Proc. Natl. Acad. Sci.

USA
Vol. 96, pp. 59735977, May 1999
Colloquium Paper

This paper was presented at the National Academy of Sciences colloquium Plants and Population: Is There Time?
held December 56, 1998, at the Arnold and Mabel Beckman Center in Irvine, CA.

Use of plant roots for phytoremediation and molecular farming

DOLORESSA GLEBA*, NIKOLAI V. BORISJUK*, LUDMYLA G. BORISJUK*, RALF K NEER*, A LEXANDER POULEV*,
MARINA SKARZHINSKAYA*, SLAVIK DUSHENKOV, SITHES L OGENDRA*, YURI Y. GLEBA, AND ILYA RASKIN*
*Biotech Center, Foran Hall, Cook College, Rutgers University, 59 Dudley Road, New Brunswick, NJ 08901-8520; Phytotech, Inc., 1 Deer Park Drive, Suite I,
Monmouth Junction, NJ 08852; and Institute of Cell Biology and Genetic Engineering, Zabolotnogo Street, 148, Kiev, DSP-22, 252650, Ukraine

Chelate-assisted phytoextraction (1) has been successfully

used to remove lead from contaminated soils using specially
selected varieties of Indian mustard (Brassica juncea L.). These
varieties combine high shoot biomass with the enhanced ability
of roots to adsorb EDTA-chelated lead from soil solution and
transport it into the shoots. The transpiration stream is likely
to be the main carrier of soluble chelated metal to the shoots,
where water is transpired while metal accumulates (5). Chelate-assisted phytoextraction was also successfully used to
phytoextract uranium (7).
One strategy for increasing the efficiency of phytoextraction
is to increase metal translocation to the shoot by increasing
plant transpiration. Earlier research showed that wind enhances metal flux to the shoots, while compounds that block
transpiration (i.e., abscisic acid) block metal accumulation in
the shoots (8). Spontaneous or chemically induced mutants
with increased stomatal transpiration were isolated from various plant species, including tomato (9), Arabidopsis (10), and
barley (11). To determine whether genetically increased transpiration would increase the efficiency of phytoextraction,
(M1) seeds of B. juncea were mutagenized with ethyl methanesulfonate (EMS), and mature plants were self-pollinated to
obtain M2 seeds.
Ten- to fourteen-day-old M2 seedlings were screened by
excising a middle leaf from each plant, laying it flat in a
well-aerated room, and visually assessing the degree of tissue
dehydration after 1 or 2 hours. Plants whose leaves wilted (lost
water) faster than others were saved and rescreened later in
hydroponics and in soil for increased transpiration to confirm
the results of the initial screen. After screening 20,000 M2
seedlings, 47 plants with significantly increased leaf transpiration rates were identified. Line M-30, in which the transpiration rate exceeded that of the wild-type plants by 130% in
soil and by 75% in hydroponics, was tested for its phytoextraction performance in lead-contaminated soil amended with
2.5 mmol of EDTA per kg of soil. This high-transpiration line
phytoextracted 104% more lead than the wild-type B. juncea,
making it a good candidate for field optimization and use.
Increased resistance to metal is another important trait that
can improve the efficiency of phytoextraction. Varieties of B.
juncea with greater metal tolerance should grow better in
metal-contaminated sites and survive longer after metal uptake is induced by chelate application to the soil. Substantial
research has been directed toward isolating genes that are
involved in metal biology, e.g., metallothioneins or transporters. Interestingly, some increases in cadmium tolerance were
observed in transgenic plants overexpressing the human metallothionein-II gene (12).

