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

REVIEW ARTICLE

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- differences among different legumes could explain the variety ing bacteria called rhizobia leads to the formation of root of nodules types and the predisposition for nodule formation nodules. Nodules are highly organized root organs that form in this plant family. In addition, the molecular studies carried in response to Nod factors produced by rhizobia, and they out under controlled conditions will need to be extended into provide rhizobia with a specialized niche to optimize nutrient the field to test whether and how phytohormone contributions exchange and nitrogen fixation. Nodule development and by host and rhizobial partners affect the long term fitness of invasion by rhizobia is locally controlled by feedback between the host and the survival and competition of rhizobia in the rhizobia and the plant host. In addition, the total number of soil. It also will be interesting to explore the interaction of nodules on a root system is controlled by a systemic mecha- hormonal signalling pathways between rhizobia and plant nism termed ’autoregulation of nodulation’. Both the local and pathogens. the systemic control of nodulation are regulated by phytohor- mones. There are two mechanisms by which phytohormone Keywords Autoregulation of nodulation . Infection thread . signalling is altered during nodulation: through direct synthe- Legume nodulation . Phytohormones . Rhizobia . Symbiosis sis by rhizobia and through indirect manipulation of the phy- tohormone balance in the plant, triggered by bacterial Nod factors. Recent genetic and physiological evidence points to a Introduction crucial role of Nod factor-induced changes in the host phyto- hormone balance as a prerequisite for successful nodule for- Most legume species are able to enter into a symbiotic rela- mation. Phytohormones synthesized by rhizobia enhance tionship with compatible strains of nitrogen-fixing rhizobia symbiosis effectiveness but do not appear to be necessary bacteria. This leads to the formation of a novel organ, called for nodule formation. This review provides an overview of the nodule, which houses the rhizobia and creates an environ- recent advances in our understanding of the roles and interac- ment suitable for nitrogen fixation (reviewed by Desbrosses tions of phytohormones and signalling peptides in the regula- and Stougaard 2011; Ferguson et al. 2010;Oldroyd2013). tion of nodule infection, initiation, positioning, development, This symbiosis evolved approximately 60 million years ago, and autoregulation. Future challenges remain to unify hor- at a time of high CO2 concentrations in the atmosphere, and mone–related findings across different legumes and to test likely gave legumes an ecological advantage over other spe- whether hormone perception, response, or transport cies by being able to utilize higher carbon availability through increased N nutrition (Sprent 2007). To maximize the effi- ciency of nodulation, it is thought that a sophistical signal B. J. Ferguson exchange between rhizobia and legume hosts has evolved. Centre for Integrative Legume Research, School of Agricultural and Food Sciences, The University of Queensland, St. Lucia, Brisbane, Most likely, a less specific symbiosis, in which rhizobia in- Queensland 4072, Australia vaded roots through crack entry and initiated nodules derived from lateral roots has evolved to a more specific interaction * U. Mathesius ( ) with highly regulated invasion via infection threads and for- Division of Plant Science, Research School of Biology, The Australian National University, Canberra, ACT 0200, Australia mation of a nodule independent of lateral roots (Sprent 2008). e-mail: [email protected] Much research has been aimed at identifying the signals J Chem Ecol (2014) 40:770–790 771 exchanged between rhizobia and legumes necessary for this multitude of plant and bacterial factors that are tightly regu- symbiosis. Most important has been the identification of lated by the host plant, giving it control over the number of flavonoids exuded by roots that stimulate host specific nodule structures it forms. The plant regulates nodule numbers rhizobia to synthesise Nod factors, lipochitin oligosaccha- locally in response to Nod factors and nitrogen availability, as rides with specific structures depending on the rhizobial well as through a systemic regulatory mechanism called ‘auto- symbiont (Oldroyd 2013). Nod factors are necessary for regulation of nodulation’ (AON) (Reid et al. 2011b; Fig. 2). triggering a cascade of signaling events in the host root, Lack of AON reduces the fitness of the symbiosis, probably culminating in the formation of nodules and their invasion by redirecting too many resources to unnecessary numbers of by rhizobia. However, Nod factor-independent nodulation nodules. Just over a decade ago, we thoroughly reviewed has been observed in some cases, and it remains largely many of the fundamental signalling components involved in unknown how the nodulation process is regulated in the the nodulation process (Ferguson and Mathesius 2003). Here, absence of Nod factors (Giraud et al. 2007). It is thought we provide an overview of key advances relating to many of that Nod factor signaling results in changes in plant hor- these signals, and discuss a number of new factors identified mone synthesis, perception, transport, and accumulation, over the last 10 years that have critical roles in the develop- which fine-tune the infection process, initiation, and devel- ment and/or regulation of legume nodules. opment of nodules and control nodule numbers (e.g., Desbrosses and Stougaard 2011; Ding and Oldroyd 2009; Mathesius 2008). In addition, rhizobia synthesize all major Abscisic Acid phytohormones (e.g., Boiero et al. 2007)andcansome- times alter phytohormone breakdown, which contributes to As reviewed by Ferguson and Mathesius (2003), early studies the effectiveness of the symbiosis (Spaepen et al. 2007). indicated that abscisic acid (ABA) had an inhibitory effect on Most research relating to the molecular mechanisms of nodulation, as exogenous application of the hormone signifi- nodulation has involved model legumes, in particular Lotus cantly reduced the number of nodules that formed on different japonicus and Glycine max (soybean), forming determinate legume species. However, the ABA content of nodules was nodules, and Medicago truncatula, a species forming indeter- elevated compared with the surrounding root tissue of various minate nodules. Determinate nodules arise from divisions in species examined. This could indicate a requirement for the the root outer cortex, which later fuse with dividing pericycle hormone in nodule development and/or functioning. These cells, and the resulting nodule develops a meristem that dif- somewhat conflicting findings led us to postulate that the role ferentiates. In contrast, indeterminate nodules arise from cell of ABA in nodulation may not be a simple one; with a positive divisions in the pericycle and inner cortex of the host root, and requirement in different tissues and/or different developmen- resulting nodules retain a meristem, resulting in elongated tal stages that becomes inhibitory when its levels are elevated, nodules (Ferguson et al. 2010; Hirsch 1992; Fig. 1). The possibly due to the induction of stress responses. nodulation process involves rhizobial infection into the root In support of the earlier conclusions, recent reports have cortex and the developing nodule, in parallel to the induction described how exogenous application of ABA can inhibit of cell division in the root to initiate a nodule. Both processes nodule development in M. truncatula (Ding et al. 2008), are regulated independently and are orchestrated by a Trifolium repens (white clover, Suzuki et al. 2004), Phaseolus

Fig. 1 Differences in nodule positioning in indeterminate and determi- divisions in outer (OC) and middle cortex cells. The dividing cells of both nate type nodules. a Indeterminate nodules are initiated in pericycle (pe) types of nodules (grey) are characterized by increased auxin and cytokinin and inner cortex cells (ic) underlying the infection site indicated by a responses. Ethylene synthesis is thought to restrict nodules to radial curled root hair (rh). ep=epidermis. b Determinate nodules arise from positions opposite the xylem poles (XP, grey striped) 772 J Chem Ecol (2014) 40:770–790

