Chaney, R.L, M. Malik, Y.M. Li, S.L. Brown, E.P. Brewer, J.S. Angle and A.J.M. Baker. 1997. Phytoremediation of soil metals. Current Opinions in Biotechnology 8:279‑284.

Phytoremediation of soil metals.

Rufus L. Chaney*†, Minnie Malik‡, Yin M. Li*, Sally L. Brown*, Eric P. Brewer‡, J. Scott Angle‡ and Alan J.M. Baker§.

*Environmental Chemistry Lab, US Department of Agriculture-Agricultural Research Service, Bldg. 007, BARC-West, Beltsville, MD  20705;
†rchaney@asrr.arsusda.gov
‡Dept. Natural Resources and Landscape Architecture, University of Maryland, College Park, MD  20742.
§Dept. Animal and Plant Sciences, University of Sheffield, Sheffield, S1O 2UO, United Kingdom.

Phytoremediation of metal contaminated soils offers a lower cost method for soil remediation and some extracted metals may be recycled for value.  Both the phytoextraction of metals and phytovolatilization of Se or Hg by plants offer great promise of commercial development.  Natural metal hyperaccumulator phenotype is much more important than high yield ability when using plants to remove metals from contaminated soils.  The hypertolerance of metals is the key plant characteristic required for hyperaccumulation; vacuolar compartmentalization appears to be the source of hypertolerance of natural hyperaccumulator plants.  Alternatively, soil Pb and Cr6+ may be inactivated in the soil by plants and soil amendments (phytostabilization).  Little molecular understanding of plant activities critical to phytoremediation has been achieved, but recent progress in characterizing Fe, Cd, and Zn uptake by Arabidopsis and yeast mutants indicates strategies for developing transgenic improved phytoremediation cultivars for commercial use.

Because the costs of growing a crop are minimal compared to those of soil removal and replacement, the use of plants to remediate hazardous soils is seen as having great promise; other recent reviews on many aspects of soil metal phytoremediation are available [1,2**,3*,4**,5*,6*,7**].  Phytoremediation is the use of plants to make soil contaminants non-toxic and is often also referred to as bioremediation, botanical-bioremediation, and  Green Remediation.  The idea of using rare plants which hyperaccumulate metals to selectively remove and recycle excessive soil metals was introduced in 1983 [8], gained public exposure in 1990 [9], and has increasingly been examined as a potential practical and more cost effective technology than soil replacement, solidification, or washing strategies presently used [2**,3*,7**].  Categories of phytoremediation include phytoextraction (the use of plants to remove contaminants from soils), phytovolatilization (the use of plants to make volatile chemical species of soil elements), rhizofiltration (the use of plant roots to remove contaminants from flowing water) and phytostabilization (the use of plants to transform soil metals to less toxic forms, but not remove the metal from the soil).  The use of plants and associated rhizosphere organisms or bioengineered plants to metabolize toxic organic compounds also appears promising (recently reviewed by Cunningham et al. [10**]).

Phytostabilization appears to have strong promise for two toxic elements, chromium and lead.  Reduction of Cr6+, which poses an environmental risk, to Cr3+ which is highly insoluble and not demonstrated to pose an environmental risk [11]), by deep rooted plants can be very effective.  Chemical species of Pb in soil are usually somewhat bioavailable if the soil is ingested by children, livestock, or wildlife [12], while a Pb phosphate mineral, chloropyromorphite is both extremely insoluble and non-bioavailable [13,14**,15,16,17**] but it is formed slowly apparently because the reactants have low solubility.  Roots of Agrostis capillaris growing in highly contaminated Pb/Zn mine wastes caused the formation of pyromorphite from soil Pb and phosphate, but the mechanism remains unknown [17**].  Although it was believed that Thlaspi rotundifolium hyperaccumulated Pb, Zea mays accumulated higher Pb levels in controlled tests if soil pH and P were low [18*].  Addition of chelating agents (e.g. HEDTA, EDTA) to such soils increased Pb solubility and mobility within plants; shoot Pb reached 1%, allowing removal of enough Pb to encourage further evaluation of this approach [18*,19**].  Methods to prevent leaching of Pb-chelates down the soil profile would be required to permit such additions in the field in regions where net infiltration occurs.  Inactivating soil Pb by use of soil amendments and revegetation to prevent erosion is increasingly seen as the promising soil Pb remediation technology [12,20].

