Lotus Japonicus Research Articles

Cyclic Isothiocyanate Goitrin Impairs Lotus japonicus Nodulation, Affects the Proteomes of Nodules and Free Mesorhizobium loti, and Induces the Formation of Caffeic Acid Derivatives in Bacterial Cultures.

The continuous release of glucosinolates into the soil by Brassicaceae root exudation is a prerequisite to maintaining toxic levels of breakdown products such as isothiocyanates (ITCs). ITCs influence plant and microbial diversity in ecosystems, while fungi and Rhizobiaceae are particularly injured. Studies explaining the molecular mechanisms of the negative effects are presently limited. Therefore, we investigated the early effects of cyclic ITC goitrin on proteomes of the host and symbiotic Mesorhizobium loti in the nodules of Lotus japonicus and of free-living bacteria. In the nodules, many host proteins had a higher abundance, among them, peroxidases and pathogenesis-related PR-10 proteins functioning in the abscisic-acid-activated signaling pathway. In the microsymbiont, transporter proteins as a prominent group are enhanced; some proteins involved in N-fixation decreased. The proteomes give a report about the loss of immunity suppression resulting in the termination of symbiosis, which initiates nodule senescence. Free-living M. loti are severely damaged, indicated, i.a., by a decrease in transporter proteins, the assumed candidates for goitrin protein complex formation, and high proteolysis. The production of chicoric acid by the accompanying bacteria is inhibitory for M. loti but connected to goitrin elimination, as confirmed by mass spectrometric (MS) analysis. In summary, the nodulation process is severely affected by goitrin, causing nodule dysfunction and failed nodule development. N deficiency conditions leads to yellowish leaves and leaf abscission.

TML1 AND TML2 SYNERGISTICALLY REGULATE NODULATION AND AFFECT ARBUSCULAR MYCORRHIZA IN MEDICAGO TRUNCATULA.

Two symbiotic processes, nodulation and arbuscular mycorrhiza, are primarily controlled by the plant's need for nitrogen (N) and phosphorus (P), respectively. Autoregulation of Nodulation (AON) and Autoregulation of Mycorrhization (AOM) both negatively regulate their respective processes and share multiple components - plants that make too many nodules usually have higher AM fungal root colonization. The protein TML (TOO MUCH LOVE) was shown to function in roots to maintain susceptibly to rhizobial infection under low N conditions and control nodule number through AON in Lotus japonicus . M. truncatula has two sequence homologs: Mt TML1 and Mt TML2. We report the generation of stable single and double mutants harboring multiple allelic variations in MtTML1 and MtTML2 using CRISPR-Cas9 targeted mutagenesis and screening of a transposon mutagenesis library. Plants containing single mutations in Mt TML1 or Mt TML2 produced 2-3 times the nodules of wild-type plants whereas plants containing mutations in both genes displayed a synergistic effect, forming 20x more nodules compared to wild type plants. Examination of expression and heterozygote effects suggest genetic compensation may play a role in the observed synergy. Plants with mutations in both TMLs only showed mild increases in AM fungal root colonization at later timepoints in our experiments, suggesting these genes may also play a minor role in AM symbiosis regulation. The mutants created will be useful tools to dissect the mechanism of synergistic action of Mt TML1 and Mt TML2 in M. truncatula symbiosis with beneficial microbes.

Zinc finger protein LjRSDL regulates arbuscule degeneration of arbuscular mycorrhizal fungi in Lotus japonicus.

