Nitrogen Journey in Plants: From Uptake to Metabolism, Stress Response, and Microbe Interaction.

Plants uptake and assimilate nitrogen from the soil in the form of nitrate, ammonium ions, and available amino acids from organic sources. Plant nitrate and ammonium transporters are responsible for nitrate and ammonium translocation from the soil into the roots. The unique structure of these transporters determines the specificity of each transporter, and structural analyses reveal the mechanisms by which these transporters function. Following absorption, the nitrogen metabolism pathway incorporates the nitrogen into organic compounds via glutamine synthetase and glutamate synthase that convert ammonium ions into glutamine and glutamate. Different isoforms of glutamine synthetase and glutamate synthase exist, enabling plants to fine-tune nitrogen metabolism based on environmental cues. Under stressful conditions, nitric oxide has been found to enhance plant survival under drought stress. Furthermore, the interaction between salinity stress and nitrogen availability in plants has been studied, with nitric oxide identified as a potential mediator of responses to salt stress. Conversely, excessive use of nitrate fertilizers can lead to health and environmental issues. Therefore, alternative strategies, such as establishing nitrogen fixation in plants through diazotrophic microbiota, have been explored to reduce reliance on synthetic fertilizers. Ultimately, genomics can identify new genes related to nitrogen fixation, which could be harnessed to improve plant productivity.

SPL12 Regulates AGL6 and AGL21 to Modulate Nodulation and Root Regeneration under Osmotic Stress and Nitrate Sufficiency Conditions in Medicago sativa.

The highly conserved plant microRNA, miR156, affects root architecture, nodulation, symbiotic nitrogen fixation, and stress response. In Medicago sativa, transcripts of eleven SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE, SPLs, including SPL12, are targeted for cleavage by miR156. Our previous research revealed the role of SPL12 and its target gene, AGL6, in nodulation in alfalfa. Here, we investigated the involvement of SPL12, AGL6 and AGL21 in nodulation under osmotic stress and different nitrate availability conditions. Characterization of phenotypic and molecular parameters revealed that the SPL12/AGL6 module plays a negative role in maintaining nodulation under osmotic stress. While there was a decrease in the nodule numbers in WT plants under osmotic stress, the SPL12-RNAi and AGL6-RNAi genotypes maintained nodulation under osmotic stress. Moreover, the results showed that SPL12 regulates nodulation under a high concentration of nitrate by silencing AGL21. AGL21 transcript levels were increased under nitrate treatment in WT plants, but SPL12 was not affected throughout the treatment period. Given that AGL21 was significantly upregulated in SPL12-RNAi plants, we conclude that SPL12 may be involved in regulating nitrate inhibition of nodulation in alfalfa by targeting AGL21. Taken together, our results suggest that SPL12, AGL6, and AGL21 form a genetic module that regulates nodulation in alfalfa under osmotic stress and in response to nitrate.

Deciphering the role of SPL12 and AGL6 from a genetic module that functions in nodulation and root regeneration in Medicago sativa.

KEY MESSAGE: Our results show that SPL12 plays a crucial role in regulating nodule development in Medicago sativa L. (alfalfa), and that AGL6 is targeted and downregulated by SPL12. Root architecture in plants is critical because of its role in controlling nutrient cycling, water use efficiency and response to biotic and abiotic stress factors. The small RNA, microRNA156 (miR156), is highly conserved in plants, where it functions by silencing a group of SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors. We previously showed that transgenic Medicago sativa (alfalfa) plants overexpressing miR156 display increased nodulation, improved nitrogen fixation and enhanced root regenerative capacity during vegetative propagation. In alfalfa, transcripts of eleven SPLs, including SPL12, are targeted for cleavage by miR156. In this study, we characterized the role of SPL12 in root architecture and nodulation by investigating the transcriptomic and phenotypic changes associated with altered transcript levels of SPL12, and by determining SPL12 regulatory targets using SPL12-silencing and -overexpressing alfalfa plants. Phenotypic analyses showed that silencing of SPL12 in alfalfa caused an increase in root regeneration, nodulation, and nitrogen fixation. In addition, AGL6 which encodes AGAMOUS-like MADS box transcription factor, was identified as being directly targeted for silencing by SPL12, based on Next Generation Sequencing-mediated transcriptome analysis and chromatin immunoprecipitation assays. Taken together, our results suggest that SPL12 and AGL6 form a genetic module that regulates root development and nodulation in alfalfa.

