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).     

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.     

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.     

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.     

Knockdown of CELL DIVISION CYCLE16 Reveals an Inverse Relationship between Lateral Root and Nodule Numbers and a Link to Auxin in Medicago truncatula

The postembryonic development of lateral roots and nodules is a highly regulated process. Recent studies suggest the existence of cross talk and interdependency in the growth of these two organs. Although plant hormones, including auxin and cytokinin, appear to be key players in coordinating this cross talk, very few genes that cross-regulate root and nodule development have been uncovered so far. This study reports that a homolog of CELL DIVISION CYCLE16 (CDC16), a core component of the Anaphase Promoting Complex, is one of the key mediators in controlling the overall number of lateral roots and nodules. A partial suppression of this gene in Medicago truncatula leads to a decrease in number of lateral roots and a 4-fold increase in number of nodules. The roots showing lowered expression of MtCDC16 also show reduced sensitivity to phytohormone auxin, thus providing a potential function of CDC16 in auxin signaling.As in all eukaryotic organisms, cell division in plants is strictly controlled by a concerted action of several key regulators, such as cyclin-dependent kinases and cyclins (De Veylder et al., 2007). The progression of the cell cycle from one phase to another requires the targeted degradation of selected cyclin molecules mediated by two ubiquitin-mediated proteolytic pathways. The SKP1-Cullin/F-Box protein (SCF) pathway acts in the G1-to-S phase transition by degrading the D-type cyclins and other substrate proteins (Yanagawa and Kimura, 2005). The second pathway, mediated by Anaphase Promoting Complex/Cyclosome (APC/C), regulates the sequential destruction of A- and B-type cyclins in a D-box or a KEN-box-dependent manner, resulting in chromosome segregation and exit from mitosis (Genschik et al., 1998; Pfleger and Kirschner, 2000). Evidence of the role of the APC/C in plant development comes from studies of the Arabidopsis (Arabidopsis thaliana) hobbit (hbt) mutant that shows defects in root growth. The HBT gene is required for both cell division and cell differentiation in root meristems and encodes CDC27, a core subunit of APC/C (Blilou et al., 2002; Perez-Perez et al., 2008). Cebolla et al. (1999) used the root nodule system of the model legume Medicago truncatula to study the function of an APC/C activator, CCS52, which is homologous to the yeast APC/C activator CDH1. A nodule-specific homolog of CCS52, CCS52A, was found to be required to initiate endoreduplication in the dividing cells, and its down-regulation affected nodule development, resulting in lower ploidy, reduced cell size, and inefficient rhizobial invasion and nodule maturation (Vinardell et al., 2003; Kondorosi et al., 2005). T-DNA insertions in the Arabidopsis CELL DIVISION CYCLE16 (CDC16) and APC2 genes result in gametophytic lethality due to the failure to degrade mitotic cyclins (Capron et al., 2003b; Kwee and Sundaresan, 2003). Although the completed Arabidopsis genome has allowed the identification of homologs of almost all vertebrate APC/C subunits in plants (Capron et al., 2003a), the functions of most of these subunits still remains largely unexplored.Direct links between root growth and auxin signaling have been well documented. Several Arabidopsis mutants with decreased auxin sensitivity often exhibit an overall defect in both primary and lateral root development (Hellmann and Estelle, 2002; Casimiro et al., 2003; Hellmann et al., 2003; Vanneste et al., 2005). A number of these auxin-resistant mutants belong to the SCF proteolysis pathway, supporting a role for the SCF pathway in auxin signaling (Teale et al., 2006; Benjamins and Scheres, 2008). Auxin appears to control lateral root development by promoting G1-to-S transition in selected xylem pericycle cells, perhaps by targeting KRP2, a cyclin-dependent kinase inhibitor, and E2F, an S phase inhibitor to SCF-mediated proteolysis (del Pozo et al., 2002; Himanen et al., 2002). Unlike SCF, the role of APC/C in auxin-mediated plant development is not clear. The only report that has so far integrated APC/C with auxin signaling pertains to the hbt mutant, which shows an increased resistance to exogenous auxin due to accumulation of Aux/IAAs in the roots (Blilou et al., 2002).As in lateral roots, auxin is an important player in the development of nodules on the roots of leguminous plants (Beveridge et al., 2007). Studies with auxin-responsive reporter gene constructs have shown auxin''s participation in cortical cell reactivation and initiation of nodule primordia (Mathesius et al., 1998). The exogenous application of Nod factor results in a transient inhibition of auxin transport capacity in roots of Vicia sativa (Boot et al., 1999) and Trifolium repens (Mathesius et al., 1998). Consistent with this, localized application of synthetic auxin transport inhibitors on alfalfa (Medicago sativa) roots induces pseudonodules (Hirsch et al., 1989). Complementing these findings, a more recent study in M. truncatula has demonstrated that increased auxin transport, caused by silencing the flavonoid pathway, results in reduced nodule formation in response to rhizobia (Wasson et al., 2006). Finally, hypernodulating mutants like sunn and skl show defective long-distance transport of auxin, further suggesting the importance of polar auxin transport, not only in regulating nodule induction but also in controlling nodule numbers (Prayitno et al., 2006; van Noorden et al., 2006).In this report, we investigated the role of the APC/C component CDC16 in root and nodule development in M. truncatula. CDC16 was identified via microarray analysis as a gene that was significantly induced in roots of M. truncatula following inoculation by Sinorhizobium meliloti and in nodules relative to uninoculated roots (Kuppusamy, 2005), thus encouraging further functional analysis of this gene. To overcome the problem of the gametophytic lethality resulting from CDC16 knockout, as seen from analysis of an insertional mutation in Arabidopsis (Kwee and Sundaresan, 2003) we undertook an RNA interference (RNAi) approach to partially suppress the expression of CDC16 gene in Agrobacterium rhizogenes-transformed roots of M. truncatula. We report that roots transformed with the CDC16 RNAi construct (hereafter called Mtcdc16i roots) displayed a decreased sensitivity to auxin, defective primary root growth, and fewer lateral roots. Interestingly, in response to S. meliloti, the Mtcdc16i roots showed almost 4-fold increase in number of nodules, suggesting that decreased sensitivity to auxin leads to a hypernodulation phenotype. Thus, this work highlights the importance of CDC16 in root and nodule development and indicates a possible role for this gene in auxin signaling.     

