Phytoremediation of Soils Contaminated with Heavy Metals and Radionuclides

-•

Annual Report Covering period: June 1, 2001 - May 31, 2002

Submitted to U.S. Agency for International Development Bureau for Global Programs, Field Support and Research; Center for Economic Growth

Phytoremediation of soils contaminated with heavy metals and radionuclides

Principallnvesligators S. Herman Lips & Moshe Sagi J. Blaustein Institute for Desert Research Ben-Gurion University of the Negev Sede 8oqer, Israel 84993

and

Zerekbay Alikulov Nazira K. Zhaparova Gumilev Euro-Asian University Institute of Botany and Phylointroduction Astana, Kazakhstan Academy of Sciences of Kazakhstan Almat Kazakhstan

Project Number CA17-018 Grant Number: TA-MOU-98-CA17-018 USAID Grant Project Officer. Project Duration: June 1, 1999 to May 31, 2003 2

Table of contents

Cover page 1

Table of contents 2

Executive Summary 3

Section 1

A) Research objectives 4

B) Research accomplishments 4 C) Scientific impact of collaboration 19

D) Description of project impact 20

E) Strengthening of Developing Country Institutions 20

F) Future Work 20

Section II

A) Managerial Issues 20

B) Budget 20

C) Special Concerns 20

D) Collaboration, Travel, Training and Publications 20

E) Request for American Embassy Tel Aviv or A.T.D. Actions 20 3

Executive Summary

The overall objective of this project is to optimize the use of plants to remove heavy metal contaminants from soils and water resources in affected areas of Kazakhstan. the former atomic testing ground of the ex-USRR.

Phytoremediation of soils and water will decrease the health hazards to local population. much of which has been affected by radionuclides. making possible the use of vast tracks of contaminated land into useful agricultural areas.

•:. Work in Israel has started with two types of plant species: (a) crops such as barley. pea. tomato and

maize and (b) Some well known HM hyperaccumulators like Typha and Phragmites. During the first year

of wOrk. we have focused on the determination of methodologies and key enzymes involved in the

production of phytochelatines in order to understand better the key metabolic systems that provide the

plant with means to chelate and accumUlate heavy metals.

•:. Work in Kazakhstan has been carried out with Amaranthus species assessing the capacity of this

species to remove heavy metals from soils and accumulate it in roots and shoots. The Kazak

investigators have also determine the changes of HM removal by Amaranthus during the life cycle of the

plant and some new techniques to enhance to HM removal capacity of the plants.

Work has been carried out in parallel at the physiological. biochemical and field levels with good integration among the different components. as evidenced in some of the results shown. Others aspects of this research integration will become more evident towards the summary of the overall work carried out in this project

All the work carried out during this year was in compliance with the objectives of the project. Collaboration between the two groups has been good with the active participation of Dr. Vladimir Kuzovlev in the past and of Dr. Zerekbay Alikulov during a good part of 2002. During the coming months an additional Kazak postdoctoral fellow (Dr. Galiya A. Shalakhmetova) will come to work with the Israeli team and extend the very promising observations made with Amaranthus and other plant species. 4

Section I A) Research Objectives

Overall aim and specific objectives

Overall aim: Removal heavy metals and radionuclides from contaminated soils by the use of selected annual crops capable of taking up and immobilizing in the plant biomass large amounts of the polluting agents. Soecific objectives: 1. Preliminary selection of appropriate endemic and/or non-endemic (a rare and endangered) annual plant populations with heavy metal accumulation potential, with anatomical characteristics which facilitate harvesting. 2. Study of the biosynthesis of metal-chelators and transporters in roots and shoots of the selected plants, and determination of optimal metal accumulation during the plant life cycle. 3. Application of simple agrotechniques such as fertilization with nitrate, sulfate and Mo to enhance biomass production and the chelating capacity of the selected plants. 4. Study of additional chelating mechanisms in the plants and determination of optimal conditions for heavy metal influx, transport and compartmentalion. This project is meant to select plant species capable of accumulating heavy and radioactive metals, to enhance their uptake and chelation in roots as well as their transport to the shoot and their storage in vacuoles. These studies will be carried out in parallel in field experiments with the selected plant species in order to devise new agrotechniques for the optimization of heavy metal removal. At a later stage of this project, production of transgenic plants reinforced by molecular insertion of foreign genes controlling heavy metal transport and chelate production is envisaged.

5) Research Accomplishments The content of lead, cadmium and zinc is generally higher in south-eastem Kazakhstan than reported in other contaminated regions of the World and they exceed maximal in many cases the allowed concentrations by several folds. Southeastm Kazakhstan includes Oskemen, Kyzilorda and Shymkent districts. Plants absorb lead and accumulation of this metal has been reported in roots, stems, leaves and root nodules, increasing with the metal level in the soil. Some plants are able to tolerate excess of lead by involving processes like exclusion, compartmentalization or synthesizing metal detoxifying peptides or organic acids. Furthermore, the metal remains largely as a surface deposit or coating of plant organs. Coating lead of foliage may contain up to 100 times the level observed in unwashed leaf surfaces of the crop. Pubescent leaves have been coated with more lead than the smooth surfaced leaves due to regular wash off the metal by the rain water from the smooth leaf surface (Karaganov et al., 1995). In higher plants the monocots have been studied in some detail for their responses to heavy metals in general than the dicots. However, new studies are needed to better understand the responses of the plants and mechanism of tolerance to the metals, or accumulation of them in several plant species, particularly in halophytes. Halophyte plants are widespread in the area of south-eastem Kazakhstan. Therefore we studied the capability of several plants, including halophyte plants, to extract heavy metals from industrially contaminated soils and an agricultural soil contaminated with metals from sewage sludge.

Experimental resulls Seeds of Amaranthus and Agropyron were obtained from the Institute of Botany and Phytointroduction of the Kazakh Academy of Sciences. Seeds of Aeluropus were collected from plants grown in sandy saline soils of the Kazali region of the Aral Sea in September-October 2001. Soils for the studies were collected from Shymkent where lead-extracting plant are located, and from Kyzilorda located near the Aral Sea. Uncontaminated control soils were collected from the Turkistan region located between the Shymkent and Kyzilorda. Efficient phytoextraction requires high biomass plants with efficient translocating properties Halophytes characteristically accumulate large quantities of salts in above ground tissue material and can have high biomass production. It has been speculated that salt-tolerant plants may also be heavy metal-tolerant and 5

further may be able to accumulate metals. In our study we compared metal uptake by a halophyte Ae/uropus Iitoralis and a glycophyte Agropyron desertorom, and also moderate salt-tolerant Amaranthus trice/or. In order to provide a better estimate of toxicity tolerance thresholds to heavy metais we studied the behavior 2 2 of Amaranthus, Agropyron and Ae/uropus exposed to different Pb + and Cd + treatment levels. In our previous experiments we studied effects of Cd, Zn and Cu on the growth of Amaranthus tricolor and A. panicu/atus, and their metal-accumulating capability. In these experiments we used Cd to compare its effect with that of Pb. We used a greenhouse screening study where seedlings of these plant species were grown in sandy soil and exposed to supplemental concentrations of soluble Pb2+ and Cd2+ salts ranging from 0 (control) to 2.5 mM. As shown in Table I, cadmium was found to be more toxic for the plants tested than lead. As shown in the Table I, the levels of endogenous metals increased with the increased exogenous lead and cadmium levels. Furthennore, the toxicity threshold of Cd and Pb was found to be 1.0 mM for the plant species.

