Perspectives of bacterial ACCdeaminase in phytoremediationMuhammad Arshad, Muhammad Saleem and Sarfraz Hussain

Institute of Soil and Environmental Sciences, University of Agriculture, Faisalabad, 38040, Pakistan

Review TRENDS in Biotechnology Vol.25 No.8

Phytoremediation of contaminated soil and waterenvironments is regulated and coordinated by the plantroot system, yet root growth is often inhibited by pollu-tant-induced stress. Prolific root growth could maximizerates of hyperaccumulation of inorganic contaminants orrhizodegradation of organic pollutants, and thus accel-erate phytoremediation. Accelerated ethylene produc-tion in response to stress induced by contaminants isknown to inhibit root growth and is considered as a majorlimitation in improving phytoremediation efficiency.Recent work shows that bacterial 1-aminocyclopro-pane-1-carboxylate (ACC) deaminase regulates ethylenelevels in plants by metabolizing its precursor ACC into a-ketobutyric acid and ammonia. Plants inoculated withACC deaminase bacteria or transgenic plants that exp-ress bacterial ACC deaminase genes can regulate theirethylene levels and consequently contribute to a moreextensive root system. Such proliferation of roots incontaminated soil can lead to enhanced uptake of heavymetals or rhizodegradation of xenobiotics.

Phytoremediation and ACC deaminasePhytoremediation is an appealing, and cost-effective,strategy for cleaning up contaminated soil. Extensive rootgrowth is a prerequisite to maximizing the effectivenessof phytoremediation processes, whether the mechanismresponsible is hyperaccumulation, rhizodegradation, phy-tostabilization or hydraulic control. Root biomass haspreviously been found to be positively correlated withphytoremediation activity [1,2]. However, previous studieshave demonstrated that most contaminants, such as heavymetals and other xenobiotics, inhibited the root growth ofplants used for phytoremediation purposes [3–5]. Plants donot, therefore, accumulate sufficient biomass (particularlyroots) in heavily contaminated soils for effective remedia-tion. Elimination of this root growth inhibition representsa major challenge to the development of successful phytor-emediation technologies.

Reduced root growth in contaminated soils might beowing to stress-induced ethylene, an established phytohor-mone [6]. The biosynthesis of ethylene at higher rates inresponse to contaminant-induced stress(es) in plants iswell established [6,7]. Thus, a potential target for normalor extreme root growth is to regulate or limit the biosyn-thesis of ethylene.

Corresponding author: Arshad, M. (bio@fsd.comsats.net.pk).Available online 18 June 2007.

www.sciencedirect.com 0167-7799/$ – see front matter � 2007 Elsevier Ltd. All rights reserve

Interestingly some plant growth-promotingrhizobacteria (PGPR) contain the enzyme 1-aminocyclo-propane-1-carboxylate (ACC) deaminase, which metab-olizes ACC (an immediate precursor of ethylene inplants) into a-ketobutyric acid and ammonia, and thusregulates the biosynthesis of ethylene in inoculated plantroots [8]. It is now established that plants inoculated withrhizobacteria containing ACC deaminase activity or trans-genic plants expressing ACC deaminase genes producelonger roots and greater root density [9,10]. The resultantincrease in root growth provided by ACC deaminase mighttherefore enhance the effectiveness of phytoremediationprocesses in contaminated soil [11].

This review highlights the role of bacterial ACCdeaminase in phytoremediation of polluted soil, an emer-ging trend in phytoremediation research.

Ecology of bacteria containing ACC deaminaseThe ACC deaminase trait has been extensively studied innumerous soilmicrobial species, that is, bacteria, fungi andendophytes; however, this trait is most common amongplant growth-promoting rhizobacteria [8,12]. Rhizobac-teria might facilitate phytoremediation directly and/orindirectly. Direct mechanisms by which rhizobacteriaenhance phytoremediation include rhizodegradation sup-ported by root exudates and biotransformation of toxicelements. Indirect mechanisms include optimizing bioa-vailability of xenobiotics and toxic metals to hyperaccu-mulators for their detoxification and inactivation [13].Interestingly, there is substantial evidence suggesting thatrhizobacteria containing ACC deaminase activity protectplants from both biotic and abiotic stresses and dramatic-ally increase plant biomass; a desirable parameter forplants to be used for phytoremediation [14].

