Proc. Natl. Acad. Sci. USAVol. 96, pp. 5973–5977, May 1999Colloquium Paper

This paper was presented at the National Academy of Sciences colloquium ‘‘Plants and Population: Is There Time?’’held December 5–6, 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 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

ABSTRACT Alternative agriculture, which expands theuses of plants well beyond food and fiber, is beginning tochange plant biology. Two plant-based biotechnologies wererecently developed that take advantage of the ability of plantroots to absorb or secrete various substances. They are (i)phytoextraction, the use of plants to remove pollutants fromthe environment and (ii) rhizosecretion, a subset of molecularfarming, designed to produce and secrete valuable naturalproducts and recombinant proteins from roots. Here wediscuss recent advances in these technologies and assess theirpotential in soil remediation, drug discovery, and molecularfarming.

Biotechnology is transforming world agriculture, adding newtraits to crop plants at a greatly accelerated rate. Plants arebecoming more efficient producers of food, fiber, medicines,and construction materials. In addition to these conventionaluses, biotechnology opens doors to unique uses of plants thatare gaining greater acceptance from the public and attentionfrom the scientific community. These so-called ‘‘value-added’’uses include phytoremediation, the use of plants to removepollutants from the environment or to render them harmless(1), and molecular farming (phytomanufacturing), the use ofplants for the production of valuable organic molecules andrecombinant proteins (2, 3). Because of the growing numberof commercially successful applications and the lack of seriousenvironmental concerns, both technologies are gaining accep-tance from the scientific community, the general public, andregulators.

With the exception of root crops, plant roots are less utilizedand studied than shoots. However, this situation may bechanging because of the emerging biotechnologies describedbelow that exploit the ability of plants to transport valuablemolecules into and out of their roots. These root-based tech-nologies include metal phytoextraction, a subset of phytore-mediation, which uses plants to remove toxic heavy metalsfrom soil; and rhizosecretion, a subset of molecular farming,which relies on the ability of plant roots to exude valuablecompounds. Both technologies exploit plants’ innate biologicalmechanisms for human benefit.

Phytoextraction. Giant underground networks formed bythe roots of living plants function as solar-driven pumps thatextract and concentrate essential elements and compoundsfrom soil and water. Absorbed substances are used to supportreproductive function and carbon fixation within shoots. Metalphytoextraction relies on metal-accumulating plants to trans-port and concentrate polluting metals, such as lead, uranium,and cadmium, from the soil into the harvestable abovegroundshoots (1, 4, 5). Hydroponically grown plant roots can alsodirectly absorb, precipitate, and concentrate toxic metals frompolluted effluents in a process termed rhizofiltration (6).

Chelate-assisted phytoextraction (1) has been successfullyused to remove lead from contaminated soils using speciallyselected varieties of Indian mustard (Brassica juncea L.). Thesevarieties combine high shoot biomass with the enhanced abilityof roots to adsorb EDTA-chelated lead from soil solution andtransport it into the shoots. The transpiration stream is likelyto be the main carrier of soluble chelated metal to the shoots,where water is transpired while metal accumulates (5). Che-late-assisted phytoextraction was also successfully used tophytoextract uranium (7).

One strategy for increasing the efficiency of phytoextractionis to increase metal translocation to the shoot by increasingplant transpiration. Earlier research showed that wind en-hances metal f lux to the shoots, while compounds that blocktranspiration (i.e., abscisic acid) block metal accumulation inthe shoots (8). Spontaneous or chemically induced mutantswith increased stomatal transpiration were isolated from var-ious plant species, including tomato (9), Arabidopsis (10), andbarley (11). To determine whether genetically increased tran-spiration would increase the efficiency of phytoextraction,(M1) seeds of B. juncea were mutagenized with ethyl meth-anesulfonate (EMS), and mature plants were self-pollinated toobtain M2 seeds.

Ten- to fourteen-day-old M2 seedlings were screened byexcising a middle leaf from each plant, laying it f lat in awell-aerated room, and visually assessing the degree of tissuedehydration after 1 or 2 hours. Plants whose leaves wilted (lostwater) faster than others were saved and rescreened later inhydroponics and in soil for increased transpiration to confirmthe results of the initial screen. After screening 20,000 M2seedlings, 47 plants with significantly increased leaf transpi-ration rates were identified. Line M-30, in which the transpi-ration rate exceeded that of the wild-type plants by 130% insoil and by 75% in hydroponics, was tested for its phytoex-traction performance in lead-contaminated soil amended with2.5 mmol of EDTA per kg of soil. This high-transpiration linephytoextracted 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 thatcan improve the efficiency of phytoextraction. Varieties of B.juncea with greater metal tolerance should grow better inmetal-contaminated sites and survive longer after metal up-take is induced by chelate application to the soil. Substantialresearch has been directed toward isolating genes that areinvolved in metal biology, e.g., metallothioneins or transport-ers. Interestingly, some increases in cadmium tolerance wereobserved in transgenic plants overexpressing the human me-tallothionein-II gene (12).