ABSTRACT
Alternative agriculture, which expands the
uses of plants well beyond food and fiber, is beginning to
change plant biology. Two plant-based biotechnologies were
recently developed that take advantage of the ability of plant
roots to absorb or secrete various substances. They are (i)
phytoextraction, the use of plants to remove pollutants from
the environment and (ii) rhizosecretion, a subset of molecular
farming, designed to produce and secrete valuable natural
products and recombinant proteins from roots. Here we
discuss recent advances in these technologies and assess their
potential in soil remediation, drug discovery, and molecular
farming.
Biotechnology is transforming world agriculture, adding new
traits to crop plants at a greatly accelerated rate. Plants are
becoming more efficient producers of food, fiber, medicines,
and construction materials. In addition to these conventional
uses, biotechnology opens doors to unique uses of plants that
are gaining greater acceptance from the public and attention
from the scientific community. These so-called value-added
uses include phytoremediation, the use of plants to remove
pollutants from the environment or to render them harmless
(1), and molecular farming (phytomanufacturing), the use of
plants for the production of valuable organic molecules and
recombinant proteins (2, 3). Because of the growing number
of commercially successful applications and the lack of serious
environmental concerns, both technologies are gaining acceptance from the scientific community, the general public, and
regulators.
With the exception of root crops, plant roots are less utilized
and studied than shoots. However, this situation may be
changing because of the emerging biotechnologies described
below that exploit the ability of plants to transport valuable
molecules into and out of their roots. These root-based technologies include metal phytoextraction, a subset of phytoremediation, which uses plants to remove toxic heavy metals
from soil; and rhizosecretion, a subset of molecular farming,
which relies on the ability of plant roots to exude valuable
compounds. Both technologies exploit plants innate biological
mechanisms for human benefit.
Phytoextraction. Giant underground networks formed by
the roots of living plants function as solar-driven pumps that
extract and concentrate essential elements and compounds
from soil and water. Absorbed substances are used to support
reproductive function and carbon fixation within shoots. Metal
phytoextraction relies on metal-accumulating plants to transport and concentrate polluting metals, such as lead, uranium,
and cadmium, from the soil into the harvestable aboveground
shoots (1, 4, 5). Hydroponically grown plant roots can also
directly absorb, precipitate, and concentrate toxic metals from
polluted effluents in a process termed rhizofiltration (6).

D.G., N.V.B., L.G.B., R.K., A.P., and M.S. contributed equally to this

work.

To whom reprint requests should be addressed. e-mail: raskin@

aesop.rutgers.edu.

PNAS is available online at www.pnas.org.

5973

5974

Colloquium Paper: Gleba et al.

Valuable metal-resistance traits can be found in metal

hyperaccumulating plants that are endemic to soils naturally
enriched with heavy metals. These plants can accumulate
exceedingly high amounts of essential and nonessential heavy
metals in their foliage, to levels that are highly toxic to most
other plants (13). For example, several Thlaspi species can
accumulate Ni and Zn, to 15% of its dry biomass. This is an
order of magnitude greater than concentrations of these
metals in the nonaccumulating plants growing nearby. The
prevention of herbivory and disease is thought to be the main
function of this unique phenomenon (14, 15). It recently has
been established that the ability of T. goesingense Halacsy to
hyperaccumulate metals is the result of high resistance to the
metals rather than the greater rates of metal uptake (16).
Unfortunately, most hyperaccumulating species are not suitable for phytoextraction for several reasons: (i) metals that are
primarily accumulated (Ni, Zn, and Cu) are not among the
most important environmental pollutants; (ii) most have very
low biomass and capricious growth habits unsuitable for
monoculture; and (iii) agronomic practices and crop protection measures for their cultivation have not been developed.
However, many metal-hyperaccumulating species belong to
Brassicaceae (mustard) family, and thus are related to B.
juncea, the preferred plant for phytoextraction of lead. Unfortunately, B. juncea, while exhibiting a high capacity for
metal uptake and translocation, is not very resistant to high
levels of lead or other heavy metals in its foliage. Therefore,
chelate-assisted phytoextraction is very toxic to B. juncea,
requiring harvesting several days after chelate application.
Unfortunately, no genes conferring metal resistance were
identified in any of the hyperaccumulating species, precluding
the possibility of direct gene transfer. Thus, an attempt was
made to introduce metal resistant traits into the high-biomass
Pb accumulator B. juncea using somatic hybridization. Thlaspi
caerulescens, a known Ni and Zn hyperaccumulator, was
selected as one of the parents for both symmetric and asymmetric hybrids in which T. caerulescens protoplasts were irradiated with x rays before fusion. Eighteen hybrids were regenerated, all showing a phenotype intermediate between those of
the parents. Two asymmetric hybrids were found to be fertile.
One of these hybrids (60/31) had vigorous growth, characteristic of B. juncea, and contained Thlaspi-specific repetitive
DNA sequences, as demonstrated by Southern hybridization.
(As expected, total DNA from B. juncea parent did not
hybridize with Thlaspi-specific probes). Hybrid 60/31 displayed
dramatically increased resistance when germinated and grown
in Pb-, Ni-, and Zn-contaminated soil (Fig. 1). The amount of
Pb that the hybrid was able to phytoextract on a dry weight
basis was similar to that of both parents. However, the total
amount of Pb phytoextracted by each hybrid plant was much
greater because of the greater biomass produced on the
contaminated soil. Interestingly, the growth habits and bio-

FIG. 1. Asymmetric somatic hybrid 60/31 (B) and its parents

Brassica juncea (A) and Thlaspi caerulescens (C) growing in soil
containing 800 mg/kg lead, 328 mg/kg nickel, and 7,600 mg/kg zinc.

