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Promotion of Plant Growth By Bacterial ACC Deaminase

Posted on: Saturday, 24 November 2007, 06:00 CST

By Glick, Bernard R Todorovic, Biljana; Czarny, Jennifer; Cheng, Zhenyu; Duan, Jin; McConkey, Brendan

To date, there has been only limited commercial use of plant growth-promoting bacteria in agriculture, horticulture, and silviculture. 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-aminocyclopropane-1- carboxylate (ACC) deaminase. This article 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. Keywords Plant growth-promoting bacteria, plant stress, ethylene, IAA

Table of Contents

I.

.................................................................228

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

A. Indirect

.................................................................228

B. Direct

.................................................................228

III. ETHYLENE AND

.................................................................229

A. Effects of Ethylene on Plants

.................................................................229

B. Genes Involved in Stress Signaling and

.................................................................229

IV. ACC

.................................................................230

A.

.................................................................230

B. Protein Structure

.................................................................231

C. Genes and

.................................................................232

D. Transcriptional Regulation

.................................................................233

V. BACTERIA WITH ACC DEAMINASE

.................................................................235

A. Plant Growth

.................................................................235

B. Amelioration of Stress

.................................................................235

C. Introducing ACC Deaminase into Other

.................................................................236

D. Bacterial Genes Activated by Root

.................................................................236

E. Plant Gene Expression Modified by Bacteria with ACC Deaminase ...............................................................237

F. Ethylene-IAA Cross-

.................................................................237

VI. CONCLUSION

.................................................................238

REFERENCES

.................................................................238

I. INTRODUCTION

Increased public concern about environmental problems caused either directly or indirectly by the use of fertilizers, pesticides, herbicides, and fungicides, has prompted researchers to consider alternatives to these established chemical strategies 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 bacteria (Brown, 1974; Kloepper et al., 1986, 1988, 1989; Davison, 1988; Glide et al., 1999; Lambert and Joos, 1989). These plantbeneficial bacteria can bind to either roots (rhizosphere bacteria), 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 nutrients exuded from the roots of many plants that can be utilized 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, under 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 phytopathogens 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 reported variability and inconsistency of results between laboratory, 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 growthpromoting bacteria. Past inconsistent and irreproducible results with plant growth-promoting bacteria may reflect variations in crops and cultivars, soil composition, the presence of indigenous soil microorganisms, weather, soil moisture content and, perhaps most importantly, an incomplete understanding 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 reproducible use of plant growth- promoting bacteria, in recent years considerable progress has been made toward developing 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 biocontrol agents against phytopathogens and thus indirectly stimulate plant growth may be the consequence of any one of a variety of mechanisms including antibiotic production, depletion of iron from the rhizosphere, induced systemic resistance, production of fungal cell wall lysing enzymes, and competition 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 antibiotics (Haas et al., 1991; Keel et al., 1992; Chet and Inbar, 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 biocontrol plant growth-promoting bacteria produce enzymes such as chitinase, beta1,3glucanase, protease, or lipase, all of which can facilitate the lysis of fungal cells (Chet and Inbar, 1994). Interestingly, the defense response of stressed plants often results in the production of the same array of proteins (i.e., chitinase, beta1,3-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 surface 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 obvious 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 metabolites (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 atmospheric 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 enhance 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 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 development 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 invoked to explain the various direct effects of plant growthpromoting bacteria on plants is the production of phytohormones, 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 enzyme 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 hydrolysis 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 initiation 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 applied 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 during developmental processes, ethylene regulates xylem formation, 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 hypertrophies, 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 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 ethylene. 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, including 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 necro trophic.

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 response (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 synergy 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 members 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 various 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 different stress response networks, where ethylene signaling plays a pivotal role.

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 a- ketobutyrate. This multimeric enzyme is a common component 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 demonstrated that these putative ACC deaminase genes encode an enzyme with ACC deaminase activity.

A. Biochemistry

The pioneering work in elaborating the biochemical properties 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 deaminase 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 K^sub m^ of various ACC deaminases for ACC indicate that the enzyme does not bind the substrate with a high affinity; K^sub m^ 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 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 K^sub m^ 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 or-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 reaction starts with conversion from an internal aldimine between the enzyme and PLP to an external aldimine between the substrate ACC and PLP. In most other PLP-dependent reactions, the next step is the nucleophilic abstraction of either an or-proton or an a-carboxylate group. However, in this regard the ACC deaminase catalyzed reaction is considered as a special case, since the substrate does not contain a a-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 C^sub beta^ methylene position followed by beta-proton abstraction and (ii) direct beta-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 basis 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 (Pseudomonas sp. ACP) and yeast (Hansenula saturnus) ACC deaminase and an ACC deaminase homologue without this activity (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 mutagenesis studies, have allowed for identification of the essential 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 alpha/beta structure similar to the beta-subunit of the PLP-dependent enzyme tryptophan synthetase.

An amino acid sequence alignment of several enzymes identified 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 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. saturnus 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 enzymatic residues involved in nucleophilic addition and beta-proton abstraction. Lys51 (Ose et al., 2003) and Tyr294 (Karthikeyan et al, 2004a,b) are two major candidates proposed to be involved 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 beta-proton abstraction, thus far, only Lys51 has been proposed to be involved (Zhao et al., 2003).