PNAS is available online at www.pnas.org.

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

¶To whom reprint requests should be addressed. e-mail: raskin@aesop.rutgers.edu.

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Valuable metal-resistance traits can be found in metalhyperaccumulating plants that are endemic to soils naturallyenriched with heavy metals. These plants can accumulateexceedingly high amounts of essential and nonessential heavymetals in their foliage, to levels that are highly toxic to mostother plants (13). For example, several Thlaspi species canaccumulate Ni and Zn, to 1–5% of its dry biomass. This is anorder of magnitude greater than concentrations of thesemetals in the nonaccumulating plants growing nearby. Theprevention of herbivory and disease is thought to be the mainfunction of this unique phenomenon (14, 15). It recently hasbeen established that the ability of T. goesingense Halacsy tohyperaccumulate metals is the result of high resistance to themetals rather than the greater rates of metal uptake (16).Unfortunately, most hyperaccumulating species are not suit-able for phytoextraction for several reasons: (i) metals that areprimarily accumulated (Ni, Zn, and Cu) are not among themost important environmental pollutants; (ii) most have verylow biomass and capricious growth habits unsuitable formonoculture; and (iii) agronomic practices and crop protec-tion measures for their cultivation have not been developed.However, many metal-hyperaccumulating species belong toBrassicaceae (mustard) family, and thus are related to B.juncea, the preferred plant for phytoextraction of lead. Un-fortunately, B. juncea, while exhibiting a high capacity formetal uptake and translocation, is not very resistant to highlevels 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 wereidentified in any of the hyperaccumulating species, precludingthe possibility of direct gene transfer. Thus, an attempt wasmade to introduce metal resistant traits into the high-biomassPb accumulator B. juncea using somatic hybridization. Thlaspicaerulescens, a known Ni and Zn hyperaccumulator, wasselected as one of the parents for both symmetric and asym-metric hybrids in which T. caerulescens protoplasts were irra-diated with x rays before fusion. Eighteen hybrids were regen-erated, all showing a phenotype intermediate between those ofthe parents. Two asymmetric hybrids were found to be fertile.One of these hybrids (60/31) had vigorous growth, character-istic of B. juncea, and contained Thlaspi-specific repetitiveDNA sequences, as demonstrated by Southern hybridization.(As expected, total DNA from B. juncea parent did nothybridize with Thlaspi-specific probes). Hybrid 60/31 displayeddramatically increased resistance when germinated and grownin Pb-, Ni-, and Zn-contaminated soil (Fig. 1). The amount ofPb that the hybrid was able to phytoextract on a dry weightbasis was similar to that of both parents. However, the totalamount of Pb phytoextracted by each hybrid plant was muchgreater because of the greater biomass produced on thecontaminated soil. Interestingly, the growth habits and bio-

mass of B. juncea and the 60/31 hybrid did not differ muchwhen the plants were grown in noncontaminated fertile soil(data not shown).

Rhizosecretion. Phytoextraction exploits the ability of plantroots to remove unwanted contaminants from their environ-ment. But could the reverse of this process also be exploited?Could roots make valuable compounds and deliver them intotheir environment? At present, most of the recombinantproteins or valuable natural products used as fine chemicals,pharmaceuticals, crop protection compounds, cosmetic ingre-dients, etc. are extracted from plants by using solvents. Thismethod requires expensive purification of the active ingredi-ents from complex mixtures of organic molecules and proteins,making downstream processing and purification of individualcomponents difficult and costly. Extracting plants is also a‘‘batch’’ process whereby the plant is harvested, and its con-tinual ability to synthesize chemicals is not utilized. Naturalrubber and maple syrup are rare examples of continuousmanufacturing processes, which produce much larger amountsof valuable plant product over the lifetime of the plant.