Proc. Natl. Acad. Sci. USA 96 (1999)

mass of B. juncea and the 60/31 hybrid did not differ much
when the plants were grown in noncontaminated fertile soil
(data not shown).
Rhizosecretion. Phytoextraction exploits the ability of plant
roots to remove unwanted contaminants from their environment. But could the reverse of this process also be exploited?
Could roots make valuable compounds and deliver them into
their environment? At present, most of the recombinant
proteins or valuable natural products used as fine chemicals,
pharmaceuticals, crop protection compounds, cosmetic ingredients, etc. are extracted from plants by using solvents. This
method requires expensive purification of the active ingredients from complex mixtures of organic molecules and proteins,
making downstream processing and purification of individual
components difficult and costly. Extracting plants is also a
batch process whereby the plant is harvested, and its continual ability to synthesize chemicals is not utilized. Natural
rubber and maple syrup are rare examples of continuous
manufacturing processes, which produce much larger amounts
of valuable plant product over the lifetime of the plant.
Rhizosecretion of Natural Products. In addition to accumulating biologically active chemicals, plant roots continuously produce and secrete compounds into their immediate
environment (rhizosphere). While up to 10% of photosynthetically fixed carbon is secreted from the roots (17, 18), the
systematic study of chemical composition of root exudates
from diverse plant species has not been undertaken. Not
surprisingly, few compounds that were identified in root
exudates were shown to play an important role in several
biological processes. For example, isoflavonoids and flavonoids present in the root exudates of a variety of legume
plants activate the Rhizobium genes responsible for the nodulation process (19, 20) and, possibly, for vesiculararbuscular
mycorrhiza (VAM) colonization (21). Strigol, a germination
stimulant for the parasitic plant Striga asiatica, has been found
in the root exudates of many cereals (22). A variety of plants
produce herbicidal allelochemicals that may inhibit growth and
germination of neighboring plants (2325). In addition, rootsecreted compounds called phytosiderophores may be involved in the acquisition of essential plant nutrients from soils
(2628) and in defense against toxic metals such as aluminum
(29).
Intuition and limited published data (30) suggest that rootsecreted compounds should have a wide spectrum of biological
activities including protection against biotic and abiotic
stresses. Survival of delicate and physically unprotected root
cells may depend on their continuous underground chemical
warfare against a hostile and constantly changing environment teeming with bacteria and fungi preying on any organic
material in soil. The unexplored chemical diversity of root
exudates is an obvious place to search for novel biologically
active compounds including antimicrobials. Our biochemical
analysis of root exudates from 120 plant species can be
summarized as follows: (i) each plant species studied exuded
a distinct set of compounds, which is a unique biochemical
fingerprint for a given species (Fig. 2 AC); (ii) root exudates
are relatively simple mixtures, in comparison to solvent extracts of plant tissue, making the isolation of the active
molecules an easier task; (iii) root exudates are devoid of
pigments and tannins, known to interfere in activity screens,
and do not contain large quantities of biologically inert structural compounds; and (iv) the chemical composition of root
exudates is very different from that of conventional methanolic
extracts of root tissue.
We have also observed that exudate chemical diversity can
be greatly increased by the elicitation process, which is known
to alter secondary metabolism in plants exposed to various
physical and chemical treatments. Phytoalexins, antimicrobial
compounds produced in plants and tissue cultures in response
to disease causing agents or their chemical components, are

Colloquium Paper: Gleba et al.

