Perspectives for genetic engineering of poplars for enhancedphytoremediation abilities

Rakesh Yadav • Pooja Arora • Sandeep Kumar •

Ashok Chaudhury

Accepted: 1 September 2010 / Published online: 17 September 2010

� Springer Science+Business Media, LLC 2010

Abstract Phytoremediation potential has been widely

accepted as highly stable and dynamic approach for

reducing eco-toxic pollutants. Earlier reports endorse

remediation abilities both in herbaceous plants as well as

woody trees. Poplars are dominant trees to the ecosystem

structure and functioning in riparian forests of North

America Rivers and also to other part of the world.

Understanding of the fact that how genetic variation in

primary producer structures communities, affects species

distribution, and alters ecosystem-level processes, attention

was paid to investigate the perspectives of genetic modi-

fication in poplar. The present review article furnishes

documented evidences for genetic engineering of Populus

tree for enhanced phytoremediation abilities. The versatil-

ity of poplar as a consequence of its distinct traits, rapid

growth rates, extensive root system, high perennial biomass

production, and immense industrial value, bring it in the

forefront of phytoremediation. Furthermore, remediative

capabilities of Populus can be significantly increased by

introducing cross-kingdom, non-resident genes encoding

desirable traits. Available genome sequence database of

Populus contribute to the determination of gene functions

together with elucidating phytoremediation linked meta-

bolic pathways. Adequate understanding of functional

genomics in merger with physiology and genetics of poplar

offers distinct advantage in identifying and upgrading

phytoremediation potential of this model forest tree species

for human welfare.

Keywords Functional genomics � Genetic engineering �Phytoremediation � Pollutants � Populus

Introduction

Phytoremediation is a highly versatile, solar driven in situ

pollutant extraction system for removal of ecosystem

trembling contaminants from soil, water, sediments, and

air. It signifies highly perceptive and promising field of

bioresources technology. It is considerably less-expensive

and effective against metals, pesticides, solvents, explo-

sives, and crude oil contaminations. Eco-toxication with

heavy-metals and xenobiotic compounds is a global threat

that has aroused from mining, industrial, agricultural and

military practices (Nriagu and Pacyna 1988). Several pol-

lutants accumulate in the food chain and intimidate health

of human and other organisms. In rich industrialized world,

contamination is often highly localized, and the pressure to

use contaminated land and water for agroforestry system

and food production can hardly ever be ignored. Soil and

water contamination is a global phenomenon and is dra-

matically increasing in large parts of the developing

countries including India (Meharg 2004) and China (Cheng

2003). Although the environmental hygiene is exploited

eternally in the modern world, very little effort has been

put forward for the avoidance of pollution in the past.

Everyday practices for scavenging and cleaning of con-

taminated soils and waters are very costly, and even no

fastidious technologies are yet available for numerous

xenobiotics and pollutants. The natural forests have been

R. Yadav � P. Arora � A. Chaudhury (&)

Department of Bio and Nano Technology, Guru Jambheshwar

University of Science and Technology, Hisar 125001,

Haryana, India

e-mail: ashokchaudhury@hotmail.com

S. Kumar

Division of Biochemistry, Directorate of Rapeseed-Mustard

Research, ICAR, Sewar, Bharatpur 321303, Rajasthan, India

123

Ecotoxicology (2010) 19:1574–1588

DOI 10.1007/s10646-010-0543-7

found to be highly efficient for clean-up of the pollutants

via phytoremediation process in situ. It has emerged as a

productive research field in the past decade and could be

the cost-effective environmental clean-up process ever-

known (Salt et al. 1998; Kramer and Chardonnens 2001;

McGrath and Zhao 2003).

Populus is a genus of deciduous flowering trees comprised

of*20–35 species which are unevenly distributed in all parts

of the world particularly in the Northern Hemisphere. Dif-

ferent species has been broadly classified under three major

groups including poplars, aspens, and cottonwoods. Cotton-

woods also called black poplars reside at temperate region

such as North America, Europe, and Western Asia. Important

species of cottonwoods are P. deltoides, P. fremontii, P. nigra,

P. canadensis (P. nigra 9 P. deltoides). Second major group

of aspens which are also designated as white poplar belongs to

circumpolar subarctic, cool temperate, and mountains farther

south. The species of white poplars are P. tremula, P. ade-

nopoda, P. alba, P. canescens (P. alba 9 P. tremula),

P. davidiana, P. grandidentata, P. sieboldii, P. tremuloides.

Third major group of balsam poplars resides at cool temperate

zones of North America, and Asia which comprises many

species such as P. angustifolia, P. balsamifera, P. cathayana,

P. koreana, P. laurifolia, P. maximowiczii, P. simonii,

P. trichocarpa, P. tristis, P. ussuriensis, and P. yunnanensis.

Some other species comes under the category of Mexicans

poplars, subtropical poplars, and bigleaf poplars. In India,

poplar is very prominent taxonomical group in eco-forestry

systems. Amongst all, P. ciliata, P. gamblei, P. jacquemontii,

P. rotundifolia, P. euphratica and P. alba are indigenous

poplar species, whereas, a few commercially important spe-

cies and hybrids of poplars including P. deltoides, P. nigra,

P. x euramericana and P. x berolinensis are exotic and were

introduced in late 1950s from USA.

Poplar, being a multipurpose hardwood tree, has been

known to be an excellent candidate for phytoremediation

and reducing environmental pollutants as a result of its

highly efficient potential of photosynthesis (Soudek et al.

2004). Poplar can be cultivated with rapid growth rates and

produce a large biomass of *90.6 Mg ha-1 at a short

period of 5–8 years (Das and Chaturvedi 2005). The

characteristic extensive root systems of poplar ensure

efficient uptake of ground water containing the pollutants.

