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Introduction: Plants strictly control root endosymbioses with nutrient-scavenging arbuscular endomycorrhizal fungi or nodule inducing diazotrophic bacteria. The GRAS-type transcriptional regulator NODULATION SIGNALING PATHWAY 2 (NSP2) is a conserved hub in this process. The NSP2 -regulated transcriptional network is instrumental in balancing nutrient homeostasis with symbiotic interactions. NSP2 activity is modulated post-transcriptionally by a specific microRNA. Overriding this control mechanism by ectopic expression of a miRNA-resistant NSP2 transgene enhances the symbiotic permissiveness to arbuscular endomycorrhizal fungi. Such engineered plants may possess enhanced capacities for nutrient uptake. However, the trade-off of this strategy on plant development or other symbiotic interactions, like nodulation, is yet to be fully understood. Method: We used the nodulating Cannabaceae species Parasponia andersonii as an experimental system to study the effect of ectopic NSP2 expression. Parasponia and legumes (Fabaceae) diverged 100 million years ago, providing a unique comparative system to dissect the nodulation trait. Results: Six independent transgenic Parasponia lines were generated that differed in the level of NSP2 expression in the root from 6 to 95-fold higher when compared to the empty vector control plants. Analysis of these plants revealed a positive correlation between mycorrhization and the NSP2 expression level, as well as with the expression of the symbiosis transcription factor CYCLOPS and the rate-limiting enzyme in the carotenoid biosynthetic pathway PHYTOENE SYNTHASE1 (PSY1). Yet ectopic expression of NSP2 affected plant architecture and root nodule organogenesis. Discussion: This indicates a significant trade-off when leveraging NSP2 over-expression to enhance endomycorrhization. [ABSTRACT FROM AUTHOR]

Cell-cell communication is essential for both unicellular and multicellular organisms. Secreted peptides that act as diffusive ligands are utilized by eukaryotic organisms to transduce information between cells to coordinate developmental and physiological processes. In plants, The CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) genes encode a family of secreted small peptides which play pivotal roles in stem cell homeostasis in various types of meristems. Accumulated evidence has revealed that CLE peptides mediate trans-kingdom interactions between plants and microbes, including pathogens and symbionts. This review highlights the emerging roles of CLE peptide signaling in plant-microbe interactions, focusing on their involvement in nodulation, immunity, and symbiosis with arbuscular mycorrhizal fungi. Understanding these interactions provides insights into the sophisticated regulatory networks to balance plant growth and defense, enhancing our knowledge of plant biology and potential agricultural applications. [ABSTRACT FROM AUTHOR]

Introduction: The chickpea (Cicer arietinum L.) is well-known for having climate resilience and atmospheric nitrogen fixation ability. Global demand for nitrogenous fertilizer is predicted to increase by 1.4% annually, and the loss of billions of dollars in farm profit has drawn attention to the need for alternative sources of nitrogen. The ability of chickpea to obtain sufficient nitrogen via its symbiotic relationship with Mesorhizobium ciceri is of critical importance in determining the growth and production of chickpea. Methods: To support findings on nodule formation in chickpea and to map the genomic regions for nodulation, an association panel consisting of 271 genotypes, selected from the global chickpea germplasm including four checks at four locations, was evaluated, and data were recorded for nodulation and 12 yield-related traits. A genome-wide association study (GWAS) was conducted using phenotypic data and genotypic data was extracted from whole-genome resequencing data of chickpea by creating a hap map file consisting of 602,344 single-nucleotide polymorphisms (SNPs) in the working set with best-fit models of association mapping. Results and Discussion: The GWAS panel was found to be structured with sufficient diversity among the genotypes. Linkage disequilibrium (LD) analysis showed an LD decay value of 37.3 MB, indicating that SNPs within this distance behave as inheritance blocks. A total of 450 and 632 stringent marker-trait associations (MTAs) were identified from the BLINK and FarmCPU models, respectively, for all the traits under study. The 75 novel MTAs identified for nodulation traits were found to be stable. SNP annotations of associated markers were found to be related to various genes including a few auxins encoding as well as nod factor transporter genes. The identified significant MTAs, candidate genes, and associated markers have the potential for use in marker-assisted selection for developing high-nodulation cultivars after validation in the breeding populations. [ABSTRACT FROM AUTHOR]

Various species of rhizobium establish compatible symbiotic relationships with soybean (Glycinemax) leading to the formation of nitrogen-fixing nodules in roots. The formation of functional nodules is mediated through complex developmental and transcriptional reprogramming that involves the activity of thousands of plant genes. However, host transcriptome that differentiate between functional or nonfunctional nodules remain largely unexplored. In this study, we investigated differential compatibilities between rhizobium strains (Bradyrhizobium diazoefficiens USDA110 Bradyrhizobium sp. strain LVM105) and cultivated and wild soybeans. The nodulation assays revealed that both USDA110 and LVM105 strains effectively nodulate G. soja but only USDA110 can form symbiotic relationships with Williams 82. LVM105 formed pseudonodules on Williams 82 that consist of a central nodule-like mass that are devoid of any rhizobia. RNA-seq data revealed that USDA110 and LVM105 induce distinct transcriptome programing in functional mature nodules formed on G. soja roots, where genes involved in nucleosome assembly, DNA replication, regulation of cell cycle, and defense responses play key roles. Transcriptome comparison also suggested that activation of genes associated with cell wall biogenesis and organization and defense responses together with downregulation of genes involved in the biosynthesis of isoprenoids and antioxidant stress are associated with the formation of non-functional nodules on Williams 82 roots. Moreover, our analysis implies that increased activity of genes involved in oxygen binding, amino acid transport, and nitrate transport differentiates between fully-developed nodules in cultivated versus wild soybeans. [ABSTRACT FROM AUTHOR]

Introduction: Drought is one of the biggest problems for crop production and also affects the survival and persistence of soil rhizobia, which limits the establishment of efficient symbiosis and endangers the productivity of legumes, the main source of plant protein worldwide. Aim: Since the biodiversity can be altered by several factors including abiotic stresses or cultural practices, the objective of this research was to evaluate the effect of water availability, plant genotype and agricultural management on the presence, nodulation capacity and genotypic diversity of rhizobia. Method: A field experiment was conducted with twelve common bean genotypes under irrigation and rain-fed conditions, both in conventional and organic management. Estimation of the number of viable rhizobia present in soils was performed before the crop establishment, whereas the crop yield, nodule number and the strain diversity of bacteria present in nodules were determined at postharvest. Results: Rainfed conditions reduced the number of nodules and of isolated bacteria and their genetic diversity, although to a lesser extent than the agrochemical inputs related to conventional management. In addition, the effect of water scarcity on the conventional management soil was greater than observed under organic conditions. Conclusions: The preservation of diversity will be a key factor to maintain crop production in the future, as problems caused by drought will be exacerbated by climate change and organic management can help to maintain the biodiversity of soil microbiota, a fundamental aspect for soil health and quality. [ABSTRACT FROM AUTHOR]

IntroductionPlants strictly control root endosymbioses with nutrient-scavenging arbuscular endomycorrhizal fungi or nodule inducing diazotrophic bacteria. The GRAS-type transcriptional regulator NODULATION SIGNALING PATHWAY 2 (NSP2) is a conserved hub in this process. The NSP2-regulated transcriptional network is instrumental in balancing nutrient homeostasis with symbiotic interactions. NSP2 activity is modulated post-transcriptionally by a specific microRNA. Overriding this control mechanism by ectopic expression of a miRNA-resistant NSP2 transgene enhances the symbiotic permissiveness to arbuscular endomycorrhizal fungi. Such engineered plants may possess enhanced capacities for nutrient uptake. However, the trade-off of this strategy on plant development or other symbiotic interactions, like nodulation, is yet to be fully understood.MethodWe used the nodulating Cannabaceae species Parasponia andersonii as an experimental system to study the effect of ectopic NSP2 expression. Parasponia and legumes (Fabaceae) diverged 100 million years ago, providing a unique comparative system to dissect the nodulation trait.ResultsSix independent transgenic Parasponia lines were generated that differed in the level of NSP2 expression in the root from 6 to 95-fold higher when compared to the empty vector control plants. Analysis of these plants revealed a positive correlation between mycorrhization and the NSP2 expression level, as well as with the expression of the symbiosis transcription factor CYCLOPS and the rate-limiting enzyme in the carotenoid biosynthetic pathway PHYTOENE SYNTHASE1 (PSY1). Yet ectopic expression of NSP2 affected plant architecture and root nodule organogenesis.DiscussionThis indicates a significant trade-off when leveraging NSP2 over-expression to enhance endomycorrhization.

