RESUMEN

Nitrogen is an essential macronutrient critical for plant growth and productivity. Plants have the capacity to uptake inorganic nitrate and ammonium, with nitrate playing a crucial role as a signaling molecule in various cellular processes. The availability of nitrate and the signaling pathways involved finely tune the processes of nitrate uptake and assimilation. NIN-like proteins (NLPs), a group of transcription factors belonging to the RWP-RK gene family, act as major nitrate sensors and are implicated in the primary nitrate response (PNR) within the nucleus of both non-leguminous and leguminous plants through their RWP-RK domains. In leguminous plants, NLPs are indispensable for the initiation and development of nitrogen-fixing nodules in symbiosis with rhizobia. Moreover, NLPs play pivotal roles in plant responses to abiotic stresses, including drought and cold. Recent studies have identified NLP homologs in oomycete pathogens, suggesting their potential involvement in pathogenesis and virulence. This review article delves into the conservation of RWP-RK genes, examining their significance and implications across different plant species. The focus lies on the role of NLPs as nitrate sensors, investigating their involvement in various processes, including rhizobial symbiosis in both leguminous and non-leguminous plants. Additionally, the multifaceted functions of NLPs in abiotic stress responses, developmental processes, and interactions with plant pathogens are explored. By comprehensively analyzing the role of NLPs in nitrate signaling and their broader implications for plant growth and development, this review sheds light on the intricate mechanisms underlying nitrogen sensing and signaling in various plant lineages.

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Legumes form root mutualistic symbioses with some soil microbes promoting their growth, rhizobia, and arbuscular mycorrhizal fungi (AMF). A conserved set of plant proteins rules the transduction of symbiotic signals from rhizobia and AMF in a so-called common symbiotic signaling pathway (CSSP). Despite considerable efforts and advances over the past 20 years, there are still key elements to be discovered about the establishment of these root symbioses. Rhizobia and AMF root colonization are possible after a deep cell reorganization. In the interaction between the model legume Lotus japonicus and Mesorhizobium loti, this reorganization has been shown to be dependent on a SCAR/Wave-like signaling module, including Rho-GTPase (ROP in plants). Here, we studied the potential role of ROP3 in the nitrogen-fixing symbiosis (NFS) as well as in the arbuscular mycorrhizal symbiosis (AMS). We performed a detailed phenotypic study on the effects of the loss of a single ROP on the establishment of both root symbioses. Moreover, we evaluated the expression of key genes related to CSSP and to the rhizobial-specific pathway. Under our experimental conditions, rop3 mutant showed less nodule formation at 7- and 21-days post inoculation as well as less microcolonies and a higher frequency of epidermal infection threads. However, AMF root colonization was not affected. These results suggest a role of ROP3 as a positive regulator of infection thread formation and nodulation in L. japonicus. In addition, CSSP gene expression was neither affected in NFS nor in AMS condition in rop3 mutant. whereas the expression level of some genes belonging to the rhizobial-specific pathway, like RACK1, decreased in the NFS. In conclusion, ROP3 appears to be involved in the NFS, but is neither required for intra-radical growth of AMF nor arbuscule formation.

RESUMEN

Corn and common bean have been cultivated together in Mesoamerica for thousands of years in an intercropping system called "milpa," where the roots are intermingled, favoring the exchange of their microbiota, including symbionts such as rhizobia. In this work, we studied the genomic expression of Rhizobium phaseoli Ch24-10 (by RNA-seq) after a 2-h treatment in the presence of root exudates of maize and bean grown in monoculture and milpa system under hydroponic conditions. In bean exudates, rhizobial genes for nodulation and degradation of aromatic compounds were induced; while in maize, a response of genes for degradation of mucilage and ferulic acid was observed, as well as those for the transport of sugars, dicarboxylic acids and iron. Ch24-10 transcriptomes in milpa resembled those of beans because they both showed high expression of nodulation genes; some genes that were expressed in corn exudates were also induced by the intercropping system, especially those for the degradation of ferulic acid and pectin. Beans grown in milpa system formed nitrogen-fixing nodules similar to monocultured beans; therefore, the presence of maize did not interfere with Rhizobium-bean symbiosis. Genes for the metabolism of sugars and amino acids, flavonoid and phytoalexin tolerance, and a T3SS were expressed in both monocultures and milpa system, which reveals the adaptive capacity of rhizobia to colonize both legumes and cereals. Transcriptional fusions of the putA gene, which participates in proline metabolism, and of a gene encoding a polygalacturonase were used to validate their participation in plant-microbe interactions. We determined the enzymatic activity of carbonic anhydrase whose gene was also overexpressed in response to root exudates.

