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Symbiotic Leghemoglobins Are Crucial for Nitrogen Fixation in Legume Root Nodules but Not for General Plant Growth and Development
Abstract
Hemoglobins are ubiquitous in nature and among the best-characterized proteins. Genetics has revealed crucial roles for human hemoglobins, but similar data are lacking for plants. Plants contain symbiotic and nonsymbiotic hemoglobins; the former are thought to be important for symbiotic nitrogen fixation (SNF). In legumes, SNF occurs in specialized organs, called nodules, which contain millions of nitrogen-fixing rhizobia, called bacteroids. The induction of nodule-specific plant genes, including those encoding symbiotic leghemoglobins (Lb), accompanies nodule development. Leghemoglobins accumulate to millimolar concentrations in the cytoplasm of infected plant cells prior to nitrogen fixation and are thought to buffer free oxygen in the nanomolar range, avoiding inactivation of oxygen-labile nitrogenase while maintaining high oxygen flux for respiration. Although widely accepted, this hypothesis has never been tested in planta. Using RNAi, we abolished symbiotic leghemoglobin synthesis in nodules of the model legume Lotus japonicus. This caused an increase in nodule free oxygen, a decrease in the ATP/ADP ratio, loss of bacterial nitrogenase protein, and absence of SNF. However, LbRNAi plants grew normally when fertilized with mineral nitrogen. These data indicate roles for leghemoglobins in oxygen transport and buffering and prove for the first time that plant hemoglobins are crucial for symbiotic nitrogen fixation.
... One of the most studied plant---bacteria relationships is that of symbiotic nitrogen fixation by rhizobia while nodulating Fabaceae 9 . For effective atmospheric nitrogen fixation, rhizobia-colonizing nodules require leghemoglobin to lower the intranodular oxygen concentration, conferring a pink color to functional nodules 8 . In addition to the rhizobia symbiont, other bacteria can improve the nodulation and plant nitrogen fixation rate through diverse mechanisms; these bacteria have been named rhizobia-helper-bacteria (RHB) 15 . ...
... Nodules were classified as pink-reddish or as white-yellowing according to a color scale with representative colors registered in produced nodules ( Fig. 2A). Previous works have shown that whiteyellowing nodules are deficient in leghemoglobin, and are ineffective at fixing nitrogen 8 . In contrast, nodules that are pink-reddish contain leghemoglobin, a characteristic that indicates nitrogen-fixing effectiveness in M. truncatula 13 . ...
... Récemment, certaines souches de Bradyrhizobium ont été identifiées comme capables de fixer l'azote en vie libre, mais aussi lors de l'interaction avec des plantes autres que des légumineuses, comme le sorgho (Hara et al., 2019). (Udvardi et al. 2005 La luzerne est une légumineuse fourragère (genre Medicago) largement cultivée pour son utilité dans l'alimentation animale. L'espèce la plus connue est la luzerne Medicago sativa. ...
... La leghémoglobine présente une forte affinité pour l'oxygène et est responsable de la coloration rose des nodosités fixatrices. La capture de l'O2 par la leghémoglobine va permettre de transporter celui-ci vers les mitochondries pour alimenter la chaîne de transfert d'électrons (Ott et al., 2005). ...
Le monoxyde d’azote (NO) est une petite molécule gazeuse extrêmement réactive intervenant dans de nombreux processus biologiques. Dans les interactions hôte-pathogènes, le NO peut être produit par les deux partenaires et fait partie de l'arsenal de défense de l'hôte tout comme il fait aussi partie des armes d'attaque du pathogène. Les pathogènes se sont aussi adaptés en mettant en place des systèmes de réponse au NO produit par l'hôte. Dans les interactions symbiotiques plante-microorganismes, du NO a été également détecté. C’est le cas durant la symbiose fixatrice d’azote entre la bactérie Ensifer meliloti et la légumineuse Medicago truncatula. Dans cette interaction il a été montré que non seulement le NO est important lors de l'infection par le symbionte, mais qu'il peut aussi avoir un rôle à des étapes plus tardives comme lors de la fixation d'azote ou de la senescence nodulaire. Dans les nodules, le NO est produit par les deux partenaires avec une contribution d’environ 30% pour la bactérie. Chez la plante, des travaux récents ont montré que la synthèse de NO reposait en grande partie sur des nitrate réductases couplées à la chaine mitochondriale de transfert d'électrons. Coté bactérien, seule la voie de dénitrification était connue comme voie de synthèse possible du NO. Ce travail de thèse pose deux questions centrales : Quelles sont les voies de synthèse du NO chez E. meliloti ? et quel est le rôle du NO produit par les bactéries dans l'interaction symbiotique ? Nous avons montré que E. meliloti ne possède pas de NO synthase capable de produire du NO comme il en existe chez certaines bactéries pathogènes. Seule la voie de dénitrification est responsable de la synthèse du NO chez E. meliloti en vie libre et durant la symbiose. Nous avons aussi montré que la bactérie possède une voie assimilatrice du nitrate fonctionnelle, permettant de contribuer à la production de NO en augmentant la quantité de nitrite disponible pour alimenter la voie de dénitrification. La voie assimilatrice est active aussi bien en aérobie qu'en conditions micro-aérobiques mais ne contribuerait pas à la production de NO dans les nodules symbiotiques. Des résultats préliminaires montrent que cette voie serait régulée par un système à deux composants mais aussi, de façon originale, par un ARN non codant. Enfin nous avons montré que le NO produit par la bactérie ne présente pas de rôle essentiel, ni durant les étapes précoces, ni dans les étapes plus tardives de la symbiose. L'ensemble des résultats nous permet de proposer un modèle des voies de production de NO chez E. meliloti et suggère que dans les interactions symbiotiques le NO bactérien ne joue pas un rôle aussi déterminant que dans les interactions hôtes-pathogènes.
... Thus, bacteroids require high rate flux of O 2 to enable high rates of ATP synthesis, but this must be achieved whilst maintaining a very low concentration of free O 2 to avoid inactivation of O 2 -labile nitrogenase. These conditions exist due to the presence of an O 2 diffusion barrier and the synthesis of nodule-specific leghemoglobins, which accumulate to millimolar concentrations in the cytoplasm of infected cells prior to nitrogen fixation and buffer the free O 2 concentration at around 7-11 nM, while maintaining high O 2 flux for respiration (Appleby, 1984;Downie, 2005;Ott et al., 2005). The unique low-O 2 environment provided for the bacteroid is a key signal in bacteroid metabolism, inducing a regulatory cascade controlling gene expression of the nitrogenase complex and the microaerobic respiratory enzymes of the bacteroid. ...
... Leghemoglobin (Lb) is also a general physiological marker of SIS. As mentioned before, Lb has a crucial role in nodule functioning (Ott et al., 2009;Ott et al., 2005). Decrease of Lb content has been shown during drought stress (Gogorcena et al., 1995;Gordon et al., 1997;Guerin et al., 1990), salt stress Mhadhbi et al., 2011) and dark stress (Gogorcena et al., 1997;Matamoros et al., 1999). ...
... Therefore, it is important to keep O 2 levels low enough in the central nodule region, which is characterised by N 2 -fixing symbiotic cells. In nodules of Lotus japonicus and Medicago sativa , a steep O 2 gradient from the surface to the innermost part was observed, ranging from 250 μM to 10-40 nM and characterised by high bacterial and mitochondrial respiration rates (Ott et al., 2009(Ott et al., , 2005Soupène et al., 1995). Legume nodules have evolved mechanisms to maintain low O 2 levels by differentiating an O 2 diffusion barrier and expressing symbiotic plant leghemoglobin (Lb), which regulates the rapid transport of O 2 to the site of respiration . ...
... Legume nodules have evolved mechanisms to maintain low O 2 levels by differentiating an O 2 diffusion barrier and expressing symbiotic plant leghemoglobin (Lb), which regulates the rapid transport of O 2 to the site of respiration . Indeed, knockdown of Lb by RNA interference in L. japonicus increased the level of free O 2 in the nodule, dramatically reduced the amount of nitrogenase protein and suppress N 2 -fixation (Ott et al., 2005). At the same time, nodules must maintain a high level of ATP for nitrogenase and N 2 fixation activities, which are very energy demanding. ...
Group VII Ethylene Response Factors (ERF-VII) are plant-specific transcription factors (TFs) known for their role in the activation of hypoxia-responsive genes under low oxygen stress but also in plant endogenous hypoxic niches. However, their function in the microaerophilic nitrogen-fixing nodules of legumes has not yet been investigated. We investigated regulation and the function of the two Medicago truncatula ERF-VII TFs ( MtERF74 and MtERF75 ) in roots and nodules, MtERF74 and MtERF75 in response to hypoxia stress and during the nodulation process using an RNA interference strategy and targeted proteolysis of MtERF75. Knockdown of MtERF74 and MtERF75 partially blocked the induction of hypoxia-responsive genes in roots exposed to hypoxia stress. In addition, a significant reduction in nodulation capacity and nitrogen fixation activity was observed in mature nodules of double knockdown transgenic roots. Overall, the results indicate that MtERF74 and MtERF75 are involved in the induction of MtNR1 and Pgb1.1 expression for efficient Phytogb-NO respiration in the nodule.
... Haag et al., 2011 ;Afin de protéger la nitrogénase bactérienne vis-à-vis de l'oxygène, la plante hôte régule la diffusion de l'oxygène dans les nodosités par la production de leghémoglobine (Lb), une protéine capable de fixer l'oxygène atmosphérique et de réduire son taux dans la nodosité. La leghémoglobine confère aux nodosités une pigmentation rose qui est un indicateur d'un état fixateur d'azote des nodosités(Ott et al., 2005).Le maintien de la viabilité des bactéroïdes et le bon fonctionnement des nodosités requiert des gènes capables de réprimer le système immunitaire des nodosités (Wang et al., 2016 ; Gourion et al., 2015 ; Berrabah et al. ; 2014a ; Bourcy et al., 2013 ; Veereshlingam et al., 2004). Plusieurs gènes symbiotiques sont impliqués dans ce processus comme par perturbe le mécanisme de protection des bactéroïdes du système immunitaire de la plante hôte. ...
... In 397 the same vein, the Leghemoglobin Lb120-1 was strongly accumulated in the cytosolic 398 fraction (F3) of WT plant. This oxygen carrier produced by legumes has been shown, in 399 another legume species, Lotus japonicus, to be crucial for nitrogen fixation (Ott et al., 2005). 400 Both nitrogenase and leghemoglobin are often used as markers of nodule functionality. ...
Les légumineuses en milieu carencé en azote, établissent une relation symbiotique avec les bactéries du sol appelées rhizobia. Cette interaction conduit à la formation d’un nouvel organe racinaire, la nodosité. Au sein de celle-ci, les rhizobia se différencient en bactéroïdes fixant l’azote atmosphérique au profit de la plante. La colonisation massive et chronique des cellules symbiotiques de nodosités par les rhizobia ne déclenche aucune réaction de défense visible. Au laboratoire nous avons isolé deux mutants symbiotiques développant des réactions de défense dans les nodosités de Medicago truncatula indiquant qu’il existe un contrôle strict de l’immunité dans cet organe. L’objectif de cette thèse est de comprendre comment l’immunité symbiotique contrôle les voies de signalisation hormonales de défense afin d’héberger le partenaire symbiotique et de trouver de nouveaux outils pour mieux comprendre les mécanismes de défense. Pour cela, des approches moléculaires, pharmacologiques et génétiques sont utilisées. Les résultats obtenus dans cette thèse suggèrent que les mécanismes de défense adoptés par M. truncatula varient en fonction de l’écotype de la plante. L’écotype A17 exploite deux voies de résistance : la voie de la sénescence et la voie des réactions de défense. Cependant, l’écotype R108 n’exploite que la voie des réactions de défense. Ce travail suggère également que chez M. truncatula A17, la protéine SymCRK réprime la voie de signalisation de l’acide jasmonique qui conduit au déclenchement de la sénescence, tandis que la protéine DNF2 réprime principalement la voie de signalisation de l’acide salicylique qui conduit au déclenchement des réactions de défense et aussi réprime secondairement la voie acide jasmonique. Chez M. truncatula R108, les deux protéines SymCRK et DNF2 contrôlent les voies de signalisation de l’acide salicylique et de l’éthylène, avec DNF2 contrôlant préférentiellement la voie acide salicylique et SymCRK contrôlant préférentiellement la voie éthylène. Cette thèse suggère aussi l’implication des protéines R dans le contrôle de l’accommodation intracellulaire des rhizobia. Un gène CNL-5 semble être impliqué dans le contrôle de l’infection des cellules symbiotiques, et deux autres gènes CNL-4 et TNL-2 semblent être impliqués dans le contrôle de l’efficacité de l’infection. Finalement, cette thèse a permis d’isoler une souche bactérienne E. adhaerens, capable se comporter comme un symbiote ou comme un pathogène pour plusieurs espèces de Medicago.
