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Polyhalite-based POLY4, a multi-nutrient source containing potassium, calcium, magnesium, and sulphur, is increasingly recognised for its potential to improve crop yields and soil health in agricultural systems. It is also been considered as a feasible approach for addressing the deficiency in potassium, calcium, and sulphur within a single application source. The present study aimed to investigate the impact of polyhalite-based POLY4 application, either as a complete or partial substitute for traditional potassium fertiliser and gypsum supplement, on the improvement of peanut (Arachis hypogaea) growth and soil quality. An extensive field study was conducted from 2021 and 2022, employing ten distinct nutrient management treatments with three replications in a randomised complete block design. The findings of the study indicated that the application of polyhalite (POLY4) in conjunction with only NP fertilisers resulted in a higher yield advantage (approximately 150-200 kg ha-1) than in plots treated with NPK + gypsum (at 500 kg ha-1) and control plots. The application of polyhalite-based fertiliser (POLY4) at a rate that was 100% equivalent to K along with NP fertilisers resulted in a significant increase in pod yield (5.3-12.8%) over NPK + gypsum and control plots. Thus, the increased crop yield led to an increase in gross returns of 4.88% and in net returns of 4.28% with the application of POLY4 (100%) + NP fertilisers over other treatments. Likewise, variable rates of conventional fertilisers along with POLY4 (100% recommended) + NP + gypsumat 310 kg/ha significantly increased the linoleic acid content (38.5%), oleic acid content, and oil content (48.1%) by reducing palmitic acid (11.96%) content in the groundnut seed. Interestingly, POLY4 use at the 50% recommended rate also resulted in yields that were comparable with those obtained with 100% NPK. Therefore, applying POLY4, a polyhalite fertiliser, in either a 100% or 50% equivalent of essential K can be an effective way of increasing the production of peanut crops and promoting agricultural sustainability. [ABSTRACT FROM AUTHOR]

IntroductionNumerous studies have reported the beneficial effects of silicon (Si) in alleviating biotic or abiotic stresses in many plant species. However, the role of Si in Fabaceae facing environmental stress is poorly documented. The aim of this study is to investigate the effect of Si on physiological traits and nodulation efficiency in Trifolium incarnatum L.MethodsSi was supplied (1.7 mM in the form of Na2SiO3) plants inoculated with Rhizobium leguminosarum bv trifolii and plant physiological traits and nodule ionomic and molecular traits were monitored over 25 days.ResultsSi supply promoted shoot biomass, the quantity of both Si and N in roots and shoots, and the number, biomass and density of nodules and their nitrogenase abundance which contribute to better dinitrogen (N2) fixation. Ionomic analysis of nodules revealed that Si supply increased the amount of several macroelements (potassium, phosphorus and sulfur) and microelements (copper, zinc and molybdenum) known to improve nodulation efficiency and N2 fixation. Finally, comparative proteomic analysis (+Si versus -Si) of nodules highlighted that Si modulated the proteome of both symbionts with 989 and 212 differentially accumulated proteins (DAPs) in the infected host root cells and their symbiont bacteria, respectively.DiscussionAmong the DAPs, the roles of those involved in nodulation and N2 fixation are discussed. For the first time, this study provides new insights into the effects of Si on both nodular partners and paves the way for a better understanding of the impact of Si on improving nodule function, and more specifically, on the nodules’ N2-fixing capacity.