Rhizosecretion of Natural Products. In addition to accu-mulating biologically active chemicals, plant roots continu-ously produce and secrete compounds into their immediateenvironment (rhizosphere). While up to 10% of photosynthet-ically fixed carbon is secreted from the roots (17, 18), thesystematic study of chemical composition of root exudatesfrom diverse plant species has not been undertaken. Notsurprisingly, few compounds that were identified in rootexudates were shown to play an important role in severalbiological processes. For example, isoflavonoids and fla-vonoids present in the root exudates of a variety of legumeplants activate the Rhizobium genes responsible for the nod-ulation process (19, 20) and, possibly, for vesicular–arbuscularmycorrhiza (VAM) colonization (21). Strigol, a germinationstimulant for the parasitic plant Striga asiatica, has been foundin the root exudates of many cereals (22). A variety of plantsproduce herbicidal allelochemicals that may inhibit growth andgermination of neighboring plants (23–25). In addition, root-secreted compounds called phytosiderophores may be in-volved 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 biologicalactivities including protection against biotic and abioticstresses. Survival of delicate and physically unprotected rootcells may depend on their continuous ‘‘underground chemicalwarfare’’ against a hostile and constantly changing environ-ment teeming with bacteria and fungi preying on any organicmaterial in soil. The unexplored chemical diversity of rootexudates is an obvious place to search for novel biologicallyactive compounds including antimicrobials. Our biochemicalanalysis of root exudates from 120 plant species can besummarized as follows: (i) each plant species studied exudeda distinct set of compounds, which is a unique biochemicalfingerprint for a given species (Fig. 2 A–C); (ii) root exudatesare relatively simple mixtures, in comparison to solvent ex-tracts of plant tissue, making the isolation of the activemolecules an easier task; (iii) root exudates are devoid ofpigments and tannins, known to interfere in activity screens,and do not contain large quantities of biologically inert struc-tural compounds; and (iv) the chemical composition of rootexudates is very different from that of conventional methanolicextracts of root tissue.

We have also observed that exudate chemical diversity canbe greatly increased by the elicitation process, which is knownto alter secondary metabolism in plants exposed to variousphysical and chemical treatments. Phytoalexins, antimicrobialcompounds produced in plants and tissue cultures in responseto disease causing agents or their chemical components, are

FIG. 1. Asymmetric somatic hybrid 60/31 (B) and its parentsBrassica juncea (A) and Thlaspi caerulescens (C) growing in soilcontaining 800 mg/kg lead, 328 mg/kg nickel, and 7,600 mg/kg zinc.

5974 Colloquium Paper: Gleba et al. Proc. Natl. Acad. Sci. USA 96 (1999)

probably the best studied elicited defense compounds in plants(31). Unfortunately, little is known about elicited compoundsin root exudates, with the exception of a recent report onisoflavonoid exudation from the roots of white lupine (30). Weobserved that chemical or physical elicitors stimulate roots ofvarious plants to exude an array of compounds not detected inthe ‘‘nonelicited’’ exudates (Fig. 2 D–F). On the other hand,the same elicitor will trigger the production of differentcompounds in different plant species. In addition, elicitationmay dramatically increase the quantities of certain compoundsin the exudates. It can be hypothesized that elicitors mimic theeffects of stresses on the hydroponically grown roots, activatingbiochemical defense systems and resulting in quantitative andqualitative changes in the composition of the exudates.

To demonstrate the presence of antimicrobial compounds inroot exudates, a screening protocol was designed in which 10ml of concentrated exudate solution was transferred into asmall cavity in agar poured into 24-well microtiter plates. Thetested microorganisms were plated in each well before thecavity was made. Exudates from 480 species, each treated with2–4 elicitors, were tested in this system for the inhibition ofgrowth of selected bacteria and fungi (Fig. 3). The followingpercentage of exudates showed moderate to strong activityagainst tested microorganisms: Escherichia coli (3.4%), Staph-ylococcus aureus (4.3%), Pseudomonas aeruginosa (0.4%),Penicillium notatum (0.8%), and Saccharomyces cerevisiae(0.6%).

In addition to exudates, hydroponically cultivated plantroots also provide a unique source of biologically activecompounds. We have also observed that elicitation, bothquantitatively and qualitatively, alters the HPLC profiles ofsecondary metabolites in roots of many plant species (data notshown). Most likely, these changes are subsequently reflectedby the dramatic alterations in the rhizosecreted compounds.