2 ·Table I. Toxicity thresholds of Pb + and cif+ for Amaranthus, Agropyron and Ae/uropus (percent of survival in the heavy metals).'

Heavy metals Concent mM Amaranthus Agropyron Aeluropus lead o (control) 100 100 100 Cadmium 100 100 100 lead 0.1 100 100 100 Cadmium 100 100 95 lead 0.5 100 100 100 Cadmium 93 95 84 lead 1.0 95 93 97 Cadmium 92 86 70 lead 1.5 85 65 96 Cadmium 60 57 55 lead 2.0 94 40 75 Cadmium 42 23 34 lead 2.5 90 15 55 Cadmium 17 2-5 20

'Plant seeds were genninated in sandy soil. Seedling pots were supplied with different concentrations of heavy metal after 10 d, and further grown until 25 d when their survival was assesed.

2 Table II. Effects of increasing NaCI concentrations on Pb + accumulation (mg/g DW) by different plant 2 species (1.0 mM Pb + added to NaCI solutions)'.

Plant Plant Concentrations of NaCI species parts 0 1% 2% 3% 4% 5% Pb in roots 18.7 13.3 6.0 Wilt - - Amaranthus Biomass 97 92 65 - Pb in leaves 17.0 12.7 4.8 Wilt - - Biomass 100 96 60 - Pb" in roots 14.5 10.3 4.6 Wilt - - Agropyron Biomass 95 83 35 - Pb" in leaves 18.0 12.3 6.5 Wilt - - Biomass 97 80 28 - Pb in roots 15.6 15.2 15.4 17.8 15.9 14.2 Aeluropus Biomass 100 100 102 105 94 83 Pb" in leaves 18.5 25.4 32.5 48.2 50.2 48.5 Biomass 100 103 107 106 98 87

, Seeds were genninated in sandy soil. After 10 d of seedling development solutions 2 containing 1.0 mM Pb + and increasing concentrations of NaCI were applied to the soil. Biomass (%) was estimated in control seedlings grown without Pb and NaCI; Seedling growth was inhibited and became wilty after 10 days of salinization with NaCI. Biomass and the content of lead in plant materials were measured in 35 d old plants. 6

Since the halophyte Aeluropus growth preferentially in saline soils, the effects of increased soil salinity on heavy metal accumulation by plants were tested, using Amaranthus and Agropyron as controls. Aeluropus Iitoralis grows in the presence of salinity up to 6.0% in the Aral Sea region (Sarsenbaeva, 2000). According to data published by the Institute of Botany and Phytointroduction of the Academy of SCiences of Kazakhstan, Aeluropus Iitoralis is one of the dominant plants in the grazing lands of the Aral Sea basin. All these halophytes have a remarkable ability to accumUlate large quantities of NaCI in their tissues. The mineral content of these plants, mainly NaGI, was between 30 and 50% of their dry weight (Jefferies and Rudnik, 1984). The main salt accumulating organ of Aeluropus was found to be its leaves (Sarsenbeva, 2000). Since Aeluropus is able to accumulate salts in its leaves, we tested the plant for heavy metal uptake and accumulation.

The monocots Aeluropus and Agropyron accumulated considerably more Pb in the leaves than in their roots. The halophyte transported more Pb from the root to the shoot than the glycophyte, while amaranth accumulated almost equal amount of the metal in the root and shoot. Our results suggest that the halophyte have greater potential to phytoextract Pb from contaminated soils. lead accumulation depended not only on' plant species but also on lead concentration in the growth SUbstrate. The dependence of heavy metal accumulation on concentration of lead in the medium was tested (Table 111).

Table 111. Dependence of Pb2+ -accumulation in plant parts of various species on metal concentrations applied to sandy soil (metal content in mg/g OW)

Plant species Plant parts Pb applied (mM) Pb in the plant 0.0 1.8 Roots 0.5 7.2 Amaranthus 1.0 18.5 tricolor 0.0 1.0 Shoots 0.5 8.5 1.0 15.7 0.0 1.7 Roots 0.5 12.6 Agropyron 1.0 15.0 desertorum 0.0 2.3 Shoots 0.5 12.5 1.0 17.5 0.0 1.4 Roots 0.5 9.5 Aeluropus 1.0 14.3 litoralis 0.0 2.1 Shoots 0.5 13.0 1.0 18.3

The effects of plant inoculation with the fungus Glomus was studied. No inoculation in amaranth plants with fungus was observed in these experiments (Table IV).

Table IV. Effects of plant colonization by Glomus on lead accumulation in the roots and leaves of Agropyron and Aeluropus·.

lead accumulation (mglg OW) Plant species Growth substrate Roots leaves Biomass Pb Biomass Pb Uncontaminated soil 100 0.6 100 0.1 Contaminated soil (CS) 100 5.1 100 5.6 Aeluropus GS+ NaGI 100 6.2 98 6.5 CS + inoculum 108 9.3 106 12.8 CS + NaCI + inoculum 109 12.2 107 19.7 Uncontaminated soil 100 0.4 100 02 Agropyron CS 97 4.7 100 7.L CS + inoculum 103 10.5 104 17.2 7

*Seeds were germinated in contaminated soil collected from the city of Shymkent, and from uncontaminated soils from the Turkestan area. Biomass and lead content were measured in 35 d old plants. NaCI (3%) was added to the soil on day 10 after germination. Agropyron and Ae/uropus roots and leaves of plants colonized with vascular-arbuscular micorhizae accumulated considerably high levels of lead. The level of endogenous metal increased with the exogenous lead concentrations (not shown). However, our attempts to inoculate amaranth plant were not successful. No VAM was detected in roots of Amaranth species in different regions of Kazakhstan. Another requirement for efficient phytoextraction is that the metal contaminants be in a form available for plant uptake. Chemically enhanced phytoextraction using ethylenediaminetetraacetic acid (EDTA) has been well documented. The effect of chelating agent EDTA on lead accumulation in roots and shoots of growing seedlings was studied (Table V).

Table V. Effects of increased concentrations of EDTA supplied to contaminated soil on lead accumulation by the plants*.

Plant EDTA supplied Plant parts Biomass Pi?" scecies % of control accumulated Without EDTA Roots 100 5.6 (control) Leaves 100 5.3 1 mglkg Roots 100 7.8 Leaves 100 8.0 10 mglkg Roots 100 9.5 Amaranthus Leaves 100 10.4 100 mglkg Roots 100 11.5 Leaves 100 15.2 1 g/kg Roots 94 19.4 Leaves 98 26.0 10 glkg Roots 78 19.2 Leaves 85 55.7 Without EDTA Roots 100 3.5 (control) Leaves 100 6.0 1 mglkg Roots 100 5.7 Leaves 100 7.2 10 mg/kg Roots 100 8.4 Agropyron Leaves 100 10.7 100 mglkg Roots 100 11.3 Leaves 100 16.5 1 glkg Roots 96 18.6 Leaves 100 23.6 10 glkg Roots 85 18.3 Leaves 95 23.5 Without EDTA Roots 100 4.2 (control) Leaves 100 6.0 1 mglkg Roots 100 5.3 Leaves 100 8.3 10 mg/kg Roots 100 11.4 I Leaves 100 13.2 Aeluropus I 100 mglkg Roots 100 15.8 : Leaves 100 29.4 r 1 glkg Roots 100 25.4 I Leaves 100 53.4 ! 10 glkg Roots 98 23.5 i Leaves 95 52.5 ,

*Seeds of the plants were germinated in contaminated soil collected from Shymkent city, and to the soil added increasing concentrations of EDTA. Biomass and the content of lead measured in 35 d old plants.

