promotion of plant growth by bacterial acc deaminase

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Promotion of Plant Growth by Bacterial ACC DeaminaseBernard R. Glicka; Biljana Todorovica; Jennifer Czarnya; Zhenyu Chenga; Jin Duana; BrendanMcConkeya

a Department of Biology, University of Waterloo, Waterloo, Ontario, Canada

To cite this Article Glick, Bernard R. , Todorovic, Biljana , Czarny, Jennifer , Cheng, Zhenyu , Duan, Jin and McConkey,Brendan(2007) 'Promotion of Plant Growth by Bacterial ACC Deaminase', Critical Reviews in Plant Sciences, 26: 5, 227— 242To link to this Article: DOI: 10.1080/07352680701572966URL: http://dx.doi.org/10.1080/07352680701572966

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Critical Reviews in Plant Sciences, 26:227–242, 2007Copyright © Taylor & Francis Group, LLCISSN: 0735-2689 print / 1549-7836 onlineDOI: 10.1080/07352680701572966

Promotion of Plant Growth by Bacterial ACC Deaminase

Bernard R. Glick, Biljana Todorovic, Jennifer Czarny, Zhenyu Cheng, Jin Duan,and Brendan McConkeyDepartment of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1

Table of Contents

I. INTRODUCTION ........................................................................................................................................... 228

II. MECHANISMS USED BY PLANT GROWTH-PROMOTING BACTERIA .................................................... 228A. Indirect Mechanisms .................................................................................................................................. 228B. Direct Mechanisms .................................................................................................................................... 228

III. ETHYLENE AND STRESS ............................................................................................................................. 229A. Effects of Ethylene on Plants ...................................................................................................................... 229B. Genes Involved in Stress Signaling and Response ......................................................................................... 229

IV. ACC DEAMINASE ......................................................................................................................................... 230A. Biochemistry ............................................................................................................................................. 230B. Protein Structure ....................................................................................................................................... 231C. Genes and Distribution ............................................................................................................................... 232D. Transcriptional Regulation ......................................................................................................................... 233

V. BACTERIA WITH ACC DEAMINASE .......................................................................................................... 235A. Plant Growth Promotion ............................................................................................................................. 235B. Amelioration of Stress ............................................................................................................................... 235C. Introducing ACC Deaminase into Other Bacteria .......................................................................................... 236D. Bacterial Genes Activated by Root Exudates ................................................................................................ 236E. Plant Gene Expression Modified by Bacteria with ACC Deaminase ............................................................... 237F. Ethylene-IAA Cross-talk ............................................................................................................................ 237

VI. CONCLUSION ............................................................................................................................................... 238

REFERENCES .......................................................................................................................................................... 238

To date, there has been only limited commercial use of plantgrowth-promoting bacteria in agriculture, horticulture, and sil-viculture. However, with recent progress toward understandingthe mechanisms that these organisms utilize to facilitate plantgrowth, the use of plant growth-promoting bacteria is expectedto continue to increase worldwide. One of the key mechanismsemployed by plant growth-promoting bacteria to facilitate plantgrowth is the lowering of plant ethylene levels by the enzyme 1-

Address correspondence to B.R. Glick, Department of Biology,University of Waterloo, 200 University Avenue West, Waterloo, On-tario, Canada N2L 3G1. E-mail: [email protected]

aminocyclopropane-1-carboxylate (ACC) deaminase. This articlereviews the published work on this enzyme, with an emphasis on itsbiochemistry, protein structure, genes, and regulation. In addition,this article provides some initial insights into the changes in bothplants and ACC deaminase-containing plant growth-promotingbacteria as a consequence of plant-microbe interactions. Finally, abrief discussion of how bacterial ACC deaminase and indoleaceticacid (IAA) together modulate plant growth and development isincluded.

Keywords Plant growth-promoting bacteria, plant stress, ethylene,IAA

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228 B. R. GLICK ET AL.

I. INTRODUCTIONIncreased public concern about environmental problems

caused either directly or indirectly by the use of fertilizers, pes-ticides, herbicides, and fungicides, has prompted researchersto consider alternatives to these established chemical strate-gies for facilitating plant growth in agriculture, horticulture,and silviculture. Ideally, replacements for the chemicals thatare currently in widespread use should not only enhance plantgrowth, but should also inhibit plant pathogens. One potentialalternative may be the use of plant growth-promoting bacte-ria (Brown, 1974; Kloepper et al., 1986, 1988, 1989; Davison,1988; Glick et al., 1999; Lambert and Joos, 1989). These plant-beneficial bacteria can bind to either roots (rhizosphere bacte-ria), leaves (phyllosphere bacteria), or they may exist withinplant tissues (bacterial endophytes). The highest concentrationsof these microorganisms typically exists around the roots, inthe rhizosphere, most probably due to the high levels of nu-trients exuded from the roots of many plants that can be uti-lized by bacteria to support their growth (Whipps, 1990). Alarge number of plant growth-promoting bacteria have beenisolated to date, each with one or more traits that might, un-der the appropriate conditions, enhance plant growth. Someof these bacteria may directly influence plant growth, e.g., bysynthesizing plant hormones or facilitating uptake of nutrientsfrom the soil. Others exert their beneficial effects indirectlyvia biological control, whereby they limit the growth of phy-topathogens that would otherwise inhibit plant growth (Glick,1995).

At the present time, plant growth-promoting bacteria areused commercially, albeit to a limited extent, in a numberof different countries worldwide in agriculture, horticulture,silviculture and environmental remediation (Reed and Glick,2004; Fravel, 2005). One reason for the somewhat limitedcommercial use of plant growth-promoting bacteria is the re-ported variability and inconsistency of results between labo-ratory, greenhouse, and field trials (Mishustin and Naumova,1962). In addition, to date in more developed countries, therelatively low cost of agrochemicals, including fertilizers, hasprecluded any serious consideration of the use of plant growth-promoting bacteria. Past inconsistent and irreproducible re-sults with plant growth-promoting bacteria may reflect varia-tions in crops and cultivars, soil composition, the presence ofindigenous soil microorganisms, weather, soil moisture con-tent and, perhaps most importantly, an incomplete understand-ing of the mechanisms employed by plant growth-promotingbacteria to facilitate plant growth. Notwithstanding the largenumber of potential variables involved in the effective and re-producible use of plant growth-promoting bacteria, in recentyears considerable progress has been made toward develop-ing a better understanding of many of the mechanisms thatthese organisms utilize and there is every reason to expect theuse of plant growth-promoting bacteria to continue to increaseworldwide.