In arbuscular mycorrhizal (AM) symbiosis, appropriate regulation of the formation, maintenance, and degeneration of the arbuscule are essential for plants and fungi. In this study, we identified a Cysteine-2/Histidine-2 zinc finger protein (C2H2-ZFP)-encoding gene in Lotus japonicus named Regulator of Symbiosome Differentiation-Like (LjRSDL) that is required for arbuscule degeneration. Evolutionary analysis showed that homologs of LjRSDL exist in mycorrhizal flowering plants. We obtained ProLjRSDL::GUS transgenic hairy roots and showed that LjRSDL was strongly upregulated upon AM colonization, particularly at 18 days post AM fungi inoculation and specifically expressed in arbuscular-containing cells. The mycorrhization rate increased in the ljrsdl mutant but decreased in LjRSDL overexpressed L. japonicus. Interestingly, we observed higher proportions of large arbuscule in the ljrsdl mutant but lower proportions of larger arbuscule in LjRSDL overexpressing plants. Transcriptome analyses indicated that genes involved in arbuscule degeneration were significantly changed upon the dysregulation of LjRSDL and that LjRSDL-dependent regulation in AM symbiosis is mainly via the hormone signal transduction pathway. LjRSDL, therefore, represents a C2H2-ZFP that negatively regulates AM symbiosis. Our study provides insight into understanding plant-AM fungal communication and AM symbiosis development.

Comprehensive characterization of the rhizosphere metabolome: A novel method for non-targeted analysis of Lotus japonicus root exudates

Plant root exudates play a pivotal role in shaping soil dynamics and the microbial community in the rhizosphere. The chemical composition of root exudates includes primary and secondary metabolites, including amino acids, organic acids, flavonoids, and small peptides. Comprehensive characterization of root exudates will allow for a better understanding of rhizosphere processes and interactions, but analysis of root exudates is hindered by complicated collection setups, time-consuming sample preparation, and a lack of definitive annotations within metabolomics. We present a method optimized for non-targeted analysis of primary and secondary metabolites in root exudate samples using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry. The method was tested on root exudates from Lotus japonicus, collected using distinct and well-established sampling methods: a hydroponic-soil-hybrid approach, as well as a modification of a soil-leaching method, thus exemplifying the versatility of the analysis method. The method allows for non-targeted screening of plant metabolites, and provides low detection limits (0.002–0.05 μg/mL) and high recoveries (78 ± 30%), though a matrix effect was observed for certain plant metabolites. Detection of a large number of features was achieved (670–2785) of which the majority could be putatively annotated at the compound class level. Of these, 14 features were putatively annotated to a specific structure with high confidence, three of which were confirmed with analytical reference standards. The method can be used for investigation of the overall change in root exudation, as well as for investigating significant changes in metabolites in response to intraenous and extraneous parameters.

A fungal endophyte increases plant resilience to low nutrient availabilities: a case of Fe acquisition in legumes.

Microbial inocula are considered a promising and effective alternative solution to the use of chemical fertilizers to support plant growth and productivity since they play a key role in the availability and uptake of nutrients. Here, the effect of a beneficial of a fungal root endophyte, Fusarium solani strain K (FsK), on nutrient acquisition efficiency of the legume Lotus japonicus was studied, and putative mode-of-action of the endophyte at a molecular level was determined. Plant colonization with the endophyte resulted in increased shoot and root fresh weight under Fe deficiency compared to control nutrient conditions. Plant inoculation with FsK was associated with a significant increase in macro- and micronutrient concentration in leaves at an early stage of endophyte inoculation and a replenishment of Fe content under prolonged iron starvation. The mechanistic basis of the plant growth promotion capabilities of the endophyte is exerted at the transcriptional level since we recorded changes in the expression levels of genes related to iron uptake in FsK-colonized plants under stress conditions compared to uninoculated plants. In addition, the observed changes in the ethylene biosynthesis-related genes suggest a possible implication of ethylene in the mode of action used by FsK to enhance plant response to nutrient stress conditions. Finally, we demonstrated that the endophyte possesses a reductive high-affinity Fe uptake system and identified a ferric reductase that was induced in planta under Fe deficiency conditions, indicating that this fungal Fe homeostasis mechanism may result in a benefit in nutrient acquisition for the plant as well.

Rhizobial secretion of truncated exopolysaccharides severely impairs the Mesorhizobium-Lotus symbiosis.

