Enhanced Nodulation and Nitrogen Fixation in the Abscisic Acid Low-Sensitive Mutant enhanced nitrogen fixation1 of Lotus japonicus

The phytohormone abscisic acid (ABA) is known to be a negative regulator of legume root nodule formation. By screening Lotus japonicus seedlings for survival on an agar medium containing 70 μm ABA, we obtained mutants that not only showed increased root nodule number but also enhanced nitrogen fixation. The mutant was designated enhanced nitrogen fixation1 (enf1) and was confirmed to be monogenic and incompletely dominant. The low sensitivity to ABA phenotype was thought to result from either a decrease in the concentration of the plant''s endogenous ABA or from a disruption in ABA signaling. We determined that the endogenous ABA concentration of enf1 was lower than that of wild-type seedlings, and furthermore, when wild-type plants were treated with abamine, a specific inhibitor of 9-cis-epoxycarotenoid dioxygenase, which results in reduced ABA content, the nitrogen fixation activity of abamine-treated plants was elevated to the same levels as enf1. We also determined that production of nitric oxide in enf1 nodules was decreased. We conclude that endogenous ABA concentration not only regulates nodulation but also nitrogen fixation activity by decreasing nitric oxide production in nodules.Many legumes establish nitrogen-fixing root nodules following reciprocal signal exchange between the plant and rhizobia (Hayashi et al., 2000; Hirsch et al., 2003). The host plant produces chemical compounds, frequently flavonoids, which induce rhizobial nod genes, whose products are involved in the synthesis and secretion of Nod factor. Perception of this chitolipooligosaccharide by the host plant results in the triggering of a signal transduction cascade that leads to root hair deformation and curling and subsequent cortical cell divisions, which establish the nodule primordium. The rhizobia enter the curled root hair cell and nodule primordial cells through an infection thread. Eventually, the rhizobia are released into nodule cells, enclosed within a membrane, and differentiate into nitrogen-fixing bacteroids that reduce atmospheric nitrogen into ammonia. In return, the host plant supplies photosynthetic products, to be used as carbon sources, to the rhizobia (Zuanazzi et al., 1998; Hayashi et al., 2000).The host plant is known to be important for regulating the number of nodules established on its roots. For example, hypernodulating mutants such as nitrate-tolerant symbiotic1 (nts1; Glycine max), hypernodulation aberrant root formation1 (har1; Lotus japonicus), super numeric nodules (sunn; Medicago truncatula), and symbiosis29 (sym29; Pisum sativum) disrupt the balance between supply and demand by developing excessive root nodules (Oka-Kira and Kawaguchi, 2006). Grafting experiments demonstrated that leaf tissue is a principal source of the systemic signals contributing to the autoregulation of nodulation (Pierce and Bauer, 1983; Kosslak and Bohlool, 1984; Krusell et al., 2002; Nishimura et al., 2002b; van Brussel et al., 2002; Searle et al., 2003; Schnabel et al., 2005). The Nts1, Har1, Sunn, and Sym29 genes encode a receptor-like kinase similar to CLAVATA1, which regulates meristem cell number and differentiation (Krusell et al., 2002; Nishimura et al., 2002a; Searle et al., 2003; Schnabel et al., 2005).Phytohormones are also known to regulate nodulation (Hirsch and Fang, 1994). For example, ethylene is a well-known negative regulator of nodulation, influencing the earliest stages from the perception of Nod factor to the growth of infection threads (Nukui et al., 2000; Oldroyd et al., 2001; Ma et al., 2003). The ethylene-insensitive mutant sickle1 (skl1) of M. truncatula has a hypernodulating phenotype (Penmetsa and Cook, 1997). Skl1 is homologous to Ethylene insensitive2 of Arabidopsis (Arabidopsis thaliana), which is part of the ethylene-signaling pathway (Alonso et al., 1999; Penmetsa et al., 2008). In contrast, cytokinin is a positive regulator of nodulation. The cytokinin-insensitive mutant hyperinfected1 (loss of function) of L. japonicus and the spontaneous nodule formation2 (gain of function) mutants of M. truncatula provide genetic evidence demonstrating that cytokinin plays a critical role in the activation of nodule primordia (Gonzalez-Rizzo et al., 2006; Murray et al., 2007; Tirichine et al., 2007).Abscisic acid (ABA), added at concentrations that do not affect plant growth, also negatively regulates nodulation in some legumes (Phillips, 1971; Cho and Harper, 1993; Bano et al., 2002; Bano and Harper, 2002; Suzuki et al., 2004; Nakatsukasa-Akune et al., 2005; Liang et al., 2007). Recently, M. truncatula overexpressing abscisic acid insensitive1-1, a gene that encodes a mutated protein phosphatase of the type IIC class derived from Arabidopsis and that suppresses the ABA-signaling pathway (Leung et al., 1994; Hagenbeek et al., 2000; Gampala et al., 2001; Wu et al., 2003), was shown to exhibit ABA insensitivity as well as a hypernodulating phenotype (Ding et al., 2008).In this study, we isolated a L. japonicus (Miyakojima MG20) mutant that showed an increased root nodule phenotype and proceeded to carry out its characterization. This mutant, named enhanced nitrogen fixation1 (enf1), exhibits enhanced symbiotic nitrogen fixation activity. Most legume nitrogen fixation activity mutants, such as ineffective greenish nodules1 (ign1), stationary endosymbiont nodule1, and symbiotic sulfate transporter1 (sst1), are Fix⁻ (Suganuma et al., 2003; Krusell et al., 2005; Kumagai et al., 2007).     