Cell-cell communication is essential for both unicellular and multicellular organisms. Secreted peptides that act as diffusive ligands are utilized by eukaryotic organisms to transduce information between cells to coordinate developmental and physiological processes. In plants, The CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) genes encode a family of secreted small peptides which play pivotal roles in stem cell homeostasis in various types of meristems. Accumulated evidence has revealed that CLE peptides mediate trans-kingdom interactions between plants and microbes, including pathogens and symbionts. This review highlights the emerging roles of CLE peptide signaling in plant-microbe interactions, focusing on their involvement in nodulation, immunity, and symbiosis with arbuscular mycorrhizal fungi. Understanding these interactions provides insights into the sophisticated regulatory networks to balance plant growth and defense, enhancing our knowledge of plant biology and potential agricultural applications.

IntroductionThe chickpea (Cicer arietinum L.) is well-known for having climate resilience and atmospheric nitrogen fixation ability. Global demand for nitrogenous fertilizer is predicted to increase by 1.4% annually, and the loss of billions of dollars in farm profit has drawn attention to the need for alternative sources of nitrogen. The ability of chickpea to obtain sufficient nitrogen via its symbiotic relationship with Mesorhizobium ciceri is of critical importance in determining the growth and production of chickpea.MethodsTo support findings on nodule formation in chickpea and to map the genomic regions for nodulation, an association panel consisting of 271 genotypes, selected from the global chickpea germplasm including four checks at four locations, was evaluated, and data were recorded for nodulation and 12 yield-related traits. A genome-wide association study (GWAS) was conducted using phenotypic data and genotypic data was extracted from whole-genome resequencing data of chickpea by creating a hap map file consisting of 602,344 single-nucleotide polymorphisms (SNPs) in the working set with best-fit models of association mapping.Results and DiscussionThe GWAS panel was found to be structured with sufficient diversity among the genotypes. Linkage disequilibrium (LD) analysis showed an LD decay value of 37.3 MB, indicating that SNPs within this distance behave as inheritance blocks. A total of 450 and 632 stringent marker–trait associations (MTAs) were identified from the BLINK and FarmCPU models, respectively, for all the traits under study. The 75 novel MTAs identified for nodulation traits were found to be stable. SNP annotations of associated markers were found to be related to various genes including a few auxins encoding as well as nod factor transporter genes. The identified significant MTAs, candidate genes, and associated markers have the potential for use in marker-assisted selection for developing high-nodulation cultivars after validation in the breeding populations.

Various species of rhizobium establish compatible symbiotic relationships with soybean (Glycine max) leading to the formation of nitrogen-fixing nodules in roots. The formation of functional nodules is mediated through complex developmental and transcriptional reprogramming that involves the activity of thousands of plant genes. However, host transcriptome that differentiate between functional or non-functional nodules remain largely unexplored. In this study, we investigated differential compatibilities between rhizobium strains (Bradyrhizobium diazoefficiens USDA110 Bradyrhizobium sp. strain LVM105) and cultivated and wild soybeans. The nodulation assays revealed that both USDA110 and LVM105 strains effectively nodulate G. soja but only USDA110 can form symbiotic relationships with Williams 82. LVM105 formed pseudonodules on Williams 82 that consist of a central nodule-like mass that are devoid of any rhizobia. RNA-seq data revealed that USDA110 and LVM105 induce distinct transcriptome programing in functional mature nodules formed on G. soja roots, where genes involved in nucleosome assembly, DNA replication, regulation of cell cycle, and defense responses play key roles. Transcriptome comparison also suggested that activation of genes associated with cell wall biogenesis and organization and defense responses together with downregulation of genes involved in the biosynthesis of isoprenoids and antioxidant stress are associated with the formation of non-functional nodules on Williams 82 roots. Moreover, our analysis implies that increased activity of genes involved in oxygen binding, amino acid transport, and nitrate transport differentiates between fully-developed nodules in cultivated versus wild soybeans.

IntroductionDrought is one of the biggest problems for crop production and also affects the survival and persistence of soil rhizobia, which limits the establishment of efficient symbiosis and endangers the productivity of legumes, the main source of plant protein worldwide.AimSince the biodiversity can be altered by several factors including abiotic stresses or cultural practices, the objective of this research was to evaluate the effect of water availability, plant genotype and agricultural management on the presence, nodulation capacity and genotypic diversity of rhizobia.MethodA field experiment was conducted with twelve common bean genotypes under irrigation and rain-fed conditions, both in conventional and organic management. Estimation of the number of viable rhizobia present in soils was performed before the crop establishment, whereas the crop yield, nodule number and the strain diversity of bacteria present in nodules were determined at postharvest.ResultsRainfed conditions reduced the number of nodules and of isolated bacteria and their genetic diversity, although to a lesser extent than the agrochemical inputs related to conventional management. In addition, the effect of water scarcity on the conventional management soil was greater than observed under organic conditions.ConclusionsThe preservation of diversity will be a key factor to maintain crop production in the future, as problems caused by drought will be exacerbated by climate change and organic management can help to maintain the biodiversity of soil microbiota, a fundamental aspect for soil health and quality.

Isoflavonoids constitute a well-investigated category of phenylpropanoidderived specialized metabolites primarily found in leguminous plants. They play a crucial role in legume development and interactions with the environment. Isoflavonoids usually function as phytoalexins, acting against pathogenic microbes in nature. Additionally, they serve as signaling molecules in rhizobial symbiosis. Notably, owing to their molecular structure resembling human estrogen, they are recognized as phytoestrogens, imparting positive effects on human health. This review comprehensively outlines recent advancements in research pertaining to isoflavonoid biosynthesis, transcriptional regulation, transport, and physiological functions, with a particular emphasis on soybean plants. Additionally, we pose several questions to encourage exploration into novel contributors to isoflavonoid metabolism and their potential roles in plantmicrobe interactions. [ABSTRACT FROM AUTHOR]

The unique evolutionary adaptation of legumes for nitrogen-fixing symbiosis leading to nodulation is tightly regulated by the host plant. The autoregulation of nodulation (AON) pathway negatively regulates the number of nodules formed in response to the carbon/nitrogen metabolic status of the shoot and root by longdistance signaling to and from the shoot and root. Central to AON signaling in the shoots of Medicago truncatula is SUNN, a leucine-rich repeat receptor-like kinase with high sequence similarity with CLAVATA1 (CLV1), part of a class of receptors in Arabidopsis involved in regulating stem cell populations in the root and shoot. This class of receptors in Arabidopsis includes the BARELY ANY MERISTEM family, which, like CLV1, binds to CLE peptides and interacts with CLV1 to regulate meristem development. M. truncatula contains five members of the BAM family, but only MtBAM1 and MtBAM2 are highly expressed in the nodules 48 hours after inoculation. Plants carry mutations in individual MtBAMs, and several double BAM mutant combinations all displayed wild-type nodule number phenotypes. However, Mtbam2 suppressed the sunn-5 hypernodulation phenotype and partially rescued the short root length phenotype of sunn-5 when present in a sunn-5 background. Grafting determined that bam2 suppresses supernodulation from the roots, regardless of the SUNN status of the root. Overexpression of MtBAM2 in wild-type plants increases nodule numbers, while overexpression of MtBAM2 in some sunn mutants rescues the hypernodulation phenotype, but not the hypernodulation phenotypes of AON mutant rdn1-2 or crn. Relative expression measurements of the nodule transcription factor MtWOX5 downstream of the putative bam2 sunn-5 complex revealed disruption of meristem signaling; while both bam2 and bam2 sunn-5 influence MtWOX5 expression, the expression changes are in different directions. We propose a genetic model wherein the specific root interactions of BAM2/SUNN are critical for signaling in nodule meristem cell homeostasis in M. truncatula. [ABSTRACT FROM AUTHOR]

The unique evolutionary adaptation of legumes for nitrogen-fixing symbiosis leading to nodulation is tightly regulated by the host plant. The autoregulation of nodulation (AON) pathway negatively regulates the number of nodules formed in response to the carbon/nitrogen metabolic status of the shoot and root by long-distance signaling to and from the shoot and root. Central to AON signaling in the shoots of Medicago truncatula is SUNN, a leucine-rich repeat receptor-like kinase with high sequence similarity with CLAVATA1 (CLV1), part of a class of receptors in Arabidopsis involved in regulating stem cell populations in the root and shoot. This class of receptors in Arabidopsis includes the BARELY ANY MERISTEM family, which, like CLV1, binds to CLE peptides and interacts with CLV1 to regulate meristem development. M. truncatula contains five members of the BAM family, but only MtBAM1 and MtBAM2 are highly expressed in the nodules 48 hours after inoculation. Plants carry mutations in individual MtBAMs, and several double BAM mutant combinations all displayed wild-type nodule number phenotypes. However, Mtbam2 suppressed the sunn-5 hypernodulation phenotype and partially rescued the short root length phenotype of sunn-5 when present in a sunn-5 background. Grafting determined that bam2 suppresses supernodulation from the roots, regardless of the SUNN status of the root. Overexpression of MtBAM2 in wild-type plants increases nodule numbers, while overexpression of MtBAM2 in some sunn mutants rescues the hypernodulation phenotype, but not the hypernodulation phenotypes of AON mutant rdn1-2 or crn. Relative expression measurements of the nodule transcription factor MtWOX5 downstream of the putative bam2 sunn-5 complex revealed disruption of meristem signaling; while both bam2 and bam2 sunn-5 influence MtWOX5 expression, the expression changes are in different directions. We propose a genetic model wherein the specific root interactions of BAM2/SUNN are critical for signaling in nodule meristem cell homeostasis in M. truncatula.