The symbiosis between Mesorhizobium japonicum R7A and Lotus japonicus Gifu is an important model system for investigating the role of bacterial exopolysaccharides (EPS) in plant-microbe interactions. Previously we showed that R7A exoB mutants that are affected at an early stage of EPS synthesis and in lipopolysaccharide (LPS) synthesis induce effective nodules on L. japonicus Gifu after a delay, whereas exoU mutants affected in the biosynthesis of the EPS side chain induce small uninfected nodule primordia and are impaired in infection. The presence of a halo around the exoU mutant when grown on Calcofluor-containing media suggested the mutant secreted a truncated version of R7A EPS. A non-polar ΔexoA mutant defective in the addition of the first glucose residue to the EPS backbone was also severely impaired symbiotically. Here we used a suppressor screen to show that the severe symbiotic phenotype of the exoU mutant was due to secretion of an acetylated pentasaccharide, as both monomers and oligomers, by the same Wzx/Wzy system that transports wild-type exopolysaccharide. We also present evidence that the ΔexoA mutant secretes an oligosaccharide by the same transport system, contributing to its symbiotic phenotype. In contrast, ΔexoYF, and polar exoA and exoL mutants have a similar phenotype to exoB mutants, forming effective nodules after a delay. These studies provide substantial evidence that secreted incompatible EPS is perceived by the plant leading to abrogation of the infection process.

Effects of LAZY family genes on shoot gravitropism in Lotus japonicus

Plant architecture is an important agronomic trait to determine the biomass and sward structure of forage grass. The IGT family plays a pivotal role in plant gravitropism, encompassing both the gravitropic response and the modulation of plant architecture. We have previously shown that LjLAZY3, one of the IGT genes, plays a distinct role in root gravitropism in L. japonicus. However, the function of LAZY proteins on shoot gravitropism in this species is poorly understood. In this study, we identified nine IGT genes in the L. japonicus genome, which have been categorized into four clades based on the phylogenetic relationships of IGT proteins from 18 legumes: LAZY1, NGR (NEGATIVE GRAVITROPIC RESPONSE OF ROOTS), IGT-LIKE, and TAC1. We found that LAZY genes in the first three clades have demonstrated distinct role for modulating plant gravitropism in L. japonicus with specific impacts as follows. Mutation of the LAZY1 gene, LjLAZY1, defected the gravitropic response of hypocotyl without impacting the main stem's branch angle. In contrast, the overexpression of the NGR gene, LjLAZY3, substantially modulated the shoot's gravitropism, leading to narrower lateral branch angles. Additionally, it enhanced the shoots' gravitropic response. The overexpression of another NGR gene, LjLAZY4, specifically reduced the main stem's branch angle and decreased plant stature without affecting the shoot gravitropic response. The phenotype of IGT-LIKE gene LjLAZY2 overexpression is identical to that of LjLAZY4. While overexpression of the IGT-LIKE gene LjLAZY5 did not induce any observable changes in branch angle, plant height, or gravitropic response. Furthermore, the LjLAZYs were selectively interacted with different BRXL and RLD proteins, which should the important factor to determine their different functions in controlling organ architecture in L. japonicus. Our results deepen understanding of the LjLAZY family and its potential for plant architecture improvement in L. japonicus.

Chitin nanofibers promote rhizobial symbiotic nitrogen fixation in Lotus japonicus

Chitin, an N-acetyl-D-glucosamine polymer, has multiple functions in living organisms, including the induction of disease resistance and growth promotion in plants. In addition, chitin oligosaccharides (COs) are used as the backbone of the signaling molecule Nod factor secreted by soil bacteria rhizobia to establish a mutual symbiosis with leguminous plants. Nod factor perception triggers host plant responses for rhizobial symbiosis. In this study, the effects of chitins on rhizobial symbiosis were examined in the leguminous plants Lotus japonicus and soybean. Chitin nanofiber (CNF), retained with polymeric structures, and COs elicited calcium spiking in L. japonicus roots expressing a nuclear-localized cameleon reporter. Shoot growth and symbiotic nitrogen fixation were significantly increased by CNF but not COs in L.japonicus and soybean. However, treatments with chitin and cellulose nanofiber, structurally similar polymers to CNF, did not affect shoot growth and nitrogen fixation in L.japonicus. Transcriptome analysis also supported the specific effects of CNF on rhizobial symbiosis in L.japonicus. Although chitins comprise the same monosaccharides and nanofibers share similar physical properties, only CNF can promote rhizobial nitrogen fixation in leguminous plants. Taking the advantages on physical properties, CNF could be a promising material for improving legume yield by enhancing rhizobial symbiosis.

