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Nodules harbour microbial communities composed of rhizobia and other lower-abundance bacteria. These non-rhizobial bacteria can promote plant growth. However, their genomic diversity and how this relates to their plant growth-promoting traits remain poorly investigated. Here, we isolated 14 Pseudomonas strains from the nodules of Lotus plants, sequenced their genomes, analysed their genomic and phylogenetic diversity, and assessed their ability to promote plant growth. We identified five distinct species, including a novel species named Pseudomonas monachiensis sp. nov., with strain PLb12A T , as the type strain. Genome analysis of these nodule-isolated Pseudomonas revealed an abundance of genes associated to plant growth-promoting traits, especially auxin-related genes, compared to closely related type strains. In accordance, most nodule-isolated Pseudomonas strains enhanced shoot growth of Lotus burttii, while only some promoted root growth or early onset of root hair proliferation. However, none of the strains significantly affected the ability to form nodules. Overall, our findings highlight the genotypic diversity and the plant growth-promoting potential of nodule-isolated Pseudomonas and underscore their possible applications in mixed inocula with rhizobia., (© 2025 The Author(s). Environmental Microbiology published by John Wiley & Sons Ltd.)

Seed longevity is crucial for long-term storage, but prolonged unfavorable conditions can lead to loss of viability. This study integrated theoretical and experimental techniques to elucidate the inherent mechanisms underlying the unique ability of lotus seeds to maintain stable viability over many years. Transcriptome analysis and microscopy revealed a sturdy structure of the lotus seed pericarp, which predominantly expressed cellulose synthase genes involved in cell wall biogenesis. The cotyledon serves as a nutrient source for seeds during long-term storage. Additionally, the inactivation of chlorophyll degradation pathways may allow for the retention of chlorophyll in the lotus seed plumule, potentially enhancing the environmental adaptability of lotus seedlings. Reduced abundance of transcripts corresponding to heat shock protein genes could impact protein processing and consequently diminish the vitality of aging lotus seeds. Moreover, an expansion in the number of seed maturation and defense response genes was observed in the lotus genome compared with 11 other species, which might represent an adaptive strategy against long-term adverse storage conditions. Overall, these findings are crucial for understanding the mechanisms underlying lotus seed longevity and may inform future improvements in the extended storage periods of seed crops., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Plant root development depends on signaling pathways responding to external and internal signals. In this study we demonstrate the involvement of the Lotus japonicus LjNPF4.6 gene in the ABA and nitrate root responding pathways. LjNPF4.6 expression in roots is induced by external application of both nitrate and ABA. LjNPF4.6 promoter activity is spatially localized in epidermal cell layer and vascular bundle structures with the latter pattern being controlled by externally applied ABA. LjNPF4.6 cRNA injection achieves both nitrate and ABA uptake in Xenopus laevis oocytes and the analyses of L. japonicus knock-out insertion mutants confirmed the role played by LjNPF4.6 in root nitrate uptake. The phenotypic characterization of the Ljnpf4.6 plants indicates the role played by LjNPF4.6 in the root program development in response to exogenously applied nitrate and ABA. Based on the presented data, the mode of action of this transporter is discussed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Masson SAS.)

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., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Leguminous plants provide carbon to symbiotic rhizobia in root nodules to fuel the energy-consuming process of nitrogen fixation. The carbon investment pattern from the acquired sources is crucial for shaping the growth regime of the host plants. The autoregulation of nodulation (AON) signaling pathway tightly regulates the number of nodules that form. AON disruption leads to excessive nodule formation and stunted shoot growth. However, the physiological role of AON in adjusting the carbon investment pattern is unknown. Here, we show that AON plays an important role in sustaining shoot water availability, which is essential for promoting carbon investment in shoot growth in Lotus japonicus. We found that AON-defective mutants exhibit substantial accumulation of nonstructural carbohydrates, such as sucrose. Consistent with this metabolic signature, resilience against water-deficit stress was enhanced in the shoots of the AON-defective mutants. Furthermore, the water uptake ability was attenuated in the AON-defective mutants, likely due to the increased ratio of nodulation zone, which is covered with hydrophobic surfaces, on the roots. These results increase our physiological understanding of legume-rhizobia symbiosis by revealing a trade-off between root nodule formation and shoot water availability., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

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., (© 2024. The Author(s).)