Why Root Exudates? The above observations suggest thatroot exudates represent a new and functionally enriched source

of biologically active compounds. Elicitation of hydroponicallygrown roots adds another unexplored dimension to the chem-ical diversity normally hidden in silent parts of the plantgenomes. In addition to shedding light on dark corners of plantbiology, the systematic study of root exudates may be valuableto the global pharmaceutical industry, which still heavily relieson novel sources of chemical diversity to discover new drugs inan ever-accelerating race against time. Twenty five percent ofall prescriptions dispensed from pharmacies in the UnitedStates contain active ingredients extracted from higher plants(32). However, methods of harvesting chemical diversity ofplant-derived compounds often follows hunter–gatherer strat-egies. Extracts of plant material haphazardly collected invarious places around the world are eventually acquired bypharmaceutical companies, which put them through sophisti-cated high-throughput screens that use an increasing array ofmolecular targets. This primitive prospecting process does notprovide a reliable and reproducible source of natural productsthat can be easily resupplied after a novel activity is found. Themismatch between the beginning of the drug developmentpipeline and what follows creates an opportunity for develop-ing new pharmaceutical agents from plants using more stan-dardized, scientific approaches that favor biologically active

FIG. 2. HPLC profiles of nonelicited root exudates of three plantspecies collected in distilled water (A–C) and root exudates of Brassicajuncea collected in distilled water (D) or in distilled water supple-mented with 1 mM AgNO3 (E) or 500 mM H2O2 (F) as elicitors. Plantswere grown hydroponically with roots suspended in aerated nutrientsolution. Root exudates from 4- to 6-week-old plants were collected for24 hours in 400 ml of distilled water with or without elicitors. Rootexudates were concentrated by freeze-drying, and exudate compoundswere separated on a Waters NovaPak C-18 reverse phase column usingacetic acid/acetonitrile gradient.

FIG. 3. Antimicrobial activity of root exudates. The exudatesshowing activity (indicated with red arrows) against Staphylococcusaureus ssp. aureus (A) were from Tagetes minuta (column 1, Aster-aceae) and Eriastrum densiflorum var. austromontana (column 6,Polemoniaceae) and activity against Saccharomyces cerevisiae (B) werefrom Hosta fortunea (column 6, Liliaceae). To test antibacterial/antifungal activity of exudates, the suspension of target microorgan-isms or spores was plated and spread on the surface of standard LBagar (bacteria) or potato dextrose agar (fungi) poured into 24-wellmicroplates. Twenty microliters of exudate dissolved in water waspipeted into a central hole punched in the agar. The antimicrobialactivity, visible as an area of growth inhibition (clearing) around thecentral hole was scored after 24 hours of incubating inoculated platesat 30°C.

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molecules over structural components and major metabolites.Tissue culture-based production of natural products, oftencombined with elicitation, is one of the recently developedstrategies for ‘‘increasing the size of the needle in the hay-stack.’’ However, plant tissue cultures are expensive, slowgrowing, and relatively deficient of secondary metabolites,presumably because of their nondifferentiated nature. Rhiz-osecretion, on the other hand, may produce a more cost-effective and diverse source of chemical compound mixturesfor the identification of novel biologically active compounds.In addition, rhizosecretion, a nondestructive and continuousprocess, may provide a constant supply of these compoundsover the lifetime of a plant.

Rhizosecretion of Recombinant Proteins. The ease of trans-formation and cultivation make plants suitable for manufac-turing many recombinant proteins. Indeed, numerous heter-ologous (recombinant) proteins have been produced in plantleaves, fruits, roots, tubers, and seeds (33–35), and are targetedto different subcellular compartments, such as the cytoplasm,endoplasmic reticulum (ER), or apoplastic space (36). Plantsare capable of carrying out acetylation, phosphorylation, andglycosylation as well as other posttranslational protein modi-fications required for the biological activity of many eukaryoticproteins. However, the extraction and purification of proteinsfrom biochemically complex plant tissues is a laborious andexpensive process that presents a major obstacle to large-scaleprotein manufacturing in plants. In attempts to overcome thisproblem, secretion-based systems utilizing transgenic plantcells or plant organs aseptically cultivated in vitro have beeninvestigated (37–39). However, these in vitro systems, whichinclude hairy roots, may be expensive, slow-growing, unstable,and relatively low-yielding. Until now, these disadvantagesprecluded the use of in vitro plant systems for the commercialmanufacturing of recombinant proteins.

Can rhizosecretion be used for the continuous manufactur-ing of recombinant proteins? The nondestructive rhizosecre-tion process may provide high yields of recombinant proteinsover the lifetime of a plant and facilitate their downstreampurification, combining the advantages of the whole plant andin vitro protein expression systems. Indeed, roots of livingplants are known to secrete proteins. For example, largeamounts of acid phosphatase are released from the roots ofmany plants during phosphate deficiency (40). We attemptedto ‘‘rhizosecrete’’ the following three heterologous proteins ofdifferent origins from Nicotiana tabacum L.; green fluorescent

protein (GFP) of the jellyfish Aequorea victoria, human pla-cental secreted alkaline phosphatase (SEAP), and xylanasefrom the thermophylic bacterium Clostridium thermocellum.