exhibited normal nodule numbers and a normal AON re- sponse; however, the individual nodules that formed on Beyma 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,the ABA-insensitive mutant enf1 exhibited increased nodule numbers as well as enhanced nitrogen fixation (Tominaga et al. 2009). The endogenous ABA content of enf1 was found to be reduced compared with wild-type plants, as was the production of nitric oxide in mutant nodules. In M. truncatula, use of the dominant negative allele abi1-1 from Arabidopsis to genetically regulate ABA signalling resulted in a hypernodulation phenotype, whereas the sta1 mutant, which dictates sensitivity to ABA, exhibited a reduction in Nod factor signalling and nodule numbers (Ding et al. 2008). The M. truncatula latd mutant, which is defective in meristem Fig. 2 Model for the Autoregulation Of Nodulation (AON) mechanism. formation of roots and nodules, shows an altered sensitivity 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 and to ABA, although ABA content is similar to the wild type binds to the Nodulation Autoregulation Receptor Kinase (NARK) recep- (Liang et al. 2007). Nodule primordia are initiated normally in tor. This induces the production of a shoot-derived inhibitor (dashed latd mutant roots, but later meristem formation is inhibited, arrow) signal (unidentified) that moves back to the root system to inhibit leading to small, white nodules. This points to an effect of further nodules from forming. In AON mutants (right)theNARKrecep- tor is defective, resulting in the loss of SDI synthesis and a ABA in later nodule development (Liang et al. 2007). Le- supernodulation phenotype gumes also are reported to have a unique tendency to increase their number of lateral roots following ABA treatment com- vulgaris (common bean; Khadri et al. 2006), and L. japonicus pared to non-legume species (Liang and Harris 2005). It will (Biswas et al. 2009; Suzuki et al. 2004; Tominaga et al. 2009). be interesting to determine whether this altered ABA response Additionally, treatment with abamine, an inhibitor of 9-cis- in legumes contributes to their ability to nodulate. epoxycarotenoid dioxygenase, increased nodule numbers and Abscisic acid also is synthesized by rhizobia, and this could nitrogen fixation in L. japonicus (Suzuki et al. 2004; not only alter nodulation but indirectly contribute to plant Tominaga et al. 2009). In T. repens, ABA treatment inhibited stress tolerance, as ABA mediates abiotic stress. For at the stage between root hair swelling and curling (Suzuki example, Bano et al. (2010) showed that in Rhizobium et al. 2004). Elegant work using M. truncatula revealed that leguminosarum, low synthesis of ABA, together with high ABA inhibited rhizobia infection, expression of the early synthesis of GA and auxin, was correlated with higher plant nodulation genes ENOD11 and RIP1, critical Nod factor- biomass under drought conditions. induced calcium spiking, and cytokinin-induced nodulation Collectively, the evidence predominately points to a nega- responses (Ding et al. 2008). Thus, it affects both the infection tive role for ABA in legume nodule development, with the pathway as well as the nodule development pathway, possibly potential for a more positive role for the hormone in nodule to reduce the costly establishment of nodules under stressful growth and/or functioning. Further studies are required to conditions. In addition, ABA application to one side of a split- unequivocally determine the molecular mechanisms affected root system inhibited nodulation locally not systemically, and by biologically-relevant levels of the hormone in different thus, the hormone is not considered to be directly involved in tissues and in different stages of nodule development. the Autoregulation of Nodulation (AON) pathway (Biswas et al. 2009). A positive influence of ABA also has been reported, as ABA pre-treatment improved the nitrogen fixa- Auxin tion capacity of M. sativa plants grown under salt stress conditions, and this was linked to the induction of antioxidant Auxin is a central regulator of plant development. Studies in enzymes (Palma et al. 2014). Arabidopsis have elegantly shown that auxin accumulation is A number of studies using ABA mutants also have recently a prerequisite for organ formation throughout the plant, and been reported. In L. japonicus, the ABA insensitive mutant, that the regulation of auxin transport is crucial for auxin Beyma, which has a wilty phenotype, was shown to be insen- accumulation at specific sites of organogenesis (Benková sitive to ABA-induced inhibition of germination, vegetative et al. 2003). Nodule development is no exception, and auxin growth, stomatal opening, and nodulation (Biswas et al. maxima have been observed in early developing nodule 2009). In the absence of exogenous ABA, the mutant primordia in M. truncatula (Mathesius et al. 1998a;van J Chem Ecol (2014) 40:770–790 773