Thrombospondin-1 signaling through CD47 inhibits cell cycle progression and induces senescence in endothelial cells

CD47 signaling in endothelial cells has been shown to suppress angiogenesis, but little is known about the link between CD47 and endothelial senescence. Herein, we demonstrate that the thrombospondin-1 (TSP1)-CD47 signaling pathway is a major mechanism for driving endothelial cell senescence. CD47 deficiency in endothelial cells significantly improved their angiogenic function and attenuated their replicative senescence. Lack of CD47 also suppresses activation of cell cycle inhibitors and upregulates the expression of cell cycle promoters, leading to increased cell cycle progression. Furthermore, TSP1 significantly accelerates replicative senescence and associated cell cycle arrest in a CD47-dependent manner. These findings demonstrate that TSP1-CD47 signaling is an important mechanism driving endothelial cell senescence. Thus, TSP1 and CD47 provide attractive molecular targets for treatment of aging-associated cardiovascular dysfunction and diseases involving endothelial dysregulation.Endothelial cell (EC) senescence is accompanied with vascular dysfunction, including arterial stiffening and remodeling,¹ impaired angiogenesis,^{2, 3} reduced endothelial repair capability and increased incidence of cardiovascular disease.^{4, 5, 6} Cellular senescence can occur in vivo or in vitro in response to various stressors,^{7, 8, 9, 10} leading to suppression of cell proliferation. EC senescence has been reported to contribute to the pathogenesis of age-associated vascular diseases, such as atherosclerosis.¹¹ Thus, further understanding the mechanisms of EC senescence may help to identify effective targets for antisenescence therapy and treatment aging-associated cardiovascular disorders.Previous studies have shown that the secreted matricellular protein thrombospondin-1 (TSP1) is as potent inhibitor of angiogenesis¹² and its antiangiogenic activity is mediated by its receptors, CD36^{13, 14} and CD47.^{15, 16} CD47 is a ubiquitously expressed transmembrane protein that serves as a ligand for signal regulatory protein-α and is a signaling receptor of TSP1. The TSP1-CD47 pathway has an important role in several fundamental cellular functions, including proliferation, apoptosis, inflammation and atherosclerotic response.¹⁷ Ligation of CD47 by TSP1 has been shown to inhibit nitric oxide (NO)/cGMP signaling in vascular cells, leading to suppression of angiogenic responses.¹⁶ Recently, it was reported that lack of CD47 expression in ECs may enable these cells to spontaneously gain characteristics of embryonic stem cells.¹⁸ However, the potential role of CD47 in regulation of EC senescence has not been well explored. The present study was initiated to determine the role and mechanisms of TSP1-CD47 signaling pathway in regulating cell cycle progression and replicative senescence of ECs.     