... Therefore, in the next step, we checked the impact of drought on the localization of arabinogalactan proteins (AGPs) in the structures responsible for bacteria-lupine interactions-nodules. We used JIM13 [37] and JIM8 [38] antibodies ( Figure 12). It seems that JIM13 binds more effectively than the other tested antibody since the fluorescent signal is higher ( Figure 12A,B). ...
... The first of them are present in the nodules of Fabaceae plants [36]. These hemoglobins are involved in N 2 fixation; however, they are not required for general growth developmental processes [37]. In turn, non-symbiotic hemoglobins are present in various tissues of many plant species, especially crops [38,39]. ...
We recently showed that yellow lupine is highly sensitive to soil water deficits since this stressor disrupts nodule structure and functioning, and at the same time triggers flower separation through abscission zone (AZ) activation in the upper part of the plant. Both processes require specific transformations including cell wall remodeling. However, knowledge about the involvement of particular cell wall elements in nodulation and abscission in agronomically important, nitrogen-fixing crops, especially under stressful conditions, is still scarce. Here, we used immuno-fluorescence techniques to visualize dynamic changes in cell wall compounds taking place in the root nodules and flower AZ of Lupinus luteus following drought. The reaction of nodules and the flower AZ to drought includes the upregulation of extensins, galactans, arabinans, xylogalacturonan, and xyloglucans. Additionally, modifications in the localization of high- and low-methylated homogalacturonans and arabinogalactan proteins were detected in nodules. Collectively, we determined for the first time the drought-associated modification of cell wall components responsible for their remodeling in root nodules and the flower AZ of L. luteus. The involvement of these particular molecules and their possible interaction in response to stress is also deeply discussed herein.
... Glyma.10g199100 within the 10-kb flanking region of the SNP with the highest LOD on Chr. 10 encodes a leghemoglobin-related protein, a strong indicator of symbiotic nitrogen fixation [36]. These findings support the previously reported hypothesis that the chlorophyll content might be related to the nitrogen supply in soybean leaves [37,38]. ...
Leaf photosynthesis and biological nitrogen fixation are two of the most important metabolic processes for soybean growth and development. Since they are considered closely linked, measuring chlorophyll content using non-destructive tools, such as a Soil Plant Analysis Development (SPAD) chlorophyll meter, may help determine the nodulation and nitrogen fixation status of soybean plants. This study aimed to identify single nucleotide polymorphism (SNP) markers associated with SPAD values in a global panel of 187 diverse accessions. The population structure and genome-wide association analyses were carried out using 1,243 high-quality SNPs and greenhouse data obtained in two consecutive years. The results revealed 14 SNPs significantly related to SPAD values on Chr. 5, 10, 12, 15, 17, and 18. In addition, 33 candidate genes were found in the Glyma.Wm82.a2 within 10 kb flanking regions of each significant SNP. Of these, three candidate genes on Chr. 10 and 12 encoded proteins related to photosynthesis, chlorophyll content, and nitrogen status. Overall, our data may help better understand the underlying molecular mechanisms controlling chlorophyll content in relation to nitrogen fixation in soybean.
... Interestingly, Kurdali et al. (2019) reported that Si's influence on root nodulation and nitrogen fixation in Sesbania legume was markedly enhanced under conditions of salinity and/or water stress, though these effects were lessened in the absence of such stressors. Furthermore, Garg and Singh (2018) demonstrated that Si facilitated nodule function through the upregulation of leghemoglobin production in pigeon pea genotypes, a hemoprotein critical for modulating oxygen levels within nodules, thus safeguarding the oxygen-sensitive enzyme nitrogenase and ensuring adequate oxygen for bacteroid respiration (Garg and Singh 2018;Ott et al. 2005). ...
Key message
The study unveils Si's regulatory influence by regulating DEGs, TFs, and TRs. Further bHLH subfamily and auxin transporter pathway elucidates the mechanisms enhancing root development and nodulation.
Abstract
Soybean is a globally important crop serving as a primary source of vegetable protein for millions of individuals. The roots of these plants harbour essential nitrogen fixing structures called nodules. This study investigates the multifaceted impact of silicon (Si) application on soybean, with a focus on root development, and nodulation employing comprehensive transcriptomic analyses and gene regulatory network. RNA sequence analysis was utilised to examine the change in gene expression and identify the noteworthy differentially expressed genes (DEGs) linked to the enhancement of soybean root nodulation and root development. A set of 316 genes involved in diverse biological and molecular pathways are identified, with emphasis on transcription factors (TFs) and transcriptional regulators (TRs). The study uncovers TF and TR genes, categorized into 68 distinct families, highlighting the intricate regulatory landscape influenced by Si in soybeans. Upregulated most important bHLH subfamily and the involvement of the auxin transporter pathway underscore the molecular mechanisms contributing to enhanced root development and nodulation. The study bridges insights from other research, reinforcing Si’s impact on stress-response pathways and phenylpropanoid biosynthesis crucial for nodulation. The study reveals significant alterations in gene expression patterns associated with cellular component functions, root development, and nodulation in response to Si.
Graphical abstract
... Symbiotic diazotrophs, on the other hand, are harbored in a specialized niche (i.e., nodules), in which sufficient energy supply and suitable conditions are provided by plant hosts in exchange for ammonium. Symbiotic BNF is one of the most important contributors to the global N budget in agricultural systems (Pankievicz et al., 2019), but is mainly restricted to leguminous crops (Killham, 1994;Mus et al., 2016;Ott et al., 2005;Sprent et al., 1988;Sylvia et al., 1999;Voisin et al., 2002;Wagner, 2011;Zeng et al., 2016). It is a process with high specificity: once compatible diazotrophs infect the host root hair, they induce the nodule formation and start fixing N 2 into NH 4 + , which later diffuses out of diazotroph cells for plant hosts to assimilate (Stacey, 2007). ...
Perennial tall grasses show promise as bioenergy crops due to high productivity and efficient nutrient use. Ongoing research on bioenergy grasses seeks to reduce their reliance on synthetic nitrogen (N) fertilizer, the manufacture of which relies on fossil fuel combustion. Excessive use of fertilizers also causes adverse environmental consequences and leads to the evolutionary loss of plant traits beneficial to sustainable N cycle. Notably, perennial tall grasses have exhibited the potential to maintain high biomass yield without the need for N fertilizer or causing soil N depletion. Perennial grasses can be adept at interacting with their microbial partners to facilitate N acquisition and retention via mechanisms such as biological N fixation and nitrification inhibition. These inherent N management traits should be preserved and optimized at the this early stage of bioenergy grass breeding programs. This review examines the impact of external N on bioenergy grass production and explores the potential of leveraging advantageous N‐cycling attributes of perennial tall grasses, laying groundwork for future management and research efforts. With minimized dependency on external N input, the cultivation of perennial energy grasses will pave the way toward more resilient agricultural systems and play a significant role in addressing key global energy and environmental challenges.
... The two stable transgenic lines (OE1 and OE2) showed an increase of 33.6% and 27.6% in the total nodule number, an increase of 69.9% and 57.1% in nodule fresh weight, resulting in increased signal nodule weight, and an increase of 47.8% and 32.7% in nitrogenase activity, respectively ( Figure 4A-E). On the other hand, the functional and active nodules were always pink due to the presence of the leghemoglobin (GmLbc3) protein, an oxygen carrier, required for nitrogenase activity and biological nitrogen fixation in nodules [23]. We found that the expression of GmLbc3 in nodules of two GmPAP4 OE lines was greatly increased compared with that in WT nodules ( Figure 4F). ...
Legume crops establish symbiosis with nitrogen-fixing rhizobia for biological nitrogen fixation (BNF), a process that provides a prominent natural nitrogen source in agroecosystems; and efficient nodulation and nitrogen fixation processes require a large amount of phosphorus (P). Here, a role of GmPAP4, a nodule-localized purple acid phosphatase, in BNF and seed yield was functionally characterized in whole transgenic soybean (Glycine max) plants under a P-limited condition. GmPAP4 was specifically expressed in the infection zones of soybean nodules and its expression was greatly induced in low P stress. Altered expression of GmPAP4 significantly affected soybean nodulation, BNF, and yield under the P-deficient condition. Nodule number, nodule fresh weight, nodule nitrogenase, APase activities, and nodule total P content were significantly increased in GmPAP4 overexpression (OE) lines. Structural characteristics revealed by toluidine blue staining showed that overexpression of GmPAP4 resulted in a larger infection area than wild-type (WT) control. Moreover, the plant biomass and N and P content of shoot and root in GmPAP4 OE lines were also greatly improved, resulting in increased soybean yield in the P-deficient condition. Taken together, our results demonstrated that GmPAP4, a purple acid phosphatase, increased P utilization efficiency in nodules under a P-deficient condition and, subsequently, enhanced symbiotic BNF and seed yield of soybean.
... The oxygen bound to leghemoglobin does not harm nitrogenase but is accessible to bacteroids, ensuring adequate respiration for N fixation [116,117]. Thus, leghemoglobin plays a crucial role in the N fixation process [118]. ...
The use of cold plasma (CP) seed treatment is an emerging agricultural technology that exhibits the potential to enhance nodulation and symbiotic nitrogen fixation (SNF) in legumes. CP is composed of a diverse mixture of excited atoms, molecules, ions, and radicals that have the potential to affect the physical properties of the seed and influence gene expressions that could have a lasting impact on the nodulation, SNF, growth, and yield of legumes. The direct participation of the CP in the nodulation process and its correlation with the escalation of nodules and SNF is still not fully understood. This review discussed four areas in the nodulation and SNF process that can directly or indirectly affect CP seed treatments: root–rhizobia signal exchange pathways, root/shoot growth and development, phytohormone production, and the nitrogen fixation process. We also discuss the potential challenges and future research requirements associated with plasma technology to enhance SNF in legumes.
... The presence of LegH in the root nodules of legumes is therefore necessary for the proper functioning of nitrogenase ( Figure 2). It takes up as much as 40% of the total share of soluble proteins in papillae [57,62,63]. In addition, LegH may also influence plant metabolism, and some studies suggest that it may influence plant defense responses against pathogens. ...
Iron is an essential component for the body, but it is also a major cause for the development of many diseases such as cancer, cardiovascular diseases, and autoimmune diseases. It has been suggested that a diet rich in meat products, especially red meat and highly processed products, constitute a nutritional model that increases the risk of developing. In this context, it is indicated that people on an elimination diet (vegetarians and vegans) may be at risk of deficiencies in iron, because this micronutrient is found mainly in foods of animal origin and has lower bioavailability in plant foods. This article reviews the knowledge on the use of leghemoglobin and plant ferritin as sources of iron and discusses their potential for use in vegetarian and vegan diets.
... However, the symbiosis phenotype did not alter the amount of N derived from BNF as expected, suggesting that the N fixation remained at a high level of efficiency. Leghemoglobin proteins are critical for BNF, and Leghemoglobin (Lb) genes could be used as markers of efficient BNF (Ott et al. 2005). Wang et al. (2019) showed that L. japonicus plants carry three Lb genes that act synergistically to maintain an optimal BNF. ...
The rhizobial and the arbuscular mycorrhizal symbioses are present on legume roots and lead to local and systemic transcriptional changes of common and specific plant genes. Among them, some small GTPase proteins called ROPs (Rho of plants) have been shown to be involved in the establishment of the legume-rhizobia interaction. In this study, we aimed to characterise the effects of LjROP3 knockdown in Lotus japonicus on plant physiology and expression of symbiosis-related genes after single and dual inoculation with rhizobia and arbuscular mycorrhizal fungus. In wild-type (Gifu) plants, the dual inoculation increased the shoot and root dry weight, nitrogen (derived from symbiosis) and phosphate content, and the number of arbuscules or nodules compared with single inoculation treatments. In addition, we observed a decrease in the expression of genes encoding the mycorrhizal transcription factors LjRAM1 and LjRAM2, and the downstream genes involved in ammonium (LjAMT2.2) and phosphate (LjPT4 and LjPT8) uptake by the plant at the arbuscule level when the dual inoculation was compared with fungal inoculation. An alteration in the expression of the Nod factor receptor LjNFR1, but not of LjNFR5, was measured in wild-type (Gifu) L. japonicus plants compared to rop3 plants under dual inoculation. We have also measured a reduction in the expression of genes encoding rhizobial and mycorrhizal transcription factors (LjNIN and LjRAM1), and of the downstream mycorrhizal genes involved in ammonium (LjAMT2.2) and phosphate (LjPT4 and LjPT8) uptake by the plant at the arbuscule level. In addition, the expression of AM fungal genes encoding nutrient transporters (known to be expressed at the arbuscule level) was also altered. In conclusion, despite altered expression of plant genes involved in the functioning of the symbioses, and associated with a reduction in the number of nodules and arbuscules, knockdown of LjROP3 did not alter plant growth and nutrition under dual inoculation, suggesting that the beneficial effects of the dual symbiosis were maintained.
