REVIEWARTICLE

Phytohormone Regulation of Legume-Rhizobia Interactions

Brett J. Ferguson & Ulrike Mathesius

Received: 30 March 2014 /Revised: 17 June 2014 /Accepted: 23 June 2014 /Published online: 23 July 2014# Springer Science+Business Media New York 2014

Abstract The symbiosis between legumes and nitrogen fix-ing bacteria called rhizobia leads to the formation of rootnodules. Nodules are highly organized root organs that formin response to Nod factors produced by rhizobia, and theyprovide rhizobia with a specialized niche to optimize nutrientexchange and nitrogen fixation. Nodule development andinvasion by rhizobia is locally controlled by feedback betweenrhizobia and the plant host. In addition, the total number ofnodules on a root system is controlled by a systemic mecha-nism termed ’autoregulation of nodulation’. Both the local andthe systemic control of nodulation are regulated by phytohor-mones. There are two mechanisms by which phytohormonesignalling is altered during nodulation: through direct synthe-sis by rhizobia and through indirect manipulation of the phy-tohormone balance in the plant, triggered by bacterial Nodfactors. Recent genetic and physiological evidence points to acrucial role of Nod factor-induced changes in the host phyto-hormone balance as a prerequisite for successful nodule for-mation. Phytohormones synthesized by rhizobia enhancesymbiosis effectiveness but do not appear to be necessaryfor nodule formation. This review provides an overview ofrecent advances in our understanding of the roles and interac-tions of phytohormones and signalling peptides in the regula-tion of nodule infection, initiation, positioning, development,and autoregulation. Future challenges remain to unify hor-mone–related findings across different legumes and to testwhether hormone perception, response, or transport

differences among different legumes could explain the varietyof nodules types and the predisposition for nodule formationin this plant family. In addition, the molecular studies carriedout under controlled conditions will need to be extended intothe field to test whether and how phytohormone contributionsby host and rhizobial partners affect the long term fitness ofthe host and the survival and competition of rhizobia in thesoil. It also will be interesting to explore the interaction ofhormonal signalling pathways between rhizobia and plantpathogens.

Keywords Autoregulation of nodulation . Infection thread .

Legume nodulation . Phytohormones . Rhizobia . Symbiosis

Introduction

Most legume species are able to enter into a symbiotic rela-tionship with compatible strains of nitrogen-fixing rhizobiabacteria. This leads to the formation of a novel organ, calledthe nodule, which houses the rhizobia and creates an environ-ment suitable for nitrogen fixation (reviewed by Desbrossesand Stougaard 2011; Ferguson et al. 2010; Oldroyd 2013).This symbiosis evolved approximately 60 million years ago,at a time of high CO2 concentrations in the atmosphere, andlikely gave legumes an ecological advantage over other spe-cies by being able to utilize higher carbon availability throughincreased N nutrition (Sprent 2007). To maximize the effi-ciency of nodulation, it is thought that a sophistical signalexchange between rhizobia and legume hosts has evolved.Most likely, a less specific symbiosis, in which rhizobia in-vaded roots through crack entry and initiated nodules derivedfrom lateral roots has evolved to a more specific interactionwith highly regulated invasion via infection threads and for-mation of a nodule independent of lateral roots (Sprent 2008).Much research has been aimed at identifying the signals

B. J. FergusonCentre for Integrative Legume Research, School of Agricultural andFood Sciences, The University of Queensland, St. Lucia, Brisbane,Queensland 4072, Australia

U. Mathesius (*)Division of Plant Science, Research School of Biology,The Australian National University, Canberra, ACT 0200, Australiae-mail: ulrike.mathesius@anu.edu.au

J Chem Ecol (2014) 40:770–790DOI 10.1007/s10886-014-0472-7

exchanged between rhizobia and legumes necessary for thissymbiosis. Most important has been the identification offlavonoids exuded by roots that stimulate host specificrhizobia to synthesise Nod factors, lipochitin oligosaccha-rides with specific structures depending on the rhizobialsymbiont (Oldroyd 2013). Nod factors are necessary fortriggering a cascade of signaling events in the host root,culminating in the formation of nodules and their invasionby rhizobia. However, Nod factor-independent nodulationhas been observed in some cases, and it remains largelyunknown how the nodulation process is regulated in theabsence of Nod factors (Giraud et al. 2007). It is thoughtthat Nod factor signaling results in changes in plant hor-mone synthesis, perception, transport, and accumulation,which fine-tune the infection process, initiation, and devel-opment of nodules and control nodule numbers (e.g.,Desbrosses and Stougaard 2011; Ding and Oldroyd 2009;Mathesius 2008). In addition, rhizobia synthesize all majorphytohormones (e.g., Boiero et al. 2007) and can some-times alter phytohormone breakdown, which contributes tothe effectiveness of the symbiosis (Spaepen et al. 2007).

Most research relating to the molecular mechanisms ofnodulation has involved model legumes, in particular Lotusjaponicus and Glycine max (soybean), forming determinatenodules, andMedicago truncatula, a species forming indeter-minate nodules. Determinate nodules arise from divisions inthe root outer cortex, which later fuse with dividing pericyclecells, and the resulting nodule develops a meristem that dif-ferentiates. In contrast, indeterminate nodules arise from celldivisions in the pericycle and inner cortex of the host root, andresulting nodules retain a meristem, resulting in elongatednodules (Ferguson et al. 2010; Hirsch 1992; Fig. 1). Thenodulation process involves rhizobial infection into the rootcortex and the developing nodule, in parallel to the inductionof cell division in the root to initiate a nodule. Both processesare regulated independently and are orchestrated by a

multitude of plant and bacterial factors that are tightly regu-lated by the host plant, giving it control over the number ofnodule structures it forms. The plant regulates nodule numberslocally in response to Nod factors and nitrogen availability, aswell as through a systemic regulatorymechanism called ‘auto-regulation of nodulation’ (AON) (Reid et al. 2011b; Fig. 2).Lack of AON reduces the fitness of the symbiosis, probablyby redirecting too many resources to unnecessary numbers ofnodules. Just over a decade ago, we thoroughly reviewedmany of the fundamental signalling components involved inthe nodulation process (Ferguson and Mathesius 2003). Here,we provide an overview of key advances relating to many ofthese signals, and discuss a number of new factors identifiedover the last 10 years that have critical roles in the develop-ment and/or regulation of legume nodules.

Abscisic Acid

As reviewed by Ferguson and Mathesius (2003), early studiesindicated that abscisic acid (ABA) had an inhibitory effect onnodulation, as exogenous application of the hormone signifi-cantly reduced the number of nodules that formed on differentlegume species. However, the ABA content of nodules waselevated compared with the surrounding root tissue of variousspecies examined. This could indicate a requirement for thehormone in nodule development and/or functioning. Thesesomewhat conflicting findings led us to postulate that the roleof ABA in nodulation may not be a simple one; with a positiverequirement in different tissues and/or different developmen-tal stages that becomes inhibitory when its levels are elevated,possibly due to the induction of stress responses.

In support of the earlier conclusions, recent reports havedescribed how exogenous application of ABA can inhibitnodule development in M. truncatula (Ding et al. 2008),Trifolium repens (white clover, Suzuki et al. 2004), Phaseolus

Fig. 1 Differences in nodule positioning in indeterminate and determi-nate type nodules. a Indeterminate nodules are initiated in pericycle (pe)and inner cortex cells (ic) underlying the infection site indicated by acurled root hair (rh). ep=epidermis. b Determinate nodules arise from

divisions in outer (OC) and middle cortex cells. The dividing cells of bothtypes of nodules (grey) are characterized by increased auxin and cytokininresponses. Ethylene synthesis is thought to restrict nodules to radialpositions opposite the xylem poles (XP, grey striped)

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vulgaris (common bean; Khadri et al. 2006), and L. japonicus(Biswas et al. 2009; Suzuki et al. 2004; Tominaga et al. 2009).Additionally, treatment with abamine, an inhibitor of 9-cis-epoxycarotenoid dioxygenase, increased nodule numbers andnitrogen fixation in L. japonicus (Suzuki et al. 2004;Tominaga et al. 2009). In T. repens, ABA treatment inhibitedat the stage between root hair swelling and curling (Suzukiet al. 2004). Elegant work using M. truncatula revealed thatABA inhibited rhizobia infection, expression of the earlynodulation genes ENOD11 and RIP1, critical Nod factor-induced calcium spiking, and cytokinin-induced nodulationresponses (Ding et al. 2008). Thus, it affects both the infectionpathway as well as the nodule development pathway, possiblyto reduce the costly establishment of nodules under stressfulconditions. In addition, ABA application to one side of a split-root system inhibited nodulation locally not systemically, andthus, the hormone is not considered to be directly involved inthe Autoregulation of Nodulation (AON) pathway (Biswaset al. 2009). A positive influence of ABA also has beenreported, as ABA pre-treatment improved the nitrogen fixa-tion capacity of M. sativa plants grown under salt stressconditions, and this was linked to the induction of antioxidantenzymes (Palma et al. 2014).

