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Proc Natl Acad Sci U S A. 1999 May 25; 96(11): 5973–5977. | PMCID: PMC34214 |
Copyright © 1999, The National Academy of Sciences Use of plant roots for phytoremediation and molecular farming Doloressa Gleba,*† Nikolai V. Borisjuk,†* Ludmyla G. Borisjuk,*† Ralf Kneer,*† Alexander Poulev,*† Marina Skarzhinskaya,* Slavik Dushenkov,‡ Sithes Logendra,* 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 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).
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). 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 1–5% 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.
). 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
biomass 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 vesicular–arbuscular 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 ( 23– 25). In addition, root-secreted compounds called
phytosiderophores may be involved in the acquisition of essential plant
nutrients from soils ( 26– 28) and in defense against toxic metals such
as aluminum ( 29). Intuition and limited published data ( 30) suggest that root-secreted
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.
A– C); ( 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 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. D– F). 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 μl 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 2–4 elicitors, were tested in this system
for the inhibition of growth of selected bacteria and fungi (Fig.
). 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 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 hunter–gatherer 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 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 cost-effective 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 ( 33– 35), 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 ( 37– 39). 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.
). Daily rhizosecretion of GFP,
released into fresh medium unprotected from proteolysis, reached 2
μg/g root dry weight, while SEAP rhizosecretion, quantified from its
activity, reached 20 μg/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. 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 value-added, nonagricultural
uses of plants, which will continue to expand in the new century. 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
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