Medicago truncatula Natural Resistance-Associated Macrophage Protein1 Is Required for Iron Uptake by Rhizobia-Infected Nodule Cells

Iron is critical for symbiotic nitrogen fixation (SNF) as a key component of multiple ferroproteins involved in this biological process. In the model legume Medicago truncatula, iron is delivered by the vasculature to the infection/maturation zone (zone II) of the nodule, where it is released to the apoplast. From there, plasma membrane iron transporters move it into rhizobia-containing cells, where iron is used as the cofactor of multiple plant and rhizobial proteins (e.g. plant leghemoglobin and bacterial nitrogenase). MtNramp1 (Medtr3g088460) is the M. truncatula Natural Resistance-Associated Macrophage Protein family member, with the highest expression levels in roots and nodules. Immunolocalization studies indicate that MtNramp1 is mainly targeted to the plasma membrane. A loss-of-function nramp1 mutant exhibited reduced growth compared with the wild type under symbiotic conditions, but not when fertilized with mineral nitrogen. Nitrogenase activity was low in the mutant, whereas exogenous iron and expression of wild-type MtNramp1 in mutant nodules increased nitrogen fixation to normal levels. These data are consistent with a model in which MtNramp1 is the main transporter responsible for apoplastic iron uptake by rhizobia-infected cells in zone II.SNF is carried out by the endosymbiosis between legumes and diazotrophic bacteria called rhizobia (van Rhijn and Vanderleyden, 1995). Detection of rhizobial nodulation (Nod) factors by the legume plant results in curling of a root hair around the rhizobia and development of an infection thread that will deliver the rhizobia to the developing root nodule primordium, which is also triggered by Nod factors (Kondorosi et al., 1984; Brewin, 1991; Oldroyd, 2013). Rhizobia are eventually released into the cytoplasm of host plant cells via endocytosis, resulting in an organelle-like structure known as the symbiosome, which consists of bacteria surrounded by a plant membrane called the symbiosome membrane (SM; Roth and Stacey, 1989; Vasse et al., 1990). Rhizobia within symbiosomes eventually differentiate into nitrogen-fixing bacteroids that produce and export ammonium to the plant for assimilation (Vasse et al., 1990).Two main developmental programs for nodulation have been described (Sprent, 2007). In the determinate type, e.g. in soybean (Glycine max), the nodule meristem is active only transiently, which gives rise to a spherical nodule. In the indeterminate nodules, e.g. in alfalfa (Medicago sativa) and pea (Pisum sativum), the meristem(s) remain active for much longer, resulting in cylindrical and/or branched nodules of indeterminate morphology. Indeterminate nodules can be divided in spatiotemporal zones that facilitate the study of the nodulation process. At least four zones are observed in a mature indeterminate nodule (Vasse et al., 1990). Zone I is the meristematic region that drives nodule growth. In zone II, rhizobia are released from the infection thread and differentiate into bacteroids. Zone III is the site of nitrogen fixation. Finally, Zone IV is the senescence zone, where bacteroids are degraded and nutrients are recycled. Some authors describe two more zones: the interzone, a transition zone between zones II and III (Vasse et al., 1990; Roux et al., 2014), and zone V, where saprophytic rhizobia live on the nutrients released by senescent cells (Timmers et al., 2000).Nodulation and nitrogen fixation are tightly regulated processes (for review, see Oldroyd, 2013; Udvardi and Poole, 2013; Downie, 2014) and require a relatively large supply of nutrients from the host: photosynthates, macronutrients such as phosphate and sulfate, amino acids, at least prior to nitrogen fixation, and metal micronutrients (Udvardi and Poole, 2013). Among the latter, iron is one of the most critical (Brear et al., 2013; González-Guerrero et al., 2014). The activity of some of the most abundant and important enzymes in SNF directly depends on iron as cofactor. Nitrogenase, the enzyme directly responsible for nitrogen fixation, needs iron-sulfur clusters and an iron-molybdenum cofactor to reduce N₂ (Miller et al., 1993). The hemoprotein leghemoglobin, which controls O₂ levels in the nodule (Ott et al., 2005), represents around 20% of total nodule protein (Appleby, 1984). Similarly, different types of superoxide dismutase, including an Fe-superoxide dismutase, control the free radicals produced during SNF (Rubio et al., 2007). Other ferroproteins are involved in energy transduction and recycling related to the nitrogen fixation process (Ruiz-Argüeso et al., 1979; Preisig et al., 1996).Despite its importance, iron is a growth-limiting nutrient for plants in most soils (Grotz and Guerinot, 2006), especially in alkaline soils. As a result, iron deficiency is prevalent in plants and hampers crop production and human health (Grotz and Guerinot, 2006; Mayer et al., 2008). This is even more so when legumes are nodulated (Terry et al., 1991; Tang et al., 1992). The relatively high iron demand of nodules can trigger the iron deficiency response, i.e. increase in iron reductase activities in the root epidermis and acidification of the surrounding soil (Terry et al., 1991; Andaluz et al., 2009). Consequently, knowing how iron homeostasis is maintained in nodulated legumes, including how this