A number of studies using ABAmutants also have recentlybeen reported. In L. japonicus, the ABA insensitive mutant,Beyma, which has a wilty phenotype, was shown to be insen-sitive to ABA-induced inhibition of germination, vegetativegrowth, stomatal opening, and nodulation (Biswas et al.2009). In the absence of exogenous ABA, the mutant

exhibited normal nodule numbers and a normal AON re-sponse; however, the individual nodules that formed onBeyma roots were significantly reduced in size, possibly sug-gesting a positive role for the hormone in nodule growth(Biswas et al. 2009). Additionally, in L. japonicus, theABA-insensitive mutant enf1 exhibited increased nodulenumbers as well as enhanced nitrogen fixation (Tominagaet al. 2009). The endogenous ABA content of enf1 was foundto be reduced compared with wild-type plants, as was theproduction of nitric oxide in mutant nodules. InM. truncatula,use of the dominant negative allele abi1-1 from Arabidopsisto genetically regulate ABA signalling resulted in ahypernodulation phenotype, whereas the sta1 mutant, whichdictates sensitivity to ABA, exhibited a reduction in Nodfactor signalling and nodule numbers (Ding et al. 2008). TheM. truncatula latd mutant, which is defective in meristemformation of roots and nodules, shows an altered sensitivityto ABA, although ABA content is similar to the wild type(Liang et al. 2007). Nodule primordia are initiated normally inlatd mutant roots, but later meristem formation is inhibited,leading to small, white nodules. This points to an effect ofABA in later nodule development (Liang et al. 2007). Le-gumes also are reported to have a unique tendency to increasetheir number of lateral roots following ABA treatment com-pared to non-legume species (Liang and Harris 2005). It willbe interesting to determine whether this altered ABA responsein legumes contributes to their ability to nodulate.

Abscisic acid also is synthesized by rhizobia, and this couldnot only alter nodulation but indirectly contribute to plantstress tolerance, as ABA mediates abiotic stress. Forexample, Bano et al. (2010) showed that in Rhizobiumleguminosarum, low synthesis of ABA, together with highsynthesis of GA and auxin, was correlated with higher plantbiomass under drought conditions.

Collectively, the evidence predominately points to a nega-tive role for ABA in legume nodule development, with thepotential for a more positive role for the hormone in nodulegrowth and/or functioning. Further studies are required tounequivocally determine the molecular mechanisms affectedby biologically-relevant levels of the hormone in differenttissues and in different stages of nodule development.

Auxin

Auxin is a central regulator of plant development. Studies inArabidopsis have elegantly shown that auxin accumulation isa prerequisite for organ formation throughout the plant, andthat the regulation of auxin transport is crucial for auxinaccumulation at specific sites of organogenesis (Benkováet al. 2003). Nodule development is no exception, and auxinmaxima have been observed in early developing noduleprimordia in M. truncatula (Mathesius et al. 1998a; van

Fig. 2 Model for the Autoregulation Of Nodulation (AON) mechanism.In wild type plants (left), rhizobial infection on a root triggers the synthe-sis of a signal, most likely a CLE peptide, which travels to the shoot andbinds to the Nodulation Autoregulation Receptor Kinase (NARK) recep-tor. This induces the production of a shoot-derived inhibitor (dashedarrow) signal (unidentified) that moves back to the root system to inhibitfurther nodules from forming. In AON mutants (right) the NARK recep-tor is defective, resulting in the loss of SDI synthesis and asupernodulation phenotype

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Noorden et al. 2007), L. japonicus (Pacios-Bras et al. 2003;Suzaki et al. 2012; Takanashi et al. 2011), and soybean(Turner et al. 2013). In agreement with increased auxin re-sponses in early nodule primordia, root responses to auxin andrhizobia were both very similar in a proteome analysis ofM. truncatula (van Noorden et al. 2007). Despite that, externaltreatment of roots with auxin inhibits nodule formation, asdoes treatment of roots with an auxin action inhibitor (vanNoorden et al. 2006). Most likely, the requirement for auxinlies within a certain concentration window, and also is depen-dent on the stage of nodule development, on the location inwhich auxin is required, and on the presence and perception ofother plant hormones that auxin interacts with.

In addition, auxin requirements appear to be different be-tween determinate and indeterminate nodule types (cf. Fig. 1).For example, increasing auxin sensitivity in soybean by ec-topic overexpression of the microRNA160 gene, which neg-atively regulates auxin response factor (ARF) genes, reducednodule numbers (Turner et al. 2013). In this case, noduleinitiation was not altered, but subsequent nodule developmentwas inhibited, possibly because the auxin distribution in de-veloping nodules needs specific gradients to allow the transi-tion between cell division and differentiation (Turner et al.2013). Overexpression of miRNA393, which targets an auxinreceptor and reduced auxin sensitivity, did not change nodulenumbers in soybean (Turner et al. 2013). In M. truncatula,miRNA160 overexpression reduced nodulation similarly tosoybean (Bustos-Sanmamed et al. 2013), whereas overexpres-sion of miRNA393 also reduced nodule density, suggestingthat M. truncatula requires a narrow ‘window’ of auxin sen-sitivity (Mao et al. 2013). Another study in which auxinsensitivity was reduced inM. truncatula by RNA interference(RNAi) against the cell cycle regulator CDC16 enhancednodule numbers (Kuppusamy et al. 2009). Currently, it isnot known why different legumes differ in their sensitivityto auxin.

Computational models predict that it is likely that auxinaccumulates at the site of nodule initiation through regulationof auxin transport, in particular by reducing auxin export(Deinum et al. 2012). Auxin transport occurs through thephloem from source to sink tissues and via active polar trans-port from cell to cell (Peer et al. 2010). The major auxin foundin most plants, indole-3-acetic acid (IAA), is activelytransported into cells by auxin importers of the amino acidpermease families AUX1 (Auxin resistant 1), LAX (Like-AUX1) and PGP4, a member of the MDR/PGP (Multidrugresistance/P-glycoprotein) families. Auxin export is mediatedby members of the PIN (Pin-formed) and PGP families. Thepolar localization of PIN proteins on either the basal, apical, orlateral side of the cell determines the polarity of auxin trans-port. During nodule development, three MtLAX proteins arehighly expressed in the developing primordia inM. truncatula,indicating active auxin import into these cells (de Billy et al.

2001). At the same time, PIN gene expression has beenlocalized in developing primordia (Huo et al. 2006), althoughthe exact localization of individual PIN proteins during noduledevelopment has not yet been determined. The importance ofPIN genes in auxin transport during nodulation was confirmedthrough silencing of several MtPIN genes, which caused areduction in nodule numbers (Huo et al. 2006).

PIN gene expression and intracellular cycling, importantfor PIN protein localization to specific sides of the cell, can bealtered by synthetic and natural auxin transport inhibitors, andthese have been implicated in nodulation. The synthetic auxintransport inhibitors NPA (1-N-naphthylphalamic acid) andTIBA (2,3,5-triiodobenzoic acid) can trigger pseudonoduleformation in several legumes in the absence of rhizobia,suggesting that auxin transport inhibition is sufficient to in-duce a developmental program to from a nodule (Hirsch et al.1989). Subsequent studies have confirmed that rhizobia andpurified Nod factors transiently inhibit auxin transport inlegumes forming indeterminate nodules (Boot et al. 1999;Mathesius et al. 1998b). In L. japonicus, which forms deter-minate nodules, an increase in polar auxin transport wasmeasured after inoculation (Pacios-Bras et al. 2003). This isconsistent with an increase in expression of the auxin reporterDR5:GUS in the vascular tissue around the inoculation site inL. japonicus (Li et al. 2014). These studies suggest that auxintransport regulation in response to rhizobia differs amonglegumes.

