Critical Reviews in Plant Sciences, 26:227–242, 2007

Copyright © Taylor & Francis Group, LLC

ISSN: 0735-2689 print / 1549-7836 online
DOI: 10.1080/07352680701572966

Promotion of Plant Growth by Bacterial ACC Deaminase

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

Table of Contents

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

II. MECHANISMS USED BY PLANT GROWTH-PROMOTING BACTERIA .................................................... 228

A. Indirect Mechanisms .................................................................................................................................. 228
B. Direct Mechanisms .................................................................................................................................... 228

III. ETHYLENE AND STRESS ............................................................................................................................. 229

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

IV. ACC DEAMINASE ......................................................................................................................................... 230

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

V. BACTERIA WITH ACC DEAMINASE .......................................................................................................... 235

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

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

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

aminocyclopropane-1-carboxylate (ACC) deaminase. This article

To date, there has been only limited commercial use of plant
growth-promoting bacteria in agriculture, horticulture, and sil-
viculture. However, with recent progress toward understanding
the mechanisms that these organisms utilize to facilitate plant
growth, the use of plant growth-promoting bacteria is expected
to continue to increase worldwide. One of the key mechanisms
employed by plant growth-promoting bacteria to facilitate plant
growth is the lowering of plant ethylene levels by the enzyme 1-
reviews the published work on this enzyme, with an emphasis on its
biochemistry, protein structure, genes, and regulation. In addition,
this article provides some initial insights into the changes in both
plants and ACC deaminase-containing plant growth-promoting
bacteria as a consequence of plant-microbe interactions. Finally, a
brief discussion of how bacterial ACC deaminase and indoleacetic
acid (IAA) together modulate plant growth and development is
included.

Address correspondence to B.R. Glick, Department of Biology,

University of Waterloo, 200 University Avenue West, Waterloo, On- Keywords Plant growth-promoting bacteria, plant stress, ethylene,
tario, Canada N2L 3G1. E-mail: glick@sciborg.uwaterloo.ca IAA