II. MECHANISMS USED BY PLANTGROWTH-PROMOTING BACTERIA

A. Indirect MechanismsThe ability of plant growth-promoting bacteria to act as bio-

control agents against phytopathogens and thus indirectly stim-ulate plant growth may be the consequence of any one of avariety of mechanisms including antibiotic production, deple-tion of iron from the rhizosphere, induced systemic resistance,production of fungal cell wall lysing enzymes, and competi-tion for binding sites on the root. The mechanism that is mostcommonly associated with the ability of a biocontrol strain toinhibit phytopathogens is the production of one or more an-tibiotics (Haas et al., 1991; Keel et al., 1992; Chet and In-bar, 1994; Whipps, 1997). Some biocontrol bacteria can inhibitthe growth of pathogens by synthesizing low molecular masssiderophores that bind most of the iron in the rhizosphere withan extremely high avidity, thereby thwarting the proliferation offungal pathogens in the vicinity of the host plant roots becauseof a lack of available iron (Castignetti and Smarrelli, 1986;O’Sullivan and O’Gara, 1992; Dowling et al., 1996). Some bio-control plant growth-promoting bacteria produce enzymes suchas chitinase, β1,3glucanase, protease, or lipase, all of which canfacilitate the lysis of fungal cells (Chet and Inbar, 1994). Inter-estingly, the defense response of stressed plants often results inthe production of the same array of proteins (i.e., chitinase, β1,3-glucanase, protease, and lipase) in response to pathogen inducedstress (van Loon et al., 2006). Other biocontrol plant growth-promoting bacteria can protect plants from phytopathogens byout-competing them for nutrients and for niches on the root sur-face thereby effectively preventing pathogens from binding toand infecting the plant (Kloepper et al., 1988; O’Sullivan andO’Gara, 1992; Loper et al., 1997). In addition to the more ob-vious methods of biocontrol, long-lasting and broad spectrumsystemic resistance to a variety of pathogens can be induced byvarious plant growth-promoting bacteria or microbial metabo-lites (Kessmann et al., 1994; Tuzun and Kloepper, 1994; vanLoon et al., 1997, 2006; Vallad and Goodman, 2004; Verhagenet al., 2004).

B. Direct MechanismsThere are several ways in which plant growth-promoting

bacteria can directly facilitate plant growth. They may fix at-mospheric nitrogen and supply it to plants—often a minorcomponent of the benefit that the bacterium provides to theplant; synthesize siderophores which can sequester iron fromthe soil and provide it to plant cells which can take up thebacterial siderophore-iron complex; synthesize phytohormonessuch as auxins, cytokinins and gibberelins, which can act to en-hance various stages of plant growth; solubilize minerals suchas phosphorus, making them more readily available for plantgrowth; and synthesize the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which can lower plant ethylene


PLANT GROWTH BY BACTERIAL ACC DEAMINASE 229

levels (Brown, 1974; Kloepper et al., 1986, 1989; Davison,1988; Lambert and Joos, 1989; Glick, 1995; Patten and Glick,1996). A bacterium may directly affect plant growth and devel-opment using any one or more of these mechanisms. Since manyplant growth-promoting bacteria possess several of these traits,a bacterium may utilize different traits at various times duringthe life cycle of the plant. Moreover, plant growth-promotingbacteria typically have little or no measurable effect on plantgrowth when the plants are cultivated under optimal and stress-free conditions.

Until recently, the mechanism that has been most often in-voked to explain the various direct effects of plant growth-promoting bacteria on plants is the production of phytohor-mones, and most of the attention has focused on the role of thephytohormone auxin (Brown, 1974; Tien et al., 1979; Pattenand Glick, 1996; Garcia de Salamone et al., 2005). However,in the last few years it has been found that a number of plantgrowth-promoting bacteria contain the enzyme ACC deaminase(Klee and Kishore, 1992; Jacobson et al., 1994; Glick et al.,1995, 1998; Burd et al., 1998; Kaneko et al., 2000; Belimovet al., 2001, 2005; Kaneko et al., 2002; Babalola et al., 2003;Ma et al., 2003; Ghosh et al., 2003; Dey et al., 2004; Mayaket al., 2004a; Hontzeas et al., 2005; Dell’Amico et al., 2005;Madhaiyan et al., 2006; Shaharoona et al., 2006a; Blaha et al.,2006; Saravanakumar and Samiyappan, 2006) and that this en-zyme can cleave the plant ethylene precursor ACC, and therebylower the level of the phytohormone ethylene in a developing orstressed plant. The major portion of this review article is focusedon the enzyme ACC deaminase (and its genes) and its centralrole in promoting plant growth.

III. ETHYLENE AND STRESS

A. Effects of Ethylene on PlantsIn higher plants, the enzyme S-adenosyl-L-methionine

(SAM) synthetase catalyzes the conversion of methionine toSAM (Giovanelli et al., 1980); ACC synthase catalyzes the hy-drolysis of SAM to ACC and 5’methylthioadenosine (Kende,1989); and ACC oxidase catalyzes the conversion of ACC toethylene, carbon dioxide, and hydrogen cyanide (John, 1991).The plant hormone ethylene plays an important role in root ini-tiation and elongation, nodulation, senescence, abscission andripening as well as in stress signaling (Mattoo and Suttle, 1991;Abeles et al., 1992; Arshad and Frankenberger, 2002). When ap-plied exogenously, it causes adventitious root formation and roothair initiation. It also begins the process of fruit ripening, flowerwilting, and leaf senescence. When produced endogenously dur-ing developmental processes, ethylene regulates xylem forma-tion, flowering in some plants, and induces fruit ripening as wellas flower wilting. As part of a stress response, it inhibits rootelongation, nodulation and auxin transport, induces hypertro-phies, speeds aging and promotes senescence and abscission.Along with auxin, ethylene regulates lateral root initiation andexudation of resins and gums (Abeles et al., 1992; Prayitno

et al., 2006; Sun et al., 2006). However, it should be mentionedthat there is an important distinction between ethylene responsesthat are due to an increase in ethylene concentration within planttissues and the increase in the sensitivity of plant tissues to ethy-lene. Unfortunately, the mechanisms that make cells responsiveto ethylene are still not fully understood.

B. Genes Involved in Stress Signaling and ResponsePlants have to respond to a number of environmental assaults,

which can be placed in two broad categories: abiotic stresses, in-cluding drought, flooding, cold, nutritional stress, heavy metalsand high salt; and biotic stresses, which are caused by differenttypes of pathogens including those that are either biotrophic ornecrotrophic.

Defenses against biotrophic pathogens are thought to relymostly on salicylic acid (SA) mediated responses, whereasthose against necrotrophic pathogens rely mostly on jasmonicacid (JA) and ethylene mediated responses (Glazebrook, 2005).Even symbiotic plant-microbe relationships induce a defense re-sponse (Timmusk and Wagner, 1999). In addition to this, bioticstress responses involve reactive oxygen species (ROS), calciumfluctuations, micro RNAs and many other signaling molecules(Broekaert et al., 2006; Navarro et al., 2006). Abiotic stressesinduce biosynthesis of ABA and ethylene, calcium fluctuations,ROS evolution, and activation of a host of transcription factors.