Most of the ACC deaminase bacteria are mesophilic [8];however, a psychrotolerant (cold tolerant) strain, Pseudo-monas putidaUW4, containingACCdeaminaseactivityhasalso been isolated that exhibited an ability to proliferate attemperatures as low as 4 8C [15]. It is very likely that mic-robial species exhibiting their optimum growth and ACCdeaminase activity at extreme environmental conditionsmight be useful in phytoremediation of sites occurring atenvironmental extremes. Moreover, there is a great vari-ation in ACC deaminase activity among rhizobacteria [8].

Biochemical and molecular aspects of ACCdeaminaseACC deaminase is a pyridoxal 5-phosphate(PLP)-dependent enzyme that degrades a cyclopropanoid

d. doi:10.1016/j.tibtech.2007.05.005

Review TRENDS in Biotechnology Vol.25 No.8 357

amino acid, ACC, to a-ketobutyrate and ammonia [12].Molecular mapping of ACC deaminase revealed that theremight be similar, in addition to several different, types ofdeaminase genes in different microbes. Alignment of theamino acid sequences from some of the genes indicates thatthere is conservation of key amino acid residues, forexample Lys51 and Cys162, both thought to be importantfor enzymatic activity [16]. Like other polymeric enzymes,ACC deaminase demonstrates a vast array of biochemicaland physical characteristics depending on the microbialspecies. Themolecularmass of ACC deaminase is up to 105000 Da and its subunit molecular mass ranges from 35 000to 41 800 Da.Moreover, the estimated numbers of subunitsare 2 or 3 [8].

Generally, ACC deaminase exhibits optimum activity ata pH close to 8; however, this might vary depending on themicrobial species. The Km values of this enzyme in anychemical reaction range from 1.5 to 4.6 mM, and the kcatvalues range from 146 to 290 min�1 [17–20]. The details ofthe reaction mechanisms followed by ACC deaminase toconvert ACC to a-ketobutyrate and ammonia can be foundelsewhere [21,22].

Mechanism of enhanced root growth by ACCdeaminase bacteriaSoil bacterial communities containing ACC deaminasestimulate root growth by lowering ethylene levels in plantroots.Glickandcoworkers [23]havehypothesizedamodel toexplain how ACC deaminase bacteria lower ethylene in theinoculated roots (Figure 1). A significant portion of the ACCcan be exuded from plant roots or seeds and subject tohydrolysis by ACC deaminase bacteria to produce ammoniaand a-ketobutyrate. The uptake and subsequent hydrolysisof ACC bymicrobes decreases the amount of ACC outside ofthe plant. To maintain the equilibrium between internaland external ACC levels, plantmust exude large amounts ofACC into the rhizosphere. Rhizobacteria containing ACC

Figure 1. Schematic representation of how bacteria containing ACC deaminase activity l

root elongation. Abbreviations: IAA, indole acetic acid; SAM, S-adenosyl-methionine. R

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deaminase activity stimulate ACC exudation from plantroots, providing microorganisms with a unique source ofcarbon and nitrogen (ACC). Consequently, the growth ofmicroorganisms containing ACC deaminase is accelerated,compared with other soil microorganisms, in the closevicinity of plant roots.As a result, the level ofACCdecreaseswithin the plant, which leads to a reduction in the endogen-ous biosynthesis of ethylene [23]. We have found a signifi-cant correlation between in vitroACC deaminase activity ofthe bacteria andgrowth-promotingactivity of these bacteriaunder axenic and natural (pot and field trials) conditions[24,25].

Hyperaccumulation of inorganic contaminantsBacteria containing ACC deaminase activity modulatestress-induced accelerated production of ethylene inplants, and might cause an enhanced uptake of inorganiccontaminants through modification of root architectureand of the root uptake system of the plant. Burd et al.[14] reported the greater potential of the ACC deaminasebacterium,Kluyvera ascorbata SUD165 (which is resistantto the toxic effects of nickel, lead, zinc and chromate ions),to protect canola (Brassica napus) and tomato (Lycopersi-con esculentum) seeds from the toxicity of high concen-trations of nickel chloride (NiCl2) grown under gnotobioticconditions. They attributed this effect to the ability of thebacterium to lower the level of stress ethylene induced bythe nickel. In another study, Burd and co-workers [26]recorded similar observations in the case of tomato (L.esculentum), canola (B. napus) and Indian mustard (B.napus) seeds, which were grown in soil for 25–42 daysin the presence of nickel, lead or zinc. Similarly, Belimovet al. [27] reported the potential of ACC deaminase bacteriato promote root and shoot growth of the seedlings of Indianmustard and rape (B. napus) grown in the presence of300 mM cadmium chloride (CdCl2) in the nutrient solution.Primarily in these studies, the use of ACC deaminase

ower the ethylene concentration and thereby prevent ethylene-caused inhibition of

eproduced, with permission, from Ref. [23].