Conserved cis-elements enable NODULES WITH ACTIVATED DEFENSE1 regulation by NODULE INCEPTION during nodulation.

Symbiotic nitrogen fixation within nitrogen-fixing clade (NFC) plants is thought to have arisen from a single gain followed by massive losses in the genomes of ancestral non-nodulating plants. However, molecular evidence supporting this model is limited. Here, we confirm through bioinformatic analysis that NODULES WITH ACTIVATED DEFENSE1 (NAD1) is present only in NFC plants and is thus an NFC-specific gene. Moreover, NAD1 was specifically expressed in nodules. We identified three conserved nodulation-associated cis-regulatory elements (NACE1-3) in the promoter of LjNAD1 from Lotus japonicus that are required for its nodule specific expression. A survey of NFC plants revealed that NACE1 and NACE2 are specific to the Fabales and Papilionoideae, respectively, while NACE3 is present in all NFC plants. Moreover, we found that nodule inception (NIN) directly binds to all three NACEs to activate NAD1 expression. Mutation of L. japonicus LjNAD1 resulted in the formation of abnormal symbiosomes with enlarged symbiosome space and frequent breakdown of bacteroids in nodules, resembling phenotypes reported for Medicago truncatula Mtnad1 and Mtnin mutants. These data point to NIN-NAD1 as an important module regulating rhizobial accommodation in nodules. The regulation of NAD1 by NIN in the NFC ancestor represent an important evolutionary adaptation for nodulation.

Mapping the molecular landscape of Lotus japonicus nodule organogenesis through spatiotemporal transcriptomics

Legumes acquire nitrogen-fixing ability by forming root nodules. Transferring this capability to more crops could reduce our reliance on nitrogen fertilizers, thereby decreasing environmental pollution and agricultural production costs. Nodule organogenesis is complex, and a comprehensive transcriptomic atlas is crucial for understanding the underlying molecular events. Here, we utilized spatial transcriptomics to investigate the development of nodules in the model legume, Lotus japonicus. Our investigation has identified the developmental trajectories of two critical regions within the nodule: the infection zone and peripheral tissues. We reveal the underlying biological processes and provide gene sets to achieve symbiosis and material exchange, two essential aspects of nodulation. Among the candidate regulatory genes, we illustrate that LjNLP3, a transcription factor belonging to the NIN-LIKE PROTEIN family, orchestrates the transition of nodules from the differentiation to maturation. In summary, our research advances our understanding of nodule organogenesis and provides valuable data for developing symbiotic nitrogen-fixing crops.

A quantitative assay for the efficiency of RNA-guided genome editing in plants.

RNA-guided endonucleases originating from the bacterial CRISPR/Cas system are a versatile tool for targeted gene editing. To determine the functional relevance of a gene of interest, deletion of the entire open reading frame (ORF) by two independent double-strand breaks (DSBs) is particularly attractive. This strategy greatly benefits from high editing efficiency, which is strongly influenced by the Cas endonuclease version used. We developed two reporter switch-on assays, for quantitative comparison and optimization of Cas constructs. The assays are based on four components: (i) A reporter gene, the mRNA of which carries a hairpin (HP) loop targeted by (ii) the endoribonuclease Csy4. Cleavage of the mRNA at the HP loop by Csy4 abolishes the translation of the reporter. Csy4 was used as the target for full deletion. (iii) A Cas system targeting sites flanking the Csy4 ORF with a 20-bp spacer either side to preferentially detect full-deletion events. Loss of functional Csy4 would lead to reporter gene expression, allowing indirect quantification of Cas-mediated deletion events. (iv) A reference gene for normalization. We tested these assays on Nicotiana benthamiana leaves and Lotus japonicus calli induced on hypocotyl sections, using Firefly luciferase and mCitrine as reporter genes and Renilla luciferase and hygromycin phosphotransferase II as reference genes, respectively. We observed a >90% correlation between reporter expression and full Csy4 deletion events, demonstrating the validity of these assays. The principle of using the Csy4-HP module as Cas target should be applicable to other editing goals including single DSBs in all organisms.