In addition, their green canopy fixes and stores carbon by

unique approach of photosynthesis, thereby, reduces

atmospheric carbon dioxide (CO2) chemically by electron

transfer and physically by lowering CO2 concentration in

air. Poplars undertake several phases of decontamination,

their foliage can be easily collected and the contaminated

biomass can be substantially reduced by incineration. The

harvested poplar wood can be used as valuable raw mate-

rial for pulp and paper industry, high quality fiber (Stettler

et al. 1996) and for the production of matchsticks (Dietz

and Schnoor 2001; Komives et al. 2003b). Although pop-

lars have been established to take up several inorganic

pollutants including heavy metals, such as cadmium

(Koprivova et al. 2002), mercury (Rugh et al. 1998), nickel

(Rai et al. 2003), copper (Borghi et al. 2007, 2008) and zinc

(Di Baccio et al. 2003, 2009; Bittsanszky et al. 2005;

Langer et al. 2009), their heavy metal tolerance is restricted

(Dietz and Schnoor 2001). Indeed, genetic manipulations

of poplars for introduction of exotic phytoremediation

related genes can cause multifold expansion to their

remediative capability and enhance their clean-up effi-

ciency (Bittsanszky et al. 2005). Several reports are known

for strengthening of the remediative mechanisms in dif-

ferent species of poplar such as P. deltoides, P. canescens,

P. alba and other hybrids through genetic manipulation and

transgene expression (Che et al. 2003; Bittsanszky et al.

2005; Doty et al. 2007; Iimura et al. 2007; Balestrazzi et al.

2009). Thus, cross-kingdom gene transfer technology can

be the favorite theme for researchers for introduction of

desirable traits including enhancement of the phytoreme-

diation ability in plants.

Economic significance, high environmental-friendly

value and social importance of poplars to the ecosystem

make its position known in the eco-forestry structures.

Environmental engineering via carbon storage and phyto-

remediation services rendered by poplars has acquired

perceptible worth. Unlike Arabidopsis, Poplars were not

only retrieved as favorite perennial from hardwood tree

genetics, genomics and forest biology viewpoint (Ameri-

can Society of Plant Biologists 2002), but also proved as

dependable candidate of the new era by providing envi-

ronmental and ecological services. Since poplars inhabit

almost all natural forests worldwide, little attention to

forest management progressions in permutation with

engineered perennials will be highly rewarding for soil

decontamination and phytoremediation efforts.

Mechanisms of photosynthesis driven technology

for remediation

Everyday mining, metallurgical, and agricultural practices

produce effluents that can be considered as the major

source of heavy metal adulteration of soil and water. The

environmental polluting practices were being chronically

used since Iron-Age and are potential hazards to aquatic,

animal, and human life because of their toxic, bio-accu-

mulative and non-biodegradable nature. Interestingly, for

appropriate growth and reproduction, plants needs to

absorb not only macronutrients (N, P, K?, S, Ca2?, and

Mg2?), but also essential micronutrients such as Fe2?,

Zn2?, Mn2?, Ni2?, Cu2?, and Mo2?. Highly specific

mechanisms have been developed in plants to take up,

Perspectives for genetic engineering of poplars 1575

123

translocate, and store these nutrients as illustrated in Fig. 1.

The structure and properties of membrane transporters

determine ion uptake selectivity. Metal ions are pumped in

across the membrane via specific membrane transporters

and form low molecular weight complex with phytochel-

atins (PCs). Glutathione (GSH, c-L-glutamyl-L-cysteinyl-

glycine) mediates metal-phytochelator complex (M�PC)

formation (Grill et al. 1989; Li et al. 1997). M�PC complex

actively transported to vacuole for detoxification by turning

out into high molecular weight complex and eventually

accumulated at very high concentration (Shah and Non-

gkynrih 2007). Simultaneously, various cascades such as

phenyl propanoid pathway and octadecanoid pathways are

triggered as subsequent effect of generation of activated

oxygen species. The activated kinases and cascades tempt

the transcription factor that in turn alarms numerous

defense and tolerance genes. Additionally, sensitive cyto-

mechanisms keep up intracellular concentration of metal

ions within the acceptable physiological range. Many

metals ions such as Zn?2, Mn?2, Ni?2 and Cu?2 are

essential micronutrients and serve as co-factors for

numerous enzymes negotiating key cellular metabolic

reactions. Certain metal superaccumulator plants do not

only accumulate exceptionally high amounts of essential

micronutrients (in thousands of ppm), but can also absorb

significant amounts of nonessential metals. Since metal

accumulation is ultimately an energy consuming process,

sometime plant may absorb heavy metal because of lack of

identification between the ion and its chemical analogue

such as cadmium absorption in place of zinc (Chaney et al.

1994), whereas, metal accumulation in the foliage may

allow hyper-accumulator species to evade predators

including caterpillars, fungi and bacteria (Boyd and Mar-

tens 1994; Pollard and Baker 1997). Some over-contami-

nated zones referred as Superfund sites which are polluted

to terrifyingly elevated levels. Besides this, great expen-

diture has been made every year in efforts to remediate

such polluted sites. There are several elementary ways in

which poplar plants can be employed for industrial waste

treatment and Superfund site remediation such as phy-

todegradation, detoxification, phytoaccumulation, phyto-

transformation, phytovolatilization as depicted in Fig. 2.

Fig. 1 A simplified model of the metal tolerance mechanisms that

strike in plant cell in response to metal exposure: metal ion uptake,

chelation, detoxification, translocation, sequestration, signaling and

signal transduction. The diagram illustrates uptake of metal ions via

membrane transporters by K? efflux, their sequestration by complex

formation with phytochelatins mediated by enzyme PCS and GSH

in vacuoles, the release of GSH by M�PC degradation through

peptidases, the generation of reactive oxygen intermediates species,

the involvement of Ca2? in activation of Ca2?/calmodulin kinase(s)

and MAP kinase(s) staircase pathways which lead to activation of

defense mechanism undertaking genes in nucleus, the impact of

reactive oxygen intermediates on resident plant defense trails like

octadecanoid pathway (a), phenyl propanoid pathway (b) and catalase

mediated pathway (c) that lead to cell defense gene activation in

response to the induced metal stress. GSH glutathione, MAP Kinasemitogen activated protein kinase, M�PC, metal phyochelatin complex,

M2? metal ions, PCS phyochelatin synthase, PCS* phyochelatin

synthase activated (adapted from Shah and Nongkynrih 2007)

1576 R. Yadav et al.

123

Stimulation of rhizosphere bioactivity

Typically, plants can stimulate microbe (bacteria and

fungi) bioactivity about 10–100 times higher in the root-

zone by the secretion of bio-enhancing compounds

including amino acids, carbohydrates, polysaccharides,

flavonoids, and phenols. The plant-excreted root exudates

facilitate soil microbes in bulk by providing a carbon and

nitrogen source. Specifically, it has been revealed that

flavonoids can promote the growth of polychlorinated

biphenyls (PCB)-degrading bacteria. In addition, the phe-

nols excreted by certain plants can stimulate PCB-

degrading bacteria and inhibit other microbes. Enhanced

herbicide degradation has also been found in the presence

of root exudates. Ex planta phytoremediation could be

efficiently carried out in poplar as a result of characteristic

extensive root systems and flourishing microbial associa-

tion. Besides secreting organic compounds for facilitating

the growth and activities of rhizospheric microorganisms,

plants also release certain enzymes capable of disinte-

grating organic contaminants in soils. Infection and colo-

nization of hybrid poplar grown in diesel-contaminated

soils by ectomycorrhizal fungi improved fine root pro-

duction and whole-plant biomass (Gunderson et al. 2007).