Noorden et al. 2007), L. japonicus (Pacios-Bras et al. 2003; 2001).Atthesametime,PIN gene expression has been Suzaki et al. 2012; Takanashi et al. 2011), and soybean localized in developing primordia (Huo et al. 2006), although (Turner et al. 2013). In agreement with increased auxin re- the exact localization of individual PIN proteins during nodule sponses in early nodule primordia, root responses to auxin and development has not yet been determined. The importance of rhizobia were both very similar in a proteome analysis of PIN genes in auxin transport during nodulation was confirmed M. truncatula (van Noorden et al. 2007). Despite that, external through silencing of several MtPIN genes, which caused a treatment of roots with auxin inhibits nodule formation, as reduction in nodule numbers (Huo et al. 2006). does treatment of roots with an auxin action inhibitor (van PIN gene expression and intracellular cycling, important Noorden et al. 2006). Most likely, the requirement for auxin for PIN protein localization to specific sides of the cell, can be lies within a certain concentration window, and also is depen- altered by synthetic and natural auxin transport inhibitors, and dent on the stage of nodule development, on the location in these have been implicated in nodulation. The synthetic auxin which auxin is required, and on the presence and perception of transport inhibitors NPA (1-N-naphthylphalamic acid) and other plant hormones that auxin interacts with. TIBA (2,3,5-triiodobenzoic acid) can trigger pseudonodule In addition, auxin requirements appear to be different be- formation in several legumes in the absence of rhizobia, tween determinate and indeterminate nodule types (cf. Fig. 1). suggesting that auxin transport inhibition is sufficient to in- For example, increasing auxin sensitivity in soybean by ec- duce a developmental program to from a nodule (Hirsch et al. topic overexpression of the microRNA160 gene, which neg- 1989). Subsequent studies have confirmed that rhizobia and atively regulates auxin response factor (ARF) genes, reduced purified Nod factors transiently inhibit auxin transport in nodule numbers (Turner et al. 2013). In this case, nodule legumes forming indeterminate nodules (Boot et al. 1999; initiation was not altered, but subsequent nodule development Mathesius et al. 1998b). In L. japonicus, which forms deter- was inhibited, possibly because the auxin distribution in de- minate nodules, an increase in polar auxin transport was veloping nodules needs specific gradients to allow the transi- measured after inoculation (Pacios-Bras et al. 2003). This is tion between cell division and differentiation (Turner et al. consistent with an increase in expression of the auxin reporter 2013). Overexpression of miRNA393, which targets an auxin DR5:GUS in the vascular tissue around the inoculation site in receptor and reduced auxin sensitivity, did not change nodule L. japonicus (Li et al. 2014). These studies suggest that auxin numbers in soybean (Turner et al. 2013). In M. truncatula, transport regulation in response to rhizobia differs among miRNA160 overexpression reduced nodulation similarly to legumes. soybean (Bustos-Sanmamed et al. 2013), whereas overexpres- In M. truncatula, application of TIBA or NPA can cause sion of miRNA393 also reduced nodule density, suggesting the formation of pseudonodules in a range of nodulation that M. truncatula requires a narrow ‘window’ of auxin sen- mutants, including those defective in early Nod factor percep- sitivity (Mao et al. 2013). Another study in which auxin tion and signal transduction (Rightmyer and Long 2011). This sensitivity was reduced in M. truncatula by RNA interference places the step of auxin transport inhibition downstream of (RNAi) against the cell cycle regulator CDC16 enhanced these signalling events. Natural auxin transport inhibitors that nodule numbers (Kuppusamy et al. 2009). Currently, it is could be involved in auxin transport regulation during nodule not known why different legumes differ in their sensitivity formation were suggested to be flavonoids (Hirsch et al. to auxin. 1989). Flavonoids are a group of secondary metabolites, some Computational models predict that it is likely that auxin of which can act as modulators of auxin transport (Peer and accumulates at the site of nodule initiation through regulation Murphy 2007). Flavonoids are induced in cortical cells des- of auxin transport, in particular by reducing auxin export tined to divide during nodule development in several legumes (Deinum et al. 2012). Auxin transport occurs through the (Mathesius et al. 1998a) and mimic auxin transport-related phloem from source to sink tissues and via active polar trans- changes in auxin responses (Mathesius et al. 1998b). Silenc- port from cell to cell (Peer et al. 2010). The major auxin found ing the flavonoid pathway in M. truncatula prevented auxin in most plants, indole-3-acetic acid (IAA), is actively transport inhibition by rhizobia (Wasson et al. 2006), and transported into cells by auxin importers of the amino acid flavonols were found to be the most likely sub-branch of the permease families AUX1 (Auxin resistant 1), LAX (Like- flavonoid pathway required for auxin transport control (Zhang AUX1) and PGP4, a member of the MDR/PGP (Multidrug et al. 2009). However, silencing the flavonoid pathway in resistance/P-glycoprotein) families. Auxin export is mediated soybean was not required for auxin transport control by members of the PIN (Pin-formed) and PGP families. The (Subramanian et al. 2006), which supports the idea that auxin polar localization of PIN proteins on either the basal, apical, or transport control may be regulated differently in legumes lateral side of the cell determines the polarity of auxin trans- forming determinate and indeterminate nodules port. During nodule development, three MtLAX proteins are (Subramanian et al. 2007). So far it remains unclear which highly expressed in the developing primordia in M. truncatula, other regulators of auxin transport might be required for auxin indicating active auxin import into these cells (de Billy et al. accumulation in primordia of determinate nodules. It is likely 774 J Chem Ecol (2014) 40:770–790 that the position of the cortical auxin maximum in either the produced by rhizobia enhances root growth and lateral root inner or outer cortex in indeterminate and determinate nodule number and thus creates more opportunities for the root sys- forming legumes is explained by lateral shifts in PIN protein tem to develop nodules. How this affects plant and bacterial localization in the cortex (Deinum et al. 2012), and it will be survival in the field remains an open question. interesting to find out in the future what controls lateral PIN Changes in auxin accumulation or signalling during plant- protein positioning in different legumes. pathogen interactions also could affect nodulation, although Flavonoids also could alter auxin accumulation by altering little is known about how rhizobia and plant pathogens affect auxin breakdown, through activation or inactivation of perox- legumes in co-inoculation experiments. Studies using non- idases that might be involved in auxin oxidation (Mathesius legumes have demonstrated that plants infected by pathogens 2001). Recent evidence from Arabidopsis shows that flavo- down-regulate auxin signalling, and that this enhances patho- noids can buffer auxin signaling by altering ROS accumula- gen resistance. In contrast, addition of auxin can enhance tion necessary for auxin breakdown/oxidation (Peer et al. pathogen symptoms, and auxin signalling appears to be a 2013). So far there is no clear evidence for the identity or target of R-gene mediated resistance (Mathesius 2010; Spoel activity of specific flavonoids in auxin breakdown during and Dong 2008). nodulation in vivo, and computational modelling of auxin maxima found that changes in auxin synthesis or breakdown are unlikely to result in a sufficiently strong cortical auxin maximum by themselves (Deinum et al. 2012). Brassinosteroids In addition to the regulation of local auxin transport at the site of nodule formation, long-distance auxin transport from Over the last decade, a great deal has been revealed about the the shoot to the root has been implicated in the control of biosynthesis, perception, and signalling response of nodulation, in particular in AON in M. truncatula.Measure- brassinosteroid (BR) hormones. However, there is still little ment of auxin transport showed that the AON mutant sunn1 known about the role of BRs in legume nodulation. Endoge- (Schnabel et al. 2005) has significantly increased capacity for nous BRs influence nodule development, as pea mutants shoot-to-root auxin transport compared to wild type. After altered in BR biosynthesis or perception form significantly inoculation of roots with rhizobia, shoot-to-root auxin trans- fewer nodules than wild-type plants (Ferguson et al. 2005). port was transiently found to decrease in wild type roots, Double mutant plants of the severely BR-deficient mutant of whereas transport remained unaltered in the sunn1 mutant pea, lk, and the autoregulation mutants, nod3 (Psrdn1), sym28 (van Noorden et al. 2006). Nodule numbers of sunn1 mutants (Psclv2), or sym29 (Psnark), all exhibit a supernodulation could be reduced to wild type numbers by the application of phenotype, and no evidence has been found for BRs acting an auxin transport inhibitor to the shoot-root junction, sug- downstream of these genes in the systemic AON pathway gesting that increased auxin transport is correlated with higher (Foo et al. 2014). nodule numbers in the sunn1 mutant (van Noorden et al. A number of other investigations have focused on the effect 2006). Whether a similar mechanism exists in other legumes of BR application on nodulation. Soaking seeds of Lens remains to be tested. Currently, studies in L. japonicus have culinaris with different concentrations of 28- shown higher auxin responses in roots of the AON mutant homobrassinolide resulted in reduced root lengths and nodule har1 than in wild type roots (Suzaki et al. 2012), but whether numbers, but increased nitrate reductase activity (Hayat and this is due to auxin transport changes has not been tested. Ahmad 2003). In contrast, imbibing pea seeds with 24- Auxin is also synthesised by rhizobia and a range of other epibrassinolide led to an increase in nodule numbers, nodule soil bacteria (Spaepen et al. 2007). The production of the fresh and dry weights, and nitrogenase activity (Shahid et al. auxin IAA by rhizobia is upregulated by flavonoid exudation 2011). Foliar application of epibrassinolide increased the nod- from the root (Prinsen et al. 1991), and knock-out of the ule number and weight, and the nitrogenase activity, of flavonoid-dependent IAA pathway in Rhizobium sp. NR234 Arachis hypogaea (peanut) plants (Vardhini and Rao 1999). reduced auxin content in nodules and inhibited nitrogen fixa- Foliar sprays of epibrassinolide or homobrassinolide also tion (Spaepen et al. 2007). Studies with rhizobial strains with were found to increase nodulation and nitrogenase activity altered auxin synthesis have shown that lack of auxin synthe- in Phaseolus vulgaris (French bean) plants (Upreti and Murti sis does not prevent nodulation, but can negatively affect (2004). In contrast, epibrassinolide application to soybean nitrogen fixation, whereas auxin overproduction can increase roots was found to decrease the extent of both nodulation nodulation efficiency; the mechanism of this is not understood and nitrogen fixation (Hunter 2001). Foliar application or root (Pii et al. 2007). One question that awaits investigation is the injection of brassinolide inhibited nodule formation and root combined effect of bacterial auxin on root architecture and development in a super-nodulating soybean mutant, but not in nodulation, as auxin has strong effects on lateral root