RESUMEN

Plants MADS-domain/AGL proteins constitute a large transcription factor (TF) family that controls the development of almost every plant organ. We performed a phylogeny of (ca. 500) MADS-domain proteins from Arabidopsis and four legume species. We identified clades with Arabidopsis MADS-domain proteins known to participate in root development that grouped legume MADS-proteins with similar high expression in roots and nodules. In this work, we analyzed the role of AGL transcription factors in the common bean (Phaseolus vulgaris) - Rhizobium etli N-fixing symbiosis. Sixteen P. vulgaris AGL genes (PvAGL), out of 93 family members, are expressed - at different levels - in roots and nodules. From there, we selected the PvAGL gene denominated PvFUL-like for overexpression or silencing in composite plants, with transgenic roots and nodules, that were used for phenotypic analysis upon inoculation with Rhizobium etli. Because of sequence identity in the DNA sequence used for RNAi-FUL-like construct, roots, and nodules expressing this construct -referred to as RNAi_AGL- showed lower expression of other five PvAGL genes highly expressed in roots/nodules. Contrasting with PvFUL-like overexpressing plants, rhizobia-inoculated plants expressing the RNAi_AGL silencing construct presented affection in the generation and growth of transgenic roots from composite plants, both under non-inoculated or rhizobia-inoculated condition. Furthermore, the rhizobia-inoculated plants showed decreased rhizobial infection concomitant with the lower expression level of early symbiotic genes and increased number of small, ineffective nodules that indicate an alteration in the autoregulation of the nodulation symbiotic process. We propose that the positive effects of PvAGL TF in the rhizobia symbiotic processes result from its potential interplay with NIN, the master symbiotic TF regulator, that showed a CArG-box consensus DNA sequence recognized for DNA binding of AGL TF and presented an increased or decreased expression level in roots from non-inoculated plants transformed with OE_FUL or RNAi_AGL construct, respectively. Our work contributes to defining novel transcriptional regulators for the common bean - rhizobia N-fixing symbiosis, a relevant process for sustainable agriculture.

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Symbiotic nitrogen fixation (SNF) makes atmospheric nitrogen biologically available and regulates carbon storage in many terrestrial ecosystems. Despite its global importance, estimates of SNF rates are highly uncertain, particularly in tropical forests where rates are assumed to be high. Here we provide a framework for evaluating the uncertainty of sample-based SNF estimates and discuss its implications for quantifying SNF and thus understanding of forest function. We apply this framework to field data sets from six lowland tropical rainforests (mature and secondary) in Brazil and Costa Rica. We use this data set to estimate parameters influencing SNF estimation error, notably the root nodule abundance and variation in SNF rates among soil cores containing root nodules. We then use simulations to gauge the relationship between sampling effort and SNF estimation accuracy for a combination of parameters. Field data illuminate a highly right-skewed lognormal distribution of SNF rates among soil cores containing root nodules that were rare and spanned five orders of magnitude. Consequently, simulations demonstrated that sample sizes of hundreds to even thousands of soil cores are needed to obtain estimates of SNF that are within, for example, a factor of 2 of the actual rate with 75% probability. This represents sample sizes that are larger than most studies to date. As a result of this previously undescribed uncertainty, we suggest that current estimates of SNF in tropical forests are not sufficiently constrained to elucidate forest stand-level controls of SNF, which hinders our understanding of the impact of SNF on tropical forest ecosystem processes.