The application of exogenous chelators increased plant uptake of lead from contaminated soils. EDTA at higher concentrations can be toxic for the plant. The chelator is not biodegradable in nature and is very costly for large-scale phytoremediation processes, limiting its usefulness to continued soil application. In this 8 respect one of the most prospective and promising directions of the phytoremediation is the use of biohumus. Biohumus contains a number of bioactive ingredients and some of them may chelate heavy metals. Though applied at approximately similar rates of application as conventional chemical fertilizers and capable of chelating heavy metals. Experiments on the use of biohumus for phytoremediation are in progress at the moment.

Study on physiological and biochemical mechanisms of lead. Seed germination and early seedling stand are the initial events in the life of a plant projecting the extent of future physiological and biochemical processes. Seed germination is initiated with regulation of enzymatic reaction which activates catabolic and anabolic processes in the storage tissue (cotyledons and endosperm) and in the embryonic axis. Germination is inhibited if even one component of these processes is affected. The inhibitory effect of lead on seedling growth may arise from interference of lead with auxin (indole acetic acid, 1M) regulated cell elongation (Van Assche and Clijsters, 1990). Our results showed that molybdenum containing 'enzymes are inhibited in roots of monocots by the metal but in the leaves a differential effects are observed in monocot species and dicots as well. The oxidation of indole acetaldehyde (IMld) and abscisic aldehyde (ABAld) to indole acetic acid (1M) and abscisic acid (ABA), respectively, were catalyzed by the molybdenum containing enzyme aldehyde oxidase (Koshiba et aI., 1996). Much attention has' recently focused on plant aldehyde oxidase (AO, EC 1.2.3.1) because of its involvement in the adaptation processes of plants to the environmental stresses (Omarov et aI., 1998). It is proposed that the main function of plant XDH is the production of uric acid, which plays an important role in ROS (reactive oxygen species) scavenging formed during oxidative stress. Lead treatment of seedlings can create water stress to the plants ( Burzynski and Grabowski, 1984), i.e. it causes the oxidative stress in plant cells. Formation of reactive oxygen species (ROS, highly reactive oxygen radicals) is a general reaction during several kinds of stress including heavy metals. 2 NR inhibition in the root due to Pb • is primarily the result of interference of the metal to de novo_synthesis of 2 NR molecules. Induction of NR activity was Significantly inhibited by Pb • (0.5 mM) in the Agro~yron leaves. Among various heavy metals Zn2• and Cu2• to some extent negated the inhibitory effect of Pb· on both in vivo and in vitro NR activity (data not shown).

Toxicity of Pb2+ on Mo-enzyme activities could be ameliorated by MoO.... The response of NR activity to lead was different in in vivo and in vitro - while the in vivo NR activity is generally inhibited (Table VI), the in vitro activity was unaffected (data not shown).

Table VI. Effect of 1.0 mM Pb2• supplied to sandy soil on the activities of plant Mo-enzymes*.

Plant Plant MoCo AO XDH NR species parts Cont. +Pb Cont. +Pb Cont. +Pb Cont. +Pb Amaranth Roots 1.8 1.4 43.0 39.5 1.2 1.0 2.7 2.0 Leaves 2,1 1.6 70.5 60.3 2.9 2.6 5.0 3.8 Agropyron Roots 2.4 1.8 67.4 61.0 2.5 2.2 5.4 4.2 Leaves 1.8 1.5 40.3 38.5 1.6 1.4 3.7 3.2 Aeluropus Roots 3.0 2.4 80.6 75.8 3.4 3.2 5.6 4.5 Leaves 2.4 1.6 66.2 62.7 2.1 1.8 2.7 2.0 ... . *Activltles: MoCo In /UTlol NO 2 formed by nit-1 NR mg-1 protein hour-1; AO in nmol DCIP mg-1 protein min-1; XDH in I1mol NADH mg-1 hour-1; NR in I1mol NO'2 mg-1 protein hour-1.

The side chain of MoCo contains vicinal thiol groups (dithiol) (Rajagopalan, 1992). Our experiments with active MoCo isolated from wheat embryos showed that heavy metals such as Hg2., Pb2• and Cd2+ have high 2 affinity to the dithiol group of MoCo. The presence of relative high concentration of Mo0 • under reducing conditions protected MoCo dithiols against heavy metal binding (manuscript in preparation). Probably, in Me deficiency and the presence of the heavy metals lead to the inactivation of newly synthesizing MoCo by heavy metals in vivo. Seed priming with molybdate and tungstate was used to test these hypotheses. Imbibition of the seeds in molybdate solutions up to 50 mM did not inhibit their germination. In special experiments the effects of infiltration of 50 mM ammonium molybdate into the seeds was studied during 30 h imbibition. TIle content of ammonium ions in the seeds determined with Nessler reagent was relatively high. Some effacts of seed priming with molybdate (or tungstate) on the activities of Me-enzymes in plant seedlings grown in the presence of Pb have been summarized (Table VII). 9

Table VII. Effects of seed priming on the activities of Mo-enzymes in 5 days old seedlings.

Amaranthus tricolor Seed priming Plant Biomass MoCo AO XDH NR conditions parts Control (dry seeds) Roots 100 1.6 43.5 1.4 3.0 Leaves 100 2.3 68.6 2.9 5.4 Control (dry seeds). Roots 96 1.3 39.0 12 2.2 grown with Pb2+ Leaves 100 1.6 61.6 2.6 3.4 Seeds + priming in H2O Roots 106 1.8 45.4 1.7 3.3 Leaves 109 2.6 70.2 2.8 5.7 Seeds + primin,a in H2O. Roots 100 1.3 40.2 1.4 2.4 Grown with Pb Leaves 103 1.5 63.0 3.0 3.6 Seeds + priming in Roots 83 0.6 18.7 1.0 1.2 Pb2+.grown without Pb Leaves 86 0.4 37.5 2.7 1.2 Seeds + priming in Pb • Roots 45 0.4 13.7 0.7 0.5 grown with Pb2+ Leaves 40 0.3 23.6 1.8 0.4 Seeds + priming in MoO Roots 111 2.1 48.8 1.9 3.6 Leaves 112 2.8 73.0 2.9 6.2 Seeds + primin!!. in MoO Roots 107 2.2 47.8 1.9 3.5 .grown with Pb Leaves 106 2.8 72.5 2.8 6.0 Seeds + priming in WO Roots 100 1.7 17.0 1.5 1.8 Leaves 100 2.4 23.0 1.9 1.2 Seeds + prim in!!. in WO- i Roots 57 1.2 0.9 1.2 0.4