358 Review TRENDS in Biotechnology Vol.25 No.8

bacteria exhibiting resistance to heavy metals was to testtheir potential to facilitate plant growth under stressconditions created by heavy metals. They concluded thatACC deaminase bacteria offer a great potential to be usedfor development of bacterial inocula for improvement ofplant growth under unfavorable environmental conditions,particularly for hyperaccumulators.

Belimov et al. [28] were the first to report a positivecorrelation between in vitro ACC deaminase activity of thebacteria and enhanced accumulation of cadmium in planttissues through enhanced root growth caused by bacteriacontaining ACC deaminase activity. They suggested thatACCdeaminase bacteria could be used for the developmentof plant-inoculant systems for phytoremediation of con-taminated soil environment. Recently, Wu et al. [29] alsofound that inoculation with a rhizobacterium, Pseudomo-nas putida 06909, caused a marked decrease in cadmiumphytotoxicity and increased metal accumulation in thesunflower plant root by up to 40%. However, they didnot investigate the rhizobacterium for ACC deaminaseactivity. It is very likely that in addition to other traits,this rhizobacterium might have ACC deaminase activitythat helped in reducing phytotoxicity of metal andenhanced its accumulation in plant roots. Likewise, Liet al. [30] have also reported that tobacco plants inoculatedwith Pseudomonas putida UW4 (containing ACC deami-nase activity) showed good growth and accumulated asubstantial amount of metal from nickel-contaminatedsoil.

Similar studies under controlled and natural conditionshave been conducted to assess the potential of transgenicplants and bacteria expressing ACC deaminase genes forhyperaccumulation of heavy metals. Such examples havebeen elaborated in the following sections.

Rhizodegradation and phyto-accumulation of organiccontaminantsRecent studies have indicated that biostimulation ofmicrobial activity in the rhizosphere of plants can accelerate

Figure 2. Relative removal of creosote from soil by using different approaches inclu

Bacteria containing ACC deaminase activity were used for inoculation in all cases exce

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the removal of various xenobiotics through variousbiochemical mechanisms commonly known as rhizodegra-dation or rhizoremediation [31,32]. Given that regulation ofethylene by ACC deaminase leads to a more extensive rootsystem of the plant, increased exploration of soil by rootsmight consequently enhance rates of rhizoremediation.Huang et al. [11] investigated the potential of different grassspecies inoculated with PGPR containing ACC deaminasefor phytoremediation of a creosote-contaminated soil envi-ronment. Enhanced biomass production, in terms of rootandshootdensities in response toACCdeaminaseactivityofPGPR, resulted in plants acting as an efficient sink forcreosote. In another study, Huang et al. [33] developedan improved multi-process phytoremediation system forremoval of polycyclic aromatic hydrocarbons from contami-nated soil. Creosote was selected as a test contaminant,and a multi-process phytoremediation system comprisedphysical (volatilization), photochemical (photooxidation),microbial remediation, and phytoremediation (plant-assi-sted remediation) approaches. The strategies used to realizethese processes included land-farming (aeration and lightexposure), bioaugmentation, PGPR containing ACC deami-nase activity, and plant growth of contaminant-tolerant tallfescue (Festuca arundinacea). After an experimental periodof four months, the average efficiency of removal of 16polyaromatic hydrocarbons (PAHs) by the multi-processremediation system was twice that of land-farming alone,50% more than bioremediation alone, and 45% more thanphytoremediation by itself. In this approach, bacteria con-taining ACC deaminase activity (Pseudomonas putidaUW3, Azospirillum brasilense Cd and Enterobactor cloacaeCAL2) played a major part because they enabled plants togrow under higher concentrations of PAHs by alleviatingstress on them. Such synergistic application of theseapproaches caused a rapid and massive biomass accumu-lation of plant tissue and consequently more uptake ofcontaminants from the soil environment (Figure 2). Veryrecently, Liu et al. [32] demonstrated that inoculation ofalfalfa withComamonas sp. strain CNB-1 not only removed

ding land farming, bioremediation, phytoremediation and multi-process system.

pt land farming. Reproduced, with permission, from Ref. [33].