Whole genome analysis of Gluconacetobacter azotocaptans DS1 and its beneficial effects on plant growth.

Plant-associated bacteria play an important role in the enhancement of plant growth and productivity. Gluconacetobacter azotocaptans is an exceptional bacterium considering that till today it has been isolated and reported only from Mexico and Canada. It is a plant growth-promoting bacterium and can be used as biofertilizer for different crops and vegetables. The objective of the current study was to evaluate the inoculation effect of Gluconacetobacter azotocaptans DS1, Pseudomonas putida CQ179, Azosprillium zeae N7, Azosprillium brasilense N8, and Azosprillium canadense DS2, on the growth of vegetables including cucumber, sweet pepper, radish, and tomato. All strains increased the vegetables' growth; however, G. azotocaptans DS1 showed better results as compared to other inoculated and control plants and significantly increased the plant biomass of all vegetables. Therefore, the whole genome sequence of G. azotocaptans DS1 was analyzed to predict genes involved in plant growth promotion, secondary metabolism, antibiotics resistance, and bioremediation of heavy metals. Results of genome analysis revealed that G. azotocaptans DS1 has a circular chromosome with a size of 4.3 Mbp and total 3898 protein-coding sequences. Based on functional analysis, genes for nitrogen fixation, phosphate solubilization, indole acetic acid, phenazine, siderophore production, antibiotic resistance, and bioremediation of heavy metals including copper, zinc, cobalt, and cadmium were identified. Collectively, our findings indicated that G. azotocaptans DS1 can be used as a biofertilizer and biocontrol agent for growth enhancement of different crops and vegetables. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02996-1.

Evaluating the biocontrol potential of Canadian strain Bacillus velezensis 1B-23 via its surfactin production at various pHs and temperatures.

BACKGROUND: Microorganisms, including Bacillus species are used to help control plant pathogens, thereby reducing reliance on synthetic pesticides in agriculture. Bacillus velezensis strain 1B-23 has been shown to reduce symptoms of bacterial disease caused by Clavibacter michiganensis subsp. michiganensis in greenhouse-grown tomatoes, with in vitro studies implicating the lipopeptide surfactin as a key antimicrobial. While surfactin is known to be effective against many bacterial pathogens, it is inhibitory to a smaller proportion of fungi which nonetheless cause the majority of crop diseases. In addition, knowledge of optimal conditions for surfactin production in B. velezensis is lacking. RESULTS: Here, B. velezensis 1B-23 was shown to inhibit in vitro growth of 10 fungal strains including Candida albicans, Cochliobolus carbonum, Cryptococcus neoformans, Cylindrocarpon destructans Fusarium oxysporum, Fusarium solani, Monilinia fructicola, and Rhizoctonia solani, as well as two strains of C. michiganensis michiganensis. Three of the fungal strains (C. carbonum, C. neoformans, and M. fructicola) and the bacterial strains were also inhibited by purified surfactin (surfactin C, or [Leu7] surfactin C15) from B. velezensis 1B-23. Optimal surfactin production occurred in vitro at a relatively low temperature (16 °C) and a slightly acidic pH of 6.0. In addition to surfactin, B. velenzensis also produced macrolactins, cyclic dipeptides and minor amounts of iturins which could be responsible for the bioactivity against fungal strains which were not inhibited by purified surfactin C. CONCLUSIONS: Our study indicates that B. velezensis 1B-23 has potential as a biocontrol agent against both bacterial and fungal pathogens, and may be particularly useful in slightly acidic soils of cooler climates.

Characterization and genomic analysis of a diesel-degrading bacterium, Acinetobacter calcoaceticus CA16, isolated from Canadian soil.

BACKGROUND: With the high demand for diesel across the world, environmental decontamination from its improper usage, storage and accidental spills becomes necessary. One highly environmentally friendly and cost-effective decontamination method is to utilize diesel-degrading microbes as a means for bioremediation. Here, we present a newly isolated and identified strain of Acinetobacter calcoaceticus ('CA16') as a candidate for the bioremediation of diesel-contaminated areas. RESULTS: Acinetobacter calcoaceticus CA16 was able to survive and grow in minimal medium with diesel as the only source of carbon. We determined through metabolomics that A. calcoaceticus CA16 appears to be efficient at diesel degradation. Specifically, CA16 is able to degrade 82 to 92% of aliphatic alkane hydrocarbons (CnHn + 2; where n = 12-18) in 28 days. Several diesel-degrading genes (such as alkM and xcpR) that are present in other microbes were also found to be activated in CA16. CONCLUSIONS: The results presented here suggest that Acinetobacter strain CA16 has good potential in the bioremediation of diesel-polluted environments.

