Molecular Microbial Ecology of the Rhizosphere (de Bruijn/Molecular Microbial Ecology of the Rhizosphere) || Bacterial Biosynthesis of Indole-3-Acetic Acid: Signal Messenger Service

Chapter 29

Bacterial Biosynthesis ofIndole-3-Acetic Acid: SignalMessenger Service

Mandira KocharNanobiotechnology Centre, Biotechnology and Bioresources Division, The Energy andResources Institute (TERI), India Habitat Centre, India

A Vaishnavi, Anamika Upadhyay, and Sheela SrivastavaDepartment of Genetics, University of Delhi South Campus, India

29.1 INTRODUCTION

The rhizosphere is a hub of immense metabolic activitiesdue to the presence of highly versatile microorganisms.Several rhizosphere-inhabiting microorganisms fulfillimportant ecological functions, such as nutrient recyclingand pathogen control, that often translates into better plantgrowth and health (Couillerot et al., 2009). Additionally,this microenvironment is described as a “microbialhot-spot” where diverse interactions between organisms,beneficial as well as pathogenic, take place (Whipps,2001). The former group of strains comprises the plant-growth-promoting bacteria (PGPB) that influence plantgrowth by producing phytohormones or enhancing theavailability of nutrients by inducing systemic resistance inplants and by antagonizing pathogenic bacteria (Bashanand de-Bashan, 2005; Lugtenberg and Kamilova, 2009;Raaijmakers et al., 2009; Spaepen et al., 2009a; Upadhyayand Srivastava, 2010a; Fibach-Paldi et al., 2011; seeChapter 53). It is also clear that some PGPB are capableof exerting multiple plant growth promoting (PGP)mechanisms (Upadhyay and Srivastava, 2008, 2010b;Bashan and de-Bashan, 2010). Successful utilizationof PGPB in agriculture, therefore, requires a thoroughunderstanding of the mechanisms that enable them to

Molecular Microbial Ecology of the Rhizosphere, Volume 1, First Edition. Edited by Frans J. de Bruijn. 2013 John Wiley & Sons, Inc. Published 2013 by John Wiley & Sons, Inc.

colonize the rhizosphere and of the factors that lead tostimulation of their beneficial effects. Of the differentfunctions carried out by PGPB, phytohormone synthesishas received a lot of attention as it could directly andbeneficially influence root growth. Although rhizospherebacteria are reported to synthesize a whole variety ofphytohormones, auxins, and more so indole-3-acetic acid(IAA), have been studied extensively.

Auxins influence a range of plant developmentalprocesses, as documented in earlier reports (Davies, 1995;Pagnussat et al., 2009; Santner and Estelle, 2009; Vannesteand Friml, 2009; Zhao, 2010). Although, both, plants andPGPB are reported to produce IAA, many aspects of auxinbiology, especially in plants, remain poorly understood.Auxins synthesized in young plant tissues are transportedto other tissues where they are perceived by members ofthe transport inhibitor response 1 (TIR1) auxin receptorfamily (Ljung et al., 2005). Although our understandingof auxin transport and signaling in plants has increased(Quint and Gray, 2006; Vieten et al., 2007), the pathwaysof auxin synthesis and their regulation still remain rela-tively unclear. Within this domain, much less well knownis the manner in which microbially produced auxins areutilized and regulated to improve plant growth (Costacurtaand Vanderleyden, 1995; Patten and Glick, 1996; see also

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310 Chapter 29 Bacterial Biosynthesis of Indole-3-Acetic Acid: Signal Messenger Service

Chapters 27, 28). In their interaction with plants for thispurpose, microbial responses can be, both, detrimental aswell as beneficial. While in the former, microbes interferewith plant development by disturbing the auxin balancein plants, as is the case with phytopathogenic bacteriasuch as Agrobacterium spp. and Pseudomonas savastanoipv. savastanoi, causing tumors and galls, respectively(Jameson 2000; Mole et al., 2007). Alternatively, theycan supplement the IAA pool, as is done by PGPB suchas Azospirillum spp., which results in improved plant rootdevelopment (Persello-Cartieaux et al., 2003; Malhotraand Srivastava, 2006; Spaepen et al., 2007a).

Microbial biosynthesis of phytohormones and plantgrowth regulators (PGR), namely auxins, cytokinins, andgibberellins, biomolecules such as nitric oxide, and othermetabolites/enzymes that interfere with plant ethylenesynthesis, such as 1-aminocyclopropane-1-carboxylate(ACC)-deaminase, jasmonates, and polyamines (Bottiniet al., 1989; Glick, 1995; Garcia de Salamone et al.,2001; Persello-Cartieaux et al., 2003; Creus et al., 2005)have been well documented for PGPB (see Chapters27, 28). Rhizosphere bacteria are likely to thrive onsubstrates present in root exudates or in the rhizospherebecause of microbial/plant activity and convert them intogrowth hormones to be used by the plant partner.

In recent years, the role of IAA as a signalingmolecule has been fully established for some microor-ganisms (Lambrecht et al., 2000; Spaepen et al., 2007a).The studies carried out over several years have madeit very clear that auxins can have a major impact onmicroorganism–plant interactions. While the main themeaddressed in this chapter is the biosynthesis of IAA byPGPB and its role in plant interactions, related recentfindings on auxin signaling will also be discussed, owingto their importance in this field of research.

29.2 IAA BIOSYNTHESIS:MULTIPLE PATHWAYS, ONEEND-PRODUCT; THE WELL-WORKEDOUT PGR

A wide range of free-living as well as plant-associatedbacteria, both symbiotic and phytopathogenic, have beendocumented to produce IAA (Costacurta and Vanderley-den 1995; Tsavkelova et al., 2006; Gravel et al., 2007).The functions performed by the IAA in relation to sym-biosis, plant-growth promotion, in planta defense, andas signaling molecules have been described by severalauthors (Malhotra and Srivastava, 2006, 2008a; Pieterseet al., 2009; Spaepen and Vanderleyden, 2010; Fu andWang, 2011; see Chapters 27, 28).

Multiple IAA biosynthesis pathways in bacteria havebeen unraveled in the past decade or so, nevertheless

many steps and molecular components still remainundefined (Patten and Glick, 1996; Spaepen et al., 2007a;Spaepen and Vanderleyden, 2010). These have been clas-sified as tryptophan (Trp)-dependent and Trp-independentpathways.