FIG. 2. HPLC profiles of nonelicited root exudates of three plant

species collected in distilled water (AC) and root exudates of Brassica
juncea collected in distilled water (D) or in distilled water supplemented with 1 mM AgNO3 (E) or 500 mM H2O2 (F) as elicitors. Plants
were grown hydroponically with roots suspended in aerated nutrient
solution. Root exudates from 4- to 6-week-old plants were collected for
24 hours in 400 ml of distilled water with or without elicitors. Root
exudates were concentrated by freeze-drying, and exudate compounds
were separated on a Waters NovaPak C-18 reverse phase column using
acetic acid/acetonitrile gradient.

probably the best studied elicited defense compounds in plants

(31). Unfortunately, little is known about elicited compounds
in root exudates, with the exception of a recent report on
isoflavonoid exudation from the roots of white lupine (30). We
observed that chemical or physical elicitors stimulate roots of
various plants to exude an array of compounds not detected in
the nonelicited exudates (Fig. 2 DF). On the other hand,
the same elicitor will trigger the production of different
compounds in different plant species. In addition, elicitation
may dramatically increase the quantities of certain compounds
in the exudates. It can be hypothesized that elicitors mimic the
effects of stresses on the hydroponically grown roots, activating
biochemical defense systems and resulting in quantitative and
qualitative changes in the composition of the exudates.
To demonstrate the presence of antimicrobial compounds in
root exudates, a screening protocol was designed in which 10
ml of concentrated exudate solution was transferred into a
small cavity in agar poured into 24-well microtiter plates. The
tested microorganisms were plated in each well before the
cavity was made. Exudates from 480 species, each treated with
24 elicitors, were tested in this system for the inhibition of
growth of selected bacteria and fungi (Fig. 3). The following
percentage of exudates showed moderate to strong activity
against tested microorganisms: Escherichia coli (3.4%), Staphylococcus aureus (4.3%), Pseudomonas aeruginosa (0.4%),
Penicillium notatum (0.8%), and Saccharomyces cerevisiae
(0.6%).
In addition to exudates, hydroponically cultivated plant
roots also provide a unique source of biologically active
compounds. We have also observed that elicitation, both
quantitatively and qualitatively, alters the HPLC profiles of
secondary metabolites in roots of many plant species (data not
shown). Most likely, these changes are subsequently reflected
by the dramatic alterations in the rhizosecreted compounds.
Why Root Exudates? The above observations suggest that
root exudates represent a new and functionally enriched source

Proc. Natl. Acad. Sci. USA 96 (1999)

5975

FIG. 3. Antimicrobial activity of root exudates. The exudates

showing activity (indicated with red arrows) against Staphylococcus
aureus ssp. aureus (A) were from Tagetes minuta (column 1, Asteraceae) and Eriastrum densiflorum var. austromontana (column 6,
Polemoniaceae) and activity against Saccharomyces cerevisiae (B) were
from Hosta fortunea (column 6, Liliaceae). To test antibacterial/
antifungal activity of exudates, the suspension of target microorganisms or spores was plated and spread on the surface of standard LB
agar (bacteria) or potato dextrose agar (fungi) poured into 24-well
microplates. Twenty microliters of exudate dissolved in water was
pipeted into a central hole punched in the agar. The antimicrobial
activity, visible as an area of growth inhibition (clearing) around the
central hole was scored after 24 hours of incubating inoculated plates
at 30C.

of biologically active compounds. Elicitation of hydroponically

grown roots adds another unexplored dimension to the chemical diversity normally hidden in silent parts of the plant
genomes. In addition to shedding light on dark corners of plant
biology, the systematic study of root exudates may be valuable
to the global pharmaceutical industry, which still heavily relies
on novel sources of chemical diversity to discover new drugs in
an ever-accelerating race against time. Twenty five percent of
all prescriptions dispensed from pharmacies in the United
States contain active ingredients extracted from higher plants
(32). However, methods of harvesting chemical diversity of
plant-derived compounds often follows huntergatherer strategies. Extracts of plant material haphazardly collected in
various places around the world are eventually acquired by
pharmaceutical companies, which put them through sophisticated high-throughput screens that use an increasing array of
molecular targets. This primitive prospecting process does not
provide a reliable and reproducible source of natural products
that can be easily resupplied after a novel activity is found. The
mismatch between the beginning of the drug development
pipeline and what follows creates an opportunity for developing new pharmaceutical agents from plants using more standardized, scientific approaches that favor biologically active

5976

Colloquium Paper: Gleba et al.