MyC Factor Analogue CO5 Promotes the Growth of Lotus japonicus and Enhances Stress Resistance by Activating the Expression of Relevant Genes.

The symbiotic relationship between arbuscular mycorrhizal fungi (AMF) and plants is well known for its benefits in enhancing plant growth and stress resistance. Research on whether key components of the AMF colonization process, such as MyC factors, can be directly utilized to activate plant symbiotic pathways and key functional gene expression is still lacking. In this paper, we found that, using a hydroponics system with Lotus japonicus, MyC factor analogue chitin oligomer 5 (CO5) had a more pronounced growth-promoting effect compared to symbiosis with AMF at the optimal concentration. Additionally, CO5 significantly enhanced the resistance of Lotus japonicus to various environmental stresses. The addition of CO5 activated symbiosis, nutrient absorption, and stress-related signaling pathways, like AMF symbiosis, and CO5 also activated a higher and more extensive gene expression profile compared to AMF colonization. Overall, the study demonstrated that the addition of MyC factor analogue CO5, by activating relevant pathways, had a superior effect on promoting plant growth and enhancing stress resistance compared to colonization by AMF. These findings suggest that utilizing MyC factor analogues like CO5 could be a promising alternative to traditional AMF colonization methods in enhancing plant growth and stress tolerance in agriculture.

Characterization of DREB family genes in Lotus japonicus and LjDREB2B overexpression increased drought tolerance in transgenic Arabidopsis

BackgroundDrought stress affects plant growth and development. DREB proteins play important roles in modulating plant growth, development, and stress responses, particularly under drought stress. To study the function of DREB transcription factors (TFs), we screened key DREB-regulating TFs for drought in Lotus japonicus.ResultsForty-two DREB TFs were identified, and phylogenetic analysis of proteins from L. japonicus classified them into five subfamilies (A1, A2, A4, A5, A6). The gene motif composition of the proteins is conserved within the same subfamily. Based on the cis-acting regulatory element analysis, we identified many growth-, hormone-, and stress-responsive elements within the promoter regions of DREB. We further analyzed the expression pattern of four genes in the A2 subfamily in response to drought stress. We found that the expression of most of the LjDREB A2 subfamily genes, especially LjDREB2B, was induced by drought stress. We further generated LjDREB2B overexpression transgenic Arabidopsis plants. Under drought stress, the growth of wild-type (WT) and overexpressing LjDREB2B (OE) Arabidopsis lines was inhibited; however, OE plants showed better growth. The malondialdehyde content of LjDREB2B overexpressing lines was lower than that of the WT plants, whereas the proline content and antioxidant enzyme activities in the OE lines were significantly higher than those in the WT plants. Furthermore, after drought stress, the expression levels of AtP5CS1, AtP5CS2, AtRD29A, and AtRD29B in the OE lines were significantly higher than those in the WT plants.ConclusionsOur results facilitate further functional analysis of L. japonicus DREB. LjDREB2B overexpression improves drought tolerance in transgenic Arabidopsis. These results indicate that DREB holds great potential for the genetic improvement of drought tolerance in L. japonicus.

Dynamic modulation of nodulation factor receptor levels by phosphorylation-mediated functional switch of a RING-type E3 ligase during legume nodulation

The precise control of receptor levels is crucial for initiating cellular signaling transduction in response to specific ligands; however, such mechanisms regulating nodulation factor (NF) receptor (NFR)-mediated perception of NFs to establish symbiosis remain unclear. In this study, we unveil the pivotal role of the NFR-interacting RING-type E3 ligase 1 (NIRE1) in regulating NFR1/NFR5 homeostasis to optimize rhizobial infection and nodule development in Lotus japonicus. We demonstrated that NIRE1 has a dual function in this regulatory process. It associates with both NFR1 and NFR5, facilitating their degradation through K48-linked polyubiquitination before rhizobial inoculation. However, following rhizobial inoculation, NFR1 phosphorylates NIRE1 at a conserved residue, Tyr-109, inducing a functional switch in NIRE1, which enables NIRE1 to mediate K63-linked polyubiquitination, thereby stabilizing NFR1/NFR5 in infected root cells. The introduction of phospho-dead NIRE1Y109F leads to delayed nodule development, underscoring the significance of phosphorylation at Tyr-109 in orchestrating symbiotic processes. Conversely, expression of the phospho-mimic NIRE1Y109E results in the formation of spontaneous nodules in L. japonicus, further emphasizing the critical role of the phosphorylation-dependent functional switch in NIRE1. In summary, these findings uncover a fine-tuned symbiotic mechanism that a single E3 ligase could undergo a phosphorylation-dependent functional switch to dynamically and precisely regulate NF receptor protein levels.