29.2.1 Tryptophan-DependentPathways of IAA BiosynthesisThree main biosynthetic routes involving indole-3-pyruvicacid (IPyA), tryptamine (TAM), and indole-3-acetonitrile(IAN) (Fig. 29.1) have been studied in higher plants,as well as plant-associated bacteria, with the latter con-taining an additional indole-3-acetamide (IAM) pathway.The IPyA pathway (Fig. 29.1a) is operational in plantsand plant-beneficial bacteria, such as Azospirillum andEnterobacter cloacae, and is subjected to extremelytight regulation (Koga et al., 1991; Costacurta et al.,1994; Costacurta and Vanderleyden, 1995; Patten andGlick, 1996; Van de Broek et al., 2005; Malhotra andSrivastava, 2008a; see subsequent sections). In thispathway, the transamination of tryptophan to IPyA occursfollowed by decarboxylation to indole-3-acetaldehyde(IAAld) by the enzyme indole-3-pyruvate decarboxylase(IPDC or phenylpyruvate decarboxylase, PPDC) and thenfinally oxidation of IAAld to IAA (Fig. 29.1a). In thisreaction, tryptophan aminotransferase, that catalyzes thetransamination reaction, is neither specific for tryptophannor does it prefer tryptophan as its substrate (Koga et al.,1994; Soto-Urzua et al., 1996; Spaepen et al., 2007b).Recent reports also indicate that this pathway may beregulated by TyrR that regulates aromatic amino acidtransport and metabolism (Ryu and Patten, 2008).

IAA is synthesized via a constitutive pathwayinvolving the production of IAM in phytopathogens,and the biocontrol strain, Pseudomonas fluorescens Psd(Escobar and Dandekar, 2003; Kochar et al., 2011). Inthe first step, tryptophan is converted to IAM followedby hydrolysis of IAM to IAA and ammonia in thesecond step. The key enzymes catalyzing these stepsinclude tryptophan mono oxygenase (encoded by theiaaM gene) and IAM hydrolase (encoded by the iaaHgene), respectively (Fig. 29.1d). In spite of the belief thatIAM is a predominantly microbial pathway, evidence isgrowing that this pathway also exists in plants such asArabidopsis (Pollmann et al., 2002, 2003). The tryptophanside-chain oxidase (TSO) pathway (Fig. 29.1f) has beenshown to co-exist in P. fluorescens CHA0 along withthe anthranilate pathway of Trp degradation (Oberhansliet al., 1991; Upadhyay and Srivastava, 2010b). Whilethe TAM pathway (Fig. 29.1b) is widely prevalent inplants (Bartel et al., 2001) and fungi (Frankenberger andArshad, 1995), scattered information is available aboutits presence in selected plant-associated bacteria, such as

29.3 Rhizobacterial IAA: Benefitting Roots Directly 311

Chorismate

Anthranilate

Indole-3-glycerol phosphate

Indole + serine

Tryptophan

Indole-3-pyruvic acid

Indole-3-lactic acid

Tryptamine

TSO

TDC

Indole-3-acetamide

TMO

IAH

TOLoxidase

NitrilaseTryptophol

Indole-3-acetaldehyde

Indole-3-acetic acid

IPDC/PPDC

Indole-3-acetonitrile

(d )

(c )

(e)

(a)(b)

(f)

Figure 29.1 Overview of the IAA biosynthesis pathways known: (a) IPyA pathway involving IPDC/PPDC, (b) TAM pathway involving Trpdecarboxylase, (c) IAN pathway involving the conversion of naturally-occurring indole-3-acetaldoxime (IAAldOx) or certain glucosinolates, (d)IAM pathway involving TMO and IAH, (e) TOL pathway where IAAld is reduced to form TOL, and (f) TSO pathway.

Azospirillum brasilense and Bacillus cereus (Hartmannet al., 1983). The IAN route of IAA biosynthesis,predominantly functioning in plants, has also beenobserved in microbes to work through two enzymaticsteps (Kobayashi et al., 1992; Fig. 29.1c). This pathwayalso involves the conversion of naturally-occurringindole-3-acetaldoxime or certain glucosinolates in plantsby myrosinase or indoleacetaldoxime hydratase to formIAN and its subsequent conversion to IAA by nitrilase,as demonstrated in cabbage, radish, grasses, and banana(Kobayashi et al., 1995). Another pathway is the tryp-tophol (TOL) pathway where IAAld is reduced to formTOL in plants, as well as in bacteria such as Azospirillaand Paenibacillus (Costacurta et al., 1994; Lebuhn andHartmann, 1994). In plants, TOL serves as a storageform and is converted to active IAA by TOL oxidase(Dobbelaere et al., 2003; Fig. 29.1e). IAAld oxidase hasbeen implicated in plants, such as cucumber, barley, andmaize, and its cDNA has been cloned from Arabidopsisthaliana (Sekimoto et al., 1998). A current overviewof different tryptophan-dependent IAA biosynthesispathways is summarized in Figure 29.1.

29.2.2 Tryptophan-IndependentPathways of IAA BiosynthesisBesides the Trp-dependent pathways described so far,biosynthesis of IAA without Trp as a precursor has beenshown in the plants Lemna gibba and A. thaliana (Baldiet al., 1991; Ouyang et al., 2000). The only report inbacteria was provided by Prinsen et al. (1993), whodemonstrated that in A. brasilense SpF94, 90% of theIAA is synthesized in a de novo, Trp-independent mannerand there may be a strong regulatory interaction betweenboth types of pathways. However, the prevalence of thisroute of IAA biosynthesis has not been conclusivelyproven in bacteria. The different genes and proteinsidentified to play a role in bacterial IAA biosynthesis aresummarized in Table 29.1.

29.3 RHIZOBACTERIAL IAA:BENEFITTING ROOTS DIRECTLY

Our understanding of phytohormone biosynthesis byPGPB as a direct mechanism of plant growth enhancement


Table 29.1 Summary of bacterial genes and proteins involved in production of indole-3-acetic acid (IAA) and theirnature of expression

Gene Pathway ProteinFunctional

Product ExpressionReference

Strains Source

IAA Biosynthesistat ? IPyA AAT Aromatic

amino acidtransferase

Mostly con-stitutive

AzospirillumbrasilenseUAP 14,Enterobactercloacae

Soto-Urzua et al.(1996); Kogaet al. (1994);Pedraza et al.(2004)

ipdC/ppdC IPyA IPDC/PPDC Indole-3-pyruvatedecarboxy-lase/phenylpyruvatedecarboxy-lase

Regulated Enterobactercloacae,Pantoeaagglomerans,Azospirillumbrasilense,Pseudomonasputida GR12

Koga et al. (1991);Costacurta et al.(1994); Pattenand Glick(2002a); Van deBroek et al.(2005); Ryu andPatten (2008);Malhotra andSrivastava(2008a, 2008b);Chalupowiczet al. (2009)

Iad1 IPyA IAD1 Indole-3-acetaldehydedehydroge-nase

Constitutive Ustilago maydis(phy-topathogenicfungus)

Basse et al. (1996)

iaaM, tms-1 IAM TMO Tryptophan-2-monooxy-genase

Regulated PseudomonassyringaeEW2009,Agrobacteriumtumefaciens(phy-topathogenicbacteria)