Proc. Natl. Acad. Sci. USA 96 (1999)

FIG. 4. Rhizosecretion of jellyfish green fluorescent protein (GFP)(A), human placental alkaline phosphatase (SEAP)(B), and bacterial
(Clostridium thermocellum) xylanase (C) from the roots of transgenic Nicotiana tabacum L. (A) To direct GFP into the secretory pathway,
GFP-coding sequence was fused to the signal peptide derived from the resident ER protein calreticulin, and the resulting fusion placed in correct
orientation between the mannopine synthase (mas29) promoter (provided by Stanton Gelvin, Purdue University, West Lafayette, IN) and nos
terminator. GFP rhizosecretion from the hydroponically cultivated aseptic roots was visualized after illuminating the hydroponic medium contacting
roots with near-UV light. Media from nontransformed plants showed no fluorescence (data not shown). (B) Visualization of SEAP rhizosecretion
in the native gel. In transformed tobacco, coding sequence of SEAP with its own signal peptide was controlled by the cauliflower mosaic virus 35S
promoter (CaMV35S). Thirty micrograms of total protein concentrated from root exudates of transgenic and nontransformed plants was separated
on native PAGE, and SEAP activity was localized using the alkaline phosphatase isoenzymes procedure (Sigma). Lanes 1 and 2, transgenic tobacco
plants; lanes 3 and 4, nontransformed tobacco. (C) Rhizosecretion of bacterial xylanase from transgenic tobacco seedlings germinated on the
RBB-xylane-containing agar medium (dark blue), which becomes colorless when cleaved by xylanase (photographed upside down). Nontransformed
plants did not change the color of the medium (data not shown). Seeds of tobacco expressing a truncated C. thermocellum xylanase gene controlled
by the CaMV35S promoter and targeted to the apoplast by proteinase inhibitor II ER signal peptide were provided by Uwe Sonnewald.

molecules over structural components and major metabolites.

Tissue culture-based production of natural products, often
combined with elicitation, is one of the recently developed
strategies for increasing the size of the needle in the haystack. However, plant tissue cultures are expensive, slow
growing, and relatively deficient of secondary metabolites,
presumably because of their nondifferentiated nature. Rhizosecretion, on the other hand, may produce a more costeffective and diverse source of chemical compound mixtures
for the identification of novel biologically active compounds.
In addition, rhizosecretion, a nondestructive and continuous
process, may provide a constant supply of these compounds
over the lifetime of a plant.
Rhizosecretion of Recombinant Proteins. The ease of transformation and cultivation make plants suitable for manufacturing many recombinant proteins. Indeed, numerous heterologous (recombinant) proteins have been produced in plant
leaves, fruits, roots, tubers, and seeds (3335), and are targeted
to different subcellular compartments, such as the cytoplasm,
endoplasmic reticulum (ER), or apoplastic space (36). Plants
are capable of carrying out acetylation, phosphorylation, and
glycosylation as well as other posttranslational protein modifications required for the biological activity of many eukaryotic
proteins. However, the extraction and purification of proteins
from biochemically complex plant tissues is a laborious and
expensive process that presents a major obstacle to large-scale
protein manufacturing in plants. In attempts to overcome this
problem, secretion-based systems utilizing transgenic plant
cells or plant organs aseptically cultivated in vitro have been
investigated (3739). However, these in vitro systems, which
include hairy roots, may be expensive, slow-growing, unstable,
and relatively low-yielding. Until now, these disadvantages
precluded the use of in vitro plant systems for the commercial
manufacturing of recombinant proteins.
Can rhizosecretion be used for the continuous manufacturing of recombinant proteins? The nondestructive rhizosecretion process may provide high yields of recombinant proteins
over the lifetime of a plant and facilitate their downstream
purification, combining the advantages of the whole plant and
in vitro protein expression systems. Indeed, roots of living
plants are known to secrete proteins. For example, large
amounts of acid phosphatase are released from the roots of
many plants during phosphate deficiency (40). We attempted
to rhizosecrete the following three heterologous proteins of
different origins from Nicotiana tabacum L.; green fluorescent