Host plants benefit from legume root nodule symbiosis with nitrogen-fixing bacteria under nitrogen-limiting conditions. In this interaction, the hosts must regulate nodule numbers and distribution patterns to control the degree of symbiosis and maintain root growth functions. The host response to symbiotic bacteria occurs discontinuously but repeatedly at the region behind the tip of the growing roots. Here, live-imaging and transcriptome analyses revealed oscillating host gene expression with approximately 6-hour intervals upon bacterial inoculation. Cytokinin response also exhibited a similar oscillation pattern. Cytokinin signaling is crucial to maintaining the periodicity, as observed in cytokinin receptor mutants displaying altered infection foci distribution. This periodic regulation influences the size of the root region responsive to bacteria, as well as the nodulation process progression.

Background: Lotus (Nelumbo nucifera G.) is an important aquatic plant with high ornamental, economic, cultural and ecological values, but abiotic stresses seriously affect its growth and distribution. Q-type C2H2 zinc finger proteins (ZFPs) play an important role in plant growth development and environmental stress responses. Although the Q-type C2H2 gene family has been identified in some plants, limited reports has been carried out it in lotus., Results: In this study, we identified 45 Q-type NnZFP members in lotus. Based on the phylogenetic tree, these Q-type NnZFP gene family members were divided into 4 groups, including C1-1i, C1-2i, C1-3i and C1-4i. Promoter cis-acting elements analysis indicated that most Q-type NnZFP gene family members in lotus were associated with response to abiotic stresses. Through collinearity analyses, no tandem duplication gene pairs and 14 segmental duplication gene pairs were identified, which showed that duplication events might play a key role in the expansion of the Q-type NnZFP gene family. The synteny results suggested that 54 and 28 Q-type NnZFP genes were orthologous to Arabidopsis and rice, respectively. The expression patterns of these Q-type NnZFP genes revealed that 30 Q-type NnZFP genes were expressed in at least one lotus tissue. Nn5g30550 showed relatively higher expression levels in all tested tissues. 12 genes were randomly selected with at least one gene from each phylogenetic clade, and the expression of these selected genes were confirmed by qRT-PCR (quantitative real-time polymerase chain reaction). The results indicated that Q-type NnZFP genes were extensively involved in cadmium, drought, salt and cold stresses responses. Among them, 11 genes responded to at least three different stress treatments, especially Nn2g12894, which induced by all four treatments., Conclusions: These results could increase our understanding of the characterization of the Q-type NnZFP gene family and provide relevant information for further functional analysis of Q-type NnZFP genes in plant development, and abiotic stress tolerance in lotus., (© 2024. The Author(s).)

The qRT-PCR technique has been regarded as an important tool for assessing gene expression diversity. Selection of appropriate reference genes is essential for validating deviation and obtaining reliable and accurate results. Lotus (Nelumbo nucifera Gaertn) is a common aquatic plant with important aesthetic, commercial, and cultural values. Twelve candidate genes, which are typically used as reference genes for qRT-PCR in other plants, were selected for this study. These candidate reference genes were cloned with, specific primers designed based on published sequences. In particular, the expression level of each gene was examined in different tissues and growth stages of Lotus. Notably, the expression stability of these candidate genes was assessed using the software programs geNorm and NormFinder. As a result, the most efficient reference genes for rootstock expansion were TBP and UBQ. In addition, TBP and EF-1α were the most efficient reference genes in various floral tissues, while ACT and GAPDH were the most stable genes at all developmental stages of the seed. CYP and GAPDH were the best reference genes at different stages of leaf development, but TUA was the least stable. Meanwhile, the gene expression profile of NnEXPA was analyzed to confirm the validity of the findings. It was concluded that, TBP and GAPDH were identified as the best reference genes. The results of this study may help researchers to select appropriate reference genes and thus obtain credible results for further quantitative RT-qPCR gene expression analyses in Lotus., (© 2024. The Author(s).)