Introduction: Nitrogen fertilizer intake promotes soybean growth before the formation of nodules, but excess nitrogen has an inhibitory effect on soybean nodulation. It is important to balance nitrogen levels to meet both growth and nodulation needs. Methods: the nitrogen level suitable for soybean growth and nodulation was studied, the role of humic acid (HA) in alleviating the inhibition of high nitrogen on soybean nodulation was analyzed, and transcriptomic analysis was performed to understand its mechanism. Results: The results showed that a lower level of nitrogen with 36.4 mg urea per pot could increase the number of nodules of soybean, and a higher level of nitrogen with 145.9 mg urea per pot (U4 group) had the best growth indicators but inhibited nodulation significantly. HA relieved the inhibitory effect at high nitrogen level, and the number of nodules increased by 122.1% when 1.29 g HA was added (H2 group) compared with the U4 group. The transcriptome analysis was subsequently performed on the H2 and U4 groups, showing that there were 2995 differentially expressed genes (DEGs) on the 25th day, accounting for 6.678% of the total annotated genes (44,848) under the test conditions. These DEGs were enriched in mitogen-activated protein kinase signaling pathwayplant, flavonoid biosynthesis, and plant hormone signal transduction based on the –log10 (Padjusted) value in the Kyoto Encyclopedia of Genes and Genomes pathway (KEGG). Discussion: HA balanced the nitrogen level through the above pathways in soybean planting to control the number of nodules. [ABSTRACT FROM AUTHOR]

Arbuscular mycorrhizal (AM) fungi and rhizobia form two of the most important plant-microbe associations for the assimilation of phosphorus (P) and nitrogen (N). Symbiont-derived signals are able to coordinate the infection process by triggering multiple responses in the plant root, such as calcium influxes and oscillations, increased reactive oxygen species (ROS), cytoskeletal rearrangements and altered gene expression. An examination was made of the role of tetraspanins, which are transmembrane proteins that self-organize into tetraspanin web regions, where they recruit specific proteins into platforms required for signal transduction, membrane fusion, cell trafficking, and ROS generation. In plant cells, tetraspanins are scaffolding proteins associated with root radial patterning, biotic and abiotic stress responses, cell fate determination, plasmodesmata and hormonal regulation. Some plant tetraspanins, such as Arabidopsis thaliana TETRASPANIN 8 and TETRASPANIN 9 (AtTET8 and AtTET9) are associated with exosomes during inter-kingdom communication. In this study, a homolog of AtTET8, PvTET8-1, in common bean (Phaseolus vulgaris L. var. Negro Jamapa) was examined in roots during interactions with Rhizobium tropici and Rhizophagus irregularis. The promoter of PvTET8-1 contained several cis-acting regulatory DNA elements potentially related to mutualistic interactions, and PvTET8-1 was transcriptionally activated during AM fungal and rhizobial associations. Silencing it decreased the size and number of nodules, nitrogen fixation, and mycorrhizal arbuscule formation, whereas overexpressing it increased the size and number of nodules, and mycorrhizal arbuscule formation but decreased nitrogen fixation. PvTET8-1 appears to be an important element in both of these mutualistic interactions, perhaps through its interaction with NADPH oxidase and the generation of ROS during the infection processes. [ABSTRACT FROM AUTHOR]

Drought is the most detrimental abiotic stress in agriculture, limiting crop growth and yield and, currently, its risk is increasing due to climate change. Thereby, ensuring food security will be one of the greatest challenges of the agriculture in the nearest future, accordingly it is essential to look for sustainable strategies to cope the negative impact of drought on crops. Inoculation of pulses with biostimulants such as rhizobium strains with high nitrogen fixation efficiency and drought-tolerance, has emerged as a promising and sustainable production strategy. However, some commercial inoculums are not effective under field conditions due to its lower effectiveness against indigenous rhizobium strains in the establishment of the symbiosis. Thus, in the present study, we evaluated the ability to improve drought tolerance in common bean plants of different indigenous rhizobia strains isolated from nearby crop fields in the Basque Country either affected by drought or salinity. The plants in this trial were grown in a climatic chamber under controlled conditions and exposed to values of 30% relative soil water content at the time of harvest, which is considered a severe drought. From the nine bacteria strains evaluated, three were found to be highly efficient under drought (namely 353, A12 and A13). These strains sustained high infectiveness (nodulation capacity) and effectiveness (shoot biomass production) under drought, even surpassing the plants inoculated with the CIAT899 reference strain, as well as the chemically Nfertilized plants. The tolerance mechanisms developed by plants inoculated with 353, A12 and A13 strains were a better adjustment of the cell wall elasticity that prevents mechanical damages in the plasma membrane, a higher WUE and an avoidance of the phenological delay caused by drought, developing a greater number of flowers. These results provide the basis for the development of efficient common bean inoculants able to increase the yield of this crop under drought conditions in the Northern Spain and, thus, to be used as biostimulants. In addition, the use of these efficient nitrogen fixation bacteria strains is a sustainable alternative to chemical fertilization, reducing cost and minimizing its negative impact on environment. [ABSTRACT FROM AUTHOR]

Arbuscular mycorrhizal (AM) fungi and rhizobia form two of the most important plant-microbe associations for the assimilation of phosphorus (P) and nitrogen (N). Symbiont-derived signals are able to coordinate the infection process by triggering multiple responses in the plant root, such as calcium influxes and oscillations, increased reactive oxygen species (ROS), cytoskeletal rearrangements and altered gene expression. An examination was made of the role of tetraspanins, which are transmembrane proteins that self-organize into tetraspanin web regions, where they recruit specific proteins into platforms required for signal transduction, membrane fusion, cell trafficking, and ROS generation. In plant cells, tetraspanins are scaffolding proteins associated with root radial patterning, biotic and abiotic stress responses, cell fate determination, plasmodesmata and hormonal regulation. Some plant tetraspanins, such as Arabidopsis thaliana TETRASPANIN 8 and TETRASPANIN 9 (AtTET8 and AtTET9) are associated with exosomes during inter-kingdom communication. In this study, a homolog of AtTET8, PvTET8-1, in common bean (Phaseolus vulgaris L. var. Negro Jamapa) was examined in roots during interactions with Rhizobium tropici and Rhizophagus irregularis. The promoter of PvTET8-1 contained several cis-acting regulatory DNA elements potentially related to mutualistic interactions, and PvTET8-1 was transcriptionally activated during AM fungal and rhizobial associations. Silencing it decreased the size and number of nodules, nitrogen fixation, and mycorrhizal arbuscule formation, whereas overexpressing it increased the size and number of nodules, and mycorrhizal arbuscule formation but decreased nitrogen fixation. PvTET8-1 appears to be an important element in both of these mutualistic interactions, perhaps through its interaction with NADPH oxidase and the generation of ROS during the infection processes.

Drought is the most detrimental abiotic stress in agriculture, limiting crop growth and yield and, currently, its risk is increasing due to climate change. Thereby, ensuring food security will be one of the greatest challenges of the agriculture in the nearest future, accordingly it is essential to look for sustainable strategies to cope the negative impact of drought on crops. Inoculation of pulses with biostimulants such as rhizobium strains with high nitrogen fixation efficiency and drought-tolerance, has emerged as a promising and sustainable production strategy. However, some commercial inoculums are not effective under field conditions due to its lower effectiveness against indigenous rhizobium strains in the establishment of the symbiosis. Thus, in the present study, we evaluated the ability to improve drought tolerance in common bean plants of different indigenous rhizobia strains isolated from nearby crop fields in the Basque Country either affected by drought or salinity. The plants in this trial were grown in a climatic chamber under controlled conditions and exposed to values of 30% relative soil water content at the time of harvest, which is considered a severe drought. From the nine bacteria strains evaluated, three were found to be highly efficient under drought (namely 353, A12 and A13). These strains sustained high infectiveness (nodulation capacity) and effectiveness (shoot biomass production) under drought, even surpassing the plants inoculated with the CIAT899 reference strain, as well as the chemically N-fertilized plants. The tolerance mechanisms developed by plants inoculated with 353, A12 and A13 strains were a better adjustment of the cell wall elasticity that prevents mechanical damages in the plasma membrane, a higher WUE and an avoidance of the phenological delay caused by drought, developing a greater number of flowers. These results provide the basis for the development of efficient common bean inoculants able to increase the yield of this crop under drought conditions in the Northern Spain and, thus, to be used as biostimulants. In addition, the use of these efficient nitrogen fixation bacteria strains is a sustainable alternative to chemical fertilization, reducing cost and minimizing its negative impact on environment.