.grown with Pb . Leaves , 55 I 2.0 0.4 0.8 0.0

Agropyron desertorum

Control (dry seeds) Roots 100 2.5 68.0 2.4 5.0

- Leaves 100 1.8 41.0 1.8 3.6 , Control (dry seeds). Roots 95 1.7 60.5 2.2 4.0 grown with Pb2+ Leaves 92 1.3 36.0 1.6 3.0 i Seeds + priming in Roots 110 2.8 70.2 2.4 5.2 I H20.grown in water Leaves 107 2.0 43.3 1.9 3.5 i Seeds + priming in Roots 97 2.9 68.2 2.3 4.8 ! H20. grown with Pb2+ Leaves 94 2.2 42.0 2.0 3.3 i Seeds + priming in Roots 85 2.6 62.3 2.0 3.8 i Pb2+.grown in water Leaves 82 2.2 36.6 1.6 2.7 I Seeds + primirw in Pb • Roots 48 1.8 39.4 1.5 1.8 i grown with Pb Leaves 52 1.5 20.5 1.0 0.8 I Seeds + priming in Roots 113 3.2 73.4 2.6 5.8 i MoO". grown in water Leaves 109 2.3 45.7 2.1 3.6 I Seeds + priming in Roots 105 3.3 72.7 2.6 5.3 2 MoO". grown with Pb + Leaves 108 2.2 46.3 2.3 32 Seeds + priming in WO- Roots 97 2.9 28.4 1.8 1.9 Leaves 100 1.2 19.0 1.2 1.4 : Seeds + primirw in WO • Roots 53 1.7 , 20.5 1.3 0.8 . grown with Pb Leaves 57 1.3 , 13.5 0.8 0.4 10

Aeluropus litoralis

Control (dry seeds) Roots 100 3.2 81.0 3.2 5.4 Leaves 100 2.5 65.7 2.5 2.7 Control (dry seeds), Roots 100 2.6 75.0 3.4 4.4 grown with Pb2+ Leaves 100 1.6 63.0 2.3 1.8 Seeds + priming in H2O, Roots 105 3.5 82.3 3.1 4.8 grown in water Leaves 115 2.5 66.3 2.7 3.0 Seeds + priming in Roots 103 3.5 78.8 2.8 3.5 H20, grown with Pb2+ Leaves 107 2.5 64.0 2.6 2.3 Seeds + priming in Pb~, Roots 100 3.2 75.2 2.8 2.5 grown in water Leaves 100 2.3 65.0 2.5 2.3 Seeds + primirw in Pb<', Roots 90 2.2 52.0 1.8 0.9 grown with Pb Leaves 95 1.6 53.0 1.5 1.0 Seeds + priming in MoO- Roots 110 3.7 85.0 3.6 6.0 Leaves 118 2.8 67.8 2.6 3.2 ! Seeds + primirw in MoO- Roots 108 3.6 80.6 3.0 5.4 i grown with Pb Leaves 112 , 2.5 64.4 2.8 2.9 I Seeds + priming in WO Roots 100 3.3 i 30.2 2.0 0.6 Leaves 100 2.2 • 27.3 1.2 0.3 Seeds + priming in WO , Roots 55 I 2.0 21.3 1.5 0.2 grown with Pb" I Leaves 62 1.6 18.8 0.8 0.2

Seed priming with molybdate resulted in a considerably increase of plant biomass (Table VII). Since an efficient uptake of heavy metals requires high biomass production of plants, seed priming of metal accumulating plants may play a significant role in soil phytoremediation. Seed priming also improved seedling stand size resulting in higher yield, and therefore it is expected that the plants grown from Me primed seeds will have a higher Pb-accumulating capacity. Saturation of the seeds with Mo using their priming allows to escape the inhibition of Mo-uptake in radicles and roots by soil sulfate. In Table VIII shows that seed priming with molybdate considerable increased lead accumulation by the plants from Pb-contaminated and relatively sulfate-rich soil.

Table VIII. Effect of seed priming with molybdate on metal accumulation by the plants grown from primed seeds'.

Pb accumulated in the seedlings (mg/g OW) Plant species Plant Plants grown from Plants grown from parts seeds primed in H2O seeds primed with M004+ Roots 7.4 8.7 Amaranthus Leaves 8.0 9.7 Roots 4.4 5.4 Agropyron Leaves 6.5 7.3 Roots 5.2 6.5 Aeluropus Leaves 6.7 7.6 'Pnmed seeds were germmated In conlamlnated soli collected from Shymkent Lead content was determined in 35 d old plants.

Foliar fertilizer provides nutrition to the cells in the leaf when the demand by the plant is grealer than their uptake capacity, or when there is a competitive uptake of ions. The effects of foliar fertilization with 2 molybdate on Mo-enzyme activities in plant parts and their Pb • -accumulating capacity was studied (Table IX). 11

Table IX: Foliar fertilization wHh molybdate on the activities of Mo-enzymes 2 and on Pb + accumulation in organs of different plant species".

Plant species Plant parts AO XDH NR Pb" accumulated Cont. FF Cont. FF Cont. FF Cent. FF Amaranthus Roots 40.2 42.2 1.2 1.3 1.8 1.8 18.0 17.7 Leaves 58.7 61.5 2.5 2.5 3.5 4.6 15.3 15.6 A\lropyron Roots 61.5 63.8 2.3 2.4 4.2 4.4 14.8 15.0 Leaves 37.8 40.2 1.3 1.2 3.0 3.8 17.0 16.5 Aeluropus Roots 74.7 76.2 3.2 3.4 4.2 4.2 14.2 15.0 Leaves 63.0.. 68.6 1.7 1.8 1.8 2.5 19.0 20.3 ·Cont. - control plants; FF - foliar fertilized plants. Plants were grown in sandy soil containing 1.0 mM Pb2+. Leaves were sprayed every 3 dayswHh 10 mM molybdate solution from day 10 after germination. Enzyme activities and lead content were determined in 35 d old plants.

Foliar supplied Mo increased the activities of leaf Mcrenzymes but plant uptake of Pb did not increase accordingly. However, unlike seed priming, foliar fertilization with molybdate did not increase biomass of plant parts. Only leaf biomass of amaranth slightly increased 35 days after fertilization (data not shown).

The following studies are presently in progress: 1. Effects of biohumus supply to the soil on metal-accumulating capabilities of the plants; 2. Radionuclide-accumuaHing capability of Suaeda plant. We observed that drought tolerant halophyte Suaeda grows well in highly radioactive soil near former uranum extracting plant in Akmola district of Central Kazakhstan.