micronutrient is delivered to the nodule, is important for understanding and improving SNF.Taking advantage of state-of-the-art metal visualization methods, the pathway for iron delivery to the nodule has been elucidated (Rodríguez-Haas et al., 2013). Synchrotron-based x-ray fluorescence studies on Medicago truncatula indeterminate nodules indicate that most of the iron is delivered by the vasculature to the apoplast of zone II. In zone III, iron is mostly localized within bacteroids. Therefore, a number of transporters must exist that move iron through the plasma membrane of plant cells and the SM of infected cells. Several transporters have been hypothesized to mediate iron transport through the SM. Soybean Divalent Metal Transporter1 (GmDMT1) is a nodule-induced Natural Resistance-Associated Macrophage Protein (Nramp) that was found in the soybean SM using specific antibodies (Kaiser et al., 2003). However, biochemical studies on Nramp transporters suggest that they transport substrates into the cytosol (Nevo and Nelson, 2006), rather than outwards or into symbiosomes. More recently, the study of stationary endosymbiont nodule1 (sen1) mutants in Lotus japonicus indicated that SEN1, a yeast (Saccharomyces cerevisiae) Cross Complements CSG1/Arabidopsis (Arabidopsis thaliana) Vacuolar Iron Transporter1 homolog, could play a role in delivering iron across the SM (Hakoyama et al., 2012), albeit this is merely based on the mutant plant phenotype and the role of members of this family in other organisms.Very little is known about the molecular identity of transporters that mediate iron uptake from the nodule apoplast. Based on known plant metal transporters and their biochemistry, the most likely candidates are members of the Nramp and Zinc-Regulated Transporter1, Iron-Regulated Transporter1-Like Protein (ZIP) families, because these can transport divalent metals into the cytosol (Vert et al., 2002; Nevo and Nelson, 2006). Moreover, given that the expression of at least one Nramp transporter (GmDMT1) is activated by nodulation (Kaiser et al., 2003), it is possible that members of this family might mediate iron uptake into rhizobia-containing cells. Nramp transporters are ubiquitous divalent transition metal importers (Nevo and Nelson, 2006). Phenotypical and electrophysiological studies indicate that they have a wide range of possible biological (Fe²⁺, Mn²⁺, Zn²⁺, Cu²⁺, Co²⁺, and Ni²⁺) and nonbiological (Pb²⁺ and Cd²⁺) substrates (Belouchi et al., 1997; Curie et al., 2000; Thomine et al., 2000; Mizuno et al., 2005; Rosakis and Köster, 2005; Cailliatte et al., 2009). In plants, Nramp transporters have been associated with a number of biological roles, such as Fe²⁺ and Mn²⁺ uptake from soil (Curie et al., 2000; Cailliatte et al., 2010), Mn²⁺ long-distance trafficking (Yamaji et al., 2013), metal remobilization during germination (Lanquar et al., 2005), Cd²⁺ and Ni²⁺ tolerance (Mizuno et al., 2005; Cailliatte et al., 2009), and the immune response (Segond et al., 2009), in addition to participating in SNF (Kaiser et al., 2003).In this study, M. truncatula MtNramp1 (Medtr3g088460) was identified as the Nramp transporter gene expressed at the highest levels in nodules. MtNramp1 protein was localized in the plasma membrane of nodule cells in zone II, where the expression reached its maximum. Its role in iron uptake and its importance for SNF were established using a loss-of-function mutant, nramp1-1. This work adds to our understanding of how apoplastic metals are imported into nodule cells.     

Overexpression of Flavodoxin in Bacteroids Induces Changes in Antioxidant Metabolism Leading to Delayed Senescence and Starch Accumulation in Alfalfa Root Nodules

Sinorhizobium meliloti cells were engineered to overexpress Anabaena variabilis flavodoxin, a protein that is involved in the response to oxidative stress. Nodule natural senescence was characterized in alfalfa (Medicago sativa) plants nodulated by the flavodoxin-overexpressing rhizobia or the corresponding control bacteria. The decline of nitrogenase activity and the nodule structural and ultrastructural alterations that are associated with nodule senescence were significantly delayed in flavodoxin-expressing nodules. Substantial changes in nodule antioxidant metabolism, involving antioxidant enzymes and ascorbate-glutathione cycle enzymes and metabolites, were detected in flavodoxin-containing nodules. Lipid peroxidation was also significantly lower in flavodoxin-expressing nodules than in control nodules. The observed amelioration of the oxidative balance suggests that the delay in nodule senescence was most likely due to a role of the protein in reactive oxygen species detoxification. Flavodoxin overexpression also led to high starch accumulation in nodules, without reduction of the nitrogen-fixing activity.Symbiotic nodules have a limited functional life that varies among different legume species. Nodule senescence is the sequence of structural, molecular, biochemical, and physiological events taking place in the process that a mature and functional nodule undergoes leading to the loss of the nitrogen-fixing activity and culminating in cell death of symbiotic tissue (Swaraj and Bishnoi, 1996; Puppo et al., 2005; Van de Velde et al., 2006).Various models have been proposed to explain the mechanisms that trigger the process of natural or stress-induced nodule senescence. However, it is generally