Symbiotic nitrogen fixation is an important factor affecting the yield and quality of leguminous crops. Nodulation is regulated by a complex network comprising several transcription factors. Here, we functionally characterized the role of a TOC1 family member, GmTOC1b, in soybean (Glycine max) nodulation. RT-qPCR assays showed that GmTOC1b is constitutively expressed in soybean. However, GmTOC1b was also highly expressed in nodules, and GmTOC1 localized to the cell nucleus, based on transient transformation in Nicotiana benthamiana leaves. Homozygous Gmtoc1b mutant plants exhibited increased root hair curling and produced more infection threads, resulting in more nodules and greater nodule fresh weight. By contrast, GmTOC1b overexpression inhibited nodulation. Furthermore, we also showed that GmTOC1b represses the expression of nodulation-related genes including GmNIN2a and GmENOD40-1 by binding to their promoters. We conclude that GmTOC1b functions as a transcriptional repressor to inhibit nodulation by repressing the expression of key nodulation-related genes including GmNIN2a, GmNIN2b, and GmENOD40-1 in soybean. [ABSTRACT FROM AUTHOR]

Symbiotic nitrogen fixation is an important component in the nitrogen cycle and is a potential solution for sustainable agriculture. It is the result of the interactions between the plant host, mostly restricted to legume species, and the rhizobial symbiont. From the first encounter between the host and the symbiont to eventual successful nitrogen fixation, there are delicate processes involved, such as nodule organogenesis, rhizobial infection thread progression, differentiation of the bacteroid, deregulation of the host defense systems, and reallocation of resources. All these processes are tightly regulated at different levels. Recent evidence revealed that non-coding RNAs (ncRNAs), including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), participate in these processes by controlling the transcription and translation of effector genes. In general, ncRNAs are functional transcripts without translation potential and are important gene regulators. MiRNAs, negative gene regulators, bind to the target mRNAs and repress protein production by causing the cleavage of mRNA and translational silencing. LncRNAs affect the formation of chromosomal loops, DNA methylation, histone modification, and alternative splicing to modulate gene expression. Both lncRNAs and circRNAs could serve as target mimics of miRNA to inhibit miRNA functions. In this review, we summarized and discussed the current understanding of the roles of ncRNAs in legume nodulation and nitrogen fixation in the root nodule, mainly focusing on their regulation of hormone signal transduction, the autoregulation of nodulation (AON) pathway and nutrient homeostasis in nodules. Unraveling the mediation of legume nodulation by ncRNAs will give us new insights into designing higher-performance leguminous crops for sustainable agriculture. [ABSTRACT FROM AUTHOR]

Nitrogen (N) inhibits soybean (Glycine max L.) nodulation and N2 fixation. Isoflavones secreted by soybean roots can stimulate signal transduction for symbiotic nodules, thus playing a key role in root nodule development and N2 fixation. The relationship between the inhibition of soybean nodulation, N2 fixation and isoflavones by N is still unclear. In this study, dual-root soybean plants were prepared by grafting, and N or isoflavones were supplied to unilateral roots. The number and dry weight of the soybean nodules, nitrogenase activity, isoflavone concentrations and relative changes in the level of expression of nodulation-related genes were measured to study the response relationship between the N systemic regulation the soybean nodule N2 fixation and changes in the concentrations of isoflavones in its roots. The results showed that N supply to one side of the dual-root soybeans systematically affected the N2 fixation of root nodules on both sides, and this effect began in the early stage of nodulation. Moreover, a unilateral supply of N systematically affected the concentrations of daidzein and genistein on both sides of the roots. The concentrations of isoflavones were consistent with the change trend of soybean root nodule and nodulation-related gene expression level. Treatment with unilateral N or isoflavones affected the soybean nodule N2 fixation and its nodulation-related genes, which had the same response to the changes in concentrations of root isoflavones. N regulates soybean nodulation and N2 fixation by systematically affecting the concentrations of isoflavones in the roots. [ABSTRACT FROM AUTHOR]

Lentil is one of the essential legume crops, which provides protein for humans and animals. This legume can improve soil fertility through nitrogen fixation, which is imperative in low-fertility soils. The growth and productivity of lentil could be enhanced through improving nutrition and root revitalization. Therefore, the objective of this study was to assess the impact of root activator (RA) and phosphorus (P) application on morphological, physiological, agronomic, and quality traits of lentil under newly reclaimed low-fertility sandy soil in an arid environment. The RA was applied at four levels of 0 (RA0-untreated control), 1.25 (RA1), 2.5 (RA2), and 3.75 (RA3) l ha-1. RA contained 9% potassium humate, 1,600 ppm indole butyric acid, 200 ppm gibberellic acid, and 200 ppm naphthalene acetic acid. The recommended rate of phosphorus (P) fertilization in the newly reclaimed low-fertility sandy soil (75 kg P2O5 ha-1) was applied, and its amount was increased and decreased by 25 kg P2O5 ha-1 vs. non-added control. Thus, P rates were applied at four rates 0 (P0; control), 50 (P1), 75 (P2), and 100 (P3) kg phosphorus pentoxide (P2O5) ha-1. Our results revealed that treated lentil plants with the high levels of both treatments (RA3 and P3) exhibited superiority in root measurements (root length, total number of nodules plant-1, number of active nodules plant-1, dry weights of active nodules, and total root), nitrogenase activity, chlorophyll a and b, carotenoids, yield traits, and seed proteins and carbohydrates. However, the recommended P level (75 kg P2O5 ha-1, P2) under the high level of RA (3.75 l ha-1, RA3) displayed non-significant differences in yield traits (plant height, 1,000-seed weight, seed yield ha-1) and quality traits (protein and carbohydrate) with the high P level (100 kg P2O5 ha-1, P3). Accordingly, its recommended economically and environmentally to use this coapplication of RA3 and P3 in low-fertility soil for better lentil growth, and seed yield and quality. [ABSTRACT FROM AUTHOR]

Lentil is one of the essential legume crops, which provides protein for humans and animals. This legume can improve soil fertility through nitrogen fixation, which is imperative in low-fertility soils. The growth and productivity of lentil could be enhanced through improving nutrition and root revitalization. Therefore, the objective of this study was to assess the impact of root activator (RA) and phosphorus (P) application on morphological, physiological, agronomic, and quality traits of lentil under newly reclaimed low-fertility sandy soil in an arid environment. The RA was applied at four levels of 0 (RA0-untreated control), 1.25 (RA1), 2.5 (RA2), and 3.75 (RA3) l ha–1. RA contained 9% potassium humate, 1,600 ppm indole butyric acid, 200 ppm gibberellic acid, and 200 ppm naphthalene acetic acid. The recommended rate of phosphorus (P) fertilization in the newly reclaimed low-fertility sandy soil (75 kg P2O5 ha–1) was applied, and its amount was increased and decreased by 25 kg P2O5 ha–1 vs. non-added control. Thus, P rates were applied at four rates 0 (P0; control), 50 (P1), 75 (P2), and 100 (P3) kg phosphorus pentoxide (P2O5) ha–1. Our results revealed that treated lentil plants with the high levels of both treatments (RA3 and P3) exhibited superiority in root measurements (root length, total number of nodules plant−1, number of active nodules plant−1, dry weights of active nodules, and total root), nitrogenase activity, chlorophyll a and b, carotenoids, yield traits, and seed proteins and carbohydrates. However, the recommended P level (75 kg P2O5 ha–1, P2) under the high level of RA (3.75 l ha–1, RA3) displayed non-significant differences in yield traits (plant height, 1,000-seed weight, seed yield ha–1) and quality traits (protein and carbohydrate) with the high P level (100 kg P2O5 ha–1, P3). Accordingly, its recommended economically and environmentally to use this coapplication of RA3 and P3 in low-fertility soil for better lentil growth, and seed yield and quality.