All three of these proteins were rhizosecreted from trans-genic plants when their expression was controlled by a strongroot-expressed promoter and targeted by a secretory signalpeptide (Fig. 4). Daily rhizosecretion of GFP, released intofresh medium unprotected from proteolysis, reached 2 mg/groot dry weight, while SEAP rhizosecretion, quantified fromits activity, reached 20 mg/g root dry weight, a significantamount considering that no attempts to optimize rhizosecre-tion had been made thus far. It is likely that methods forincreasing protein expression and secretion will be developedalong with plant varieties optimized for the rhizosecretion ofrecombinant proteins.

Data suggest that plant roots can continuously produce andsecrete biologically active recombinant proteins of differentorigins. The rhizosecretion system offers a simplified methodfor the isolation of recombinant proteins from simple hydro-ponic medium rather than from complex plant extracts. Aswith rhizosecretion of natural products, protein rhizosecretioncan be operated continuously without destroying the plant,thus producing a higher total yield of the recombinant proteinover the life of the transgenic plant. In addition, recombinantbiopharmaceutical proteins purified from root exudates areless likely to be contaminated with pathogenic viruses that maybe present in the milk or urine of transgenic animals. Rhiz-osecretion also borrows from many well developed and testedmethods of commercial hydroponic plant cultivation, andtherefore, will be relatively easy to scale up.

CONCLUSIONS

While the evolution of plant shoots followed primarily ‘‘intro-verted’’ paths by perfecting physical barriers between them-selves and the environment, roots had to be more ‘‘extrovert-ed’’ in their relationship with soil. This requirement created aunique set of biological mechanisms, which until recently, wereunderstudied and underutilized. Phytoextraction and rhizos-ecretion are starting to change this, while allowing scientists totake a radically new look at the darkest corners of plantbiology. These technologies also open the doors to the value-added, nonagricultural uses of plants, which will continue toexpand in the new century.

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 correctorientation between the mannopine synthase (mas29) promoter (provided by Stanton Gelvin, Purdue University, West Lafayette, IN) and nosterminator. GFP rhizosecretion from the hydroponically cultivated aseptic roots was visualized after illuminating the hydroponic medium contactingroots with near-UV light. Media from nontransformed plants showed no fluorescence (data not shown). (B) Visualization of SEAP rhizosecretionin the native gel. In transformed tobacco, coding sequence of SEAP with its own signal peptide was controlled by the caulif lower mosaic virus 35Spromoter (CaMV35S). Thirty micrograms of total protein concentrated from root exudates of transgenic and nontransformed plants was separatedon native PAGE, and SEAP activity was localized using the alkaline phosphatase isoenzymes procedure (Sigma). Lanes 1 and 2, transgenic tobaccoplants; lanes 3 and 4, nontransformed tobacco. (C) Rhizosecretion of bacterial xylanase from transgenic tobacco seedlings germinated on theRBB-xylane-containing agar medium (dark blue), which becomes colorless when cleaved by xylanase (photographed upside down). Nontransformedplants did not change the color of the medium (data not shown). Seeds of tobacco expressing a truncated C. thermocellum xylanase gene controlledby the CaMV35S promoter and targeted to the apoplast by proteinase inhibitor II ER signal peptide were provided by Uwe Sonnewald.

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Neither phytoextraction nor rhizosecretion will directly con-tribute to feeding world population in the next century.However, these technologies will improve the quality of life formany people if their development continues. The futurechallenge for metal phytoextraction is to further reduce thecost and increase the spectrum of metals amenable to thistechnology. This goal can be achieved by creating superiorplant varieties for phytoextraction by using genetic engineeringto introduce valuable traits into plants, developing betteragronomic protocols for their cultivation, and designing saferand more effective soil amendments. A recent, and probablythe only, example of the successful use of genetic engineeringapplied to metal phytoremediation is the use of bacterialmercuric reductase (merA) gene to achieve mercuric ionreduction in transgenic Arabidopsis (41) and yellow poplarplants (42). Elemental mercury produced in transgenic plantsis much less toxic than ionic mercury and can be volatilizedfrom transgenic plants in a process termed phytovolatilization,which is related to phytoextraction.

The future challenge for rhizosecretion lies in the successfuldevelopment of effective and safe pharmaceuticals from thecollection of biologically active lead molecules secreted by theroots, and in large-scale, cost-effective manufacturing of re-combinant proteins. The aging population and ever-growingdemand for better pharmaceuticals should foster the use greenplants as sources of new drug discovery, biotransformation,and in some cases, manufacturing. Thus, more effective utili-zation of immense biosynthetic capacity of plants based ontheir inexpensive and renewable nature will present majoropportunities for plant researchers in the next century.

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