One of the most vulnerable phases in the plant life cycle is sexual reproduction, which depends on effective pollen transfer, but also on the thermotolerance of pollen grains. Pollen thermotolerance is temperature-dependent and may be reduced by increasing temperature associated with global warming. A growing body of research has focused on the effect of increased temperature on pollen thermotolerance in crops to understand the possible impact of temperature extremes on yield. Yet, little is known about the effects of temperature on pollen thermotolerance of wild plant species. To fill this gap, we selected Lotus corniculatus s.l. (Fabaceae), a species common to many European habitats and conducted laboratory experiments to test its pollen thermotolerance in response to artificial increase in temperature. To test for possible local adaptation of pollen thermal tolerance, we compared data from six lowland (389-451 m a.s.l.) and six highland (841-1,030 m a.s.l.) populations. We observed pollen germination in vitro at 15 °C, 25 °C, 30 °C, and 40 °C. While lowland plants maintained a stable germination percentage across a broad temperature range (15-30 °C) and exhibited reduced germination only at extremely high temperatures (40 °C), highland plants experienced reduced germination even at 30 °C-temperatures commonly exceeded in lowlands during warm summers. This suggests that lowland populations of L. corniculatus may be locally adapted to higher temperature for pollen germination. On the other hand, pollen tube length decreased with increasing temperature in a similar way in lowland and highland plants. The overall average pollen germination percentage significantly differed between lowland and highland populations, with highland populations displaying higher germination percentage. On the other hand, the average pollen tube length was slightly smaller in highland populations. In conclusion, we found that pollen thermotolerance of L. corniculatus is reduced at high temperature and that the germination of pollen from plant populations growing at higher elevations is more sensitive to increased temperature, which suggests possible local adaptation of pollen thermotolerance., Competing Interests: The authors declare that they have no competing interests., (© 2024 Jackwerth et al.)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

Legumes attract symbiotic bacteria and create de novo root organs called nodules. Nodule development consists of bacterial infection of root epidermis and subsequent primordium formation in root cortex, steps that need to be spatiotemporally coordinated. The Lotus japonicus mutant “ daphne ” has uncoupled symbiotic events in epidermis and cortex, in that it promotes excessive bacterial infection in epidermis but does not produce nodule primordia in cortex. Therefore, daphne should be useful for exploring unknown signals that coordinate these events across tissues. Here, we conducted time-course RNA sequencing using daphne after rhizobial infection. We noticed that IAA carboxyl methyltransferase 1 (IAMT1) , which encodes the enzyme that converts auxin (IAA) into its methyl ester (MeIAA), is transiently induced in wild-type roots at early stages of infection but shows different expression dynamics in daphne . IAMT1 serves an important function in shoot development of Arabidopsis , a nonsymbiotic plant, but the function of IAMT1 in roots has not been reported. Phylogenetic tree analysis suggests a gene duplication of IAMT1 in the legume lineage, and we found that one of the two IAMT1s (named IAMT1a ) was induced in roots by epidermal infection. IAMT1a knockdown inhibited nodule development in cortex; however, it had no effect on epidermal infection. The amount of root MeIAA increased with rhizobial infection. Application of MeIAA, but not IAA , significantly induced expression of the symbiotic gene NIN in the absence of rhizobial infection. Our results provide evidence for the role of auxin methylation in an early stage of root nodule development.