... La mayoría de los nódulos de C. brachystegia (43 de 46) presentaron coloración rosada interna, que indica presencia de leghemoglobina y fijación de N (Ott et al., 2005), y tuvieron tamaño promedio de 3 mm aproximadamente. Los nódulos macerados y luego estriados generaron alrededor de 55 colonias con crecimiento entre las 48 a 96 horas. ...
Rhizobia are one of the best-studied symbionts of leguminous plants, with high efficiency in biological nitrogen fixation and able to supply up to 90% of the plant nitrogen needs. Among legumes from tropical dry forests, Clitoria brachystegia Beth. (Fabaceae) is endemic to this ecosystem and its bacterial symbionts have never been reported. This study aimed to establish the diversity and nitrogen fixation potential of rhizobia isolated from C. brachystegia root nodules found in tropical dry forest remnants. The use of molecular techniques like BOX-PCR and 16S ribosomal gene sequencing, allowed the identification of 24 Rhizobium and 22 Bradyrhizobium strains which showed differential geographic distribution with most Rhizobium being recovered from Ecuador and all Bradyrhizobium from Peru. Diversity varied between sampling sites but no significant relationships were found with soil characteristics or climate conditions. Several Rhizobium and Bradyrhizobium strains showed good nitrogen fixation potential and thus may represent valuable resources in future conservation efforts of C. brachystegia, an endangered plant species.
... The evolution of symbiotic nitrogen fixation provides the microbial partner with an environment that can maintain low oxygen concentration. Leghemoglobin, a protein synthesized by the host plant, plays a unique role in lowering oxygen tension due to its advanced empathy for binding oxygen (Ott et al. 2005). ...
Nitrogen is a major element for plant life, yet environmental nitrogen is poorly available to plant, and thus classified as a ‘limiting element’. As a consequence, most plants, except the insectivorous florae, rely upon microbial partners to maintain the nitrogen supply. Nitrogen-fixing prokaryotes are widely distributed and account for the fixation of nearly 50–200 megatonnes of nitrogen per year. Nitrogen-fixing microorganisms are potent agents for applications in agricultural fields. Reduction of gaseous dinitrogen to bioavailable nitrogen is mainly done by the molybdenum-dependent nitrogenase in archaea and eubacteria. In plants, the process of nodulation has evolved from 100 million years ago, confering the nodulation capability to about 70% of leguminous plant species. The genes necessary for the nitrogen fixation evolved only after the divergence of bacteria and archaea. Furthermore, the nitrogen-fixing endosymbionts are supposed to have evolved many times in the higher plants, especially in angiosperms. This chapter reviews the diversity and evolution of nitrogen-fixing bacteria.KeywordsNitrogenNitrogen fixationEndosymbiosisRhizobiaceaeNitrogenaseSymbiotic nitrogen fixationAssociative nitrogen fixation
Nostoc
EvolutionEvolutionary analysis
... Legume hosts provide carbon and other essential resources for bacteroids to support the energetically expensive but O 2 -sensitive nitrogen-fixing reaction [2]. To allow respiration of bacteroids but avoid nitrogenase inactivation, free O 2 in host cells is modulated by leghemoglobins as low as~50 nM [11][12][13][14]. These distinct features support the rhizobium-legume symbiotic nitrogen fixation (SNF) as the most efficient biological nitrogen fixation system in nature. ...
There are ubiquitous variations in symbiotic performance of different rhizobial strains associated with the same legume host in agricultural practices. This is due to polymorphisms of symbiosis genes and/or largely unexplored variations in integration efficiency of symbiotic function. Here, we reviewed cumulative evidence on integration mechanisms of symbiosis genes. Experimental evolution, in concert with reverse genetic studies based on pangenomics, suggests that gain of the same circuit of key symbiosis genes through horizontal gene transfer is necessary but sometimes insufficient for bacteria to establish an effective symbiosis with legumes. An intact genomic background of the recipient may not support the proper expression or functioning of newly acquired key symbiosis genes. Further adaptive evolution, through genome innovation and reconstruction of regulation networks, may confer the recipient of nascent nodulation and nitrogen fixation ability. Other accessory genes, either co-transferred with key symbiosis genes or stochastically transferred, may provide the recipient with additional adaptability in ever-fluctuating host and soil niches. Successful integrations of these accessory genes with the rewired core network, regarding both symbiotic and edaphic fitness, can optimize symbiotic efficiency in various natural and agricultural ecosystems. This progress also sheds light on the development of elite rhizobial inoculants using synthetic biology procedures.
... The nodules are specialized organs consisting of bacteroids, meristems, and vascular bundles. Nitrogen fixation is enabled by a complex of nitrogenase-nitrate reductase enzymes [26] supported by leghaemoglobin proteins located in the nodules that provide oxygen for respiratory processes into the bacteroid membrane as well as reduce the oxygen concentration inside bacteroids [27]. Two types of root nodules are distinguished: determinate and indeterminate [24]. ...
Commonly studied in the context of legume–rhizobia symbiosis, biological nitrogen fixation (BNF) is a key component of the nitrogen cycle in nature. Despite its potential in plant breeding and many years of research, information is still lacking as to the regulation of hundreds of genes connected with plant–bacteria interaction, nodulation, and nitrogen fixation. Here, we compared root nodule transcriptomes of red clover (Trifolium pratense L.) genotypes with contrasting nitrogen fixation efficiency, and we found 491 differentially expressed genes (DEGs) between plants with high and low BNF efficiency. The annotation of genes expressed in nodules revealed more than 800 genes not yet experimentally confirmed. Among genes mediating nodule development, four nod-ule-specific cysteine-rich (NCR) peptides were confirmed in the nodule transcriptome. Gene duplication analyses revealed that genes originating from tandem and dispersed duplication are significantly over-represented among DEGs. Weighted correlation network analysis (WGCNA) organized expression profiles of the transcripts into 16 modules linked to the analyzed traits, such as nitrogen fixation efficiency or sample-specific modules. Overall, the results obtained broaden our knowledge about transcriptomic landscapes of red clover’s root nodules and shift the phenotypic description of BNF efficiency on the level of gene expression in situ.
... Nitrogen fixation in strict anaerobes takes place under anaerobic conditions whereas in facultative anaerobes during their anaerobic growth period. Haemoglobin, a high affinity oxygen-binding protein, provides a microaerobic environment for symbiotic nitrogen fixation and allows the diazotroph to carry out aerobic respiration along with nitrogen fixation (Ott et al. 2005). Likewise, in obligate aerobes, high respiration rates lead to oxygen consumption at the cell membrane known as the cytochrome-dependent respiratory protection mechanism, thus, maintaining low intracellular oxygen concentration, protecting the nitrogen fixation apparatus (Poole and Hill 1997). ...
Fertilizers, which are both environmentally harmful and economically costly, constitute a major part of global agriculture. In rice cultivation, nitrogen is by far the most significant nutritional input, yet it is also the most common limiting factor. Rice cultivation must be developed in a way that reduces the need for chemical N‐fertilizers, while still maintaining high yields, in a world plagued by a global energy crisis and a lack of reliable alternative energy resources. Nitrogen is a key limiting factor in plant growth and production. It is an important component of chlorophyll, the most important pigment required for photosynthesis, as well as amino acids, which are the basic building blocks of proteins. The ability of rice to fix nitrogen appears to be a useful potential system that is consistent with the ideals of resource conservation and ecological security. In this chapter, we will address both classic and innovative features of biological nitrogen fixation in rice‐based agricultural systems, using examples mostly selected from underdeveloped nations.
... Abolishing leghemoglobin synthesis in Lotus japonicus resulted to an increase in nodule free oxygen, a decrease in the ATP/ADP ratio, loss of rhizobial nitrogenase protein, and a collapse of nitrogen fixation (Ott et al., 2005). ...
The agricultural sector is the cornerstone of Kenya’s economy. It employs about 40% of Kenya’s population, more than 70% in the rural areas, and contributes to about 33% of Kenya’s gross domestic product (GDP). Kenya’s vision 2030 identifies agriculture, including common bean cultivation, as one of the main pillars that will play a role in achieving sustainable 10% annual GDP. However, the main impediment to realizing this goal is the high cost of fertilizer which is not affordable for most farmers. An alternative to nitrogenous fertilizers is the application of rhizobia for common bean production, which converts atmospheric dinitrogen to reduced ammonium, suitable for plant use. Inoculant formulations must include local elite rhizobia because they can compete with the indigenous rhizobia in the soil and tolerate the harsh conditions in Kenyan soils. I isolated three nitrogen-fixing rhizobia (B3, S2, and S3) from these soils.
In greenhouse experiments, plants infected with these rhizobia produced more biomass and accumulated more nitrogen than uninoculated plants and plants inoculated with a commercial strain CIAT899 which Kenyan farmers widely use. The biomass of the plants infected by either of the three isolates was comparable to that of plants supplied with nitrogenous fertilizer. Field experiments in Kenya demonstrated that the seed dry weight of plants inoculated with S3 was significantly higher than that of all other plants. S3 conferred more benefits to the plants than S2 and B2 and the commercial strain CIAT899
Kenyan soil faces detrimental abiotic stresses, particularly low pH and the toxic metallic ion Al. The three isolates were better adapted to low pH and Al toxicity than CIAT899. Isolate B3 grew in media with pH 4.8, a pH that was detrimental to the commercial isolate CIAT899. Short-term viability assays and long-term recovery experiments demonstrated that B3 performs better under Al stress than CIAT899. Al did not only bind to the rhizobia membrane but also interfered with its integrity. Al stress promoted leakage of ATP and the cytoplasmic marker protein mScarlet-1 out of the cell, consistent with the observation that the amino acid concentration in Al-treated cells is reduced. Compared to CIAT899, isolate B3 was more Al tolerant. Finally, the three isolates solubilized phosphorus and produced IAA.
The three isolates were characterized as R. phaseoli, although syntenic analysis indicated that their genomes had undergone significant evolutionary changes compared to the reference genome. 48.3% of the genomic sections have either been deleted, inverted, or undergone translocation. The harsh environment in Kenyan soils likely causes these chromosomal changes. Genomic and transcriptomic analyses indicated that genes involved in membrane repair, stabilization, and biogenesis might play an important role in Al tolerance. Genes involved in either active or passive transport of solutes across the membrane or extracellular polysaccharide transport were upregulated during Al stress. The latter observation correlated with the EPS level increase and biofilm formation after Al treatment. The increased activity of the GAD system and elevated amount of GABA in Al-treated cells also suggests a role of GABA in Al tolerance.
... Infected cells synthesize leghemoglobin that buffers free oxygen to the nanomolar range, preventing the inactivation of oxygen-labile nitrogenase while also maintaining high oxygen flux for respiration (Brear et al. 2013). Abolishing leghemoglobin synthesis in Lotus japonicus resulted to an increase in nodule free oxygen, a decrease in the ATP/ADP ratio, loss of rhizobial nitrogenase protein, and a collapse of nitrogen fixation (Ott et al. 2005). ...
The symbiotic interaction of rhizobia with Legume roots results in nodule formation and eventually nitrogen fixation. An alternative to nitrogenous fertilizers is the application of rhizobia for common bean production, which convert atmospheric dinitrogen to reduced ammonium, suitable for plant use. Inoculant formulations must include local elite rhizobia because they can compete with the indigenous rhizobia in the soil and tolerate the harsh conditions found in many tropical soils. I describe the symbiotic interaction between rhizobial strains with common bean roots and the threat to which they are often exposed in harsh environments (low pH; high aluminum levels in soil, drought). Local rhizobial strains are often better adapted to their soil environment and are good tool for improving common bean production in regions with contaminated soil. Endocytobiosis and Cell Research (2022) 31:39-48 Category: Review
... Por otro lado, Pentaclethra macroloba y Zygia longifolia (Caesalpinioideae) sí presentaron nódulos a pesar de tener raíces con tonalidades oscuras. Estas especies podrían tener algún mecanismo de señalización e infección particular o algunas formas distintas de leghemoglobina por la cual tengan una coloración oscura en sus raíces a pesar de ser capaces de formar nódulos [41], [42]. En este sentido, es necesario profundizar sobre el cómo los rasgos anatómicos, e incluso arquitecturales, de las raíces podrían tener relación con el establecimiento de relaciones simbióticas con bacterias fijadoras de nitrógeno. ...