227
228 B. R. GLICK ET AL.
I. INTRODUCTION
Increased public concern about environmental problems
caused either directly or indirectly by the use of fertilizers, pes-
ticides, herbicides, and fungicides, has prompted researchers
to consider alternatives to these established chemical strate-
gies for facilitating plant growth in agriculture, horticulture,
and silviculture. Ideally, replacements for the chemicals that
are currently in widespread use should not only enhance plant
growth, but should also inhibit plant pathogens. One potential
alternative 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 within
plant tissues (bacterial endophytes). The highest concentrations
of these microorganisms typically exists around the roots, in
the 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). A
large number of plant growth-promoting bacteria have been
isolated to date, each with one or more traits that might, un-
der the appropriate conditions, enhance plant growth. Some
of these bacteria may directly influence plant growth, e.g., by
synthesizing plant hormones or facilitating uptake of nutrients
from the soil. Others exert their beneficial effects indirectly
via 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 are
used commercially, albeit to a limited extent, in a number
of different countries worldwide in agriculture, horticulture,
silviculture and environmental remediation (Reed and Glick,
2004; Fravel, 2005). One reason for the somewhat limited
commercial 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, the
relatively low cost of agrochemicals, including fertilizers, has
precluded 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 of
indigenous soil microorganisms, weather, soil moisture con-
tent and, perhaps most importantly, an incomplete understand-
ing of the mechanisms employed by plant growth-promoting
bacteria to facilitate plant growth. Notwithstanding the large
number of potential variables involved in the effective and re-
producible use of plant growth-promoting bacteria, in recent
years considerable progress has been made toward develop-
ing a better understanding of many of the mechanisms that
these organisms utilize and there is every reason to expect the
use of plant growth-promoting bacteria to continue to increase
worldwide.
II. MECHANISMS USED BY PLANT
GROWTH-PROMOTING BACTERIA
A. Indirect Mechanisms
The 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 a
variety 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 most
commonly associated with the ability of a biocontrol strain to
inhibit 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 inhibit
the growth of pathogens by synthesizing low molecular mass
siderophores that bind most of the iron in the rhizosphere with
an extremely high avidity, thereby thwarting the proliferation of
fungal pathogens in the vicinity of the host plant roots because
of 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 such
as chitinase, β1,3glucanase, protease, or lipase, all of which can
facilitate the lysis of fungal cells (Chet and Inbar, 1994). Inter-
estingly, the defense response of stressed plants often results in
the production of the same array of proteins (i.e., chitinase, β1,3-
glucanase, protease, and lipase) in response to pathogen induced
stress (van Loon et al., 2006). Other biocontrol plant growth-
promoting bacteria can protect plants from phytopathogens by
out-competing them for nutrients and for niches on the root sur-
face thereby effectively preventing pathogens from binding to
and infecting the plant (Kloepper et al., 1988; O’Sullivan and
O’Gara, 1992; Loper et al., 1997). In addition to the more ob-
vious methods of biocontrol, long-lasting and broad spectrum
systemic resistance to a variety of pathogens can be induced by
various plant growth-promoting bacteria or microbial metabo-
lites (Kessmann et al., 1994; Tuzun and Kloepper, 1994; van
Loon et al., 1997, 2006; Vallad and Goodman, 2004; Verhagen
et al., 2004).
B. Direct Mechanisms
There 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 minor
component of the benefit that the bacterium provides to the
plant; synthesize siderophores which can sequester iron from
the soil and provide it to plant cells which can take up the
bacterial siderophore-iron complex; synthesize phytohormones
such as auxins, cytokinins and gibberelins, which can act to en-
hance various stages of plant growth; solubilize minerals such
as phosphorus, making them more readily available for plant
growth; and synthesize the enzyme 1-aminocyclopropane-1-
carboxylate (ACC) deaminase, which can lower plant ethylene
 PLANT GROWTH BY BACTERIAL ACC DEAMINASE
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 many
plant growth-promoting bacteria possess several of these traits,
a bacterium may utilize different traits at various times during
the life cycle of the plant. Moreover, plant growth-promoting
bacteria typically have little or no measurable effect on plant
growth 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 the
phytohormone auxin (Brown, 1974; Tien et al., 1979; Patten
and Glick, 1996; Garcia de Salamone et al., 2005). However,
in the last few years it has been found that a number of plant
growth-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; Belimov
et al., 2001, 2005; Kaneko et al., 2002; Babalola et al., 2003;
Ma et al., 2003; Ghosh et al., 2003; Dey et al., 2004; Mayak
et 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 thereby
lower the level of the phytohormone ethylene in a developing or
stressed plant. The major portion of this review article is focused
on the enzyme ACC deaminase (and its genes) and its central
role in promoting plant growth.
III. ETHYLENE AND STRESS
A. Effects of Ethylene on Plants
In higher plants, the enzyme S-adenosyl-L-methionine
(SAM) synthetase catalyzes the conversion of methionine to
SAM (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 to
ethylene, carbon dioxide, and hydrogen cyanide (John, 1991).
The plant hormone ethylene plays an important role in root ini-
tiation and elongation, nodulation, senescence, abscission and
ripening 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 root
hair initiation. It also begins the process of fruit ripening, flower
wilting, and leaf senescence. When produced endogenously dur-
ing developmental processes, ethylene regulates xylem forma-
tion, flowering in some plants, and induces fruit ripening as well
as flower wilting. As part of a stress response, it inhibits root
elongation, nodulation and auxin transport, induces hypertro-
phies, speeds aging and promotes senescence and abscission.
Along with auxin, ethylene regulates lateral root initiation and
exudation of resins and gums (Abeles et al., 1992; Prayitno
229
et al., 2006; Sun et al., 2006). However, it should be mentioned
that there is an important distinction between ethylene responses
that are due to an increase in ethylene concentration within plant
tissues and the increase in the sensitivity of plant tissues to ethy-
lene. Unfortunately, the mechanisms that make cells responsive
to ethylene are still not fully understood.
B. Genes Involved in Stress Signaling and Response
Plants 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 metals
and high salt; and biotic stresses, which are caused by different
types of pathogens including those that are either biotrophic or
necrotrophic.
Defenses against biotrophic pathogens are thought to rely
mostly on salicylic acid (SA) mediated responses, whereas
those against necrotrophic pathogens rely mostly on jasmonic
acid (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, biotic
stress responses involve reactive oxygen species (ROS), calcium
fluctuations, micro RNAs and many other signaling molecules
(Broekaert et al., 2006; Navarro et al., 2006). Abiotic stresses
induce 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 these
types of stress, yet each pathway interacts extensively with other
signaling cascades. Thus, there are multiple instances of syn-
ergy and antagonism, and responses to different stresses form
a complex network of signaling pathways that often overlap.
In turn, activation of particular signaling cascades enables a
plant to modify the nature and amount of many of its proteins
in response to the stress. Understanding these signal cascades
may be the key to unraveling the complex patterns of stress
response in plants. A list of some of the key genes involved
in stress response signaling is presented in Table 1 (Shinozaki
et al., 2003; Fujita et al., 2006). In particular, the DRE-binding
protein (DREB) family of transcription factors is responsible
for signaling the response to many abiotic stresses and mem-
bers of this family are similar to genes in the ethylene response
pathway, 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 signaling
molecules include some of the members of the protein kinase
family which act to link biotic and abiotic stress responses. For
instance, in Arabidopsis the MAP-kinases MPK6 and MPK3
are upregulated during biotic and abiotic stress and function
to phosphorylate ACC synthase stabilizing this enzyme and
thereby increasing ethylene biosynthesis (Liu and Zhang, 2004).
These examples illustrate points of convergence between differ-
ent stress response networks, where ethylene signaling plays a
pivotal role.
230 B. R. GLICK ET AL.