Different views of the potential for use of phytoremediation to clean up contaminated soils have developed among researchers.  Some have examined the naturally occurring metal hyperaccumulators, plants which can accumulate 10--500 times higher levels of elements than crops; Reeves [21] suggested a widely accepted definition of Ni-hyperaccumulators:  'a plant in which a nickel concentration of at least 1000 Fg g-1 has been recorded in the dry matter of any above-ground tissue in at least one specimen growing in its natural habitat.'  This definition can be adapted to other elements.  Most plant species suffer significant yield reduction when shoots reach 50-100 mg Ni kg-1 dry weight while Ni-hyperaccumulators tolerate at least 10-20 times the normal maximum tolerable levels; and among the smaller group of plants which can tolerate at least 1% Ni in shoots, a few can reach 5% Ni, or 500 times the shoot Ni tolerated by crop plants.  Species which accumulate over 1% Ni have been called 'hypernickelophores' by Jaffré [22].  This term seems appropriate for the plant species which accumulate over 1% of several elements (hypernickelophore, hyperzincophore, etc.) because this ability is qualitatively different than the hyperaccumulators as defined by Reeves [21].  Crop plants tolerate higher shoot Zn and Mn levels than Ni (about 300-500 mg Zn kg-1), so 'hyperaccumulators' contain > 1% shoot Zn or Mn [7].  Shoot Cd levels are usually <1 mg kg-1, so 'hyperaccumulators' must accumulate and tolerate $ 100 mg Cd kg-1; some hypertolerate > 1% Cd [23].

How do hyperaccumulators achieve this remarkable bio-accumulation of soil metals?  Research has identified several characteristics that are important: 

1) The plant must be able to tolerate high levels of the element in root and shoot cells; hypertolerance is the key property which makes hyperaccumulation possible.  Such hypertolerance is believed to result from vacuolar compartmentalization and chelation [24,25**].  The most direct demonstration used isolated vacuoles from protoplasts of tobacco cells which had accumulated high levels of Cd and Zn [24].  Whether hypertolerance in the known hyperaccumulators is due to an enhancement of these mechanisms is not yet known.  However, electron microprobe analysis [26] supports vacuolar compartmentation for Zn in leaves of the hyperaccumulator Thlaspi caerulescens.

2) A plant must have the ability to translocate an element from roots to shoots at high rates.  Normally root Zn, Cd or Ni concentrations are 10 or more times higher than shoot concentrations, but in hyperaccumulators, shoot metal concentrations can exceed root levels [27,28**,29**].  Krämer et al. [29**] recently found that although the chemical forms of Ni found in extracts of leaves of Alyssum hyperaccumulators are the chelates with malate and citrate, in the xylem exudate histidine chelates about 40% of the total Ni present; nearly all of the histidine in exudate is chelated with Ni.  Whether Ni(histidine)2, Ni2+ or a mixed chelate such as Ni(histidine, malate) is pumped into the xylem by a membrane transporter remains unknown.  Additions of histidine to nutrient solution increased Ni tolerance and transport to shoots by Alyssum montanum, a nonhyperaccumulator species.

3)  There must be a rapid uptake rate for the element at levels which occur in soil solution.  Here quite different patterns have been observed in different groups of hyperaccumulators.  Brown et al. [27] found that T. caerulescens accumulated Zn and Cd from nutrient solution only about as well as tomato and Silene vulgaris did, but tomato was severely injured at 30 FM Zn, S. vulgaris at 320 FM Zn, and T. caerulescens only at 10,000 FM Zn.  Because this species can keep tolerating and accumulating Zn and Cd at high soil solution levels, it is found in nature with 1--4% Zn while surrounding plants are << 0.05% Zn (Zn excluders).  Further, studies have shown that Zn hypertolerant genotypes of T. caerulescens require much higher solution Zn2+ (104-fold) and leaf Zn concentrations (100-300 mg kg-1 vs. 10-12 mg kg-1 in normal plants) to grow normally than do related non hyperaccumulator species [28**].  By implication, the highly effective compartmentalization to reduce the toxicity of Zn and Cd appears to require the plant to accumulate much more Zn to have adequate supply.  In contrast, the Ni-hyperaccumulator Alyssum species accumulate remarkably higher shoot Ni levels compared to other species grown at the same Ni2+ activity in solution [29**,30].  And the Se‑hyperaccumulating species similarly accumulate higher shoot Se levels and many can volatilize Se at high rates growing beside plants with more normal levels and slow volatilization [31,32].

What evolutionary advantage does metal hyperaccumulation give these species?  Boyd et al. [33,34] have demonstrated that high (but not low) Ni levels in leaves of hyperaccumulators can reduce herbivory by chewing insects and reduce the incidence of bacterial and fungal diseases.  Similar results were found for Zn in Thlaspi caerulescens (A.J. Pollard and A.J.M. Baker, unpublished data).

For effective development of phytoremediation, each element must be considered separately because of its unique soil and plant chemistry.  Both agronomic management practices and plant genetic abilities need to be optimized to develop commercially useful practices.  Some elements can be accumulated by plant roots and converted to a volatile species such as dimethylselenide [32] or Hg0 [35**].  Although many plants can volatilize dimethylselenide (or dimethyldiselenide in the case of the Se hyperaccumulators) [36], co-contaminating sulfate and salinity in Se contaminated soils commonly inhibit this process) [36,37]; very high B or salinity can kill most plants.  So growing species in normal crop rotations which can phytovolatilize soil Se, or accumulate Se into the forage biomass for sale as a Se supplement for livestock feeds are alternative approaches to treating irrigation drainage waters which are much higher in B and sulfate than the water used for irrigation [36,38].