accepted that a senescence-inducing signal from the plant causes a decrease in antioxidant levels and thus an increase in reactive oxygen species (ROS) up to a point of no return. Numerous studies have shown that ROS and antioxidant systems are involved in natural (Lucas et al., 1998; Evans et al., 1999; Hernández-Jiménez et al., 2002; Puppo et al., 2005) as well as induced (Dalton et al., 1993; Becana et al., 2000; Hernández-Jiménez et al., 2002; Matamoros et al., 2003) nodule senescence. Nitrogen fixation is very sensitive to ROS, and nitrogenase activity drastically decreases during nodule senescence (Dalton et al., 1986).Antioxidant systems that protect cells from oxidative damage have been described in symbiotic nodules (Dalton et al., 1986, 1993; Evans et al., 1999; Becana et al., 2000; Matamoros et al., 2003; Puppo et al., 2005). These include the enzymes superoxide dismutase (SOD), catalase, and peroxidase. Another enzymatic system associated with ROS detoxification is the ascorbate-glutathione pathway, which includes ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), and glutathione reductase (GR; Dalton et al., 1986, 1992; Noctor and Foyer 1998; Becana et al., 2000). Ascorbate and reduced glutathione (GSH) in this pathway can also scavenge superoxide and hydrogen peroxide.During nodule senescence, several ultrastructural alterations in the nodule tissues and cells have been observed (Lucas et al., 1998; Hernández-Jiménez et al., 2002; Puppo et al., 2005, and refs. therein; Van de Velde et al., 2006). Cytosol becomes electron dense, altered vesicles proliferate, and eventually the cytosol undergoes lysis. The number of peroxisomes increases, mitochondria form complex elongated structures, and symbiosomes change in size and shape and fuse during natural and induced senescence of nodules (Hernández-Jiménez et al., 2002). Damage of the symbiosome membrane is also detected (Puppo et al., 2005; Van de Velde et al., 2006).A strategy of delayed nodule senescence could lead to increased nitrogen fixation and legume productivity. Delayed nodule senescence together with enhanced sustainability under field conditions are among the key aims of legume improvement programs (Puppo et al., 2005). An interesting approach proposed to achieve delayed senescence is to induce nodulation in legumes using rhizobial strains with modified redox capacity (Zahran, 2001).The protein flavodoxin contains a FMN group acting as a redox center transferring electrons at low potentials (Pueyo et al., 1991; Pueyo and Gómez-Moreno, 1991). The FMN cofactor of flavodoxin can exist in three different redox states: oxidized, one-electron-reduced semiquinone, and two-electron-reduced hydroquinone. This property confers high versatility to flavodoxins in electron transport systems (Simondsen and Tollin, 1980; McIver et al., 1998). To date, flavodoxin has not been described in plants, as flavodoxin-encoding genes were lost during the transition of algae to plants (Zurbriggen et al., 2007) and, consequently, no homologs have been identified in the sequenced genome of Arabidopsis (Arabidopsis thaliana; Arabidopsis Genome Initiative, 2000). Flavodoxin is present as a constitutive or inducible protein in different microorganisms (Klugkist et al., 1986). In the nitrogen-fixing cyanobacterium Anabaena variabilis PCC 7119, flavodoxin is expressed under conditions of limited iron availability, replacing ferredoxin in the photosynthetic electron transport from PSI to NADP⁺ and in nitrogenase reduction (Sandmann et al., 1990). Reversible electron transfer from flavodoxin to NADP⁺ is catalyzed by ferredoxin NADP⁺ reductase in different pathways of oxidative metabolism (Arakaki et al., 1997). In its reduced state, flavodoxin might be able to react with ROS and revert to its original redox state in the presence of an appropriate electron source. This could probably occur without the associated molecular damage that metallic complexes in catalases or SODs suffer (Keyer et al., 1995). The presence of flavodoxin has not been documented to date in the symbiotic bacterium Sinorhizobium meliloti. In Escherichia coli, however, flavodoxin induction is linked to the oxidative stress-responsive regulon soxRS (Zheng et al., 1999). It has been suggested that flavodoxin and ferredoxin (flavodoxin) NADP⁺ reductase might be induced and have a role in reestablishing the cell redox balance under oxidative stress conditions (Liochev et al., 1994). The properties of flavodoxin suggest that its presence in the cell may have a facilitating effect on ROS detoxification. In fact, an increase in the amount of flavodoxin has been observed in some bacterial species subjected to oxidative stress (Zheng et al., 1999; Yousef et al., 2003; Singh et al., 2004), and transgenic tobacco (Nicotiana tabacum) plants expressing flavodoxin in chloroplasts show enhanced tolerance to a broad range of stresses related to oxidative damage (Tognetti et al., 2006, 2007a, 2007b).In this work, Sinorhizobium meliloti was transformed with the A. variabilis flavodoxin gene and used to nodulate alfalfa (Medicago sativa) plants. The effects of flavodoxin expression on nodulation dynamics, on nodule development and senescence processes, and on nitrogen-fixing activity were analyzed. Mechanistic insights suggesting putative roles for flavodoxin in protection from ROS and the induced delay of nodule senescence are likewise discussed.     