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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.

RESUMEN

The common bean (Phaseolus vulgaris L.) low phytic acid (lpa1) biofortified genotype produces seeds with improved nutritional characteristics and does not display negative pleiotropic effects. Here we demonstrated that lpa1 plants establish an efficient nitrogen-fixing symbiosis with Rhizobium etli CE3. The lpa1 nodules showed a higher expression of nodule-function related genes than the nodules of the parental wild type genotype (BAT 93). We analyzed the response to water stress of lpa1 vs. BAT 93 plants grown under fertilized or under symbiotic N2-fixation conditions. Water stress was induced by water withholding (up to 14% soil moisture) to fertilized or R. etli nodulated plants previously grown with normal irrigation. The fertilized lpa1 plants showed milder water stress symptoms during the water deployment period and after the rehydration recovery period when lpa1 plants showed less biomass reduction. The symbiotic water-stressed lpa1 plants showed decreased nitrogenase activity that coincides with decreased sucrose synthase gene expression in nodules; lower turgor weight to dry weight (DW) ratio, which has been associated with higher drought resistance index; downregulation of carbon/nitrogen (C/N)-related and upregulation of stress-related genes. Higher expression of stress-related genes was also observed in bacteroids of stressed lpa1 plants that also displayed very high expression of the symbiotic cbb3 oxidase (fixNd).

RESUMEN

Symbiotic nitrogen (N)-fixing trees can drive N and carbon cycling and thus are critical components of future climate projections. Despite detailed understanding of how climate influences N-fixation enzyme activity and physiology, comparatively little is known about how climate influences N-fixing tree abundance. Here, we used forest inventory data from the USA and Mexico (>125,000 plots) along with climate data to address two questions: (1) How does the abundance distribution of N-fixing trees (rhizobial, actinorhizal, and both types together) vary with mean annual temperature (MAT) and precipitation (MAP)? (2) How will changing climate shift the abundance distribution of N-fixing trees? We found that rhizobial N-fixing trees were nearly absent below 15°C MAT, but above 15°C MAT, they increased in abundance as temperature rose. We found no evidence for a hump-shaped response to temperature throughout the range of our data. Rhizobial trees were more abundant in dry than in wet ecosystems. By contrast, actinorhizal trees peaked in abundance at 5-10°C MAT and were least abundant in areas with intermediate precipitation. Next, we used a climate-envelope approach to project how N-fixing tree relative abundance might change in the future. The climate-envelope projection showed that rhizobial N-fixing trees will likely become more abundant in many areas by 2080, particularly in the southern USA and western Mexico, due primarily to rising temperatures. Projections for actinorhizal N-fixing trees were more nuanced due to their nonmonotonic dependence on temperature and precipitation. Overall, the dominant trend is that warming will increase N-fixing tree abundance in much of the USA and Mexico, with large increases up to 40° North latitude. The quantitative link we provide between climate and N-fixing tree abundance can help improve the representation of symbiotic N fixation in Earth System Models.

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RESUMEN

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.

RESUMEN

Biological nitrogen fixation (BNF) is the largest natural source of exogenous nitrogen (N) to unmanaged ecosystems and also the primary baseline against which anthropogenic changes to the N cycle are measured. Rates of BNF in tropical rainforest are thought to be among the highest on Earth, but they are notoriously difficult to quantify and are based on little empirical data. We adapted a sampling strategy from community ecology to generate spatial estimates of symbiotic and free-living BNF in secondary and primary forest sites that span a typical range of tropical forest legume abundance. Although total BNF was higher in secondary than primary forest, overall rates were roughly five times lower than previous estimates for the tropical forest biome. We found strong correlations between symbiotic BNF and legume abundance, but we also show that spatially free-living BNF often exceeds symbiotic inputs. Our results suggest that BNF in tropical forest has been overestimated, and our data are consistent with a recent top-down estimate of global BNF that implied but did not measure low tropical BNF rates. Finally, comparing tropical BNF within the historical area of tropical rainforest with current anthropogenic N inputs indicates that humans have already at least doubled reactive N inputs to the tropical forest biome, a far greater change than previously thought. Because N inputs are increasing faster in the tropics than anywhere on Earth, both the proportion and the effects of human N enrichment are likely to grow in the future.