A large part of the success of legume plants (Fabaceae) in the different ecosystems of Costa Rica could be due to their ability to establish symbiotic relationships with microorganisms of the Rhizobiaceae family and to the fixed nitrogen that these bacteria can provide them. In this work, we evaluated the presence of nodules, the tissue where nitrogen fixation is carried out, in 24 forest species of legumes belonging to four subfamilies of Fabaceae, and several morphological characteristics of the roots were determined. The presence of nodules was detected in 14 of the 24 species (58.3 %); however, there were large differences between the subfamilies. In the two subfamilies considered the most basal (Detarioideae and Cercidoideae), no nodules were detected. In the Caesalpinioideae subfamily, nodules were reported in 58.3 % of the species, mostly restricted to the clade Mimosoideae. In the Papilionoideae family, considered the most derived, the presence of nodules was observed in 87.5 % of the evaluated species. Most species with nodules had a light coloration on their roots, and species without nodules had a darker coloration, with some exceptions. These trends are related to the phylogenetic history of the subfamilies and the origins of nodulation. However, they could also respond to different infection mechanisms and could even be related to physiological characteristics of plants, such as the production of secondary inhibitory compounds of the nodulation. In this way, this study constitutes. the first step to understand better the interactions between nitrogen-fixing microorganisms with their hosts. Capacidad de nodulación en especies forestales leguminosas (Fabaceae) según su filogenia y características morfológicas
... Rhizobia are themselves unable to protect nitrogenase from oxygen and this is mainly achieved by formation of an oxygen diffusion layer formed from cells with suberised walls around the periphery of the nodule (Minchin et al., 2008). Leghemoglobin formed by the host binds oxygen for delivery to the terminal cytochrome oxidase of the bacterial electron transport chain, which is especially important in the microaerobic environment at the centre of the nodule (Ott et al., 2005). ...
Nitrogen fixing symbioses between plants and bacteria are ancient and, while not numerous, are formed in diverse lineages of plants ranging from microalgae to angiosperms. One symbiosis stands out as the most widespread one is that between legumes and rhizobia, leading to the formation of nitrogen-fixing nodules. The legume family is one of the largest and most diverse group of plants and legumes have been used by humans since the beginning of agriculture, both as high nitrogen food, as well as pastures and rotation crops. One open question is whether their ability to form a nitrogen-fixing symbiosis has contributed to legumes’ success, and whether legumes have any unique characteristics that have made them more diverse and widespread than other groups of plants.
This review examines the evolutionary journey that has led to the diversification of legumes, in particular its nitrogen-fixing symbiosis, and asks four questions to investigate which legume traits might have contributed to their success: 1. In what ways do legumes differ from other plant groups that have evolved nitrogen-fixing symbioses? In order to answer this question, the characteristics of the symbioses, and efficiencies of nitrogen fixation are compared between different groups of nitrogen fixing plants. 2. Could certain unique features of legumes be a reason for their success? This section examines the manifestations and possible benefits of a nitrogen-rich ‘lifestyle’ in legumes. 3. If nitrogen fixation was a reason for such a success, why have some species lost the symbiosis? Formation of symbioses has trade-offs, and while these are less well known for non-legumes, there are known energetic and ecological reasons for loss of symbiotic potential in legumes. 4. What can we learn from the unique traits of legumes for future crop improvements? While exploiting some of the physiological properties of legumes could be used to improve legume breeding, our increasing molecular understanding of the essential regulators of root nodule symbioses raise hope of creating new nitrogen fixing symbioses in other crop species.
... Full differentiation is achieved in the N-fixation zone where differentiated bacteria, called bacteroids, reduce atmospheric N (N 2 ) into ammonium thanks to the nitrogenase enzymatic complex. In nodules, both expression and functioning of the nitrogenase rely on the microaerobic environment of the N fixation zone, partly achieved through oxygen scavenging by plant leghemoglobins specifically expressed in this zone and responsible for the pink colour of nodules (Ott et al., 2005). ...
Senescence determines plant organ lifespan depending on aging and environmental cues. During the endosymbiotic interaction with rhizobia, legume plants develop a specific organ, the root nodule, which houses nitrogen (N)‐fixing bacteria. Unlike earlier processes of the legume–rhizobium interaction (nodule formation, N fixation), mechanisms controlling nodule senescence remain poorly understood. To identify nodule senescence‐associated genes, we performed a dual plant‐bacteria RNA sequencing approach on Medicago truncatula‐Sinorhizobium meliloti nodules having initiated senescence either naturally (aging) or following an environmental trigger (nitrate treatment or salt stress). The resulting data allowed the identification of hundreds of plant and bacterial genes differentially regulated during nodule senescence, thus providing an unprecedented comprehensive resource of new candidate genes associated with this process. Remarkably, several plant and bacterial genes related to the cell cycle and stress responses were regulated in senescent nodules, including the rhizobial RpoE2‐dependent general stress response. Analysis of selected core nodule senescence plant genes allowed showing that MtNAC969 and MtS40, both homologous to leaf senescence‐associated genes, negatively regulate the transition between N fixation and senescence. In contrast, overexpression of a gene involved in the biosynthesis of cytokinins, well‐known negative regulators of leaf senescence, may promote the transition from N fixation to senescence in nodules. This article is protected by copyright. All rights reserved.
... Thus, expression of the cysteine proteinase at elevated levels (FC = 16.9) may explain the observed early senescence of the bacA mutant nodule. Many downregulated genes have known functions in nodule development, including 10 leghemoglobin genes responsible for delivering oxygen in root nodules (Ott et al. 2005) and key nitrogen metabolic enzymes, such as glutamine synthetase (Silva and Carvalho 2013) and asparagine synthetase (Garcia-Calderon et al. 2017). However, the most prominent group of downregulated genes are those encoding for nodulespecific proteins or late nodulin proteins, 36 in total, and many of which show sequence similarity with the functionally characterized NCR peptides (Supplementary Table S5). ...
Legumes in the inverted repeat-lacking clade (IRLC) each produce a unique set of nodule-specific cysteine-rich (NCR) peptides, which act in concert to determine the terminal differentiation of nitrogen-fixing bacteroid. IRLC legumes differ greatly in their numbers of NCR and sequence diversity. This raises the significant question how bacteroid differentiation is collectively controlled by the specific NCR repertoire of an IRLC legume. Astragalus sinicus is an IRLC legume that forms indeterminate nodules with its microsymbiont Mesorhizobium huakuii 7653R. Here, we performed transcriptome analysis of root and nodule samples at 3, 7, 14, 28 days postinoculation with M. huakuii 7653R and its isogenic ∆ bacA mutant. BacA is a broad-specificity peptide transporter required for the host-derived NCRs to target rhizobial cells. A total of 167 NCRs were identified in the RNA transcripts. Comparative sequence and electrochemical analysis revealed that A. sinicus NCRs (AsNCRs) are dominated by a unique cationic group (termed subgroup C), whose mature portion is relatively long (>60 amino acids) and phylogenetically distinct and possessing six highly conserved cysteine residues. Subsequent functional characterization showed that a 7653R variant harboring AsNCR083 (a representative of subgroup C AsNCR) displayed significant growth inhibition in laboratory media and formed ineffective white nodules on A. sinicus with irregular symbiosomes. Finally, bacterial two-hybrid analysis led to the identification of GroEL1 and GroEL3 as the molecular targets of AsNCR067 and AsNCR076. Together, our data contribute to a systematic understanding of the NCR repertoire associated with the A. sinicus and M. huakuii symbiosis.
[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .
... Infected cells synthesize leghemoglobin that is thought to buffer free oxygen in the nanomolar range, avoiding the inactivation of oxygen-labile nitrogenase while maintaining high oxygen flux for respiration [25]. Abolishing leghemoglobin synthesis in Lotus japonicus caused an increase in nodule free oxygen, a decrease in the ATP/ADP ratio, loss of rhizobial nitrogenase protein, and an absence of nitrogen fixation [26]. ...
Grain legumes play a significant role in smallholder farming systems in Africa because of their contribution to nutrition and income security and their role in fixing nitrogen. Biological Nitrogen Fixation (BNF) serves a critical role in improving soil fertility for legumes. Although much research has been conducted on rhizobia in nitrogen fixation and their contribution to soil fertility, much less is known about the distribution and diversity of the bacteria strains in different areas of the world and which of the strains achieve optimal benefits for the host plants under specific soil and environmental conditions. This paper reviews the distribution, characterization, and commer-cialization of elite rhizobia strains in Africa.
... The activities of enzymes involved in the ascorbate-glutathione pathway are higher in N-fixing nodules with high SNF capacity than in nodules with lower SNF capacity (Dalton, Langeberg, & Treneman, 1993). Similar results were obtained in L. japonicus lines with leghemoglobin-RNA interference (Gunther, Schlereth, Udvardi, & Ott, 2007;Ott et al., 2005). ...
Reactive oxygen species (ROS) are essential regulators of plant growth, development and defense. The regulated compartment-specific production and processing of ROS are central to the reduction-oxidation (redox) balance of the intracellular and extracellular environments. ROS generation has often been considered to be harmful, because of the potential collateral damage, but their high chemical reactivity is essential to their roles as pro-life signals. Nevertheless, literature evidence demonstrates that the ability to mount a robust antioxidant defense is integral to stress tolerance in soybean as in other plant species. Moreover, the integration of redox signaling with reactive nitrogen species and molecular oxygen signaling is crucial to the functioning of many plant organs, such as seeds and nodules, which are maintained in a state of developmental hypoxia. Here, we describe ROS production, processing and signaling in soybean, particularly in relation to nodule development and protection against environmental stresses. We also consider the interactions between ROS and nitric oxide (NO), highlighting gaps in current knowledge and considering future research directions in exploring redox metabolism in soybean.
... Bacteroid respiration necessitates a high O 2 flux, but this must be done while maintaining a very low free oxygen concentration. Leghemoglobin, a family of O 2 binding heme proteins that functions similarly to myoglobin, is typically used by legume nodules to maintain a low O 2 concentration (Downie, 2005;Ott et al., 2005;Senthilkumar, Amaresan, & Sankaranarayanan, 2021). A low oxygen environment required as nitrogenase is very oxygen sensitive. ...
Nitrogen is the most essential macronutrient for the growth of plants. The current scenario of soil engineering is based on synthetic nitrogen fertilizer and imposing economical as well as environmental stress. Biological nitrogen fixation (BNF) is the only strategy to fulfill the nitrogen demand of plants in a sustainable manner. BNF provides N, which is economic and ecofriendly, reduces the use of chemical fertilizers, and improves the internal resources by engineering the rhizosphere. Indigenous diazotrophs increase the crop productivity, availability or uptake of nutrients through hormonal action or antibiosis. The main objective of rhizosphere engineering is reducing our dependence on agrochemicals by replacing them with ecofriendly beneficial native microbes or introducing the genetically engineered microbes, biostimulants in the soil. Hence, it will help us in developing environment-friendly sustainable agriculture.
... Cumulative evidence suggests that Irr binds DNA targets under low-iron conditions (16), while RirA functions under high-iron conditions (29,31,(33)(34)(35). Given the high level of iron-containing nitrogenase and leghemoglobin in nodules (19,42,43), nitrogen-fixing rhizobia in nodule cells are supposed to be under iron-replete conditions (18). Then, it could be hypothesized that rhizobial RirA, rather than Irr, might be active in nodules. ...
Iron homeostasis is strictly regulated in cellular organisms. The Rhizobiales order enriched with symbiotic and pathogenic bacteria has evolved a lineage-specific regulator, RirA, responding to iron fluctuations. However, the regulatory role of RirA in bacterium-host interactions remains largely unknown. Here, we report that RirA is essential for mutualistic interactions of Sinorhizobium fredii with its legume hosts by repressing a gene cluster directing biosynthesis and transport of petrobactin siderophore. Genes encoding an inner membrane ABC transporter (fat) and the biosynthetic machinery (asb) of petrobactin siderophore are sporadically distributed in Gram-positive and Gram-negative bacteria. An outer membrane siderophore receptor gene (fprA) was naturally assembled with asb and fat, forming a long polycistron in S. fredii. An indigenous regulation cascade harboring an inner membrane protease (RseP), a sigma factor (FecI), and its anti-sigma protein (FecR) were involved in direct activation of the fprA-asb-fat polycistron. Operons harboring fecI and fprA-asb-fat, and those encoding the indigenous TonB-ExbB-ExbD complex delivering energy to the outer membrane transport activity, were directly repressed by RirA under iron-replete conditions. The rirA deletion led to upregulation of these operons and iron overload in nodules, impaired intracellular persistence, and symbiotic nitrogen fixation of rhizobia. Mutualistic defects of the rirA mutant can be rescued by blocking activities of this naturally "synthetic" circuit for siderophore biosynthesis and transport. These findings not only are significant for understanding iron homeostasis of mutualistic interactions but also provide insights into assembly and integration of foreign machineries for biosynthesis and transport of siderophores, horizontal transfer of which is selected in microbiota.
IMPORTANCE Iron is a public good explored by both eukaryotes and prokaryotes. The abundant ferric form is insoluble under neutral and basic pH conditions, and many bacteria secrete siderophores forming soluble ferric siderophore complexes, which can be then taken up by specific receptors and transporters. Siderophore biosynthesis and uptake machineries can be horizontally transferred among bacteria in nature. Despite increasing attention on the importance of siderophores in host-microbiota interactions, the regulatory integration process of transferred siderophore biosynthesis and transport genes is poorly understood in an evolutionary context. By focusing on the mutualistic rhizobium-legume symbiosis, here, we report how a naturally synthetic foreign siderophore gene cluster was integrated with the rhizobial indigenous regulation cascade, which is essential for maintaining mutualistic interactions.