In M. truncatula, application of TIBA or NPA can causethe formation of pseudonodules in a range of nodulationmutants, including those defective in early Nod factor percep-tion and signal transduction (Rightmyer and Long 2011). Thisplaces the step of auxin transport inhibition downstream ofthese signalling events. Natural auxin transport inhibitors thatcould be involved in auxin transport regulation during noduleformation were suggested to be flavonoids (Hirsch et al.1989). Flavonoids are a group of secondary metabolites, someof which can act as modulators of auxin transport (Peer andMurphy 2007). Flavonoids are induced in cortical cells des-tined to divide during nodule development in several legumes(Mathesius et al. 1998a) and mimic auxin transport-relatedchanges in auxin responses (Mathesius et al. 1998b). Silenc-ing the flavonoid pathway in M. truncatula prevented auxintransport inhibition by rhizobia (Wasson et al. 2006), andflavonols were found to be the most likely sub-branch of theflavonoid pathway required for auxin transport control (Zhanget al. 2009). However, silencing the flavonoid pathway insoybean was not required for auxin transport control(Subramanian et al. 2006), which supports the idea that auxintransport control may be regulated differently in legumesforming determinate and indeterminate nodules(Subramanian et al. 2007). So far it remains unclear whichother regulators of auxin transport might be required for auxinaccumulation in primordia of determinate nodules. It is likely

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that the position of the cortical auxin maximum in either theinner or outer cortex in indeterminate and determinate noduleforming legumes is explained by lateral shifts in PIN proteinlocalization in the cortex (Deinum et al. 2012), and it will beinteresting to find out in the future what controls lateral PINprotein positioning in different legumes.

Flavonoids also could alter auxin accumulation by alteringauxin breakdown, through activation or inactivation of perox-idases that might be involved in auxin oxidation (Mathesius2001). Recent evidence from Arabidopsis shows that flavo-noids can buffer auxin signaling by altering ROS accumula-tion necessary for auxin breakdown/oxidation (Peer et al.2013). So far there is no clear evidence for the identity oractivity of specific flavonoids in auxin breakdown duringnodulation in vivo, and computational modelling of auxinmaxima found that changes in auxin synthesis or breakdownare unlikely to result in a sufficiently strong cortical auxinmaximum by themselves (Deinum et al. 2012).

In addition to the regulation of local auxin transport at thesite of nodule formation, long-distance auxin transport fromthe shoot to the root has been implicated in the control ofnodulation, in particular in AON in M. truncatula. Measure-ment of auxin transport showed that the AON mutant sunn1(Schnabel et al. 2005) has significantly increased capacity forshoot-to-root auxin transport compared to wild type. Afterinoculation of roots with rhizobia, shoot-to-root auxin trans-port was transiently found to decrease in wild type roots,whereas transport remained unaltered in the sunn1 mutant(van Noorden et al. 2006). Nodule numbers of sunn1mutantscould be reduced to wild type numbers by the application ofan auxin transport inhibitor to the shoot-root junction, sug-gesting that increased auxin transport is correlated with highernodule numbers in the sunn1 mutant (van Noorden et al.2006). Whether a similar mechanism exists in other legumesremains to be tested. Currently, studies in L. japonicus haveshown higher auxin responses in roots of the AON mutanthar1 than in wild type roots (Suzaki et al. 2012), but whetherthis is due to auxin transport changes has not been tested.

Auxin is also synthesised by rhizobia and a range of othersoil bacteria (Spaepen et al. 2007). The production of theauxin IAA by rhizobia is upregulated by flavonoid exudationfrom the root (Prinsen et al. 1991), and knock-out of theflavonoid-dependent IAA pathway in Rhizobium sp. NR234reduced auxin content in nodules and inhibited nitrogen fixa-tion (Spaepen et al. 2007). Studies with rhizobial strains withaltered auxin synthesis have shown that lack of auxin synthe-sis does not prevent nodulation, but can negatively affectnitrogen fixation, whereas auxin overproduction can increasenodulation efficiency; the mechanism of this is not understood(Pii et al. 2007). One question that awaits investigation is thecombined effect of bacterial auxin on root architecture andnodulation, as auxin has strong effects on lateral root forma-tion and root elongation. Thus, it might be possible that auxin

produced by rhizobia enhances root growth and lateral rootnumber and thus creates more opportunities for the root sys-tem to develop nodules. How this affects plant and bacterialsurvival in the field remains an open question.

Changes in auxin accumulation or signalling during plant-pathogen interactions also could affect nodulation, althoughlittle is known about how rhizobia and plant pathogens affectlegumes in co-inoculation experiments. Studies using non-legumes have demonstrated that plants infected by pathogensdown-regulate auxin signalling, and that this enhances patho-gen resistance. In contrast, addition of auxin can enhancepathogen symptoms, and auxin signalling appears to be atarget of R-gene mediated resistance (Mathesius 2010; Spoeland Dong 2008).

Brassinosteroids

Over the last decade, a great deal has been revealed about thebiosynthesis, perception, and signalling response ofbrassinosteroid (BR) hormones. However, there is still littleknown about the role of BRs in legume nodulation. Endoge-nous BRs influence nodule development, as pea mutantsaltered in BR biosynthesis or perception form significantlyfewer nodules than wild-type plants (Ferguson et al. 2005).Double mutant plants of the severely BR-deficient mutant ofpea, lk, and the autoregulation mutants, nod3 (Psrdn1), sym28(Psclv2), or sym29 (Psnark), all exhibit a supernodulationphenotype, and no evidence has been found for BRs actingdownstream of these genes in the systemic AON pathway(Foo et al. 2014).

A number of other investigations have focused on the effectof BR application on nodulation. Soaking seeds of Lenscu l inar i s wi th d i f f e ren t concen t ra t ions of 28-homobrassinolide resulted in reduced root lengths and nodulenumbers, but increased nitrate reductase activity (Hayat andAhmad 2003). In contrast, imbibing pea seeds with 24-epibrassinolide led to an increase in nodule numbers, nodulefresh and dry weights, and nitrogenase activity (Shahid et al.2011). Foliar application of epibrassinolide increased the nod-ule number and weight, and the nitrogenase activity, ofArachis hypogaea (peanut) plants (Vardhini and Rao 1999).Foliar sprays of epibrassinolide or homobrassinolide alsowere found to increase nodulation and nitrogenase activityin Phaseolus vulgaris (French bean) plants (Upreti and Murti(2004). In contrast, epibrassinolide application to soybeanroots was found to decrease the extent of both nodulationand nitrogen fixation (Hunter 2001). Foliar application or rootinjection of brassinolide inhibited nodule formation and rootdevelopment in a super-nodulating soybean mutant, but not inits wild-type parent (Terakado et al. 2005). In contrast, theapplication of brassinazole (an inhibitor of BR biosynthesis)

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to the leaves, or the culture media, increased the nodulenumber of wild-type soybean plants.

In all of these studies, the differences observed may simplyrelate to the methods or species used, in addition to the typeand concentration of BR applied. Furthermore, in many in-stances, it is difficult to ascertain which effects of the hormoneare direct, and which are indirect. To date, there continues tobe no molecular evidence pertaining to the role of BRs innodule formation.

Cytokinins

Cytokinins were long suspected to act as regulators of celldivision in nodulation, and this has been confirmed with theidentification of a number of cytokinin mutants over the lastfew years. Cytokinin response genes are activated in eitherinner or outer cortex cells during nodule initiation in indeter-minate and determinate nodules, respectively (e.g., Held et al.2014; Lohar et al. 2004; Plet et al. 2011), similar to the auxinresponses observed in these cells. Whether this is due toincreased cytokinin concentration or sensitivity or both hasnot been directly determined yet. Cytokinin synthesis by thecytokinin 5’-monophosphate phosphoribohydrolase LOG(LONELY GUY) recently has been shown to be requiredwithin a certain expression window for nodule initiation,control of nodule numbers, and nodule differentiation inM. truncatula (Mortier et al. 2014), suggesting that de novocytokinin synthesis is required for nodule development. TheMtLOG1 gene is expressed in the dividing cortex cells, whichcould indicate that cytokinin synthesis is increased after theinitial increase in cytokinin perception in the cortex (Mortieret al. 2014). The perception of cytokinin in the root cortex iscrucial for nodule initiation. Legumes defective in cytokininperception are unable to form nodule primordia, as shown inboth the L. japonicus hit1-1 (lhk1) (Murray et al. 2007) andthe M. truncatula cre1 mutants (Gonzalez-Rizzo et al. 2006;Plet et al. 2011), although in both legume mutants a lowfrequency of delayed nodules was observed, suggesting thatother cytokinin receptors are required for nodule initiation.This recently was confirmed in L. japonicus with the identifi-cation of additional LHK genes that are expressed in dividingmiddle and outer cortical cells and that are required for noduleinitiation (Held et al. 2014).

Infection thread formation was not abolished in the cytoki-nin insensitive mutants, although their progress through theepidermis and cortex often appeared to be looped and aborted,presumably because of the absence of underlying noduleprimordia (Gonzalez-Rizzo et al. 2006; Murray et al. 2007).Interestingly, the formation of nodules in the L. japonicus lhkmutants that formed occasionally in a delayed fashion couldbe completely abolished by mutations preventing bacterialentry (Held et al. 2014). This suggests a model in which

bacterial entry provides a signal to the cortex that bypasses ahypothetical signal between epidermis and cortex, mediatedby LHK1 (Held et al. 2014).