Isolation and characterization of Burkholderia cenocepacia CR318, a phosphate solubilizing bacterium promoting corn growth.

Plant-growth promoting rhizobacteria benefit crop health and growth through various mechanisms including phosphate and potassium solubilisation, and antimicrobial activity. Previously, we sequenced the genome of bacterial strain Burkholderia cenocepacia CR318, which was isolated from the roots of the starch corn (Zea mays L.) in London, Ontario, Canada. In this work, the species identity of this isolate is confirmed by recA phylogeny and in silico DNA-DNA hybridization (isDDH), and its plant-growth promoting characteristics are described. B. cenocepacia CR318 exhibited strong activity of inorganic phosphate and potassium solubilization. It significantly promoted the growth of corn plants and roots by solubilizing inorganic tricalcium phosphate under greenhouse conditions. Functional analysis of the complete B. cenocepacia CR318 genome revealed genes associated with phosphate metabolism such as pstSCAB encoding a high affinity inorganic phosphate-specific transporter, and the pqqABCDE gene cluster involved in the biosynthesis of pyrroloquinoline quinone (PQQ), which is a required cofactor for quinoprotein glucose dehydrogenase (Gdh). However, it appears that B. cenocepacia CR318 lacks the quinoprotein Gdh which can produce gluconic acid to solubilize inorganic phosphate. Overall, these findings provide an important step in understanding the molecular mechanisms underlying the plant growth promotion trait of B. cenocepacia CR318.

Isolation and characterization of novel bacterial strains for integrated solar-bioelectrokinetic of soil contaminated with heavy petroleum hydrocarbons.

This study investigated the isolation and characterization of three novel bacterial strains; Acinetobacter calcoaceticus, Sphingobacterium multivorum, and Sinorhizobium, isolated form agriculture land. From three hundred strains of bacteria, the three isolates were identified for their superior diesel degradation ability by a series of bench-scale tests. The isolates were further investigated in bench tests for their ability to grow in different diesel fuel concentrations, temperature and pH; degrade diesel fuel in vitro; and for the identification of functional genes. Semi-pilot bioelectrokinetic tests were conducted in three electrokinetic cells. An innovative electrode configuration was adopted to stabilize the soil pH and water content during the test. The genes expressed in the diesel degradation process including Lipases enzymes Lip A, LipB, Alk-b2, rubA, P450, and 1698/2041 were detected in the three isolates. The results showed that the solar panel voltage output is in agreement with the trapezoid model. The temperatures in the cells were found to be 5-7 °C higher than the ambient temperature. The electrode configuration succeeded in stabilizing the soil pH and water content, preventing the development of a pH gradient, important progress for the survival of bacteria. The diesel degradation in the soil after bioelectrokinetic tests were 20-30%, compared to 10-12% in the controls. The study succeeded in developing environmentally friendly technology employing novel bacterial strains to degrade diesel fuel and utilizing solar panels to produce renewable energy for bioelectrokinetics during the winter season.

Bacteria in Cancer Therapeutics: A Framework for Effective Therapeutic Bacterial Screening and Identification.

By 2030, the global incidence of cancer is expected to increase by approximately 50%. However, most conventional therapies still lack cancer selectivity, which can have severe unintended side effects on healthy body tissue. Despite being an unconventional and contentious therapy, the last two decades have seen a significant renaissance of bacterium-mediated cancer therapy (BMCT). Although promising, most present-day therapeutic bacterial candidates have not shown satisfactory efficacy, effectiveness, or safety. Furthermore, therapeutic bacterial candidates are available to only a few of the approximately 200 existing cancer types. Excitingly, the recent surge in BMCT has piqued the interest of non-BMCT microbiologists. To help advance these interests, in this paper we reviewed important aspects of cancer, present-day cancer treatments, and historical aspects of BMCT. Here, we provided a four-step framework that can be used in screening and identifying bacteria with cancer therapeutic potential, including those that are uncultivable. Systematic methodologies such as the ones suggested here could prove valuable to new BMCT researchers, including experienced non-BMCT researchers in possession of extensive knowledge and resources of bacterial genomics. Lastly, our analyses highlight the need to establish and standardize quantitative methods that can be used to identify and compare bacteria with important cancer therapeutic traits.