Whether metal hyperaccumulation in shoots or high shoot biomass is more important in phytoremediation of soil metals has been debated [2**,3*,7**]. A quantitative example may provide clarity:  presume that a high-biomass crop plant is grown on a contaminated soil and the soil pH is lowered to increase Zn uptake to attain Zn phytotoxicity with 50% yield reduction; Zea mays and Brassica juncea are examples of such annual crops.  Under favorable conditions, these plants can reach 20 t dry biomass ha-1.  In the case of the usual Zn and Cd co‑contamination at 100 mg Zn:1 mg Cd, crop plants suffer significant yield reduction when the shoots have about 500 mg Zn kg-1 at harvest.  Because Cd is not 100-times more toxic than Zn, soil Zn phytotoxicity is the factor controlling plant yield.  At 50% yield reduction (10 t ha-1), dry biomass contains 500 mg kg-1 (500 g Zn t-1); one removes only 5 kg of Zn ha-1 year-1.  T. caerulescens, which can remove both soil Zn and Cd, has a low yield compared to the above species, but can hypertolerate up to 25,000 mg Zn kg-1 (25 kg t-1) [39] without yield reduction.  Even with a low yield of 5 t ha-1 at the point of incipient yield reduction, Zn removal would be 125 kg ha-1.  We conclude that the ability to hyperaccumulate and hypertolerate the metals to be phytoremediated is of greater importance than high biomass.  Some authors have suggested that yield of a crop would be two orders of magnitude higher than that for hyperaccumulators such as T. caerulescens, but pot and field studies show that such perennial species grown as a crop can attain as high as 5 t ha-1 before breeding to increase the combination of yield and shoot metal concentration [27,28**].  Further, recycling of shoot metals in commerce may provide value for the ash from metal hyperaccumulators such that there is need to pay for safe disposal.  Continuing the above model, biomass ash contains 20-40% Zn for T. caerulescens, but only 0.5% for Zea mays; the former is a rich ore, while the latter is a phytotoxic waste requiring disposal.  Increasing the yield of a crop could give a linear increase in phytoremediation capacity with increasing yield.  But increasing from 'normal' tolerance to 'hypertolerance' and hyperaccumulation increases the potential annual removal of the soil contaminant 25--400 fold.  Even for elements which have little value in the biomass, the higher the concentration, the less expensive the disposal of the phytoremediation crop residue or ash (e.g., 137Cs; As; U) will be.  Thus, we have emphasized the importance of domestication of metal hyperaccumulator plants and breeding of improved cultivars [7**,28**], the characterization of mechanisms used by hyperaccumulators to accumulate, translocate, and hypertolerate metals, and, eventually, the cloning and use of these genes to convert high biomass agronomic plants into special phytoremediation cultivars if this is required for some elements (7**).

Remediation of other elements (e.g. As, Cu, Cs, Sr, U) from soils by hyperaccumulator crops has not been demonstrated, but is expected to be possible if creative research is applied [2**,3*,7**,35**].  In some cases, the phytoremediation of an element may require soil amendments such as chelating agents because soil or plant chemistry reduces element uptake or translocation to shoots [18*,19**].

Use of biotechnology to improve phytoremediation:
Biotechnology approaches to develop phytoremediation plants have been examined.  Traditional plant breeding can only use available genetic diversity within a species to combine the characteristics needed for successful phytoremediation.  Researchers expected that increasing the concentrations of metal binding proteins or peptides in plant cells would increase metal binding capacity and tolerance.  Although plant cell cultures expressing mammalian metallothioneins (MTs) [40] or phytochelatins (PCs) [41**] are more tolerant of acute Cd toxicity, the transfer of mammalian metallothionein genes to higher plants appear to provide no benefit for phytoremediation.  Further, when natural metal hypertolerant plants were examined, the concentration of PCs showed no difference, suggesting that hypertolerance to Cd and Zn in these plants were not due to the hyperaccumulation of PC peptides [42,43].  The evidence for the role of PCs is that their presence does correlate with normal levels of metal tolerance, since mutations that abolished PC production in Arabidopsis and fission yeast resulted in hypersensitivity to Cd [41**,44*,45*].  Cd-sensitive (hypotolerant) single gene mutants cad1 [44*] and cad2 [45*] of Arabidopsis thaliana have been identified and studied (blocked in glutathione synthesis or PC synthesis).  For a plant species with normal tolerance (A. thaliana), PCs were essential for the normal level of tolerance.  

Interestingly, when these researchers tested genotypes without and with effective phytochelatin biosynthesis, the outcome was a surprise in that the sensitive mutants (low PCs) had a significantly lower degree of transport of Cd to shoots than the wildtype [45*].  A similar result was observed in corn inbreds which differed substantially in shoot Cd; higher levels of PCs were associated with higher shoot Cd [46].