Polyhalite-based POLY4, a multi-nutrient source containing potassium, calcium, magnesium, and sulphur, is increasingly recognised for its potential to improve crop yields and soil health in agricultural systems. It is also been considered as a feasible approach for addressing the deficiency in potassium, calcium, and sulphur within a single application source. The present study aimed to investigate the impact of polyhalite-based POLY4 application, either as a complete or partial substitute for traditional potassium fertiliser and gypsum supplement, on the improvement of peanut (Arachis hypogaea) growth and soil quality. An extensive field study was conducted from 2021 and 2022, employing ten distinct nutrient management treatments with three replications in a randomised complete block design. The findings of the study indicated that the application of polyhalite (POLY4) in conjunction with only NP fertilisers resulted in a higher yield advantage (approximately 150–200 kg ha-1) than in plots treated with NPK + gypsum (at 500 kg ha-1) and control plots. The application of polyhalite-based fertiliser (POLY4) at a rate that was 100% equivalent to K along with NP fertilisers resulted in a significant increase in pod yield (5.3–12.8%) over NPK + gypsum and control plots. Thus, the increased crop yield led to an increase in gross returns of 4.88% and in net returns of 4.28% with the application of POLY4 (100%) + NP fertilisers over other treatments. Likewise, variable rates of conventional fertilisers along with POLY4 (100% recommended) + NP + gypsum at 310 kg/ha significantly increased the linoleic acid content (38.5%), oleic acid content, and oil content (48.1%) by reducing palmitic acid (11.96%) content in the groundnut seed. Interestingly, POLY4 use at the 50% recommended rate also resulted in yields that were comparable with those obtained with 100% NPK. Therefore, applying POLY4, a polyhalite fertiliser, in either a 100% or 50% equivalent of essential K can be an effective way of increasing the production of peanut crops and promoting agricultural sustainability.

Nitrogen (N) and phosphorus (P) are two major plant nutrients, and their deficiencies often limit plant growth and crop yield. The uptakes of N or P affect each other, and consequently, understanding N–P interactions is fundamental. Their signaling mechanisms have been studied mostly separately, and integrating N–P interactive regulation is becoming the aim of some recent works. Lupins are singular plants, as, under N and P deficiencies, they are capable to develop new organs, the N2-fixing symbiotic nodules, and some species can also transform their root architecture to form cluster roots, hundreds of short rootlets that alter their metabolism to induce a high-affinity P transport system and enhance synthesis and secretion of organic acids, flavonoids, proteases, acid phosphatases, and proton efflux. These modifications lead to mobilization in the soil of, otherwise unavailable, P. White lupin (Lupinus albus) represents a model plant to study cluster roots and for understanding plant acclimation to nutrient deficiency. It tolerates simultaneous P and N deficiencies and also enhances uptake of additional nutrients. Here, we present the structural and functional modifications that occur in conditions of P and N deficiencies and lead to the organogenesis and altered metabolism of nodules and cluster roots. Some known N and P signaling mechanisms include different factors, including phytohormones and miRNAs. The combination of the individual N and P mechanisms uncovers interactive regulation pathways that concur in nodules and cluster roots. L. albus interlinks N and P recycling processes both in the plant itself and in nature.

Nitrogen (N) and phosphorus (P) are two major plant nutrients, and their deficiencies often limit plant growth and crop yield. The uptakes of N or P affect each other, and consequently, understanding N–P interactions is fundamental. Their signaling mechanisms have been studied mostly separately, and integrating N–P interactive regulation is becoming the aim of some recent works. Lupins are singular plants, as, under N and P deficiencies, they are capable to develop new organs, the N2-fixing symbiotic nodules, and some species can also transform their root architecture to form cluster roots, hundreds of short rootlets that alter their metabolism to induce a high-affinity P transport system and enhance synthesis and secretion of organic acids, flavonoids, proteases, acid phosphatases, and proton efflux. These modifications lead to mobilization in the soil of, otherwise unavailable, P. White lupin (Lupinus albus) represents a model plant to study cluster roots and for understanding plant acclimation to nutrient deficiency. It tolerates simultaneous P and N deficiencies and also enhances uptake of additional nutrients. Here, we present the structural and functional modifications that occur in conditions of P and N deficiencies and lead to the organogenesis and altered metabolism of nodules and cluster roots. Some known N and P signaling mechanisms include different factors, including phytohormones and miRNAs. The combination of the individual N and P mechanisms uncovers interactive regulation pathways that concur in nodules and cluster roots. L. albus interlinks N and P recycling processes both in the plant itself and in nature. [ABSTRACT FROM AUTHOR]

