khadka (2003)

1 d.w –dry weight; 2 Shoot; - not reported; 3in 6 re-harvests of the leaf of the same plants; 4in 4 re-harvests of the leaf of the same plants

The accumulation of Cd in Allium schoenoprasum leaves in a single harvest was less than in Thlaspi caerulescens and Silene vulgaris. However,

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after 6 successive harvests A. schoenoprasum accumulated Cd at the highest level found in the cited references (Table 2). The accumulation of the metal in the root was also higher than in most other species.

The potential of chives for phytoremediation of heavy metals from contaminated water may become a handicap when grown for food on soil or water containing toxic metals. The accumulated metals may end-up on the consumers’ plate. Thus it would be advisable to test the metal content in chives and other crop plants tolerant to heavy metals.

The chives model was good for the study of plant tolerance to heavy metals because of their richness in sulphur compounds. Some of them, such as cysteine and glutathione can serve as reductants or as scavengers to prevent oxidative damage of heavy metals. We have recently reported an increase in total SH-containing compounds in the root of chives under Cd stress conditions (Barazani et al., 2004). This was accompanied by a reduction in glutathione concentration in root, which raises the question about the identity of the newly synthesized SH compounds induced under the Cd treatment.

3.2 Iron Fortification of Food Plants

The other side of the coin of plant tolerance to heavy metals and phytoremediation is fortification of tolerant food crops with essential metals (Martinez-Navarrete, 2002). Iron deficiency is considered to be the commonest worldwide nutritional deficiency that affects approximately 20% of the world population. Women and children are especially at risk (http://www.who.int/nut/research1.htm). Promoting iron-rich foods as well as iron-fortified foods is necessary to reduce iron deficiency, as most of the people of the world get their iron from eating plant products. The tolerance of chives (Allium schoenoprasum) to heavy metals was reflected also in its ability to accumulate iron at approx. 800 mg/kg dry weight, when the medium was supplemented by 24 mg/L iron (data not shown).

4. DISCUSSION

Our preliminary findings show an almost linear correlation between iron concentration in the medium and its accumulation in the shoot. This taken together with the reported high concentration of vitamin C in chives, which improves iron availability in the diet, opens a new potential for promoting chives as an iron fortified health food.

Israel exports 1,500 tons of chives a year, representing 30% of the annual production. The remaining 70% is deemed inadequate for export. We

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484 Avi Golan-Goldhirsh

propose that fortified chive production in Israel, especially the un-exportable yield, be dried, powdered and/or encapsulated and sold as a food additive/supplement. However, a critical question remains open concerning the nutritional quality of the fortified product in terms of the availability of the iron.

4.1 Metal Catalyzed Modification of Protein Amino

Acids as Structural Signals

It is a well established fact that reactive oxygen species (ROS) can cause extensive cell injury, but in recent years it has become clear that they also play a role in plant metabolism, cellular signaling and defense (Davletova et al., 2005). Several researchers have reported the inactivation of enzymatic activities by the free radical forming ascorbate-Cu2+ system (Shinar et al., 1983; Nakamura and Stadtman, 1984; Golan-Goldhirsh et al., 1992 and others). This non-enzyme catalyzed reaction was site directed toward histidine residues of proteins and to a lesser extent toward methionine (Golan-Goldhirash et al., 1992). These results led to the proposed hypothetical ‘structural signal’ mechanism in response to metal stress (Figure 3).

Figure 3. Schematic presentation of Ascorbate- Cu2+ catalyzed modification of histidine residues in protein leading to structural change and signaling to induce proteases, chaperons

and antioxidative systems.

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Higher specificity to histidine may be explained by the tight complex formation of Cu2+ with histidine compared to other amino acids (Golan-Goldhirsh et al., 1982). The proposed mechanism involves ascorbate anion combining with Cu2+ and O2 forming a ternary complex on His, that dissociates to semidehydroascorbate radical, Cu2+ and the peroxy radical. Strong oxidants can be formed in the vicinity of the histidine residues of the protein leading to its modification into aspartate and possibly other degradation products (Golan-Goldhirsh et al., 1992). The modification of His leads to a structural change, which may mark proteins for degradation (Stadtman and Levine, 2003) and plausibly be a structural signal to induce proteases, chaperons and antioxidative systems in response to metal stress. Such a response can be envisioned as an “alarm” mechanism to metal excess and stress. It can thus be hypothesized that metal tolerant/hyperaccumulator plants would have more histidines in their proteins as a selective advantage in coping with metal stress. This hypothetical mechanism can also be partially verified in vivo by testing for induction of proteases, chaperons and antioxidative systems upon metal stress. More research is needed to understand the interactions of cupric ion and other metal ions with ascorbic acid and proteins and the role of ensuing structural changes of proteins on metal tolerance and homeostasis in plants.

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