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As leguminosas forrageiras, além de contribuírem para o aumento da oferta de forragem com alto teor de proteína, também são importantes como cobertura vegetal de solos. Entre as leguminosas com grande potencial forrageiro, espécies do gênero Lotus têm se destacado. Este trabalho visou à seleção de rizóbios nativos eficientes em Lotus glaber a partir de amostras de solo de cinco localidades do Rio Grande do Sul. Obtiveram-se 259 isolados de rizóbios, que foram avaliados quanto à morfologia colonial e produção de melanina. Destes, 15 isolados foram selecionados para avaliação da eficiência na fixação simbiótica de nitrogênio em experimento em casa de vegetação e caracterizados geneticamente por comparação do perfil eletroforético dos produtos de amplificação do DNA genômico, por PCR com os oligonucleotídeos iniciadores BOX e ERIC. Apenas cinco isolados produziram melanina. Nove isolados foram mais eficientes do que a estirpe SEMIA 830, que é autorizada para a produção de inoculante para L. glaber no país. Na caracterização genética, observou-se que nenhum isolado apresentou identidade com as estirpes recomendadas, o que demonstra que, nos solos do Rio Grande do Sul, existem rizóbios autóctones eficientes na fixação simbiótica de nitrogênio com L. glaber, podendo ser recomendados para estudos a campo, visando a uma futura produção de inoculantes para estas leguminosas no Brasil.

Forage legumes besides contributing to increase the supply of high protein content forage are also good cover for the soil surface. Among the legumes with high potential forage, the genus Lotus has been outstanding. This research aimed to selectnative rhizobia efficient in fixing nitrogen with Lotus glaber, from soil samples of five localities of Rio Grande do Sul. A total of 259 rhizobia isolates were obtained, which were evaluated based on colony morphology and in vitro melanin production. Among them, 15 isolates were selected for the evaluation of the symbiotic nitrogen fixation efficiency in greenhouse experiments and genetically characterized by genomic DNA fingerprinting, PCR amplification with BOX and ERIC primers. Only five isolates produced melanin. Nine isolates were more efficient than strain SEMIA 830, which is authorized for the production of commercial inoculants of L. glaber in Brazil. In relation to the genetic characterization, the isolates have shown no similarity with the commercial strains, indicating that in the soils of Rio Grande do Sul there are autochthonous rhizobia capable of efficient symbiotic nitrogen fixation with L. glaber, which might be recommended for field evaluations, aiming a future production of inoculants for this legume in Brazil.

RESUMEN

Forage legumes besides contributing to increase the supply of high protein content forage are also good cover for the soil surface. Among the legumes with high potential forage, the genus Lotus has been outstanding. This research aimed to selectnative rhizobia efficient in fixing nitrogen with Lotus glaber, from soil samples of five localities of Rio Grande do Sul. A total of 259 rhizobia isolates were obtained, which were evaluated based on colony morphology and in vitro melanin production. Among them, 15 isolates were selected for the evaluation of the symbiotic nitrogen fixation efficiency in greenhouse experiments and genetically characterized by genomic DNA fingerprinting, PCR amplification with BOX and ERIC primers. Only five isolates produced melanin. Nine isolates were more efficient than strain SEMIA 830, which is authorized for the production of commercial inoculants of L. glaber in Brazil. In relation to the genetic characterization, the isolates have shown no similarity with the commercial strains, indicating that in the soils of Rio Grande do Sul there are autochthonous rhizobia capable of efficient symbiotic nitrogen fixation with L. glaber, which might be recommended for field evaluations, aiming a future production of inoculants for this legume in Brazil.