Nitrogen is a key macronutrient to plant growth. We found previously that increased expression of phospholipase Dε (PLDε), which hydrolyzes phospholipids into phosphatidic acid (PA), enhanced plant growth under nitrogen deficiency in Brassicaceae species Arabidopsis and canola. The present study investigated the effect of AtPLDε -overexpression (OE) on soybean (Glycine max), a species capable of symbiotic nitrogen fixation. AtPLDε -OE soybean plants displayed increased root length and leaf size, and the effect of AtPLDε -ΟΕ on leaf size was greater under nitrogen-deficient than -sufficient condition. Under nitrogen deficiency, AtPLDε -OE soybean plants had a higher chlorophyll content and activity of nitrogen assimilation-related enzymes than wild-type soybean plants. AtPLDε -OE led to a higher level of specific PA species in roots after rhizobium inoculation than wild type. AtPLDε -OE soybean plants also increased seed production under nitrogen deprivation with and without nodulation and decreased seed germination in response to high humidity storage and artificial aging. These results suggest that PLDε promotes nitrogen response and affects adversely seed viability during storage. [ABSTRACT FROM AUTHOR]

Nodule organogenesis in legumes is regulated temporally and spatially through gene networks. Genome-wide transcriptome, proteomic, and metabolomic analyses have been used previously to define the functional role of various plant genes in the nodulation process. However, while significant progress has been made, most of these studies have suffered from tissue dilution since only a few cells/root regions respond to rhizobial infection, with much of the root non-responsive. To partially overcome this issue, we adopted translating ribosome affinity purification (TRAP) to specifically monitor the response of the root cortex to rhizobial inoculation using a cortex-specific promoter. While previous studies have largely focused on the plant response within the root epidermis (e.g., root hairs) or within developing nodules, much less is known about the early responses within the root cortex, such as in relation to the development of the nodule primordium or growth of the infection thread. We focused on identifying genes specifically regulated during early nodule organogenesis using roots inoculated with Bradyrhizobium japonicum. A number of novel nodulation gene candidates were discovered, as well as soybean orthologs of nodulation genes previously reported in other legumes. The differential cortex expression of several genes was confirmed using a promoter-GUS analysis, and RNAi was used to investigate gene function. Notably, a number of differentially regulated genes involved in phytohormone signaling, including auxin, cytokinin, and gibberellic acid (GA), were also discovered, providing deep insight into phytohormone signaling during early nodule development. [ABSTRACT FROM AUTHOR]

In response to colonization by rhizobia bacteria, legumes are able to form nitrogen-fixing nodules in their roots, allowing the plants to grow efficiently in nitrogen-depleted environments. Legumes utilize a complex, long-distance signaling pathway to regulate nodulation that involves signals in both roots and shoots. We measured the transcriptional response to treatment with rhizobia in both the shoots and roots of Medicago truncatula over a 72-h time course. To detect temporal shifts in gene expression, we developed GeneShift, a novel computational statistics and machine learning workflow that addresses the time series replicate the averaging issue for detecting gene expression pattern shifts under different conditions. We identified both known and novel genes that are regulated dynamically in both tissues during early nodulation including leginsulin, defensins, root transporters, nodulin-related, and circadian clock genes. We validated over 70% of the expression patterns that GeneShift discovered using an independent M. truncatula RNA-Seq study. GeneShift facilitated the discovery of condition-specific temporally differentially expressed genes in the symbiotic nodulation biological system. In principle, GeneShift should work for time-series gene expression profiling studies from other systems. [ABSTRACT FROM AUTHOR]

Nitrogen is a key macronutrient to plant growth. We found previously that increased expression of phospholipase Dε (PLDε), which hydrolyzes phospholipids into phosphatidic acid (PA), enhanced plant growth under nitrogen deficiency in Brassicaceae species Arabidopsis and canola. The present study investigated the effect of AtPLDε-overexpression (OE) on soybean (Glycine max), a species capable of symbiotic nitrogen fixation. AtPLDε-OE soybean plants displayed increased root length and leaf size, and the effect of AtPLDε-ΟΕ on leaf size was greater under nitrogen-deficient than -sufficient condition. Under nitrogen deficiency, AtPLDε-OE soybean plants had a higher chlorophyll content and activity of nitrogen assimilation-related enzymes than wild-type soybean plants. AtPLDε-OE led to a higher level of specific PA species in roots after rhizobium inoculation than wild type. AtPLDε-OE soybean plants also increased seed production under nitrogen deprivation with and without nodulation and decreased seed germination in response to high humidity storage and artificial aging. These results suggest that PLDε promotes nitrogen response and affects adversely seed viability during storage.

In response to colonization by rhizobia bacteria, legumes are able to form nitrogen-fixing nodules in their roots, allowing the plants to grow efficiently in nitrogen-depleted environments. Legumes utilize a complex, long-distance signaling pathway to regulate nodulation that involves signals in both roots and shoots. We measured the transcriptional response to treatment with rhizobia in both the shoots and roots of Medicago truncatula over a 72-h time course. To detect temporal shifts in gene expression, we developed GeneShift, a novel computational statistics and machine learning workflow that addresses the time series replicate the averaging issue for detecting gene expression pattern shifts under different conditions. We identified both known and novel genes that are regulated dynamically in both tissues during early nodulation including leginsulin, defensins, root transporters, nodulin-related, and circadian clock genes. We validated over 70% of the expression patterns that GeneShift discovered using an independent M. truncatula RNA-Seq study. GeneShift facilitated the discovery of condition-specific temporally differentially expressed genes in the symbiotic nodulation biological system. In principle, GeneShift should work for time-series gene expression profiling studies from other systems.

Commercial inoculants are often used to inoculate field-grown soybean in Europe. However, nodulation efficiencies in these areas are often low. To enhance biological nitrogen (N) fixation and increase domestic legume production, indigenous strains that are adapted to local conditions could be used to develop more effective inoculants. The objective of this study was to assess the ability of locally isolated Bradyrhizobium strains to enhance soybean productivity in different growing conditions of Northeast Germany. Three indigenous Bradyrhizobium isolates (GMF14, GMM36, and GEM96) were tested in combination with different soybean cultivars of different maturity groups and quality characteristics in one field trial and two greenhouse studies. The results showed a highly significant strain × cultivar interactions on nodulation response. Independent of the Bradyrhizobium strain, inoculated plants in the greenhouse showed higher nodulation, which corresponded with an increased N uptake than that in field conditions. There were significantly higher nodule numbers and nodule dry weights following GMF14 and GMM36 inoculation in well-watered soil, but only minor differences under drought conditions. Inoculation of the soybean cultivar Merlin with the strain GEM96 enhanced nodulation but did not correspond to an increased grain yield under field conditions. USDA110 was consistent in improving the grain yield of soybean cultivars Sultana and Siroca. On the other hand, GMM36 inoculation to Sultana and GEM96 inoculation to Siroca resulted in similar yields. Our results demonstrate that inoculation of locally adapted soybean cultivars with the indigenous isolates improves nodulation and yield attributes. Thus, to attain optimal symbiotic performance, the strains need to be matched with specific cultivars. [ABSTRACT FROM AUTHOR]

Commercial inoculants are often used to inoculate field-grown soybean in Europe. However, nodulation efficiencies in these areas are often low. To enhance biological nitrogen (N) fixation and increase domestic legume production, indigenous strains that are adapted to local conditions could be used to develop more effective inoculants. The objective of this study was to assess the ability of locally isolated Bradyrhizobium strains to enhance soybean productivity in different growing conditions of Northeast Germany. Three indigenous Bradyrhizobium isolates (GMF14, GMM36, and GEM96) were tested in combination with different soybean cultivars of different maturity groups and quality characteristics in one field trial and two greenhouse studies. The results showed a highly significant strain × cultivar interactions on nodulation response. Independent of the Bradyrhizobium strain, inoculated plants in the greenhouse showed higher nodulation, which corresponded with an increased N uptake than that in field conditions. There were significantly higher nodule numbers and nodule dry weights following GMF14 and GMM36 inoculation in well-watered soil, but only minor differences under drought conditions. Inoculation of the soybean cultivar Merlin with the strain GEM96 enhanced nodulation but did not correspond to an increased grain yield under field conditions. USDA110 was consistent in improving the grain yield of soybean cultivars Sultana and Siroca. On the other hand, GMM36 inoculation to Sultana and GEM96 inoculation to Siroca resulted in similar yields. Our results demonstrate that inoculation of locally adapted soybean cultivars with the indigenous isolates improves nodulation and yield attributes. Thus, to attain optimal symbiotic performance, the strains need to be matched with specific cultivars.