Some proteins involved in plant stress responses
Transcription factor family
DRE-binding protein (DREB) family
Ethylene-responsive element binding
factor (EREBP) family
Some zinc-finger proteins
Some WRKY transcription factors
Some MYB transcription factors
Basic helix-loop-helix (bHLH) family
Basic-domain leucine zipper (bZIP)
family
NAC family
Other signaling proteins
Some MAP-kinases (i.e.,
MPK6/MPK3)
DEAD-box helicases
Glutathione
TABLE 1
Inducing stress
Cold, drought, high salt, osmotic
High salt, drought, cold, wounding,
pathogen infection and ethylene
treatment
Cold, drought
Cold, drought, biotic
Botrytis infection, cold, drought
Cold, drought
Cold, drought
JA, H2 O2 , pathogen infection, drought,
high salinity and ABA treatment
Wounding, osmotic shock, UV, salinity,
drought, ozone, temperature extremes,
and pathogen infection
Abiotic stress
Abiotic and biotic stress
Function
Bind to dehydration responsive
elements (DRE) within the promoters
abiotic stress genes
Binds to the GCC box of ethylene
inducible genes
Interact with bZIP proteins, enhances
freezing tolerance
Bind to elements within pathogenesis
related genes
Bind (with AtMYC2) to a cis-element in
the dehydration-inducible RD22 gene.
May also mediate biotic and abiotic
stress through ROS
Multiple hormone signaling pathways
such as ABA, JA and JA/Ethylene
mediated pathogen defense
Involved in ABA signalling
Turns on genes involved in ROS
signaling and detoxification, defense
and senescence
Induce ethylene biosynthesis through an
increase in ACC synthase activity
Unwinding of energetically stable DNA
or RNA duplexes
Responds to changes in photosynthesis