forma- its wild-type parent (Terakado et al. 2005). In contrast, the tion and root elongation. Thus, it might be possible that auxin application of brassinazole (an inhibitor of BR biosynthesis) J Chem Ecol (2014) 40:770–790 775 to the leaves, or the culture media, increased the nodule bacterial entry provides a signal to the cortex that bypasses a number of wild-type soybean plants. hypothetical signal between epidermis and cortex, mediated In all of these studies, the differences observed may simply by LHK1 (Held et al. 2014). relate to the methods or species used, in addition to the type Cytokinin is not only necessary but also sufficient for the and concentration of BR applied. Furthermore, in many in- induction of cortical cell divisions, as demonstrated in the lhk1 stances, it is difficult to ascertain which effects of the hormone gain-of-function mutant (snf2)inL. japonicus, which forms are direct, and which are indirect. To date, there continues to spontaneous nodules (Tirichine et al. 2007). In some legumes, be no molecular evidence pertaining to the role of BRs in external application of cytokinin to roots is sufficient to induce nodule formation. nodules, and cytokinin was shown to act downstream of the common signalling pathway activated by Nod factors, but upstream of the NSP1, NSP2, and NIN transcription factors Cytokinins necessary for nodulation (e.g., Heckmann et al. 2011). Mature nodules formed in the cre1 mutant showed an Cytokinins were long suspected to act as regulators of cell altered zonation, suggesting that cytokinin is required at an division in nodulation, and this has been confirmed with the early, as well as a later, stage during nodulation (Plet et al. identification of a number of cytokinin mutants over the last 2011). Cytokinin also is involved in activation of AON, as few years. Cytokinin response genes are activated in either cytokinin signalling through cre1 is required for induction of inner or outer cortex cells during nodule initiation in indeter- the MtCLE13 peptide mediating AON in M. truncatula minate and determinate nodules, respectively (e.g., Held et al. (Mortier et al. 2012; see section on CLE peptides below). 2014; Lohar et al. 2004;Pletetal.2011), similar to the auxin The action of cytokinin is linked with the regulation of responses observed in these cells. Whether this is due to auxin during nodulation. For example, the cre1 mutant increased cytokinin concentration or sensitivity or both has showed increased auxin transport and PIN gene expression, not been directly determined yet. Cytokinin synthesis by the and lacks auxin transport control after infection with rhizobia cytokinin 5’-monophosphate phosphoribohydrolase LOG (Plet et al. 2011). Similarly, in Arabidopsis, cytokinin affects (LONELY GUY) recently has been shown to be required lateral root development through action on PIN gene expres- within a certain expression window for nodule initiation, sion (Marhavy et al. 2011). It remains to be shown exactly control of nodule numbers, and nodule differentiation in how cytokinin signalling acts on PIN genes and/or PIN pro- M. truncatula (Mortier et al. 2014), suggesting that de novo tein localization during nodulation. In L. japonicus, auxin cytokinin synthesis is required for nodule development. The responses were detected in spontaneously induced nodules MtLOG1 gene is expressed in the dividing cortex cells, which of the lhk1 gain-of-function mutant (snf2) (Suzaki et al. could indicate that cytokinin synthesis is increased after the 2012), further strengthening the idea that cytokinin signalling initial increase in cytokinin perception in the cortex (Mortier is required for downstream auxin accumulation. Whether this et al. 2014). The perception of cytokinin in the root cortex is occurs through alteration of auxin transport or other mecha- crucial for nodule initiation. Legumes defective in cytokinin nisms in determinate nodules is not known. On the other hand, perception are unable to form nodule primordia, as shown in there also is evidence for cytokinin regulation in response to both the L. japonicus hit1-1 (lhk1) (Murray et al. 2007)and changes in auxin sensitivity. In soybean, increasing auxin the M. truncatula cre1 mutants (Gonzalez-Rizzo et al. 2006; sensitivity through overexpression of miRNA160 caused a Plet et al. 2011), although in both legume mutants a low reduction in cytokinin-induced gene expression, suggesting frequency of delayed nodules was observed, suggesting that a feedback loop between auxin and cytokinin signalling other cytokinin receptors are required for nodule initiation. (Turner et al. 2013). This recently was confirmed in L. japonicus with the identifi- Rhizobia also synthesize cytokinin, and cytokinin synthe- cation of additional LHK genes that are expressed in dividing sis by Rhizobium nodulation mutants has been shown to be middle and outer cortical cells and that are required for nodule sufficient to induce cortical cell divisions in alfalfa (M. sativa) initiation (Held et al. 2014). (Cooper and Long 1994). However, a survey of cytokinin Infection thread formation was not abolished in the cytoki- production in four different Rhizobium species showed that nin insensitive mutants, although their progress through the there was no correlation between the types and amounts of epidermis and cortex often appeared to be looped and aborted, cytokinins produced by rhizobia and their ability to induce presumably because of the absence of underlying nodule nodules (Kisiala et al. 2013). In the interaction between primordia (Gonzalez-Rizzo et al. 2006;Murrayetal.2007). Bradyrhizobium sp. strain ORS285, which can form nodules Interestingly, the formation of nodules in the L. japonicus lhk on the legume Aeschynomene in a Nod factor-independent mutants that formed occasionally in a delayed fashion could way, cytokinin production by the bacteria was found to accel- be completely abolished by mutations preventing bacterial erate nodule formation, and to alter nodule number and size, entry (Held et al. 2014). This suggests a model in which but cytokinin deficient mutants were still able to induce 776 J Chem Ecol (2014) 40:770–790 nodules (Podlešáková et al. 2013). This negates the earlier et al. 2004;PenmetsaandCook1997). In M. truncatula, this is hypothesis that purine derivatives, suggested to include cyto- not mediated through an effect of ethylene on AON as dem- kinins, could contribute to nodulation in the absence of Nod onstrated by grafting (Penmetsa et al. 2003; Prayitno et al. factors (Giraud et al. 2007). 2006b). Interestingly, L. japonicus EIN2a mutants showed slightly reduced, not increased, numbers of nodules (Chan et al. 2013), even though external application of ethylene also Ethylene reduces nodule numbers in L. japonicus wild-type plants (Lohar et al. 2009). In addition, nodule numbers were con- The gaseous hormone ethylene plays a role as a negative regu- trolled by both the shoot and the root in LjEIN2a mutants lator of nodulation, and acts on different processes during nodule (Chan et al. 2013). It is possible that this discrepancy is due to formation, including regulation of total nodule numbers, infec- duplication and differentiation of L. japonicus EIN2 genes tion thread formation, nodule morphology, and nodule position- (Chan et al. 2013; Desbrosses and Stougaard 2011). A sepa- ing (Guinel and Geil 2002). Ethylene is produced in the plant in rate study in L. japonicus identified two EIN2 genes in response to rhizobial infection (e.g., Ligero et al. 1986). Its L. japonicus, and their combined silencing led to increased actions likely are due to its effects on plant defense (Penmetsa nodule numbers (Miyata et al. 2013). It also is possible that et al. 2008;Prayitnoetal.2006a), as well as its involvement in again there is a difference in the sensitivity or response of auxin transport (Prayitno et al. 2006b) and interaction with ABA legumes forming determinate and indeterminate nodules, as (Chan et al. 2013), among other developmental signals. As ethylene did not consistently inhibit nodulation in soybean, reviewed previously, application of ethylene reduces nodule which forms determinate nodules like L. japonicus (Schmidt numbers in several legumes, while ethylene synthesis or sensing et al. 1999). inhibitors generally increase nodule numbers (Ferguson and Ethylene may be involved in auxin transport regulation Mathesius 2003). In the last decade, identification of a number during nodule development and this may influence total nod- of ethylene-insensitive mutants in different legumes has provi- ule numbers. The Mtskl mutant showed increased PIN1 and ded genetic evidence for the involvement of ethylene signalling PIN2 expression after inoculation with rhizobia, and auxin in nodulation (Gresshoff et al. 2009). accumulation above the infection site was exaggerated Very early during the nodulation process, ethylene inter- (Prayitno et al. 2006b). In addition, shoot-to-root auxin trans- feres with calcium signalling and negatively regulates infec- port, which has been associated with AON in M. truncatula tion thread formation (Oldroyd et al. 2001). Inhibition of (van Noorden et al. 2006), was found to be insensitive to spontaneous nodulation in the lhk1 gain-of-function mutants rhizobia in the skl mutant, in contrast to wild type plants of L. japonicus by ethylene precursors additionally places (Prayitno et al. 2006b). While it has long been known that ethylene inhibition of nodulation upstream of cytokinin sig- ethylene can act as an auxin transport inhibitor (Morgan and nalling (Tirichine et al. 2006). Ethylene insensitive mutants or Gausman 1966), its mechanism of action remains unclear. transgenic lines, including the M. truncatula sickle (skl)mu- One possibility is that ethylene could act via the induction of tant defective in a gene homologous to Arabidopsis EIN2 flavonoids, which then modulate auxin transport (Buer et al. (Ethylene Insensitive 2) (Penmetsa and Cook 1997; 2006), but this has not directly been tested during nodulation. Penmetsa et al. 2008), transgenic L. japonicus plants express- While ethylene appears to act upstream of auxin transport ing the melon ethylene receptor gene Cm-ERS1 (Nukui et al. regulation, there also is evidence that ethylene action is re- 2004)andL. japonicus transgenic lines expressing a dominant quired for auxin transport changes to take place. For example, etr1-1 gene from Arabidopsis (Lohar et al. 2009)showan NPA or TIBA were unable to cause pseudonodules in the skl increased number of infection threads. As an exception, the mutant (Rightmyer and Long 2011). In the rel3 mutant of L. japonicus enigma1 (LjEIN2a) mutant displays a reduced L. japonicus, which is defective in the formation of trans- number of infection threads (Chan et al. 2013). acting RNAs targeting the auxin response factors ARF3a, The sites of ethylene synthesis in the root are found in cells ARF3b, and ARF4, nodulation was significantly inhibited overlying the phloem poles, thus generating a gradient of (Li et al. 2014). Nodulation in rel3 mutants was restored to ethylene that is thought to restrict nodules radially to positions levels similar to wild type by application of ethylene inhibi- opposite the xylem poles (Heidstra et al. 1997). This position- tors. This suggests that increase ethylene synthesis or percep- ing effect of ethylene has been confirmed in a number of tion in rel3 mutants is a result of altered auxin signalling and/ ethylene insensitive mutants, in which nodule positioning or transport (Li et al. 2014). looses the radial restriction (Chan et al. 2013; Lohar et al. Finally, ethylene also has been observed to alter nodule 2009;PenmetsaandCook1997). morphology and bacteroid numbers in L. japonicus (Lohar Total nodule numbers also are partially controlled by eth- et al. 2009), and while the number of nodule primordia were ylene signalling, as ethylene insensitive