Comai and Kosuge(1980); Inzeet al. (1984)

iaaH, tms-2,bam

IAM IAH Indole-3-acetamidehydrolase

Regulated Same as above,Bradyrhizo-biumjaponicum,Pantoeaagglomerans

Comai andKosuge (1980);Kaneshiro et al.(1983); Inzeet al. (1984);Chalupowiczet al. (2009)

ND TSO TSO Tryptophan-2′-dioxygenase

Regulated PseudomonasfluorescensCHA0

Narumiya et al.(1979);Oberhansli et al.(1991)

ND TAM TDC Tryptophandecarboxy-lase

Regulated Azospirillumbrasilense,Lactobacilluscurvatus

Hartmann et al.(1983);Carreno-Lopezet al. (2000);Bover-Cid et al.(2001)

Nit ? IAN ND Nitrilase Regulated Alcaligenesfaecalis

Nagasawa et al.(1990);Kobayashi et al.(1992)

29.4 Auxin (IAA) in Symbiosis 313

Table 29.1 (Continued)

Gene Pathway ProteinFunctional

Product ExpressionReference

Strains Source

ND IAN NH Nitrilehydratase

ND Rhodococcus Brady et al.(2004);Kobayashi et al.(1992)

trpE Trp operon TrpE Anthranilatesynthase

Regulated Azospirillumbrasilense Sp7

Zimmer et al.,1991

trpG Trp operon TrpG Glutamine ami-dotransferase

Constitutive Same as above Same as above

trpD Trp operon TrpD Phosphoribosylanthranilatetransferase

Constitutive Same as above Same as above

trpC Trp operon TrpC Indole-3-glycerolphosphatesynthase

Regulated Same as above Same as above

iaaspH IAA-conjugate IAASPH IAA-aspartatehydrolase

Storageform

Enterobacteragglomerans

Chou et al. (1996)

ND, not determined or identified.

has increased based on molecular approaches appliedto analyze microbial and plant mutants altered in theirability to synthesize or respond to specific phytohormones(Bloemberg and Lugtenberg, 2001; Persello-Cartieauxet al., 2003; Bottini et al., 2004; Malhotra and Srivastava,2008b). IAA has been detected in as many as 80% ofnon-symbiotic PGPB and rhizosphere bacteria (Loperand Schroth, 1986; Dobbelaere et al., 1999), in symbioticnitrogen-fixing cyanobacteria (Sergeeva et al., 2002),in the actinomycete, Frankia (Wheeler et al., 1984),and in rhizobia (Keford et al., 1960). Moreover, theenhancement of plant growth by root-colonizing Bacillusand Paenibacillus strains has been well documented(Kloepper et al., 2004; Timmusk and Wagner, 1999; Yaoet al., 2006), but information on the prevalence of IAAproduction by Gram-positive free-living soil bacteriais still lacking. The majority of the reports focus onGram-negative bacteria (Spaepen and Vanderleyden,2010 and references therein).

IAA has been considered to be the predominant causeof growth improvement, as opposed to the N2-fixingcapacity of diazotrophic PGPB strains such as Azospiril-lum (Bashan et al., 1989; Malhotra and Srivastava, 2006,2009). Although many species of Pseudomonas have beenclassified as PGPB, only some information is availableon IAA biosynthesis by these bacteria (Oberhansli et al.,1991; Patten and Glick, 2002a; Kochar et al., 2011).

The majority of PGPB use the IPyA pathway tosynthesize IAA. Patten and Glick (2002a) demonstratedthat a mutation in the P. putida ipdC gene resulted in a

significant reduction of primary root growth in canolaseedlings. Results obtained in our laboratory show thatknocking out the A. brasilense strain SM ipdC generesults in a 50% reduction in IAA level and a consequentdecrease in the root growth-promoting response (Mal-hotra and Srivastava, 2009). Dobbelaere et al. (1999)have reported a 90% reduction in IAA level in case ofAzospirillum strain Sp6. This may reflect the presence andcontribution of different IAA biosynthesis pathways orthe genetic composition of the strain studied. In additionto PGPB, symbiotic Rhizobia are also able to synthesizeIAA (Sridevi and Mallaiah, 2007; Kumari et al., 2009).It has also been speculated that rhizobia alter the rootauxin balance as a pre-requisite for nodule formation,and nodule numbers are regulated by shoot-to-root auxintransport (Mathesius, 2008). In Rhizobium meliloti andR. leguminosarum, the production of IAA is regulated ina host-specific manner. The production is enhanced inthe presence of Nod-inducing flavonoids (Prinsen et al.,1991; Hassan and Mathesius, 2012; see Chapter 51).

29.4 AUXIN (IAA) IN SYMBIOSIS

Unlike the majority of plants, legumes have the capacityto interact with Rhizobia to develop a beneficial rootendosymbiosis (see Chapters 44, 45). Bacterial endosym-bionts are also found with some non-legumes, wherean actinomycete, Frankia, is involved (Mathesius, 2008;Desbrosses and Stougaard, 2011; Perrine-Walker et al.,2011). Several studies have converged on the fact that


auxin is the key regulator of, both, lateral root develop-ment and symbiotic nodule development (Fukaki et al.,2007). Lateral roots have been an integral part of theplant root system ever since plants evolved. Nodulation,however, is considered to be a comparatively more recentevent (∼60 million years), perhaps as a trigger for thelack of N2 in a CO2-rich environment (Sprent, 2008).Thus, this shift from a normal root to a nodulated onewould have involved a bacterium-assisted manipulationof a major developmental program. The process could beenvisaged to comprise two tightly coordinated steps: nod-ule organogenesis, and infection and bacterial colonization(Madsen et al., 2010; Desbrosses and Stougaard, 2011).

Nodulation involves a host–symbiont signal ex-change that leads to the secretion of flavanoids by thehost (Hassan and Mathesius, 2012; see Chapter 51)stimulating the symbionts to synthesize Nod factors, thatin turn, induce rapid reprogramming of root pericycle andcortical cells (see Chapter 45). This leads to the creationof a nodule primordium based on a new meristematiccenter comprising the nodule founder cells. At this site,rapid cell divisions are initiated by two crucial planthormones, auxins and cytokinins (Mathesius, 2008;Desbrosses and Stougaard, 2011).