Plants have developed response mechanisms for each of thesetypes of stress, yet each pathway interacts extensively with othersignaling cascades. Thus, there are multiple instances of syn-ergy and antagonism, and responses to different stresses forma complex network of signaling pathways that often overlap.In turn, activation of particular signaling cascades enables aplant to modify the nature and amount of many of its proteinsin response to the stress. Understanding these signal cascadesmay be the key to unraveling the complex patterns of stressresponse in plants. A list of some of the key genes involvedin stress response signaling is presented in Table 1 (Shinozakiet al., 2003; Fujita et al., 2006). In particular, the DRE-bindingprotein (DREB) family of transcription factors is responsiblefor signaling the response to many abiotic stresses and mem-bers of this family are similar to genes in the ethylene responsepathway, i.e., the ethylene-responsive element binding proteins(EREBP) which also show altered expression in response to var-ious stresses (Broekaert et al., 2006). Other important signalingmolecules include some of the members of the protein kinasefamily which act to link biotic and abiotic stress responses. Forinstance, in Arabidopsis the MAP-kinases MPK6 and MPK3are upregulated during biotic and abiotic stress and functionto phosphorylate ACC synthase stabilizing this enzyme andthereby increasing ethylene biosynthesis (Liu and Zhang, 2004).These examples illustrate points of convergence between differ-ent stress response networks, where ethylene signaling plays apivotal role.



TABLE 1Some proteins involved in plant stress responses

Transcription factor family Inducing stress Function

DRE-binding protein (DREB) family Cold, drought, high salt, osmotic Bind to dehydration responsiveelements (DRE) within the promotersabiotic stress genes

Ethylene-responsive element bindingfactor (EREBP) family

High salt, drought, cold, wounding,pathogen infection and ethylenetreatment

Binds to the GCC box of ethyleneinducible genes

Some zinc-finger proteins Cold, drought Interact with bZIP proteins, enhancesfreezing tolerance

Some WRKY transcription factors Cold, drought, biotic Bind to elements within pathogenesisrelated genes

Some MYB transcription factors Botrytis infection, cold, drought Bind (with AtMYC2) to a cis-element inthe dehydration-inducible RD22 gene.May also mediate biotic and abioticstress through ROS

Basic helix-loop-helix (bHLH) family Cold, drought Multiple hormone signaling pathwayssuch as ABA, JA and JA/Ethylenemediated pathogen defense

Basic-domain leucine zipper (bZIP)family

Cold, drought Involved in ABA signalling

NAC family JA, H2O2, pathogen infection, drought,high salinity and ABA treatment

Turns on genes involved in ROSsignaling and detoxification, defenseand senescence

Other signaling proteinsSome MAP-kinases (i.e.,MPK6/MPK3)

Wounding, osmotic shock, UV, salinity,drought, ozone, temperature extremes,and pathogen infection

Induce ethylene biosynthesis through anincrease in ACC synthase activity

DEAD-box helicases Abiotic stress Unwinding of energetically stable DNAor RNA duplexes

Glutathione Abiotic and biotic stress Responds to changes in photosynthesis

IV. ACC DEAMINASEThe enzyme ACC deaminase (E.C. 4.1.99.4) cleaves ACC,

the immediate precursor of ethylene in plants, to form ammoniaand α-ketobutyrate. This multimeric enzyme is a common com-ponent of many soil microorganisms, both bacteria and fungi. Ithas also been suggested, based largely on sequence similarities,that some plants may contain ACC deaminase genes (Sterkyet al., 1998). However, it has not yet been unequivocably demon-strated that these putative ACC deaminase genes encode an en-zyme with ACC deaminase activity.

A. BiochemistryThe pioneering work in elaborating the biochemical proper-

ties of ACC deaminase is largely the results of studies carriedout by Honma and his co-workers (Honma and Shimomura,1978; Walsh et al., 1981; Honma, 1985; Honma et al., 1993a, b; Minami et al., 1998; Jia et al., 1999; Ose et al., 2003).However, a few biochemical studies of this enzyme have also

been reported by other laboratories (Liu et al., 1984; Jacobsonet al., 1994; Li et al., 1996; Zhao et al., 2003; Hontzeas et al.,2004a).

ACC deaminase is a multimeric enzyme (homodimeric orhomotrimeric) with a subunit molecular mass of approximately35-42 kDa. It is a sulfhydryl enzyme in which one moleculeof the essential co-factor pyridoxal phosphate (PLP) is tightlybound to each subunit. Interestingly, the enzyme ACC deami-nase is cytoplasmically localized (Jacobson et al., 1994) so thatthe substrate ACC must be exuded by plant tissues (Penroseet al., 2001; Penrose and Glick, 2001) and subsequently takenup by an ACC deaminase-containing microorganism before itis cleaved (Glick, 1998).

Measurement of the Km of various ACC deaminases for ACCindicate that the enzyme does not bind the substrate with ahigh affinity; Km values range from 1.5 to 17.5 mM (Honmaand Shimomura, 1978; Klee and Kishore, 1992; Honma, 1993;Hontzeas et al., 2004a). This has been interpreted as indicatingthat in order to compete with ACC oxidase for ACC, ACC



deaminase must be present in much greater amounts, i.e., from100- to 1000-fold (Glick, 1998). Moreover, it was suggestedthat since plant ACC levels are usually in the micromolar range,the amount of substrate will nearly always be lower than the Km

and for every increase in the plant ACC concentration there willbe a parallel increase in the rate of ACC cleavage (Glick, 2005).

The PLP-dependent enzymes catalyze a wide variety ofbiochemical reactions, many of which are involved in themetabolism of amino acids. In most of these reactions two basicchemical properties of the PLP have been conserved: (i) PLPforms an external aldimine between its aldehyde group and theα-amino group of the substrates and (ii) PLP acts as an electronsink, withdrawing electrons from the substrate (John, 1995). Asa PLP-dependent enzyme, the ACC deaminase ring opening re-action starts with conversion from an internal aldimine betweenthe enzyme and PLP to an external aldimine between the sub-strate ACC and PLP. In most other PLP-dependent reactions, thenext step is the nucleophilic abstraction of either an α-proton oran α-carboxylate group. However, in this regard the ACC deam-inase catalyzed reaction is considered as a special case, sincethe substrate does not contain a α-proton and the carboxylategroup is retained in the product, ruling out the usual mechanism.As such, studies of this unusual enzyme have been conductedwith the intent to unravel the mechanism of the ring opening.Walsh et al. (1981) proposed two possible routes for the ringfragmentation: (i) nucleophilic addition at the Cβ methylene po-sition followed by β-proton abstraction and (ii) direct β-protonabstraction leading to the cyclopropane ring cleavage. The exactmechanism is still unknown, with data to support both routes(i) and (ii) (Ose et al., 2003; Karthikeyan et al., 2004a, b). Thisnotwithstanding, Hontzeas et al. (2006) have argued, on the ba-sis of theoretical considerations and following the mechanisticstudies of Li et al. (1996), that mechanism (i) is most likely tobe operative in the cleavage of ACC.