mutants form many increased in L. japonicus expressing the CmERS1 gene, the more nodules than wild-type plants (Lohar et al. 2009;Nukui number of mature nodules was similar to wild type plants, J Chem Ecol (2014) 40:770–790 777 suggesting that the effect of ethylene changes at different findings indicate that some characteristics of na mutant plants stages of nodulation (Nukui et al. 2004). could be associated in part with elevated ethylene levels Rhizobia also contribute to the levels of ethylene during produced as a result of a low GA content, but that GAs nodulation. Many rhizobial species encode ACC deaminase, themselves are still required for proper nodule formation which degrades the ethylene precursor 1-aminocyclopropoane- (Ferguson et al. 2011). Moreover, double mutants of na and 1-carboxylic acid (ACC), and overexpression of ACC deami- the autoregulation mutants, nod3 (Psrdn1), sym28 (Psclv2), or nase in rhizobia has been shown to increase nodule numbers, sym29 (Psnark), all exhibit a supernodulation phenotype with increase the competitiveness of rhizobia (e.g., Confonte et al. nodules that are abnormal in their development (Ferguson 2010), and enhance environmental stress tolerance of legumes, et al. 2011). This indicates that GAs likely act independently for example under saline conditions (e.g., Brigido et al. 2013). of the autoregulation of nodulation pathway, and further sup- However, knockout of ACC deaminase has not produced con- ports a requirement for the hormone as an essential factor for sistent results, with some studies showing decreased nodula- proper nodule formation. tion, whereas other studies examining different rhizobial spe- An optimum concentration of GA appears to be required cies did not find any deleterious effect of knocking out ACC for nodulation to be successful. For example, pea mutants deaminase in rhizobia (Murset et al. 2012). having reduced GA levels form fewer nodules, but so do Collectively, these studies show that while ethylene plays a constitutive GA signalling mutants, as well as sln mutant negative role during nodulation in most legumes, there is seedlings, which have an elevated GA content (Ferguson some difference among legume species and among different et al. 2005, 2011). This is consistent with findings that low ethylene insensitive mutants. Thus, it is likely that receptors or levels of bioactive GA applied to wild-type pea increased other regulators of ethylene signalling could have diversified nodule development, whereas higher levels inhibited it in different legumes. (Ferguson et al. 2005). Exogenous GA application, or the constitutive over-expression of the SLEEPY1 gene that acts in GA signalling, both inhibited nodulation in L. japonicus Gibberellins (Maekawa et al. 2009). Likewise, treatment with various GA biosynthesis inhibitors reduced lateral root-based nodulation Gibberellins (gibberellic acids; GA) are involved in a wide in Sesbania rostrata, as did the application of bioactive GA range of biological processes, including cell elongation, seed (Lievens et al. 2005). Down-regulation of the monolignol germination, and flowering. They appear to be required at biosynthetic enzyme, HCT, in alfalfa (Medicago sativa)re- different stages of nodulation, with their content tightly regu- sulted in an elevated GA content and an increase in nodule lated to achieve successful nodule development (reviewed in numbers, surprisingly accompanied by a reduction in root Hayashi et al. 2014). A number of early studies reported growth (Gallego-Giraldo et al. 2014). Collectively, these out- elevated GA levels in nodules compared with root tissue comes suggest a positive role for GAs in nodule organogen- (reviewed in Ferguson and Mathesius 2003). Additional early esis, but that too little, or too much, is inhibitory to the process. investigations focusing on GA application found both positive The optimum level likely varies depending on the species, and negative changes in nodule numbers, depending on the stage of nodule development and growing conditions. species examined, growing conditions, application technique, A number of GA biosynthesis genes have been shown to be and the type and concentration of GA applied (reviewed in up-regulated during legume nodulation. In Sesbania rostrata, Ferguson and Mathesius 2003). the SrGA20ox1 gene, which encodes a key component of the More recently, Ferguson et al. (2005, 2011)demonstrated GA biosynthetic pathway, was significantly up-regulated dur- that GA deficient mutants of pea developed fewer nodules ing lateral root-base nodulation (Lievens et al. 2005). In than wild-type plants. Mutant and grafting studies showed that soybean, recent transcriptome studies identified several up- these reductions were due to the reduced GA level of the roots regulated GA biosynthesis genes during early nodulation, (Ferguson et al. 2005). The few nodules that did form on the including GmGA3ox 1a and also GmGA20ox a, which shares severely GA-deficient na mutant were small, white, and aber- a high sequence similarity to SrGA20ox1 (Hayashi et al. rant in structure. Mutant na plants evolve approximately twice 2012). Transcripts of the GmGA20ox gene were detected only as much ethylene as wild type plants (Ferguson et al. 2011), in soybean roots induced to form nodules, indicating the gene which, as discussed above, is a potent inhibitor of nodulation. is nodulation-specific (Hayashi et al. 2012). Moreover, the Application of the ethylene biosynthesis inhibitor, amino- GA20ox genes of S. rostrata and soybean were both Nod ethoxyvinyl glycine (AVG), partially increased the number factor-dependent in their expression (Hayashi et al. 2012; of nodules formed on these mutants, but was unable to rescue Lievens et al. 2005). The expression of GmGA3ox 1a and their aberrant phenotype (Ferguson et al. 2011). In contrast, GmGA20ox a in soybean peaked at 12 h post inoculation and the application of bioactive GA restored both the number and declined thereafter (Hayashi et al. 2012). This indicates that morphology of na nodules (Ferguson et al. 2005). These the genes may act together to increase GA biosynthesis, and 778 J Chem Ecol (2014) 40:770–790 also that the GA content is tightly regulated during nodulation. expression of the nodulation gene, LjNIN (Nakagawa and GA biosynthesis genes also are reported to be up-regulated in Kawaguchi 2006). In contrast, infection thread formation plants engaging in symbiosis with mycorrhizal fungi (García- and nodulation are both reported to be increased in Garrido et al. 2010;Ortuetal.2012). The regulation of these L. japonicus plants grown under low red/far-red light and genes was reliant on CCaMK (Ortu et al. 2012), a key signal- treated with JA (Suzuki et al. 2011). Moreover, the diminished ling component shared by the nodulation and mycorrhization nodulation phenotype of a phyB mutant of L. japonicus that pathways. has reduced photoassimilate and JA-Ile levels can be restored In addition to being expressed at very early time points, the following the application of JA (Suzuki et al. 2011). GA20ox-encoding genes were also elevated in expression in Genes involved in JA biosynthesis and response were more mature nodules of S. rostrata (Lievens et al. 2005)and regulated in the leaves of soybean plants in a GmNARK- soybean (Hayashi et al. 2012), as was a GA20ox-encoding dependent manner following the induction of nodulation gene identified in L. japonicus (Kouchi et al. 2004). Indeed, (Kinkema and Gresshoff 2008; Seo et al. 2006). Moreover, Lievens et al. (2005) identified two patterns of SrGA20ox1 JA levels were elevated in the leaves of a supernodulating expression in S. rostrata, one related to intercellular infection Gmnark mutant (Seo et al. 2006) and foliar application of an events, and a second associated with the nodule meristem. inhibitor of JA biosynthesis reduced nodulation, specifically Transcripts of an LjGA2ox-encoding gene involved in GA in a Gmnark mutant (Kinkema and Gresshoff 2008). catabolism also were detected in maturing nodules of In support of a positive role for JAs in nodulation, L. japonicus, further indicative of a tight regulation of the jasmonates stimulated the expression of nod genes in Rhizobi- GA content during nodule organogenesis (Kouchi et al. 2004). um leguminosarum (Rosas et al. 1998)andBradyrhizobium The up-regulation of GA biosynthesis genes at different japonicum (Mabood and Smith 2005), and increased Nod stages of nodule development suggests a transient requirement Factor production in B. japonicum (Mabood et al. 2006). Pre- for the hormone during nodulation, occurring both temporally inoculation of B. japonicum (Mabood and Smith 2005)or and spatially. This pattern of expression is consistent with R. leguminosarum bv. phaseoli (Poustini et al. 2005)with findings that GAs affect early infection events, such as root jasmonates also was found to enhance nodulation and nitrogen hair curling and infection thread development, as well as more fixation in soybean and bean plants, respectively. Interestingly, mature stages of nodulation, including nodule primordium JA also is involved in tripartite interactions between legumes, development and the correct establishment of nodulation rhizobia, and herbivores. For example, rhizobial inoculation zones (Ferguson et al. 2011; Lievens et al. 2005;Maekawa altered the composition of volatile organic compounds against et al. 2009). Based on the above mentioned findings, Hayashi herbivores induced by JA (Ballhorn et al. 2013). et al. (2014) proposed a model where GAs are required for Collectively, the findings appear to indicate that JAs can act rhizobia infection, and also for the establishment of the nodule as either positive or negative regulators of nodulation and primordium and the maturation of the nodule structure. nitrogen fixation, depending on the legume species, the type GAs also can be synthesized by rhizobia, although the of JA used, and when, where, and how the hormone is applied. effects on nodulation have not been fully determined yet and Clearly more research is required to understand the precise might depend on environmental factors. Interestingly, the roles that JAs have in legume nodulation. gene encoding enzymes of the ent-kaurene biosynthesis path- way that leads to GA biosynthesis is present only in rhizobia that infect legumes forming determinate nodules, not those Nitric Oxide forming indeterminate nodules. The significance of this find- ing remains to be investigated (Hershey et al. 2014). Nitric oxide (NO) is a gaseous signal molecule having broad roles in various aspects of plant development and stress response. Its roles in nodulation recently have been Jasmonic Acid thoroughly reviewed by Boscari et al. (2013), Meilhoc et al. (2011), and Puppo et al. (2013). Essentially, NO appears to be Jasmonic acid (JA) has well-established roles in plant defense present from early nodulation through to nitrogen-fixation and and wound response. Supplying JA to the growing medium of nodule senescence, thus suggesting it acts at different devel- M. truncatula plants suppressed nodule formation, including opmental stages of the symbiosis. Its production has been their responsiveness to Nod factor, by interfering with Ca reported in infection threads and nodule primordia (del spiking and the expression of MtRIP1 and MtENOD11 (Sun Guidice et al. 2011), as well as in mature, nitrogen-fixing et al. 2006). Similarly, application of methyl jasmonate nodules of M. truncatula, M. sativa, G. max, and L. japonicus (MeJA) to the shoots of L. japonicus plants suppressed nodule (Baudouin et al. 2006; Pii et al. 2007; Sánchez et al. 2010; development in both wild-type and hypernodulation mutant Shimoda et al. 2009), with both symbiotic partners capable of (Ljhar1) plants, inhibiting infection thread formation and the synthesizing the signal (Horchani et al. 2011; Sánchez et al. J Chem Ecol (2014) 40:770–790 779