The determination of the three-dimensional X-ray crystallographic structure of the ACC deaminase homologue from Pyrococcus horikoshii has allowed for further insight into what makes the ACC deaminase reaction unique. The Pyrococcus horikoshii enzyme was originally predicted to be ACC deaminase 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 Dand L-serine, producing pyruvate as a result of catalysis (Fujino et al, 2004). The overall topology of the ACC deaminase homologue 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 electron 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 pyridine 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 homologue, 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 beta-subunit (TRPSbeta) family, which includes TRPSbeta, O- acetylserine sulfhydrylase, threonine deaminase, and threonine synthase, such that the ACC deaminase homologue 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 are expected to have a leucine residue (Leu323 for H. saturnus) in close proximity to the Glu296 residue. In the ACC deaminase 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; Jacobson et al., 1994; Glick et al., 1995,1998; Campbell and Thomson, 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; Shaharoona et al., 2006a, b; Saravanakumar and Samiyappan, 2006; Hameeda et al., 2006). Moreover, it is found at a relatively high frequency in many rhizosphere soils (Klee and Kishore, 1992; Glick et al., 1995; Blaha et al., 2006; Duan et al., unpublished results). Interestingly, within a particular genus and species of microorganism, some strains have ACC deaminase activity, and others often do not. Thus, for example, while some strains of Azospirillum have recently been reported to contain an acdS gene (Blaha et al., 2006) under the transcriptional control of the regulatory gene acdR (Moenne-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 phylogenetic 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 experimentally 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 addition, 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 Betaproteobacterium. 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 Actinobacteria. 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 undergone 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 Mesorhizobium loti strain R7A. Similarly, a recently sequenced Rhizobium 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 protein, 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 leguminosarum bv. trifolii strain NZP514 has an acdS gene on plasmid pRtr514a with an acdR located upstream of acdS. Generally speaking, it is easier, both in the laboratory and in the environment, 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 protein (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; Moenne-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 encoding this protein has been termed acdR (i.e., ACC deaminase regulatory gene). A detailed model, described below, of transcriptional 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, located 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 transcription of acdS. Upon synthesis of ACC deaminase (AcdS), ACC is cleaved to form ammonia and alpha- 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 nevertheless still able to produce active enzyme (Glick et al., unpublished results). At this point, whether the AcdB-ACC complex is sufficient 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 responsible 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 nodules from synthesizing ethylene in response to the stress of depleting local energy resources to fuel energy intensive nitrogen 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 bacteria bind to the surface of either the seed or root of a developing 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 stimulate 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 exuded and taken up by the ACC deaminase-containing bacteria. This ACC is cleaved by ACC deaminase to form ammonia and alpha-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 bacteria act as a sink for ACC.

Direct consequences of this interaction are significantly increased plant root and shoot length, an increase in biomass, and protection of plants from inhibitory effects of ethylene synthesized 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 inhibition 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 growthpromoting 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., 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 including 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 various 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 deaminase 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 instances, transgenic plants that express the enzyme ACC deaminase 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 deaminase genes into bacterial strains that lack this activity. Transformation 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 pseudomonads 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 expression of many acdS genes (see section IV-D) may not be operative 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 bacterial 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 varieties 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 surprising since the utilization of the major plant root components, such as amino acids and organic acids, by Pseudomonas fluorescence 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 biosynthesis 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 possible 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 colonization 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 differentially 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 electrophoresis (2-D DIGE) analysis (Cheng, Glick, and McConkey, unpublished data). Out of a total number of 1,757 proteins detected on the analytical gels, the expression levels of 172 (9.8%) proteins and 220 (12.5%) proteins were significantly increased or decreased (P /= 1.5, or Ratio

Integration of the differential expression data with protein cellular functions sheds some light on the biochemical mechanisms and regulatory systems mediating plant-microbe interactions 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 colonize 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 without 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 minus 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 deaminase, some plant stress response genes are no longer turned on by plant growth-promoting bacteria.

Microarray studies of plant responses to plant growthpromoting 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 increase in transcription of stress response genes (defense genes), 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) decrease in the transcription of ethylene response factors (EREBP) genes, that levels off to a 2-fold decrease by day seven (Verhagen et al, 2004). This change is not consistent within studies of shoot tissue, since EREBP transcripts have also been observed to increase (Czamy 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 bacteria 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 functioning 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), me 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 mought 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 transcription of ACC synthase (Kende, 1993; Kende and Zeevaart, 1997; Kim et al., 1992), it is less well known that ethylene may inhibit 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 synthesis and/or functioning limits the amount of ACC synthase, ACC and, ultimately, ethylene following every stressful event in me life of the plant. When an ACC deaminase- containing plant growth-promoting bacterium lowers the ethylene concentration 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 facilitate plant growth by (i) decreasing emylene inhibition of various plant processes and (ii) permitting IAA stimulation of cell proliferation and elongation without the negative effects of increasing 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 mese organisms. While there is still a lot that is not understood regarding the functioning of mese organisms, the major impediments to the increased commercial use of these bacteria are economic. Thus, in countries of me world (generally more developed countries) where agricultural productivity is high and much of this productivity is based on the extensive use of relatively inexpensive agricultural chemicals, there is little immediate economic incentive to alter existing agricultural practice. On the other hand, in many of the less developed countries of the world where agricultural 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 me 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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Source: Critical Reviews in Plant Sciences

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