Review TRENDS in Biotechnology Vol.25 No.8 359

4-chloronitrobenzene (4-CNB) completelywithin 1 or 2 daysfrom soil but also eliminated the phytotoxicity of 4-CNB toalfalfa plants. The authors did not investigate ACC deami-nase activity in this bacterium. It is very likely that thebacterium might have ACC deaminase activity in additionto someother specific traits,whichpromotedplant toleranceagainst the toxicity of 4-CNB.

Very little work has been conducted on the use of ACCdeaminase containing microorganisms in rhizoremedia-tion of organics-contaminated soil, but this novel approachcarries a great potential to be exploited.

Transgenic bacteria and plants with ACC deaminaseexpressionSophisticated tools of genetic engineering, togetherwith appreciation of biotechnological methods, are highlyeffective in facilitating the genetic manipulation of manycharacteristics into a single organism for achieving a defi-ned set of goals and objectives. To achieve the goal ofsuccessful bioremediation, scientists are striving hard tointroduce specific genes from one organism into others toaccelerate the vital process of phytoremediation. In thisdirection, the introduction of genes responsible for expres-sion of ACC deaminase in plants has received more atten-tion than in microorganisms. The major reasons for littlefocus on developing genetically engineered bacteria exp-ressing ACC deaminase could include: (i) the ACC deami-nase trait is already widely found among indigenous soilbacterial species; (ii) the degree of success in transformingthe ACC deaminase genes into plants has been extremelysuccessful, hence less emphasis has been placed on geneticengineering of microbial species; and/or (iii) the survivaland proliferation of transgenic bacteria in natural soilenvironments is often reduced.

Recently, Reed and Glick [34] compared the efficiency oftransgenic bacteria that carry ACC deaminase with controlbacteria in promoting seed germination and root elongationunder stress conditions caused by copper or PAHs in con-taminated soils. They reported that both native and trans-formedPseudomonas aspleniiACwere equally useful in thepromotion of seed germination and root elongation understress conditions caused by copper or PAH contamination.This could be because the efficiency of transgenic inoculatedstrains is determined by several biotic and abiotic factorssuch as soil pH, temperature, moisture content and theircompetition with native soil microflora and microfauna.Under such conditions, the use of geneticallymodified endo-phytes that exhibit ACC deaminase activity coupled withxenobiotic-degrading characteristics might yield morepromising results than non-endophytes [35]. As describedelsewhere, the ACC deaminase trait has also been found insome endophytes. Therefore, the selection of endophyteshaving both ACCdeaminase and specific degradation genescould also be a useful approach for developing a successfulphytoremediation strategy.

The plant growth promotion observed in response toinoculation with bacteria containing ACC-deaminasehas prompted scientists to develop transgenic plantsthat express ACC deaminase genes [36]. To date, manyplant species have been genetically engineered withACC deaminase expression to protect the plant against

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multiple biotic and abiotic stresses [37–40]. Grichko et al.[41] expressed bacterial ACC deaminase in tomato(Lycopersicum esculentum) cv. Heinz 902 under the tran-scriptional control of either two tandem 35S cauliflowermosaic virus promoters (constitutive expression), the rolDpromoter from Agrobacterium rhizogenes (root-specificexpression) or the pathogenesis-related prb-1b promoterfrom tobacco. The growth of transgenic tomato plants inthe presence of cadmium, copper, cobalt, magnesium,nickel, lead or zinc was monitored. Parameters testedwere metal accumulation and ACC deaminase activityin both plant shoots and roots, root and shoot develop-ment, and leaf chlorophyll content. Transgenic tomatoplants expressing ACC deaminase particularly controlledby the prb-1b promoter accumulated larger amounts ofmetals within the plant tissues. However, because thetomato (L. esculentum) plants are unlikely to be used inthe phytoremediation of contaminated sites, Nie et al. [42]expressed ACC deaminase genes in canola (B. napus)plants and tested their potential to grow in the presenceof high levels of arsenate in the soil formetal accumulationin plant tissues. They also tested the ability of the plantgrowth-promoting bacteriumE. cloacaeCAL2 to facilitatethe growth of both non-transformed and ACC deaminase-expressing canola (B. napus) plants for developing a suc-cessful phytoremediation strategy. In all cases, transgeniccanola (B. napus) expressing ACC deaminase genes accu-mulated larger amounts of arsenate from the contami-nated soil than non-transformed canola plants. Recently,Stearns et al. [9] reported similar results in the case ofphytoremediation of a nickel-contaminated soil environ-ment. They observed that the growth of transgenic plantsconstructed through rolD promoters demonstrated moregrowth under high nickel concentration compared withcontrol (non-transgenic) and other transgenic plants con-structed through (CaMV) 35 S and prb-1b promotersexpressing ACC deaminase genes (Figure 3).