Cytokinin is not only necessary but also sufficient for theinduction of cortical cell divisions, as demonstrated in the lhk1gain-of-function mutant (snf2) in L. japonicus, which formsspontaneous nodules (Tirichine et al. 2007). In some legumes,external application of cytokinin to roots is sufficient to inducenodules, and cytokinin was shown to act downstream of thecommon signalling pathway activated by Nod factors, butupstream of the NSP1, NSP2, and NIN transcription factorsnecessary for nodulation (e.g., Heckmann et al. 2011).

Mature nodules formed in the cre1 mutant showed analtered zonation, suggesting that cytokinin is required at anearly, as well as a later, stage during nodulation (Plet et al.2011). Cytokinin also is involved in activation of AON, ascytokinin signalling through cre1 is required for induction ofthe MtCLE13 peptide mediating AON in M. truncatula(Mortier et al. 2012; see section on CLE peptides below).

The action of cytokinin is linked with the regulation ofauxin during nodulation. For example, the cre1 mutantshowed increased auxin transport and PIN gene expression,and lacks auxin transport control after infection with rhizobia(Plet et al. 2011). Similarly, in Arabidopsis, cytokinin affectslateral root development through action on PIN gene expres-sion (Marhavy et al. 2011). It remains to be shown exactlyhow cytokinin signalling acts on PIN genes and/or PIN pro-tein localization during nodulation. In L. japonicus, auxinresponses were detected in spontaneously induced nodulesof the lhk1 gain-of-function mutant (snf2) (Suzaki et al.2012), further strengthening the idea that cytokinin signallingis required for downstream auxin accumulation. Whether thisoccurs through alteration of auxin transport or other mecha-nisms in determinate nodules is not known. On the other hand,there also is evidence for cytokinin regulation in response tochanges in auxin sensitivity. In soybean, increasing auxinsensitivity through overexpression of miRNA160 caused areduction in cytokinin-induced gene expression, suggestinga feedback loop between auxin and cytokinin signalling(Turner et al. 2013).

Rhizobia also synthesize cytokinin, and cytokinin synthe-sis by Rhizobium nodulation mutants has been shown to besufficient to induce cortical cell divisions in alfalfa (M. sativa)(Cooper and Long 1994). However, a survey of cytokininproduction in four different Rhizobium species showed thatthere was no correlation between the types and amounts ofcytokinins produced by rhizobia and their ability to inducenodules (Kisiala et al. 2013). In the interaction betweenBradyrhizobium sp. strain ORS285, which can form noduleson the legume Aeschynomene in a Nod factor-independentway, cytokinin production by the bacteria was found to accel-erate nodule formation, and to alter nodule number and size,but cytokinin deficient mutants were still able to induce

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nodules (Podlešáková et al. 2013). This negates the earlierhypothesis that purine derivatives, suggested to include cyto-kinins, could contribute to nodulation in the absence of Nodfactors (Giraud et al. 2007).

Ethylene

The gaseous hormone ethylene plays a role as a negative regu-lator of nodulation, and acts on different processes during noduleformation, including regulation of total nodule numbers, infec-tion thread formation, nodule morphology, and nodule position-ing (Guinel and Geil 2002). Ethylene is produced in the plant inresponse to rhizobial infection (e.g., Ligero et al. 1986). Itsactions likely are due to its effects on plant defense (Penmetsaet al. 2008; Prayitno et al. 2006a), as well as its involvement inauxin transport (Prayitno et al. 2006b) and interaction with ABA(Chan et al. 2013), among other developmental signals. Asreviewed previously, application of ethylene reduces nodulenumbers in several legumes, while ethylene synthesis or sensinginhibitors generally increase nodule numbers (Ferguson andMathesius 2003). In the last decade, identification of a numberof ethylene-insensitive mutants in different legumes has provi-ded genetic evidence for the involvement of ethylene signallingin nodulation (Gresshoff et al. 2009).

Very early during the nodulation process, ethylene inter-feres with calcium signalling and negatively regulates infec-tion thread formation (Oldroyd et al. 2001). Inhibition ofspontaneous nodulation in the lhk1 gain-of-function mutantsof L. japonicus by ethylene precursors additionally placesethylene inhibition of nodulation upstream of cytokinin sig-nalling (Tirichine et al. 2006). Ethylene insensitive mutants ortransgenic lines, including the M. truncatula sickle (skl) mu-tant defective in a gene homologous to Arabidopsis EIN2(Ethylene Insensitive 2) (Penmetsa and Cook 1997;Penmetsa et al. 2008), transgenic L. japonicus plants express-ing the melon ethylene receptor gene Cm-ERS1 (Nukui et al.2004) and L. japonicus transgenic lines expressing a dominantetr1-1 gene from Arabidopsis (Lohar et al. 2009) show anincreased number of infection threads. As an exception, theL. japonicus enigma1 (LjEIN2a) mutant displays a reducednumber of infection threads (Chan et al. 2013).

The sites of ethylene synthesis in the root are found in cellsoverlying the phloem poles, thus generating a gradient ofethylene that is thought to restrict nodules radially to positionsopposite the xylem poles (Heidstra et al. 1997). This position-ing effect of ethylene has been confirmed in a number ofethylene insensitive mutants, in which nodule positioninglooses the radial restriction (Chan et al. 2013; Lohar et al.2009; Penmetsa and Cook 1997).

Total nodule numbers also are partially controlled by eth-ylene signalling, as ethylene insensitive mutants form manymore nodules than wild-type plants (Lohar et al. 2009; Nukui

et al. 2004; Penmetsa and Cook 1997). InM. truncatula, this isnot mediated through an effect of ethylene on AON as dem-onstrated by grafting (Penmetsa et al. 2003; Prayitno et al.2006b). Interestingly, L. japonicus EIN2a mutants showedslightly reduced, not increased, numbers of nodules (Chanet al. 2013), even though external application of ethylene alsoreduces nodule numbers in L. japonicus wild-type plants(Lohar et al. 2009). In addition, nodule numbers were con-trolled by both the shoot and the root in LjEIN2a mutants(Chan et al. 2013). It is possible that this discrepancy is due toduplication and differentiation of L. japonicus EIN2 genes(Chan et al. 2013; Desbrosses and Stougaard 2011). A sepa-rate study in L. japonicus identified two EIN2 genes inL. japonicus, and their combined silencing led to increasednodule numbers (Miyata et al. 2013). It also is possible thatagain there is a difference in the sensitivity or response oflegumes forming determinate and indeterminate nodules, asethylene did not consistently inhibit nodulation in soybean,which forms determinate nodules like L. japonicus (Schmidtet al. 1999).

Ethylene may be involved in auxin transport regulationduring nodule development and this may influence total nod-ule numbers. The Mtskl mutant showed increased PIN1 andPIN2 expression after inoculation with rhizobia, and auxinaccumulation above the infection site was exaggerated(Prayitno et al. 2006b). In addition, shoot-to-root auxin trans-port, which has been associated with AON in M. truncatula(van Noorden et al. 2006), was found to be insensitive torhizobia in the skl mutant, in contrast to wild type plants(Prayitno et al. 2006b). While it has long been known thatethylene can act as an auxin transport inhibitor (Morgan andGausman 1966), its mechanism of action remains unclear.One possibility is that ethylene could act via the induction offlavonoids, which then modulate auxin transport (Buer et al.2006), but this has not directly been tested during nodulation.While ethylene appears to act upstream of auxin transportregulation, there also is evidence that ethylene action is re-quired for auxin transport changes to take place. For example,NPA or TIBAwere unable to cause pseudonodules in the sklmutant (Rightmyer and Long 2011). In the rel3 mutant ofL. japonicus, which is defective in the formation of trans-acting RNAs targeting the auxin response factors ARF3a,ARF3b, and ARF4, nodulation was significantly inhibited(Li et al. 2014). Nodulation in rel3 mutants was restored tolevels similar to wild type by application of ethylene inhibi-tors. This suggests that increase ethylene synthesis or percep-tion in rel3 mutants is a result of altered auxin signalling and/or transport (Li et al. 2014).

Finally, ethylene also has been observed to alter nodulemorphology and bacteroid numbers in L. japonicus (Loharet al. 2009), and while the number of nodule primordia wereincreased in L. japonicus expressing the CmERS1 gene, thenumber of mature nodules was similar to wild type plants,

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suggesting that the effect of ethylene changes at differentstages of nodulation (Nukui et al. 2004).