Virgilia divaricata is a tree legume that grows in the Cape Floristic Region (CFA) in poor nutrient soils. A comparison between high and low phosphate growth conditions between roots and nodules was conducted and evaluated for the plants ability to cope under low phosphate stress conditions in V. divaricata. We proved that the plant copes with low phosphate stress through an increased allocation of resources, reliance on BNF and enhanced enzyme activity, especially PEPC. Nodules had a lower percentage decline in P compared to roots to uphold its metabolic functions. These strategies partly explain how V. divaricata can sustain growth despite LP conditions. Although the number of nodules declined with LP, their biomass remained unchanged in spite of a plant decline in dry weight. This is achieved via the high efficiency of BNF under P stress. During LP, nodules had a lower % decline at 34% compared to the roots at 88%. We attribute this behavior to P conservation strategies in LP nodules that imply an increase in a metabolic bypass that operates at the PEP branch point in glycolysis. The enhanced activities of nodule PEPC, MDH, and ME, whilst PK declines, suggests that under LP conditions an adenylate bypass was in operation either to synthesize more organic acids or to mediate pyruvate via a non-adenylate requiring metabolic route. Both possibilities represent a P-stress adaptation route and this is the first report of its kind for legume trees that are indigenous to low P, acid soils. Although BNF declined by a small percentage during LP, this P conservation was evident in the unchanged BNF efficiency per weight, and the increase in BNF efficiency per mol of P. It appears that legumes that are indigenous to acid soils, may be able to continue their reliance on BNF via increased allocation to nodules and also due to increase their efficiency for BNF on a P basis, owing to P-saving mechanisms such as the organic acid routes.

Actinorhizal nodules are structurally different from legume nodules and show a greater similarity to lateral roots. Because of the important role of auxins in lateral root and nodule formation, auxin profiles were examined in roots and nodules of the actinorhizal species Datisca glomerata and the model legume Medicago truncatula. The auxin response in roots and nodules of both species was analyzed in transgenic root systems expressing a beta-glucuronidase gene under control of the synthetic auxin-responsive promoter DR5. The effects of two different auxin on root development were compared for both species. The auxin present in nodules at the highest levels was phenylacetic acid (PAA). No differences were found between the concentrations of active auxins of roots vs. nodules, while levels of the auxin conjugate indole-3-acetic acid-alanine were increased in nodules compared to roots of both species. Because auxins typically act in concert with cytokinins, cytokinins were also quantified. Concentrations of cis -zeatin and some glycosylated cytokinins were dramatically increased in nodules compared to roots of D. glomerata , but not of M. truncatula. The ratio of active auxins to cytokinins remained similar in nodules compared to roots in both species. The auxin response, as shown by the activation of the DR5 promoter, seemed significantly reduced in nodules compared to roots of both species, suggesting the accumulation of auxins in cell types that do not express the signal transduction pathway leading to DR5 activation. Effects on root development were analyzed for the synthetic auxin naphthaleneacetic acid (NAA) and PAA, the dominant auxin in nodules. Both auxins had similar effects, except that the sensitivity of roots to PAA was lower than to NAA. However, while the effects of both auxins on primary root growth were similar for both species, effects on root branching were different: both auxins had the classical positive effect on root branching in M. truncatula , but a negative effect in D. glomerata. Such a negative effect of exogenous auxin on root branching has previously been found for a cucurbit that forms lateral root primordia in the meristem of the parental root; however, root branching in D. glomerata does not follow that pattern. [ABSTRACT FROM AUTHOR]