B. Protein StructureTo gain further insight into the functioning of this PLP-

dependent enzyme, the crystal structures of a bacterial (Pseu-domonas sp. ACP) and yeast (Hansenula saturnus) ACC deam-inase and an ACC deaminase homologue without this activ-ity (from Pyrococcus horikoshii) have been determined (Yaoet al., 2000, Ose et al., 2003; Karthikeyan et al., 2004a; Fujinoet al., 2004). The crystal structures, along with site-specific mu-tagenesis studies, have allowed for identification of the essen-tial amino acid residues for catalysis and substrate recognition.These studies have indicated that ACC deaminase folds to formtwo domains, each of which has an open twisted α/β structuresimilar to the β-subunit of the PLP-dependent enzyme trypto-phan synthetase.

An amino acid sequence alignment of several enzymes iden-tified as ACC deaminase and some putative ACC deaminasesis shown in Figure 1. The alignment illustrates that most ofthe amino acid residues that are known to be important are

conserved, and the H. saturnus and Pseudomonas sp. ACCdeaminase active sites are virtually identical. The lysine residuethat binds PLP, the tyrosine residue that stacks with the pyridinering, and residues important in recognizing the substrate ACCare all conserved. In addition, mutational studies with H. satur-nus ACC deaminase have confirmed the importance of Lys51,Ser78, Tyr295 and Glu296, as changes to any of these aminoacid residues leads to complete loss of activity. In addition,substitution of Tyr269 leads to a reduction in specific activity toless than 10% of the wild type enzyme (Ose et al., 2003). Thesame results have been obtained for the mutational analysis atresidues Ser78 and Tyr294 (H. saturnus corresponding residueis Try295) of the Pseudomonas sp. enzyme (Karthikeyanet al., 2004b). However, the precise role that some of the abovementioned residues play in the catalysis still remains to beelucidated. Recent work has focused on determining the enzy-matic residues involved in nucleophilic addition and β-protonabstraction. Lys51 (Ose et al., 2003) and Tyr294 (Karthikeyanet al., 2004a,b) are two major candidates proposed to be in-volved in nucleophilic addition; however, it has been suggestedthat Ser78 also plays a role in nucleophilic addition (Zhaoet al., 2003). As far as direct β-proton abstraction, thus far, onlyLys51 has been proposed to be involved (Zhao et al., 2003).

The determination of the three-dimensional X-ray crystallo-graphic structure of the ACC deaminase homologue from Py-rococcus horikoshii has allowed for further insight into whatmakes the ACC deaminase reaction unique. The Pyrococcushorikoshii enzyme was originally predicted to be ACC deam-inase due to its sequence similarity to other ACC deaminaseenzymes. But while the ACC deaminase homologue preservesthe key amino acid residues (Figure 1), it does not have ACCdeaminase activity. Instead, this enzyme shows specificity for D-and L-serine, producing pyruvate as a result of catalysis (Fujinoet al., 2004). The overall topology of the ACC deaminase ho-mologue is very similar to that of H. saturnus and Pseudomonassp. ACC deaminases; the residue arrangement in the active siteis very similar between ACC deaminase from H. saturnus andthe ACC deaminase homologue, with only a few substitutions.These include a change from Gln77 in H. saturnus to His80 inthe homologue, a change from Glu296 in H. saturnus to Thr283in the homologue and a change from Leu323 in H. saturnus toThr308 in the homologue. The inertness of the ACC deaminasehomologue towards ACC is explained by the change in the elec-tron density of the ACC cyclopropane ring, which is influencedby the pyridine ring of PLP within the active site (Fujino et al.,2004). The charge density of the pyridine ring is modulated byresidues in the vicinity of the pyridine nitrogen atom. The pyri-dine nitrogen atom of H. saturnus ACC deaminase exists withinhydrogen bonding distance to the side-chain oxygen atom ofGlu296, whereas in the P. horikoshii ACC deaminase homo-logue, the hydroxyl group of Thr308 side-chain approaches thecorresponding nitrogen atom (Figure 2). The threonine or serineside-chain at this position has been observed in other membersof the trypthophan synthase β-subunit (TRPSβ) family, which



FIG. 1. Amino acid sequence alignment of the annotated ACC deaminases, including the putative ACC deaminases. The conserved lysine residue that binds thePLP cofactor and other conserved residues are marked with a black box. The residues that seem to approach the pyridine nitrogen atom of PLP and thus play aregulatory role are marked with a black arrow. The alignment was created using MUSCLE (Edgar, 2004) with default parameters.

includes TRPSβ, O-acetylserine sulfhydrylase, threonine deam-inase, and threonine synthase, such that the ACC deaminase ho-mologue is more similar to these enzymes at these two positions(Hyde et al., 1988; Burkhard et al., 1998; Gallagher et al., 1998;Thomazeau et al., 2001). In addition, true ACC deaminases areexpected to have a leucine residue (Leu323 for H. saturnus)in close proximity to the Glu296 residue. In the ACC deami-nase homologue, this position is occupied by another threonineresidue. In the three-dimensional structure, the leucine residueprovides space for the long side-chain of the glutamate residueby orienting itself in the opposite direction (Figure 2.; Fujinoet al., 2004). Hence, the putative ACC deaminases that containthreonine residues at these positions are expected to be unableto utilize ACC as a substrate, however, this still remains to beconfirmed experimentally with enzymes other than the ACCdeaminase homologue.

C. Genes and DistributionACC deaminase activity has been found to be associated

with a large number of different soil microorganisms (Kleeet al., 1991; Sheehy et al., 1991; Klee and Kishore, 1992; Jacob-son et al., 1994; Glick et al., 1995, 1998; Campbell and Thom-son, 1996; Burd et al., 1998; Jia et al., 1999; Kaneko et al., 2000,2002; Belimov et al., 2001, 2005; Babalola et al., 2003; Maet al., 2003; Ghosh et al., 2003; Mayak et al., 2004a; Khalidet al., 2004; Dey et al., 2004; Hontzeas et al., 2005; Dell’Amicoet al., 2005; Blaha et al., 2006; Madhaiyan et al., 2006; Shaha-roona et al., 2006a, b; Saravanakumar and Samiyappan, 2006;Hameeda et al., 2006). Moreover, it is found at a relatively highfrequency in many rhizosphere soils (Klee and Kishore, 1992;

Glick et al., 1995; Blaha et al., 2006; Duan et al., unpublishedresults). Interestingly, within a particular genus and speciesof microorganism, some strains have ACC deaminase activity,and others often do not. Thus, for example, while some strainsof Azospirillum have recently been reported to contain an acdSgene (Blaha et al., 2006) under the transcriptional control of

FIG. 2. Superimposed active site residues for P. horikoshii ACC deaminasehomologue (carbon atoms are yellow), H. saturnus (carbon atoms are green),and Pseudomonas sp. ACP (carbon atoms are cyan) ACC deaminase. The aminoacid residues are labeled in that order, from top to bottom.



the regulatory gene acdR (Moenne-Loccoz et al., submitted forpublication), other strains of Azospirillum do not contain ACCdeaminase (Holguin and Glick, 2001). These observations,along with a phylogenetic analysis of known ACC deaminasegenes performed by Hontzeas et al. (2005) suggested that thesegenes might be inherited horizontally (laterally) rather thanvertically. In fact, there is some evidence that ACC deaminasegenes may not always be an integral part of the chromosomalDNA of a microorganism, but rather are present on largerelatively stable plasmids.