IV. ACC DEAMINASE
The enzyme ACC deaminase (E.C. 4.1.99.4) cleaves ACC,
the immediate precursor of ethylene in plants, to form ammonia
and α-ketobutyrate. This multimeric enzyme is a common com-
ponent of many soil microorganisms, both bacteria and fungi. It
has also been suggested, based largely on sequence similarities,
that some plants may contain ACC deaminase genes (Sterky
et 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. Biochemistry
The pioneering work in elaborating the biochemical proper-
ties of ACC deaminase is largely the results of studies carried
out by Honma and his co-workers (Honma and Shimomura,
1978; Walsh et al., 1981; Honma, 1985; Honma et al., 1993
a, 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; Jacobson
et al., 1994; Li et al., 1996; Zhao et al., 2003; Hontzeas et al.,
2004a).
ACC deaminase is a multimeric enzyme (homodimeric or
homotrimeric) with a subunit molecular mass of approximately
35-42 kDa. It is a sulfhydryl enzyme in which one molecule
of the essential co-factor pyridoxal phosphate (PLP) is tightly
bound to each subunit. Interestingly, the enzyme ACC deami-
nase is cytoplasmically localized (Jacobson et al., 1994) so that
the substrate ACC must be exuded by plant tissues (Penrose
et al., 2001; Penrose and Glick, 2001) and subsequently taken
up by an ACC deaminase-containing microorganism before it
is cleaved (Glick, 1998).
Measurement of the Km of various ACC deaminases for ACC
indicate that the enzyme does not bind the substrate with a
high affinity; Km values range from 1.5 to 17.5 mM (Honma
and Shimomura, 1978; Klee and Kishore, 1992; Honma, 1993;
Hontzeas et al., 2004a). This has been interpreted as indicating
that in order to compete with ACC oxidase for ACC, ACC
 PLANT GROWTH BY BACTERIAL ACC DEAMINASE
deaminase must be present in much greater amounts, i.e., from
100- to 1000-fold (Glick, 1998). Moreover, it was suggested
that 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 will
be a parallel increase in the rate of ACC cleavage (Glick, 2005).
The PLP-dependent enzymes catalyze a wide variety of
biochemical reactions, many of which are involved in the
metabolism of amino acids. In most of these reactions two basic
chemical properties of the PLP have been conserved: (i) PLP
forms an external aldimine between its aldehyde group and the
α-amino group of the substrates and (ii) PLP acts as an electron
sink, withdrawing electrons from the substrate (John, 1995). As
a PLP-dependent enzyme, the ACC deaminase ring opening re-
action starts with conversion from an internal aldimine between
the enzyme and PLP to an external aldimine between the sub-
strate ACC and PLP. In most other PLP-dependent reactions, the
next step is the nucleophilic abstraction of either an α-proton or
an α-carboxylate group. However, in this regard the ACC deam-
inase catalyzed reaction is considered as a special case, since
the substrate does not contain a α-proton and the carboxylate
group is retained in the product, ruling out the usual mechanism.
As such, studies of this unusual enzyme have been conducted
with the intent to unravel the mechanism of the ring opening.
Walsh et al. (1981) proposed two possible routes for the ring
fragmentation: (i) nucleophilic addition at the Cβ methylene po-
sition followed by β-proton abstraction and (ii) direct β-proton
abstraction leading to the cyclopropane ring cleavage. The exact
mechanism is still unknown, with data to support both routes
(i) and (ii) (Ose et al., 2003; Karthikeyan et al., 2004a, b). This
notwithstanding, Hontzeas et al. (2006) have argued, on the ba-
sis of theoretical considerations and following the mechanistic
studies of Li et al. (1996), that mechanism (i) is most likely to
be operative in the cleavage of ACC.
B. Protein Structure
To 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 (Yao
et al., 2000, Ose et al., 2003; Karthikeyan et al., 2004a; Fujino
et 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 form
two domains, each of which has an open twisted α/β structure
similar 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 deaminases
is shown in Figure 1. The alignment illustrates that most of
the amino acid residues that are known to be important are
231
conserved, and the H. saturnus and Pseudomonas sp. ACC
deaminase active sites are virtually identical. The lysine residue
that binds PLP, the tyrosine residue that stacks with the pyridine
ring, and residues important in recognizing the substrate ACC
are 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 amino
acid residues leads to complete loss of activity. In addition,
substitution of Tyr269 leads to a reduction in specific activity to
less than 10% of the wild type enzyme (Ose et al., 2003). The
same results have been obtained for the mutational analysis at
residues Ser78 and Tyr294 (H. saturnus corresponding residue
is Try295) of the Pseudomonas sp. enzyme (Karthikeyan
et al., 2004b). However, the precise role that some of the above
mentioned residues play in the catalysis still remains to be
elucidated. Recent work has focused on determining the enzy-
matic residues involved in nucleophilic addition and β-proton
abstraction. Lys51 (Ose et al., 2003) and Tyr294 (Karthikeyan
et al., 2004a,b) are two major candidates proposed to be in-
volved in nucleophilic addition; however, it has been suggested
that Ser78 also plays a role in nucleophilic addition (Zhao
et al., 2003). As far as direct β-proton abstraction, thus far, only
Lys51 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 what
makes the ACC deaminase reaction unique. The Pyrococcus
horikoshii enzyme was originally predicted to be ACC deam-
inase due to its sequence similarity to other ACC deaminase
enzymes. But while the ACC deaminase homologue preserves
the key amino acid residues (Figure 1), it does not have ACC
deaminase activity. Instead, this enzyme shows specificity for D-
and L-serine, producing pyruvate as a result of catalysis (Fujino
et al., 2004). The overall topology of the ACC deaminase ho-
mologue is very similar to that of H. saturnus and Pseudomonas
sp. ACC deaminases; the residue arrangement in the active site
is very similar between ACC deaminase from H. saturnus and
the ACC deaminase homologue, with only a few substitutions.
These include a change from Gln77 in H. saturnus to His80 in
the homologue, a change from Glu296 in H. saturnus to Thr283
in the homologue and a change from Leu323 in H. saturnus to
Thr308 in the homologue. The inertness of the ACC deaminase
homologue towards ACC is explained by the change in the elec-
tron density of the ACC cyclopropane ring, which is influenced
by the pyridine ring of PLP within the active site (Fujino et al.,
2004). The charge density of the pyridine ring is modulated by
residues in the vicinity of the pyridine nitrogen atom. The pyri-
dine nitrogen atom of H. saturnus ACC deaminase exists within
hydrogen bonding distance to the side-chain oxygen atom of
Glu296, whereas in the P. horikoshii ACC deaminase homo-
logue, the hydroxyl group of Thr308 side-chain approaches the
corresponding nitrogen atom (Figure 2). The threonine or serine
side-chain at this position has been observed in other members
of the trypthophan synthase β-subunit (TRPSβ) family, which
232 B. R. GLICK ET AL.

FIG. 1. Amino acid sequence alignment of the annotated ACC deaminases, including the putative ACC deaminases. The conserved lysine residue that binds the
PLP 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 a
regulatory 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- Glick et al., 1995; Blaha et al., 2006; Duan et al., unpublished
inase, and threonine synthase, such that the ACC deaminase ho- results). Interestingly, within a particular genus and species
mologue is more similar to these enzymes at these two positions of microorganism, some strains have ACC deaminase activity,
(Hyde et al., 1988; Burkhard et al., 1998; Gallagher et al., 1998; and others often do not. Thus, for example, while some strains
Thomazeau et al., 2001). In addition, true ACC deaminases are of Azospirillum have recently been reported to contain an acdS
expected to have a leucine residue (Leu323 for H. saturnus) gene (Blaha et al., 2006) under the transcriptional control of
in close proximity to the Glu296 residue. In the ACC deami-
nase homologue, this position is occupied by another threonine
residue. In the three-dimensional structure, the leucine residue
provides space for the long side-chain of the glutamate residue
by orienting itself in the opposite direction (Figure 2.; Fujino
et al., 2004). Hence, the putative ACC deaminases that contain
threonine residues at these positions are expected to be unable
to utilize ACC as a substrate, however, this still remains to be
confirmed experimentally with enzymes other than the ACC
deaminase homologue.