Micro-RNAs from legume plants are emerging as relevant regulators of the rhizobia nitrogen-fixing symbiosis. In this work we functionally characterized the role of the node conformed by micro-RNA319 (miR319) – TEOSINTE BRANCHED/CYCLOIDEA/PCF (TCP) transcription factor in the common bean (Phaseolus vulgaris) – Rhizobium tropici symbiosis. The miR319d, one of nine miR319 isoforms from common bean, was highly expressed in root and nodules from inoculated plants as compared to roots from fertilized plants. The miR319d targets TCP10 (Phvul.005G067950), identified by degradome analysis, whose expression showed a negative correlation with miR319d expression. The phenotypic analysis of R. tropici-inoculated composite plants with transgenic roots/nodules overexpressing or silencing the function of miR319d demonstrated the relevant role of the miR319d/TCP10 node in the common bean rhizobia symbiosis. Increased miR319d resulted in reduced root length/width ratio, increased rhizobial infection evidenced by more deformed root hairs and infection threads, and decreased nodule formation and nitrogenase activity per plant. In addition, these plants with lower TCP10 levels showed decreased expression level of the jasmonic acid (JA) biosynthetic gene: LOX2. The transcription of LOX2 by TCPs has been demonstrated for Arabidopsis and in several plants LOX2 level and JA content have been associate with TCP levels. On this basis, we propose that in roots/nodules of inoculated common bean plants TCP10 could be the transcriptional regulator of LOX2 and the miR319d/TCP10 node could affect nodulation through JA signaling. However, given the complexity of nodulation, the participation of other signaling pathways in the phenotypes observed cannot be ruled out.

Micro-RNAs from legume plants are emerging as relevant regulators of the rhizobia nitrogen-fixing symbiosis. In this work we functionally characterized the role of the node conformed by micro-RNA319 (miR319) – TEOSINTE BRANCHED/CYCLOIDEA/PCF (TCP) transcription factor in the common bean (Phaseolus vulgaris) – Rhizobium tropici symbiosis. The miR319d, one of nine miR319 isoforms from common bean, was highly expressed in root and nodules from inoculated plants as compared to roots from fertilized plants. The miR319d targets TCP10 (Phvul.005G067950), identified by degradome analysis, whose expression showed a negative correlation with miR319d expression. The phenotypic analysis of R. tropici -inoculated composite plants with transgenic roots/nodules overexpressing or silencing the function of miR319d demonstrated the relevant role of the miR319d/TCP10 node in the common bean rhizobia symbiosis. Increased miR319d resulted in reduced root length/width ratio, increased rhizobial infection evidenced by more deformed root hairs and infection threads, and decreased nodule formation and nitrogenase activity per plant. In addition, these plants with lower TCP10 levels showed decreased expression level of the jasmonic acid (JA) biosynthetic gene: LOX2. The transcription of LOX2 by TCPs has been demonstrated for Arabidopsis and in several plants LOX2 level and JA content have been associate with TCP levels. On this basis, we propose that in roots/nodules of inoculated common bean plants TCP10 could be the transcriptional regulator of LOX2 and the miR319d/TCP10 node could affect nodulation through JA signaling. However, given the complexity of nodulation, the participation of other signaling pathways in the phenotypes observed cannot be ruled out. [ABSTRACT FROM AUTHOR]

The efficient use of resources such as water and nutrients by plants is increasingly important as the world population food demand continues to grow. With the increased production of lentil in the temperate zones of North America, improvement in yield needs to be maintained. The use of wild lentil genotypes as sources of genetic diversity for introgression into cultivated lentil is an important breeding strategy, but little is known about their root systems. We evaluated the root systems of five wild lentil species and Lens culinaris under fully watered conditions. Plants were grown in 60 cm tubes containing equal volumes of soil collected from the reconstructed A, B, and C horizons. Significant differences were observed for root traits and fine root distribution between and within species and the proportion of root biomass partitioned into each soil layer was unique for each genotype. We also observed variability in nodule number and nodule shape within and between genotypes. Some genotypes more efficiently used water for either biomass or seed production. The allocation of resources to seed production also varied between genotypes. These observations could have impact on the design of future lentil breeding in the context of strategies for managing changes in rainfall amount and distribution for lentil production ecosystems.