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Karaganov KD, Nekrasova TF, Asanbaev II<, Akhanov GU. 1995. Heavy metal content in the ~ils of s0uth eastern region of Kazakhstan. Reports of the Minitry of Sciences - Academy of Sciences of Kazakhstan Republic. N3: 47-61. Kasymbekov B. 2000. Widespread plant species in South Kazakhstan colonized with arbuscular mycorrhizal fungi. Doct. Thesis. Almaty. Koshiba T, Saito E, Ono N, Yamomoto N, M. Sato. 1996. Purification and properties of a fJavin- and molybdenum-containing aldehyde oxidase from coleoptiies of maize. Plant Physiol. 110: 781-789. McMillan B.G., Juniper S., Abbott L.K. 1998. Inhibition of hyphal growth of a vesicular-arbuscular mycorrhizal containing sodium chloride of infection from fungus in soil limits the spread spores. Soil. BioI. Biochem. 30(13): 1639-1646. Ning J. and J.R.Cumming. 2001. Arbuscular mycorrhizal fungi alter phosphorus relations of broomsedge (Andropogon virginicus L.) plants. J. Exp. Bot. 52(362): 1883-1891. Omarov R. T, Sagi M, and H.Lips. 1998. Regulation of aldehyde oxidase and nitrate reductase in roots of barley (Hordeum vulgare L.) by nitrogen source and salinity. J.Exp. Bot. 49: 897-902. Rajagopalan KV. 1992. Novel aspects of the biochemistry of the molybdenum cofactor. Adv. Enzym. Relat. Areas Mol. BioI. (A.Meister,ed). 64: 215-290. Ruiz-Lozano J.M. Azcon R. and Gomez M. 1996. Alleviation of salt-stress by arbuscular-mycorrhizal Glomus species in Lactuca sativa plants. Physio!. Plant. 98: 767-772. Sarsenbaeva Zh. 2000. Some halophyte plants in highly saline soils in Aral Sea region of Kazakhstan. Diploma thesis. The L.N.Gumilev Eurasian State university. Astana Savidov N.A., Alikulov Z, H.Lips. 1998. Identification of an endogenous NADPH-regenera:ing system coupled to nitrate reduction in vitro and fungal crude extracts. Plant Sci. 133(1): 33-45. Simons SS, Chakraborti PK, Cavanaugh AH. 1990. Arsenite and cadmium as probes of glucocorticoid receptor structure and function. J. BioI. Chem. 265(4): 1938-1943. Singh RP, Bharti N, Kumar G. 1994. Differential toxicity of heavy metals to growth and nitrate reductase activity of Sesamum indicum seedlings. Phytochemistry. 35(5): 1153-1156. Sinha SK, Srivastava HS, Mishra SN. 1988. Nitrate assimilation in intact and excised maize leaves in the presence of lead. Bull. Environ. Cont. Toxico!. 41: 419-426. Smith WH. 1971. Lead contamination of road sides white pines. Forest Sci. 17(2): 195-198. Triplett EW, Blevins DG, and DD. Randall. 1982. Purification and properties of soybean nodule xanthine dehydrogenase. Arch.Biochem.Biophys. 219: 39-46. Van Assche F. and Clijsters H. 1971. Effects of metals on enzyme activity in plants. Plant Cel!. Environ. 13: 195-206. Vunkova-Radeva R, Schieman J, Mendel RR, Salcheva G. Georgieva D. 1988. Stress and activity of molybdenum containing complex (molybdenum cofactor) in winter wheat seeds. Plant Physiol. 87: 533- 535. Wahl RC, Hageman RV, Rajagopalan KV. 1984. The relationship of Mo, molybdopterin, and cyanolysable sulfur in the Mo-cofactor. Arch. Biochem. Biophys. 230: 264-273. Zheng W, Maiorino RM, Brendel K, Aposhian HV. 1990. Determination and metabolism of dithiol chelating agents: VII. Biliary excretion of dithiols and their interactions with cadmium and metallothionein. Fundam. App!. Toxico!. 14(3): 598-607. 13

Field experiments Higher concentrations of Zn showed the same effects on plant growth and development in laboratory and field experiments. The height of aerial parts, length of roots and biomass production of flowering A. panicu/atus grown in soil containing 300 mg/kg OS. Zn content was 105%, 90% and 108% of control plants, respectively. The heavy metal inhibited root growth, but not of aerial organs during later stages of plant development Biomass production of A. tricolor under the same conditions dramatically increasedto 182% of the control plants. Amaranth plants grown with high concentrations of Zn had dark-green leaves with larger area and thicker stems as compared to control plants grown without the metal. The main organs accumulating Zn under field conditions were the roots and the leaves for both amaranth subspecies. A. tricolor showed more effective accumulation of Zn than A. panicu/atus under laboratory experiments and field conditions. The content of Zn in the roots and leaves increased gradually during plant development. A. tricolor and A. panicu/atus accumulated 78 and 60 mg Znlkg OW during the vegetative stage and '144 and 123 mg/kg OW during flowering. Effective uptake and accumulation of Zn were observed amaranth during the vegetative and bud formation stages. The increase of metal accumulation during flowering was negligible. A. panicu/atus reduced the soil content of Zn to from 300 to 183 mglkg OS during vegetati.... e growth A. tricolor decreased soil content to 165 mglkg ·OS. During flowering Zn soil content decreased further to 98 and 74 mglkg OS, respectively for the species mentioned. Maximum accumulation of Zn by amaranth plants was observed during bud formation and at the beginning of flowering. All the plants showed better growth and development under field conditions than in the laboratory. Cross-sections of the root, stem and leaf tissue were treated with the heavy metal-coloring reagent 4-{2- pyridilazo)-resorcine. All the organs of amaranth plants grown with heavy metals showed the presence of heavy metals in their tissues, while in those of control plants grown without heavy metals the coloring by the reagent was not observed (Picture 2 a, band 3 a,b). Detailed histochemical analysis of these colored microscope sections showed that Cd accumulated considerably more in the roots of A. panicu/atus than in its leaves (Picture 2b and 3b). Cd accumulated in roots mostly in the cells of the rhizodermis and endodermis (Picture 2, a and b). The histochemical analysis confirmed that in root tissues of amaranth grown in the presence of heavy metals the roots accumulated more than the stems and leaves. Heavy metal accumulation ability of amaranth as affected by the removal of main apexes. Removal of stem apex induces intensive growth of lateral branches. Biomass production of amaranth plants was increased using this method (Safarov et al. 1992). Stem apexes of amaranth plants in field experiments were removed at the end of the vegetative stage. The main stem apex was cut 1 em below the apical bud. This operation resulted in an increase of density and growth rate of lateral branches of amaranth, and as a consequence the biomass production also increased considerably. Following apex removal, the total plant biomass of A. panicu/atus increased to 121% compared to the control plants at the end of bud formation stage, while A. tricolor increased to 133% of controls. Removal of the main apex of amaranth plants at the beginning of the bud formation stage resulted in the increase of Zn accumulation in the roots and leaves. The roots of A. tricoIorwithout apex accumulated Zn up to 175 mglkg OW during flowering. Leaves accumulated up to 160 mg Zn/kg OW. Roots of untreated A. tricolor and A. paniculatus accumulated up to 145 and 123 mg Zn /kg OW, respectively. Under the same conditions the roots and leaves of A. panicu/atus accumulated up to 150 and 125 mg ZnJkg OW, respectively. The content of Zn in pot soil was decreased more effectively by amaranth without apexes. A. tricolor without apex decreased Zn content in the soil during flowering up to 40 mg ZnJkg OS. but A. panicutfus decreased up to 65 mg Znlkg OS. Untreated amaranth plants accumulated 75 and 100 mg ZnJkg OS. respectively. Thus, the removal of the apex of amaranth plants significantly increases Zn accumulation and decreases the metal content in the soil. The following experiments are in progress during the summer of 2001: Seeds of two amaranth subspecies were primined in the presence of key minerals such as Mo, K. Ca, SO•. and NO"". Their germination ability, growth rate, metal accumulating ability. •:. Two amaranth subspecies are grown in higher concentrations of Cd and Cu. Effects of pre-sowing treatments on the accumulation of these metals . •:. Effects of EOTA application to the soil on heavy metal uptake and accumulation by the amaranth plants. •:. Experiments of 1, 2 and 3 are being carried out in different treatment combinations: (a) seeds were primed separately in water and Mo-, K-, Ca-, SO-4, and NO"" solutions and untreated control plants.; (b) the primed seeds were then planted in soils containing Zn, Cu and Cd; (c) all these treatments 14

are applied to plants during their development while regularly irrigated with water with or without EDTA.