C. Genes and Distribution

ACC deaminase activity has been found to be associated
with a large number of different soil microorganisms (Klee
et 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; Ma
et al., 2003; Ghosh et al., 2003; Mayak et al., 2004a; Khalid
et al., 2004; Dey et al., 2004; Hontzeas et al., 2005; Dell’Amico
et al., 2005; Blaha et al., 2006; Madhaiyan et al., 2006; Shaha- FIG. 2. Superimposed active site residues for P. horikoshii ACC deaminase
roona et al., 2006a, b; Saravanakumar and Samiyappan, 2006; homologue (carbon atoms are yellow), H. saturnus (carbon atoms are green),
Hameeda et al., 2006). Moreover, it is found at a relatively high and Pseudomonas sp. ACP (carbon atoms are cyan) ACC deaminase. The amino
frequency in many rhizosphere soils (Klee and Kishore, 1992; acid residues are labeled in that order, from top to bottom.
 PLANT GROWTH BY BACTERIAL ACC DEAMINASE
the regulatory gene acdR (Moënne-Loccoz et al., submitted for
publication), other strains of Azospirillum do not contain ACC
deaminase (Holguin and Glick, 2001). These observations,
along with a phylogenetic analysis of known ACC deaminase
genes performed by Hontzeas et al. (2005) suggested that these
genes might be inherited horizontally (laterally) rather than
vertically. In fact, there is some evidence that ACC deaminase
genes may not always be an integral part of the chromosomal
DNA of a microorganism, but rather are present on large
relatively stable plasmids.
Given the extensive sequence database that currently exists
for microbial genes, we have undertaken a thorough phyloge-
netic analysis of known and putative bacterial ACC deaminase
genes. Of the 154 available putative bacterial ACC deaminase
structural gene (acdS) sequences, only a relatively small number
of the proteins encoded by these genes have been experimen-
tally shown to have ACC deaminase activity; the others have
been annotated as acdS on the basis of sequence similarity to
some of the more well characterized acdS genes. Moreover, the
phylogentic tree shown in Figure 3 includes only 86 sequences
since many acdS genes are essentially identical to ones already
present and are not shown for simplicity of presentation. In addi-
tion, when multiple nucleotide sequence alignments showed that
19 sequences, annotated as ACC deaminase, compared poorly
with established acdS sequences, they were removed from the
dataset.
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 from
the Betaproteobacteria, as well as two Gammaproteobacteria.
Group IV includes Alphaproteobacteria and a small group of
Betaproteobacteria, Group V consists entirely of Actinobacte-
ria. Group VI consists of Beta- and Gammaproteobacteria. Ach.
xylosoxidans BM1 was not included within Group V because it
diverged significantly from the other sequences in this group.
Enterobacter sp., Pseudomonas sp., and Achromobacter sp.
ACC deaminase gene sequences are distributed throughout the
tree. In addition, within group III, Ralstonia eutropha was not
grouped with other Ralstonia sp., instead it was clustered with
Burkholderia tropica BM273. These observations are consistent
with the suggestion that ACC deaminase genes did not evolve
exclusively vertically but instead some of these genes have un-
dergone horizontal gene transfer (Hontzeas et al., 2005; Blaha
et al., 2006).
Sullivan et al. (2002) reported that acdS is located within a
symbiotic 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 putative
acdS gene on one of its plasmids, pRL10 (Young et al., 2006).
One strain of Sinorhizobium meliloti has an acdS gene on an
accessory 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 not
contain acdS at all (Ma et al., 2004). Likewise, Rhizobium legu-
233
minosarum bv. trifolii strain NZP514 has an acdS gene on plas-
mid pRtr514a with an acdR located upstream of acdS. Generally
speaking, it is easier, both in the laboratory and in the environ-
ment, to transfer plasmid DNA than to transfer chromosomal
DNA from one organism to another (Bertolla et al., 1999; Kay
et al., 2003; Mercier et al., 2006). And, if many acdS genes are
plasmid encoded, it is likely that at least in some bacteria they
are inherited by horizontal gene transfer.
D. Transcriptional Regulation
Many 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 base
pairs upstream of the start of the ACC deaminase structural
gene (acdS) and transcribed in the direction opposite to acdS
(Grichko and Glick, 2000; Li and Glick, 2001; Moënne-Loccoz
et al., submitted for publication; Cheng et al., submitted for
publication; Duan et al., unpublished results). Since the leucine
responsive regulatory protein has been shown to be involved
in the regulation of the transcription of acdS, the gene encod-
ing this protein has been termed acdR (i.e., ACC deaminase
regulatory 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 other
systems (Leonard et al., 2001), the active form of the leucine
responsive regulatory protein is an octamer. When an excess
amount of LRP is present, this protein binds to an LRP box, lo-
cated on the DNA sequence immediately upstream of the acdR
gene, preventing further transcription of this gene. Alternatively,
the LRP octamer can bind to a complex of ACC and another
protein, 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), ACC
is cleaved to form ammonia and α-ketobutyrate (a precursor of
branched chain amino acids such as leucine), and when the cell
accumulates high levels of leucine, this amino acid binds to the
LRP octamer causing it to dissociate into an inactive dimeric
form thereby shutting down further transcription of acdS. This
complex mode of regulation ensures that ACC deaminase is
synthesized only when it is needed and, most likely, only in
somewhat limited amounts.
DNA sequence analysis indicates that some bacterial strains
appear to have lost all or part of the acdR gene but are neverthe-
less still able to produce active enzyme (Glick et al., unpublished
results). At this point, whether the AcdB-ACC complex is suf-
ficient to activate transcription of the acdS gene in the absence
of LRP is very much an open question.
In some strains of Rhizobia the acdS gene has been found to
be 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 (Uchiumi
et al., 2004; Nukui et al., 2006). One may speculate that ACC
deaminase under the transcriptional control of nifA may prevent
234 B. R. GLICK ET AL.