protein (GFP) of the jellyfish Aequorea victoria, human placental secreted alkaline phosphatase (SEAP), and xylanase
from the thermophylic bacterium Clostridium thermocellum.
All three of these proteins were rhizosecreted from transgenic plants when their expression was controlled by a strong
root-expressed promoter and targeted by a secretory signal
peptide (Fig. 4). Daily rhizosecretion of GFP, released into
fresh medium unprotected from proteolysis, reached 2 mg/g
root dry weight, while SEAP rhizosecretion, quantified from
its activity, reached 20 mg/g root dry weight, a significant
amount considering that no attempts to optimize rhizosecretion had been made thus far. It is likely that methods for
increasing protein expression and secretion will be developed
along with plant varieties optimized for the rhizosecretion of
recombinant proteins.
Data suggest that plant roots can continuously produce and
secrete biologically active recombinant proteins of different
origins. The rhizosecretion system offers a simplified method
for the isolation of recombinant proteins from simple hydroponic medium rather than from complex plant extracts. As
with rhizosecretion of natural products, protein rhizosecretion
can be operated continuously without destroying the plant,
thus producing a higher total yield of the recombinant protein
over the life of the transgenic plant. In addition, recombinant
biopharmaceutical proteins purified from root exudates are
less likely to be contaminated with pathogenic viruses that may
be present in the milk or urine of transgenic animals. Rhizosecretion also borrows from many well developed and tested
methods of commercial hydroponic plant cultivation, and
therefore, will be relatively easy to scale up.

CONCLUSIONS
While the evolution of plant shoots followed primarily introverted paths by perfecting physical barriers between themselves and the environment, roots had to be more extroverted in their relationship with soil. This requirement created a
unique set of biological mechanisms, which until recently, were
understudied and underutilized. Phytoextraction and rhizosecretion are starting to change this, while allowing scientists to
take a radically new look at the darkest corners of plant
biology. These technologies also open the doors to the valueadded, nonagricultural uses of plants, which will continue to
expand in the new century.

Colloquium Paper: Gleba et al.

Neither phytoextraction nor rhizosecretion will directly contribute to feeding world population in the next century.
However, these technologies will improve the quality of life for
many people if their development continues. The future
challenge for metal phytoextraction is to further reduce the
cost and increase the spectrum of metals amenable to this
technology. This goal can be achieved by creating superior
plant varieties for phytoextraction by using genetic engineering
to introduce valuable traits into plants, developing better
agronomic protocols for their cultivation, and designing safer
and more effective soil amendments. A recent, and probably
the only, example of the successful use of genetic engineering
applied to metal phytoremediation is the use of bacterial
mercuric reductase (merA) gene to achieve mercuric ion
reduction in transgenic Arabidopsis (41) and yellow poplar
plants (42). Elemental mercury produced in transgenic plants
is much less toxic than ionic mercury and can be volatilized
from transgenic plants in a process termed phytovolatilization,
which is related to phytoextraction.
The future challenge for rhizosecretion lies in the successful
development of effective and safe pharmaceuticals from the
collection of biologically active lead molecules secreted by the
roots, and in large-scale, cost-effective manufacturing of recombinant proteins. The aging population and ever-growing
demand for better pharmaceuticals should foster the use green
plants as sources of new drug discovery, biotransformation,
and in some cases, manufacturing. Thus, more effective utilization of immense biosynthetic capacity of plants based on
their inexpensive and renewable nature will present major
opportunities for plant researchers in the next century.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.

Salt, D. E., Smith, R. D. & Raskin, I. (1998) Annu. Rev. Plant

Physiol. Plant Mol. Biol. 49, 643668.
Nawrath, C., Poirier, Y. & Somerville, C. (1995) Mol. Breeding 1,
105122.
Franken, E., Teuschel, U. & Hain, R. (1997) Curr. Opin. Biotechnol. 8, 411416.
Kumar, P. B. A. N., Dushenkov, V., Motto, H. & Raskin, I. (1995)
Environ. Sci. Technol. 29, 12321238.
Vassil, A. D., Kapulnik, Y., Raskin, I. & Salt, D. E. (1998) Plant.
Physiol. 117, 447453.
Dushenkov, V., Kumar, P. B. A. N., Motto, H. & Raskin, I. (1995)
Env. Sci. Technol. 29, 12391245.
Huang, J. W., Blaylock, M. J., Kapulnik, Y. & Ensley, B. D. (1998)
Environ. Sci. Technol. 32, 20042008.
Salt, D. E., Prince, R. C. Pickering, I. J. & Raskin, I. (1995) Plant
Physiol. 109, 14271433.
Tal, M. (1966) Plant Physiol. 41, 13871391.
Koornneef, M., Reuling, G. & Karssen, C. M. (1984) Physiol.
Plant. 61, 377383.
Raskin, I. & Ladyman, J. A. R. (1988) Planta 173, 7378.
Misra, S. & Gedamu, L. (1989) Theor. Appl. Genet. 78, 161168.