Aldehyde oxidase in Phragmites australis under stress Phragmites has been extensively used in phytoremediation although relatively little is known about the mechanism and strategies used by these plants to cope with the accumulation heavy metals in its tissues. Part of our work has focused on a better understanding of the biochemical and genetic mechanisms of these plants as a possible source of information for the development of more efficient heavy metal accumUlators in the Mure. Aldehyde oxidase (AD, EC 1.2.3.1) activity increased in Phragmites plants during all the stress conditions applied in our experiments: salinity (100 mM NaCI), nitrogen supply (2mM NH. + as single nitrogen source) and heavy, metal treatments for increasing periods of time (0-7 days) and concentration (0- 300 IJM Cd). Plant responses did not correlate with the severity of Cd stress. The highest activity of AD in response to stress, was observed in roots after 3 days of treatments. After this time, the activity diminished. AD and one of its products, ABA, are involved in stress responses, tolerance and post-stress recovery, AD increase representing an early event of adaptation to s.tress. Aldehyde oxidase (AD, E.C 1.2.3.1), among other processes, is involved in the final step of ABA synthesis, catalyzing the oxidation of abscisic aldehyde to abscisic acid (Buchanan et aI., 2000, Akaba et aI., 1998, Sagi et aI., 1999). AD is one of the enzymes that regulates and controls ABA biosynthesis (Seo and Koshiba, 2002). The active enzyme contains FAD, Fe and Mo as cofactor (MoGo), AD belonging to the family of molybdoenzymes (Mendel and Schwarz, 1999). The involvement of AD in ABA synthesis was deduced from the results of experiments with MoCo deficient mutants of barley, tobacco, tomato, Arabidopsis in which AD activity was inSignificant or absent and, consequently, the synthesis of ABA very low (Seo et aI., 1998). The concomitant increase of AD activity and ABA synthesis is also a proof for this role of the enzyme. Addition of Mo to nutrient solution results in the increase of ABA content, proving that Mo is required for ABA synthesis (Omarov et aI., 1999). AD showed a wide substrate specificity, catalyzing the oxidation of many aliphatic and aromatic aldehydes, N-methyl-nicotinamide and xanthine (Seo et aI., 1998, Akaba et aI., 1998). Frequently used substrates in the study of AD activity are: indole-3-aldehyde, heptaldehyde, naphtaldehyde, acetaldehyde, benzaldehyde. AO was intensely studied in animals and microorganisms and less in plants. During stress, in plants was showed increased AO activity with consequent ABA accumulation, suggesting the involvement of AO in stress responses (Omarov et aI., 1998). ABA controls physiological and molecular processes of plants reactions to stress, from the signaling of environmental changes, till the induction of specific stress responses (Iuchi et aI., 2000, Rock, 2000, Jia and Zhang, 2000). The processes regulated by ABA are involved in the development of stress tolerance and post-stress recovery, processes with great importance in stress biology and biotechnology of resistant plants (Chandler and Robertson, 1994).

100 528 460 172 100 8.4 91 54 ..Relative density(%) EJ .. .,. I,,, .. .. 0 NH4 Cd NaCI 0 NH4 Cd NaCI

100 38.6 59.6 160 100 248 519 533

EJ'o";: NH4 C~'i<'~·~· Fig. L AO activity of Plzragmites plants as affected by different stress conditions (2mM NH. +, lOO!JM Cd, 100 mM NaCl) after 3 and 7 days of treatments, the substarte used was indole-3-aldehyde,R=root, S=shoot, each lane was loaded with extract containingl00jlg protein for roots or 200 jlg proteins for shoot. 15

The present work represents a part of the studies concerning Phragmites plants responses and resistance to stress, for estimating their usefulness in phytoremediation (Fediuc and Erdei, 2002), which requires stress resistant plants. We checked AO activity in different stress conditions: salinity, suboptimal nitrogen supply and especially heavy metal stress with different duration and intensity. The increase of AO activity contributes to higher stress resistance, due to higher ABA level. AO activity was analyzed in native gels, using two substrates for the enzymatic reaction, for the visualization of AO bands: indole-3-aldehyde and heptaldehyde. Enzymatic activity was compared in different stress conditions: salinity (100 mM NaCI), ammonium as single nitrogen source (2mM NH. + ), Cd treatments with different duration and intensity. As shown in figure 1, after 3 days of treatments with different stress factors, AO activity increased significantly in roots. The relative density of AO bands showed values of 528% in the case of treatment with ammonium, 460% after Cd treatment (100 11M) and 170% in saline condition, comparing to the control (100%).

After 7 days of treatments Significant stimulation were recorded in shoots (240% after ammonium treatment 519% after Cd treatment and 533 % in saline condition). Short term treatments (0- 48 h) with Cd (100 11M) showed a slight stimulation (20%) of AO activity of roots already after 6 hours (fig. 2). After 12 h of treatment the slight stimulation is maintained and after 24 h the stimulation was higher (50%). In shoots the intensification of AO band was showed after 12 h of Cd treatment

100 155 100 120 100 98 .- Relative density (%) EJ h

100 116 100 123 100 150 100 105 ~-< ..., ...... •, .... Cd . '-=-=--J (a) ,))f"~ Cd s R S R

100 131 100 73 ,----

Fig. 2. AO activity in Phragmites plants subjected to 100 JJM Cd for 0- 48 h, R=roots, S=shoots, the substrate was indole-3-aldehyde, each lane was loaded with extract containing 100 Ilg proteins for roots and 200 Ilg proteins for shoots

Using heptaldehyde as substrate in native gel, the effect of different Cd concentrations (0- 300 11M) on AO activity in Phragmites plants was analyzed after 3 and 7 days of treatments (fig. 3). Beside a major band, which intenSity is influenced by Cd treatments, 3 minor bands can be seen, if using heptaldehyde. After 3 days of treatment the major AO band intenSity in roots was doubled, as an effect of 100- 300 11M Cd. In shoots, AO activity was stimulated after 3 days of treatments, more by 100 11M Cd and less by 200 11M Cd. After 7 days, AO activity was stimulated by 200 11M Cd. Most obvious responses to Cd were recorded in roots, the stimulation being higher after 3 days. AO activity increased during all the stress conditions applied in our experiments: Cd treatments. salinity and ammonium nutrition. AO activity during stress is modulated at transcriptional and postranscriptional levels 16

(Sea and Koshiba, 2002). Using indole-3-aldehyde as substrate, one AO band is visible or. native gels loaded with Phragmites root and shoot crude proteic extracts (fig. 1 and 2). Using heptaldehyde. three minor bands with lower intensity were found beside one major band, influenced by stress (fig. 3). Data from literature have shown in different plant species 3- 4 AO bands (isoforms), with different mobility in native gel (Koshiba et aI., 1996, Sec and Koshiba, 2002, Sec et aI., 1998, Omarov et aI., 1999, Barabas at aI., 2000). AO activity is stimulated in saline stress and nutrition with ammonium, effects showed previously also on other plant species (barley, maize, pea) (Omarov et aI., 1998, Barabas et aI., 2000, Zdunek et ai., 2000). We found also stimulation of AO activity during Cd stress. The intensity of response did not correlate with the applied Cd concentration between 0- 300 IJM (fig. 3).