Available information on nodulation has suggestedthat initiation of nodules requires a lowering of auxin-to-cytokinin ratios. This is in contrast to lateral root develop-ment that is promoted by a high auxin-to-cytokinin ratio,indicating the bifurcation of these two developmental pro-cesses. The reduction in the levels of auxin can be broughtabout, both, by inhibition of auxin transport by Nod fac-tors as well as by cytokinin signaling (Mathesius et al.,1998). Once the organ determination is specified, auxincan play a similar role in activating cell cycle progres-sion in both types of primordia. Auxin remains a centralregulator of vascular differentiation and meristem activa-tion (Mathesius, 2008). With the initial report by Thimann(1936) that root nodules have a higher IAA content andthat auxin accumulates in the nodule during its formation(Mathesius et al., 1998), many rhizobial strains capable ofproducing IAA have been reported (Prinsen et al., 1991;Theunis et al., 2004). However, the exact benefit of IAAto the bacterial partner in the legume–Rhizobium symbio-sis has not yet been elucidated. Screening of a transposonmutant library of Rhizobium etli identified four genes thatappear to be regulated by IAA. These genes are likely tobe involved in plant signal processing, motility, or attach-ment to plant roots, all of which could implicate IAA inhost–microbe interaction (Spaepen et al., 2009b).

The auxin-responsive plant promoters, GH3 andDR5, have been monitored for their spatial and temporalchanges in auxin accumulation during nodulation inlegumes forming determinate and indeterminate nodules.In white clover, which is known to form indeterminate

nodules, Rhizobia were found to cause an initial dropin GH3 activation around the site of infection withina couple of hours (Mathesius et al., 1998), followedby a subsequent increase. According to Huo et al.(2006), in Medicago truncatula, DR5 expression appearsto be intermittent, below the nodule initiation site,whereas it was induced in the nodule-forming site. GH3expression was observed in the dividing cortical cells ofa nodule in white clover (Mathesius et al., 1998) andM. truncatula (Van Noorden et al., 2006). GH3 alsodisplayed an increased expression pattern in the earlynodule primordium of white clover. It remained active inthe meristem part and the vascular bundles (Mathesiuset al., 1998). These studies show that the changes inauxin distribution contribute to, both, nodule formationas well as lateral root development. The genetics ofnodulation and plant–microbe interaction has progressedslowly because of a paucity of mutants in the hormonalpathways. In the future, this gap in knowledge shouldbe filled with the analysis of novel mutants and genomesequencing to identify the genes and their function in thiscomplex pathway. This effort will prove worthwhile notonly to understand the basis of symbiotic plant–microbeinteraction but will also serve as a model to study themolecular signaling pathway between the two partners.

29.5 IAA IN PHYTOPATHOGENS

IAA production plays an important role in numerousplant–pathogen interactions (Yamada, 1993; Jameson,2000). The potential of IAA production by pathogenicbacteria has been identified as a major pathogenicitydeterminant in gall- and knot-forming bacterial speciessuch as P. savastanoi, Agrobacterium tumefaciens,and Pantoea agglomerans. These bacteria synthesizelarge amounts of IAA mostly via the IAM pathway(Comai and Kosuge, 1982; Yamada et al., 1985; Jameson2000; Persello-Cartieaux et al., 2003; Fig. 29.1d). InP. agglomerans pv. gypsophilae, both, the IAM andthe IPyA (Fig. 29.1a) pathways coexist (Spaepen andVanderleyden, 2010). Synthesis of IAA by the Grampositive phytopathogen, Rhodococcus fascians, has alsobeen reported (Vandeputte et al., 2005).

The genes involved in the pathway, iaaM andiaaH (Table 29.1), have been characterized from manyphytopathogens (Clark et al., 1993). The genes may belocated either on plasmids or on the chromosome. Forinstance, in A. tumefaciens, these genes are located in theT-DNA region of the pTi plasmid together with the ipt(isopentenyltransferase) gene (Table 29.1). In A. tumefa-ciens, plant-derived signals such as amino acids, organicacids, sugars, and so forth lead to activation of vir genes.After the transfer of T-DNA into the plant genome, the

29.6 Regulation of IAA Levels 315

massive accumulation of auxin and cytokinin broughtabout by the activities of the iaaM, iaaH, and ipt codedenzymes is the primary driver of tumorigenesis (Escobarand Dandekar, 2003. In P. savastanoi pv. savastanoi,these genes are located on plasmid pIAA1 (Comai andKosuge, 1982) and clustered in an iaa operon that isresponsible for IAA production in free-living cultures.The strains that do not harbor the pIAA1 plasmid areno longer capable of inducing gall formation on hostplants such as olive and oleander (Comai and Kosuge,1982). An earlier report by Smith and Kosuge (1978)demonstrated that an IAA-deficient mutant of Pseu-domonas syringae pv. savastanoi failed to induce gallformation in oleander. In P. savastanoi pv. savastanoi,some intermediates of IAA biosynthesis pathways werefound, which contribute to the stability of bacterial IAAinside the host plant by providing protection againstseveral plant enzymes such as hydrolases. The gene,iaaL, encoding indole-3-acetic acid-lysine synthetase ispresent on plasmid pIAA1 along with IAA biosynthesisgenes. This enzyme catalyzes the formation of an amidebond between carboxyl group of IAA and the epsilonamino group of Lys (Glass and Kosuge, 1988). AniaaL-deficient mutant lost the ability to accumulate IAAin the medium and, hence, the virulence was affected.In P. syringae pv. syringae, the IAA biosynthesis genesare located on the chromosome, resulting in a lower IAAproduction that could be enhanced as much as four-foldby incorporation of a low copy number plasmid carryingthe IAA biosynthesis operon (Mazzola and White, 1994;Patten and Glick, 1996; Brandl and Lindow, 1996).Auxin signaling has also been shown to promote diseasesusceptibility of Arabidopsis to P. syringae (Navarroet al., 2006).

This indicates that IAA has a direct role in the devel-opment of symptoms caused by tumorigenic bacteria andthe expression of genes involved in virulence (Yamada,1993).

29.5.1 Contribution of IAA toVirulence of PhytopathogensA number of mechanisms have been proposed toexplain the direct contribution of IAA to pathogenicityof the phytopathogen (Fu and Wang, 2011). Cell wall,the primary barrier against phytopathogens, is amongthe first targets of IAA. Rigidity of the cell wall is per-turbed by some proteins such as endo-β-1,4-glucanases,xyloglucan endotransglycosylases, and expansins. Theseproteins mediate the cell wall extension, facilitating theentry of pathogens. A few reports have described auxininduction of these proteins (Fry et al., 1992; Brummellet al., 1994; Catala et al., 1997). While the functionof the first two categories of these proteins is coupled

to the depolymerization of cell-wall polysaccharidesby hydrolysis, the expansins function by breaking thehydrogen bonds between cellulose microfibrils and cellwall polysaccharides, leading to a permanent extensionof the cell wall (McQueen-Mason and Cosgrove, 1995).IAA is also reported to mediate the overexpressionof expansins, thus making the plant vulnerable to thebiotic intruders. The expression of LeExp2, a tomatoexpansin gene, is also upregulated by IAA (Catala et al.,2000). Another mechanism for eliciting pathogenicityis the suppression of salicylic acid (SA)-mediated plantdefenses. P. syringae type III effector AvrRpt2, a serineprotease, cleaves a number of host proteins leadingto alterations in IAA homeostasis within the host. Itsuppresses the pathogenesis-related (PR) gene expressionthat is activated in SA-dependent defense responses(Chen et al., 2007).