Remodeling of the Infection Chamber before Infection Thread Formation Reveals a Two-Step Mechanism for Rhizobial Entry into the Host Legume Root Hair

In many legumes, root entry of symbiotic nitrogen-fixing rhizobia occurs via host-constructed tubular tip-growing structures known as infection threads (ITs). Here, we have used a confocal microscopy live-tissue imaging approach to investigate early stages of IT formation in Medicago truncatula root hairs (RHs) expressing fluorescent protein fusion reporters. This has revealed that ITs only initiate 10 to 20 h after the completion of RH curling, by which time major modifications have occurred within the so-called infection chamber, the site of bacterial entrapment. These include the accumulation of exocytosis (M. truncatula Vesicle-Associated Membrane Protein721e)- and cell wall (M. truncatula EARLY NODULIN11)-associated markers, concomitant with radial expansion of the chamber. Significantly, the infection-defective M. truncatula nodule inception-1 mutant is unable to create a functional infection chamber. This underlines the importance of the NIN-dependent phase of host cell wall remodeling that accompanies bacterial proliferation and precedes IT formation, and leads us to propose a two-step model for rhizobial infection initiation in legume RHs.Legumes possess the remarkable capacity to improve their nutrition by establishing a nitrogen-fixing root nodule symbiosis (RNS) with soil bacteria collectively called rhizobia. In many legumes such as Medicago truncatula, rhizobia penetrate across the root epidermis and outer cortex to reach the differentiating nodule tissues via sequentially constructed transcellular compartments known as infection threads (ITs; Gage, 2004). It is now well established that this mode of entry through specialized infection compartments, often referred to as accommodation, is shared with the more ancient arbuscular mycorrhizal (AM) symbiosis, from which the legume-Rhizobium RNS is thought to have evolved (Parniske, 2008; Markmann and Parniske, 2009). Furthermore, strong evidence indicates that the signaling and cellular mechanisms underlying IT formation in legumes are closely related to those used for infection compartment formation during AM infection of epidermal and outer cortical tissues (Bapaume and Reinhardt, 2012; Oldroyd, 2013).Rhizobial infection is set in motion after an initial molecular dialogue between symbiotic partners, in which rhizobial lipo-chitooligosaccharide (LCO) Nod factors (NFs) are key signaling molecules (for review, see Oldroyd, 2013). Host responses to NF signaling include rapid and sustained nuclear-associated Ca²⁺ oscillations (Ca²⁺ spiking; Ehrhardt et al., 1996; Oldroyd and Downie, 2006; Sieberer et al., 2009; Capoen et al., 2011) and the rapid expression of early epidermal marker genes such as M. truncatula EARLY NODULIN11 (Charron et al., 2004). The activation of nuclear Ca²⁺ spiking is one of the most characteristic features of the so-called common symbiotic signaling pathway, common to both RNS and AM (Kistner and Parniske, 2002; Singh and Parniske, 2012). Whereas these preinfection responses to NFs are observed in the majority of elongating root hairs (RHs) early after rhizobial inoculation (Journet et al., 2001; Wais et al., 2002), ITs are only formed in a small subset of RHs, and MtENOD11 expression is strongly activated at these rhizobial infection sites (Journet et al., 2001; Boisson-Dernier et al., 2005).ITs are tubular plant-derived structures delimited by a membrane that is contiguous with the RH plasmalemma and a layer of cell wall-like material, thus isolating the rhizobia from the host cell cytoplasm (Gage, 2004). These apoplastic infection compartments are progressively constructed along the length of the RH with their growing tip connected via a cytoplasmic bridge to the migrating RH nucleus. This broad cytoplasmic column provides the cell machinery for tip growth, which involves targeted exocytosis of membrane and extracellular materials to the growing apex of the IT (Oldroyd et al., 2011; Bapaume and Reinhardt, 2012). It is presumed that this cytoplasmic bridge shares an equivalent role to the prepenetration apparatus (PPA) formed at the onset of AM fungal infection (Genre et al., 2005, 2008). We now know that the IT tip region is formed in advance of rhizobial colonization and is progressively populated by dividing rhizobia that also physically move down the thread (Gage, 2004; Fournier et al., 2008). It has been proposed that the matrix of the growing IT tip is initially in a fluid or gel-like state compatible with bacterial growth and movement (Brewin, 2004; Fournier et al., 2008). This relative plasticity could result in part from the presence of atypical extracellular (glyco) proteins such as the repetitive Pro-rich proteins MtENOD11/MtENOD12 because their low Tyr content is presumed to limit cross linking to other wall components (Scheres et al., 1990; Pichon et al., 1992; Journet et al., 2001).Nevertheless, the mechanism by which rhizobial IT formation is initiated in RHs is not clear. Whereas AM fungal hyphae form contact structures called hyphopodia on the exposed surface of nonhair epidermal cells prior to PPA formation and perifungal infection compartment formation (Genre et al., 2005), rhizobial entry requires that the bacteria first become entrapped between RH walls. Attachment of rhizobia close to a growing RH tip induces a continuous reorientation of tip growth, most likely the result of localized NF production (Esseling et al., 2003), eventually leading to RH curling and subsequent bacterial entrapment within a closed chamber in the center of the curl (Catoira et al., 2001; Geurts et al., 2005). Rhizobial entrapment can also occur between the cell walls of two touching RHs (Dart, 1974; Gage, 2004).The closed chamber in curled RHs has often been termed the infection pocket (e.g. Murray, 2011; Guan et al., 2013). However, because this term is also used to designate a quite different and larger structure formed in root subepidermal tissues of legumes during intercellular infection after crack entry and involving localized cell death (Goormachtig et al., 2004), we propose to use the term infection chamber to describe the unique enclosure formed during rhizobial RH infection.After entrapment, it has been proposed that rhizobia multiply to form a so-called microcolony (Gage et al., 1996; Limpens et al., 2003), and that IT polar growth initiates in front of this microcolony by local invagination of the RH plasmalemma combined with exocytosis of extracellular materials (Gage, 2004). Furthermore, it has been suggested that localized degradation of the chamber wall would allow the rhizobia to access the newly formed IT (Callaham and Torrey, 1981; Turgeon and Bauer, 1985). However, a detailed investigation of this particular stage of rhizobial infection is lacking, particularly concerning when and where the rhizobia/cell wall interface becomes modified. Such studies have been limited until now, notably because ITs develop only in a low proportion of curled RHs (Dart, 1974).To attempt to answer this question, we have used a live-tissue imaging approach developed for in vivo confocal microscopy in M. truncatula (Fournier et al., 2008; Cerri et al., 2012; Sieberer et al., 2012) and particularly well adapted to time-lapse studies of the initial stages of rhizobial infection, including RH curling and IT formation. To investigate modifications occurring at the RH interface with the enclosed rhizobia during these early stages, we prepared M. truncatula plants expressing fluorescent protein fusions aimed at detecting both exocytosis activity and cell wall remodeling during the initial construction of the IT apoplastic compartment. To this end, we made use of the M. truncatula Vesicle-Associated Membrane Protein721e (MtVAMP721e; Ivanov et al., 2012), recently shown to label exocytosis sites both in growing RHs and during AM colonization (Genre et al., 2012), as well as the infection- and cell wall-associated MtENOD11 Pro-rich glycoprotein (Journet et al., 2001). Our experiments have revealed that IT development in curled RHs only initiates after a lengthy interval of 10 to 20 h, during which sustained exocytosis and MtENOD11 secretion to the infection chamber are associated with radial expansion as well as remodeling of the surrounding walls. Importantly, it was found that the infection-defective M. truncatula nodule inception-1 (Mtnin-1) mutant (Marsh et al., 2007) is impaired in chamber remodeling. Our findings led us to propose a new model for IT formation in which the infection chamber first differentiates into a globular apoplastic compartment displaying similarities to the future IT, and in which the enclosed rhizobia multiply. This is then followed by a switch from radial to tubular growth corresponding to tip-driven IT growth and associated movement of rhizobia into the extending thread. Importantly, this two-step model no longer requires that the host cell wall is degraded to allow access of the colonizing rhizobia to the newly initiated IT.     