Roots have a fundamental role in plant growth and adaptation to different environments. Diversity in root morphology and architecture enables plants to acquire water and nutrients in contrasting substrate conditions, resist biotic and abiotic stress, and develop symbiotic associations. At its most fundamental level, morphology is determined by discrete changes in tissue patterning. Differences in the number and arrangement of the cell layers in the root can change tissue structure, as well as root length and girth, affecting important productivity traits. Therefore, understanding the molecular mechanisms controlling variation in developmental patterning is an important goal in biology. The ground tissue (GT) system is an ideal model to study the genetic basis of morphological diversity because it displays great interspecific variability in cell layer number. In addition, the genetic circuit controlling GT patterning in Arabidopsis thaliana has been well described, although little is known about species with more complex root anatomies. In this review, we will describe the Arabidopsis model for root radial patterning and present recent progress in elucidating the genetic circuitry controlling GT patterning in monocots and the legume Medicago truncatula (Mt) , species that develop roots with more complex anatomies and multilayered cortex. [ABSTRACT FROM AUTHOR]

To support the adaption of soybean [Glycine max (L) Merrill] cultivation across Central Europe, the availability of compatible soybean nodulating Bradyrhizobia (SNB) is essential. Little is known about the symbiotic potential of indigenous SNB in Central Europe and the interaction with an SNB inoculum from commercial products. The objective of this study was to quantify the capacity of indigenous and inoculated SNB strains on the symbiotic performance of soybean in a pot experiment, using soils with and without soybean history. Under controlled conditions in a growth chamber, the study focused on two main factors: a soybean cropping interval (time since the last soybean cultivation; SCI) and inoculation with commercial Bradyrhizobia strains. Comparing the two types of soil, without soybean history and with 1–4 years SCI, we found out that plants grown in soil with soybean history and without inoculation had significantly more root nodules and higher nitrogen content in the plant tissue. These parameters, along with the leghemoglobin content, were found to be a variable among soils with 1–4 years SCI and did not show a trend over the years. Inoculation in soil without soybean history showed a significant increase in a nodulation rate, leghemoglobin content, and soybean tissue nitrogen concentration. The study found that response to inoculation varied significantly as per locations in soil with previous soybean cultivation history. An inoculated soybean grown on loamy sandy soils from the location Müncheberg had significantly more nodules as well as higher green tissue nitrogen concentration compared with non-inoculated plants. No significant improvement in a nodulation rate and tissue nitrogen concentration was observed for an inoculated soybean grown on loamy sandy soils from the location Fehrow. These results suggest that introduced SNB strains remained viable in the soil and were still symbiotically competent for up to 4 years after soybean cultivation. However, the symbiotic performance of the SNB remaining in the soils was not sufficient in all cases and makes inoculation with commercial products necessary. The SNB strains found in the soil of Central Europe could also be promising candidates for the development of inoculants and already represent a contribution to the successful cultivation of soybeans in Central Europe.

Bradyrhizobium elkanii utilizes the type III effector Bel2-5 for nodulation in host plants in the absence of Nod factors (NFs). In soybean plants carrying the Rj4 allele, however, Bel2-5 causes restriction of nodulation by triggering immune responses. Bel2-5 shows similarity with XopD of the phytopathogen Xanthomonas campestris pv. vesicatoria and possesses two internal repeat sequences, two ethylene (ET)-responsive element-binding factor-associated amphiphilic repression (EAR) motifs, a nuclear localization signal (NLS), and a ubiquitin-like protease (ULP) domain, which are all conserved in XopD except for the repeat domains. By mutational analysis, we revealed that most of the putative domains/motifs in Bel2-5 were essential for both NF-independent nodulation and nodulation restriction in Rj4 soybean. The expression of soybean symbiosis- and defense-related genes was also significantly altered by inoculation with the bel2-5 domain/motif mutants compared with the expression upon inoculation with wild-type B. elkanii, which was mostly consistent with the phenotypic changes of nodulation in host plants. Notably, the functionality of Bel2-5 was mostly correlated with the growth inhibition effect of Bel2-5 expressed in yeast cells. The nodulation phenotypes of the domain-swapped mutants of Bel2-5 and XopD indicated that both the C-terminal ULP domain and upstream region are required for the Bel2-5-dependent nodulation phenotypes. These results suggest that Bel2-5 interacts with and modifies host targets via these multiple domains to execute both NF-independent symbiosis and nodulation restriction in Rj4 soybean.

Bradyrhizobium elkanii utilizes the type III effector Bel2-5 for nodulation in host plants in the absence of Nod factors (NFs). In soybean plants carrying the Rj4 allele, however, Bel2-5 causes restriction of nodulation by triggering immune responses. Bel2-5 shows similarity with XopD of the phytopathogen Xanthomonas campestris pv. vesicatoria and possesses two internal repeat sequences, two ethylene (ET)-responsive element-binding factor-associated amphiphilic repression (EAR) motifs, a nuclear localization signal (NLS), and a ubiquitin-like protease (ULP) domain, which are all conserved in XopD except for the repeat domains. By mutational analysis, we revealed that most of the putative domains/motifs in Bel2-5 were essential for both NF-independent nodulation and nodulation restriction in Rj4 soybean. The expression of soybean symbiosis- and defense-related genes was also significantly altered by inoculation with the bel2-5 domain/motif mutants compared with the expression upon inoculation with wild-type B. elkanii , which was mostly consistent with the phenotypic changes of nodulation in host plants. Notably, the functionality of Bel2-5 was mostly correlated with the growth inhibition effect of Bel2-5 expressed in yeast cells. The nodulation phenotypes of the domain-swapped mutants of Bel2-5 and XopD indicated that both the C-terminal ULP domain and upstream region are required for the Bel2-5-dependent nodulation phenotypes. These results suggest that Bel2-5 interacts with and modifies host targets via these multiple domains to execute both NF-independent symbiosis and nodulation restriction in Rj4 soybean. [ABSTRACT FROM AUTHOR]

To support the adaption of soybean [ Glycine max (L) Merrill] cultivation across Central Europe, the availability of compatible soybean nodulating Bradyrhizobia (SNB) is essential. Little is known about the symbiotic potential of indigenous SNB in Central Europe and the interaction with an SNB inoculum from commercial products. The objective of this study was to quantify the capacity of indigenous and inoculated SNB strains on the symbiotic performance of soybean in a pot experiment, using soils with and without soybean history. Under controlled conditions in a growth chamber, the study focused on two main factors: a soybean cropping interval (time since the last soybean cultivation; SCI) and inoculation with commercial Bradyrhizobia strains. Comparing the two types of soil, without soybean history and with 1–4 years SCI, we found out that plants grown in soil with soybean history and without inoculation had significantly more root nodules and higher nitrogen content in the plant tissue. These parameters, along with the leghemoglobin content, were found to be a variable among soils with 1–4 years SCI and did not show a trend over the years. Inoculation in soil without soybean history showed a significant increase in a nodulation rate, leghemoglobin content, and soybean tissue nitrogen concentration. The study found that response to inoculation varied significantly as per locations in soil with previous soybean cultivation history. An inoculated soybean grown on loamy sandy soils from the location Müncheberg had significantly more nodules as well as higher green tissue nitrogen concentration compared with non-inoculated plants. No significant improvement in a nodulation rate and tissue nitrogen concentration was observed for an inoculated soybean grown on loamy sandy soils from the location Fehrow. These results suggest that introduced SNB strains remained viable in the soil and were still symbiotically competent for up to 4 years after soybean cultivation. However, the symbiotic performance of the SNB remaining in the soils was not sufficient in all cases and makes inoculation with commercial products necessary. The SNB strains found in the soil of Central Europe could also be promising candidates for the development of inoculants and already represent a contribution to the successful cultivation of soybeans in Central Europe. [ABSTRACT FROM AUTHOR]

Auxin Response Factors (ARFs) constitute a large family of transcription factors that mediate auxin-regulated developmental programs in plants. ARF2, ARF3, and ARF4 are post-transcriptionally regulated by the microRNA390 (miR390)/trans-acting small interference RNA 3 (TAS3) module through the action of TAS3-derived trans-acting small interfering RNAs (ta-siRNA). We have previously reported that constitutive activation of the miR390/TAS3 pathway promotes elongation of lateral roots but impairs nodule organogenesis and infection by rhizobia during the nitrogen-fixing symbiosis established between Medicago truncatula and its partner Sinorhizobium meliloti. However, the involvement of the targets of the miR390/TAS3 pathway, i.e., MtARF2, MtARF3, MtARF4a, and MtARF4b, in root development and establishment of the nitrogen-fixing symbiosis remained unexplored. Here, promoter:reporter fusions showed that expression of both MtARF3 and MtARF4a was associated with lateral root development; however, only the MtARF4a promoter was active in developing nodules. In addition, up-regulation of MtARF2, MtARF3, and MtARF4a/b in response to rhizobia depends on Nod Factor perception. We provide evidence that simultaneous knockdown of MtARF2, MtARF3, MtARF4a, and MtARF4b or mutation in MtARF4a impaired nodule formation, and reduced initiation and progression of infection events. Silencing of MtARF2, MtARF3, MtARF4a, and MtARF4b altered mRNA levels of the early nodulation gene nodulation signaling pathway 2 (MtNSP2). In addition, roots with reduced levels of MtARF2, MtARF3, MtARF4a, and MtARF4b, as well as arf4a mutant plants exhibited altered root architecture, causing a reduction in primary and lateral root length, but increasing lateral root density. Taken together, our results suggest that these ARF members are common key players of the morphogenetic programs that control root development and the formation of nitrogen-fixing nodules.