2010). It is a strong inhibitor of nitrogenase activity and contrast, SA levels did not increase in roots of wild-type plants nitrogen fixation (e.g., Baudouin et al. 2006;Katoetal. inoculated with a compatible rhizobia strain (Blilou et al. 1999; 2010; Shimoda et al. 2009), and can inhibit rhizobia growth Martínez-Abarca et al. 1998). Collectively, these findings sug- (Meilhoc et al. 2010). gest a negative role for SA in nodulation, and the possible need Nitric Oxide also may play a positive role in nodulation, as for compatible rhizobia and/or their Nod factor signal to sup- its depletion delayed nodule development and reduced nodule press an SA-dependent defence mechanism to enable the entry numbers (del Guidice et al. 2011). It also is reported to play a and successful establishment of the bacteria in the host plant. beneficial role in nodule energy metabolism and in the regu- lation of nitrogen metabolism in root nodules (Horchani et al. 2011;Meloetal.2011). Additional evidence suggests that NO Strigolactones might act as a developmental signal to trigger the induction of nodule senescence (reviewed in Boscari et al. 2013; Meilhoc Strigolactones (SL) have diverse roles in regulating auxin et al. 2011; Puppo et al. 2013). Thus, NO may have multiple transport, stimulating parasitic weed germination, coordinat- signalling roles in legume nodulation, acting as either a pos- ing mycorrhizal symbiosis, and defining shoot and root itive or negative regulator of a broad range of processes architecture. They were first reported to have a role in associated with nodule development and functioning. regulating nodulation by Soto et al. (2010), where application of the synthetic strigolactone analogue, GR24, positively af- fected alfalfa (Medicago sativa) nodulation. Silencing of the Salicylic Acid SL biosynthesis gene in L. japonicus, LjCCD7,resultedin slightly fewer nodules formed, with no alterations observed in Salicylic acid (SA) is involved in systemic acquired resistance nodule development and morphology (Liu et al. 2013). Inter- and plant defense responses to pathogens. Its role in legume estingly, the SL biosynthesis mutant of pea, rms1 (Psccd8), nodulation is not well defined, with some conflicting results produced fewer nodules than its wild type, but the SL re- reported. As with many of the factors reviewed here, the sponse mutant, rms4 (Psmax2), did not (Foo and Davies experimental outcomes likely vary considerably depending 2011; Foo et al. 2013). Double mutant plants of rms1 and on the concentration and frequency of SA applied, as well as the autoregulation mutants, nod3 (Psrdn1), sym28 (Psclv2), or the legume species, application method, and growing condi- sym29 (Psnark), all exhibit a supernodulation phenotype, tions used. indicating that the hormone does not act downstream of these Exogenous application of SA is reported to reduce nodu- genes in nodulation control, and instead likely influences the lation in the indeterminate nodule-forming Vicia sativa, promotion of nodulation (Foo et al. 2014). M. sativa, T. repens and P. sativum, but not in the determinate Establishing a direct effect of SL on nodulation is compli- nodule-forming L. japonicus, P. vulgaris,andGlycine soja cated due to the hormone’s role in regulating other critical (van Spronsen et al. 2003). However, additional studies inves- processes, such as auxin transport and shoot and root devel- tigating the determinate nodule-forming soybean have report- opment, which can considerably influence resource allocation. ed an inhibitory effect of SA treatment on nodulation (Lian Moreover, SLs influence the development of lateral roots and et al. 2000;Satoetal.2002). This inhibition was found to be root hairs (Kapulnik et al. 2011), which are essential structures less pronounced in supernodulating soybean mutants than in for rhizobia infection. Thus, more research is required to better wild-type plants (Sato et al. 2002). Additional work ectopi- understand the role of SLs in nodulation, including establish- cally over-expressing the bacterial SA hydroxylase gene, ing any direct effects they may have on factors specific to NahG, elegantly demonstrated that reduced endogenous SA nodule development. Interestingly, the GRAS-type transcrip- levels correlated with increased rhizobia infections and nodule tion factors, NODULATION SIGNALING PATHWAY1 numbers in both the indeterminate nodule-forming (NSP1) and NSP2, which are critical for Nod-factor induced M. truncatula and the determinate nodule-forming nodulation, also were found to be necessary for SL biosyn- L. japonicus (Stacey et al. 2006). As well as inhibiting nodu- thesis in Medicago truncatula and non-nodulating rice (Liu lation, SA can suppress rhizobia growth (Stacey et al. 2006), et al. 2011). The authors suggest that these transcription which could have considerable impact on studies investigating factors fulfil a dual regulatory function to regulate down- the effects of SA application on nodulation. stream targets of both nodulation and SL biosynthesis. Inoculation of M. sativa (Martínez-Abarca et al. 1998)or pea (Blilou et al. 1999) with incompatible or Nod factor- deficient mutant strains of rhizobia resulted in an accumulation Signaling Peptides of SA in the roots. Moreover, inoculation of the non- nodulation pea mutant, Pssym30, with a compatible rhizobia Moreandmorepeptideshavebeenidentifiedthathavecritical strain increased the root SA content (Blilou et al. 1999). In roles in legume nodulation. The number is likely to rise further 780 J Chem Ecol (2014) 40:770–790 as peptides frequently are found to act as ligands for receptors (Penterman et al. 2014). The ecological and evolutionary to trigger aspects of plant growth and development, similar to consequences of this host control mechanism over its bacterial the action of classical phytohormones. Peptides also interact symbiont remain an interesting question for the future. with phytohormone signalling pathways to alter development. The following peptides have been identified as having key roles in nodulation. CLE Peptides