... Rhizobia are obligate aerobic microorganisms that require oxygen for their metabolism, but they also need the level of O2 to be precisely regulated to protect the nitrogen fixation process. Leghemoglobin is one of the key players required for efficient nitrogenase activity, limiting free O2 levels while also maintaining an adequate O2 flow to support vigorous respiration by bacteroids and host mitochondria [47]. In the current study, the transcript accumulation of leghemoglobin in silenced roots did not increase as it did in control roots at 14 dpi ( Figure S12), pointing to a transcriptional relationship between PvMT1A and leghemoglobin during root nodule symbiosis in P. vulgaris. ...
Metallothioneins (MTs) constitute a heterogeneous family of ubiquitous metal ion-binding proteins. In plants, MTs participate in the regulation of cell growth and proliferation, protection against heavy metal stress, oxidative stress responses, and responses to pathogen attack. Despite their wide variety of functions, the role of MTs in symbiotic associations, specifically nodule-fabacean symbiosis, is poorly understood. Here, we analyzed the role of the PvMT1A gene in Phaseolus vulgaris-Rhizobium tropici symbiosis using bioinformatics and reverse genetics approaches. Using in silico analysis, we identified six genes encoding MTs in P. vulgaris, which were clustered into three of the four classes described in plants. PvMT1A transcript levels were significantly higher in roots inoculated with R. tropici at 7 and 30 days post inoculation (dpi) than in non-inoculated roots. Functional analysis showed that downregulating PvMT1A by RNA interference (RNAi) reduced the number of infection events at 7 and 10 dpi and the number of nodules at 14 and 21 dpi. In addition, nodule development was negatively affected in PvMT1A:RNAi transgenic roots, and these nodules displayed a reduced nitrogen fixation rate at 21 dpi. These results strongly suggest that PvMT1A plays an important role in the infection process and nodule development in P. vulgaris during rhizobial symbiosis.
... Les témoins déterrés ne possèdent pas de nodules. Les nodules obtenus sont de forme indéterminée, sont presque de la même taille (figure 23), de couleur rose (dû à la présence de la léghémoglobine) signe de l'efficience des souches (Downie, 2005 ;Ott et al., 2005) (figure 24). Toutes les souches isolées (sauf la S12) ont nodulé leur plante hôte, ce qui indique leur infectivité et leur appartenance aux BNL. ...
Les symbioses végétales sont une composante fondamentale de la stabilité et de la durabilité des écosystèmes. Dans des sites dégradés et fortement appauvris en éléments nutritifs, l’introduction de légumineuses associées à leurs auxiliaires microbiens en l’occurrence les rhizobiums et les champignons mycorhiziens sont impliqués respectivement dans la fixation biologique de l’azote et dans la mobilisation entre autres du phosphore. Cette symbiose est une condition primordiale pour réussir l’installation de plants en milieu particulièrement contraignant tels que les sites de carrière après exploitation sous climat méditerranéen.
Le choix des plantes symbiotiques utilisables pour la revégétalisation repose, d’une part, sur la facilité de la multiplication de l’espèce des légumineuses et d’autre part, sur la sélection et la production de rhizobiums partenaires efficaces. L’enjeu est d’identifier les meilleures souches de Rhizobium associées à Acacia saligna, puis de les associer avec des partenaires mycorhiziens en pépinière pour les transférer dans le milieu dégradé où elles exprimeront leur potentiel. C’est ainsi que 25 isolats ont été obtenus à partir des nodules racinaires d’Acacia saligna. Ces isolats présentent des morphologies comparables à celle des rhizobia décrites par plusieurs auteurs sauf un isolat qui n’a pas nodulé la plante hôte.
Le choix de la sélection des souches pour la revégétalisation de la sablière de Terga s’est basé uniquement sur leur performance symbiotique dans des conditions contrôlées étant donné que les analyses physico-chimiques n’ont révélé aucune contrainte au développement des rhizobiums (sol à pH neutre et non salé), et de l’Acacia saligna (sol de texture sablonneuse), et d’autre part sur la concentration de l’inoculum afin de pallier au problème de compétitivité avec les souches locales. Ces dernières sont inoculées à de jeunes plants d’Acacia saligna en pépinière avec leur partenaire mycorhizien obtenu in-natura des racines fraiches d’Acacia saligna et dont la fréquence de mycorhization est égale à 100%.
... Leghemoglobin maintains the low O 2 cellular environment required for nitrogenase activity, while facilitating the flux of O 2 toward respiration sites in infected nodule cells (Appleby, 1984;Downie, 2005). The production of leghemoglobin is essential for effective N 2 fixation, and its diminished accumulation, in white nodules, is associated with a Fix − phenotype (Ott et al., 2009;Ott et al., 2005). To confirm that white nodules indeed contain low amounts of leghemoglobin (i.e., are Fix − ), we compared leghemoglobin levels in wild-type and mutant white nodules formed at 30 dpi. ...
De novo purine biosynthesis is required for the incorporation of fixed nitrogen in ureide exporting nodules, as formed on soybean [Glycine max (L.) Merr.] roots. However, in many cases, the enzymes involved in this pathway have been deduced strictly from genome annotations with little direct genetic evidence, such as mutant studies, to confirm their biochemical function or importance to nodule development. While efforts to develop large mutant collections of soybean are underway, research on this plant is still hampered by the inability to obtain mutations in any specific gene of interest. Using a forward genetic approach, as well as CRISPR/Cas9 gene editing via Agrobacterium rhizogenes‐mediated hairy root transformation, we identified and characterized the role of GmUOX (Uricase) and GmXDH (Xanthine Dehydrogenase) in nitrogen fixation and nodule development in soybean. The gmuox knockout soybean mutants displayed nitrogen deficiency chlorosis and early nodule senescence, as exemplified by the reduced nitrogenase (acetylene reduction) activity in nodules, the internal greenish‐white internal appearance of nodules, and diminished leghemoglobin production. In addition, gmuox1 nodules showed collapsed infected cells with degraded cytoplasm, aggregated bacteroids with no discernable symbiosome membranes, and increased formation of poly‐β‐hydroxybutyrate granules. Similarly, knockout gmxdh mutant nodules, generated with the CRISPR/Cas9 system, also exhibited early nodule senescence. These genetic studies confirm the critical role of the de novo purine metabolisms pathway not only in the incorporation of fixed nitrogen but also in the successful development of a functional, nitrogen‐fixing nodule. Furthermore, these studies demonstrate the great utility of the CRISPR/Cas9 system for studying root‐associated gene traits when coupled with hairy root transformation.
... According to Escuredo and co-workers [28] leghaemoglobin accounts for over 21% of the soluble proteins in the root nodules. It provides a micro-anaerobic environment inside the nodule, which is essential for the proper nitrogen fixation [29]. Since leghaemoglobin is a mostly α-helical protein, it can be suggested that the high amount α-helical proteins in the nodules from plants treated with purified biofertilizer is linked with a high amount of leghaemoglobin. ...
Multimodal spectroscopic imaging methods such as Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI MSI), Fourier Transform Infrared spectroscopy (FT-IR) and Raman spectroscopy were used to monitor the changes in distribution and to determine semi quantitatively selected metabolites involved in nitrogen fixation in pea root nodules. These approaches were used to evaluate the effectiveness of nitrogen fixation by pea plants treated with biofertilizer preparations containing Nod factors. To assess the effectiveness of biofertilizer, the fresh and dry masses of plants were determined. The biofertilizer was shown to be effective in enhancing the growth of the pea plants. In case of metabolic changes, the biofertilizer caused a change in the apparent distribution of the leghaemoglobin from the edges of the nodule to its centre (the active zone of nodule). Moreover, the enhanced nitrogen fixation and presumably the accelerated maturation form of the nodules were observed with the use of a biofertilizer.
... L'expression de la nitrogénase démarre en zone II, mais cette enzyme n'est fonctionnelle qu'en condition de microoxie, c'est-à-dire en zone III. La mise en place de l'environnement microoxique en zone III est réalisée grâce d'une part à la présence dans le cortex d'une barrière à l'O2, qui limite la diffusion des gaz extérieurs à l'intérieur de la nodosité (Batut and Boistard, 1994), et, d'autre part, à la présence, en grande quantité, d'une protéine végétale affine pour l'O2 de la famille des hémoglobines, la leghémoglobine, qui facilite la diffusion de l'O2 vers les mitochondries pour alimenter la chaîne de transfert d'électrons (Ott et al., 2005). Afin de réduire la concentration en oxygène en zone III, une respiration mitochondriale de type « phytoglobine-oxyde nitreux (NO)» est mise en place. ...
Lors de la symbiose fixatrice d’azote entre la légumineuse modèle Medicago truncatula et la rhizobiacée Sinorhizobium meliloti, les bactéries, différenciées en bactéroïdes dans la nodosité, réduisent l’azote atmosphérique (N2) en ammoniac (NH3), directement assimilable par la plante. En contrepartie, la plante fournit à son symbiote une niche écologique et des substrats carbonés. Tout au long de l’interaction, la bactérie est confrontée à des microenvironnements changeants depuis la vie libre dans le sol, l’infection et l’invasion des cellules végétales, la différenciation en bactéroïde fixateur et finalement la rupture symbiotique. Afin de mieux comprendre les mécanismes d‘adaptation rapide développés par S. meliloti lors de la symbiose, nous nous sommes intéressées au rôle des systèmes Toxine-Antitoxine (TA) bactériens de type VapBC dans ce processus. Ces modules, composés d’une toxine et de son antitoxine apparentée, sont associés chez les bactéries pathogènes à la réponse aux stress et à l’adaptation à la vie intracellulaire. La toxine, de par son activité RNase site-spécifique, permet une inhibition globale ou partielle de la traduction. Chez S. meliloti, 11 systèmes VapBC chromosomiques putatifs ont été identifiés et deux seulement (VapC4 et VapC5) ont été caractérisés en interaction symbiotique. Au cours de cette thèse, nous avons tout d’abord démontré, par une approche de validation fonctionnelle, que les neuf systèmes VapBC prédits sont des modules Toxine/Antitoxine. Puis, afin d’évaluer le rôle de chaque système dans la symbiose, des mutants d’invalidation du gène de la toxine VapC ont été construits et analysés en interaction avec M. truncatula. Trois mutants (vapC3, vapC7 et vapC10) présentent, à 6 semaines post-inoculation, une amélioration de la capacité fixatrice d’azote des nodosités par rapport au sauvage. Les mutants vapC3 et vapC7 présentent également un nombre et une taille de nodosités supérieurs. Ces mutants, en augmentant l’apport azoté global de la plante, conduisent à une augmentation du rendement végétal. Le mutant vapC10, quant à lui, présente une amélioration de la viabilité bactérienne associée à une sénescence retardée, sans amélioration du rendement végétal en raison d’un nombre inférieur de nodosités par plante. Ainsi, nous démontrons que la bactérie, par l’intermédiaire des systèmes VapBC, participe à la régulation de l’interaction symbiotique. Les toxines VapC3 et VapC7, dans un contexte sauvage, limitent la prolifération et/ou l’infection bactérienne tandis que la toxine VapC10 diminue la viabilité bactéroïdienne. Afin d’identifier les cibles moléculaires des ribonucléases VapC7 et VapC10, nous avons développé une analyse de MORE RNA-seq (Mapping by Overexpression of an RNase in E. coli). Cette technique permet de déterminer, par leur extrémité 5’, les ARN clivés après surexpression chez E. coli des toxines de S. meliloti. Les toxines VapC7 et VapC10, clivent respectivement l’ARNtfMet et deux ARNtSer d’E. coli permettant ainsi, une inhibition globale ou partielle de la traduction. Des ARNt homologues ont été identifiés chez S. meliloti par analyse bio-informatique. Il en résulte que le gène spécifiant l’ARNtfMet initiateur de la traduction, jusqu’alors non formellement identifié chez S. meliloti, serait présent en trois copies sur les trois loci rrn chromosomiques. En conclusion, les toxines VapC7 et VapC10 de S. meliloti sont des tRNAses impliquées dans la reprogrammation métabolique de S. meliloti lors de la symbiose et intervenant dans la régulation de l’interaction symbiotique, probablement en réponse aux besoins azotés, énergétiques et spécifiques de sa plante hôte.
Legumes house nitrogen-fixing endosymbiotic rhizobia in specialised polyploid cells within root nodules. This results in a mutualistic relationship whereby the plant host receives fixed nitrogen from the bacteria in exchange for dicarboxylic acids. This plant-microbe interaction requires the regulation of multiple metabolic and physiological processes in both the host and symbiont in order to achieve highly efficient symbiosis. Recent studies have showed that the success of symbiosis is influenced by the circadian clock of the plant host. Medicago and soybean plants with altered clock mechanisms showed compromised nodulation and reduced plant growth. Furthermore, transcriptomic analyses revealed that multiple genes with key roles in recruitment of rhizobia to plant roots, infection and nodule development were under circadian control, suggesting that appropriate timing of expression of these genes may be important for nodulation. There is also evidence for rhythmic gene expression of key nitrogen fixation genes in the rhizobium symbiont, and temporal coordination between nitrogen fixation in the bacterial symbiont and nitrogen assimilation in the plant host may be important for successful symbiosis. Understanding of how circadian regulation impacts on nodule establishment and function will identify key plant-rhizobial connections and regulators that could be targeted to increase the efficiency of this relationship.