Given the extensive sequence database that currently existsfor microbial genes, we have undertaken a thorough phyloge-netic analysis of known and putative bacterial ACC deaminasegenes. Of the 154 available putative bacterial ACC deaminasestructural gene (acdS) sequences, only a relatively small numberof the proteins encoded by these genes have been experimen-tally shown to have ACC deaminase activity; the others havebeen annotated as acdS on the basis of sequence similarity tosome of the more well characterized acdS genes. Moreover, thephylogentic tree shown in Figure 3 includes only 86 sequencessince many acdS genes are essentially identical to ones alreadypresent and are not shown for simplicity of presentation. In addi-tion, when multiple nucleotide sequence alignments showed that19 sequences, annotated as ACC deaminase, compared poorlywith established acdS sequences, they were removed from thedataset.

From the phylogenetic tree, six acdS groups were defined.The first group consists of Gammaproteobacteria and one Be-taproteobacterium. Group II and III contain sequences fromthe Betaproteobacteria, as well as two Gammaproteobacteria.Group IV includes Alphaproteobacteria and a small group ofBetaproteobacteria, Group V consists entirely of Actinobacte-ria. Group VI consists of Beta- and Gammaproteobacteria. Ach.xylosoxidans BM1 was not included within Group V because itdiverged significantly from the other sequences in this group.

Enterobacter sp., Pseudomonas sp., and Achromobacter sp.ACC deaminase gene sequences are distributed throughout thetree. In addition, within group III, Ralstonia eutropha was notgrouped with other Ralstonia sp., instead it was clustered withBurkholderia tropica BM273. These observations are consistentwith the suggestion that ACC deaminase genes did not evolveexclusively vertically but instead some of these genes have un-dergone horizontal gene transfer (Hontzeas et al., 2005; Blahaet al., 2006).

Sullivan et al. (2002) reported that acdS is located within asymbiotic island (i.e., a cluster of symbiotic genes) in Mesorhi-zobium loti strain R7A. Similarly, a recently sequenced Rhi-zobium leguminosarum bv. viciae strain 3841 has a putativeacdS gene on one of its plasmids, pRL10 (Young et al., 2006).One strain of Sinorhizobium meliloti has an acdS gene on anaccessory plasmid, pSmeSM11a, and a putative regulatory pro-tein, encoded by acdR, is located upstream of the deduced acdS(Stiens et al., 2006), while another S. meliloti strain does notcontain acdS at all (Ma et al., 2004). Likewise, Rhizobium legu-

minosarum bv. trifolii strain NZP514 has an acdS gene on plas-mid pRtr514a with an acdR located upstream of acdS. Generallyspeaking, it is easier, both in the laboratory and in the environ-ment, to transfer plasmid DNA than to transfer chromosomalDNA from one organism to another (Bertolla et al., 1999; Kayet al., 2003; Mercier et al., 2006). And, if many acdS genes areplasmid encoded, it is likely that at least in some bacteria theyare inherited by horizontal gene transfer.

D. Transcriptional RegulationMany of the ACC deaminase genes that have been examined

in some detail also have a leucine responsive regulatory pro-tein (LRP) gene located from about 50 to a few hundred basepairs upstream of the start of the ACC deaminase structuralgene (acdS) and transcribed in the direction opposite to acdS(Grichko and Glick, 2000; Li and Glick, 2001; Moenne-Loccozet al., submitted for publication; Cheng et al., submitted forpublication; Duan et al., unpublished results). Since the leucineresponsive regulatory protein has been shown to be involvedin the regulation of the transcription of acdS, the gene encod-ing this protein has been termed acdR (i.e., ACC deaminaseregulatory gene). A detailed model, described below, of tran-scriptional regulation of acdS has been developed (Glick et al.,2007). Briefly, it is assumed that, on the basis of data from othersystems (Leonard et al., 2001), the active form of the leucineresponsive regulatory protein is an octamer. When an excessamount of LRP is present, this protein binds to an LRP box, lo-cated on the DNA sequence immediately upstream of the acdRgene, preventing further transcription of this gene. Alternatively,the LRP octamer can bind to a complex of ACC and anotherprotein, termed AcdB (Cheng et al., submitted for publication).Together, LRP and the AcdB-ACC complex activate transcrip-tion of acdS. Upon synthesis of ACC deaminase (AcdS), ACCis cleaved to form ammonia and α-ketobutyrate (a precursor ofbranched chain amino acids such as leucine), and when the cellaccumulates high levels of leucine, this amino acid binds to theLRP octamer causing it to dissociate into an inactive dimericform thereby shutting down further transcription of acdS. Thiscomplex mode of regulation ensures that ACC deaminase issynthesized only when it is needed and, most likely, only insomewhat limited amounts.

DNA sequence analysis indicates that some bacterial strainsappear to have lost all or part of the acdR gene but are neverthe-less still able to produce active enzyme (Glick et al., unpublishedresults). At this point, whether the AcdB-ACC complex is suf-ficient to activate transcription of the acdS gene in the absenceof LRP is very much an open question.

In some strains of Rhizobia the acdS gene has been found tobe under the control of a nifA promoter (the promoter responsi-ble for activating the transcription of all nif, or nitrogen fixation,genes and to be expressed within legume nodules (Uchiumiet al., 2004; Nukui et al., 2006). One may speculate that ACCdeaminase under the transcriptional control of nifA may prevent



FIG. 3. Maximum likelihood phylogenetic tree of ACC deaminase nucleotide sequences. Numbers next to the branches represent bootstrap values using NeighborJoining (first number), bootstrap values using Maximum Parsimony (second number) and Bayesian posterior probabilities (third number). “�” represents thenumber 100/100/1.00 for the three methods. Branches with an asterisk had less than 50% support in that analysis. When both Neighbor Joining and MaximumParsimony had less than 50% bootstrap values at one branch then only Bayesian posterior probability is shown. Accession numbers are given in brackets.



nodules from synthesizing ethylene in response to the stressof depleting local energy resources to fuel energy intensive ni-trogen fixation. However, this conjecture remains to be testedexperimentally.

V. BACTERIA WITH ACC DEAMINASE

A. Plant Growth PromotionA model was previously developed to explain the role of

bacterial ACC deaminase in the promotion of plant growth bybacteria that have this activity (Glick et al., 1998). In this model,the ACC deaminase-containing plant growth-promoting bacte-ria bind to the surface of either the seed or root of a devel-oping plant—some endophytic bacteria are also located insideof the plant root. In response to root exudates, including theamino acid tryptophan, the bacteria synthesize indoleacetic acid(IAA). Plant cells take up some of the IAA that is secreted by thebacteria and, together with the endogenous plant IAA, can stim-ulate plant cell proliferation and/or elongation as well as inducethe synthesis of the enzyme ACC synthase. Some of the ACC,either already present or newly synthesized by the plant, is ex-uded and taken up by the ACC deaminase-containing bacteria.This ACC is cleaved by ACC deaminase to form ammonia andα-ketobutyrate, both of which are readily metabolized by thebacteria. As a consequence of lowering the level of ACC within

a plant, the amount of ethylene that can form is reduced. Thus,the net result of the interaction of ACC deaminase-containingplant growth-promoting bacteria with plant cells is that the bac-teria act as a sink for ACC.