Rhizobia also contribute to the levels of ethylene duringnodulation. Many rhizobial species encode ACC deaminase,which degrades the ethylene precursor 1-aminocyclopropoane-1-carboxylic acid (ACC), and overexpression of ACC deami-nase in rhizobia has been shown to increase nodule numbers,increase the competitiveness of rhizobia (e.g., Confonte et al.2010), and enhance environmental stress tolerance of legumes,for example under saline conditions (e.g., Brigido et al. 2013).However, knockout of ACC deaminase has not produced con-sistent results, with some studies showing decreased nodula-tion, whereas other studies examining different rhizobial spe-cies did not find any deleterious effect of knocking out ACCdeaminase in rhizobia (Murset et al. 2012).

Collectively, these studies show that while ethylene plays anegative role during nodulation in most legumes, there issome difference among legume species and among differentethylene insensitive mutants. Thus, it is likely that receptors orother regulators of ethylene signalling could have diversifiedin different legumes.

Gibberellins

Gibberellins (gibberellic acids; GA) are involved in a widerange of biological processes, including cell elongation, seedgermination, and flowering. They appear to be required atdifferent stages of nodulation, with their content tightly regu-lated to achieve successful nodule development (reviewed inHayashi et al. 2014). A number of early studies reportedelevated GA levels in nodules compared with root tissue(reviewed in Ferguson and Mathesius 2003). Additional earlyinvestigations focusing on GA application found both positiveand negative changes in nodule numbers, depending on thespecies examined, growing conditions, application technique,and the type and concentration of GA applied (reviewed inFerguson and Mathesius 2003).

More recently, Ferguson et al. (2005, 2011) demonstratedthat GA deficient mutants of pea developed fewer nodulesthan wild-type plants. Mutant and grafting studies showed thatthese reductions were due to the reduced GA level of the roots(Ferguson et al. 2005). The few nodules that did form on theseverely GA-deficient namutant were small, white, and aber-rant in structure. Mutant na plants evolve approximately twiceas much ethylene as wild type plants (Ferguson et al. 2011),which, as discussed above, is a potent inhibitor of nodulation.Application of the ethylene biosynthesis inhibitor, amino-ethoxyvinyl glycine (AVG), partially increased the numberof nodules formed on these mutants, but was unable to rescuetheir aberrant phenotype (Ferguson et al. 2011). In contrast,the application of bioactive GA restored both the number andmorphology of na nodules (Ferguson et al. 2005). These

findings indicate that some characteristics of namutant plantscould be associated in part with elevated ethylene levelsproduced as a result of a low GA content, but that GAsthemselves are still required for proper nodule formation(Ferguson et al. 2011). Moreover, double mutants of na andthe autoregulation mutants, nod3 (Psrdn1), sym28 (Psclv2), orsym29 (Psnark), all exhibit a supernodulation phenotype withnodules that are abnormal in their development (Fergusonet al. 2011). This indicates that GAs likely act independentlyof the autoregulation of nodulation pathway, and further sup-ports a requirement for the hormone as an essential factor forproper nodule formation.

An optimum concentration of GA appears to be requiredfor nodulation to be successful. For example, pea mutantshaving reduced GA levels form fewer nodules, but so doconstitutive GA signalling mutants, as well as sln mutantseedlings, which have an elevated GA content (Fergusonet al. 2005, 2011). This is consistent with findings that lowlevels of bioactive GA applied to wild-type pea increasednodule development, whereas higher levels inhibited it(Ferguson et al. 2005). Exogenous GA application, or theconstitutive over-expression of the SLEEPY1 gene that actsin GA signalling, both inhibited nodulation in L. japonicus(Maekawa et al. 2009). Likewise, treatment with various GAbiosynthesis inhibitors reduced lateral root-based nodulationin Sesbania rostrata, as did the application of bioactive GA(Lievens et al. 2005). Down-regulation of the monolignolbiosynthetic enzyme, HCT, in alfalfa (Medicago sativa) re-sulted in an elevated GA content and an increase in nodulenumbers, surprisingly accompanied by a reduction in rootgrowth (Gallego-Giraldo et al. 2014). Collectively, these out-comes suggest a positive role for GAs in nodule organogen-esis, but that too little, or toomuch, is inhibitory to the process.The optimum level likely varies depending on the species,stage of nodule development and growing conditions.

A number of GA biosynthesis genes have been shown to beup-regulated during legume nodulation. In Sesbania rostrata,the SrGA20ox1 gene, which encodes a key component of theGA biosynthetic pathway, was significantly up-regulated dur-ing lateral root-base nodulation (Lievens et al. 2005). Insoybean, recent transcriptome studies identified several up-regulated GA biosynthesis genes during early nodulation,including GmGA3ox 1a and also GmGA20ox a, which sharesa high sequence similarity to SrGA20ox1 (Hayashi et al.2012). Transcripts of the GmGA20ox gene were detected onlyin soybean roots induced to form nodules, indicating the geneis nodulation-specific (Hayashi et al. 2012). Moreover, theGA20ox genes of S. rostrata and soybean were both Nodfactor-dependent in their expression (Hayashi et al. 2012;Lievens et al. 2005). The expression of GmGA3ox 1a andGmGA20ox a in soybean peaked at 12 h post inoculation anddeclined thereafter (Hayashi et al. 2012). This indicates thatthe genes may act together to increase GA biosynthesis, and

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also that the GA content is tightly regulated during nodulation.GA biosynthesis genes also are reported to be up-regulated inplants engaging in symbiosis with mycorrhizal fungi (García-Garrido et al. 2010; Ortu et al. 2012). The regulation of thesegenes was reliant on CCaMK (Ortu et al. 2012), a key signal-ling component shared by the nodulation and mycorrhizationpathways.

In addition to being expressed at very early time points, theGA20ox-encoding genes were also elevated in expression inmore mature nodules of S. rostrata (Lievens et al. 2005) andsoybean (Hayashi et al. 2012), as was a GA20ox-encodinggene identified in L. japonicus (Kouchi et al. 2004). Indeed,Lievens et al. (2005) identified two patterns of SrGA20ox1expression in S. rostrata, one related to intercellular infectionevents, and a second associated with the nodule meristem.Transcripts of an LjGA2ox-encoding gene involved in GAcatabolism also were detected in maturing nodules ofL. japonicus, further indicative of a tight regulation of theGA content during nodule organogenesis (Kouchi et al. 2004).

The up-regulation of GA biosynthesis genes at differentstages of nodule development suggests a transient requirementfor the hormone during nodulation, occurring both temporallyand spatially. This pattern of expression is consistent withfindings that GAs affect early infection events, such as roothair curling and infection thread development, as well as moremature stages of nodulation, including nodule primordiumdevelopment and the correct establishment of nodulationzones (Ferguson et al. 2011; Lievens et al. 2005; Maekawaet al. 2009). Based on the above mentioned findings, Hayashiet al. (2014) proposed a model where GAs are required forrhizobia infection, and also for the establishment of the noduleprimordium and the maturation of the nodule structure.

GAs also can be synthesized by rhizobia, although theeffects on nodulation have not been fully determined yet andmight depend on environmental factors. Interestingly, thegene encoding enzymes of the ent-kaurene biosynthesis path-way that leads to GA biosynthesis is present only in rhizobiathat infect legumes forming determinate nodules, not thoseforming indeterminate nodules. The significance of this find-ing remains to be investigated (Hershey et al. 2014).

Jasmonic Acid

Jasmonic acid (JA) has well-established roles in plant defenseand wound response. Supplying JA to the growing medium ofM. truncatula plants suppressed nodule formation, includingtheir responsiveness to Nod factor, by interfering with Caspiking and the expression of MtRIP1 and MtENOD11 (Sunet al. 2006). Similarly, application of methyl jasmonate(MeJA) to the shoots of L. japonicus plants suppressed noduledevelopment in both wild-type and hypernodulation mutant(Ljhar1) plants, inhibiting infection thread formation and the

expression of the nodulation gene, LjNIN (Nakagawa andKawaguchi 2006). In contrast, infection thread formationand nodulation are both reported to be increased inL. japonicus plants grown under low red/far-red light andtreated with JA (Suzuki et al. 2011). Moreover, the diminishednodulation phenotype of a phyB mutant of L. japonicus thathas reduced photoassimilate and JA-Ile levels can be restoredfollowing the application of JA (Suzuki et al. 2011).

Genes involved in JA biosynthesis and response wereregulated in the leaves of soybean plants in a GmNARK-dependent manner following the induction of nodulation(Kinkema and Gresshoff 2008; Seo et al. 2006). Moreover,JA levels were elevated in the leaves of a supernodulatingGmnark mutant (Seo et al. 2006) and foliar application of aninhibitor of JA biosynthesis reduced nodulation, specificallyin a Gmnark mutant (Kinkema and Gresshoff 2008).