The virulence of phytopathogens may also beattributed to some pathogen-associated molecular patterns(PAMPs). The bacterial protein, flagellin, Flg22, is anevolutionary conserved PAMP and acts by repressingmRNA levels of TIR1, the transport inhibitor response1 protein (Abramovitch et al., 2006). Flg22 also leadsto an increase in the transcription levels of miR393targeting F-box mRNAs (TIR1, AFB2, and AFB3),thereby leading to downregulation of auxin receptorproteins and an increased resistance to P. syringae inArabidopsis (Navarro et al., 2006).

29.6 REGULATION OF IAA LEVELS

The mechanisms by which microbes regulate the levels ofIAA are complex and not fully understood. Besides thenumber of IAA biosynthesis pathways operating in onebacterium, the problem is further compounded by a multi-tude of factors that interplay with each other to control theIAA secreted by a microbe. IAA biosynthesis and secre-tion by bacteria is, therefore, multipronged and intricatelyregulated, as outlined in Figure 29.2. It is clear that besidesthe genetic regulation, the expression of IAA biosynthe-sis genes needs to be fine-tuned to encounter a diversearray of environmental stress conditions (Spaepen et al.,2007b; Malhotra and Srivastava, 2008a, 2009). Some ofthe factors and their detailed contribution to the regulationof IAA levels are detailed subsequently (Table 29.2). Foradditional information on this subject, the authors recom-mend recent reviews (Spaepen et al., 2007a, b; Spaepenand Vanderleyden, 2010).

29.6.1 Synthesis of IAAConjugatesOne level of regulation appears to be the synthesis andhydrolysis of IAA conjugates, which function both in


Table 29.2 Overview of the factors and mechanisms involved in regulating IAA biosynthesis

Regulatory Factora Mechanism References

Trp level In the absence of Trp, intracellular anthranilate inhibitsconversion of Trp to IAA; and as anthranilate synthase isactive in the absence of Trp, the Trp biosynthesis genesare transcribed and translated. As a result, while the levelof anthranilate increases, to prevent a loss of Trp by thecells, it inhibits IAA biosynthesis. When exogenous Trpis present, the intracellular anthranilate level is lowbecause of feedback inhibition exerted by Trp, thusenabling IAA biosynthesis. In addition, Trp levels maybe increased as it is a component of root exudates andcellular material released from dying bacterial cells

Omay et al. (1993); Patten andGlick (1996); Dakora et al.(2002); Kravchenko et al.(2004)

Type ofbiosynthesispathway

IpyA and IAN are inducible in nature; IAM pathway genesare constitutively expressed. Coexistence andsimultaneous involvement of more than one biosynthesispathway in microbes has been established(Bradyrhizobium japonicum, Azospirillum brasilense)

Sekine et al. (1988); Hartmannand Zimmer (1994);Kobayashi et al. (1995);Patten and Glick (1996);Kochar et al. (2011)

Multiple pathways Involvement of more than one pathway in the same straineither naturally or by heterologous expression whereinnot only IAA levels improve but also the nature of IAAexpression changes

Clark et al. (1993); Sekineet al. (1988); Hartmann andZimmer (1994); Malhotraand Srivastava (2006)

Nutrient limitation Reduction in the growth rate may trigger while aerobicconditions inhibit IAA biosynthesis

Ona et al. (2005); Malhotraand Srivastava (2009)

Rhizosphere pH Change in pH of rhizosphere by proton extrusion throughroot cell membranes and pH inducibility of IAAbiosynthesis

Bashan (1990); Van de Broeket al. (2005); Malhotra andSrivastava (2009)

RpoS, GacS, IAA Mediation of growth phase and cell-density-dependentinduction of certain bacterial genes (ipdC).Overexpression of IAA was achieved by improving thecopy number of rpoS or gacS genes. In α-proteobacteria,RpoN or RpoH may possibly regulate IAA expression

Van de Broek et al. (1999);Saleh and Glick (2001);Patten and Glick (2002b);Spaepen et al. (2007a)

TyrR TyrR-regulated genes involved in auxin transport andmetabolism. It is required for IAA production and ipdCtranscription. TyrR is also responsive to tryptophan,phenylalanine, and tyrosine

Ryu and Patten (2008)

σ54-Dependentpromoters

Auxin-responsive gene expression that requires specifictranscription factors or enhancer binding proteins toinitiate transcription. Such promoters may containfeatures such as auxin-responsive elements and othersequence features that aid in binding of the specifictranscription factors or enhancer binding proteins

Barrios et al. (1999);Lambrecht et al. (1999); Vande Broek et al. (2005);Malhotra and Srivastava(2008a)

IAA conjugates Permanent inactivation and temporary storage of auxin inaddition to transport, and compartmentalization of auxinsas well as to detoxify excess IAA and protect the freeacid against peroxidative degradation. A group ofbacterial aminoacylases that hydrolyze acetylated aminoacids, include IAA-Asp hydrolase (IAASPH) that waspurified from Enterobacter agglomerans

Cohen and Bandurski (1982);Chou et al. (1996)

a Besides the factors mentioned here, in symbiotic bacteria (Rhizobium), plant extracts/specific compounds such as flavonoids may accumulate in therhizosphere and stimulate IAA production via a specific regulatory cascade (Prinsen et al., 1991; Theunis et al., 2004). In addition, signals from theplant surface may be involved in transcriptional regulation of genes involved in IAA biosynthesis (Spaepen et al., 2007a).

29.6 Regulation of IAA Levels 317

Figure 29.2 Factors affecting the IAA level in the rhizosphere.Both, bacteria and plants, are involved in enriching the rhizosphereIAA pool and utilizing the IAA for improved plant growth response.The conditions influencing microbial IAA biosynthesis are alsomentioned.

the permanent inactivation and temporary storage ofauxins. Both, in plants and microbes, IAA may notonly be utilized as such, but may also be converted intosoluble metabolites. In addition to de novo synthesis,IAA can also be released from its conjugates whenrequired (Fig. 29.2). Such conjugates have been proposedto have roles in storage, transport, and compartmental-ization of auxins, thus protecting the free acid againstperoxidative degradation (Cohen and Bandurski, 1982;Seidel et al., 2006). The nature of conjugates variesamong different groups of bacteria: they may be storageforms of IAA (IAA–Ala, –Leu) or catabolites of IAA(IAA-Asp, and IAA-Glu) (Tam et al., 2000). A groupof bacterial aminoacylases that specifically hydrolyzesacetylated amino acids, including an indole-3-aceticacid-Asp hydrolase (IAASPH; Table 29.1) has beenpurified from P. agglomerans (formerly Enterobacteragglomerans) and showed approximately 20% identityto the Arabidopsis amidohydrolases (Chou et al., 1996;LeClere et al., 2002). The other report of iaaL gene fromP. savastanoi has been discussed earlier in this chapter.These results suggest that synthesis of IAA conjugatescan be a generalized mechanism to regulate IAA levels.