The fungi, growing in symbiotic association with the plant,

have unique enzyme-mediated metabolic pathways, similar

to white rot fungus enzymes, which facilitate to degrade

organic pollutants that could not be transformed exclu-

sively by bacteria. Microbial titers of denitrifiers, pseudo-

monads, and monoaromatic petroleum hydrocarbon (BTX)

degraders were reported to be considerably higher in

poplar-tree-rhizosphere soil samples despite of agricultural

soil, whereas, atrazine degraders were only verified in

rhizosphere sample. Hence, the poplar rhizosphere can

enhance the progression of microbial populations that

contribute in natural bioremediation without wielding

selection pressure for them (Jordhal et al. 1997). Well

known ectomycorrhizal fungi (Hebeloma crustuliniforme,

Paxillus involutus and Pisolithus tinctorius) in association

with roots of poplar plant (P. canadensis) led to an abun-

dant increase in Cd2? uptake, translocation and accumu-

lation particularly in the leaves, which is significantly

higher than willows (Sell et al. 2005).

Enzymes-mediated precipitation, binding

and degradation of aromatic pollutants

Plants have adapted to tolerate elevated heavy metal con-

centrations in soil and water through the specific enzymes

driven mechanisms. The potentiality of dehalogenase

enzyme has been revealed from degradation of certain

xenobiotics by poplar trees (Schnoor et al. 1995), which

catalyzes oxidation of alkanes, alkenes, and methanes and

their halogenated analogues. Dehalogenase(s) will eventu-

ally mineralize trichloroethylene (TCE) to CO2 through an

oxidative pathway and releases it into air. Several other

enzymes such as glutathione-S-transferases (GST), peroxi-

dases, peroxygenases, cytochrome P450, carboxylesterases,

O-glucosyltransferases, O-malonyltransferases, N-glucos-

yltransferases, and N-malonyltransferases are involved in

the oxidation of pollutants within plant cells, transport of

intermediates, and other physiological and biochemical

processes (Sandermann 1994; Macek et al. 2000; Kramer

and Chardonnens 2001). Primarily, peroxidases mediate the

metal accumulation and detoxification processes in plant

tissue. Phytoremediation potential is strongly associated

with the ability of the plant to discharge noxious levels of the

toxic form of the pollutant and the active oxygen species

generated in the pollutant exposed tissue. GST enzymes

have been identified to mediate the process of detoxification

of active oxygen species (Komives et al. 2003b). Carreira

(1996) reported that nitroreductase and laccase enzymes

completely degraded 2,4,6-trinitrotoluene (TNT) to form

new plant biomass like lignin. An apparent increase in

superoxide dismutase activity was reported in P. nigra

grown in heavy metal deposited soil from a copper smelter

(Stobrawa and Lorenc-Plucinska 2007). Moreover, physio-

logical responses in the form of stimulated activities of

guaiacol peroxidase (GPX) and polyphenol oxidase (PPO)

Fig. 2 Schematic representation of potential fates of pollutants

during phytoremediation in Populus plant: the pollutant (denoted by

filled circles) can be stabilized, detoxified or degraded in the

rhizosphere, while it can be up taken and translocated to the above

ground tissue, where it has been sequestered, volatilized or degraded

inside the tissue

Perspectives for genetic engineering of poplars 1577

123

were known to cope with the toxicity of Mn in P. cathayana

(Lei et al. 2007). Thus, various enzyme-mediated metabolic

pathways regulate the phytoremediation ability in poplar

and other plants.

Phytostabilization and phytovolatilization

Plants are known for several modes of in situ stabilization

of pollutants by sequestering them in roots or in the above

ground tissues. Binding of metallic ions like Cu?2, Ni?2,

Cd?2, Cr?3, Pb?2, and Zn?2 to shoot biomass of alfalfa has

been studied and found to possess highest binding affinity

in short term studies at pH 5 with 19.7, 4.11, 7.1, 7.7, 43,

and 4.9 mg metal bound per gram of biomass (Tiemann

et al. 1997). A specific nature of stabilization of metals

employing high hydraulic pumping pressure to prevent

migration of contaminants, have also been reported in

poplars (Schnoor 2000). Such biochemical binding of

metals or charged pollutants lead to their reduction in the

environment and are maintained below critical limits.

Plant can convert solid or dissolved forms of compounds

from soil or groundwater into gaseous forms that are

excreted from the plant. Indigenous metabolic processes

within the plants and associated microflora can convert a

fraction of the contaminants to volatile materials. Plants

can also volatilize certain metals, like highly toxic mercury

(Hg2?) and methyl mercury. They reduce them using the

enzyme, mercuric reductase, to the volatile state and con-

vert them to less harmful Hg (0). Poplar trees were tested

for the movement and upshot of volatile organic com-

pounds (VOCs), and found that benzene, toluene, ethyl-

benzene, xylene (BTEX), TCE, and chlorinated benzene

effectively transported through the plant and volatilized

into the environment (Burken and Schnoor 1996). No

doubt, members of the Brassica genus are particularly

excellent volatilizers (Terry et al. 1992) but Populus has

also been largely exploited genus for phytovolatilization of

VOCs because of its high transpiration rate, which facili-

tates the translocation of these compounds by the plant into

the atmosphere.