Direct consequences of this interaction are significantly in-creased plant root and shoot length, an increase in biomass, andprotection of plants from inhibitory effects of ethylene synthe-sized as a direct consequence of a variety of biotic and abioticstresses.

B. Amelioration of StressDuring periods of environmental stress, plants produce high

levels of “stress ethylene.” Moreover, much of the growth inhi-bition that occurs as a consequence of an environmental stressis the result of the response of the plant to the increased levels ofstress ethylene which exacerbates the response to the stressor.In addition, inhibitors of ethylene synthesis can significantlydecrease the severity of some environmental stresses. Thus, ifACC deaminase-containing bacteria can lower plant ethylenelevels, treatment of plants with these organisms should providesome protection against the inhibitory effects of these stresses.

In practice, ACC deaminase-containing plant growth-promoting bacteria have been used to protect plants againstgrowth inhibition caused by: flooding, both in the laboratory(Grichko and Glick, 2001a) and in the field (Farwell et al.,

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−FIG. 3. A asterisk next to the strain name indicates that it has been shown to have ACC deaminase activity. Abbreviations: Ac., Acidovorax; Ach.,Achromobacter; Ag., Agrobacterium; Az. Azospirillum; Br., Bradyrhizobium; Bre., Brevibacterium; Bu., Burkholderia; En., Enterobacter; Fu., Fulvimarina;Ma., Marine; Me., Mesorhizobium; Met., Methylibium; My., Mycobacterium; Ph., Phyllobacterium; Po., Polaromonas; Ps., Pseudomonas; R., Rhizobium;Ra., Ralstonia; Rh., Rhodococcus; Ro., Roseovarius; Sa., Sagittula; Sac., Saccharopolyspora; Se., Serratia; Si., Sinorhizobium; St. Stappia; Va., Variovo-rax. Strains shown include: Ach. sp. CM1∗ (AY604541), Ach. xylosoxidans A551∗ (AY604539), Ach. xylosoxidans BM1∗ (AY604540), Ach. xylosoxidans∗(DQ133504), Ac. facilis 4P6∗ (AY604529), Ac. sp. JS42 (NC 008782), Ag. tumefaciens d3∗ (AF315580), Az. lipoferum 4B∗ (DQ125242), Az. lipoferum CN1∗(DQ125253), Az. lipoferum TVV3∗ (DQ125257), Br. japonicum USDA 110 (BA000040), Br. sp. BTAi1 (AALJ01000006), Br. sp. ORS278 (NC 009445),Bre. linens BL2 (NZ AAGP01000039), Bu. ambifaria MC40-6 (NZ AAUZ01000006), Bu. caledonica LMG19076 (DQ125247), Bu. cenocepacia AU1054(NC 008061), Bu. cenocepacia PC184 (NZ AAKX01000119), Bu. cepacia AMMD (NC 008391), Bu. cepacia LMG1222∗ (DQ125251), Bu. dolosa AUO158(NZ AAKY01000194), Bu. graminis LMG18924 (DQ125249), Bu. mallei ATCC 23344 (CP000011), Bu. multivorans ATCC 17616 (NZ AAVB01000002),Bu. pseudomallei S13 (NZ AAHW02000043), Bu. phenazinium LMG2247∗ (DQ125252), Bu. phymatum STM815 (NZ AAUG01000006), Bu. phytofirmansPsJN∗ (NZ AAUH01000001), Bu. pseudomallei 668 (NC 009075), Bu. pseudomallei 1710b (CP000125), Bu. sp. 383 (CP000152), Bu. thailandensis E264(NC 007650), Bu. tropica BM273∗ (DQ125254), Bu. vietnamiensis G4 (NC 009255), Bu. xenovorans LB400 (NC 007952), En. aerogenes Cal3∗ (AY604544),En. cloacae CAL2∗ (AF047840), Fu. pelagi HTCC2506 (AATP01000002), Ma. actinobacterium PHSC20C1 (AAOB01000003), Me. loti R7A (AL672114), Me.loti MAFF303099∗ (BA000012), Met. petroleiphilum PM1 (NC 008825), My. smegmatis MC2 155 (CP000480), Ph. brassicacearum STM196∗ (EF452620), Po.sp. JS666 (NC 007948), Ps. 6G5∗ (M80882), Ps. brassicacearum Am3∗ (AY604528), Ps. fluorescens 17∗ (U37103), Ps. fluorescens CM1’A2∗ (DQ125246), Ps.fluorescens P97.30∗ (DQ125248), Ps. fluorescens PITR2∗ (DQ125244), Ps. marginalis∗ (AY604542), Ps. plecoglossicida AM10 (EF011162), Ps. putida AM15(EF011160), Ps. putida Bm3∗ (AY604533), Ps. putida UW4∗ (AY823987), Ps. sp. ACP ∗ (M73488), Ps. sp. AT14 (EF011161), Ps. sp. PNSL (DQ830987),Ps. syringae GR12-2∗ (AY604545), Ps. syringae pv. phaseolicola 1448A (NC 005773), Ps. syringae pv. syringae B728a (CP000075), Ps. syringae pv. tomatoDC3000 (AE016853), Ra. eutropha H16 (AM260480), Ra. pickettii 12J (NZ AAWK01000011), Ra. solanacearum GMI1000∗ (AL646053), Ra. solanacearumUW551 (NZ AAKL01000018), R. gallicum PB2∗ (EF525234), R. leguminosarum 99A1∗ (AY604535), R. leguminosarum bv. viciae 128C53K∗ (AF421376),R. leguminosarum bv. viciae 3841 (AM236084), R. leguminosarum PB45∗ (EF525235), R. leguminosarum PB163∗ (EF525253), R. sullae ATCC 43676∗(AY604534), Rh. sp. 4N4∗ (AY604538), Rh. sp. Fp2∗ (AY604537), Ro. sp. HTCC2601 (AATQ01000052), Sac. erythraea NRRL2338 (AM420293), Sa. stellataE-37 (AAYA01000005), Se. proteamaculans SUD∗ (AY604543), Si. medicae WSM419 (AATG01000018), Si. meliloti pSmeSM11a∗ (DQ145546), St. aggregataIAM 12614 (AAUW01000003), Va. paradoxus 2C1∗ (AY604530), Va. paradoxus 3C3∗ (AY604532) and Va. paradoxus 5C2∗ (AY604531). The multiple nu-cleotide sequence alignments were generated using ClustalW (Chenna et al., 2003) and optimized manually with BioEdit (Hall, 1999). Phylogenetic analyses wereperformed using PAUP v4.10b (Swofford, 2002) and MrBayes (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003). Appropriate evolutionarymodels were chosen for Maximum Likelihood analysis using Modeltest 3.7 (Posada and Crandall, 1998). The chosen substitution model was GTR+I+G (numberof substitutions [NST] = 6; general time reversible with invariant sites and a gamma rate distribution). Nodal support in Maximum Parsimony (MP) and NeighborJoining (NJ) was evaluated by 1000 bootstrap pseudoreplications. The topology of the ML tree was evaluated by calculating posterior probabilities in the Bayesiananalysis. The phylogram was generated with TreeView (Page, 1996).