FIG. 3. Maximum likelihood phylogenetic tree of ACC deaminase nucleotide sequences. Numbers next to the branches represent bootstrap values using Neighbor
Joining (first number), bootstrap values using Maximum Parsimony (second number) and Bayesian posterior probabilities (third number). “” represents the
number 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 Maximum
Parsimony had less than 50% bootstrap values at one branch then only Bayesian posterior probability is shown. Accession numbers are given in brackets.
 PLANT GROWTH BY BACTERIAL ACC DEAMINASE 235

nodules from synthesizing ethylene in response to the stress
of depleting local energy resources to fuel energy intensive ni-
trogen fixation. However, this conjecture remains to be tested
experimentally.
V. BACTERIA WITH ACC DEAMINASE
A. Plant Growth Promotion
A model was previously developed to explain the role of
bacterial ACC deaminase in the promotion of plant growth by
bacteria 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 inside
of the plant root. In response to root exudates, including the
amino acid tryptophan, the bacteria synthesize indoleacetic acid
(IAA). Plant cells take up some of the IAA that is secreted by the
bacteria and, together with the endogenous plant IAA, can stim-
ulate plant cell proliferation and/or elongation as well as induce
the 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 and
α-ketobutyrate, both of which are readily metabolized by the
bacteria. 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-containing
plant 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, and
protection of plants from inhibitory effects of ethylene synthe-
sized as a direct consequence of a variety of biotic and abiotic
stresses.
B. Amelioration of Stress
During 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 stress
is the result of the response of the plant to the increased levels of
stress ethylene which exacerbates the response to the stressor.
In addition, inhibitors of ethylene synthesis can significantly
decrease the severity of some environmental stresses. Thus, if
ACC deaminase-containing bacteria can lower plant ethylene
levels, treatment of plants with these organisms should provide
some protection against the inhibitory effects of these stresses.
In practice, ACC deaminase-containing plant growth-
promoting bacteria have been used to protect plants against
growth 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. phytofirmans
PsJN∗ (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. tomato
DC3000 (AE016853), Ra. eutropha H16 (AM260480), Ra. pickettii 12J (NZ AAWK01000011), Ra. solanacearum GMI1000∗ (AL646053), Ra. solanacearum
UW551 (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. stellata
E-37 (AAYA01000005), Se. proteamaculans SUD∗ (AY604543), Si. medicae WSM419 (AATG01000018), Si. meliloti pSmeSM11a∗ (DQ145546), St. aggregata
IAM 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 were
performed using PAUP v4.10b (Swofford, 2002) and MrBayes (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003). Appropriate evolutionary
models were chosen for Maximum Likelihood analysis using Modeltest 3.7 (Posada and Crandall, 1998). The chosen substitution model was GTR+I+G (number
of substitutions [NST] = 6; general time reversible with invariant sites and a gamma rate distribution). Nodal support in Maximum Parsimony (MP) and Neighbor
Joining (NJ) was evaluated by 1000 bootstrap pseudoreplications. The topology of the ML tree was evaluated by calculating posterior probabilities in the Bayesian
analysis. The phylogram was generated with TreeView (Page, 1996).
236 B. R. GLICK ET AL.
2007); the presence of organic toxicants such as polyaromatic
hydrocarbons (PAHs), polycyclic biphenyls (PCBs) and total
petroleum hydrocarbons (TPHs) both in the laboratory (Glick,
2003; Saleh et al., 2004; Huang et al., 2004a, b, 2005; Reed
and Glick, 2005; Reed et al., 2005) and in the field (Greenberg
et al., 2006); the presence of a variety of different metals includ-
ing nickel, lead, zinc, copper, cadmium, cobalt and arsenic, both
in 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 (Mayak
et al., 2004b; Saravanakumar and Samiyappan, 2006; Cheng
et 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; Shaharoona
et al., 2006a).
As an alternative to the use of bacteria, several transgenic
plants (tomato, canola, and tobacco) that express ACC deami-
nase have been engineered. These transgenic plants have been
reported to be tolerant of certain pathogens (Lund et al., 1998;
Robison et al., 2001), metals (Grichko et al., 2000; Stearns
et 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 with
ACC deaminase-containing plant growth-promoting bacteria.
Not only does there not appear to be any intrinsic advantage
of using transgenic plants as compared to plants treated with
plant growth-promoting bacteria, the bacterially-treated plants
generally outperform the transgenic plants probably reflecting
the fact that plant growth-promoting bacteria do more for plants
than merely lower their ethylene levels.
C. Introducing ACC Deaminase into Other Bacteria
It 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 activity
with isolated acdS genes and their regulatory regions has been
shown to improve their usefulness. For example, E. coli and
Pseudomonas strains that lack ACC deaminase but have been