Heavy metal super-accumulation in the plant tissue

(phytoextraction, rhizofiltration)

Peroxidase activity stimulation in root and leaf tissue is

evidently linked with tissue concentration of heavy metals

and growth. Miteva and Peycheva (1999) examined per-

oxide synthesis activity at different stages of green bean

(Phaseolus vulgaris) and tomato (Lycopersicum esculen-

tum) development after treatment with arsenide (As)

showed high phytoextraction abilities of Phaseolus. Dye

tolerance has also been verified to be coupled with

enhanced peroxidase activity. Peroxidase activity appears

in several physiological developments such as lignification,

wound healing, aromatic compound degradation, pathogen

defense, and stiffening (Zheng et al. 2000). The enhanced

peroxidase activities boost super-accumulation properties

of the plants. Populus canadensis Moench was studied for

uptake and translocation of As by treating with soil

amendments including 30 mg per gram of As along with

other heavy metals and found to have fairly high average

concentration in the root bark, root core, trunk, branch, and

leaves of 4.25, 10.51, 2.27, and 6.31 mg/kg of tissue,

respectively (Yu et al. 1996). Similarly, association of

poplar roots with ectomycorrhizal fungi can enhance

metal accumulation tendency of above ground tissue to

manifolds.

Populus in the sphere of phytoremediation

Advantages of populus sp. in phytoremediation

Genus Populus has been established as model system for

plant biology (Taylor 2002; Jansson and Douglas 2007)

and favorite perennial for genomics and forest biology

amongst tree species. Concurrently, domino effects of

several unique features in poplar tree depict its factuality

for phytoremediation. There are more than 25 Populus

species virtually available in all habitats worldwide. This

fast growing (3–5 meters/year) tree species lives a long life

and possesses exorbitant transpiration rates of approxi-

mately 100 l/day. Interestingly, a recent investigation

determined air pollution monitoring capabilities of poplar

tree (P. nigra L.) bark for urban industrial regions (Berli-

zov et al. 2008). Such capabilities strongly permits living

organization particularly tree species to be exploited as

pollution regulating agents over traditional instrumental

methods. Great potential of poplar for efficient accretion

and retention of aerosol particles is considered to be a

propitious direction in air pollution monitoring. In addition

to this, a separate study revealed inverse correlations amid

herbicide phytotoxicity and the activity of the plant’s GSH

conjugating system which signifies the tolerance of poplars

against chloroacetanilide herbicide and highlights plant’s

capability to detoxify these herbicides via GSH conjuga-

tion (Komives et al. 2003a). Above-ground tissues of

P. marilandica were determined with considerable super-

accumulation efficiency for metals, and it has been rec-

ommended as a tree for land reclamation (Rachwal et al.

1992; Lukaszewski et al. 1993; Giniyatullin et al. 1998).

Owing to its extensive root system, hydroponic cultures of

hybrid poplar tree were reported to be potential phyto-

remediator for organic pollutants (Aitchison et al. 2000).

Encouragingly, a simple root mass estimation protocol has

been developed in young hybrid poplar trees using the

1578 R. Yadav et al.

123

electrical capacitance method. The technique has been

fabricated as a result of the polarization of biological

membranes in the root system that enable root mass of trees

to be estimated when grown in mineral rich soils. The

measurements and interpretations of electrical capacitance

by employing this technique are very easy, non-intrusive,

and reassuring (Preston et al. 2004). This prompt field-

application method may be highly competent of providing

adequate estimates of root mass for young trees in context

of phytoremediation examinations.

Poplars are extensively planted across the United States

as ornamentals and windbreaks, as well as significant worth

as a woody timber species. Several horticultural hybrids

have been depicted, with P. deltoides (eastern cottonwood)

crossed by one of the common forms, P. nigra. The extre-

mely rapid growth characteristics of these hybrids and their

deep root systems have caught attention in the phytoreme-

diation community. Different studies have advocated that

they can efficiently eliminate organic contaminants such as

aromatic hydrocarbons, herbicides, and solvents (Burken

and Schnoor 1998; Aitchison et al. 2000). Tree trunk can be

used in paper, pulp and timber industries or as biomass for

energy production such that the accumulated pollutant has

been consequentially dumped at anodyne site. Even though,

Populus tissue culture is not new (Ahuja 1983); tremendous

efforts have been made in direction of in vitro culture, high

frequency shoot regeneration and micropropagation of this

woody species (Ahuja 1983; Thakur and Sharma 2006;

Peternel et al. 2009; Yadav et al. 2009), over the years. In

vitro cultures are subject to somaclonal variation; as a

result, new plants with improved phytoremediation traits

can be recovered and perpetuated inexpensively (Flokstra

et al. 2008). It has also been well accustomed for sufficient

high frequency transformation protocols intended for

improved remediation (Bittsanszky et al. 2005; Doty et al.

2007; Brentner et al. 2008) and become ideal choice in the

agroforestry systems. Poplar intercropping is permissible

with no critical effect of the tree on field grown crops. To

date, several genetically modified P. deltoides, P. canes-

cens, P. alba have been reported with improved phyto-

remediation efficiency.

Physiology and genetics of poplar in phytoremediation

context

There is an increased interest in the application of genus

Populus in phytoremediation studies. Genome sequence of

poplar, if compared by employing a large-scale genomics,

proteomics and systems biology approaches with other

plant species such as Arabidopsis, Oryza sativa, Chla-

mydomonas, C. elegance, will enable us to sort out several

unresolved skepticism related to toxic compound metabo-

lism, metal accumulation and tolerance in plant cells

through a universal analysis of gene expression. It is now

feasible to identify gene segments that enclose quantitative

trait loci (QTLs) involved in phytoremediation. Recently,

Brentner et al. (2008) demonstrated the advantage of

functional genomics information from Arabidopsis, in

identifying essential detoxification genes for TNT in the

model phytoremediation species, P. trichocarpa. Making

use of Arabidopsis thaliana sequence, 12 genes were

identified in Populus that were found to be TNT induced.