Leaf Senescence Signaling: The Ca2+-Conducting Arabidopsis Cyclic Nucleotide Gated Channel2 Acts through Nitric Oxide to Repress Senescence Programming

Ca²⁺ and nitric oxide (NO) are essential components involved in plant senescence signaling cascades. In other signaling pathways, NO generation can be dependent on cytosolic Ca²⁺. The Arabidopsis (Arabidopsis thaliana) mutant dnd1 lacks a plasma membrane-localized cation channel (CNGC2). We recently demonstrated that this channel affects plant response to pathogens through a signaling cascade involving Ca²⁺ modulation of NO generation; the pathogen response phenotype of dnd1 can be complemented by application of a NO donor. At present, the interrelationship between Ca²⁺ and NO generation in plant cells during leaf senescence remains unclear. Here, we use dnd1 plants to present genetic evidence consistent with the hypothesis that Ca²⁺ uptake and NO production play pivotal roles in plant leaf senescence. Leaf Ca²⁺ accumulation is reduced in dnd1 leaves compared to the wild type. Early senescence-associated phenotypes (such as loss of chlorophyll, expression level of senescence-associated genes, H₂O₂ generation, lipid peroxidation, tissue necrosis, and increased salicylic acid levels) were more prominent in dnd1 leaves compared to the wild type. Application of a Ca²⁺ channel blocker hastened senescence of detached wild-type leaves maintained in the dark, increasing the rate of chlorophyll loss, expression of a senescence-associated gene, and lipid peroxidation. Pharmacological manipulation of Ca²⁺ signaling provides evidence consistent with genetic studies of the relationship between Ca²⁺ signaling and senescence with the dnd1 mutant. Basal levels of NO in dnd1 leaf tissue were lower than that in leaves of wild-type plants. Application of a NO donor effectively rescues many dnd1 senescence-related phenotypes. Our work demonstrates that the CNGC2 channel is involved in Ca²⁺ uptake during plant development beyond its role in pathogen defense response signaling. Work presented here suggests that this function of CNGC2 may impact downstream basal NO production in addition to its role (also linked to NO signaling) in pathogen defense responses and that this NO generation acts as a negative regulator during plant leaf senescence signaling.Senescence can be considered as the final stage of a plant’s development. During this process, nutrients will be reallocated from older to younger parts of the plant, such as developing leaves and seeds. Leaf senescence has been characterized as a type of programmed cell death (PCD; Gan and Amasino, 1997; Quirino et al., 2000; Lim et al., 2003). During senescence, organelles such as chloroplasts will break down first. Biochemical changes will also occur in the peroxisome during this process. When the chloroplast disassembles, it is easily observed as a loss of chlorophyll. Mitochondria, the source of energy for cells, will be the last cell organelles to undergo changes during the senescence process (Quirino et al., 2000). At the same time, other catabolic events (e.g. protein and lipid breakdown, etc.) are occurring (Quirino et al., 2000). Hormones may also contribute to this process (Gepstein, 2004). From this information we can infer that leaf senescence is regulated by many signals.Darkness treatment can induce senescence in detached leaves (Poovaiah and Leopold, 1973; Chou and Kao, 1992; Weaver and Amasino, 2001; Chrost et al., 2004; Guo and Crawford, 2005; Ülker et al., 2007). Ca²⁺ can delay the senescence of detached leaves (Poovaiah and Leopold, 1973) and leaf senescence induced by methyl jasmonate (Chou and Kao, 1992); the molecular events that mediate this effect of Ca²⁺ are not well characterized at present.Nitric oxide (NO) is a critical signaling molecule involved in many plant physiological processes. Recently, published evidence supports NO acting as a negative regulator during leaf senescence (Guo and Crawford, 2005; Mishina et al., 2007). Abolishing NO generation in either loss-of-function mutants (Guo and Crawford, 2005) or transgenic Arabidopsis (Arabidopsis thaliana) plants expressing NO degrading dioxygenase (NOD; Mishina et al., 2007) leads to an early senescence phenotype in these plants compared to the wild type. Corpas