2007); the presence of organic toxicants such as polyaromatichydrocarbons (PAHs), polycyclic biphenyls (PCBs) and totalpetroleum hydrocarbons (TPHs) both in the laboratory (Glick,2003; Saleh et al., 2004; Huang et al., 2004a, b, 2005; Reedand Glick, 2005; Reed et al., 2005) and in the field (Greenberget al., 2006); the presence of a variety of different metals includ-ing nickel, lead, zinc, copper, cadmium, cobalt and arsenic, bothin the laboratory (Burd et al., 1998, 2000; Belimov et al., 2001,2005; Nie et al., 2002; Glick, 2003; Reed and Glick, 2005;Reed et al., 2005; Dell’Amico et al., 2005; Safranova et al.,2006) and in the field (Farwell et al., 2006); high salt (Mayaket al., 2004b; Saravanakumar and Samiyappan, 2006; Chenget al., 2007); phytopathogens (Wang et al., 2000); drought(Mayak et al., 2004a); and infection of plant roots by vari-ous strains of Rhizobia (Ma et al., 2003a, b, 2004; Shaharoonaet al., 2006a).

As an alternative to the use of bacteria, several transgenicplants (tomato, canola, and tobacco) that express ACC deami-nase have been engineered. These transgenic plants have beenreported to be tolerant of certain pathogens (Lund et al., 1998;Robison et al., 2001), metals (Grichko et al., 2000; Stearnset al., 2005; Farwell et al., 2006), high salt (Sergeeva et al.,2006), and flooding (Grichko and Glick, 2001b). In these in-stances, transgenic plants that express the enzyme ACC deami-nase responded similarly to non-transformed plants treated withACC deaminase-containing plant growth-promoting bacteria.Not only does there not appear to be any intrinsic advantageof using transgenic plants as compared to plants treated withplant growth-promoting bacteria, the bacterially-treated plantsgenerally outperform the transgenic plants probably reflectingthe fact that plant growth-promoting bacteria do more for plantsthan merely lower their ethylene levels.

C. Introducing ACC Deaminase into Other BacteriaIt is generally quite straightforward to introduce ACC deam-

inase genes into bacterial strains that lack this activity. Trans-formation of bacterial strains lacking ACC deaminase activitywith isolated acdS genes and their regulatory regions has beenshown to improve their usefulness. For example, E. coli andPseudomonas strains that lack ACC deaminase but have beentransformed to express a Pseudomonas acdS gene are able topromote the elongation of canola roots in growth pouches (Shahet al., 1998). The effectiveness of some biocontrol pseudomon-ads was also significantly enhanced following the introductionof a Pseudomonas acdS gene (Wang et al., 2000). However, thecomplex transcriptional regulatory system that controls the ex-pression of many acdS genes (see section IV-D) may not be op-erative in all bacteria. When Azospirillum strains lacking ACCdeaminase were transformed with a Pseudomonas acdS geneunder the control of the regulatory acdR gene, ACC deaminasewas not expressed (Holguin and Glick, 2001). However, whenthe native regulatory region of the Pseudomonas acdS gene wasreplaced by either the E. coli lac promoter or the tet promoter,

ACC deaminase was expressed at a high level and the growth-promoting activity of the transformed Azospirillum strain wassignificantly improved (Holguin and Glick, 2001, 2003). Finally,transformation of a strain of Sinorhizobium meliloti with an acdSgene from Rhizobium leguminosarum enables the transformedbacterium to nodulate alfalfa plants and stimulate their growthby 35–40% more than the native (non-transformed) strain ofSinorhizobium meliloti can (Ma et al., 2004).

D. Bacterial Genes Activated by Root ExudatesWhile on the one hand, plant growth-promoting bacteria can

alter gene expression in plants, the nutrients released by plantroots as exudates can both attract bacteria and modify bacte-rial physiology as a consequence of the amount and types ofnutrients provided in the exudates (Lynch and Whipps, 1991).However, only a very small number of studies have examinedthe influence of plant exudates on bacterial gene expression.

In one study, the influence of root exudates from two va-rieties of sugarbeet on the Pseudomonas aeruginosa PAO1transcriptome were examined using microarray analyses (Market al., 2005). P. aeruginosa PAO1 is an opportunistic pathogenof humans but is also capable of plant root colonization (Market al., 2005). Among the genes with significantly upregulatedexpression are those that encode proteins involved in energygeneration and amino acid biosynthesis. This finding is not sur-prising since the utilization of the major plant root components,such as amino acids and organic acids, by Pseudomonas fluo-rescence WCS365 was shown to be essential for this biocontrolbacterium to colonize tomato roots (Lugtenberg et al., 1999).On the contrary, some genes involved in alginate biosynthe-sis and twitching motility were downregulated in response tothese root exudates. When a group of genes with putative orunknown function showed altered expression patterns, the pos-sible roles of these genes in plant colonization were examinedusing a panel of mutants with targeted disruptions. While eachseparately inoculated mutant appeared to have a similar colo-nization ability to the wild-type, some of these mutants had areduced ability to compete with the wild-type when they wereco-inoculated. In addition, homologues of some of these differ-entially expressed genes were identified in the genomes of bothbeneficial and pathogenic root-associated bacteria, suggestingtheir involvement in biocontrol, plant growth promotion, and/orplant pathogenesis (Mark et al., 2005).

In another study, the effect of canola root exudates on theplant growth-promoting bacterium Pseudomonas putida UW4was investigated using two-dimensional difference in-gel elec-trophoresis (2-D DIGE) analysis (Cheng, Glick, and McConkey,unpublished data). Out of a total number of 1,757 proteins de-tected on the analytical gels, the expression levels of 172 (9.8%)proteins and 220 (12.5%) proteins were significantly increasedor decreased (P ≤ 0.05, Ratio ≥ 1.5, or Ratio ≤ –1.5), respec-tively. Proteins with significant differences in expression levelsfollowing exposure to canola root exudates were identified by



mass spectrometry. The annotation of the proteins that havebeen identified so far reveals that many proteins involved in cellmovement, nutrient transportation, signal transduction, energyproduction, protein synthesis and transcriptional regulation areupregulated, while some proteins involved in cell growth anddivision are downregulated. The participation of many of theseproteins in plant-microbe interactions through various mecha-nisms was verified by individual functional analysis. In addition,some previously uncharacterized proteins were also found to bedifferentially expressed, and the implication that these proteinsare involved in plant-microbe interactions needs further con-firmation. The differential expression profiles generated frommicroarray and proteomic studies show significant overlap withone another, with the expression of many of the same proteinschanging (Mark et al., 2005).