transformed to express a Pseudomonas acdS gene are able to
promote the elongation of canola roots in growth pouches (Shah
et al., 1998). The effectiveness of some biocontrol pseudomon-
ads was also significantly enhanced following the introduction
of a Pseudomonas acdS gene (Wang et al., 2000). However, the
complex 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 ACC
deaminase were transformed with a Pseudomonas acdS gene
under the control of the regulatory acdR gene, ACC deaminase
was not expressed (Holguin and Glick, 2001). However, when
the native regulatory region of the Pseudomonas acdS gene was
replaced 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 was
significantly improved (Holguin and Glick, 2001, 2003). Finally,
transformation of a strain of Sinorhizobium meliloti with an acdS
gene from Rhizobium leguminosarum enables the transformed
bacterium to nodulate alfalfa plants and stimulate their growth
by 35–40% more than the native (non-transformed) strain of
Sinorhizobium meliloti can (Ma et al., 2004).
D. Bacterial Genes Activated by Root Exudates
While on the one hand, plant growth-promoting bacteria can
alter gene expression in plants, the nutrients released by plant
roots as exudates can both attract bacteria and modify bacte-
rial physiology as a consequence of the amount and types of
nutrients provided in the exudates (Lynch and Whipps, 1991).
However, only a very small number of studies have examined
the 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 PAO1
transcriptome were examined using microarray analyses (Mark
et al., 2005). P. aeruginosa PAO1 is an opportunistic pathogen
of humans but is also capable of plant root colonization (Mark
et al., 2005). Among the genes with significantly upregulated
expression are those that encode proteins involved in energy
generation 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 biocontrol
bacterium 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 to
these root exudates. When a group of genes with putative or
unknown function showed altered expression patterns, the pos-
sible roles of these genes in plant colonization were examined
using a panel of mutants with targeted disruptions. While each
separately inoculated mutant appeared to have a similar colo-
nization ability to the wild-type, some of these mutants had a
reduced ability to compete with the wild-type when they were
co-inoculated. In addition, homologues of some of these differ-
entially expressed genes were identified in the genomes of both
beneficial and pathogenic root-associated bacteria, suggesting
their involvement in biocontrol, plant growth promotion, and/or
plant pathogenesis (Mark et al., 2005).
In another study, the effect of canola root exudates on the
plant growth-promoting bacterium Pseudomonas putida UW4
was 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 increased
or decreased (P ≤ 0.05, Ratio ≥ 1.5, or Ratio ≤ –1.5), respec-
tively. Proteins with significant differences in expression levels
following exposure to canola root exudates were identified by
 PLANT GROWTH BY BACTERIAL ACC DEAMINASE
mass spectrometry. The annotation of the proteins that have
been identified so far reveals that many proteins involved in cell
movement, nutrient transportation, signal transduction, energy
production, protein synthesis and transcriptional regulation are
upregulated, while some proteins involved in cell growth and
division are downregulated. The participation of many of these
proteins in plant-microbe interactions through various mecha-
nisms was verified by individual functional analysis. In addition,
some previously uncharacterized proteins were also found to be
differentially expressed, and the implication that these proteins
are involved in plant-microbe interactions needs further con-
firmation. The differential expression profiles generated from
microarray and proteomic studies show significant overlap with
one another, with the expression of many of the same proteins
changing (Mark et al., 2005).
Integration of the differential expression data with protein
cellular 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 on
bacterial protein expression pattern. That is, bacterial proteins
involved in nutrient uptake and utilization, energy production,
and protein synthesis are upregulated in response to plant root
exudates, which may enhance the bacterium’s ability to colo-
nize the host root. However, a more complete understanding of
the effects of root exudates on bacterial gene expression awaits
additional analysis of this complex data set.
E. Plant Gene Expression Modified by Bacteria with
ACC Deaminase
The effect of plant growth-promoting bacteria on plant gene
expression has been assessed using differential display PCR,
randomly amplified PCR, microarrays, or a proteomic approach,
in each case following growth under specific conditions. Using
differential display PCR, Timmusk and Wagner (1999) observed
that plants respond to a plant growth-promoting bacterium with-
out ACC deaminase as if it were a mild biotic stressor, and this
response appears to protect the plant against subsequent stresses.
On the other hand, using randomly amplified PCR, Hontzeas
et al. (2004b) compared a wild-type ACC deaminase-containing
plant growth-promoting bacterium with an ACC deaminase mi-
nus mutant of this strain and found that the wild-type bacterium
lowered the stress level in the plant so that the plant no longer