et al. (2004) showed that endogenous NO is mainly accumulated in vascular tissues of pea (Pisum sativum) leaves. This accumulation is significantly reduced in senescing leaves (Corpas et al., 2004). Corpas et al. (2004) also provided evidence that NO synthase (NOS)-like activity (i.e. generation of NO from l-Arg) is greatly reduced in senescing leaves. Plant NOS activity is regulated by Ca²⁺/calmodulin (CaM; Delledonne et al., 1998; Corpas et al., 2004, 2009; del Río et al., 2004; Valderrama et al., 2007; Ma et al., 2008). These studies suggest a link between Ca²⁺ and NO that could be operating during senescence.In animal cells, all three NOS isoforms require Ca²⁺/CaM as a cofactor (Nathan and Xie, 1994; Stuehr, 1999; Alderton et al., 2001). Notably, animal NOS contains a CaM binding domain (Stuehr, 1999). It is unclear whether Ca²⁺/CaM can directly modulate plant NOS or if Ca²⁺/CaM impacts plant leaf development/senescence through (either direct or indirect) effects on NO generation. However, recent studies from our lab suggest that Ca²⁺/CaM acts as an activator of NOS activity in plant innate immune response signaling (Ali et al., 2007; Ma et al., 2008).Although Arabidopsis NO ASSOCIATED PROTEIN1 (AtNOA1; formerly named AtNOS1) was thought to encode a NOS enzyme, no NOS-encoding gene has yet been identified in plants (Guo et al., 2003; Crawford et al., 2006; Zemojtel et al., 2006). However, the AtNOA1 loss-of-function mutant does display reduced levels of NO generation, and several groups have used the NO donor sodium nitroprusside (SNP) to reverse some low-NO related phenotypes in Atnoa1 plants (Guo et al., 2003; Bright et al., 2006; Zhao et al., 2007). Importantly, plant endogenous NO deficiency (Guo and Crawford, 2005; Mishina et al., 2007) or abscisic acid/methyl jasmonate (Hung and Kao, 2003, 2004) induced early senescence can be successfully rescued by application of exogenous NO. Addition of NO donor can delay GA-elicited PCD in barley (Hordeum vulgare) aleurone layers as well (Beligni et al., 2002).It has been suggested that salicylic acid (SA), a critical pathogen defense metabolite, can be increased in natural (Morris et al., 2000; Mishina et al., 2007) and transgenic NOD-induced senescent Arabidopsis leaves (Mishina et al., 2007). Pathogenesis related gene1 (PR1) expression is up-regulated in transgenic Arabidopsis expressing NOD (Mishina et al., 2007) and in leaves of an early senescence mutant (Ülker et al., 2007).Plant cyclic nucleotide gated channels (CNGCs) have been proposed as candidates to conduct extracellular Ca²⁺ into the cytosol (Sunkar et al., 2000; Talke et al., 2003; Lemtiri-Chlieh and Berkowitz, 2004; Ali et al., 2007; Demidchik and Maathuis, 2007; Frietsch et al., 2007; Kaplan et al., 2007; Ma and Berkowitz, 2007; Urquhart et al., 2007; Ma et al., 2009a, 2009b). Arabidopsis “defense, no death” (dnd1) mutant plants have a null mutation in the gene encoding the plasma membrane-localized Ca²⁺-conducting CNGC2 channel. This mutant also displays no hypersensitive response to infection by some pathogens (Clough et al., 2000; Ali et al., 2007). In addition to involvement in pathogen-mediated Ca²⁺ signaling, CNGC2 has been suggested to participate in the process of leaf development/senescence (Köhler et al., 2001). dnd1 mutant plants have high levels of SA and expression of PR1 (Yu et al., 1998), and spontaneous necrotic lesions appear conditionally in dnd1 leaves (Clough et al., 2000; Jirage et al., 2001). Endogenous H₂O₂ levels in dnd1 mutants are increased from wild-type levels (Mateo et al., 2006). Reactive oxygen species molecules, such as H₂O₂, are critical to the PCD/senescence processes of plants (Navabpour et al., 2003; Overmyer et al., 2003; Hung and Kao, 2004; Guo and Crawford, 2005; Zimmermann et al., 2006). Here, we use the dnd1 mutant to evaluate the relationship between leaf Ca²⁺ uptake during plant growth and leaf senescence. Our results identify NO, as affected by leaf Ca²⁺ level, to be an important negative regulator of leaf senescence initiation. Ca²⁺-mediated NO production during leaf development could control senescence-associated gene (SAG) expression and the production of molecules (such as SA and H₂O₂) that act as signals during the initiation of leaf senescence programs.