250 -- i - 200 +.. __ ._-

i.. 150 i- .;-= 100 !--- .. ..-- J 50 ;--. o ,_ .. = 3 7 days

... 160 • 140 - 120 i 100 ~ U 80 ~ u 60 .t.. 40 .I 20· U If o· 3 7 days

Fig. 3. AO activity of Phragmites plants subjected to different Cd concentrations for 3 and 7 days, A roots, B shoots, the substrate used was beptaldehyde, each lane was loaded with extract cotaining 100 flg proteins for roots and 200 flg proteins for shoots

Highest activity (5x of control) was observed in roots, after 3 days of stress (fig. 1). Before the third day of experiment the stimulation of AO was slight (20-50 %) (fig. 2). After 7 days of treatments, stimulation of AO activity was recorded also in shoots (fig. 1). In some experiments high increase of AO activity in shoots was noticed already after 3 days of experiment (fig, 3). The use of heptaldehyde as substrate for enzymatic reaction confirmed the results obtained with indole-3-aldehyde, on plants subjected to stress; but heptaldehyde is a better substrate for visualization of AO isoforms in the case of Phragmites plants. Studies concerning aldehyde oxidase activity and thus synthesis of abscisic acid in different plant species subjected to stress conditions highlighted the involvement of AO and ABA in adaptation processes, their intensification being an early event of adaptation to stress (Omarov et aI., 1998, Barabas et aI., 2000, Leung 17

that limited survival of plants in stress is and Giraudat, 1998). Aguilar and coworkers (2000) have shown showed the involvement of ABA (and related to their low content of ABA. Moya and his coworkers (1995) stress. They reported that addition of ABA also giberelic acid) in the response of rice plants to heavy metal plants. let to transitory recovery of net photosynthesis rate in the case of Cd-treated was recorded also in Phragmites rcots During stress, in parallel with AO activation, high ABA synthesis were found in roots, showing that the main (Fediuc et al., 2000). Significant changes of AO and ABA level is transported via xylem in shoots location of ABA synthesis is in roots, from where the phytohormone are in agreement with the findings of (Omarov et aI., 1999, 1998, Barabas et aI., 2000). Our results to heavy metal treatments were Talanova et al. (2000), who reported that cucumber plants responses low Cd concentration led to increase of accompanied by accumulation of ABA. Pretreatment of plants with of Cd. ABA, which enabled plants to tolerate the subsequent high concentration contributes to their ability to tolerate The increase of AO activity in Phragmites plants during stress suboptimal' conditions, a feature which make them useful in phytoremediation.

The efficiency of Phragmites and Typha in removal of Cd for the remediation of eutrophic lakes, Wetland plant species as Phragmites and Typha are useful tools were subjected to long-term (56/140 waste waters and contaminated soils. Plants in these experiments to the metal and their ability to days) Cd treatments (0-100 jJM) in order to estimate their resistance time and metal concentration. Interspecific accumulate and transport it. Our results showed dependence on severe stress Typha showed higher differences were found between Typha and Phragmites. During of the toxic metal to shoots are also resistance. Higher efficiency in Cd removal and in translocation observed in Typha, which uses plants for the removal of Phytoremediation- "the green technology" - is a new field of life science, (Gleba 1999, Salt et aI., 1998). Plants pollutants from the environment and their change into harmless forms to their metal removing capacity: possess many physiological, biochemical and genetic features contributing or biochemical transformation of the ability of take up, transfer and store metal ions, the decomposition soils and waters. Phytoremediation offers xenobiotics, which make them ideal agents for bioremediation of all over the world, polluted by human an efficient way to recover large extensions of soils and waters alternative to remediation employing activities. This technology is a cost-effective and ecological sound physical and chemical methods (Chaney et aI., 1997, Meagher, 2000). and mineralized in plants, elemental Apart from the organic pollutants, which can be completely decomposed their remediation constitutes a special pollutants are immutable by biological, physical processes and and radionuclides (As, Cd, Cs, Cr, Pb, problem (Meagher, 2000). This category includes toxic heavy metal et aI., 1998). Cadmium is an important Hg., U) and represents a major target for phytoremediation (Salt or near smeHers and sewage sludge areas. environmental pollutant, present in areas with heavy road traffiC, heavily polluted areas a content of 1-6 ppm Cd is present also as impurities in phosphate fertilizers. In some Cd was reported (Haag-Kerwer et aI., 1999). Trin. ex Steud. and cattail Typha latifolia L. Wetland species like common reed Phragmites australis (Cav.) waste waters and contaminated soils are supposed to be useful for the cleaning of eutrophic lakes, work is a comparative study of Typha (McNaughton et aI., 1974, Taylor and Crowder, 1984). The present Cd and their efficiency in the removal and Phragmites plants, concerning their ability to uptake and transfer of toxic ions in hydroponic experimental models. applied treatment (fig. 1). Quantities of mg The accumUlation of Cd depends on time and the severity of the shoots during severe cadmium treatments order were accumulated in roots in all experiments (fig 1) and in mild stress (0.1-1 jJM Cd) (fig. 1). Typha (10-100 jJM Cd), while jJg amounts were recorded in shoots during However after 14 and 56 days the accumulates in time higher amounts of Cd in both roots and shoots. Typha and Phragmites rcots subjected to determination of Cd content indicated small differences between species, The highest value of accumulation 100 jJM Cd, the pattems of accumUlation are different in the two of treatment and maintained with small in Typha roots was 12±0.6 mg Cdl g OW, aChieved after 20 days of Cd in Typha plants continued changes till 75 days of experiment Further increases in the accumulation rate in shoot was 1.01mg Cd( g DW in through the 140 days of the experiment The maximum accumulated Generally, in Typha the high rate of Typha, achieved gradually by the day 75 of the experiment. and maintained during the experiment, accumulation is achieved in short time after the start of treatments lower and changeable in time. while in Phragmites the accumulated amount of Cd is significantly content of plants considered 100%) showed The percents of Cd transferred to the shoots (from the total Cd to Phragmites. In Typha the percent of much higher ability of translocation in the case of Typha comparing value was 25.3% during the long-term Cd transported to shoot was gradually increased in time; the highest in Typha shoots showeo much lower treatment of plants with 10 jJM Cd. During mild stress the translocation subjected to severe stress the highest value percents, without increasing with the time factor. In Phragmites 18

101M Cd. Later the translocation is diminished. was 3.6% of Cd transferred after 28 days of treatment with 100 Phragmites plants led also \0 higher Cd Long-term treatments with low Cd concentration (0.1 101M) of translocation percent to shoot (4.3%).

----~--. - shoots

6,, ;;: c 5 " CD 4 : e 3' ;; 0 2' ~ 1 0 0 14 20 28 56 75 time (day)

roots .'.~.