The NRAMP (natural resistance-associated macrophage protein) family of genes has been widely characterized in organisms ranging from bacteria to yeast, plants, mice, and humans. This gene family plays vital roles in divalent metal ion transport across cellular membranes. As yet, comprehensive analysis of NRAMP family genes has not been reported for soybean. In this study, bioinformatics analysis was conducted to identify 13 soybean NRAMP genes, along with their gene structures, phylogenetic relationships, and transmembrane domains. Expression analysis suggests that GmNRAMP genes function in numerous tissues and development stages. Moreover, soybean NRAMP genes were differentially regulated by deficiencies of N, P, K, Fe, and S, along with toxicities of Fe, Cu, Cd, and Mn. These results indicate that GmNRAMP genes function in many nutrient stress pathways, and might be involved in crosstalk among nutrient stress pathways. Subcellular localization analysis in Arabidopsis protoplasts confirmed the tonoplast or plasma membrane localization of selected soybean NRMAP proteins. Protein-protein interaction analysis found that the networks of three GmNRAMP proteins which putatively interact with nodulin-like proteins, almost distinct from the network that is common to the other 10 soybean NRAMP proteins. Subsequent qRT-PCR results confirmed that these three GmNRMAP genes exhibited enhanced expression in soybean nodules, suggesting potential functions in the transport of Fe or other metal ions in soybean nodules. Overall, the systematic analysis of the GmNRAMP gene family reported herein provides valuable information for further studies on the biological roles of GmNRAMPs in divalent metal ion transport in various soybean tissues under numerous nutrient stresses and soybean-rhizobia symbiosis.

The efficient use of resources such as water and nutrients by plants is increasingly important as the world population food demand continues to grow. With the increased production of lentil in the temperate zones of North America, improvement in yield needs to be maintained. The use of wild lentil genotypes as sources of genetic diversity for introgression into cultivated lentil is an important breeding strategy, but little is known about their root systems. We evaluated the root systems of five wild lentil species and Lens culinaris under fully watered conditions. Plants were grown in 60 cm tubes containing equal volumes of soil collected from the reconstructed A, B, and C horizons. Significant differences were observed for root traits and fine root distribution between and within species and the proportion of root biomass partitioned into each soil layer was unique for each genotype. We also observed variability in nodule number and nodule shape within and between genotypes. Some genotypes more efficiently used water for either biomass or seed production. The allocation of resources to seed production also varied between genotypes. These observations could have impact on the design of future lentil breeding in the context of strategies for managing changes in rainfall amount and distribution for lentil production ecosystems. [ABSTRACT FROM AUTHOR]

Highlights from the article: Keywords: nitrogen fixation; nodules; metabolic adjustment; gene expression reprogramming; long-range signals; legume; rhizobia The symbiosis involves compatible legume-rhizobia species to recognize one another in the rhizosphere, followed by the bacteria infecting the host and initiating the formation of nodules that would eventually house the bacteria (Ferguson et al., [2]). Sogawa et al. studied the role of SNARE proteins that mediate membrane trafficking in eukaryotic cells, showing that both LjVAMP72a and LjVAMP72b facilitate the legume-rhizobia symbioses and root hair development by affecting the secretory pathway in the host plant. Nitrogen fixation, nodules, metabolic adjustment, gene expression reprogramming, long-range signals, legume, rhizobia.