Genome-wide occupancy of histone H3K27 methyltransferases CURLY LEAF and SWINGER in Arabidopsis seedlings.

The Polycomb Group (PcG) proteins form two protein complexes, PcG Repressive Complex 1 (PRC1) and PRC2, which are key epigenetic regulators in eukaryotes. PRC2 represses gene expression by catalyzing the trimethylation of histone H3 lysine 27 (H3K27me3). In Arabidopsis (Arabidopsis thaliana), CURLY LEAF (CLF) and SWINGER (SWN) are two major H3K27 methyltransferases and core components of PRC2, playing essential roles in plant growth and development. Despite their importance, genome-wide binding profiles of CLF and SWN have not been determined and compared yet. In this study, we generated transgenic lines expressing GFP-tagged CLF/SWN under their respective native promoters and used them for ChIP-seq analyses to profile the genome-wide distributions of CLF and SWN in Arabidopsis seedlings. We also profiled and compared the global H3K27me3 levels in wild-type (WT) and PcG mutants (clf, swn, and clf swn). Our data show that CLF and SWN bind to almost the same set of genes, except that SWN has a few hundred more targets. Two short DNA sequences, the GAGA-like and Telo-box-like motifs, were found enriched in the CLF and SWN binding regions. The H3K27me3 levels in clf, but not in swn, were markedly reduced compared with WT; and the mark was undetectable in the clf swn double mutant. Further, we profiled the transcriptomes in clf, swn, and clf swn, and compared that with WT. Thus this work provides a useful resource for the plant epigenetics community for dissecting the functions of PRC2 in plant growth and development.

RNA polymerase II-independent recruitment of SPT6L at transcription start sites in Arabidopsis.

SPT6 is a conserved elongation factor that is associated with phosphorylated RNA polymerase II (RNAPII) during transcription. Recent transcriptome analysis in yeast mutants revealed its potential role in the control of transcription initiation at genic promoters. However, the mechanism by which this is achieved and how this is linked to elongation remains to be elucidated. Here, we present the genome-wide occupancy of Arabidopsis SPT6-like (SPT6L) and demonstrate its conserved role in facilitating RNAPII occupancy across transcribed genes. We also further demonstrate that SPT6L enrichment is unexpectedly shifted, from gene body to transcription start site (TSS), when its association with RNAPII is disrupted. Protein domains, required for proper function and enrichment of SPT6L on chromatin, are subsequently identified. Finally, our results suggest that recruitment of SPT6L at TSS is indispensable for its spreading along the gene body during transcription. These findings provide new insights into the mechanisms underlying SPT6L recruitment in transcription and shed light on the coordination between transcription initiation and elongation.

Mitigation of soil contaminated with diesel fuel using bioelectrokinetics.

This study investigated the effectiveness of bioelectrokinetics in rehabilitating a silty clayey sand contaminated with diesel fuel using three novel bacterial strains; Acinetobacter calcoaceticus, Sphingobacterium multivorum, and Sinorhizobium, isolated form agriculture land. Three electrokinetic bioremediation cells were used to conduct the tests and a novel electrode configuration technique was used to stabilize pH and water content in the soil specimen. Solar photovoltaic panels were used to generate sustainable energy for the process. The tests were carried out in outdoors for 55 days. Applied voltage, current passing through the electrokinetic cell, and the temperature of the soil specimen were recorded periodically during the test. The pH, water content, and diesel concentration were determined at the end of the tests. Over the test period, the voltage typically increased from zero before sunrise, remained relatively stabilized for about 4 h, and then started to decrease and dropped to zero by sunset. The temperatures in the cells were found to be 5-7 °C higher than the ambient temperature. The innovative electrode configuration succeeded in keeping the pH of soil to remain the same and thereby prevented the development of a pH gradient in the soil, an important development for survival of the bacteria. The diesel degradation in the soil after bioelectrokinetics were 20-30%, compared to 10-12% in the control test. The study was successful in developing environmentally friendly technology employing novel bacterial strains to degrade diesel fuel and utilizing solar panel to produce renewable energy for bioelectrokinetic during the winter season.

Characterization and complete genome analysis of the surfactin-producing, plant-protecting bacterium Bacillus velezensis 9D-6.