A Nodule-Specific Lipid Transfer Protein AsE246 Participates in Transport of Plant-Synthesized Lipids to Symbiosome Membrane and Is Essential for Nodule Organogenesis in Chinese Milk Vetch

Rhizobia in legume root nodules fix nitrogen in symbiosomes, organelle-like structures in which a membrane from the host plant surrounds the symbiotic bacteria. However, the components that transport plant-synthesized lipids to the symbiosome membrane remain unknown. This study identified and functionally characterized the Chinese milk vetch (Astragalus sinicus) lipid transfer protein AsE246, which is specifically expressed in nodules. It was found that AsE246 can bind lipids in vitro. More importantly, AsE246 can bind the plant-synthesized membrane lipid digalactosyldiacylglycerol in vivo. Immunofluorescence and immunoelectron microscopy showed that AsE246 and digalactosyldiacylglycerol localize in the symbiosome membrane and are present in infection threads. Overexpression of AsE246 resulted in increased nodule numbers; knockdown of AsE246 resulted in reduced nodule numbers, decreased lipids contents in nodules, diminished nitrogen fixation activity, and abnormal development of symbiosomes. AsE246 knockdown also resulted in fewer infection threads, nodule primordia, and nodules, while AsE246 overexpression resulted in more infection threads and nodule primordia, suggesting that AsE246 affects nodule organogenesis associated with infection thread formation. Taken together, these results indicate that AsE246 contributes to lipids transport to the symbiosome membrane, and this transport is required for effective legume-rhizobium symbiosis.Legume crops can act as hosts for nitrogen-fixing soil Rhizobium spp. bacteria, which induce and occupy a specialized organ, the root nodule (Limpens et al., 2009). This endosymbiotic relationship is mutualistic for both the host plant and the Rhizobium spp.; the plant receives a crucial supply of reduced nitrogen from the bacteria and the nodule bacteria receive reduced carbon and other nutrients (Held et al., 2010).Symbiosis requires specialized host-symbiont communication and cellular development. Plant roots are exposed to various microorganisms in the soil, but their strong protective barriers, including cell walls, prevent the entry of most harmful species. To bypass these barriers, the invasion of plant roots by rhizobia begins with a reciprocal exchange of signals that allow the bacteria to enter through the plant root hair cells (Jones et al., 2007). The rhizobia enter the root hair and underlying cells via an infection thread (IT), from which they are eventually released into cortical cells via endocytosis. Each bacterial cell is endocytosed by a target cell into an individual, unwalled membrane compartment that originates from the IT. The bacteria are surrounded by a membrane of plant origin; this membrane is variously termed the endocytic, peribacteroid, or symbiosome membrane. Also, the entire unit is known as the symbiosome (Verma and Hong, 1996; Jones et al., 2007), and the space between the bacteria and the membrane is called the peribacteroid space.The symbiosome membrane forms the structural and functional interface between the host plant and the rhizobia. First, it prevents direct contact between the host plant cell cytoplasm and the invading prokaryote, which may otherwise interfere with host cell metabolism and may provoke host defense responses. Second, the symbiosome membrane controls the exchange of substrate and signal molecules between host plant cell and the bacteria (Verma and Hong, 1996; Gaude et al., 2004). The host plant makes the symbiosome membrane, which has similar properties to the plant vacuolar membrane, but contains several nodule-specific proteins. For example, the syntaxin MtSYP132, a Medicago truncatula homolog of Arabidopsis (Arabidopsis thaliana) SYNTAXIN OF PLANTS132, occurs on M. truncatula symbiosomes throughout their development (Verma and Hong, 1996; Whitehead and Day, 1997; Catalano et al., 2007). The symbiosome membrane also contains a nonphosphorus galactoglycerolipid, digalactosyldiacylglycerol (DGDG), which is also found in chloroplast, extraplastidic, tonoplast, and plasma membranes (Gaude et al., 2004; Benning, 2009). The symbiosome membrane also contains saturated (16:0, palmitic acid; 18:0, stearic acid) and unsaturated fatty acids (16:1^Δ3trans, palmitoleic acid; 18:1^Δ9cis, oleic acid 18:2^Δ9,12, linoleic acid; 18:3^Δ9,12,15, α-linolenic