Although these studies have allowed cloning of genes involved in acute Cd tolerance, and characterization or confirmation of metabolic pathways, the environmental relevance of findings from such acute Cd exposure has not been established.  An alternative view of Cd-catalyzed PC biosynthesis is that chelation of PCs with Cd alleviates the feedback inhibition of the PC‑synthase; as long as Cd activity in the cytoplasm is high, an enzyme supports more transfer to form more PCs and longer PCs.  Because the level of Zn present in nearly all environments is 100 times higher than that of Cd, if an acutely toxic Cd dose is provided, the plants would be killed by Zn.  Even the formation of the sulfide-stabilized high molecular weight Cd-PC complex in vacuoles [4**,25**,41**] may result from the acutely toxic Cd supply without Zn.  Further, the finding that the hmt1 vacuolar membrane pump protein (which restored Cd hypertolerance to mutant fission yeast) transported both Cd-PCs and PCs without Cd, raises questions about how the pump works to induce Cd hypertolerance in vivo.  Cd phytotoxicity in soil is a recent anthropogenic effect, whereas Zn phytotoxicity and co-accumulation of trace levels of Cd which are normal biogeochemical phenomena.  We believe that scientists should place less emphasis on 'Cd hypertolerance' in plants.  It seems increasingly likely that the Cd hypertolerance mechanisms are incidental biochemical phenomena.  Although Cd-PCs can be found at low levels in plants in the environment, they account for only a small fraction of the tissue Cd [47,48,49*].

Another goal of developing transgenic plants with increased metal binding capacity was to use these metal-binding factors to keep Cd in plant roots, thus reducing Cd movement to the food chain or into tobacco [50,51,52,53].  Vacuolar compartmentation of Cd only in roots may reduce Cd translocation to shoots; expression in plants of the hmt1 vacuolar pump for Cd-PCs from fission yeast [25**] has not yet been successful, and modification of gene sequences may be required before its effectiveness can be tested (similar to the mercury reductase gene sequence changes [35**]).  The expression of MT as the whole protein, the Cd binding '"-domain' part of the protein, or a fusion protein with $-glucuronidase, under several promoters [50,51,52,53] increased Cd tolerance of tobacco and other plants, but had little effect on Cd transport to shoots.  Recently use of the improved 35S2 promoter may have increased the ability of MT to keep Cd in roots [53]; tests have not yet progressed to soil studies which must be the important measure of success.  Many of the studies noted here have used acutely toxic levels of Cd, such that the study results do not model metal-contaminated soils in the environment.  Rauser and Meuwly [49] used non toxic levels of Cd (3 FM, 30-times the level generally found in soil solution) to study PC physiology in Zea mays, and found that in the short term PCs bound only a small fraction of cell Cd, but, over time, over 90% of root Cd was bound to PCs.  McKenna and Chaney [54] used chelator-buffered [55**] Cd to grow lettuce at Cd levels relevant to food chain safety and found no evidence of Cd-PCs in lettuce leaves.

Possible use of 'metallophores' to aid phytoextraction of soil metals
Because Poaceae species secrete mugineic acid family phytosiderophores (chelating agents) to solubilize soil Fe, and accumulate the intact chelate into root cells [56**], Raskin [5*] suggested that transgenic plants could be developed to secrete metal selective ligands into the rhizosphere which could specifically solubilize elements of phytoremediation interest.  Although this approach holds promise, phytosiderophores obtain their specificity not by chelation specifically only of Fe in soils, but from their uptake of nearly only Fe-phytosiderophores by a membrane carrier [55**,56**,57*].  Finding other simple biosynthetic molecules with selective chelation ability, that plants can make and secrete into the rhizosphere at adequate concentrations, and simultaneously creating a selective transport protein for the metal chelate seems difficult, but worth examination to develop unique phytoremediation tools.  Regulatory control of phytosiderophore secretion in barley was induced by Fe-deficiency, but not Mn, Zn, or Cu deficiency [58*] in contrast with other reports which indicated that Zn deficiency also induced the biosynthesis and secretion of phytosiderophores.

Lastly, extensive progress has recently been achieved in identifying genes and proteins involved in uptake of Fe by yeast and plants [59**,60**,61*,62**]; high affinity Zn [63] and Cu membrane transporters have also been found in yeast.  A fundamental understanding of both uptake and translocation processes in normal plants and metal hyperaccumulators, regulatory control of these activities, and the use of tissue specific promoters offers great promise that use of molecular biology tools can give scientists the ability to develop effective and economic phytoremediation plants for soil metals.

Conclusion
Extensive progress has been made in characterizing soil chemistry management needed for phytoremediation, and physiology of plants which hyperaccumulate and hypertolerate metals.  It is increasingly clear that hypertolerance is fundamental to hyperaccumulation, and high rates of uptake and translocation are observed in hyperaccumulator plants.  Fundamental characterization of mechanisms, and cloning of genes required for phytoremediation has begun with the mercuric ion reductase [35**], and hmt1 [25] expression in higher plants is expected soon.  Improved hyperaccumulator plants and agronomic technology to improve the annual rate of phytoextraction and to allow recycling of soil toxic metals accumulated in plant biomass is very likely to support commercial environmental remediation which society can afford in contrast with present practices.  Although most phytoremediation systems are still in development, or in plant breeding to improve the cultivars for field use, application for Se phytovolatilization has already begun.  Many opportunities have been identified for research and development to improve the efficiency of phytoremediation.  Progress had been hindered by limited funds for research and development for 12 years since the first report of the model for phytoremediation [8].  New commercial firms are moving into this field and phytoremediation technologies will be increasingly applied commercially in the near term.