29.6.2 Regulation of the IAABiosynthesis Pathway GenesThe pathways involving IPyA and IAN are induciblein nature whereas the IAM pathway genes are con-stitutively expressed in most of the microbes studiedtill now (Abramovitch et al., 2006). TMO (Fig. 29.1)

may be inhibited, both, by IAM and IAA, whichsuggests that TMO may be involved in the regulationof IAA biosynthesis (Hutcheson and Kosuge, 1985;Table 29.2). Trp supplementation drastically increasesthe IAA synthesized via the IPyA pathway in E. cloacae,Rhizobium phaseoli, and different strains of A. brasilense(Patten and Glick, 1996; Malhotra and Srivastava, 2006,2008a). On the other hand, nitrile hydratase (Fig. 29.1)is induced by IAM in Agrobacterium and Rhizobiumsp. (Kobayashi et al., 1995). The co-existence andsimultaneous involvement of more than one pathwaywith differential contribution to the total IAA producedby microbes (Fig. 29.1) has also been established inErwinia herbicola pv. gypsophilae (Clark et al., 1993),Bradyrhizobium japonicum (Sekine et al., 1988), and A.brasilense (Hartmann and Zimmer, 1994; Malhotra andSrivastava, 2008b; Kochar and Srivastava, 2012).

29.6.3 Regulation of Trp LevelTrp-dependent IAA production is well known inA. brasilense, P. fluorescens, and Bacillus amyloliquefa-ciens (Malhotra and Srivastava, 2006; Idris et al., 2007;Kochar et al., 2011). As Trp is the substrate for the major-ity of the pathways (Fig. 29.1), its level will need to beregulated such that sufficient amount of Trp is availableintracellularly for IAA biosynthesis (Table 29.2). In theabsence of Trp, the intracellular anthranilate levels inhibitthe conversion of Trp to IAA (Hartmann and Zimmer,1994). However, when exogenous Trp is present, theintracellular anthranilate level is low because of feedbackinhibition exerted by Trp, thus enabling IAA biosynthesis(Patten and Glick, 1996; Table 29.2). PGPB can obtainTrp from different sources and channel it toward IAAproduction. First, Trp has been shown to be a componentof the root exudates secreted by plants with which suchPGPB are associated (Dakora et al., 2002). The othersources of Trp are the proteins and cellular materialreleased from dying bacterial cells. A significant increasein IAA biosynthesis in the stationary growth phase ofbacteria is in accordance with this proposition (Pattenand Glick, 1996). Contribution of Trp from dead anddecomposing organic matter can also not be ruled out.

29.6.4 Effect of the EnvironmentPGPB can benefit plant growth if optimum conditions areencountered in the rhizosphere. They have the ability tomodulate their physiological properties under fluctuatingconditions to survive in the rhizosphere (Fig. 29.2). IAAbiosynthesis via the IPyA pathway may be elevated underanaerobic conditions, as reported in Azospirillum, but thiseffect of O2 fluctuation has not received much support.


Similarly, Heckman and Strick (1996) proposed that nutri-ents absorbed by the roots are in the form of cations andanions, the most important being NO3

− and NH4+. The

differential uptake of these ions can affect the pH of therhizosphere. While the absorption of NH4

+ promotes theefflux of H+ ions and reduces the pH in the rhizosphere,the absorption of NO3

− promotes the efflux of OH− anda consequent increase in the rhizosphere pH. Moreover,such a change in pH is localized to rhizospheric soil anddoes not affect the bulk soil pH (Bashan, 1990). Localchanges in pH and temperature have been found to inducethe expression of ipdC in A. brasilense strain SM, therebyimpacting IAA levels (Malhotra and Srivastava, 2008a;Fig. 29.2; Table 29.2).

29.6.5 Mediation of Growth Phaseand Cell-Density-DependentInductionGrowth phase and cell-density-dependent induction of cer-tain bacterial genes is known to be mediated by smalldiffusible signal molecules. For example, weak acids actas a signal to activate the sigma factor, RpoS, that inturn activates other growth-phase-specific genes. Growth-phase-dependent expression of A. brasilense ipdC geneand IAA levels have been observed (Van de Broek et al.,1999; Malhotra and Srivastava, 2008a, 2009; Fig. 29.2).

29.6.6 Other RegulatoryElements/ComponentsDNA sequence and transcriptional analysis of the ipdCgene in A. brasilense Sp245 has identified iaaC as acomponent of a bicistronic operon with ipdC, althoughthe former can also be transcribed monocistronically. The222-residue IPDC protein has significant homology witha variety of proteins of the DJ-1/PfpI superfamily, whichincludes chaperones, proteases, RNA-binding proteins,and hypothetical proteins with unknown functions.Such proteins belong to varied bacterial groups, fromEscherichia coli, Pseudomonas aeruginosa, P. fluo-rescens, Magnetospirillum, Salmonella enterica, Yersiniapestis, to humans and zebrafish (Van de Broek et al.,2005; Malhotra and Srivastava 2008a). As the expressionof ipdC is auxin responsive (Van de Broek et al., 1999),a short “cis” element in the promoters of auxin-inducedgenes (auxin-responsive element, AuxRE TGTCNC) hasbeen reported not only in plants (Ulmasov et al., 1995)but also in A. brasilense strains Sp245 and SM (Lam-brecht et al., 1999; Van de Broek et al., 2005; Malhotraand Srivastava, 2008a). Certain sequence elements in thevicinity of the AuxRE of the ipdC promoter of strainSp245 have been identified to be responsible for IAAinducibility of ipdC (Van de Broek et al., 2005). Bacterial

IAA may influence plant processes in a spatio-temporalway and this depends on bacterial inoculum size, as wellas mode of colonization and the regulation of IAA biosyn-thesis at the plant and microbial level (Spaepen et al.,2007a; Spaepen and Vanderleyden, 2010). Auxins areinvolved in regulating the expression of certain genes inplants (Sitbon and Perrot-Rechenmann, 1997; Hagen andGuilfoyle, 2002) and have also been implicated as a recip-rocal signaling molecule in plant–microbe interactions(Lambrecht et al., 2000; Spaepen et al., 2007a, b).