Nitrogenase is an oxygen labile enzyme. Microaerobic conditions within the infected zone of nodules are maintained primarily by an oxygen diffusion barrier located in the nodule cortex. Flexibility of the oxygen diffusion barrier is important for the acclimation processes of nodules in response to changes in external oxygen concentration. The hypothesis of the present study was that there are additional molecular mechanisms involved. Nodule activity of Medicago truncatula plants were continuously monitored during a change from 21 to 25 or 30 % oxygen around root nodules by measuring nodule H2 evolution. Within about two minutes of the increase in oxygen concentration, a steep decline in nitrogenase activity occurred. A quick recovery commenced about eight minutes later. A qPCR-based analysis of the expression of genes for nitrogenase components showed a tendency towards upregulation during the recovery. The recovery resulted in a new constant activity after about 30 minutes, corresponding to approximately 90 % of the pre-treatment level. An RNAseq-based comparative transcriptome profiling of nodules at that point in time revealed that genes for nodule-specific cysteine-rich (NCR) peptides, defensins, leghaemoglobin and chalcone and stilbene synthase were significantly upregulated when considered as a gene family. A gene for a nicotianamine synthase-like protein (Medtr1g084050) showed a strong increase in count number. The gene appears to be of importance for nodule functioning, as evidenced by its consistently high expression in nodules and a strong reaction to various environmental cues that influence nodule activity. A Tnt1-mutant that carries an insert in the coding sequence (cds) of that gene showed reduced nitrogen fixation and less efficient acclimation to an increased external oxygen concentration. It was concluded that sudden increases in oxygen concentration around nodules destroy nitrogenase, which is quickly counteracted by an increased neoformation of the enzyme. This reaction might be induced by increased formation of NCR peptides and necessitates an efficient iron supply to the bacteroid, which is probably mediated by nicotianamine.

Aluminum (Al) toxicity is widespread in acidic soils where the common bean (Phaseolus vulgaris), the most important legume for human consumption, is produced and it is a limiting factor for crop production and symbiotic nitrogen fixation. We characterized the nodule responses of common bean plants inoculated with Rhizobioum tropici CIAT899 and the root responses of nitrate-fertilized plants exposed to excess Al in low pH, for long or short periods. A 43 - 50% reduction in nitrogenase activity indicates that Al toxicity highly affected nitrogen fixation in common bean. Bean roots and nodules showed characteristic symptoms for Al toxicity. In mature nodules Al accumulation and lipoperoxidation were observed in the infected zone, while callose deposition and cell death occurred mainly in the nodule cortex. Regulatory mechanisms of plant responses to metal toxicity involve microRNAs (miRNAs) along other regulators. Using a miRNA-macroarray hybridization approach we identified 28 (14 up-regulated) Al toxicity nodule-responsive miRNAs. We validated (qRT-PCR) the expression of eight nodule responsive miRNAs in roots and in nodules exposed to high Al for long or short periods. The inverse correlation between the target and miRNA expression ratio (stress:control) was observed in every case. Generally, miRNAs showed a higher earlier response in roots than in nodules. Some of the common bean Alt responsive miRNAs identified have also been reported as differentially expressed in other plant species subjected to similar stress condition. miRNA/target nodes analyzed in this work are known to be involved in relevant signaling pathways, thus we propose that the participation of miR164/NAC1 and miR393/TIR1 in auxin and of miR170/SCL in gibberellin signaling is relevant for common bean response/adaptation to Al stress. Our data provide a foundation for evaluating the individual roles of miRNAs in the response of common bean nodules to Al toxicity.