References and recommended reading:
Papers of particular interest, published within the annual period of review, have been highlighted as:

*		of special interest
**	of outstanding interest

1.	Raskin I, Kumar PBAN, Dushenkov S, Salt DE: Bioconcentration of heavy metals by plants. Curr Opin Biotech 1994, 5:285‑290.

2.	Salt DE, Blaylock M, Kumar PBAN, Dushenkov S, Ensley BD, Chet I,
**	Raskin I: Phytoremediation: A novel strategy for the removal of toxic metals from the environment using plants. Bio/Tech 1996, 13:468-474.
A thorough review with opinions about approaches for commercially useful phytoremediation. The authors stress the importance of higher biomass plants for use in phytoremediation, and the biochemistry of metal uptake and hypertolerance.

3.	Cunningham SD, Berti WR, Huang JW: Phytoremediation of
*	contaminated soils. Trends Biotechnol 1995, 13:393‑397.

4.	Cunningham SD, Ow DW: Promises and prospects for
**	phytoremediation. Plant Physiol 1996, 110:715‑719.
A review of phytoremediation from the perspective of plant biochemists and molecular biologists. It considers fruitful areas of research to understand better the fundamental processes in metal tolerance and accumulation by higher plants.

5.	Raskin I: Plant genetic engineering may help with environmental 
*	cleanup (Commentary). Proc Natl Acad Sci USA 1996, 93:3164‑3166.
A commentary published in same issue as Rugh et al. paper [34] on transgenic Arabidopsis expressing Hg reductase. Discusses the application of phytoremediation and presently discussed approaches to development of phytoremediation cultivars using novel biotechnology methods.

6.	Moffat A: Plants proving their worth in toxic metal cleanup. Science
*	1995, 269:302‑303.
A review of phytoremediation of metals which notes promising approaches to develop metal phytoremediation technologies.

7.	Chaney RL, Brown SL, Li YM, Angle JS, Homer FA, Green CE:
**	Potential use of metal hyperaccumulators. Mining Environmental Management 1995, 3(3):9-11.
This paper describes use of Zn and Cd hyperaccumulator plants in the remediation of contaminated soils, including strategies to make Zn and Cd remediation cultivars of T. caerulescens.

8.	Chaney, R.L. 1983. Plant uptake of inorganic waste constituents. pp 50‑76. In James F. Parr, Paul B. Marsh and Joanne M. Kla (eds.) Land Treatment of Hazardous Wastes. Noyes Data Corp., Park Ridge, NJ.

9.	Anonymous. 1990. NEA dumps on science art. Science 250:1515.

10.	Cunningham SD, Anderson TA, Schwab AP, Hsu FC: Phytoremediation
**	of soils contaminated with organic pollutants. Advances in Agronomy 1996, 56:55‑114.
A timely and thorough review of many aspects of the biodegradation of organics in soils by plants or rhizosphere microbes supported by plants.

11.	James BR: The challenge of remediating chromium-contaminated soils.
*	Environ. Sci. Technol. 1996, 30:248A-251A.
Research has shown that if chromate is reduced to chromic by chemical or biological methods, the inertness and insolubility of chromic oxides in soil will limit formation of chromate and limit environmental risk. Phytoremediation offers the ability to reduce chromate below the tilled soil layer which is not provided by identified soil amendments to reduce chromate.

12.	Chaney RL, Ryan JA: Risk Based Standards for Arsenic, Lead and Cadmium in Urban Soils. DECHEMA, Frankfurt. 1994: 1-130.

13.	Cotter-Howells JD, Champness PE, Charnock JM, Pattrick RAD:
Identification of pyromorphite in mine-waste contaminated soils by ATEM and EXAFS. Euro J Soil Sci 1994, 45:393‑402.

14.	Ma QY, Logan TJ, Traina SJ: Lead immobilization from aqueous
**	solutions and contaminated soils using phosphate rocks. Environ Sci Technol 1995, 29:1118‑1126.
This paper examines use of inexpensive phosphate rock as the phosphate amendment to inactivate soil Pb, the alternative to phytoremediation.

15.	Ruby MV, Davis A, Nicholson A: In situ formation of lead phosphates in soils as a method to immobilize lead. Environ Sci Technol 1994, 28:646‑654.

16.	Cotter-Howells J: Lead phosphate formation in soils. Environ Pollut 1996, 93:9‑16.

17.	Cotter-Howells JD, Caporn S: Remediation of contaminated land by
**	formation of heavy metal phosphates. Appl Geochem 1996, 11:335‑342.
This paper reports on the use of phosphate soil amendment to promote inactivation of soil Pb, and a brief report of finding pyromorphite in particles of rhizosphere soils which were not observed before growing a Pb/Zn-tolerant plant on the soil.