Auxin Response Factors (ARFs) constitute a large family of transcription factors that mediate auxin-regulated developmental programs in plants. ARF2 , ARF3 , and ARF4 are post-transcriptionally regulated by the microRNA390 (miR390)/ trans -acting small interference RNA 3 (TAS3) module through the action of TAS3 -derived trans - acting small interfering RNAs (ta-siRNA). We have previously reported that constitutive activation of the miR390/ TAS3 pathway promotes elongation of lateral roots but impairs nodule organogenesis and infection by rhizobia during the nitrogen-fixing symbiosis established between Medicago truncatula and its partner Sinorhizobium meliloti. However, the involvement of the targets of the miR390/ TAS3 pathway, i.e., MtARF2, MtARF3, MtARF4a , and MtARF4b , in root development and establishment of the nitrogen-fixing symbiosis remained unexplored. Here, promoter:reporter fusions showed that expression of both MtARF3 and MtARF4a was associated with lateral root development; however, only the MtARF4a promoter was active in developing nodules. In addition, up-regulation of MtARF2 , MtARF3 , and MtARF4a/b in response to rhizobia depends on Nod Factor perception. We provide evidence that simultaneous knockdown of MtARF2, MtARF3, MtARF4a , and MtARF4b or mutation in MtARF4a impaired nodule formation, and reduced initiation and progression of infection events. Silencing of MtARF2, MtARF3, MtARF4a , and MtARF4b altered mRNA levels of the early nodulation gene nodulation signaling pathway 2 (MtNSP2). In addition, roots with reduced levels of MtARF2, MtARF3, MtARF4a , and MtARF4b , as well as arf4a mutant plants exhibited altered root architecture, causing a reduction in primary and lateral root length, but increasing lateral root density. Taken together, our results suggest that these ARF members are common key players of the morphogenetic programs that control root development and the formation of nitrogen-fixing nodules. [ABSTRACT FROM AUTHOR]

Symbiotic nitrogen fixation by legume nodules provides an abundant nitrogen source for plants, and understanding this process is key for developing green agriculture. Circular RNA (circRNA), a type of endogenous RNA produced by reverse splicing of mRNA precursors, plays important regulatory roles in plants at the transcriptional and post-transcriptional levels. However, the relationship between circRNAs and legume–rhizobium is unknown. Here, we performed comprehensive identification and expression profiling of circRNAs during nodulation in common bean (Phaseolus vulgaris) compared to uninoculated roots of corresponding ages by constructing circRNA-seq and mRNA-seq libraries. We identified 8,842 high-confident circRNAs, 3,448 of which were specifically produced during symbiosis, with the highest number at the nitrogen-fixing stage. Significantly, more circRNAs were derived from exons than from intergenic regions or introns in all samples. The lengths and GC contents of the circRNAs were similar in roots and nodules. However, circRNAs showed specific spatiotemporal expression patterns during nodule and root development. GO and other functional annotation of parental genes of differentially expressed circRNAs indicated their potential involvement in different biological processes. The expression of major circRNAs during symbiosis is independent of parental genes’ expression to a certain degree, while expression of the remaining minor circRNAs showed positive correlation to parental genes. Functional annotation of the targeted mRNAs in the circRNA–miRNA–mRNA network showed that circRNAs may be involved in transmembrane transport and positive regulation of kinase activity during nodulation and nitrogen fixation as miRNA sponges. Our comprehensive analysis of the expression profile of circRNAs and their potential functions suggests that circRNAs may function as new post-transcriptional regulators in legume–rhizobium symbiosis.

Nodulation process in legume plants is essential for biological nitrogen fixation during which process a large amount of phosphorus (P) is required. Under P deficiency, nodule formation is greatly affected, and induction of purple acid phosphatases (PAPs) is an adaptive strategy for nodules to acquire more P. However, regulation roles of PAPs in nodules remain largely understood. In this study, by transcriptome sequencing technology, five PAP genes were found to be differentially expressed, which led to the greatly increased acid phosphatase (APase) and phytase activities in soybean mature nodules under P starvation conditions; and among the five PAP genes, GmPAP12 had the highest transcript level, and RT-PCR indicated expression of GmPAP12 was gradually increasing during nodule development. GUS activity driven by GmPAP12 promoter was also significantly induced in low phosphorus conditions. Further functional analysis showed that under low phosphorus stress, overexpression of GmPAP12 resulted in higher nodule number, fresh weight, and nitrogenase activity as well as the APase activity than those of control plant nodules, whereas the growth performance and APase activity of nodules on hairy roots were greatly lower when GmPAP12 was suppressed, indicating that GmPAP12 may promote P utilization in soybean nodules under low P stress, which thus played an important role in nodulation and biological nitrogen fixation. Moreover, P1BS elements were found in the promoter of GmPAP12, and yeast one-hybrid experiment further proved the binding of P1BS by transcription factor GmPHR1 in the promoter of GmPAP12. At last, overexpression and suppression of GmPHR1 in nodules indeed caused highly increased and decreased expression of GmPAP12, respectively, indicating that GmPAP12 is regulated by GmPHR1 in soybean nodules. Taken together, these data suggested that GmPAP12 was a novel soybean PAP involved in the P utilization and metabolism in soybean root nodules and played an important role in the growth and development of root nodules and biological nitrogen fixation.

Symbiotic nitrogen fixation carried out by the interaction between legumes and diazotrophic bacteria known as rhizobia requires relatively large levels of transition metals. These elements are cofactors of many key enzymes involved in this process. Metallic micronutrients are obtained from soil by the roots and directed to sink organs by the vasculature, in a process mediated by a number of metal transporters and small organic molecules that facilitate metal delivery in the plant fluids. Among the later, nicotianamine is one of the most important. Synthesized by nicotianamine synthases (NAS), this molecule forms metal complexes participating in intracellular metal homeostasis and long-distance metal trafficking. Here we characterized the NAS2 gene from model legume Medicago truncatula. MtNAS2 is located in the root vasculature and in all nodule tissues in the infection and fixation zones. Symbiotic nitrogen fixation requires of MtNAS2 function, as indicated by the loss of nitrogenase activity in the insertional mutant nas2-1, phenotype reverted by reintroduction of a wild-type copy of MtNAS2. This would result from the altered iron distribution in nas2-1 nodules shown with X-ray fluorescence. Moreover, iron speciation is also affected in these nodules. These data suggest a role of nicotianamine in iron delivery for symbiotic nitrogen fixation.

Symbiotic nitrogen fixation by legume nodules provides an abundant nitrogen source for plants, and understanding this process is key for developing green agriculture. Circular RNA (circRNA), a type of endogenous RNA produced by reverse splicing of mRNA precursors, plays important regulatory roles in plants at the transcriptional and post-transcriptional levels. However, the relationship between circRNAs and legume–rhizobium is unknown. Here, we performed comprehensive identification and expression profiling of circRNAs during nodulation in common bean (Phaseolus vulgaris) compared to uninoculated roots of corresponding ages by constructing circRNA-seq and mRNA-seq libraries. We identified 8,842 high-confident circRNAs, 3,448 of which were specifically produced during symbiosis, with the highest number at the nitrogen-fixing stage. Significantly, more circRNAs were derived from exons than from intergenic regions or introns in all samples. The lengths and GC contents of the circRNAs were similar in roots and nodules. However, circRNAs showed specific spatiotemporal expression patterns during nodule and root development. GO and other functional annotation of parental genes of differentially expressed circRNAs indicated their potential involvement in different biological processes. The expression of major circRNAs during symbiosis is independent of parental genes' expression to a certain degree, while expression of the remaining minor circRNAs showed positive correlation to parental genes. Functional annotation of the targeted mRNAs in the circRNA–miRNA–mRNA network showed that circRNAs may be involved in transmembrane transport and positive regulation of kinase activity during nodulation and nitrogen fixation as miRNA sponges. Our comprehensive analysis of the expression profile of circRNAs and their potential functions suggests that circRNAs may function as new post-transcriptional regulators in legume–rhizobium symbiosis. [ABSTRACT FROM AUTHOR]