Substantial increase of two GST gene expression was

quantified and validated upon TNT exposure of hydro-

ponics poplar cultures. Selected Populus genotypes

exhibited elevation in total tree Cl- ion concentration and

increase in biomass, when irrigated with municipal solid

waste landfill leachate (Zalesny et al. 2008). PCBs dis-

persed in disposal are highly volatile congeners which

adulterate air, soils and sediments. Municipal wastes

treatment could be designed by uptake, translocation and

distribution of PCBs in whole poplar plants (Liu and

Schnoor 2008). Heavy metal polluted environment stimu-

lates the overproduction of free radicals in fine roots and

induces superoxide dismutase (SOD) activity, whereas,

declines the guaiacol peroxidase activity (Stobrawa and

Lorenc-Plucinska 2007). Besides this, high metal-accu-

mulating P. alba clone AL35 exhibited a significantly

higher concentration of free and conjugated putrescine

as a result of high genetic dissimilarity index even

within populations. This signifies possibility of identifying

best suited genotypes for soil clean-up, and physiologi-

cal markers associated with metal hyper-accumulation

(Castiglione et al. 2009). Inference of genetic basis for

molecular, chemical and physiological characterization of

response to environmental pollution tolerance in P. nigra

was studied by Gaudet et al. (2008) and has eventually

determined the correlations among allelic variants as well

as the phenotypic expression of traits. A recent study by

Berlizov et al. (2008) revealed strong biomonitoring

physiology of black poplar-tree (P. nigra L.) bark against

atmospheric air pollution by chemical elements. An epi-

phytic lichens based powerful analytical techniques pro-

vided a complementary tool to traditional instrumental

methods of studying the atmospheric pollution by airborne

substances. Similarly, P. alba can be used to monitor the

level of trace elements in riparian ecosystem especially for

Cd2? and Zn2? ion concentrations (Madejona et al. 2004).

In addition to water stress and recovery conditions (Berta

et al. 2009), rise in Cd2? and Zn2? ion concentrations also

trigger expression of metallothioneins (MTs) genes in

various tissues of poplar (Hassinen et al. 2009). Alteration

in physiology and array of enzymatic activity of poplar

upon exposure to pollutants is implicated to environmental

inductive effect on phytoremediation linked gene regula-

tion and expression. The phytoremediation inductive

Perspectives for genetic engineering of poplars 1579

123

response mechanisms advocate suitability of poplar tree for

remediation competence.

Phytoremediation: need for selection of versatile

candidate with numerous potential applications

The availability of suitable plant material is a vital factor

for efficient application of phytoremediation to diverse

forms of contaminated substrate (soil, water, sludge). Metal

mobilization to the above ground tissues is a critical bio-

chemical process in a commercial exploitation of plants to

remediate contaminated sites. Indeed, a high efficiency

translocation of metals from root mass to the above ground

tissues could shrink the detrimental consequences exerted

by the pollutants on root anatomy, physiology, and bio-

chemistry. This would enhance the efficacy of plant metal

uptake permitting metal removal from the polluted sub-

strate over time. Thus, metal tolerance, bioaccumulation

and translocation ability must be considered simulta-

neously to explore species, genotypes or individuals with

remarkable perspectives in phytoremediation. Members of

genus Populus, due to their distinctive growth, genetic and

cultural characteristics, are potential candidates in the

remediation of contaminated substrates. High metal toler-

ance, consequently the protection of the integrity, func-

tionality of the primary physiological and metabolic

processes (Pietrini et al. 2003), are necessary credentials

for a plant to be employed in phytoremediation. The cost of

phytoremediation has been estimated as US$ 25–100 per

ton of soil, and US$ 0.60–6.00 per 1000 gallons of polluted

water with remediation of organics being cheaper than

remediation of metals (Movahed and Maeiyat 2009).

Though technologies have been identified for clean-up of

heavy-metal-polluted soils but are very expensive (Iskan-

dar and Adriano 1997). Considerable advancement has

been made in this direction by converting metal hyperac-

cumulator wild poplar plants into commercial phytoreme-

diation systems. Amelioration of soils prior to the

introduction of tolerant poplar species may lead to bigger

success in future.

An investigation was performed on the in vitro grown

aspen plantlets exposed to the aqueous solutions having

concentrations 0.1 mM of Pb2? ion. The accumulation

capacity for aspen was reported about 70% of Pb2? origi-

nally present in the solution with no negative effect on

growth of cultures (Spirochova et al. 2003). Besides, P. alba

exhibited a metal hyperaccumulation efficiency of

42.50 ppm for Pb2?, 234.50 ppm for Zn2?, 3.00 ppm for

Cd2?, 28.50 ppm for Cu2?, 10.00 ppm for Ni2?, and

307.5 ppm for Fe2? (Chehregani and Malayeri 2007).

Recently, Zacchini et al. (2009) reported remarkable ability

of poplars for bio-concentrating high cadmium extent in the

root system. Even though phytoremediation in poplars were

almost exclusively confined to roots; the bio-accumulation

was almost double to that of willow clones. Therefore,

poplars could be proficiently used in the remediation of

polluted water (rhizofiltration) or contaminated sites to

regulate metal percolating to the water layer (Phytostabili-

zation). Much acclimatized poplar plants against heavy

metal contamination impart the best suitable approach for

metal reduction and hyperaccumulation from Superfund

sites, and render harmless environmental contaminants

(Sebastiani et al. 2004; Giachetti and Sebastiani 2006; Saier

Jr. and Trevors 2010).

Engineering phytoremediation: a biotechnological

breakthrough

Novel advancements in phytoremediation

During the last fifteen years, phytoremediation has secured

recognition as a technology and has been accredited as an

area of advanced research. There has been a significant

increase in our acumen of the mechanisms that lead to the

uptake, translocation, and detoxification of pollutants by

plants. However, huge disparities in our understanding

await advance research. Recent improvements in phyto-

remediation methods have focused on multidisciplinary

approach for studying the numerous pathways from

remediation of contaminant from the particle to the eco-

system. Online databases of living organisms particularly

plant species may fill the knowledge-gap for cleanup of

different types of pollutants. An interesting advancement in

phytoremediation is its inclusion with topographic archi-

tectural planning. Remediation of metropolitan sites can be

united with a delightful design so that the area may be kept

in use by the community all through and afterward the

remediation process carried out for the duration of the

reducing hazard (Kirkwood 2001). Other polluted sites that

are phytoremediated may be rehabilitated into wildlife

sanctuaries. A different promising advancement in phyto-

remediation is the application of genetically engineered

plants. Understanding of plant metabolic and molecular

studies led to the development of some capable transgenics

that embody great tolerance, bioaccumulation, and degra-

dation capacity for various pollutants. On the horizon,

computational analysis of the online database and avail-

ability of new genomic technologies will lead to the

identification of novel genes focal point for pollutant

remediation, including regulatory factors and tissue-tar-

geted transporters. The introduction and expression of

these genes through genetic engineering in high biomass

species like poplar will stringently cleanup the substrate

and biosphere. Since several tailor made transgenics have

already been reported in the poplar, the genus will be

1580 R. Yadav et al.

123

amongst the top contenders for upcoming remediation-

capable transgenics.