The legume-rhizobium symbiosis represents as a unique model within the realm of plant-microbe interactions. Unlike typical cases of pathogenic invasion, the infection of rhizobia and their residence within symbiotic cells do not elicit a noticeable immune response in plants. Nevertheless, there is still much to uncover regarding the mechanisms through which plant immunity influences rhizobia symbiosis. In this study, we identify an important player in this intricate interplay: the Lotus japonicus PRP1, which serves as a positive regulator of plant immunity but also exhibits the capacity to decrease rhizobial colonization and nitrogen fixation within nodules. The PRP1 gene encodes an uncharacterized protein and is named as Pathogenesis-Related Protein1, owing to its ortholog in Arabidopsis thaliana, a pathogenesis-related family protein (At1g78780). The PRP1 gene displays high expression levels in nodules compared to other tissues. We observed an increase in rhizobium infection in the L. japonicus prp1 mutants, while PRP1-overexpressing plants exhibited a reduction in rhizobium infection compared to control plants. Intriguingly, L. japonicus prp1 mutants produced nodules with a pinker color compared to wild-type controls, accompanied by elevated levels of leghemoglobin and an increased proportion of infected cells within the prp1 nodules. The Nodule Inception (NIN) could directly bind to the PRP1 promoter, activating PRP1 gene expression. Furthermore, we found that PRP1 is a positive mediator of innate immunity in plants. In summary, our study provides clear evidence of the intricate relationship between plant immunity and symbiosis. PRP1, acting as a positive regulator of plant immunity, simultaneously exerts suppressive effects on rhizobial infection and colonization within nodules.
The model legume Medicago truncatula establishes a symbiosis with soil bacteria (rhizobia) that carry out symbiotic nitrogen fixation (SNF) in plant root nodules. SNF requires the exchange of nutrients between the plant and rhizobia in the nodule that occurs across a plant-derived symbiosome membrane. One iron transporter, belonging to the Vacuolar iron Transporter-Like (VTL) family, MtVTL8, has been identified as essential for bacteria survival and therefore SNF. In this work we investigated the spatial expression of MtVTL8 in nodules and addressed whether it could be functionally interchangeable with a similar nodule-expressed iron transporter, MtVTL4. Using a structural model for MtVTL8 and the previously hypothesized mechanism for iron transport in a phylogenetically-related Vacuolar Iron Transporter (VIT), EgVIT1 with known crystal structure, we identified critical amino acids and obtained their mutants. Mutants were tested in planta for complementation of an SNF defective line and in an iron sensitive mutant yeast strain. An extended phylogenetic assessment of VTLs and VITs showed that amino acids critical for function are conserved differently in VTLs vs. VITs. Our studies showed that some amino acids are essential for iron transport leading us to suggest a model for MtVTL8 function, one that is different for other iron transporters (VITs) studied so far. This study extends the understanding of iron transport mechanisms in VTLs as well as those used in SNF.
Legume nodules express multiple leghemoglobins (Lbs) and nonsymbiotic hemoglobins (Glbs), but how they are regulated is unclear. Here, we study the regulation of all Lbs and Glbs of Lotus japonicus in different physiologically-relevant conditions and mutant backgrounds. We quantified hemoglobin expression, localized reactive oxygen species (ROS) and nitric oxide (NO) in nodules, and deployed mutants deficient in Lbs and in the transcription factors NLP4 (associated with nitrate sensitivity) and NAC094 (associated with senescence). Expression of Lbs and class 2 Glbs was supressed by nitrate, whereas expression of class 1 and 3 Glbs was positively correlated with external nitrate concentrations. Nitrate-responsive elements were found in the promoters of several hemoglobin genes. Mutant nodules without Lbs showed accumulation of ROS and NO, and alterations of antioxidants and senescence markers. NO accumulation occurred by a nitrate-independent pathway, probably due to the virtual disappearance of Glb1-1 and the deficiency of Lbs. We conclude that hemoglobins are regulated in a gene-specific manner during nodule development and in response to nitrate and dark stress. Mutant analyses reveal that nodules lacking Lbs experience nitro-oxidative stress and that there is compensation of expression between Lb1 and Lb2. They also show modulation of hemoglobin expression by NLP4 and NAC094.
Five non-symbiotic hemoglobins (nsHb) have been identified in rice (Oryza sativa). Previous studies have shown that stress conditions can induce their overexpression, but the role of those globins is still unclear. To better understand the functions of nsHb, the reactivity of rice Hb1 toward hydrogen peroxide (H2O2) has been studied in vitro. Our results show that recombinant rice Hb1 dimerizes through dityrosine cross-links in the presence of H2O2. By site-directed mutagenesis, we suggest that tyrosine 112 located in the FG loop is involved in this dimerization. Interestingly, this residue is not conserved in the sequence of the five rice non-symbiotic hemoglobins. Stopped-flow spectrophotometric experiments have been performed to measure the catalytic constants of rice Hb and its variants using the oxidation of guaiacol. We have shown that Tyrosine112 is a residue that enhances the peroxidase activity of rice Hb1, since its replacement by an alananine leads to a decrease of guaiacol oxidation. In contrast, tyrosine 151, a conserved residue which is buried inside the heme pocket, reduces the protein reactivity. Indeed, the variant Tyr151Ala exhibits a higher peroxidase activity than the wild type. Interestingly, this residue affects the heme coordination and the replacement of the tyrosine by an alanine leads to the loss of the distal ligand. Therefore, even if the amino acid at position 151 does not participate to the formation of the dimer, this residue modulates the peroxidase activity and plays a role in the hexacoordinated state of the heme.
Symbiotic nitrogen fixation carried out by the interaction between legumes and rhizobia is the main source of nitrogen in natural ecosystems and in sustainable agriculture. For the symbiosis to be viable, nutrient exchange between the partners is essential. Transition metals are among the nutrients delivered to the nitrogen‐fixing bacteria within the legume root nodule cells. These elements are used as cofactors for many of the enzymes controlling nodule development and function, including nitrogenase, the only known enzyme able to convert N2 into NH3. In this review, we discuss the current knowledge on how iron, zinc, copper, and molybdenum reach the nodules, how they are delivered to nodule cells, and how they are transferred to nitrogen‐fixing bacteria within.
Legume nodules produce large quantities of heme required for the synthesis of leghemoglobin (Lb) and other hemoproteins. Despite the crucial function of Lb in nitrogen fixation and the toxicity of free heme, the mechanisms of heme homeostasis remain elusive.
Biochemical, cellular, and genetic approaches were used to study the role of heme oxygenases (HOs) in heme degradation in the model legume Lotus japonicus. Heme and biliverdin were quantified and localized, HOs were characterized, and knockout LORE1 and CRISPR/Cas9 mutants for LjHO1 were generated and phenotyped.
We show that LjHO1, but not the LjHO2 isoform, is responsible for heme catabolism in nodules and identify biliverdin as the in vivo product of the enzyme in senescing green nodules. Spatiotemporal expression analysis revealed that LjHO1 expression and biliverdin production are restricted to the plastids of uninfected interstitial cells. The nodules of ho1 mutants showed decreased nitrogen fixation, and the development of brown, rather than green, nodules during senescence. Increased superoxide production was observed in ho1 nodules, underscoring the importance of LjHO1 in antioxidant defense.
We conclude that LjHO1 plays an essential role in degradation of Lb heme, uncovering a novel function of nodule plastids and uninfected interstitial cells in nitrogen fixation.
Globally, the increase in human population continues to threaten the sustainability of agricultural systems. Despite the fast-growing population in Sub-Saharan Africa (SSA) and the efforts in improving the productivity of crops, the increase in the yield of crops per unit area is still not promising. The productivity of crops is primarily constrained by inadequate levels of soil nutrients to support optimum crop growth and development. However, smallholder farmers occasionally use fertilizers, and the amount applied is usually small and does not meet plant requirements. This is due to the unaffordability of the cost of fertilizers, which is enough to suffice the crop requirement. Therefore, there is a need for alternative affordable and effective fertilization methods for sustainable intensification and improvement of the smallholder farming system's productivity. This study was designed to evaluate the symbiotic performance of indigenous soybean nodulating rhizobia in selected agricultural soils of Tanzania. In total, 217 rhizobia isolates were obtained from three agroecological zones, i.e., eastern, northern, and southern highlands. The isolates collected were screened for N2 fixing abilities under in vitro (nitrogen-free medium) and screen house conditions. The results showed varying capabilities of isolates in nitrogen-fixing both under in vitro and screen house conditions. Under in vitro experiment, 22% of soybean rhizobia isolates were identified to have a nitrogen-fixing capability on an N-free medium, with the highest N2-fixing diameter of 1.87 cm. In the screen house pot experiment, results showed that soybean rhizobia isolate significantly (P < 0.001) influenced different plant growth and yield components, where the average shoot dry weight ranged from 2.49 to 10.98 g, shoot length from 41 to 125.27 cm whilst the number of leaves per plant ranged from 20 to 66. Furthermore, rhizobia isolates significantly (P = 0.038) increased root dry weight from 0.574 to 2.17 g. In the case of symbiotic parameters per plant, the number of nodules was in the range of 0.33–22, nodules dry weight (0.001–0.137 g), shoot nitrogen (2.37–4.97%), total nitrogen (53.59–6.72 g), and fixed nitrogen (46.878–0.15 g) per plant. In addition, the results indicated that 51.39% of the tested bacterial isolates in this study were ranked as highly effective in symbiosis, suggesting that they are promising as potential alternative biofertilizers for soybean production in agricultural soils of Tanzania to increase productivity per unit area while reducing production cost.
Innovative technology to increase efficient nitrogen (N) use while avoiding environmental damages is needed because of the increasing food demand of the rapidly growing global population. Soybean (Glycine max) has evolved a complex symbiosis with N-fixing bacteria that forms nodules to fix N. Herein, foliar application of 10 mg L-1 Fe7(PO4)6 and Fe3O4 nanomaterials (NMs) (Fe-based NMs) promoted soybean growth and root nodulation, thus improving the yield and quality over that of the unexposed control, EDTA-control, and 1 and 5 mg L-1 NMs. Mechanistically, flavonoids, key signaling molecules at the initial signaling steps in nodulation, were increased by more than 20% upon exposure to 10 mg L-1 Fe-based NMs, due to enhanced key enzyme (phenylalanine ammonia-lyase, PAL) activity and up-regulation of flavonoid biosynthetic genes (GmPAL, GmC4H, Gm4CL, and GmCHS). Accumulated flavonoids were secreted to the rhizosphere, recruiting rhizobia for colonization. Fe7(PO4)6 NMs increased Allorhizobium by 87.3%, and Fe3O4 NMs increased Allorhizobium and Mesorhizobium by 142.2% and 34.9%, leading to increased root nodules by 50.0% and 35.4% over the unexposed control, respectively. Leghemoglobin content was also noticeably improved by 8.2-46.5% upon Fe-based NMs. The higher levels of nodule number and leghemoglobin content resulted in enhanced N content by 15.5-181.2% during the whole growth period. Finally, the yield (pod number and grain biomass) and quality (flavonoids, soluble protein, and elemental nutrients) were significantly increased more than 14% by Fe-based NMs. Our study provides an effective nanoenabled strategy for inducing root nodules to increase N use efficiency, and then both yield and quality of soybean.
Group VII Ethylene Response Factors (ERF‐VII) are plant‐specific transcription factors (TFs) known for their role in the activation of hypoxia‐responsive genes under low oxygen stress but also in plant endogenous hypoxic niches. However, their function in the microaerophilic nitrogen‐fixing nodules of legumes has not yet been investigated. We investigated regulation and the function of the two Medicago truncatula ERF‐VII TFs (MtERF74 and MtERF75) in roots and nodules, MtERF74 and MtERF75 in response to hypoxia stress and during the nodulation process using an RNA interference strategy and targeted proteolysis of MtERF75. Knockdown of MtERF74 and MtERF75 partially blocked the induction of hypoxia‐responsive genes in roots exposed to hypoxia stress. In addition, a significant reduction in nodulation capacity and nitrogen fixation activity was observed in mature nodules of double knockdown transgenic roots. Overall, the results indicate that MtERF74 and MtERF75 are involved in the induction of MtNR1 and Pgb1.1 expression for efficient Phytogb‐NO respiration in the nodule. This article is protected by copyright. All rights reserved.