Recently, Farwell et al. [43] reported the successfulutilization of transgenic canola (rolD) and plant growth-promotingbacteria,P.putida strainUW4andPseudomonasputida strain HS-2, in phytoremediation of a nickel-con-taminated soil in situ. They investigated the potential ofseedlings of non-transformed and transgenic canola (B.napus) (rolD) in the presence and absence of eitherP. putidastrain UW4 orP. putida strain HS-2 in phytoremediation ofa nickel-contaminated field site. The most interesting fea-ture of this study was that ACC deaminase bacteriaenhanced plant growth of both transgenic and non-trans-formed canola, resulting in an approximate 10% increase intotal nickel per plant compared with plants that were notinoculated. In another study, Farwell et al. [44] comparedthe growth of transgenic canola (B. napus) expressing ACCdeaminase with non-transformed canola, both inoculatedwith the plant growth-promoting bacterium P. putidaUW4containing ACC deaminase activity, under multiple stres-ses, such as flooding and elevated soil nickel concentration,in situ. They reported that flooding reduced the growth ofcanola; however,nickelaccumulation in transgenicandnon-transgenic plant tissues was increased.

Results of these studies strongly encourage intensivefuture research on the role of transgenic plants expressing

Figure 3. Three-week-old canola plants. (a) Non-transformed plant in unspiked soil. (b) 35S plant in unspiked soil. (c) rolD plant in unspiked soil. (d) Non-transformed plant

in nickel-spiked soil. (e) 35S plant in nickel-spiked soil. (f) rolD plant in nickel-spiked soil. Reproduced, with permission, from Ref. [9].

360 Review TRENDS in Biotechnology Vol.25 No.8

ACC deaminase genes and ACC deaminase bacteria inphytoremediation under different soil conditions.

Future prospects and recommendationsAnthropogenic environmental contamination mightincrease in the future owing to rapid population growthand, consequently, a boost in industrialization, urbaniz-ation and intensive agriculture around the globe. Tomitigate the ill-effects of soil contamination caused byxenobiotic chemicals and heavy metals, phytoremediationmight be a more effective and affordable approach tocleaning up polluted soil ecosystems. However, there is aneed for further intensive research to develop successfulphytoremediation technologies. In particular, attentionshould be paid to the following aspects.

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oncerted efforts should be focused on increasing thepopulation of soil microbial communities containingACC deaminase activity in polluted soil environmentsby inoculation of roots or seeds of hyperaccumulators.

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enetic manipulation of hyperaccumulating plantsexpressing ACC deaminase and other contaminant-specific genes [45–46] might be a new breakthrough in

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terms of accelerated removal of heavy metals andxenobiotics and rhizodegradation of xenobiotics in thesoil environment.

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ikewise, selection of native or engineered endophytesexpressing both ACCdeaminase and other contaminant-specific genes [47] should be more intensively exploitedfor developing an effective phytoremediation strategy.

Concluding remarksVigorous growth of hyperaccumulators caused by ACCdeaminase bacteria could help not only in acceleratingthe rhizodegradation of xenobiotics but might also enhancethe uptake of heavy metals and xenobiotics. Using trans-genic hyperaccumulator plants expressing ACC deaminasecould also be an excellent strategy for phytoremediation ofcontaminated soil environments. The studies describedhave unequivocally demonstrated the validity of this novelapproach for tackling the problem of soil and environmentalcontamination by heavy metals and xenobiotics success-fully. However, intensive future research is needed toexplore various aspects of this approach to make it moreuseful.

Review TRENDS in Biotechnology Vol.25 No.8 361

AcknowledgementsFinancial support for this study was provided by Higher EducationCommission (HEC), Islamabad, Pakistan. We also thank Mary BethLeigh, Assistant Professor of Microbiology, Department of Biology,University of Alaska Fair Banks, USA, and Norman T. Uphoff, CornellInternational Institute for Food, Agriculture and Development (CIIFAD),Cornell University, Ithaca, NY, USA, for editing and technical input andguidance during the preparation of this manuscript.

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47 Barac, T. et al. (2004) Engineered endophytic bacteria improvephytoremediation of water-soluble, volatile, organic pollutants.Nature Biotechnol. 22, 583–588

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