Transgenic poplar with enhanced phytoremediation

trait

Genetic transformation is the most promising tool for

engineering phytoremediation in poplars and other plants

as well (Abhilash et al. 2009; Doty 2008). There are several

reports of enhancement of phytoremediation potential in

poplars and other plants via genetic modification and

transgene inclusion as shown in Table 1. Two key advan-

tages of this technology over conventional breeding are: (a)

specific gene encoding enviable proteins can be virtually

‘copied’ from any living organism such as bacteria, higher

plants, or animals and transferred into poplars, hence,

broadening the array of genes available even outside the

compatibility barriers of the genus; (b) particular com-

mercial genotypes can be genetically customized for a few

well defined traits while upholding the bits and pieces of

the genome to be intact.

Up-regulation of the genes involved in metabolism,

uptake or transport of specific pollutants in transgenic plants

is thought to be a proficient approach for enhancing the

phytoremediation potential. Agrobacterium tumefaciens-

mediated plant transformation provides a rapid tool for

genetic modification in several plants including woody tree

species. c-glutamylcysteine synthetase (c-ECS) is the rate-

limiting regulatory enzyme in the biosynthesis of the

ubiquitous tripeptide thiol compound c-L-glutamyl-L-cys-

teinyl-glycine (GSH) encoded by bacterial gene. The

overexpression of this bacterial gene coding for c-ECS in

transgenic poplars play a role in the antioxidative protection

of plant cells against oxidative stress caused by indigenous

phytoremediation processes (Noctor et al. 1998). Overex-

pression of c-ECS in hybrid poplars (P. tremula 9 P. alba)

treated with cadmium also showed elevated amounts of

phytochelatins and accumulated higher amounts of cad-

mium. Although the overexpression of c-ECS allows

greater accumulation of cadmium in above ground tissue, it

has only a subsidiary effect on cadmium tolerance (Ren-

nenberg and Will 2000; Koprivova et al. 2002). Another

bacterial gene merA (coding for mercuric reductase) was

expressed in transgenic yellow poplar, phytovolatilization

efficiency has been elevated up to ten-times the rate of the

untransformed plant for elemental mercury (Meagher

2000). Phytoremediation ability can also be increased

through genetically engineering plants with a mammalian

cytochrome P450 enzyme known to control the rate-limit-

ing step in the metabolism of numerous environmental

pollutants, including TCE, carbon tetrachloride, chloro-

form, benzene, vinyl chloride, and ethylene dibromide.

Furthermore, the mammalian enzymes were verified to

function competently in plants without the requirement of

cascade of mammalian genes or inclusion of the other

enzymes such as oxidoreductase and cytochrome b5, iden-

tified for full function of mammalian P450s. Doty et al.

(2007, 2008) reported an unambiguous 100-fold enhance-

ment of phytoremediation potential by the over-expression

of rabbit P450 2E1 gene in hybrid poplar (P. tremula 9

P. alba). A dramatic enhancement in the phytovolatilization

ability of poplar has been achieved for benzene and TCE up

to 79% in the short-term exposure of pollutants for one-

week. Calligari et al. (2008) strongly advocates the suit-

ability of interspecific Populus euramericana hybrids for

introduction of useful traits including improved phyto-

remediation potential, resistance to pests and diseases,

altered wood properties and composition, herbicide toler-

ance and growth rate. Rugh et al. (1998) examined the

ability of in vitro cultures and plantlets of yellow poplar

(Liriodendron tulipifera) to express modified mercuric

reductase (merA) gene constructs successfully. Thus, typi-

cal expression of merA gene in transgenic poplar would

certainly be beneficial among ecologically compatible

remediation substitutes. The different genes that could be

introduced into poplars for improved phytoremediation in

future have been enumerated in Fig. 3. Various genes for

improving biomass production, proliferating root system,

and increasing growth rate permit superfluous deposition of

pollutants while the others for increasing chelation render

plants with hyperaccumulating capabilities. The genes

expressing for biodegradative enzymes, metal transporters,

metallothioneins, and transportation, enhancing rhizosphere

activity, sequestering enzyme, detoxifying enzyme, phyto-

volatilization, and changing oxidation state of metal ions

could enhance remediation potential of a plant through

altered molecular mechanisms. By transforming rapidly

growing poplar trees with enhanced capacities to chelate or

metabolize toxic metals may lead to commercial plantation

in near future (Liu et al. 2000).

Preliminary hurdles in the phytoremediation

technology exploration

Though phytoremediation technologies are still at infancy

platform of research and development phases, diverse

applications have publicized potential for success. This has

led to strengthened interest and research in both public as

well as private sectors, and facilitated to explore phyto-

remediation progress into a commercially viable industry.

Few nominal hurdles that must be taken into account for an

industry to develop and grow are (a) identifying more

species those enjoy remediative capabilities and their mass

multiplication through tissue culture applications, (b)

optimizing phytoremediation processes by selection of

Perspectives for genetic engineering of poplars 1581

123

suitable plant and agronomic practices, (c) understanding

pathways about how plants uptake, mobilize, chelate,

volatilize and metabolize pollutants, (d) identifying genes

responsible for metabolic degradation or conversion of

contaminants to appropriate biomass, (e) reducing time

needed for phytoremediation episode accomplishment, (f)

Table 1 Genes introduced into various Populus spp. and other plants blotching effects of their expression on pollutant tolerance, accumulation,

volatilization or phytoremediation potential

Gene Product Source Plant sp. Observed effect Medium References

In poplar

merA Mercuric ion

reductase

Bacteria Populusdeltoides

Resistant to toxic levels of Hg(II) Hydroponic Che et al.