acid), all of which are typically found in higher plants (Whitehead and Day, 1997; Gaude et al., 2004).During nodule development, the number of rhizobia in infected cells increases dramatically. This increase requires membrane biosynthesis in the bacteria as part of cell division and also requires membrane biosynthesis by the plant to produce symbiosome membrane to enclose the bacteria (Verma, 1992; Gaude et al., 2004). Thus, symbiosis requires large amounts of lipid, but how plant-synthesized lipids are transported to the symbiosome membrane remains unclear.In eukaryotic cells, vesicular and nonvesicular transport mechanisms mediate intracellular lipid trafficking (Voelker, 1990; Lev, 2010). Large amounts of lipids were thought to be transported between organelles only by vesicular transport. However, lipid transport can occur even when vesicular transport is blocked by ATP depletion, by reduction in temperature, or by treatment with specific drugs such as brefeldin A and colchicine (Kaplan and Simoni, 1985; Vance et al., 1991). Lipid transport can also occur between organelles that are not connected to the vesicular transport machinery, e.g. chloroplasts, mitochondria, and peroxisomes (Levine, 2004; Holthuis and Levine, 2005; Benning, 2009). These observations suggest that nonvesicular transport mechanisms have an important role in intracellular lipid trafficking. However, to date, no symbiosome-targeting plant lipid transport proteins have been characterized.Nonvesicular lipid transport could occur, in principle, by spontaneous desorption of a lipid monomer from a bilayer and free diffusion through the cytosol, but this process is slow and insufficient to support substantial transport of most lipids. Lipid transfer proteins (LTPs) can facilitate lipid transport between membranes in vitro (Kader et al., 1984; Shin et al., 1995; Lee et al., 1998; Lascombe et al., 2008). LTPs were initially discovered as soluble factors that accelerate the exchange or net transfer of lipid species between membranes in vitro. Since then, many LTPs have been isolated, characterized, and crystallized. LTPs have been identified in eukaryotes and in bacteria and have been subdivided into different families according to their protein sequences and structures (D’Angelo et al., 2008).Plant LTPs are cationic peptides, with molecular masses of around 7 to 10 kD and can reversibly bind and transport hydrophobic molecules in vitro. Plant LTPs are subdivided into two families, LTP1 and LTP2 (Carvalho et al., 2007). Both families possess conserved patterns of eight Cys residues, and their three-dimensional structure reveals an internal hydrophobic cavity that forms the lipid binding site. NMR data have demonstrated that a wheat (Triticum aestivum) LTP forms a complex with prostaglandin B2, suggesting that LTPs can accommodate large hydrophobic compounds (Tassin-Moindrot et al., 2000). Also, an LTP from Arabidopsis, LTPg1, is required for normal export of wax to the cuticle (Debono et al., 2009).Chinese milk vetch (Astragalus sinicus) is a winter-growing green manure legume. It can establish a specific endosymbiosis with Mesorhizobium huakuii 7653R and form indeterminate-type N₂-fixing root nodules, which are cylindrical and consist of a gradient of developmental zones with a persistent apical meristem (zone I), an infection zone (zone II), and a fixation zone (zone III). In mature nodules, a senescence zone (zone IV) is established proximal to zone III. As the nodule ages, this zone gradually moves in a proximal-distal direction until it reaches the apical part and the nodule degenerates (Monahan-Giovanelli et al., 2006; Van de Velde et al., 2006). Chinese milk vetch is mainly distributed in China, Japan, and Korea, where it is widely planted in rice fields to increase soil fertility (Li et al., 2008).To investigate whether LTPs function in symbiosis membrane deposition and nodule organogenesis in Chinese milk vetch, we previously identified two candidate LTP genes, AsE246 and AsIB259, via suppressive subtractive hybridization. AsE246 is specifically expressed in nodules, but AsIB259 is expressed in both uninfected roots and nodules, indicating that only AsE246 is specific to nodulation (Chou et al., 2006). Here, we report the functional characterization of AsE246, a nodule-specific lipid transfer protein that localizes on the symbiosome membrane and plays an essential role in symbiosome membrane biogenesis and effective symbiosis.     