18.	Huang JW, Cunningham SD: Lead phytoextraction: Species variation in
*	 lead uptake and translocation. New Phytol 1996, 134:75‑84.
This paper shows the difficulty of phytoextraction of soil Pb using plants studied to date. Even species with unusual ability to accumulate soil Pb require low soil phosphate and low soil pH to facilitate soil Pb phytoavailability, but only reach a few hundred mg Pb per kg. The addition of chelating agents, however, could make soil Pb soluble and keep it from being precipitated by phosphate in roots.

19.	Blaylock MJ, Salt DE, Dushenkov S, Zakharova O, Gussman C,
**	Kapulnik Y, Ensley BD, Raskin I: Enhanced accumulation of Pb in Indian mustard by soil-applied chelating agents. Environ Sci Technol 1997, 31:860-865.
** Reports the effect of several chelating agents on uptake and translocation to shoots of 5 metals. Pb transfer to shoots was most increased by EDTA additions, and Cd in shoots by EGTA additions, showing that the chelation specificity of the agent added strongly affects which metals have increased uptake and transfer to shoots.

20.	Berti WR, Cunningham SD: In‑place inactivation of Pb in Pb-contaminated soils. Environ Sci Technol 1997, 31: In press (May).

21.	Reeves RD: The hyperaccumulation of nickel by serpentine plants. In The Vegetation of Ultramafic (Serpentine) Soils. Edited by Baker AJM, Proctor J, Reeves RD. Intercept Ltd., Andover, Hampshire, UK, 1992: 253-277.

22.	Jaffré T, Schmid M: Accumulation du nickel par une Rubiacée de Nouvelle Calédonia: Psychotria douarrei (G. Beauvisage) Däniker.  C. R. Acad. Sci. Paris, 1974, Ser. D-278:1727-1730.

23.	Baker AJM, Reeves RD, Hajar ASM: Heavy metal accumulation and tolerance in British populations of the metallophyte Thlaspi caerulescens J.&C. Presl (Brassicaceae). New Phytol 1994, 127:61‑68.

24.	Vogeli-Lange R, Wagner GJ: Subcellular localization of cadmium and cadmium-binding peptides in tobacco leaves: Implication of a transport function for cadmium binding peptides. Plant Physiol 1990, 92:1086-1093. 

25.	Ortiz DF, Ruscitti T, McCue KF, Ow DW: Transport of metal-binding
**	peptides by HMT1, a fission yeast ABC-type vacuolar membrane protein. J Biol Chem 1995, 270:4721‑4728.
This paper describes the expression of a metal hypertolerance gene as a vacuolar membrane pump for Cd-PCs. First unequivocal demonstration of pumping the Cd-PCs into a storage location by Cd hypertolerant cells.

26.	Vázquez MD, Poschenreider C, Barceló J, Baker AJM, Hatton P, Cope GH: Compartmentation of zinc in roots and leaves of the zinc hyperaccumulator Thlaspi caerulescens J&C Presl. Bot Acta 1994, 107:243‑250.

27.	Brown SL, Chaney RL, Angle JS, Baker AJM: Zinc and cadmium uptake of Thlaspi caerulescens grown in nutrient solution. Soil Sci Soc Am J 1995, 59:125‑133.

28.	Li, YM, Chaney RL, Angle JS, Chen KY, Kerschner BA, Baker AJM:
**	Genotypical differences in zinc and cadmium hyperaccumulation in Thlaspi caerulescens [abstract]. Agron Abstr 1996, 27.
The authors report the comparison of 20 diverse genotypes of Thlaspi caerulescens in high Zn and Cd nutrient solutions and field plots. Extensive genetic variation was found in Zn hypertolerance, Zn requirement, and Cd uptake relative to Zn. Such genetic diversity may support the breeding of improved cultivars, and determination of the inheritance of these different properties.

29.	Krämer U, Cotter-Howells JD, Charnock JM, Baker AJM, Smith JAC:
**	Free histidine as a metal chelator in plants that accumulate nickel. Nature 1996, 379:635‑638.
The first demonstration of a specific ligand which may be involved in the high translocation of a metal to shoots of Alyssum Ni-hyperaccumulator species. Further, addition of histidine to the nutrient solution increased tolerance and uptake of Ni by a nonhyperaccumulator species. Causality remains unsettled because only 40% of the xylem exudate Ni was chelated by histidine; this may have resulted from the long exudate collection period because it has been shown that the levels of ligands and nutrients in xylem exudate change by 1 hr after severing the stem.

30.	Baker AJM, Brooks RR: Terrestrial higher plants which hyperaccumulate metal elements  A review of their distribution, ecology, and phytochemistry. Biorecovery 1989, 1:81-126.

31.	Banuelos GS, Meek DW: Selenium uptake by different species in selenium enriched soils. J Environ Qual 1990, 19:772-777.