A number of CLAVATA/ESR-related (CLE) peptide hormones NCR Peptides recently were identified as negative regulators of nodulation, including LjCLE-RS1 and 2 in L. japonicus (Okamoto et al. A fascinating class of peptides, called nodule-specific cysteine- 2009), MtCLE12 and 13 in M. truncatula (Mortier et al. 2010; rich (NCR) peptides, mediate the terminal, intracellular differ- Saur et al. 2011),andGmRIC1and2insoybean(Limetal. entiation of bacteroids in legumes of the inverted repeat–lack- 2011;Reidetal.2011a)andPvRIC1and2inP. vulgaris ing clade (IRLC), such as M. truncatula, M. sativa, P. sativum, (Ferguson et al. 2014). These peptides typically are 12–13 Astragalus sinicus, T. repens,andVicia faba (reviewed in amino acids in length and are located at or near the C-terminus Kondorosi et al. 2013). Over 600 NCR-encoding genes are of their prepropeptide. They act systemically in the autoregula- predicted to be scattered across the M. truncatula genome. tion of nodulation pathway to control nodule numbers (reviewed Most are composed of two exons, one encoding a conserved in Reid et al. 2011b). The genes encoding these peptides are all signal peptide domain, and the second encoding the mature induced in the root following inoculation with compatible peptide, which is typically 30–50 amino acids in length and rhizobia species. Over-expression of these genes inhibits, and contains either 4 or 6 cysteines residues at highly conserved can even completely abolish, nodule organogenesis. positions (Alunni et al. 2007;Fedorovaetal.2002;Kondorosi The rhizobia-induced CLE peptides, or prepropeptides, are all et al. 2013; Mergaert et al. 2003). predicted to be transported in the xylem to the shoot where they NCR peptides can control the differentiation of are perceived by a leucine-rich repeat receptor kinase, called Sinorhizobium meliloti bacteroids, the compatible LjHAR1/MtSUNN/GmNARK, possibly in a complex with oth- microsymbiont of M. truncatula (Van de Velde et al. 2010; er receptors, such as CLAVATA2 and KLAVIER (Krusell et al. Wang et al. 2010). They appear to do so by manipulating its 2011; Miyazawa et al. 2010). Indeed, recent findings by cell cycle, possibly indicating that the rhizobia and/or bacteroids Okamoto et al. (2013) elegantly demonstrated that LjCLE-RS2 have receptors to perceive the plant-derived factors. They are is a post-translationally arabinosylated glycopeptide that can be targeted to the bacteria and enter the bacterial membrane and detected in xylem sap and directly bind to LjHAR1. Perception cytosol. A subunit of a signal peptidase complex (SPC) of of these peptides leads to the production of a new signal, called M. truncatula, called MtDNF1, is highly expressed in nodules the shoot-derived inhibitor (SDI), which is transported back and is essential for removing the signal peptide from the proteins down to the root to suppress further nodulation events (Lin (Van de Velde et al. 2010;Wangetal.2010). This occurs in the et al. 2010, 2011;Fig.2). Other factors acting after the perception endoplasmic reticulum, and is required for correct targeting of of the nodulation-suppressive CLE peptides includes a ubiquitin the mature peptides to the bacteria. Mtdnf1 mutants were found fusion degradation protein that is significantly up-regulated in to contain unprocessed NCR peptides that remained in the expression, called GmUFD1a (Reid et al. 2012). endoplasmic reticulum and failed to reach the bacteria, resulting Key domains and amino acid residues required for the nodule in undifferentiated bacteroids unable to fix nitrogen (Van de suppressive activity of the soybean CLEs were recently identified Velde et al. 2010;Wangetal.2010). Some NCR peptides also using domain-swap and site-directed mutagenesis techniques, partially mimicked S. meliloti bacteroid differentiation in respectively (Reid et al. 2013). Additional studies in M. truncatula L. japonicus, a non-IRLC legume (Van de Velde et al. 2010). revealed that MtCLE13 expression depends on cytokinin, which, NCR peptides are similar to defensin-type antimicrobial as outlined above, is essential for nodule development and indi- peptides (AMPs), and some exhibit strong antimicrobial ac- cates that the autoregulation of nodulation may be induced tivity against S. meliloti and other bacteria species, possibly by concomitant to nodule primordia formation (Mortier et al. 2012). increasing their cell permeabilization (Haag et al. 2011, 2012; Soybean has an additional nodulation-suppressive CLE Tiricz et al. 2013; Van de Velde et al. 2010). Sub-lethal levels peptide, called GmNIC1, which is highly similar in sequence of an NCR peptide recently were shown to block cell division to GmRIC1 and 2 (Reid et al. 2011a). However, GmNIC1 is and antagonize Z-ring function, partially by altering the ex- induced by nitrate, not rhizobia, and acts locally through pression of master cell-cycle regulators and genes critical to GmNARK in the root, not systemically via the shoot. Thus, cell division (Penterman et al. 2014). Thus, NCR peptides GmNIC1 appears to have a role in nitrate-regulation of nod- may target regulatory pathways of S. meliloti to cause ulation (reviewed in Reid et al. 2011b). Recent findings using endoreduplication and differentiation during symbiosis soybean also have shown that acidic growing conditions can J Chem Ecol (2014) 40:770–790 781 inhibit nodulation systemically through GmNARK in the dividing nodule cells, possibly as a means of generating a shoot, possibly suggesting that an acid-induced CLE pep- carbon sink, as both Peptides A and B have been shown to tide(s) exists that acts similarly to the nodulation-suppressive bind with sucrose synthase (Röhrig et al. 2002). The binding CLE peptides to inhibit nodule organogenesis (Ferguson et al. of Peptide A enhanced the breakdown of sucrose, which could 2013; Lin et al. 2012). increase the carbon supply to cells. Due to its wide distribution across both legume and non-legume species, its expression in plant organs other than nodules, and its role in other processes ENOD40 such as mycorrhization (Staehelin et al. 2001), nematode infec- tion (Favery et al. 2002), and lateral root formation (Mathesius ENOD40 is an enigmatic signal. The gene encoding it is well et al. 2000), ENOD40 likely functions broadly in plant develop- conserved across the plant kingdom, including non-legumes, ment, with general roles in organ and tissue formation. and it has roles in plant development aside from nodulation. Originally identified as an early nodulin gene (hence the name ENOD), ENOD40 lacks a long open reading frame (Crespi Ralf et al. 1994;Yangetal.1993); however, it does encode two short peptides. Peptide A is 12–13 amino acids in length, and Rapid Alkalinisation Factor (RALF) peptides originate from Peptide B, which partially overlaps with the open reading members of a highly conserved gene family found through- frame of Peptide A, is roughly 24–27 amino acids long out the plant kingdom (Bedinger et al. 2010). They are (Röhrig et al. 2002; Sousa et al. 2001). Unlike the other expressed in an array of tissues, including shoots, leaves, peptides described here, ENOD40 peptides do not appear to flowers, roots, and nodules. Most are processed from the C- result from proteolytic cleavage of a larger protein product, terminus of their prepropeptide to produce a mature peptide and instead are produced directly from their mRNA. Interest- roughly 50 amino acids in length (Bedinger et al. 2010). ingly, the role of the peptides may be to stabilize and form a Application of synthetic RALF arrested root growth in complex with the ENOD40 mRNA transcript, which exhibits Arabidopsis and tomato plants (Pearce et al. 2001). Recent a conserved secondary structure and may function as an un- evidence supports a common role for RALF peptides in translated signal molecule (Crespi et al. 1994; Sousa et al. regulating cell expansion, regardless of divergence in their 2001). Indeed, in M. truncatula, ENOD40 mRNA induced the tissue specificity and gene expression patterns (Morato do cytoplasmic localization of a nuclear RNA binding protein, Canto et al. 2014). called MtRBP1, and interacted with the RNA-binding peptides, In M. truncatula, the RALF-encoding gene, MtRALF1,is MtSNARP1 and MtSNARP2, that appear to sustain bacteroids up-regulated following Nod factor application (Combier et al. during symbiosis (Campalans et al. 2004; Laporte et al. 2010). 2008). Over-expression of MtRALF1 increased the number of Following rhizobia inoculation, ENOD40 is expressed in infection threads that aborted, leading to a reduction in the the root pericycle and in dividing cells of the nodule primor- number of nodules that formed (Combier et al. 2008). More- dium (e.g., Crespi et al. 1994; Mathesius et al. 2000; Yang over, the nodules that did form were small and poorly colo- et al. 1993). It also can be triggered by factors such as Nod nized by their symbiotic partner (Combier et al. 2008). These factors (Fang and Hirsch 1998; Minami et al. 1996)and findings suggest a role for the peptide in both rhizobia infec- cytokinin (Fang and Hirsch 1998; Mathesius et al. 2000), tion and nodule organogenesis, possibly as a negative regula- consistent with it having a role in cell division and nodule tor, akin to the reported function of RALF peptides in root primordium formation. Likewise, over-expression of development. ENOD40 can induce cortical cell divisions and promote nod- ule primordium formation, whereas down-regulation of the gene can hinder nodule and bacteroid development (Charon CEP et al. 1997, 1999;Crespietal.1994; Wan et al. 2007). In mature nodules, ENOD40 is expressed in nodule vascular Members of the CEP (C-terminally encoded peptide) signal- bundles, uninfected cells of the central tissues and the infec- ing peptide family are widespread among flowering plants, tion zone of indeterminate nodules (e.g., Crespi et al. 1994; but are absent from some primitive species (Roberts et al. Wan et al. 2007;Yangetal.1993). Interestingly, many legume 2013). The mature peptides are around 15 amino acids in species have more than one copy of ENOD40, which can length, and are processed from their full-length protein at or exhibit slightly different patterns of expression during nodu- near the C-terminus, as their name implies (Delay et al. 2013; lation (e.g., Fang and Hirsch 1998; Wan et al. 2007). Ohyama et al. 2008). CEP peptides appear to have distinct ENOD40 likely acts along with other factors to activate the roles in orchestrating root and shoot growth. They are regu- cell cycle and induce the formation of nodule primordia. It lated by environmental cues and typically act as negative also may have a role in phloem unloading in mature and regulators of plant development (Delay et al. 2013). 782 J Chem Ecol (2014) 40:770–790