Symbiotic nitrogen fixation carried out by the interaction between legumes and diazotrophic bacteria known as rhizobia requires relatively large levels of transition metals. These elements are cofactors of many key enzymes involved in this process. Metallic micronutrients are obtained from soil by the roots and directed to sink organs by the vasculature, in a process mediated by a number of metal transporters and small organic molecules that facilitate metal delivery in the plant fluids. Among the later, nicotianamine is one of the most important. Synthesized by nicotianamine synthases (NAS), this molecule forms metal complexes participating in intracellular metal homeostasis and long-distance metal trafficking. Here we characterized the NAS2 gene from model legume Medicago truncatula. MtNAS2 is located in the root vasculature and in all nodule tissues in the infection and fixation zones. Symbiotic nitrogen fixation requires of MtNAS2 function, as indicated by the loss of nitrogenase activity in the insertional mutant nas2-1, phenotype reverted by reintroduction of a wild-type copy of MtNAS2. This would result from the altered iron distribution in nas2-1 nodules shown with X-ray fluorescence. Moreover, iron speciation is also affected in these nodules. These data suggest a role of nicotianamine in iron delivery for symbiotic nitrogen fixation. [ABSTRACT FROM AUTHOR]

Interestingly, these two miRNAs showed opposite expression patterns in response to I B. japonicum i infection (Figure 1A); gma-miR171o expression was suppressed upon bacterial infection, while gma-miR171q was induced". Graph: Figure 1 Expression patterns of gma-miR171o and gma-miR171q during symbiosis with B. japonicum. [Extracted from the article]

Organisms rely on symbiotic associations for metabolism, protection, and energy. However, these intimate partnerships can be vulnerable to exploitation. What prevents microbial mutualists from parasitizing their hosts? In legumes, there is evidence that hosts have evolved sophisticated mechanisms to manage their symbiotic rhizobia, but the generality and evolutionary origins of these control mechanisms are under debate. Here, we focused on the symbiosis between Parasponia hosts and N2-fixing rhizobium bacteria. Parasponia is the only non-legume lineage to have evolved a rhizobial symbiosis and thus provides an evolutionary replicate to test how rhizobial exploitation is controlled. A key question is whether Parasponia hosts can prevent colonization of rhizobia under high nitrogen conditions, when the contribution of the symbiont becomes nonessential. We grew Parasponia andersonii inoculated with Bradyrhizobium elkanii under four ammonium nitrate concentrations in a controlled growth chamber. We measured shoot and root dry weight, nodule number, nodule fresh weight, nodule volume. To quantify viable rhizobial populations in planta , we crushed nodules and determined colony forming units (CFU), as a rhizobia fitness proxy. We show that, like legumes and actinorhizal plants, P. andersonii is able to control nodule symbiosis in response to exogenous nitrogen. While the relative host growth benefits of inoculation decreased with nitrogen fertilization, our highest ammonium nitrate concentration (3.75 mM) was sufficient to prevent nodule formation on inoculated roots. Rhizobial populations were highest in nitrogen free medium. While we do not yet know the mechanism, our results suggest that control mechanisms over rhizobia are not exclusive to the legume clade. [ABSTRACT FROM AUTHOR]

Root nodulation results from a symbiotic relationship between a plant host and Rhizobium bacteria. Synchronized gene expression patterns over the course of rhizobial infection result in activation of pathways that are unique but overlapping with the highly conserved pathways that enable mycorrhizal symbiosis. We performed RNA sequencing of 30 Medicago truncatula root maturation zone samples at five distinct time points. These samples included plants inoculated with Sinorhizobium medicae and control plants that did not receive any Rhizobium. Following gene expression quantification, we identified 1,758 differentially expressed genes at various time points. We constructed a gene co-expression network (GCN) from the same data and identified link community modules (LCMs) that were comprised entirely of differentially expressed genes at specific time points post-inoculation. One LCM included genes that were up-regulated at 24 h following inoculation, suggesting an activation of allergen family genes and carbohydrate-binding gene products in response to Rhizobium. We also identified two LCMs that were comprised entirely of genes that were down regulated at 24 and 48 h post-inoculation. The identity of the genes in these modules suggest that down-regulating specific genes at 24 h may result in decreased jasmonic acid production with an increase in cytokinin production. At 48 h, coordinated down-regulation of a specific set of genes involved in lipid biosynthesis may play a role in nodulation. We show that GCN-LCM analysis is an effective method to preliminarily identify polygenic candidate biomarkers of root nodulation and develop hypotheses for future discovery.

MicroRNAs (miRNAs) control expression of endogenous target genes through transcript cleavage or translational inhibition. Legume plants can form a specialized organ, the nodule, through interaction with nitrogen fixing soil bacteria. To understand the regulatory roles of miRNAs in the nodulation process, we functionally validated gma-miR171o and gma-miR171q and their target genes in soybean. These two miRNA sequences are identical in sequence but their miRNA genes are divergent and show unique, tissue-specific expression patterns. The expression levels of the two miRNAs are negatively correlated with that of their target genes. Ectopic expression of these miRNAs in transgenic hairy roots resulted in a significant reduction in nodule formation. Both gma-miR171o and gma-miR171q target members of the GRAS transcription factor superfamily, namely GmSCL-6 and GmNSP2. Transient interaction of miRNAs and their target genes in tobacco cells further confirmed their cleavage activity. The results suggest that gma-miR171o and gma-miR171q regulate GmSCL-6 and GmNSP2, which in turn, influence expression of the Nodule inception (NIN), Early Nodulin 40 (ENOD40), and Ethylene Response Factor Required for Nodulation (ERN) genes during the Bradyrhizobium japonicum-soybean nodulation process. Collectively, our data suggest a role for two miRNAs, gma-miR171o and gma-miR171q, in regulating the spatial and temporal aspects of soybean nodulation.

Plant hormones play key roles in nodulation and arbuscular mycorrhizal (AM) associations. These two agriculturally and ecologically important symbioses enable plants to gain access to nutrients, in particular, nitrogen in the case of nodulation and phosphorous in the case of AM. Work over the past few decades has revealed how symbioses with nitrogen-fixing rhizobia, restricted almost exclusively to legumes, evolved in part from ancient AM symbioses formed by more than 80% of land plants. Although overlapping, these symbiotic programs also have important differences, including the de novo development of a new organ found only in nodulation. One emerging area of research is the role of two plant hormone groups, the gibberellins (GAs) and brassinosteroids (BRs), in the development and maintenance of these symbioses. In this review, we compare and contrast the roles of these hormones in the two symbioses, including potential interactions with other hormones. This not only focuses on legumes, most of which can host both symbionts, but also examines the role of these in AM development in non-legumes. GA acts by suppressing DELLA, and this regulatory module acts to negatively influence both rhizobial and mycorrhizal infection but appears to promote nodule organogenesis. While an overall positive role for BRs in nodulation and AM has been suggested by studies using mutants disrupted in BR biosynthesis or response, application studies indicate that BR may play a more complex role in nodulation. Given the nature of these symbioses, with events regulated both spatially and temporally, future studies should examine in more detail how GAs and BRs may influence precise events during these symbioses, including interactions with other hormone groups.

Transcription factors of the Nuclear Factor Y (NF-Y) family play essential functions in plant development and plasticity, including the formation of lateral root organs such as lateral root and symbiotic nodules. NF-Ys mediate transcriptional responses by acting as heterotrimers composed of three subunits, NF-YA, NF-YB, and NF-YC, which in plants are encoded by relatively large gene families. We have previously shown that, in the Phaseolus vulgaris × Rhizobium etli interaction, the PvNF-YC1 subunit is involved not only in the formation of symbiotic nodules, but also in the preference exhibited by the plant for rhizobial strains that are more efficient and competitive in nodule formation. PvNF-YC1 forms a heterotrimer with the PvNF-YA1 and PvNF-YB7 subunits. Here, we used promoter:reporter fusions to show that both PvNF-YA1 and PvNF-YB7 are expressed in symbiotic nodules. In addition, we report that knock-down of PvNF-YA1 and its close paralog PvNF-YA9 abolished nodule formation by either high or low efficient strains and arrested rhizobial infection. On the other hand, knock-down of PvNF-YB7 only affected the symbiotic outcome of the high efficient interaction, suggesting that other symbiotic NF-YB subunits might be involved in the more general mechanisms of nodule formation. More important, we present functional evidence supporting that both PvNF-YA1 and PvNF-YB7 are part of the mechanisms that allow P. vulgaris plants to discriminate and select those bacterial strains that perform better in nodule formation, most likely by acting in the same heterotrimeric complex that PvNF-YC1.