In silico analysis identified bZIP transcription factors genes responsive to abiotic stress in Alfalfa (Medicago sativa L.)

BackgroundAlfalfa (Medicago sativa L.) is the most cultivated forage legume around the world. Under a variety of growing conditions, forage yield in alfalfa is stymied by biotic and abiotic stresses including heat, salt, drought, and disease. Given the sessile nature of plants, they use strategies including, but not limited to, differential gene expression to respond to environmental cues. Transcription factors control the expression of genes that contribute to or enable tolerance and survival during periods of stress. Basic-leucine zipper (bZIP) transcription factors have been demonstrated to play a critical role in regulating plant growth and development as well as mediate the responses to abiotic stress in several species, including Arabidopsis thaliana, Oryza sativa, Lotus japonicus and Medicago truncatula. However, there is little information about bZIP transcription factors in cultivated alfalfa.ResultIn the present study, 237 bZIP genes were identified in alfalfa from publicly available sequencing data. Multiple sequence alignments showed the presence of intact bZIP motifs in the identified sequences. Based on previous phylogenetic analyses in A. thaliana, alfalfa bZIPs were similarly divided and fell into 10 groups. The physico-chemical properties, motif analysis and phylogenetic study of the alfalfa bZIPs revealed high specificity within groups. The differential expression of alfalfa bZIPs in a suite of tissues indicates that bZIP genes are specifically expressed at different developmental stages in alfalfa. Similarly, expression analysis in response to ABA, cold, drought and salt stresses, indicates that a subset of bZIP genes are also differentially expressed and likely play a role in abiotic stress signaling and/or tolerance. RT-qPCR analysis on selected genes further verified these differential expression patterns.ConclusionsTaken together, this work provides a framework for the future study of bZIPs in alfalfa and presents candidate bZIPs involved in stress-response signaling.

Synergistic effects of plant genotype and soil microbiome on growth in Lotus japonicus.

The biological interactions between plants and their root microbiomes are essential for plant growth, and even though plant genotype [G], soil microbiome [M], and growth conditions (environment) [E] are the core factors shaping root microbiome, their relationships remain unclear. In this study we investigated the effects of G, M, and E and their interactions on the Lotus root microbiome and plant growth using an in vitro cross-inoculation approach which reconstructed the interactions between nine Lotus accessions and four soil microbiomes under two different environmental conditions. Results suggested that a large proportion of the root microbiome composition is determined by M and E, while G-related (G, G × M, and G × E) effects were significant but small. In contrast, the interaction between G and M had a more pronounced effect on plant shoot growth than M alone. Our findings also indicated that most microbiome variations controlled by M have little effect on plant phenotypes, whereas G × M interactions have more significant effects. Plant genotype-dependent interactions with soil microbes warrant more attention to optimize crop yield and resilience.

RinRK1 enhances NF receptors accumulation in nanodomain-like structures at root-hair tip

Legume-rhizobia root-nodule symbioses involve the recognition of rhizobial Nod factor (NF) signals by NF receptors, triggering both nodule organogenesis and rhizobial infection. RinRK1 is induced by NF signaling and is essential for infection thread (IT) formation in Lotus japonicus. However, the precise mechanism underlying this process remains unknown. Here, we show that RinRK1 interacts with the extracellular domains of NF receptors (NFR1 and NFR5) to promote their accumulation at root hair tips in response to rhizobia or NFs. Furthermore, Flotillin 1 (Flot1), a nanodomain-organizing protein, associates with the kinase domains of NFR1, NFR5 and RinRK1. RinRK1 promotes the interactions between Flot1 and NF receptors and both RinRK1 and Flot1 are necessary for the accumulation of NF receptors at root hair tips upon NF stimulation. Our study shows that RinRK1 and Flot1 play a crucial role in NF receptor complex assembly within localized plasma membrane signaling centers to promote symbiotic infection.