32.	Terry N, Carlson C, Raab TK, Zayed AM: Rates of selenium volatilization among crop species. J Environ Qual 1992, 21:341‑344.

33.	Boyd RS, Martens SN: Nickel hyperaccumulated by Thlaspi montanum var. montanum is acutely toxic to an insect herbivore. Oikos 1994, 70:21-25.

34.	Boyd RS, Shaw JJ, Martens SN: Nickel hyperaccumulation defends Streptanthus polygaloides (Brassicaceae) against pathogens. Am J Bot 1994, 81:294‑300.

35.	Rugh CL, Wilde HD, Stack NM, Thompson DM, Summers AO, Meagher
**	RB: Mercuric ion reduction and resistance in transgenic Arabidopsis thaliana plants expressing a modified bacterial merA gene. Proc Natl Acad Sci USA 1996, 93:3182‑3187.
This paper describes the first transgenic phytoremediation plant that achieves the removal of Hg from soils. The bacterial gene had to be modified to obtain expression in plants. This team is working to obtain a methyl-Hg hydrolase gene in the same way so that these plants can reduce the risk of methyl-Hg so much that emission of Hg0 vapor is environmentally acceptable.

36.	Terry N, Zayed AM: Selenium volatilization in plants. In Selenium in the Environment. Edited by Frankenberger Jr WT, Benson S. Marcel Dekker, New York; 1994:343-367.

37.	Bell PF, Parker DR, Page AL: Contrasting selenate-sulfate interactions in selenium-accumulating and nonaccumulating plant species. Soil Sci Soc Am J 1992, 56:1818‑1824.

38.	Banuelos GS, Cardon G, Mackey B, Ben-Asher J, Wu L, Beuselinck P, Akohoue S, Zambrzuski S: Boron and selenium removal in boron-laden soils by four sprinkler irrigated plant species. J Environ Qual 1993, 22:786-792.

39.	Brown SL, Chaney RL, Angle JS, Baker AJM: Phytoremediation potential of Thlaspi caerulescens and bladder campion for zinc- and cadmium-contaminated soil. J Environ Qual 1994, 23:1151‑1157.

40.	Robinson NJ, Tommey AM, Kuske C, Jackson PJ: Plant metallothioneins. Biochem J 1994, 295:1-10.

41.	Rauser WE: Phytochelatins and related peptides: Structure,
**	biosynthesis and function. Plant Physiol 1995, 109:1141‑1149.
An updated review on metal binding peptides by a leader in the field. In contrast with other research groups, Rauser used levels of Cd that were not acutely phytotoxic, and his view of the literature reflects a more physiologically or environmentally relevant evaluation of PCs.

42.	de Knecht JA, Koevoets PLM, Verkleij JAC, Ernst WHO: Evidence against a role for phytochelatins in naturally selected increased cadmium tolerance in Silene vulgaris (Moench) Garcke. New Phytol 1992, 122:681-688.

43.	Harmens H, Den Hartog PR, Ten Bookum WM, Verkleij JAC: Increased zinc tolerance in Silene vulgaris (Moench) Garcke is not due to increased production of phytochelatins. Plant Physiol 1993, 103:1305‑1309.

44.	Howden R, Goldsbrough PB, Andersen CR, Cobbett CS: Cadmium-
*	sensitive, cad1 mutants of Arabidopsis thaliana are phytochelatin deficient. Plant Physiol 1995, 107:1059‑1066.
The characterization of a Cd-sensitive mutant which was altered by the mutagenesis of a protein required for PC biosynthesis. It was also reported that Cd uptake and translocation by the mutants was much lower than the wild type, questioning the model whereby increased phytochelatins were assumed to both increase Cd tolerance and keep it from being translocated.

45.	Howden R, Andersen CR, Goldsbrough PB, Cobbett CS: A cadmium-
*	sensitive, glutathione-deficient mutant of Arabidopsis thaliana. Plant Physiol 1995, 107:1067‑1073.
The characterization of another mutant in PC synthesis.  In this case, addition of glutathione alleviated the PC deficiency.  Together, these two papers indicate that for mutants selected for sensitivity to Cd (hypotolerance), PCs are essential to that phenotype.

46.	Florijn PJ, de Knecht JA, van Beusichem ML: Phytochelatin concentrations and binding state of Cd in roots of maize genotypes differing in shoot/root Cd partitioning. J Plant Physiol 1993, 142:537‑542.

47.	Schat H, Kalff MMA: Are phytochelatins involved in differential metal tolerance or do they merely reflect metal-imposed strain? Plant Physiol. 1992, 99:1475‑1480.

48.	Ahner BA, Price NM, Morel FMM: Phytochelatin production by marine phytoplankton at low free metal ion concentrations: Laboratory studies and field data from Massachusetts Bay. Proc. Natl. Acad. Sci. USA, 1994, 91:8433-8436.