2003

gshI c-glutamylcysteine

synthetase

Bacteria P. canescens(P. tremula 9

P. alba)

Significant accumulation of Cd, Cr,

and particularly Cu

Agar Bittsanszky

et al. 2005

merB Organomercurial

lyase

Bacteria Populusdeltoides

Increase tenfold in roots length and

twofold fresh shoot-weight with

enhanced PMA uptake

Hydroponic Che et al.

2006

CYP2E1 Cytochrome P450

2E1

Rabbit P. tremula 9

P. albaEnhancement of *79%

phytovolatilization and 100 benzene

and TCE metabolism

Hydroponic Doty et al.

2007

MnP Manganese-

dependent

peroxidase (MnP)

Fungus P. seiboldii 9

P. gradientataBisphenol A (BPA)-removing

activities doubled

Agar Iimura et al.

2007

cys1 O-acetylserine

(thiol) lyase

Wheat P. seiboldii 9

P. gradientataReduced foliar damage in transgenic

plants in response to H2S or SO2

Agar Nakamura

et al. 2009

PSMTA1 Metallothioneins

(PSMTA1)

Pea Populus alba Significant reduction of fourfold–

eightfold in the level of DNA

damage resulting from copper

exposure

Agar Balestrazzi

et al. 2009

In other plants

merB Organomercurial

lyase

Bacteria Arabidopsisthaliana

Enhanced tolerance to

organomercurials

Hydroponics Bizily et al.

1999

onr Pentaerythritol

tetranitrate

reductase

Enterobactercloacae

Nicotianatabacum

Enhanced denitration of glycerol

trinitrate

Hydroponic French et al.

1999

CYP2E1 Cytochrome P450

monooxygenase

Human N. tabacum Increased uptake and considerably

enhanced metabolism of

trichloroethylene

Hydroponic Doty et al.

2000

ars, gshI Arsenate reductase,

g-glutamylcysteine

synthetase

Bacteria A. thaliana Significant As accumulation Hydroponics Dhankher

et al. 2002

CYP1A1 Cytochrome P450

monooxygenase

Rat Solanumtuberosum

Increased tolerance to atrazine and

chlortoluron, assumed to be via

metabolism to less-toxic derivatives

Hydroponic Yamada et al.

2002

ars Arsenate reductase Bacteria N. tabacum 1.4-fold higher leaf Cd concentrations Hydroponics Dhankher

et al. 2003

CYP1A1 Cytochrome P450

monooxygenase

Human Oryza sativa Enhanced metabolism of atrazine,

norflurazon and chlortoluron (should

also metabolize PAHs)

Hydroponic Kawahigashi

et al. 2003

YCF1 Vacuole transporter Saccharomycescerevisiae

A. thaliana Fourfold uptake of Cd Hydroponic Song et al.

2003

SMTA Selenocysteine

methyltransferase

A. bisulcatus Brassica juncea Volatilization and fivefold Se

accumulation from SeO42-

Agar LeDuc et al.

2004

CYP1A1,CYP2B6,CYP2C19

Cytochrome P450

monooxygenases

Human O. sativa Enhanced metabolism of atrazine,

norflurazon and metol achlor from

soil (should also metabolize PAHs)

Hydroponic Kawahigashi

et al. 2006

ppk Polyphosphate

kinase

bacteria Nicotianatabacum

2.5-fold higher Hg accumulation Soil Nagata et al.

2006

1582 R. Yadav et al.

123

formulating appropriate protocols for contaminated bio-

mass disposal, particularly for xenobiotic compounds,

heavy metals and radioactive wastes, and (g) protecting

wildlife from grazing on super-accumulators.

Additionally, overall success of phytoremediation is also

determined by government regulations and political will.

Since the remediation industry is accord controlled, phy-

toremediation technologies must validate their efficiency at

meeting State and Federal regulations. According to age of

contamination and relative bioavailability of the contami-

nants, changes in regulatory status and/or continuing

technical improvements will be necessary for commer-

cialization of phytoremediation technologies. Furthermore,

like any other novel technology, the adoption and public

acceptance is not spared from criticism and negative pro-

paganda by disparate groups. Such adverse regulations and

hurdles do not allow a novel technology to inflate its wings

and make headway in serving the community at large.

Conclusions

After complete genome sequencing of poplar (Tuskan et al.

2006) and other plants, diverse group of phytoremediation-

related genes have been determined to take action promptly

on environmental pollutants. The existence of numerous

catabolic enzymes and transporter sequences in genome

recommend that plants may have prosperous potential

within their tissues and organs to muster and detoxify

contaminants in their environment. Functional genomics

and proteomics knowledge acquired from the sequenced

plant species will significantly accelerate the phytoreme-

diation abilities in situ. Taking into account and accepting

the validation of metal accumulation by plants, however,

the major concern is the extent up to which poplar eco-

systems can be intended to clean metal pollutants and

contaminants. The fundamental scientific opinions would

have a broad managing implication for global sustainable

Fig. 3 Genes that could

potentially be introduced in

Populus for enhanced

phytoremediation in future

Perspectives for genetic engineering of poplars 1583

123

development of phytoremediation industries. Better

knowledge of rhizosphere processes, metal-seizure ave-

nues, pollutant uptake, translocation, chelation, degrada-

tion, and volatilization strategies as well as their

physiology and biochemistry would ameliorate achieving

goals in this highly capable field. Genetic strategies par-

ticularly genetic engineering of poplars for enhanced

phytoremediation potential, synthesis and excretion of

natural chelators into the root-zone, expansion of the rhi-

zosphere with the mutual aid of mycorrhizae would emerge

as the potent area for the success of phytoremediation

(Schafer et al. 1998).

Professor Milton P. Gordon (University of Washington,

USA) had initiated a project for enhancing phytoremedia-

tion in poplar using transgenic approach by assuming that

genetic engineering protocols and strategies would be used

more widely and efficiently to reduce environmental pol-

lution. Later on, the project was successfully accomplished

by Doty et al. (2007). Hence, integration of mammalian

cytochrome P450 gene in poplar provided sufficient evi-

dence for its suitability for genetic engineering and stable

expression of transgene for improvement of phytoremedi-

ation potential.

High accumulation properties of poplar species over

diverse trees and shrubs, grown on soils of variable metal

bioavailability and different chemical characteristics have

been authenticated by several authors (Spirochova et al.

2003; Chehregani and Malayeri 2007; Migeon et al. 2009).