BACKGROUND: Bacillus velezensis is an endospore-forming, free-living soil bacterium with potential as a biopesticide against a broad spectrum of microbial pathogens of plants. Its potential for commercial development is enhanced by rapid replication and resistance to adverse environmental conditions, typical of Bacillus species. However, the use of beneficial microbes against phytopathogens has not gained dominance due to limitations that may be overcome with new biopesticidal strains and/or new biological knowledge. RESULTS: Here, we isolated B. velezensis strain 9D-6 and showed that it inhibits the in vitro growth of prokaryotic and eukaryotic pathogens, including the bacteria Bacillus cereus , Clavibacter michiganensis, Pantoea agglomerans, Ralstonia solanacearum, Xanthomonas campestris, and Xanthomonas euvesicatoria; and the fungi Alternaria solani, Cochliobolus carbonum, Fusarium oxysporum, Fusarium solani, Gibberella pulicaris, Gibberella zeae, Monilinia fructicola, Pyrenochaeta terrestris and Rhizoctonia solani. Antimicrobial compounds with activity against Clavibacter michiganensis were isolated from B. velezensis 9D-6 and characterized by high resolution LC-MS/MS, yielding formulae of C52H91N7O13 and C53H93N7O13, which correspond to [Leu7] surfactins C14 and C15 (also called surfactin B and surfactin C), respectively. We further sequenced the B. velezensis 9D-6 genome which consists of a single circular chromosome and revealed 13 gene clusters expected to participate in antimicrobial metabolite production, including surfactin and two metabolites that have not typically been found in this species - ladderane and lantipeptide. Despite being unable to inhibit the growth of Pseudomonas syringae DC3000 in an in vitro plate assay, B. velezensis 9D-6 significantly reduced root colonization by DC3000, suggesting that 9D-6 uses methods other than antimicrobials to control phytopathogens in the environment. Finally, using in silico DNA-DNA hybridization (isDDH), we confirm previous findings that many strains currently classified as B. amyloliquefaciens are actually B. velezensis. CONCLUSIONS: The data presented here suggest B. velezensis 9D-6 as a candidate plant growth promoting bacterium (PGPB) and biopesticide, which uses a unique complement of antimicrobials, as well as other mechanisms, to protect plants against phytopathogens. Our results may contribute to future utilization of this strain, and will contribute to a knowledge base that will help to advance the field of microbial biocontrol.

Clavibacter michiganensis ssp. michiganensis: bacterial canker of tomato, molecular interactions and disease management.

Bacterial canker disease is considered to be one of the most destructive diseases of tomato (Solanum lycopersicum), and is caused by the seed-borne Gram-positive bacterium Clavibacter michiganensis ssp. michiganensis (Cmm). This vascular pathogen generally invades and proliferates in the xylem through natural openings or wounds, causing wilt and canker symptoms. The incidence of symptomless latent infections and the invasion of tomato seeds by Cmm are widespread. Pathogenicity is mediated by virulence factors and transcriptional regulators encoded by the chromosome and two natural plasmids. The virulence factors include serine proteases, cell wall-degrading enzymes (cellulases, xylanases, pectinases) and others. Mutational analyses of these genes and gene expression profiling (via quantitative reverse transcription-polymerase chain reaction, transcriptomics and proteomics) have begun to shed light on their roles in colonization and virulence, whereas the expression of tomato genes in response to Cmm infection suggests plant factors involved in the defence response. These findings may aid in the generation of target-specific bactericides or new resistant varieties of tomato. Meanwhile, various chemical and biological controls have been researched to control Cmm. This review presents a detailed investigation regarding the pathogen Cmm, bacterial canker infection, molecular interactions between Cmm and tomato, and current perspectives on improved disease management.

Cytosolic acetyl-CoA promotes histone acetylation predominantly at H3K27 in Arabidopsis.