Large-Scale Phosphoprotein Analysis in Medicago truncatula Roots Provides Insight into in Vivo Kinase Activity in Legumes

Nitrogen fixation in legumes requires the development of root organs called nodules and their infection by symbiotic rhizobia. Over the last decade, Medicago truncatula has emerged as a major model plant for the analysis of plant-microbe symbioses and for addressing questions pertaining to legume biology. While the initiation of symbiosis and the development of nitrogen-fixing root nodules depend on the activation of a protein phosphorylation-mediated signal transduction cascade in response to symbiotic signals produced by the rhizobia, few sites of in vivo phosphorylation have previously been identified in M. truncatula. We have characterized sites of phosphorylation on proteins from M. truncatula roots, from both whole cell lysates and membrane-enriched fractions, using immobilized metal affinity chromatography and tandem mass spectrometry. Here, we report 3,457 unique phosphopeptides spanning 3,404 nonredundant sites of in vivo phosphorylation on 829 proteins in M. truncatula Jemalong A17 roots, identified using the complementary tandem mass spectrometry fragmentation methods electron transfer dissociation and collision-activated dissociation. With this being, to our knowledge, the first large-scale plant phosphoproteomic study to utilize electron transfer dissociation, analysis of the identified phosphorylation sites revealed phosphorylation motifs not previously observed in plants. Furthermore, several of the phosphorylation motifs, including LxKxxs and RxxSxxxs, have yet to be reported as kinase specificities for in vivo substrates in any species, to our knowledge. Multiple sites of phosphorylation were identified on several key proteins involved in initiating rhizobial symbiosis, including SICKLE, NUCLEOPORIN133, and INTERACTING PROTEIN OF DMI3. Finally, we used these data to create an open-access online database for M. truncatula phosphoproteomic data.Medicago truncatula has become a model for studying the biology of leguminous plants such as soybean (Glycine max), alfalfa (Medicago sativa), and clover (Trifolium spp.; Singh et al., 2007). Most members of this vast family have the ability to fix atmospheric nitrogen by virtue of an endosymbiotic association with rhizobial bacteria, through which legumes undergo nodulation, the process of forming root nodules (Jones et al., 2007). Legumes are central to modern agriculture and civilization because of their ability to grow in nitrogen-depleted soils and replenish nitrogen through crop rotation. Consequently, there is great interest in understanding the molecular events that allow legumes to recognize their symbionts, develop root nodules, and fix nitrogen. Nod factors are lipochitooligosaccharidic signals secreted by the rhizobia and are required, in most legumes, for intracellular infection and nodule development. In recent decades, an elegant combination of genetics, biochemistry, and cell biology has shown that Nod factors activate intricate signaling events within cells of legume roots, including protein phosphorylation cascades and intracellular ion fluxes (Oldroyd and Downie, 2008).Protein phosphorylation is a central mechanism of signal transfer in cells (Laugesen et al., 2006; Peck, 2006; Huber, 2007). Several characterized protein kinases are required for symbiosis signal transduction in M. truncatula roots (Lévy et al., 2004; Yoshida and Parniske, 2005; Smit et al., 2007). A recent antibody-based study of cultured M. truncatula cells observed protein phosphorylation changes at the proteomic level in response to fungal infection (Trapphoff et al., 2009); however, the target residues of the phosphorylation events were not determined. A variety of studies have determined in vitro phosphorylation sites on legume proteins and demonstrated the biological importance of the target residues by mutagenesis (Yoshida and Parniske, 2005; Arrighi et al., 2006; Lima et al., 2006; Miyahara et al., 2008; Yano et al., 2008). To our knowledge, only six sites of in vivo protein phosphorylation have been detected for M. truncatula (Laugesen et al., 2006; Lima et al., 2006; Wienkoop et al., 2008), demonstrating the need for the identification of endogenous protein phosphorylation sites in legume model organisms on a proteome-wide scale.While considerable advancements have been made in the global analysis of protein phosphorylation (Nita-Lazar et al., 2008; Macek et al., 2009; Piggee, 2009; Thingholm et al., 2009), phosphoproteomics in plants has lagged years behind that of the mammalian systems (Kersten et al., 2006, 2009; Peck, 2006), which have more fully sequenced genomes and better annotated protein predictions. Arabidopsis (Arabidopsis thaliana), the first plant genome sequenced (Arabidopsis Genome Initiative, 2000), is now predicted to have over 1,000 protein kinases (Finn et al., 2008), approximately twice as many as in human (Manning et al., 2002). Because many of the kinases in the commonly studied mammalian systems are not conserved in the plant kingdom, there is significant need for large-scale phosphoproteomic technologies to discern the intricacies of phosphorylation-mediated cell signaling in plants. With the high mass accuracy afforded by the linear ion trap-orbitrap hybrid mass spectrometer (Makarov et al., 2006; Yates et al., 2006), recent studies in Arabidopsis have reported 2,597 phosphopeptides from suspension cell culture (Sugiyama et al., 2008) and 3,029 phosphopeptides from seedlings (Reiland et al., 2009).All previous large-scale plant phosphoproteomic studies have relied solely on collision-activated dissociation (CAD) during tandem mass spectrometry (MS/MS) and have not taken advantage of the more recently developed methods (Kersten et al., 2009) electron capture dissociation (Kelleher et al., 1999) or electron transfer dissociation (ETD; Coon et al., 2004; Syka et al., 2004). Mapping sites of posttranslational modifications, such as phosphorylation, is often more straightforward using electron-based fragmentation methods, as they frequently produce a full spectrum of sequence-informative ions without causing neutral loss of the modifying functional groups (Meng et al., 2005; Chi et al., 2007; Khidekel et al., 2007; Molina et al., 2007; Wiesner et al., 2008; Chalkley et al., 2009; Swaney et al., 2009). With an ETD-enabled hybrid orbitrap mass spectrometer (McAlister et al., 2007, 2008), we previously compared the performance of CAD and ETD tandem MS for large-scale identification of phosphopeptides (Swaney et al., 2009). ETD identified a greater percentage of unique phosphopeptides and more frequently localized phosphorylation sites. Still, the low overlap of identified phosphopeptides indicates that the two methods are highly complementary. With this in mind, we recently developed a decision tree-driven tandem MS algorithm to select the optimal fragmentation method for each precursor (Swaney et al., 2008).Here, we utilize this technology to map sites of in vivo protein phosphorylation in roots of M. truncatula Jemalong A17 plants. Phosphoproteins, from both whole-cell lysate and membrane-enriched fractions, were analyzed after digestion with a variety of different enzymes individually. Utilizing the complementary fragmentation methods of ETD and CAD, we report 3,404 nonredundant phosphorylation sites at an estimated false discovery rate (FDR) of 1%. Analysis of these data revealed several phosphorylation motifs not previously observed in plants. The phosphorylation sites identified provide insight into the potential regulation of key proteins involved in rhizobial symbiosis, potential consensus sequences by which kinases recognize their substrates, and critical phosphorylation events that are conserved between plant species.