Nitrogenase is an oxygen labile enzyme. Microaerobic conditions within the infected zone of nodules are maintained primarily by an oxygen diffusion barrier (ODB) located in the nodule cortex. Flexibility of the ODB is important for the acclimation processes of nodules in response to changes in external oxygen concentration. The hypothesis of the present study was that there are additional molecular mechanisms involved. Nodule activity of Medicago truncatula plants were continuously monitored during a change from 21 to 25 or 30% oxygen around root nodules by measuring nodule H2 evolution. Within about 2 min of the increase in oxygen concentration, a steep decline in nitrogenase activity occurred. A quick recovery commenced about 8 min later. A qPCR-based analysis of the expression of genes for nitrogenase components showed a tendency toward upregulation during the recovery. The recovery resulted in a new constant activity after about 30 min, corresponding to approximately 90% of the pre-treatment level. An RNAseq-based comparative transcriptome profiling of nodules at that point in time revealed that genes for nodule-specific cysteine-rich (NCR) peptides, defensins, leghaemoglobin and chalcone and stilbene synthase were significantly upregulated when considered as a gene family. A gene for a nicotianamine synthaselike protein (Medtr1g084050) showed a strong increase in count number. The gene appears to be of importance for nodule functioning, as evidenced by its consistently high expression in nodules and a strong reaction to various environmental cues that influence nodule activity. A Tnt1-mutant that carries an insert in the coding sequence (cds) of that gene showed reduced nitrogen fixation and less efficient acclimation to an increased external oxygen concentration. It was concluded that sudden increases in oxygen concentration around nodules destroy nitrogenase, which is quickly counteracted by an increased neoformation of the enzyme. This reaction might be induced by increased formation of NCR peptides and necessitates an efficient iron supply to the bacteroid, which is probably mediated by nicotianamine. [ABSTRACT FROM AUTHOR]

Aluminum (Al) toxicity is widespread in acidic soils where the common bean (Phaseolus vulgaris), the most important legume for human consumption, is produced and it is a limiting factor for crop production and symbiotic nitrogen fixation. We characterized the nodule responses of common bean plants inoculated with Rhizobioum tropici CIAT899 and the root responses of nitrate-fertilized plants exposed to excess Al in low pH, for long or short periods. A 43-50% reduction in nitrogenase activity indicates that Al toxicity (Alt) highly affected nitrogen fixation in common bean. Bean roots and nodules showed characteristic symptoms for Alt. In mature nodules Al accumulation and lipoperoxidation were observed in the infected zone, while callose deposition and cell death occurred mainly in the nodule cortex. Regulatory mechanisms of plant responses to metal toxicity involve microRNAs (miRNAs) along other regulators. Using a miRNA-macroarray hybridization approach we identified 28 (14 up-regulated) Alt nodule-responsive miRNAs. We validated (quantitative reverse transcriptase-PCR) the expression of eight nodule responsive miRNAs in roots and in nodules exposed to high Al for long or short periods. The inverse correlation between the target and miRNA expression ratio (stress:control) was observed in every case. Generally, miRNAs showed a higher earlier response in roots than in nodules. Some of the common bean Alt-responsive miRNAs identified has also been reported as differentially expressed in other plant species subjected to similar stress condition. miRNA/target nodes analyzed in this work are known to be involved in relevant signaling pathways, thus we propose that the participation of miR164/NAC1 (NAM/ATAF/CUC transcription factor) and miR393/TIR1 (TRANSPORT INHIBITOR RESPONSE 1-like protein) in auxin and of miR170/SCL (SCARECROW-like protein transcription factor) in gibberellin signaling is relevant for common bean response/adaptation to Al stress. Our data provide a foundation for evaluating the individual roles of miRNAs in the response of common bean nodules to Alt. [ABSTRACT FROM AUTHOR]

Nitrate reductase (NR) is the first enzyme of the nitrogen reduction pathway in plants, leading to the production of ammonia. However, in the nitrogen-fixing symbiosis between legumes and rhizobia, atmospheric nitrogen (N 2 ) is directly reduced to ammonia by the bacterial nitrogenase, which questions the role of NR in symbiosis. Next to that, NR is the best-characterized source of nitric oxide (NO) in plants, and NO is known to be produced during the symbiosis. In the present study, we first surveyed the three NR genes (Mt NR1 , Mt NR2 , and Mt NR3 ) present in the Medicago truncatula genome and addressed their expression, activity, and potential involvement in NO production during the symbiosis between M. truncatula and Sinorhizobium meliloti . Our results show that Mt NR1 and Mt NR2 gene expression and activity are correlated with NO production throughout the symbiotic process and that MtNR1 is particularly involved in NO production in mature nodules. Moreover, NRs are involved together with the mitochondrial electron transfer chain in NO production throughout the symbiotic process and energy regeneration in N 2 -fixing nodules. Using an in vivo NMR spectrometric approach, we show that, in mature nodules, NRs participate also in the regulation of energy state, cytosolic pH, carbon and nitrogen metabolism under both normoxia and hypoxia. These data point to the importance of NR activity for the N 2 -fixing symbiosis and provide a first explanation of its role in this process., (Copyright © 2020 Berger, Boscari, Horta Araújo, Maucourt, Hanchi, Bernillon, Rolin, Puppo and Brouquisse.)