As leguminosas forrageiras, além de contribuírem para o aumento da oferta de forragem com alto teor de proteína, também são importantes como cobertura vegetal de solos. Entre as leguminosas com grande potencial forrageiro, espécies do gênero Lotus têm se destacado. Este trabalho visou à seleção de rizóbios nativos eficientes em Lotus glaber a partir de amostras de solo de cinco localidades do Rio Grande do Sul. Obtiveram-se 259 isolados de rizóbios, que foram avaliados quanto à morfologia colonial e produção de melanina. Destes, 15 isolados foram selecionados para avaliação da eficiência na fixação simbiótica de nitrogênio em experimento em casa de vegetação e caracterizados geneticamente por comparação do perfil eletroforético dos produtos de amplificação do DNA genômico, por PCR com os oligonucleotídeos iniciadores BOX e ERIC. Apenas cinco isolados produziram melanina. Nove isolados foram mais eficientes do que a estirpe SEMIA 830, que é autorizada para a produção de inoculante para L. glaber no país. Na caracterização genética, observou-se que nenhum isolado apresentou identidade com as estirpes recomendadas, o que demonstra que, nos solos do Rio Grande do Sul, existem rizóbios autóctones eficientes na fixação simbiótica de nitrogênio com L. glaber, podendo ser recomendados para estudos a campo, visando a uma futura produção de inoculantes para estas leguminosas no Brasil.

RESUMEN

Forage legumes besides contributing to increase the supply of high protein content forage are also good cover for the soil surface. Among the legumes with high potential forage, the genus Lotus has been outstanding. This research aimed to selectnative rhizobia efficient in fixing nitrogen with Lotus glaber, from soil samples of five localities of Rio Grande do Sul. A total of 259 rhizobia isolates were obtained, which were evaluated based on colony morphology and in vitro melanin production. Among them, 15 isolates were selected for the evaluation of the symbiotic nitrogen fixation efficiency in greenhouse experiments and genetically characterized by genomic DNA fingerprinting, PCR amplification with BOX and ERIC primers. Only five isolates produced melanin. Nine isolates were more efficient than strain SEMIA 830, which is authorized for the production of commercial inoculants of L. glaber in Brazil. In relation to the genetic characterization, the isolates have shown no similarity with the commercial strains, indicating that in the soils of Rio Grande do Sul there are autochthonous rhizobia capable of efficient symbiotic nitrogen fixation with L. glaber, which might be recommended for field evaluations, aiming a future production of inoculants for this legume in Brazil.

As leguminosas forrageiras, além de contribuírem para o aumento da oferta de forragem com alto teor de proteína, também são importantes como cobertura vegetal de solos. Entre as leguminosas com grande potencial forrageiro, espécies do gênero Lotus têm se destacado. Este trabalho visou à seleção de rizóbios nativos eficientes em Lotus glaber a partir de amostras de solo de cinco localidades do Rio Grande do Sul. Obtiveram-se 259 isolados de rizóbios, que foram avaliados quanto à morfologia colonial e produção de melanina. Destes, 15 isolados foram selecionados para avaliação da eficiência na fixação simbiótica de nitrogênio em experimento em casa de vegetação e caracterizados geneticamente por comparação do perfil eletroforético dos produtos de amplificação do DNA genômico, por PCR com os oligonucleotídeos iniciadores BOX e ERIC. Apenas cinco isolados produziram melanina. Nove isolados foram mais eficientes do que a estirpe SEMIA 830, que é autorizada para a produção de inoculante para L. glaber no país. Na caracterização genética, observou-se que nenhum isolado apresentou identidade com as estirpes recomendadas, o que demonstra que, nos solos do Rio Grande do Sul, existem rizóbios autóctones eficientes na fixação simbiótica de nitrogênio com L. glaber, podendo ser recomendados para estudos a campo, visando a uma futura produção de inoculantes para estas leguminosas no Brasil.