Proc. Natl. Acad. Sci. USA 96 (1999)

13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.

5977

Baker, A. J. M. & Brooks, R. R. (1989) Biorecovery 1, 81126.

Boyd, R. S. & Martens, S. N. (1994) Oikos 70, 2125.
Boyd, R. S., Shaw J. J. & Martens, S. N. (1994) Am. J. Bot. 81,
294300.
Kramer, U., Smith, R. D., Wenzel, W. W., Raskin, I. & Salt, D. E.
(1997) Plant Physiol. 115, 16411650.
Johansson, G. (1992) Soil Biol. Biochem. 24, 427433.
Shepherd, T. & Davies, H. V. (1993) Ann. Bot. 72, 155163.
Peters, N. K. & Long, S. R. (1988) Plant Physiol. 88, 396400.
Maxwell, C. A. & Phillips, D. A. (1990) Plant Physiol. 93,
15521558.
Tsai, S. M. & Phillips, D. A. (1991) Appl. Environ. Microbiol. 57,
14851488.
Siame, B. A., Weerasuriya, Y., Wood, K., Ejeta, G. & Butler,
L. G. J. (1993) Agric. Food Chem. 41, 14861491.
Friebe, A., Schulz, M., Kuck, P. & Schnabel, H. (1995) Phytochemistry 38, 11571159.
Yu, J. Q. & Matsui, Y. J. (1994) Chem. Ecol. 20, 2131.
Inoue, M., Nishimura, H., Li, H. H. & Mizutani, J. (1992)
J. Chem. Ecol. 18, 18331840.
Miyasaka, S. C., Buta, J. G., Howell, R. K. & Foy, C. D. (1991)
Plant Physiol. 96, 737743.
Lipton, D. S. Blanchar, R. W. & Blevins, D. G. (1987) Plant
Physiol. 85, 315317.
Mori, S., Nishizawa, N., Kawai, S., Sato, Y. & Takagi, S. J. (1987)
Plant Nutr. 10, 10031011.
de la Fuente, J. B. M., Ramirez-Rodriguez, V., Cabrera-Ponce,
J. B. L. & Herrera- Estrella, L. (1997) Science 276, 15661568.
Gagnon, H. & Ibrahim, R. K. (1997) Phytochemistry 44, 1463
1467.
Dixon, R. A. (1986) Biol. Rev. 61, 239291.
Farnsworth, N. R. & Morris, R W. (1976) Am. J. Pharm. 148,
4652.
McGarvey, P. B., Hammond, J., Dienelt, M. M., Hooper, D. C.,
Fu, Z. F., Dietzschold, B., Koprowski, H. & Michaels, F. H.
(1995) Bio/Technology 14841487.
Van Engelen, F. A., Schouten, A., Molthoff, J. W., Roosien, J.,
Salinas, J., Dirkse, W. G., Schots, A., Bakker, J., Gommers, F. J.,
Jongsma, M. A., et al. (1994) Plant Mol. Biol. 26, 17011710.
Sonnewald, U., Hajirezaei, M-R., Kossmann, J., Heyer, A.,
Trethewey, R. N. & Willmitzer, L. (1997) Nat. Biotechnol. 15,
794797.
Conrad, U. & Fiedler, U. (1998) Plant. Mol. Biol. 38, 101109.
Li, J., Hegeman, E., Hanlon, R. W., Lacy, G. H., Cenbow, D. M.
& Grabau, E. A. (1997) Plant Physiol. 114, 11031111.
Firek, S., Draper, J., Owen, M. R. L., Gandecha, A., Cockburn,
B. & Whitelam, G. C. (1993) Plant Mol. Biol. 23, 861870.
Wongsamuth, R. & Doran, P. M. (1997) Biotechnol. Bioeng. 54,
401415.
Li, M. & Tadano, T. (1996) Soil Sci. Plant Nutr. 42, 753763.
Rugh, C. L., Wilde, H. D., Stack, N. M., Thompson, D. M.,
Summers, A. O. & Meagher, R. B. (1996) Proc. Natl. Acad. Sci.
USA 93, 31823187.
Rugh, C. L, Senecoff, J. F., Meagher, R. B. & Merkle, S. A. (1998)
Nat. Biotechnol. 16, 925928.