In support of a positive role for JAs in nodulation,jasmonates stimulated the expression of nod genes in Rhizobi-um leguminosarum (Rosas et al. 1998) and Bradyrhizobiumjaponicum (Mabood and Smith 2005), and increased NodFactor production in B. japonicum (Mabood et al. 2006). Pre-inoculation of B. japonicum (Mabood and Smith 2005) orR. leguminosarum bv. phaseoli (Poustini et al. 2005) withjasmonates also was found to enhance nodulation and nitrogenfixation in soybean and bean plants, respectively. Interestingly,JA also is involved in tripartite interactions between legumes,rhizobia, and herbivores. For example, rhizobial inoculationaltered the composition of volatile organic compounds againstherbivores induced by JA (Ballhorn et al. 2013).

Collectively, the findings appear to indicate that JAs can actas either positive or negative regulators of nodulation andnitrogen fixation, depending on the legume species, the typeof JA used, and when, where, and how the hormone is applied.Clearly more research is required to understand the preciseroles that JAs have in legume nodulation.

Nitric Oxide

Nitric oxide (NO) is a gaseous signal molecule having broadroles in various aspects of plant development and stressresponse. Its roles in nodulation recently have beenthoroughly reviewed by Boscari et al. (2013), Meilhoc et al.(2011), and Puppo et al. (2013). Essentially, NO appears to bepresent from early nodulation through to nitrogen-fixation andnodule senescence, thus suggesting it acts at different devel-opmental stages of the symbiosis. Its production has beenreported in infection threads and nodule primordia (delGuidice et al. 2011), as well as in mature, nitrogen-fixingnodules ofM. truncatula, M. sativa, G. max, and L. japonicus(Baudouin et al. 2006; Pii et al. 2007; Sánchez et al. 2010;Shimoda et al. 2009), with both symbiotic partners capable ofsynthesizing the signal (Horchani et al. 2011; Sánchez et al.

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2010). It is a strong inhibitor of nitrogenase activity andnitrogen fixation (e.g., Baudouin et al. 2006; Kato et al.2010; Shimoda et al. 2009), and can inhibit rhizobia growth(Meilhoc et al. 2010).

Nitric Oxide also may play a positive role in nodulation, asits depletion delayed nodule development and reduced nodulenumbers (del Guidice et al. 2011). It also is reported to play abeneficial role in nodule energy metabolism and in the regu-lation of nitrogen metabolism in root nodules (Horchani et al.2011;Melo et al. 2011). Additional evidence suggests that NOmight act as a developmental signal to trigger the induction ofnodule senescence (reviewed in Boscari et al. 2013; Meilhocet al. 2011; Puppo et al. 2013). Thus, NO may have multiplesignalling roles in legume nodulation, acting as either a pos-itive or negative regulator of a broad range of processesassociated with nodule development and functioning.

Salicylic Acid

Salicylic acid (SA) is involved in systemic acquired resistanceand plant defense responses to pathogens. Its role in legumenodulation is not well defined, with some conflicting resultsreported. As with many of the factors reviewed here, theexperimental outcomes likely vary considerably dependingon the concentration and frequency of SA applied, as well asthe legume species, application method, and growing condi-tions used.

Exogenous application of SA is reported to reduce nodu-lation in the indeterminate nodule-forming Vicia sativa,M. sativa, T. repens and P. sativum, but not in the determinatenodule-forming L. japonicus, P. vulgaris, and Glycine soja(van Spronsen et al. 2003). However, additional studies inves-tigating the determinate nodule-forming soybean have report-ed an inhibitory effect of SA treatment on nodulation (Lianet al. 2000; Sato et al. 2002). This inhibition was found to beless pronounced in supernodulating soybean mutants than inwild-type plants (Sato et al. 2002). Additional work ectopi-cally over-expressing the bacterial SA hydroxylase gene,NahG, elegantly demonstrated that reduced endogenous SAlevels correlated with increased rhizobia infections and nodulenumbers in both the indeterminate nodule-formingM. truncatula and the determinate nodule-formingL. japonicus (Stacey et al. 2006). As well as inhibiting nodu-lation, SA can suppress rhizobia growth (Stacey et al. 2006),which could have considerable impact on studies investigatingthe effects of SA application on nodulation.

Inoculation of M. sativa (Martínez-Abarca et al. 1998) orpea (Blilou et al. 1999) with incompatible or Nod factor-deficient mutant strains of rhizobia resulted in an accumulationof SA in the roots. Moreover, inoculation of the non-nodulation pea mutant, Pssym30, with a compatible rhizobiastrain increased the root SA content (Blilou et al. 1999). In

contrast, SA levels did not increase in roots of wild-type plantsinoculatedwith a compatible rhizobia strain (Blilou et al. 1999;Martínez-Abarca et al. 1998). Collectively, these findings sug-gest a negative role for SA in nodulation, and the possible needfor compatible rhizobia and/or their Nod factor signal to sup-press an SA-dependent defence mechanism to enable the entryand successful establishment of the bacteria in the host plant.

Strigolactones

Strigolactones (SL) have diverse roles in regulating auxintransport, stimulating parasitic weed germination, coordinat-ing mycorrhizal symbiosis, and defining shoot and rootarchitecture. They were first reported to have a role inregulating nodulation by Soto et al. (2010), where applicationof the synthetic strigolactone analogue, GR24, positively af-fected alfalfa (Medicago sativa) nodulation. Silencing of theSL biosynthesis gene in L. japonicus, LjCCD7, resulted inslightly fewer nodules formed, with no alterations observed innodule development and morphology (Liu et al. 2013). Inter-estingly, the SL biosynthesis mutant of pea, rms1 (Psccd8),produced fewer nodules than its wild type, but the SL re-sponse mutant, rms4 (Psmax2), did not (Foo and Davies2011; Foo et al. 2013). Double mutant plants of rms1 andthe autoregulation mutants, nod3 (Psrdn1), sym28 (Psclv2), orsym29 (Psnark), all exhibit a supernodulation phenotype,indicating that the hormone does not act downstream of thesegenes in nodulation control, and instead likely influences thepromotion of nodulation (Foo et al. 2014).

Establishing a direct effect of SL on nodulation is compli-cated due to the hormone’s role in regulating other criticalprocesses, such as auxin transport and shoot and root devel-opment, which can considerably influence resource allocation.Moreover, SLs influence the development of lateral roots androot hairs (Kapulnik et al. 2011), which are essential structuresfor rhizobia infection. Thus, more research is required to betterunderstand the role of SLs in nodulation, including establish-ing any direct effects they may have on factors specific tonodule development. Interestingly, the GRAS-type transcrip-tion factors, NODULATION SIGNALING PATHWAY1(NSP1) and NSP2, which are critical for Nod-factor inducednodulation, also were found to be necessary for SL biosyn-thesis in Medicago truncatula and non-nodulating rice (Liuet al. 2011). The authors suggest that these transcriptionfactors fulfil a dual regulatory function to regulate down-stream targets of both nodulation and SL biosynthesis.

Signaling Peptides

More and more peptides have been identified that have criticalroles in legume nodulation. The number is likely to rise further

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as peptides frequently are found to act as ligands for receptorsto trigger aspects of plant growth and development, similar tothe action of classical phytohormones. Peptides also interactwith phytohormone signalling pathways to alter development.The following peptides have been identified as having keyroles in nodulation.

NCR Peptides

A fascinating class of peptides, called nodule-specific cysteine-rich (NCR) peptides, mediate the terminal, intracellular differ-entiation of bacteroids in legumes of the inverted repeat–lack-ing clade (IRLC), such asM. truncatula,M. sativa, P. sativum,Astragalus sinicus, T. repens, and Vicia faba (reviewed inKondorosi et al. 2013). Over 600 NCR-encoding genes arepredicted to be scattered across the M. truncatula genome.Most are composed of two exons, one encoding a conservedsignal peptide domain, and the second encoding the maturepeptide, which is typically 30–50 amino acids in length andcontains either 4 or 6 cysteines residues at highly conservedpositions (Alunni et al. 2007; Fedorova et al. 2002; Kondorosiet al. 2013; Mergaert et al. 2003).

NCR peptides can control the differentiation ofSinorhizobium meliloti bacteroids, the compatiblemicrosymbiont of M. truncatula (Van de Velde et al. 2010;Wang et al. 2010). They appear to do so by manipulating itscell cycle, possibly indicating that the rhizobia and/or bacteroidshave receptors to perceive the plant-derived factors. They aretargeted to the bacteria and enter the bacterial membrane andcytosol. A subunit of a signal peptidase complex (SPC) ofM. truncatula, called MtDNF1, is highly expressed in nodulesand is essential for removing the signal peptide from the proteins(Van de Velde et al. 2010; Wang et al. 2010). This occurs in theendoplasmic reticulum, and is required for correct targeting ofthe mature peptides to the bacteria.Mtdnf1 mutants were foundto contain unprocessed NCR peptides that remained in theendoplasmic reticulum and failed to reach the bacteria, resultingin undifferentiated bacteroids unable to fix nitrogen (Van deVelde et al. 2010; Wang et al. 2010). Some NCR peptides alsopartially mimicked S. meliloti bacteroid differentiation inL. japonicus, a non-IRLC legume (Van de Velde et al. 2010).