The root nodule is a complexed symbiotic nitrogen fixation factory, in which cells are highly heterogeneous. However, the differentiation and interconnection of nodule cells are still largely unknown. Here, we set up a modified protocol for nodule protoplast preparation and report on a single-cell RNA sequencing assay of the indeterminate nodule type represented by Medicago truncatula. We designated 13 cell clusters with specific expression patterns in 14-dpi (days post inoculation) nodules and described a spatial and functional cellular map by experimental and bioinformatic methods. Pseudotime analysis revealed that two groups of apical meristematic cells develop into symbiotic and un-symbiotic fate cells along their particular trajectories. Biofunction analysis of each cell cluster revealed their particularity and interrelation, especially that the un-infected cells in nitrogen fixation zone are involved in nitrogen assimilation as well by undertaking the asparagine synthesis pathway. Our results offer an important resource for investigating the mechanism of nodule organogenesis and symbiotic nitrogen fixation.
Host/symbiont compatibility is a hallmark of the symbiotic nitrogen‐fixing interaction between rhizobia and legumes, mediated in part by plant‐produced nodule‐specific cysteine‐rich (NCR) peptides and the bacterial BacA membrane protein that can act as a NCR peptide transporter. In addition, the genetic and metabolic properties supporting symbiotic nitrogen fixation often differ between compatible partners, including those sharing a common partner, highlighting the need for multiple study systems. Here, we report high‐quality nodule transcriptome assemblies for Medicago sativa cv. Algonquin and Melilotus officinalis, two legumes able to form compatible symbioses with Sinorhizobium meliloti. The compressed M. sativa and M. officinalis assemblies consisted of 79,978 and 64,593 contigs, respectively, of which 33,341 and 28,278 were assigned putative annotations, respectively. As expected, the two transcriptomes showed broad similarity at a global level. We were particularly interested in the NCR peptide profiles of these plants, as these peptides drive bacterial differentiation during the symbiosis. A total of 412 and 308 NCR peptides were predicted from the M. sativa and M. officinalis transcriptomes, respectively, with approximately 9% of the transcriptome of both species consisting of NCR transcripts. Notably, transcripts encoding highly cationic NCR peptides (isoelectric point > 9.5), which are known to have antimicrobial properties, were ∼2‐fold more abundant in M. sativa than in M. officinalis, and ∼27‐fold more abundant when considering only NCR peptides in the six‐cysteine class. We hypothesize that the difference in abundance of highly cationic NCR peptides explains our previous observation that some rhizobial bacA alleles which can support symbiosis with M. officinalis are unable to support symbiosis with M. sativa.
Nitrogen is an essential nutrient in plant growth. The ability of a plant to supply its requirements from biological nitrogen fixation through interactions with associative, endophytic and endosymbiotic symbionts, is of tremendous importance to the environment and world agriculture. Symbiotic nitrogen fixation is part of a mutualistic relationship, in which plants provide a niche and fixed carbon to bacteria in exchange for fixed nitrogen, and relies on complex regulatory circuits between both partners. This process is restricted mainly to legumes in agricultural systems, and there is considerable interest in exploring whether similar symbioses can be developed in non-legumes.
Host/symbiont compatibility is a hallmark of the symbiotic nitrogen-fixing interaction between rhizobia and legumes, mediated in part by plant produced nodule-specific cysteine-rich (NCR) peptides and the bacterial BacA membrane protein that can act as a NCR peptide transporter. In addition, the genetic and metabolic properties supporting symbiotic nitrogen fixation often differ between compatible partners, including those sharing a common partner, highlighting the need for multiple study systems. Here, we report high quality nodule transcriptome assemblies for Medicago sativa cv. Algonquin and Melilotus officinalis , two legumes able to form compatible symbioses with Sinorhizobium meliloti . The compressed M. sativa and M. officinalis assemblies consisted of 79,978 and 64,593 contigs, respectively, of which 33,341 and 28,278 were assigned putative annotations, respectively. As expected, the two transcriptomes showed broad similarity at a global level. We were particularly interested in the NCR peptide profiles of these plants, as these peptides drive bacterial differentiation during the symbiosis. A total of 412 and 308 NCR peptides were predicted from the M. sativa and M. officinalis transcriptomes, respectively, with approximately 9% of the transcriptome of both species consisting of NCR transcripts. Notably, transcripts encoding highly-cationic NCR peptides (isoelectric point > 9.5), which are known to have antimicrobial properties, were ~2-fold more abundant in M. sativa than in M. officinalis , and ~27-fold more abundant when considering only NCR peptides in the six-cysteine class. We hypothesize that the difference in abundance of highly-cationic NCR peptides explains our previous observation that some rhizobial bacA alleles which can support symbiosis with M. officinalis are unable to support symbiosis with M. sativa .
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Tian L, Liu L, Xu S, Deng R, Wu P, Jiang H, Wu G, Chen Y. 2022. A d-pinitol transporter, LjPLT11, regulates plant growth and nodule development in Lotus japonicus. Journal of Experimental Botany 73, 351–365.
Haemoglobin has previously been recorded in plants only in the nitrogen-fixing nodules formed by symbiotic association between Rhizobium or Frankia and legume or non-legume hosts. Structural similarities amongst these and animal haemoglobins at the protein and gene level suggested a common evolutionary origin. This suggests that haemoglobin genes, inherited from an ancestor common to plants and animals, might be present in all plants. We report here the isolation of a haemoglobin gene from Trema tomentosa, a non-nodulating relative of Parasponia (Ulmaceae). The gene has three introns located at positions identical to those in the haemoglobin genes of nodulating plant species, strengthening the case for a common origin of all plant haemoglobin genes. The data argue strongly against horizontal haemoglobin gene transfer from animals to plants. The Trema gene has a tissue-specific pattern of transcription and translation, producing monomeric haemoglobin in Trema roots. We have also found that the Parasponia haemoglobin gene is transcribed in roots of non-nodulated plants. These results suggest that haemoglobin has a role in the respiratory metabolism of root cells of all plant species. We propose that its special role in nitrogen-fixing nodules has required adaptation of the haemoglobin-gene regulation pathway, to give high expression in the specialized environment of the nodule.
We have applied gene counting and restric- viduals with HbH disease. In two of these tion endonuclease mapping techniques to individuals, the chromosome containing the the study of two American black families in single a gene could have originated by which there were one or more cases of crossing over between mispaired a genes, HbH disease. We found deletions of three resulting in a deletion of about 4.2 kilo- of the four normal a-globin genes in mdi- bases (kb). T HE a-THALASSEMIA SYNDROMES are inherited anemias caused by decreased function of one or more a-globin genes. When one, two, three, or all four of the normal ct-globin genes are nonfunctional, the respective disease states are termed silent carrier (a-thal-2), a-thalassemia trait (ct-thal-l), HbH disease, and othalassemial Patients with ct-thalassemia are usually of Asian, Mediterranean, or black descent. Gene counting experiments using liquid hybridization suggest that one or more a-globin genes are deleted in most, but not all, Asian subjects with a-thalassemia.2�5 These deletions in Asians have been confirmed by restriction endonuclease patterns.68 Recently, both techniques have been used to document genetic compounds of deletions as well as more complex defects in Mediterraneans with HbH disease.9 HbH disease is rare in blacks, and affected individuals are reported to have relatively lower percentages of HbH (2%-7%) and a milder condition than Asians with HbH disease.'#{176}" Recently, Dozy et al. reported that one-quarter ofa screened black population had a-thal-2 due to deletion of a single a gene. However, none of the 15 black a-thal-l individuals studied had deletions of both a-loci on one chromosome.�2 We report quantitative and qualitative studies of the a genes in two black families in which HbH disease is present. Our results indicate that in family members, HbH disease, a-thal-l, and a-thal-2 are due to deletions of 3, 2, and I ct-globin genes, respectively. Furthermore, at least one of these deletions may have originated by unequal crossing over.
We have identified a nuclear-encoded Hb from plants (GLB3) that has a central domain similar to the "truncated" Hbs of bacteria, protozoa, and algae. The three-dimensional structure of these Hbs is a 2-on-2 arrangement of alpha-helices, distinct from the 3-on-3 arrangement of the standard globin fold [Pesce, A., Couture, M., Dewilde, S., Guertin, M., Yamauchi, K., Ascenzi, P., Moens, L. & Bolognesi, M. (2000) EMBO J. 19, 2424-2434]. GLB3-like genes are not found in animals or yeast, but our analysis reveals that they are present in a wide range of Angiosperms and a Bryophyte. Although cyanobacteria and Chlamydomonas have 2-on-2 Hbs (GLBN), GLB3 is more likely related to GLBO-type 2-on-2 Hbs from bacteria. Consequently, GLB3 is unlikely to have arisen from a horizontal transfer between the chloroplast and nuclear genomes. Arabidopsis thaliana GLB3 protein exhibits unusual concentration-independent binding of O(2) and CO. The absorbance spectrum of deoxy-GLB3 is unique; the protein forms a transient six-coordinate structure after reduction and deoxygenation, which slowly converts to a five-coordinate structure. In A. thaliana, GLB3 is expressed throughout the plant but responds to none of the treatments that induce plant 3-on-3 Hbs. Our analysis of the sequence, ligand interactions, and expression profile of GLB3 indicates that this protein has unique biochemical properties, evolutionary history, and, most likely, a function distinct from those of other plant Hbs.
We have investigated the consequences of endogenous limitations in oxygen delivery for phloem transport in Ricinus communis. In situ oxygen profiles were measured directly across stems of plants growing in air (21% [v/v] oxygen), using a microsensor with a tip diameter of approximately 30 microm. Oxygen levels decreased from 21% (v/v) at the surface to 7% (v/v) in the vascular region and increased again to 15% (v/v) toward the hollow center of the stem. Phloem sap exuding from small incisions in the bark of the stem was hypoxic, and the ATP to ADP ratio (4.1) and energy charge (0.78) were also low. When 5-cm stem segments of intact plants were exposed to zero external oxygen for 90 min, oxygen levels within the phloem decreased to approximately 2% (v/v), and ATP to ADP ratio and adenylate energy charge dropped further to 1.92 and 0.68, respectively. This was accompanied by a marked decrease in the phloem sucrose (Suc) concentration and Suc transport rate, which is likely to be explained by the inhibition of retrieval processes in the phloem. Germinating seedlings were used to analyze the effect of a stepwise decrease in oxygen tension on phloem transport and energy metabolism in more detail. Within the endosperm embedding the cotyledons-next to the phloem loading sites-oxygen decreased from approximately 14% (v/v) in 6-d-old seedlings down to approximately 6% (v/v) in 10-d-old seedlings. This was paralleled by a similar decrease of oxygen inside the hypocotyl. When the endosperm was removed and cotyledons incubated in a 100 mM Suc solution with 21%, 6%, 3%, or 0.5% (v/v) oxygen for 3 h before phloem sap was analyzed, decreasing oxygen tensions led to a progressive decrease in phloem energy state, indicating a partial inhibition of respiration. The estimated ratio of NADH to NAD(+) in the phloem exudate remained low (approximately 0.0014) when oxygen was decreased to 6% and 3% (v/v) but increased markedly (to approximately 0.008) at 0.5% (v/v) oxygen, paralleled by an increase in lactate and ethanol. Suc concentration and translocation decreased when oxygen was decreased to 3% and 0.5% (v/v). Falling oxygen led to a progressive increase in amino acids, especially of alanine, gamma-aminobutyrat, methionine, and isoleucine, a progressive decrease in the C to N ratio, and an increase in the succinate to malate ratio in the phloem. These results show that oxygen concentration is low inside the transport phloem in planta and that this results in adaptive changes in phloem metabolism and function.
Leguminous plants have both symbiotic and nonsymbiotic hemoglobin (sym- and nonsym-Hb) genes. Three symbiotic (LjLb1, 2, 3) and one nonsymbiotic (LjNSG1) Hb genes were isolated from a genomic library of Lotus japonicus MG20 Miyakojima. Two motif sequences (AAAGAT and CTCTT) critical for nodule specific expression were conserved on the 5'-upstream sequence of LjLb1, 2 and 3. The 5'-upstream region of LjNSG1 contained the sequence consensus for nonsym-Hb. RT-PCR with specific primer sets for each LjLb gene showed that all the sym-Hb genes (LjLb1, 2, 3) were expressed specifically and strongly in root nodules. The expression of LjLb1, 2 and 3 could not be detected in root, leaf or stem of a mature plant, whereas low level expression was detected in seedlings by RT-PCR. This suggests that sym-Hbs may have another unknown function besides being oxygen transporter for the microsymbiont in symbiotic nitrogen fixation. The expression of LjNSG1, examined with RT-PCR, was detected at low level in root, leaf and stem. The expression of LjNSG1 was enhanced in root nodules, whereas it was repressed in roots colonized by mycorrhizal fungi Glomus sp. R10. The repression of the nonsym-Hb gene was also observed in the roots of Medicago sativa colonized by Glomus sp. R10.
THE similarity in tertiary structure of mammalian haemoglobins1-3 and myoglobins4,5 and of haemoglobins from invertebrates6,7 shown by X-ray analysis has been particularly important for our understanding of the biological evolution of these proteins. We have therefore studied the structure of leghaemoglobins-plant haemoglobins found in root nodules of leguminous plants-and compared the structures of seemingly unrelated, in terms of evolution, haemoglobins.