Although a lot has been unraveled in the auxinsignal transduction pathway in plants (Guilfoyle et al.,1998), the information is still incomplete in bacteriaas many components and steps remain undefined. Thedifferences observed in the IAA levels, their regulation,and the nature of expression of the ipdC gene betweendifferent A. brasilense strains (Sp245, Sp7, and SM) canbe assigned to variations not only in the 5′ upstreamregions but also those in the 3′ regions of the iaaCgene (coding for an IAA/ipdC-controlling protein). Thesestrains may harbor a full or a truncated copy of iaaC (Vande Broek et al., 2005; Malhotra and Srivastava, 2008a).Additional differences could also be derived from thenumber of functional pathways. For example, the IAMpathway has been reported for strain Sp7 (Bar and Okon,1993), but the same is absent in strain SM (Malhotraand Srivastava, 2006). Thus, while in strain Sp7 twoTrp-dependent pathways are operating, strain SM reliesonly on the IPyA pathway. This property may servean important role in realizing effective plant–microbialinteractions by the PGPB strains.

Some other secondary metabolites, such as biocon-trol antibiotic, 2,4-diacetyl phloroglucinol (DAPG; seeChapter 56), enhances expression of a wide range ofgenes in A. brasilense Sp245, including those involvedin phytostimulation (ppdC, flgE, nirK, and nifX-nifB).These genes are upregulated during root association, inthe presence of DAPG-producing strain, P. fluorescensF113. DAPG, therefore, can act as a signal by whichsome beneficial pseudomonads may stimulate plant-beneficial activities of Azospirillum. This has been furtherdemonstrated by comparing it with phl-negative mutantof P. fluorescens F113 (Combes-Meynet et al., 2011).Furthermore, when Azospirillum is exposed to IAA, itadapts itself to the rhizosphere by changing its arsenal oftransport and cell-surface proteins. For example, a type VIsecretion system that is specific for bacteria–eukaryotichost interactions is induced. It has been shown thatipdC inactivation or exogenous IAA addition may resultin broader transcriptional changes rather than only thepredictable ones (Van Puyvelde et al., 2011).

The actual IAA concentration available to a plantis, thus, a combined result of de novo biosynthesis,release of IAA-conjugates, polar transport of auxins,

29.7 Cross Talk Between IAA and Some Other PGRS: Signal Messaging Service (SMS) 319

and catabolism of bound IAA, with an important con-tribution coming from plant-associated bacteria. Theavailable IAA in plants is under strict control and isnot only dependent upon the developmental stages butalso the prevailing environmental factors and microbialassociation (Fig. 29.3).

29.7 CROSS TALK BETWEEN IAAAND SOME OTHER PGRS: SIGNALMESSAGING SERVICE (SMS)

In plants, genetic screens have been useful in identifyinggenes involved in phytohormone signaling. More oftenthan not, mutations in such genes confer changes on thesensitivity to more than one hormone/PGR (Gazzarriniand McCourt, 2003). The modulation of this sensitivitymay change the biosynthesis of such hormones. Screensfor genes involved in phytohormone signaling are, how-ever, not yet available for bacteria. In this part of thechapter, we will try and analyze how different PGRs pro-duced by bacteria interact with each other to regulate thefunctions involved in plant root growth and development(Fig. 29.3).

29.7.1 Interaction of PGR with NOIAA and nitric oxide (NO) have been hypothesizedto share common steps in the phytohormone signaltransduction pathways, because both elicit similar plantresponses. Auxin-induced development of adventitiousroots has been demonstrated in cucumber to be partlydependent on NO (Pagnussat et al., 2002). Zimmer et al.(1988) reported that nitrite produced by Azospirillummay have hormonal effects in plants. The competitiveauxin inhibitor, p-chlorophenoxyisobutyric acid (PCIB),leads to a reduced stimulatory effect produced byAzospirillum on lateral root formation (LRF), suggestingthat auxins are involved in triggering an increase in NOconcentration (Pagnussat et al., 2002; Fig. 29.3) and thatAzospirillum-promoted LRF also requires NO in additionto IAA (Molina-Favero et al., 2008; Cohen et al., 2010).

IAA treatment has been shown to induce a transientincrease in the level of NO in the basal region of thehypocotyls in cucumber explants, where the new rootmeristems develop (Pagnussat et al., 2002). This local-ized NO increase might stimulate the guanylyl cyclase(GC) catalyzed synthesis of cGMP, as occurs in mam-malian systems (McDonald and Murad, 1995). An earlierreport in tobacco has described the activation of defensegenes by NO-induced cGMP (Durner et al., 1998) which,in turn, may regulate Ca2+ levels in plants (Allen et al.,1995). The latter is translated into a signal transductionpathway leading to the initiation of mitotic processes and

differentiation of roots. NO can also act via a cGMP-independent pathway, activating phosphatases and proteinkinases including MAPKs (Klessig et al., 2000). Interest-ingly, a rapid and transient increase of MAPK activity inresponse to low levels of auxins was reported in Arabidop-sis seedling roots (Mockaitis and Howell, 2000). How-ever, further light needs to be shed on whether bacterialIAA influences MAPK’s activity and/or whether bacterialMAPKs affect NO synthesis, as they do in plants. Auxinand NO have been reported to positively affect indeter-minate nodule formation with an increased expression ofan auxin efflux carrier in roots possessing nodules withhigher IAA and NO content. This result supports a modelof nodule formation that involves auxin transport, regu-lation, and NO synthesis (Pii et al., 2007). It has beenshown that NO is produced in root nodules of Medicagotruncatula and M. sativa and its level further increasesin IAA-overproducing nodules. Both plant and bacterialactivities have been implicated in this process, with NOsynthesis by plant being locally enhanced by bacterialIAA (Fig. 29.3). In plant tissues, NO can be generatedby enzymatic and non-enzymatic systems, while rhizobiaunder anaerobic conditions produce NO via the denitrifi-cation pathway (Watmough et al., 1999; Crawford et al.,2006). Pii et al. (2007) observed that aerobically grown,IAA-overproducing Sinorhizobium meliloti produces NOand possesses NO synthase-like activity. These resultsidentify a role of IAA and NO in indeterminate noduleformation. Moreover, the NO scavenger, cPTIO, reducingnodule formation, supports the involvement of NO in theauxin-signaling pathway controlling nodule formation.

Furthermore, a mutant Faj009 of A. brasilense Sp245,negative for IPDC activity, displayed a 90% reduction inIAA synthesis (Costacurta et al., 1994), but produced thesame amount of NO as strain Sp245 when growing onnitrate, confirming that the defect in IAA biosynthesis didnot affect the activity of the periplasmic nitrate reductase(Molina-Favero et al., 2008). The mechanism by whichIAA induces NO formation is not known. In plants, anacidic environment activates a plasma membrane-boundenzyme, the nitrite:NO reductase (NI-NOR), reported tobe involved in NO formation in roots (Stohr et al., 2001).Whether IAA triggers acidification and/or regulates NI-NOR expression at the transcriptional or post-translationallevel leading to an increase in NO production is yet to beestablished.