3000 ,.~ t ,. J 1 ! ;: 2500 1 c 2000 J C> ;; 1500 ~O to) 1000 - -0-0.1 C> __ 1 500 ~ a ..~:;; ~~S;;::G:;~i:::~ -e-O ' a 14 20 28 56 75 -0-0.1 , -"-1 time (days) ) 1- __ _ I Fig. 1. Cadmium accumulation in time during mild Cd stress in Tvoha (circles) and Phraamites(sauares) olants I

as plants subjected to severe stress (100 Typha proved to be more resistant to Cd comparing to Phragmites, only 56 days, in the cond,tions of our 101M Cd) survived 140 days, while in the case of Phragmites the highest Cd concentration after the days experiments. Leaves dried and the roots browned completely at indicated. the average Cd amount taken up by plants The efficiency of Cd removal was expressed as a ratio between of data highlights that the efficiency of and the concentration applied (loIg Cd g>1 OW /IoIM Cd). The analysis both species. The efficiency is in positive removal decreases as the applied concentration increases, in of Phragmites. In Typha, during mild stress correlation with the time factor in all the treatments in the case of severe stress (10-100 101M Cd) (01-1 101M Cd) efficiency showed small changes in time, while in the case in the case of Typha plants (maximum the efficiency increased with time. Much higher efficiency was found to Phragmites (800-1000 IoIg Cd g-' values ranging around 2000-30001ol9 Cd g>1 OW/101M Cd) comparing OW/101M Cd). Cd treatments in order to estimate their Phragmites and Typha plants were subjected to long-term translocate it. Our results represent total resistance to the metal and also their ability to accumulate and absorption of the metal. For efficient cadmium content of plants, resulted by both adsorption and (Meagher, 2000» In most plant species phytoremedition both absorbed and adsorbed fractions are important is translocated in shoots (Trivedi and Erdei, the main part of Cd is retained in roots and only a small percent aI., 1998). Our results support these 1992, Rauser and Meuwly, 1995, Lozano-Rodriguez et aI., 1997, Ye et according to our measurements are data (fig. 1, 2, 3). Accumulation and translocation of Cd in Phragmites, 19 in agreement with data published by other research teams, showing high ability of retaining the metal in roots (Ye at aI., 1997, Lakatos et aI., 1999). Typha follows also this tendency, but accumulates higher Cd amounts and transports higher quantity to shoots comparing to Phragmites (fig. 1. 2 and 3). The reason of Cd accumulation in roots is the binding of the heavy metal in phytochelatin complexes in cytosol and especially in vacuoles (Rauser, 2000, Cakmak et al. 2000). Literature data showed that 75-88% of Cd in most of the plants is retained in this form in roots. Roots, immersed in nutrient solution, are in immediate contact with Cd, while shoots accumulate the metal taken up by the roots and transported through the xylem (Hart et aI., 1998). This explains the differences in Cd content of roots and shoots and subsequently the differences in their responses to stress .: Fediuc and Lips, submitted, Fediuc and Erdei, 2002, Fediuc et aI., 2000). Interspecific differences between Typha and Phragmites have been showed previously. conceming their ability of take up and translocate Pb and Mb (Kufel, 1991). Different plant species as well as different genotypes of the same species manifested variability io Cd and other metals uptake and transport (Cakmak et al. 2000). One basic required characteristic of plants used in phytoremediation is their tolerance to stress as growth inhibition in contaminated environments leads to low cleaning up efficiency (Zhu et al.. 1999). Our previous results showed that both species possess protective mechanisms that enable them to resist stress conditions (Fediuc et aI., 2000, Fediuc and Erdei, 2002). Naturally occurring Cd concentrations in polluted areas (0.1-10 !-1M) are tolerated by both Typha and Phragmites during long-term experiments. DUring very severe stress Typha showed a higher tolerance. Typha also showed higher Cd removal efficiency. Concerning the usefulness of Phragmites and Typha in phytoremediation we can suggest that naturally occurring plants can be used in rhizofiltration (technique which use roots for the removal of contaminants from aqueous enVironment), while for phytoextraction (which requires the transport and accumulation of contaminants in harvestable organs, usually shoots, Chaney et aI., 1997, Chardonens et aI., 1999) Typha seems to be more feasible. Our results and suggestions are in agreement with those reported by Dinka (1986), based on Cd bioaccumulation in the same plants. Hyperaccumulators are able to accumUlate higher Cd amounts in shoots. The efficiency of removal using Typha and Phragmites might be increased by genetiC engineering for achieving higher accumulation and better translocation to shoot. These methods are more and more known and developed in our days (Gleba et aI., 1999, Francova et al.. 2001).

General conclusions of the present stage of research: .:. Key compounds in stress defense mechanisms such as cysteine synthase (OASTL), glutathione and total free thiol pool responded to increased levels of heavy metals in the nutrient solution of Typha and Phragmites plants subjected to stress conditions . •:. OASTL activity varied with time and the following heavy metal concentrations in the nutrient solution: Cd (2.5-100 !-1M), NaCI (100 mM) and NH: (2 mM) as the only nitrogen source . •:. AO activity correlated well with OASTL under stress conditions. They were regulated by the same factors (salinity, ammonium ions, heavy metals).

Short term plans To summarize the results of experiments undergoing at this time and start preparing a comprehensive final report for the project. Complete training of Kazakh researchers to enable them to continue work on phytoremediation after the end of the AID/CDR/CAR project Long term plans Prepare material for the formulation of a large scale project for phytoremediation of heavy metals in contaminated soils and water resources of Kazakhstan, seeking funding from national and intemational funding agencies. Reclamation of contaminated areas in a larger scale for their eventual retum to agricultural production, creating a sustainable system of environmentally safe agricutture for farmers affected by unemployment and poverty. C. Scientific Impact of Collaboration Dr. Vladimir Kuzovlev from the Kazak laboratory has been working in Sede Boqer from August 1999 to 2001. During this period he has leamed a number of biochemical techniques and has become proficient in the use of HPLC and other equipment. Dr. Zerekbay Alikulov spent several long periods in the Sede Boqer laboratories preparing in collaboration with the Israeli investigators most of the experimental work to be carried out in Astana, the initial results of which are shown here. Dr. Galiya A. Shalekhmetova will arrive the Sede Boqer lab from the beginning of 2003 for one year to integrate the Israeli team on physiological and biochemical aspects of heavy metal removal from contaminated soils and water. 20 i" D. Description of Project Impact We have been determining ways to enhance the HM accumulation capacity of plant species. During the second and third year of the program, field and pot experiments to test the regulation of HM removal from soils by selected plant species will be carried out. The project impact on HM removal from Kazak soils will be assessed only then. However, we can already see that the capacity (practical and scientific) to achieve more effective technologies will be well developed with the Kazak group. E. Strengthening of Developing Country Institutions Dr. Zerekbay Alikulov has been recently appointed as Head of the Plant Physiology Department of the State University of Astana, the new capital of Kazakhstan. The present project will allow him to initiate the introduction of modern equipment and will contribute to the development of new scientific skills to solve many of the numerous and crop production problems produced by unfavourable environmental conditions. The principal investigators will also assist their Kazak collaborators in the preparation of large scale projects in cooperation with local and national governments. F. Future Work Summarize the project, prepare Final Report to AID, prepare data obtained for publication and seek funding for large scale phytoremediation operations in Kazakhstan's polluted areas .. Extend work in Kazakhstan in cooperation with UNDP and other agencies funding environmental work in Central Asia.

Section II

A) Managerial Issues None B) Budget None that we know of C) Special Concerns None D) Collaboration, Travel, Training and Publications During the year 2002 Zerekbay Alikulov came for a few months to Sede Boqer. During the same year, the Israeli scientists will travel to Kazakhstan. Preliminary material for publications is in preparation although further data is in preparation. E) Request for American Embassy Tel Aviv or A.T.D. Actions None