Systemic long-distance sulfur transport and its role in symbiotic root nodule protein turnover

Sulfur is an essential nutrient for all plants, but also crucial for the nitrogen fixing symbiosis between legumes and rhizobia. Sulfur limitation can hamper nodule development and functioning. Until now, it remained unclear whether sulfate uptake into nodules is local or mainly systemic via the roots, and if long-distance transport from shoots to roots and into nodules occurs. Therefore, this work investigates the systemic regulation of sulfur transportation in the model legume Lotus japonicus by applying stable isotope labeling to a split-root system. Metabolite and protein extraction together with mass spectrometry analyses were conducted to determine the plants molecular phenotype and relative isotope protein abundances. Data show that treatments of varying sulfate concentrations including the absence of sulfate on one side of a nodulated root was not affecting nodule development as long as the other side of the root system was provided with sufficient sulfate. Concentrations of shoot metabolites did not indicate a significant stress response caused by a lack of sulfur. Further, we did not observe any quantitative changes in proteins involved in biological nitrogen fixation in response to the different sulfate treatments. Relative isotope abundance of 34S confirmed a long-distance transport of sulfur from one side of the roots to the other side and into the nodules. Altogether, these results provide evidence for a systemic long-distance transport of sulfur via the upper part of the plant to the nodules suggesting a demand driven sulfur distribution for the maintenance of symbiotic N-fixation.

Nitrogen and Nod factor signaling determine Lotus japonicus root exudate composition and bacterial assembly

Symbiosis with soil-dwelling bacteria that fix atmospheric nitrogen allows legume plants to grow in nitrogen-depleted soil. Symbiosis impacts the assembly of root microbiota, but it is unknown how the interaction between the legume host and rhizobia impacts the remaining microbiota and whether it depends on nitrogen nutrition. Here, we use plant and bacterial mutants to address the role of Nod factor signaling on Lotus japonicus root microbiota assembly. We find that Nod factors are produced by symbionts to activate Nod factor signaling in the host and that this modulates the root exudate profile and the assembly of a symbiotic root microbiota. Lotus plants with different symbiotic abilities, grown in unfertilized or nitrate-supplemented soils, display three nitrogen-dependent nutritional states: starved, symbiotic, or inorganic. We find that root and rhizosphere microbiomes associated with these states differ in composition and connectivity, demonstrating that symbiosis and inorganic nitrogen impact the legume root microbiota differently. Finally, we demonstrate that selected bacterial genera characterizing state-dependent microbiomes have a high level of accurate prediction.

Comparative Genomics of Lotus japonicus Reveals Insights into Proanthocyanidin Accumulation and Abiotic Stress Response.

Lotus japonicus, is an important perennial model legume, has been widely used for studying biological processes such as symbiotic nitrogen fixation, proanthocyanidin (PA) biosynthesis, and abiotic stress response. High-quality L. japonicus genomes have been reported recently; however, the genetic basis of genes associated with specific characters including proanthocyanidin distribution in most tissues and tolerance to stress has not been systematically explored yet. Here, based on our previous high-quality L. japonicus genome assembly and annotation, we compared the L. japonicus MG-20 genome with those of other legume species. We revealed the expansive and specific gene families enriched in secondary metabolite biosynthesis and the detection of external stimuli. We suggested that increased copy numbers and transcription of PA-related genes contribute to PA accumulation in the stem, petiole, flower, pod, and seed coat of L. japonicus. Meanwhile, According to shared and unique transcription factors responding to five abiotic stresses, we revealed that MYB and AP2/ERF play more crucial roles in abiotic stresses. Our study provides new insights into the key agricultural traits of L. japonicus including PA biosynthesis and response to abiotic stress. This may provide valuable gene resources for legume forage abiotic stress resistance and nutrient improvement.