NCR peptides are similar to defensin-type antimicrobialpeptides (AMPs), and some exhibit strong antimicrobial ac-tivity against S. meliloti and other bacteria species, possibly byincreasing their cell permeabilization (Haag et al. 2011, 2012;Tiricz et al. 2013; Van de Velde et al. 2010). Sub-lethal levelsof an NCR peptide recently were shown to block cell divisionand antagonize Z-ring function, partially by altering the ex-pression of master cell-cycle regulators and genes critical tocell division (Penterman et al. 2014). Thus, NCR peptidesmay target regulatory pathways of S. meliloti to causeendoreduplication and differentiation during symbiosis

(Penterman et al. 2014). The ecological and evolutionaryconsequences of this host control mechanism over its bacterialsymbiont remain an interesting question for the future.

CLE Peptides

A number of CLAVATA/ESR-related (CLE) peptide hormonesrecently were identified as negative regulators of nodulation,including LjCLE-RS1 and 2 in L. japonicus (Okamoto et al.2009), MtCLE12 and 13 in M. truncatula (Mortier et al. 2010;Saur et al. 2011), and GmRIC1 and 2 in soybean (Lim et al.2011; Reid et al. 2011a) and PvRIC1 and 2 in P. vulgaris(Ferguson et al. 2014). These peptides typically are 12–13amino acids in length and are located at or near the C-terminusof their prepropeptide. They act systemically in the autoregula-tion of nodulation pathway to control nodule numbers (reviewedin Reid et al. 2011b). The genes encoding these peptides are allinduced in the root following inoculation with compatiblerhizobia species. Over-expression of these genes inhibits, andcan even completely abolish, nodule organogenesis.

The rhizobia-induced CLE peptides, or prepropeptides, are allpredicted to be transported in the xylem to the shoot where theyare perceived by a leucine-rich repeat receptor kinase, calledLjHAR1/MtSUNN/GmNARK, possibly in a complex with oth-er receptors, such as CLAVATA2 and KLAVIER (Krusell et al.2011; Miyazawa et al. 2010). Indeed, recent findings byOkamoto et al. (2013) elegantly demonstrated that LjCLE-RS2is a post-translationally arabinosylated glycopeptide that can bedetected in xylem sap and directly bind to LjHAR1. Perceptionof these peptides leads to the production of a new signal, calledthe shoot-derived inhibitor (SDI), which is transported backdown to the root to suppress further nodulation events (Linet al. 2010, 2011; Fig. 2). Other factors acting after the perceptionof the nodulation-suppressive CLE peptides includes a ubiquitinfusion degradation protein that is significantly up-regulated inexpression, called GmUFD1a (Reid et al. 2012).

Key domains and amino acid residues required for the nodulesuppressive activity of the soybean CLEswere recently identifiedusing domain-swap and site-directed mutagenesis techniques,respectively (Reid et al. 2013).Additional studies inM. truncatularevealed thatMtCLE13 expression depends on cytokinin, which,as outlined above, is essential for nodule development and indi-cates that the autoregulation of nodulation may be inducedconcomitant to nodule primordia formation (Mortier et al. 2012).

Soybean has an additional nodulation-suppressive CLEpeptide, called GmNIC1, which is highly similar in sequenceto GmRIC1 and 2 (Reid et al. 2011a). However, GmNIC1 isinduced by nitrate, not rhizobia, and acts locally throughGmNARK in the root, not systemically via the shoot. Thus,GmNIC1 appears to have a role in nitrate-regulation of nod-ulation (reviewed in Reid et al. 2011b). Recent findings usingsoybean also have shown that acidic growing conditions can

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inhibit nodulation systemically through GmNARK in theshoot, possibly suggesting that an acid-induced CLE pep-tide(s) exists that acts similarly to the nodulation-suppressiveCLE peptides to inhibit nodule organogenesis (Ferguson et al.2013; Lin et al. 2012).

ENOD40

ENOD40 is an enigmatic signal. The gene encoding it is wellconserved across the plant kingdom, including non-legumes,and it has roles in plant development aside from nodulation.Originally identified as an early nodulin gene (hence the nameENOD), ENOD40 lacks a long open reading frame (Crespiet al. 1994; Yang et al. 1993); however, it does encode twoshort peptides. Peptide A is 12–13 amino acids in length, andPeptide B, which partially overlaps with the open readingframe of Peptide A, is roughly 24–27 amino acids long(Röhrig et al. 2002; Sousa et al. 2001). Unlike the otherpeptides described here, ENOD40 peptides do not appear toresult from proteolytic cleavage of a larger protein product,and instead are produced directly from their mRNA. Interest-ingly, the role of the peptides may be to stabilize and form acomplex with the ENOD40 mRNA transcript, which exhibitsa conserved secondary structure and may function as an un-translated signal molecule (Crespi et al. 1994; Sousa et al.2001). Indeed, inM. truncatula, ENOD40 mRNA induced thecytoplasmic localization of a nuclear RNA binding protein,calledMtRBP1, and interacted with the RNA-binding peptides,MtSNARP1 and MtSNARP2, that appear to sustain bacteroidsduring symbiosis (Campalans et al. 2004; Laporte et al. 2010).

Following rhizobia inoculation, ENOD40 is expressed inthe root pericycle and in dividing cells of the nodule primor-dium (e.g., Crespi et al. 1994; Mathesius et al. 2000; Yanget al. 1993). It also can be triggered by factors such as Nodfactors (Fang and Hirsch 1998; Minami et al. 1996) andcytokinin (Fang and Hirsch 1998; Mathesius et al. 2000),consistent with it having a role in cell division and noduleprimordium formation. Likewise, over-expression ofENOD40 can induce cortical cell divisions and promote nod-ule primordium formation, whereas down-regulation of thegene can hinder nodule and bacteroid development (Charonet al. 1997, 1999; Crespi et al. 1994; Wan et al. 2007). Inmature nodules, ENOD40 is expressed in nodule vascularbundles, uninfected cells of the central tissues and the infec-tion zone of indeterminate nodules (e.g., Crespi et al. 1994;Wan et al. 2007; Yang et al. 1993). Interestingly, many legumespecies have more than one copy of ENOD40, which canexhibit slightly different patterns of expression during nodu-lation (e.g., Fang and Hirsch 1998; Wan et al. 2007).

ENOD40 likely acts along with other factors to activate thecell cycle and induce the formation of nodule primordia. Italso may have a role in phloem unloading in mature and

dividing nodule cells, possibly as a means of generating acarbon sink, as both Peptides A and B have been shown tobind with sucrose synthase (Röhrig et al. 2002). The bindingof Peptide A enhanced the breakdown of sucrose, which couldincrease the carbon supply to cells. Due to its wide distributionacross both legume and non-legume species, its expression inplant organs other than nodules, and its role in other processessuch as mycorrhization (Staehelin et al. 2001), nematode infec-tion (Favery et al. 2002), and lateral root formation (Mathesiuset al. 2000), ENOD40 likely functions broadly in plant develop-ment, with general roles in organ and tissue formation.

Ralf

Rapid Alkalinisation Factor (RALF) peptides originate frommembers of a highly conserved gene family found through-out the plant kingdom (Bedinger et al. 2010). They areexpressed in an array of tissues, including shoots, leaves,flowers, roots, and nodules. Most are processed from the C-terminus of their prepropeptide to produce a mature peptideroughly 50 amino acids in length (Bedinger et al. 2010).Application of synthetic RALF arrested root growth inArabidopsis and tomato plants (Pearce et al. 2001). Recentevidence supports a common role for RALF peptides inregulating cell expansion, regardless of divergence in theirtissue specificity and gene expression patterns (Morato doCanto et al. 2014).

In M. truncatula, the RALF-encoding gene, MtRALF1, isup-regulated following Nod factor application (Combier et al.2008). Over-expression ofMtRALF1 increased the number ofinfection threads that aborted, leading to a reduction in thenumber of nodules that formed (Combier et al. 2008). More-over, the nodules that did form were small and poorly colo-nized by their symbiotic partner (Combier et al. 2008). Thesefindings suggest a role for the peptide in both rhizobia infec-tion and nodule organogenesis, possibly as a negative regula-tor, akin to the reported function of RALF peptides in rootdevelopment.