Virgilia divaricata is a tree legume that grows in the Cape Floristic Region (CFA) in poor nutrient soils. A comparison between high and low phosphate growth conditions between roots and nodules was conducted and evaluated for the plants ability to cope under low phosphate stress conditions in V. divaricata . We proved that the plant copes with low phosphate stress through an increased allocation of resources, reliance on BNF and enhanced enzyme activity, especially PEPC. Nodules had a lower percentage decline in P compared to roots to uphold its metabolic functions. These strategies partly explain how V. divaricata can sustain growth despite LP conditions. Although the number of nodules declined with LP, their biomass remained unchanged in spite of a plant decline in dry weight. This is achieved via the high efficiency of BNF under P stress. During LP, nodules had a lower % decline at 34% compared to the roots at 88%. We attribute this behavior to P conservation strategies in LP nodules that imply an increase in a metabolic bypass that operates at the PEP branch point in glycolysis. The enhanced activities of nodule PEPC, MDH, and ME, whilst PK declines, suggests that under LP conditions an adenylate bypass was in operation either to synthesize more organic acids or to mediate pyruvate via a non-adenylate requiring metabolic route. Both possibilities represent a P-stress adaptation route and this is the first report of its kind for legume trees that are indigenous to low P, acid soils. Although BNF declined by a small percentage during LP, this P conservation was evident in the unchanged BNF efficiency per weight, and the increase in BNF efficiency per mol of P. It appears that legumes that are indigenous to acid soils, may be able to continue their reliance on BNF via increased allocation to nodules and also due to increase their efficiency for BNF on a P basis, owing to P-saving mechanisms such as the organic acid routes.

The NRAMP (natural resistance-associated macrophage protein) family of genes has been widely characterized in organisms ranging from bacteria to yeast, plants, mice, and humans. This gene family plays vital roles in divalent metal ion transport across cellular membranes. As yet, comprehensive analysis of NRAMP family genes has not been reported for soybean. In this study, bioinformatics analysis was conducted to identify 13 soybean NRAMP genes, along with their gene structures, phylogenetic relationships, and transmembrane domains. Expression analysis suggests that GmNRAMP genes function in numerous tissues and development stages. Moreover, soybean NRAMP genes were differentially regulated by deficiencies of N, P, K, Fe, and S, along with toxicities of Fe, Cu, Cd, and Mn. These results indicate that GmNRAMP genes function in many nutrient stress pathways, and might be involved in crosstalk among nutrient stress pathways. Subcellular localization analysis in Arabidopsis protoplasts confirmed the tonoplast or plasma membrane localization of selected soybean NRMAP proteins. Protein-protein interaction analysis found that the networks of three GmNRAMP proteins which putatively interact with nodulin-like proteins, almost distinct from the network that is common to the other 10 soybean NRAMP proteins. Subsequent qRT-PCR results confirmed that these three GmNRMAP genes exhibited enhanced expression in soybean nodules, suggesting potential functions in the transport of Fe or other metal ions in soybean nodules. Overall, the systematic analysis of the GmNRAMP gene family reported herein provides valuable information for further studies on the biological roles of GmNRAMPs in divalent metal ion transport in various soybean tissues under numerous nutrient stresses and soybean-rhizobia symbiosis.