perceived a mild stress. Thus, in the presence of ACC deami-
nase, some plant stress response genes are no longer turned on
by plant growth-promoting bacteria.
Microarray studies of plant responses to plant growth-
promoting bacteria have been utilized to provide a bird’s eye
perspective of the transcriptional trends, within plant tissues,
that occur during plant-microbe interactions. Many interesting
changes in gene expression occur within genes related to the
stress response, auxin responses, cellular metabolism and the
ethylene response. For example, workers have observed an in-
crease in transcription of stress response genes (defense genes),
237
genes involved in wounding and pathogenesis signaling, and
genes involved in auxin signaling. In many cases there are
changes in the expression of many of the transcription factor
families 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-like
genes; Myb and WRKY transcription factors; genes involved in
translation and protein folding as well as nitrogen metabolism
and catabolism. In the roots of plants treated with ISR-inducing
bacteria, 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 of
shoot tissue, since EREBP transcripts have also been observed
to increase (Czarny and Glick, unpublished results), and stay
the same (Cartieaux et al., 2003). The different responses of
EREBP genes may reflect the interaction of the plant with bac-
teria containing a variety of different physiological traits such
as the presence of ACC deaminase, the production of IAA or the
synthesis of siderophores. The enzyme ACC deaminase, when
present in the plant growth-promoting bacteria that are found in
the rhizosphere of many plants, can lower the stress perceived
by the plant and also derepress the expression of auxin response
genes in the shoots (Glick et al., 2007). In addition, bacteria that
contain ACC deaminase can suppress the expression or func-
tioning of other plant signaling molecules such as jasmonic acid
and giberellin (Czarny and Glick, unpublished results). This is
of course a consequence of the lowering of plant ethylene levels
by the action of the ACC deaminase.
Since ethylene has been found to be required for the induction
in plants of systemic resistance elicited by rhizobacteria (van
Loon et al., 1997), the question arises whether treating plants
with ethylene-lowering bacteria might prevent this induction.
However, in practice, “lowering of ethylene levels by bacterial
ACC deaminase does not appear to be incompatible with the
induction of systemic resistance. Indeed, some bacterial strains
possessing ACC deaminase also induce systemic resistance”
(van Loon and Glick, 2004). This may reflect the fact that there
is an initial very small peak of ethylene close in time to the
onset of a stress and then a second much larger peak some time
later (Glick et al., 2007). The first peak is thought to initiate a
defensive response by the plant (systemic resistance) while the
second ethylene peak is so large that processes inhibitory to plant
growth are initiated. Immediately following an environmental
stress, the pool of ACC in a plant is low as is the level of ACC
deaminase in the associated bacterium and only some of the
ACC will be cleaved by the bacterial enzyme with the remainder
being converted into the first small ethylene peak.
F. Ethylene-IAA Cross-talk
While 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,
238 B. R. GLICK ET AL.
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 event
in the life of the plant. When an ACC deaminase-containing
plant growth-promoting bacterium lowers the ethylene concen-
tration in plant roots, this relieves the ethylene repression of
auxin response factor synthesis, and indirectly increases plant
growth (see also Dharmasiri and Estelle, 2004). Thus, ACC
deaminase-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 cell
proliferation and elongation without the negative effects of in-
creasing ACC synthase and plant ethylene levels.
VI. CONCLUSION
Our understanding of some of the mechanisms used by plant
growth-promoting bacteria has come a long way in the past 10 to
20 years, and with this increased understanding there has been a
concomitant increase in the commercial application of these or-
ganisms. While there is still a lot that is not understood regarding
the functioning of these organisms, the major impediments to the
increased 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 inexpensive
agricultural 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 and
high chemical costs provide a situation where the use of plant
growth-promoting bacteria provides an attractive commercial
possibility. In addition, with recent public attention worldwide
being directed to environmental issues, many parts of the more
developed world are more actively pursuing “green” strategies
which could include the replacement of some agrochemicals
with plant growth-promoting bacteria.
Scientifically, the use of plant growth-promoting bacteria
appears, in many cases, to present a superior alternative to the
use of transgenic plants. In particular, plant growth-promoting
bacteria can help plants to tolerate a range of biotic and abiotic
stresses so that it is not necessary to genetically engineer
all cultivars of all plants to be tolerant to a large number of
different stresses.
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