Table 1 Overview of major nodulation processes affected by phytohormones and interacting peptides - denotes a negative effect on the process, + a positive effect Infection thread ABA – formation 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)

Application of the MtCEP1 peptide, or over-expression of the DVL1/ROT4 gene encoding it, results in an inhibition of lateral roots, an enhancement of nodulation, and the induction of periodic Devil/rotundifolia (ROT)-Four-Like (DVL/RTFL) peptides circumferential root swellings in M. truncatula plants that are approximately 30 amino acids in length and, like CLE, show increased auxin response (Imin et al. 2013). The in- CEP, and RALF peptides, are derived from the C-terminus of crease in nodulation is partially tolerant to high levels of their full length proteins (Valdivia et al. 2013). The function of nitrate that normally suppress nodule development (Imin many DVL/RTFL peptides is largely thought to be redundant; et al. 2013). Collectively, these findings appear to indicate however, they likely exert specialized control in development that MtCEP1 differentially modulates lateral root and nodule via divergent expression patterns (Wen et al. 2004). Determin- development in M. truncatula. ing the function of DVL/RTFL peptides has been hindered J Chem Ecol (2014) 40:770–790 783

Table 1 (continued) Nodule ABA + differentiation and Auxin (synthesized by rhizobia ) + growth BR + Cytokinin + GA + (correct ‘level and window’ required) NO + RALF - Nodule number Auxin + control via AON JA + CLE peptides + Cytokinin (induces CLE) +

Nitrogen fixation ABA – N2 JA + NO - NH3

Bacteroid NCR + (indeterminate nodules) differentiation

Nodule senescence NO +

largely by the absence of clear phenotypes in loss-of-function increased the number of infection threads that aborted, mutants and silencing lines (Valdivia et al. 2013; Wen et al. resulting in a diminished number of nodules that formed 2004). However, over-expression studies indicate that they (Combier et al. 2008). However, unlike what was observed may control aspects of growth and development by regulating with MtRALF1, over-expression of MtDVL1 did not impair cell proliferation (e.g., Wen et al. 2004). the development of the nodules that did form, or their ability to In M. truncatula roots, a DVL1/ROT4 peptide-encoding fix nitrogen. This led Combier et al. (2008)toproposearole gene, called MtDVL1, was transiently up-regulated following for the peptide in infection thread formation and progression, treatment with Nod factor (Combier et al. 2008). As was possibly via regulating the differentiation of pre-infection observed with MtRALF1, over-expression of the gene threads. 784 J Chem Ecol (2014) 40:770–790

Conclusion and Future Prospectives legumes, Navia-Gine et al. 2009 and Zhang et al. 2009). Future studies also are necessary to study, in a common Molecular studies and the identification of hormone and other system, the parallel changes in stress hormone related changes signalling mutants in legumes have started to elucidate many in response to a range and combination of symbiotic and of the signalling interactions that control nodule development pathogenic organisms to identify overlapping response path- and rhizobial infection at different stages during the interac- ways (De Vos et al. 2005). While rhizobia are symbionts, they tion (Table 1). At the current time, it is difficult to construct a also carry common microbe-associated molecular patterns detailed model of hormone interactions during nodulation (MAMPs) and trigger some early plant defense responses, because studies have been done in different legume species although their MAMPs may have been modified to down- with different methods and under different conditions. To regulate plant defense responses during successful interac- unify this area, we first need controlled studies in model tions (Zamioudis and Pieterse 2012). It would be interesting legumes that systematically study interactions between hor- to investigate to what extent plant pathogens and rhizobia mones utilizing existing hormone mutants. This should in- trigger overlapping hormone responses in the root, and how clude accurate localized measurements of hormone concen- rhizobia have evolved to modulate these response pathways trations during nodulation in time and space, and across a thus avoiding plant defenses. For example, the master activa- range of legumes in order to resolve the sometimes conflicting tor of SA-response genes required for plant immunity, NPR1, findings on nodulation responses to external hormone appli- also functions in the infection of roots by rhizobia (Peleg- cation. Since hormones often carry location-specific informa- Grossman et al. 2009). Similarly, a central regulator of ethyl- tion, their sites of action will be crucial to integrate into current ene signalling, EIN2, is targeted by both pathogens and genetic models of hormones action. It also will be important to rhizobia in legumes (Penmetsa et al. 2008). In addition to clarify how signalling peptides interact with the classical the traditional stress hormones, auxin signalling has emerged phytohormone pathways. Second, it will be interesting to as a common target in both symbiont and pathogen interac- extend our findings from model legumes to the diverse range tions, and this has reshaped our thinking of auxin as a devel- of legumes that display different types of nodules. Third, our opmental signal. Instead, it may play roles in both develop- knowledge of the contribution of phytohormones produced by ment and defense, although its role in defense regulation rhizobia remains sketchy, and is partially limited by the avail- during nodulation remains unstudied. So far, little is known ability of rhizobial hormone mutants. Overall, rhizobially- about the hormonal cross-talk that fine-tunes nodulation in contributed phytohormones appear to give rhizobia additional legumes, but the increasing identification of legume hormone benefits for successful symbiotic establishment, especially mutants could be further utilized to help address this question. under stressful environmental conditions, but they do not Finally, an unsolved question remains whether legumes appear to be essential for the symbiosis. However, it is unclear have a predisposition for nodule development because of whether the production of phytohormones by rhizobia has altered developmental responses to phytohormones, as indi- effects not only on nodule number and functioning, but on cated, for example, by the different responses of legumes and overall fitness of the host. non-legumes to ABA (Liang and Harris 2005). It would be Very little attention has been paid to extending many of the interesting to investigate whether legumes have evolved spe- studies involving bacterial or plant hormone mutants to the cific hormone receptors specifically for controlling nodula- field and to investigating effects on tripartite interactions with tion. Comparisons of hormone responses between nodulating other organisms or the abiotic environment. While many and non-nodulating legumes, and between legumes and non- hormone studies have been carried out under controlled con- legumes could inform future studies attempting to extend ditions, plants living in the real world are colonized by many nodulation to non-legumes. In that context, it is perhaps different organisms, while at the same time adjusting to fluc- noteworthy that a whole genome duplication occurred in the tuating abiotic conditions. In general, it appears that stress- Papillionoid legume subfamily at about the time of the evolu- related hormones such as ABA, JA, ethylene, and SA, have tion of nodulation in legumes, and this may have enabled negative impacts on nodulation, and this may well reflect the legumes to evolve hormonal response pathways specifically need of the plant to limit nodulation under stressful situations. for nodulation (Op den Camp et al. 2011). In addition, these hormone response pathways are up- regulated in response to pathogen and herbivore attack, and Acknowledgments Due to the large size of this research field, a num- this may influence the level of nodulation. Since most studies ber of publications were undoubtedly overlooked. We thank Peter on pathogen and insect responses have been carried out on Gresshoff for careful reading of the manuscript. Financial support was aboveground organs and in non-legumes, it would be inter- provided to BJF by the Australian Research Council Discovery Project esting to test whether infection by shoot pathogens has a grants (DP130103084 and DP130102266) as well as University of Queensland strategic funds. UM was supported by a Future Fellowship systemic effect on nodulation in the roots of legumes (e.g., (FT100100669) and a Discovery Project grant (DP120102970) from the for belowground organs, Rasmann et al. 2005,andfor Australian Research Council. J Chem Ecol (2014) 40:770–790 785

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