49.	Rauser WE, Meuwly P: Retention of cadmium in roots of maize
*	seedlings: Role of complexation by phytochelatins and related thiol peptides. Plant Physiol 1995, 109:195‑202.
This paper shows the complexity of PCs in relation to chronic nonphytotoxic Cd exposures. Three families of PCs are present in maize, and the length of the peptides is affected by the intensity and longevity of exposure. In the short term, PCs bound only a small part of the absorbed Cd, but after several days, PCs could bind all plant Cd. Did not include 100 times as much Zn as Cd, levels which would co-occur with Cd in the environment.

50.	Brandle JE, Labbe H, Hattori J, Miki BL: Field performance and heavy metal concentrations in transgenic flue-cured tobacco expressing a mammalian metallothionein-$-glucuronidase gene fusion. Genome 1993, 36:255-260.

51.	Pan A, Tie F, Duau Z, Yang M, Wang Z, Li L, Chen Z, Ru B: "-Domain of human metallothionein I-A can bind to metals in transgenic tobacco plants. Mol Gen Genet 1994, 242:666‑674.

52.	Yeargan R, Maiti IB, Nielsen MT, Hunt AG, Wagner GJ: Tissue partitioning of cadmium in transgenic tobacco seedlings and field grown plants expressing the mouse metallothionein I gene. Transgenic Res 1992, 1:261‑267.

53.	Elmayan T, Tepfer M: Synthesis of a bifunctional metallothionein/$-glucuronidase fusion protein in transgenic tobacco plants as a means of reducing leaf cadmium levels. Plant J 1994, 6:433‑440.

54.	McKenna IM, Chaney RL: Characterization of a cadmium-zinc complex
*	in lettuce leaves. Biol Trace Element Res 1995, 48:13‑29.
The authors grew lettuce with chelator buffering to provide controlled activity of Zn and Cd to the plants over time, and used an Fe-chelate which is stable with added Zn and Cd. Only the leaves were examined, and no evidence of PCs was found.

55.	Parker DR, Chaney RL, Norvell WA: Equilibrium computer models:
**	Applications to plant nutrition research. In Chemical Equilibrium and Reaction Models. Edited by Loeppert RH, Schwab AP, Goldberg S. Madison, WI; 1995,163‑200.
This paper reviews the problems of controlling metal phytoavailability in nutrient solutions due to the presence of chelated metals. Shows Fe-chelates that minimize artifacts in nutrient solutions, and discusses how to use chelator buffering to make the different metals in a test system independent.

56.	Ma JF, Nomoto K: Effective regulation of iron acquisition in
**	graminaceous plants. The role of mugineic acids as phytosiderophores. Physiol Plant 1996, 97:609‑617.
A review of phytosiderophores, the chelating amino acids secreted by grasses to obtain soil Fe, and possibly other elements. Details of the biosynthesis have been worked out, and some genes have been identified. Intact Fe-chelates with mugineic acid are transported into the root, thereby providing the Fe3+ specificity by uptake rather than chelation.

57. Yehuda Z, Shenker M, Römheld V, Marschner H, Hadar Y, Chen Y: The
*	role of ligand exchange in the uptake of iron from microbial siderophores by gramineous plants. Plant Physiol 1996, 112:1273‑1280.
This paper clarifies literature that is full of artifacts due to the exchange of Fe among different ligands in a nutrient solution. It shows that Fe added as microbial siderophores must be exchanged to phytosiderophores before uptake by grasses, rather than grasses being able to obtain Fe from microbial siderophores. These issues were debated for over 10 years before this clean demonstration of the actual mechanism used was found.

58.	Gries D, Brunn S, Crowley DE, Parker DR: Phytosiderophore release in
*	relation to micronutrient metal deficiencies in barley. Plant Soil 1995, 172:299:308.
This paper indicates that only Fe deficiency induces the secretion of phytosiderophores by barley, in contrast with other's observations that Zn deficiency might also induce biosynthesis and secretion of these ligands.

59.	Askwith CC, de Silva D, Kaplan J: Molecular biology of iron acquisition
**	in Saccharomyces cerevisiae. Molec Microbiol 1996, 20:27‑34.
A timely review of a rapidly progressing research area closely related to absorption of heavy metals by plants.

60.	Yamaguchi-Iwai Y, Stearman R, Dancis A, Klausner RD: Iron-regulated
**	DNA binding by the AFT1 protein controls the iron regulon in yeast. EMBO J 1996, 15:3377‑3384.
This paper describes the discovery of a family of genes, the expression of which is regulated by a protein in response to the Fe status of cells.

61.	Eide D, Broderius M, Fett J, Guerinot ML: A novel iron-regulated metal
*	transporter from plants identified by functional expression in yeast. Proc Natl Acad Sci USA 1996, 93:5624‑5628.
The authors describe the first gene involved in Fe uptake by dicots isolated for study.

62.	Yi Y, Guerinot, ML: Genetic evidence that induction of root Fe(III) chelate reductase activity is necessary for iron uptake under iron deficiency. Plant J 1996, 10:835‑844.

63.	Zhao H, Eide D: The yeast ZRT1 gene encodes the zinc transporter protein of a high affinity system induced by zinc limitation. Proc Natl Acad Sci USA 1996, 93:2455‑2458.