Overall, the IAA-induced NO accumulation in rootsmay result in a bifurcated signal transduction pathway inwhich NO mediates a cGMP-dependent or-independentincrease of cytosolic Ca2+. The latter, in turn, triggerschanges in plant gene expression leading to the auxinresponse. However, it still remains to be tested whetherNO-mediated MAPKs activation is involved in suchbacterial IAA-induced processes and if bacterial NO acts


PlantsImproved plant

growth

Nitric oxide

Cytokinins frombacterial origin

Gibberellins frombacterial origin

Improvedrhizospheric

IAA pool

Microbiallyproduced IAA

PGPR

Rhizosphere/bacteria

NH3

Excess ACC releasedfrom plant into

rhizosphore catalyzed byACC deaminase of PGPR

Root mucilage, N and Csources, acidification of

rhizosphere, aminoacids like Trp

Exudation

ACC and SAM

In plant

Ethylene productionin plant Plant root

elongation/proliferationof lateral roots

Enhancednutrient/mineral

uptake

Complexnutrient andPhosphate

solubilization/N2 fixation

Nitrification

Induction signalslike IAA,Trp

Figure 29.3 Schematic representation of the interplay involving indole-3-acetic acid (IAA) and other bacterial PGR in relation to their effecton plants. Schematic representation of the signaling involving the phytohormones—auxins, cytokinins (CK), gibberellins (GA), and NO, andtheir effects on plant growth. Under stress conditions, phytohormone signal transduction pathways are activated. The G-protein transducesextracellular signals in plants which are likely modulated by bacterial auxins, GAs, and CKs. NO-mediated inhibition of ethylene biosynthesishas also been observed. Microbially produced IAA, in addition to directly influencing plant root proliferation, also tends to stimulate ACCsynthase activity, thereby leading to more breakdown of ACC by ACC deaminase activity of PGPR. The NH3 released during ACC breakdownand IAA biosynthesis in the rhizosphere (by IAM pathway) is fixed by N2-fixing PGPR, which, along with biomineralization of complexnutrient sources, allows enhanced mineral/nutrient uptake leading to improved plant growth. NO acts as a cross-route in PGR signaling totrigger metabolic and physiological responses in plants by three distinct mechanisms involving (i) a cGMP-dependent pathway involvingchanges in cytosolic ADP ribose-mediated Ca2+ concentration, (ii) a cGMP-independent route involving a direct NO action on Ca2+ channelsvia protein nitrosylation, and (iii) NO action on MAPK activities, which are involved in many physiological adaptation and developmentalprocesses (for further details refer Lamattina et al., 2003). IAA is known to induce its own production which may be initiated by signals fromplant root exudates and root mucilage and recent studies have shown that IAA and NO may cocontribute toward the plant-growth-promoting

ability of a PGPR strain. ( ) represents induction of biosynthesis, ( ) shows inhibition of processes, and ( ) identify the mechanismsthat beneficially influence plant growth. The oval area marked in gray shows the processes influencing plants and taking place within the plantswhile the rest of the area shows processes taking place in the rhizosphere mediated by bacteria.

in other IAA-mediated plant physiological responses(Lamattina et al., 2003).

29.7.2 Interactions of IAA,Cytokinins, and EthyleneRecent evidence from P. agglomerans pv. gypsophilae hasshed some light on the involvement of IAA and cytokininsin the regulation of the quorum sensing system and hrp

regulatory genes that control gall formation. Some genes,such as pagI, pagR, hrpL, and hrpS, showed reducedtranscription in iaaH (IAM pathway) and etz (cytokininbiosynthesis) mutants. However, an increased expressionof these genes was observed in case of an ipdC (IPyApathway) mutant (Chalupowicz et al., 2009).

Ethylene, a gaseous phytohormone, is transientlyinduced and interacts with auxin transport (Fig. 29.3).

References 321

It has been hypothesized that a high concentration ofbacterial IAA may induce the expression of the enzyme1-aminocyclopropane-1-carboxylate synthase (ACS),thereby increasing ethylene biosynthesis and causingroot growth (Kende, 1993; Abel et al., 1995; Abel andTheologis, 1996). However, it is less well known whetherethylene inhibits IAA transport and signal transduction(Prayitno et al., 2006). PGPB take up ACC exuded fromthe plants and degrade it via ACC-deaminase releasingammonia and α-ketobutyrate in the process, and reducingthe amount of ACC in the rhizosphere. As a result, moreACC is released by the plant to maintain its equilibriumand consequently less ACC is then available for ethylenebiosynthesis (Glick et al., 1998). This feedback loop ofethylene inhibition of IAA synthesis and/or functioninglimits the amount of ACS, ACC, and ethylene in theplant. With a decrease in the ethylene level, such bacterialassociations lead not only to improved root growth butalso to the alleviation of the inhibitory effects of ethylenestress (Glick et al., 1994, 2007; Ma et al., 2003). InP. putida GR12-2, Glick et al. (1998) proposed a likelycounterplay between ethylene produced by plants andIAA produced by the PGPB. Further, it also relieves theethylene repression of auxin response factor synthesis,and indirectly increases plant growth (Dharmasiri andEstelle, 2004). A general model to integrate all thebacterial PGRs in relation to their effect on plants hasbeen generated and depicted in Figure 29.3. Nevertheless,there are signaling molecules that still need identificationand characterization. This information can aid in theidentification of the important genes involved in thesignal cross talk, which can subsequently be used astargets to strengthen bacteria–plant association andimprove plant productivity.

29.8 CONCLUSIONS

The plant–rhizobacterial commune or the continuum ofbacteria in the rhizosphere that are either free-living orassociative include a variety of PGPB that can provide arange of beneficial effects to plants with which they areassociated. It is now possible to metabolically engineera PGPB by introducing specific pathway genes, or refinethe beneficial functions, or add new desirable functions,all aimed toward improving the health of plants. Althoughthe mechanism of the auxin cross talk with other hormonesand the integration of IAA signaling pathways have beendeciphered to a large extent in plants, yet the interrelation-ships with and between the bacterial PGRs have not beenunraveled. However, one may be tempted to extrapolatecertain plant auxin-signaling mechanisms to be operatingin case of plant–bacteria associations as well. It is impor-tant to note that the role of IAA is becoming clear in such

diverse functions as root growth promotion, symbiosis,pathogenesis, and plant defense. A better understandingof these processes will facilitate better exploitation ofthese roles toward improved plant growth and productiv-ity. Further studies should address the cis- and trans-actingregulatory elements that control the PGRs and the mod-ulatory role that plant root components may play in theintimate association of bacteria with its plant partner.

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Molecular Microbial Ecology of the Rhizosphere (de Bruijn/Molecular Microbial Ecology of the Rhizosphere) || Bacterial Biosynthesis of Indole-3-Acetic Acid: Signal Messenger Service