In view of the speedy growth of poplar species along with

several magnificent features, the results from various

studies indicate that poplar suits best for long term man-

agement of phytoextraction of heavy metals and phyto-

remediation of pollutants. Moreover, genetic variations in

poplars are venerable and could offer a model source for

the collection of high-accumulating clones for phytoex-

traction (Pulford and Watson 2003). Finally, short-rotation

covert cultures of poplar generate high perennial biomass

over a short period of time and favor economically low-

cost reclamation systems and significant reduction of

contamination. Eventually, contaminated biomass can be

valorized either via paper, plywood, and timber production

or through recovery of metals after combustion.

Recommendations and future prospects

Photosynthetic power-driven pollutant-scavenging proper-

ties are alleged to form the basis of the remediative con-

sistency of plants attributed to phytoremediation that have

recently been documented. The comprehensive phyto-

remediation market revenue of 2005 is estimated around

US$ 235–400 million. This is presumed to continue to show

strong market growth in the new decade (Glass 2000). In

future, it may have highest probability to attract scientific

attention and perhaps the global remediation market would

grow progressively. Still, phytoremediation effectiveness is

restricted by a deficiency of knowledge of many vital plant

metabolic processes and plant–microbe interactions. There

is a need for further phytoremediation field studies to val-

idate the success of the technology and increase its indus-

trial public acceptance. Hyper-accumulator poplar species

not only accumulate exceptionally high amounts of essen-

tial metal ions, but can also absorb considerable amounts of

nonessential metal ions and critically reduce the toxic level

of contaminants. It utilizes photosynthetic potential to take

up ions from the soil, to subsist its nutritional requirements

and to congregate them in biomass. When present at

undesirably high levels, contaminants which are essential or

nonessential micronutrients are able to move across the

roots to trunk section of higher plants by virtue of being

chemical analogue to other nutrient ions. Phytoextraction is

aimed to exploit the nutrient acquisition system of plants in

order to achieve maximum of heavy metals and other

contaminants uptaken in the above-ground tissues. In the

industrialized world, above-ground biomass with high

degree accumulation is subsequently harvested after several

successive growth periods, thereby, removing the pollutant

from heavy-metal contaminated sites. Plant material can be

cindered by ignition and perhaps be recycled in metal

smelting, or deposited in unique dumps like timber and

furniture. Poplar meets the criteria of all mandatory

necessities if grown at Superfund sites such as (a) high

growth rate, (b) accumulate several trace elements in the

trunk region, (c) exhibit a high rate of biomass production,

(d) develop an extensive root system, (e) used in wood

industries and (f) amenable to in vitro culture, genetic

manipulation, and genetic mapping. Poplars are best known

candidates for cleaning up and detoxifying several organic

pollutants, such as chlorinated hydrocarbon solvents, the

herbicide atrazine, and a range of explosives (Komives and

Gullner 2000). It has also been reveled that Populus confers

remarkably high levels of the endogenous reducing and

detoxifying agent GSH (Noctor et al. 1998) and an efficient

Phase II detoxification construct made of the endogenous

co-substrate GSH and the enzyme system GST that cata-

lyzes the metabolism of potentially toxic electrophilic

compounds conjugation reaction (Komives et al. 2003b).

Genetic engineering approach could enhance phytoaccu-

mulation and phytoextraction potential of Populus by

introduction of novel indispensable traits. This genetics

based tree improving tool will definitely promote future

phytoremediation industries and Populus will prove a

model candidate for serving the ecosystem and the envi-

ronment. Photosynthesis fuelled cheaper alternatives of

environmental decontamination strongly facilitates com-

mercialization of phytoremediation.

1584 R. Yadav et al.

123

A second generation approach for toxic metal tolerance

in some metal hyperaccumulators is compartmentalization

of metals in the vacuole. Vacuoles perform as a main

storage site for metals in yeast and plant cells and the

phytochelatin binds to metals in cytoplasm to give phyto-

chelatin-metal complexes which are pumped into the vac-

uole in fission yeast (Schizosaccharomyces pombe)

(Kramer et al. 2000) and in plants (Ortiz et al. 1995). YCF1

is the most common vacuolar transporters and channels

involved in metal tolerance from Saccharomyces cerevisi-

ae. It is an ATP powered glutathione S-conjugate trans-

porter responsible for vacuolar sequestration of organic

compounds after their S-binding with glutathione, as well

as GSH–metal complexes. It mediates the transport of

cadmium, arsenic and mercury complexes into vacuoles

(Li et al. 1997). An overexpression of YCF1 in Arabidopsis

thaliana revealed that vacuoles of YCF1-transgenic plants

had a fourfold higher uptake of cadmium than those of

wild-type plants (Song et al. 2003). Thus, understanding of

metal uptake and translocation processes in higher plants at

genetic and molecular levels could provide a real break-

through on the way to engineering the ideal phytoreme-

diator.

The blend of advanced disciplines and technologies

could congregate valuable multitraits in poplar and evolve

it as an imperative component of future forests. Although,

poplar holds unique characteristics of heavy metal

absorption and translocation but no direct remediation

technology is available commercially. It is clear that the

exploitation of the remarkable potential of green plants to

bioaccumulate elements and compounds from the envi-

ronment and to execute biochemical transformations is

becoming a new frontier of plant biology. It is envisaged

that afforestation with remediation of engineered poplars

would undeniably improve biomass production, easy forest

product availability, and phytoremediation affinity. Future

plantation with transgenic poplars with several desirable

engineered traits, would modernize the scenario of the

forests, environment, ecology, and finally of the phyto-

remediation industries. Poplars are the top alternatives for

such investigations as they represent a model tree in for-

estry with a fully sequenced genome and are usually

employed in the phytoextraction of industry polluted zones

(Laureysens et al. 2004, 2005; Giachetti and Sebastiani

2006).

Acknowledgments Authors thank Dr. Suchitra Banerjee for critical

reading, valuable comments and suggestions on an earlier version

of this manuscript. This work was supported by the Department of

Biotechnology, Ministry of Science and Technology, Government of

India, New Delhi as a research project to Professor A. Chaudhury.

The financial support given as JRF by the Department of Biotech-

nology, Government of India to Mr. Rakesh Yadav is also gratefully

acknowledged.

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