Integration of the differential expression data with proteincellular functions sheds some light on the biochemical mecha-nisms and regulatory systems mediating plant-microbe interac-tions by indicating the overall effects of plant root exudates onbacterial protein expression pattern. That is, bacterial proteinsinvolved in nutrient uptake and utilization, energy production,and protein synthesis are upregulated in response to plant rootexudates, which may enhance the bacterium’s ability to colo-nize the host root. However, a more complete understanding ofthe effects of root exudates on bacterial gene expression awaitsadditional analysis of this complex data set.

E. Plant Gene Expression Modified by Bacteria withACC Deaminase

The effect of plant growth-promoting bacteria on plant geneexpression has been assessed using differential display PCR,randomly amplified PCR, microarrays, or a proteomic approach,in each case following growth under specific conditions. Usingdifferential display PCR, Timmusk and Wagner (1999) observedthat plants respond to a plant growth-promoting bacterium with-out ACC deaminase as if it were a mild biotic stressor, and thisresponse appears to protect the plant against subsequent stresses.On the other hand, using randomly amplified PCR, Hontzeaset al. (2004b) compared a wild-type ACC deaminase-containingplant growth-promoting bacterium with an ACC deaminase mi-nus mutant of this strain and found that the wild-type bacteriumlowered the stress level in the plant so that the plant no longerperceived a mild stress. Thus, in the presence of ACC deami-nase, some plant stress response genes are no longer turned onby plant growth-promoting bacteria.

Microarray studies of plant responses to plant growth-promoting bacteria have been utilized to provide a bird’s eyeperspective of the transcriptional trends, within plant tissues,that occur during plant-microbe interactions. Many interestingchanges in gene expression occur within genes related to thestress response, auxin responses, cellular metabolism and theethylene response. For example, workers have observed an in-crease in transcription of stress response genes (defense genes),

genes involved in wounding and pathogenesis signaling, andgenes involved in auxin signaling. In many cases there arechanges in the expression of many of the transcription factorfamilies of genes, some of which are summarized in Table 1(Cartieaux et al., 2003; Verhagen et al., 2004; Wang et al., 2005).Other changes that have been reported include: nodulin-likegenes; Myb and WRKY transcription factors; genes involved intranslation and protein folding as well as nitrogen metabolismand catabolism. In the roots of plants treated with ISR-inducingbacteria, there is a rapid (three-day), very large (> 20-fold) de-crease in the transcription of ethylene response factors (EREBP)genes, that levels off to a 2-fold decrease by day seven (Verha-gen et al., 2004). This change is not consistent within studies ofshoot tissue, since EREBP transcripts have also been observedto increase (Czarny and Glick, unpublished results), and staythe same (Cartieaux et al., 2003). The different responses ofEREBP genes may reflect the interaction of the plant with bac-teria containing a variety of different physiological traits suchas the presence of ACC deaminase, the production of IAA or thesynthesis of siderophores. The enzyme ACC deaminase, whenpresent in the plant growth-promoting bacteria that are found inthe rhizosphere of many plants, can lower the stress perceivedby the plant and also derepress the expression of auxin responsegenes in the shoots (Glick et al., 2007). In addition, bacteria thatcontain ACC deaminase can suppress the expression or func-tioning of other plant signaling molecules such as jasmonic acidand giberellin (Czarny and Glick, unpublished results). This isof course a consequence of the lowering of plant ethylene levelsby the action of the ACC deaminase.

Since ethylene has been found to be required for the inductionin plants of systemic resistance elicited by rhizobacteria (vanLoon et al., 1997), the question arises whether treating plantswith ethylene-lowering bacteria might prevent this induction.However, in practice, “lowering of ethylene levels by bacterialACC deaminase does not appear to be incompatible with theinduction of systemic resistance. Indeed, some bacterial strainspossessing ACC deaminase also induce systemic resistance”(van Loon and Glick, 2004). This may reflect the fact that thereis an initial very small peak of ethylene close in time to theonset of a stress and then a second much larger peak some timelater (Glick et al., 2007). The first peak is thought to initiate adefensive response by the plant (systemic resistance) while thesecond ethylene peak is so large that processes inhibitory to plantgrowth are initiated. Immediately following an environmentalstress, the pool of ACC in a plant is low as is the level of ACCdeaminase in the associated bacterium and only some of theACC will be cleaved by the bacterial enzyme with the remainderbeing converted into the first small ethylene peak.

F. Ethylene-IAA Cross-talkWhile it is well known that IAA can activate the transcrip-

tion of ACC synthase (Kende, 1993; Kende and Zeevaart, 1997;Kim et al., 1992), it is less well known that ethylene may in-hibit IAA transport and signal transduction (Burg and Burg,



1966; Morgan and Gausman, 1966; Suttle, 1988; Prayitno et al.,2006). This feedback loop of ethylene inhibition of IAA syn-thesis and/or functioning limits the amount of ACC synthase,ACC and, ultimately, ethylene following every stressful eventin the life of the plant. When an ACC deaminase-containingplant growth-promoting bacterium lowers the ethylene concen-tration in plant roots, this relieves the ethylene repression ofauxin response factor synthesis, and indirectly increases plantgrowth (see also Dharmasiri and Estelle, 2004). Thus, ACCdeaminase-containing plant growth-promoting bacteria facili-tate plant growth by (i) decreasing ethylene inhibition of vari-ous plant processes and (ii) permitting IAA stimulation of cellproliferation and elongation without the negative effects of in-creasing ACC synthase and plant ethylene levels.

VI. CONCLUSIONOur understanding of some of the mechanisms used by plant

growth-promoting bacteria has come a long way in the past 10 to20 years, and with this increased understanding there has been aconcomitant increase in the commercial application of these or-ganisms. While there is still a lot that is not understood regardingthe functioning of these organisms, the major impediments to theincreased commercial use of these bacteria are economic. Thus,in countries of the world (generally more developed countries)where agricultural productivity is high and much of this pro-ductivity is based on the extensive use of relatively inexpensiveagricultural chemicals, there is little immediate economic in-centive to alter existing agricultural practice. On the other hand,in many of the less developed countries of the world where agri-cultural productivity is not as high, relatively cheap labor andhigh chemical costs provide a situation where the use of plantgrowth-promoting bacteria provides an attractive commercialpossibility. In addition, with recent public attention worldwidebeing directed to environmental issues, many parts of the moredeveloped world are more actively pursuing “green” strategieswhich could include the replacement of some agrochemicalswith plant growth-promoting bacteria.

Scientifically, the use of plant growth-promoting bacteriaappears, in many cases, to present a superior alternative to theuse of transgenic plants. In particular, plant growth-promotingbacteria can help plants to tolerate a range of biotic and abioticstresses so that it is not necessary to genetically engineerall cultivars of all plants to be tolerant to a large number ofdifferent stresses.

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promotion of plant growth by bacterial acc deaminase

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