Recent years have seen rapid growth in amino acid sequence data on globins and nucleotide sequence data on haemoglobin genes and pseudogenes, and cladistic analysis1 of these data continues to reveal new facets of globin evolution. Our present findings demonstrate: (1) avian and mammalian embryonic α genes (π and ξ, respectively) had a monophyletic origin involving an α locus duplication about 400 Myr ago soon after the duplication which separated α and β genes; (2) much later in phylogeny, independent β-gene duplications produced the embryonic ρ locus of birds and embryonic ε and fetal γ loci of mammals. This parallels the earlier finding2 that myoglobins evolved more than once from generalized globin ancestors. Here we support the view2 that such globin evolution resulted from natural selection acting on mutations in duplicated genes. Thus, our evidence contradicts the neutralist view3,4 in which almost all amino acid substitutions in descent to extant globins evaded positive selection.
Rhizobium is able to induce the formation of a new organ on roots of leguminous plants, the root nodule, in which the penetrated bacteria fix atmospheric nitrogen. This process is initiated by specific lipo-oligosaccharides, called Nod factors, secreted by the bacterium. Nodule formation proceeds through distinct steps like infection thread formation and activation of mitotic activity in cortical cells. During these steps specific plant genes, nodulin genes, are induced and several of these have been identified and characterized. Nodulin genes are used now as markers to study Nod factor perception and signal transduction.
Vitreoscilla, a filamentous bacterium in the Beggiatoa family, synthesizes a soluble haem-protein which has two identical subunits of relative molecular mass 15,775 and two b haems per molecule. It is synthesized in relatively large quantities when the organism, a strict aerobe, is grown under hypoxic conditions. It forms a relatively stable oxygenated form which is spectrally similar to oxymyoglobin (oxyHb) and oxyhaemoglobin (oxyHb). The amino acid sequence of this protein has been determined and aligned to fit the helical regions of several animal and plant globins. This alignment is consistent with its being a structural homologue of the eucaryotic haemoglobins although it diverged from the others in the N-terminal region and may lack an A-helix. It showed the maximum sequence homology (24%) with lupin leghaemoglobin (Lb). Vitreoscilla Hb is the first bacterial haemoglobin to be sequenced. It may function to enable the organism to survive in oxygen-limited environments by acting as an oxygen storage-trap or to facilitate oxygen diffusion.
1. The finding that the plant is the genetic determinant of leghaemoglobin production in legume nodules was further tested by inoculating snake beans with two strains of Rhizobium selected to give large genetic differences. Carbohydrate requirement patterns, immunological techniques and DNA base ratio determinations were used to demonstrate genetic differences between the two rhizobial strains. 2. Partially purified preparations of the haemoglobins from the nodules produced by the two strains showed no differences when examined by electrophoresis, isoelectric focusing or ion-exchange chromatography. 3. Two different leghaemoglobins from each type of nodule were separated by chromatography on DEAE-cellulose. One of these was isolated in the Fe(3+) form and accounted for two-thirds of the total leghaemoglobin. When it was examined in the analytical ultracentrifuge and by amino acid analysis, this major component did not vary with the inoculant rhizobial strain. The molecule had an s(20,w) of 1.88S, a diffusion coefficient of 10.7x10(-7)cm(2).s(-1) and a mol. wt. of 16700. 4. These results strongly support the hypothesis that the mRNA for leghaemoglobin is transcribed from plant DNA.
Infection of legume roots with Rhizobium species results in the development of a root nodule structure in which the bacteria form an intracellular symbiosis with the plant. We report here that the infection of soybean (Glycine max L.) roots with Rhizobium japonicum results in the synthesis by the plant of at least 18-20 polypeptides other than leghemoglobin during the development of root nodules. Identification of these "nodule-specific" host polypeptides (referred to as nodulins) was accomplished by two-dimensional gel analysis of the immunoprecipitates formed by a "nodule-specific" antiserum with in vitro translation products of root-nodule polysomes that are free of bacteroidal contaminations. Nodulins account for 7-11% of the total 35S-methionine-labeled protein synthesized in the host cell cytoplasm, and the majority of them are of 12,000-20,000 molecular weight. These proteins are absent from the uninfected roots, bacteroids and free-living Rhizobium, and appear to be coded for the plant genes that may be obligatory for the development of symbiosis in the legume root nodules. Analysis of nodulins in ineffective (unable to fix nitrogen) nodules developed due to Rhizobium strains SM5 and 61A24 showed that their synthesis is reduced and their expression differentially influenced by mutations in rhizobia. Two polypeptides of bacterial origin were also found to be cross-reactive with the "nodule-specific" antiserum, suggesting that they are secreted by Rhizobium into the host cell cytoplasm during symbiotic nitrogen fixation.
Rhizobia are gram-negative bacteria with two distinct habitats: the soil rhizosphere in which they have a saprophytic and, usually, aerobic life and a plant ecological niche, the legume nodule, which constitutes a microoxic environment compatible with the operation of the nitrogen reducing enzyme nitrogenase. The purpose of this review is to summarize the present knowledge of the changes induced in these bacteria when shifting to a microoxic environment. Oxygen concentration regulates the expression of two major metabolic pathways: energy conservation by respiratory chains and nitrogen fixation. After reviewing the genetic data on these metabolic pathways and their response to oxygen we will put special emphasis on the regulatory molecules which are involved in the control of gene expression. We will show that, although homologous regulatory molecules allow response to oxygen in different species, they are assembled in various combinations resulting in a variable regulatory coupling between genes for microaerobic respiration and nitrogen fixation genes. The significance of coordinated regulation of genes not essential for nitrogen fixation with nitrogen fixation genes will also be discussed.
We have isolated a new hemoglobin gene from soybean. It is expressed in cotyledons, stems of seedlings, roots, young leaves, and in some cells in the nodules that are associated with the nitrogen-fixing Bradyrhizobium symbiont. This contrasts with the expression of the leghemoglobins, which are active only in the infected cells of the nodules. The deduced protein sequence of the new gene shows only 58% similarity to one of the soybean leghemoglobins, but 85-87% similarity to hemoglobins from the nonlegumes Parasponia, Casuarina, and barley. The pattern of expression and the gene sequence indicate that this new gene is a nonsymbiotic legume hemoglobin. The finding of this gene in legumes and similar genes in other species strengthens our previous suggestion that genomes of all plants contain hemoglobin genes. The specialized leghemoglobin gene family may have arisen from a preexisting nonsymbiotic hemoglobin by gene duplication.
Adaptation of rhizobia to a symbiotic life style and synthesis of the nitrogen-fixing apparatus are coordinated with nodule development by the microaerobic conditions prevailing in the central nodule tissue. Sensing and transduction of the "low oxygen' signal involves similar regulatory elements in different rhizobia, yet, these are combined in species-specific circuits.
We cloned two hemoglobin genes from Arabidopsis thaliana. One gene, AHB1, is related in sequence to the family of nonsymbiotic hemoglobin genes previously identified in a number of plant species (class 1). The second hemoglobin gene, AHB2, represents a class of nonsymbiotic hemoglobin (class 2) related in sequence to the symbiotic hemoglobin genes of legumes and Casuarina. The properties of these two hemoglobins suggest that the two families of nonsymbiotic hemoglobins may differ in function from each other and from the symbiotic hemoglobins. AHB1 is induced, in both roots and rosette leaves, by low oxygen levels. Recombinant AHB1 has an oxygen affinity so high as to make it unlikely to function as an oxygen transporter. AHB2 is expressed at a low level in rosette leaves and is low temperature-inducible. AHB2 protein has a lower affinity for oxygen than AHB1 but is similar to AHB1 in having an unusually low, pH-sensitive oxygen off-rate.
We report here on strategies aimed at improving the frequency of detectable recombination in plants by increasing the efficiency of selecting double-recombinants in transgenic calli. Gene targeting was approached on the Gln1 and the Pzfloci of Lotus japonicus, using Agrobacterium tumefaciens T-DNA replacement vectors. Large flanking regions, up to 22.9 kb, surrounding a positive selection marker were presented as substrates for homologous recombination. For easier detection of putative recombinants the negative selectable marker cytosine deaminase was inserted at the outside borders of the flanking regions offered for cross-over. A combination of positive and negative selection allowing double-recombinants to grow, while counter-selecting random insertions, was used to select putative targeting events. The more than 1000-fold enrichment observed with replacement vectors designed to minimize gene silencing demonstrated the efficiency of the negative selection. Using five different replacement vectors an estimated total of 18,974 transformation events were taken through the positive-negative selection procedure and 185 resistant calli obtained. Targeting events could not be verified in the survivors by PCR screening and Southern blot analysis. With this approach the frequency of detectable gene targeting in L. japonicus was below 5.3 x 10(-5), despite the large flanking sequences offered for recombination.
Metabolite assays are required to characterise how metabolism changes between genotypes during development and in response to environmental perturbations. They provide a springboard to identify important regulatory sites and investigate the underlying mechanisms. Due to their small size, Arabidopsis seeds pose a technical challenge for such measurements. A set of assays based on a novel enzymic cycling system between glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate oxidase have been developed and optimised for use with growing Arabidopsis seeds. In combination with existing assays they provide a suite of high throughput, sensitive assays for the immediate precursors for starch (adenine diphosphate glucose) and lipid (acetyl coenzyme A, glycerol-3-phosphate) synthesis, as well as pyrophosphate, ATP, ADP and most of the glycolytic intermediates. A method is also presented to rapidly quench intact siliques, lyophilise them and then manually separate seeds for metabolite analysis. These techniques are used to investigate changes in overall seed metabolite levels during development and maturation, and in response to a stepwise decrease of the external oxygen concentration.
Summary To overcome the detection limits inherent to DNA array-based methods of transcriptome analysis, we developed a real-time reverse transcription (RT)-PCR-based resource for quantitative measurement of transcripts for 1465 Arabidopsis transcription factors (TFs). Using closely spaced gene-specific primer pairs and SYBR Green to monitor amplification of double-stranded DNA (dsDNA), transcript levels of 83% of all target genes could be measured in roots or shoots of young Arabidopsis wild-type plants. Only 4% of reactions produced non-specific PCR products. The amplification efficiency of each PCR was determined from the log slope of SYBR Green fluorescence versus cycle number in the exponential phase, and was used to correct the readout for each primer pair and run. Measurements of transcript abundance were quantitative over six orders of magnitude, with a detection limit equivalent to one transcript molecule in 1000 cells. Transcript levels for different TF genes ranged between 0.001 and 100 copies per cell. Only 13% of TF transcripts were undetectable in these organs. For comparison, 22K Arabidopsis Affymetrix chips detected less than 55% of TF transcripts in the same samples, the range of transcript levels was compressed by a factor more than 100, and the data were less accurate especially in the lower part of the response range. Real-time RT-PCR revealed 35 root-specific and 52 shoot-specific TF genes, most of which have not been identified as organ-specific previously. Finally, many of the TF transcripts detected by RT-PCR are not represented in Arabidopsis EST (expressed sequence tag) or Massively Parallel Signature Sequencing (MPSS) databases. These genes can now be annotated as expressed.
Research on legume nodule metabolism has contributed greatly to our knowledge of primary carbon and nitrogen metabolism in plants in general, and in symbiotic nitrogen fixation in particular. However, most previous studies focused on one or a few genes/enzymes involved in selected metabolic pathways in many different legume species. We utilized the tools of transcriptomics and metabolomics to obtain an unprecedented overview of the metabolic differentiation that results from nodule development in the model legume, Lotus japonicus. Using an array of more than 5000 nodule cDNA clones, representing 2500 different genes, we identified approximately 860 genes that were more highly expressed in nodules than in roots. One-third of these are involved in metabolism and transport, and over 100 encode proteins that are likely to be involved in signalling, or regulation of gene expression at the transcriptional or post-transcriptional level. Several metabolic pathways appeared to be co-ordinately upregulated in nodules, including glycolysis, CO(2) fixation, amino acid biosynthesis, and purine, haem, and redox metabolism. Insight into the physiological conditions that prevail within nodules was obtained from specific sets of induced genes. In addition to the expected signs of hypoxia, numerous indications were obtained that nodule cells also experience P-limitation and osmotic stress. Several potential regulators of these stress responses were identified. Metabolite profiling by gas chromatography coupled to mass spectrometry revealed a distinct metabolic phenotype for nodules that reflected the global changes in metabolism inferred from transcriptome analysis.
A dimeric hemoglobin was purified from nitrogen-fixing root nodules formed by association of Rhizobium with a nonleguminous plant, Parasponia. The oxygen dissociation rate constant is probably sufficiently high to allow Parasponia hemoglobin to function in a fashion similar to that of leghemoglobin, by oxygen buffering and transport during symbiotic nitrogen
fixation. The identification of hemoglobin in a nonlegume raises important questions about the evolution of plant hemoglobin
genes.
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Control of leghae-NifD, respectively, and Fernando Carrari of the Max Planck Institute moglobin synthesis in snake beans
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A new hemoglobin gene from soybean: A role for hemoglobin in all plants.
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