CEP

Members of the CEP (C-terminally encoded peptide) signal-ing peptide family are widespread among flowering plants,but are absent from some primitive species (Roberts et al.2013). The mature peptides are around 15 amino acids inlength, and are processed from their full-length protein at ornear the C-terminus, as their name implies (Delay et al. 2013;Ohyama et al. 2008). CEP peptides appear to have distinctroles in orchestrating root and shoot growth. They are regu-lated by environmental cues and typically act as negativeregulators of plant development (Delay et al. 2013).

J Chem Ecol (2014) 40:770–790 781

Application of the MtCEP1 peptide, or over-expression of thegene encoding it, results in an inhibition of lateral roots, anenhancement of nodulation, and the induction of periodiccircumferential root swellings in M. truncatula plants thatshow increased auxin response (Imin et al. 2013). The in-crease in nodulation is partially tolerant to high levels ofnitrate that normally suppress nodule development (Iminet al. 2013). Collectively, these findings appear to indicatethat MtCEP1 differentially modulates lateral root and noduledevelopment in M. truncatula.

DVL1/ROT4

Devil/rotundifolia (ROT)-Four-Like (DVL/RTFL) peptidesare approximately 30 amino acids in length and, like CLE,CEP, and RALF peptides, are derived from the C-terminus oftheir full length proteins (Valdivia et al. 2013). The function ofmany DVL/RTFL peptides is largely thought to be redundant;however, they likely exert specialized control in developmentvia divergent expression patterns (Wen et al. 2004). Determin-ing the function of DVL/RTFL peptides has been hindered

Table 1 Overview of major nodulation processes affected by phytohormones and interacting peptides - denotes a negative effect on the process, + apositive effect

Infection thread formation

ABA –Cytokinin +/-Ethylene –DLV1/ROT4-GA +/- (correct ‘level and window’ required)JA -RALF –SA –

Nodule initiation ABA -Auxin + (correct localization and sensitivity required)BR +/-CEP1 +Cytokinin +DLV1/ROT4-ENOD40 +Ethylene –GA + (correct ‘level and window’ required)JA –RALF –NO +SA -Strigolactone +

Nodule positioning Auxin + (positioning in inner or outer cortex)Cytokinin + (positioning in inner or outer cortex) Ethylene + (radial position)

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largely by the absence of clear phenotypes in loss-of-functionmutants and silencing lines (Valdivia et al. 2013; Wen et al.2004). However, over-expression studies indicate that theymay control aspects of growth and development by regulatingcell proliferation (e.g., Wen et al. 2004).

In M. truncatula roots, a DVL1/ROT4 peptide-encodinggene, called MtDVL1, was transiently up-regulated followingtreatment with Nod factor (Combier et al. 2008). As wasobserved with MtRALF1, over-expression of the gene

increased the number of infection threads that aborted,resulting in a diminished number of nodules that formed(Combier et al. 2008). However, unlike what was observedwith MtRALF1, over-expression of MtDVL1 did not impairthe development of the nodules that did form, or their ability tofix nitrogen. This led Combier et al. (2008) to propose a rolefor the peptide in infection thread formation and progression,possibly via regulating the differentiation of pre-infectionthreads.

Table 1 (continued)

Nodule differentiation and growth

ABA +Auxin (synthesized by rhizobia ) +BR +Cytokinin +GA + (correct ‘level and window’ required)NO +RALF -

Nodule number control via AON

Auxin +JA +CLE peptides +Cytokinin (induces CLE) +

Nitrogen fixation ABA –JA +NO -

Bacteroid differentiation

NCR + (indeterminate nodules)

Nodule senescence NO +

N2

NH3

J Chem Ecol (2014) 40:770–790 783

Conclusion and Future Prospectives

Molecular studies and the identification of hormone and othersignalling mutants in legumes have started to elucidate manyof the signalling interactions that control nodule developmentand rhizobial infection at different stages during the interac-tion (Table 1). At the current time, it is difficult to construct adetailed model of hormone interactions during nodulationbecause studies have been done in different legume specieswith different methods and under different conditions. Tounify this area, we first need controlled studies in modellegumes that systematically study interactions between hor-mones utilizing existing hormone mutants. This should in-clude accurate localized measurements of hormone concen-trations during nodulation in time and space, and across arange of legumes in order to resolve the sometimes conflictingfindings on nodulation responses to external hormone appli-cation. Since hormones often carry location-specific informa-tion, their sites of action will be crucial to integrate into currentgenetic models of hormones action. It also will be important toclarify how signalling peptides interact with the classicalphytohormone pathways. Second, it will be interesting toextend our findings from model legumes to the diverse rangeof legumes that display different types of nodules. Third, ourknowledge of the contribution of phytohormones produced byrhizobia remains sketchy, and is partially limited by the avail-ability of rhizobial hormone mutants. Overall, rhizobially-contributed phytohormones appear to give rhizobia additionalbenefits for successful symbiotic establishment, especiallyunder stressful environmental conditions, but they do notappear to be essential for the symbiosis. However, it is unclearwhether the production of phytohormones by rhizobia haseffects not only on nodule number and functioning, but onoverall fitness of the host.

Very little attention has been paid to extending many of thestudies involving bacterial or plant hormone mutants to thefield and to investigating effects on tripartite interactions withother organisms or the abiotic environment. While manyhormone studies have been carried out under controlled con-ditions, plants living in the real world are colonized by manydifferent organisms, while at the same time adjusting to fluc-tuating abiotic conditions. In general, it appears that stress-related hormones such as ABA, JA, ethylene, and SA, havenegative impacts on nodulation, and this may well reflect theneed of the plant to limit nodulation under stressful situations.In addition, these hormone response pathways are up-regulated in response to pathogen and herbivore attack, andthis may influence the level of nodulation. Since most studieson pathogen and insect responses have been carried out onaboveground organs and in non-legumes, it would be inter-esting to test whether infection by shoot pathogens has asystemic effect on nodulation in the roots of legumes (e.g.,for belowground organs, Rasmann et al. 2005, and for

legumes, Navia-Gine et al. 2009 and Zhang et al. 2009).Future studies also are necessary to study, in a commonsystem, the parallel changes in stress hormone related changesin response to a range and combination of symbiotic andpathogenic organisms to identify overlapping response path-ways (De Vos et al. 2005). While rhizobia are symbionts, theyalso carry common microbe-associated molecular patterns(MAMPs) and trigger some early plant defense responses,although their MAMPs may have been modified to down-regulate plant defense responses during successful interac-tions (Zamioudis and Pieterse 2012). It would be interestingto investigate to what extent plant pathogens and rhizobiatrigger overlapping hormone responses in the root, and howrhizobia have evolved to modulate these response pathwaysthus avoiding plant defenses. For example, the master activa-tor of SA-response genes required for plant immunity, NPR1,also functions in the infection of roots by rhizobia (Peleg-Grossman et al. 2009). Similarly, a central regulator of ethyl-ene signalling, EIN2, is targeted by both pathogens andrhizobia in legumes (Penmetsa et al. 2008). In addition tothe traditional stress hormones, auxin signalling has emergedas a common target in both symbiont and pathogen interac-tions, and this has reshaped our thinking of auxin as a devel-opmental signal. Instead, it may play roles in both develop-ment and defense, although its role in defense regulationduring nodulation remains unstudied. So far, little is knownabout the hormonal cross-talk that fine-tunes nodulation inlegumes, but the increasing identification of legume hormonemutants could be further utilized to help address this question.

Finally, an unsolved question remains whether legumeshave a predisposition for nodule development because ofaltered developmental responses to phytohormones, as indi-cated, for example, by the different responses of legumes andnon-legumes to ABA (Liang and Harris 2005). It would beinteresting to investigate whether legumes have evolved spe-cific hormone receptors specifically for controlling nodula-tion. Comparisons of hormone responses between nodulatingand non-nodulating legumes, and between legumes and non-legumes could inform future studies attempting to extendnodulation to non-legumes. In that context, it is perhapsnoteworthy that a whole genome duplication occurred in thePapillionoid legume subfamily at about the time of the evolu-tion of nodulation in legumes, and this may have enabledlegumes to evolve hormonal response pathways specificallyfor nodulation (Op den Camp et al. 2011).

Acknowledgments Due to the large size of this research field, a num-ber of publications were undoubtedly overlooked. We thank PeterGresshoff for careful reading of the manuscript. Financial support wasprovided to BJF by the Australian Research Council Discovery Projectgrants (DP130103084 and DP130102266) as well as University ofQueensland strategic funds. UM was supported by a Future Fellowship(FT100100669) and a Discovery Project grant (DP120102970) from theAustralian Research Council.

784 J Chem Ecol (2014) 40:770–790

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