Acetyl-coenzyme A (acetyl-CoA) is a central metabolite and the acetyl source for protein acetylation, particularly histone acetylation that promotes gene expression. However, the effect of acetyl-CoA levels on histone acetylation status in plants remains unknown. Here, we show that malfunctioned cytosolic acetyl-CoA carboxylase1 (ACC1) in Arabidopsis leads to elevated levels of acetyl-CoA and promotes histone hyperacetylation predominantly at lysine 27 of histone H3 (H3K27). The increase of H3K27 acetylation (H3K27ac) is dependent on adenosine triphosphate (ATP)-citrate lyase which cleaves citrate to acetyl-CoA in the cytoplasm, and requires histone acetyltransferase GCN5. A comprehensive analysis of the transcriptome and metabolome in combination with the genome-wide H3K27ac profiles of acc1 mutants demonstrate the dynamic changes in H3K27ac, gene transcripts and metabolites occurring in the cell by the increased levels of acetyl-CoA. This study suggests that H3K27ac is an important link between cytosolic acetyl-CoA level and gene expression in response to the dynamic metabolic environments in plants.

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling.

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. Arabidopsis thaliana-Agrobacterium tumefaciens provides an excellent model system to study bacterial pathogenesis and plant interactions. Previous studies of plant-Agrobacterium interactions have largely relied on plant cell suspension cultures, the artificial wounding of plants, or the artificial induction of microbial virulence factors or plant defenses by synthetic chemicals. However, these methods are distinct from the natural signaling in planta, where plants and microbes recognize and respond in spatial and temporal manners. This work presents a hydroponic cocultivation system where intact plants are supported by metal mesh screens and cocultivated with Agrobacterium. In this cocultivation system, no synthetic phytohormone or chemical that induces microbial virulence or plant defense is supplemented. The hydroponic cocultivation system closely resembles natural plant-microbe interactions and signaling homeostasis in planta. Plant roots can be separated from the medium containing Agrobacterium, and the signaling and responses of both the plant hosts and the interacting microbes can be investigated simultaneously and systematically. At any given timepoint/interval, plant tissues or bacteria can be harvested separately for various "omics" analyses, demonstrating the power and efficacy of this system. The hydroponic cocultivation system can be easily adapted to study: 1) the reciprocal signaling of diverse plant-microbe systems, 2) signaling between a plant host and multiple microbial species (i.e. microbial consortia or microbiomes), 3) how nutrients and chemicals are implicated in plant-microbe signaling, and 4) how microbes interact with plant hosts and contribute to plant tolerance to biotic or abiotic stresses.

Complete Genome Sequence of Acinetobacter calcoaceticus CA16, a Bacterium Capable of Degrading Diesel and Lignin.

We report here the complete assembled genome sequence of Acinetobacter calcoaceticus CA16, which is capable of utilizing diesel and lignin as a sole carbon source. CA16 contains a 4,110,074-bp chromosome and a 5,920-bp plasmid. The assembled sequences will help elucidate potential metabolic pathways and mechanisms responsible for CA16's hydrocarbon degradation ability.

Complete Genome Sequence of Burkholderia cenocepacia CR318, a Phosphate-Solubilizing Bacterium Isolated from Corn Root.

Here, we report the complete genome sequence of the phosphate-solubilizing bacterium Burkholderia cenocepacia CR318, consisting of three circular chromosomes of 3,511,146 bp, 3,097,552 bp, and 1,056,069 bp. The data presented will facilitate further insight into the mechanisms of phosphate solubilization and its application for agricultural and ecological sustainability.

MsmiR156 affects global gene expression and promotes root regenerative capacity and nitrogen fixation activity in alfalfa.

MicroRNA156 (miR156) regulates a network of downstream genes to affect plant growth and development. We previously generated alfalfa (Medicago sativa) plants that overexpress homologous miR156 (MsmiR156OE), and identified three of its SPL target genes. These plants exhibited increased vegetative yield, delayed flowering and longer roots. In this study, we aimed to elucidate the effect of miR156 on the root system, including effect on nodulation and nitrogen fixation. We found that MsmiR156 overexpression increases root regeneration capacity in alfalfa, but with little effect on root biomass at the early stages of root development. MsmiR156 also promotes nitrogen fixation activity by upregulating expression of nitrogenase-related genes FixK, NifA and RpoH in roots inoculated with Sinorrhizobium meliloti. Furthermore, we conducted transcriptomics analysis of MsmiR156OE alfalfa roots and identified differentially expressed genes belonging to 132 different functional categories, including plant cell wall organization, peptidyl-hypusine synthesis, and response to water stress. Expression analysis also revealed miR156 effects on genes involved in nodulation, root development and phytohormone biosynthesis. The present findings suggest that miR156 regulates root development and nitrogen fixation activity. Taken together, these findings highlight the important role that miR156 may play as a tool in the biotechnological improvement of alfalfa, and potentially other crops.