Beta-proteobacteria belonging to the genus Acidovorax have been described from various environments. Many strains can interact with a range of hosts, including humans and plants, forming neutral, beneficial or detrimental associations. In the frame of this study, we investigated the genomic properties of 52 bacterial strains of the genus Acidovorax , isolated from healthy roots of Lotus japonicus, with the intent of identifying traits important for effective plant-growth promotion. Based on single-strain inoculation bioassays with L. japonicus , performed in a gnotobiotic system, we distinguished seven robust plant-growth promoting strains from strains with no significant effects on plant-growth. We showed that the genomes of the two groups differed prominently in protein families linked to sensing and transport of organic acids, production of phytohormones, as well as resistance and production of compounds with antimicrobial properties. In a second step, we compared the genomes of the tested isolates with those of plant pathogens and free-living strains of the genus Acidovorax sourced from public repositories. Our pan-genomics comparison revealed features correlated with commensal and pathogenic lifestyle. We showed that commensals and pathogens differ mostly in their ability to use plant-derived lipids and in the type of secretion-systems being present. Most free-living Acidovorax strains did not harbour any secretion-systems. Overall, our data indicate that Acidovorax strains undergo extensive adaptations to their particular lifestyle by horizontal uptake of novel genetic information and loss of unnecessary genes.

Auxin is essential for root development, and its regulatory action is exerted at different steps from perception of the hormone up to transcriptional regulation of target genes. In legume plants there is an overlap between the developmental programs governing lateral root and N 2 -fixing nodule organogenesis, the latter induced as the result of the symbiotic interaction with rhizobia. Here we report the characterization of a member of the L. japonicus TIR1/AFB auxin receptor family, LjAFB6 . A preferential expression of the LjAFB6 gene in the aerial portion of L. japonicus plants was observed. Significant regulation of the expression was not observed during the symbiotic interaction with Mesorhizobium loti and the nodule organogenesis process. In roots, the LjAFB6 expression was induced in response to nitrate supply and was mainly localized in the meristematic regions of both primary and lateral roots. The phenotypic analyses conducted on two independent null mutants indicated a specialized role in the control of primary and lateral root elongation processes in response to auxin, whereas no involvement in the nodulation process was found. We also report the involvement of LjAFB6 in the hypocotyl elongation process and in the control of the expression profile of an auxin-responsive gene.

Root hair cells form the primary interface of plants with the soil environment, playing key roles in nutrient uptake and plant defense. In legumes, they are typically the first cells to become infected by nitrogen-fixing soil bacteria during root nodule symbiosis. Here, we report a role for the CELLULOSE SYNTHASE-LIKE D1 (CSLD1) gene in root hair development in the legume species Lotus japonicus. CSLD1 belongs to the cellulose synthase protein family that includes cellulose synthases and cellulose synthase-like proteins, the latter thought to be involved in the biosynthesis of hemicellulose. We describe 11 Ljcsld1 mutant alleles that impose either short (Ljcsld1-1) or variable (Ljcsld1-2 to 11) root hair length phenotypes. Examination of Ljcsld1-1 and one variable-length root hair mutant, Ljcsld1-6, revealed increased root hair cell wall thickness, which in Ljcsld1-1 was significantly more pronounced and also associated with a strong defect in root nodule symbiosis. Lotus japonicus plants heterozygous for Ljcsld1-1 exhibited intermediate root hair lengths, suggesting incomplete dominance. Intragenic complementation was observed between alleles with mutations in different CSLD1 domains, suggesting CSLD1 function is modular and that the protein may operate as a homodimer or multimer during root hair development., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Carbonic anhydrase (CA) plays a vital role in photosynthetic tissues of higher plants, whereas its non-photosynthetic role in the symbiotic root nodule was rarely characterized. In this study, 13 CA genes were identified in the model legume Lotus japonicus by comparison with Arabidopsis CA genes. Using qPCR and promoter-reporter fusion methods, three previously identified nodule-enhanced CA genes ( Lj αCA2 , Lj αCA6 , and Lj βCA1 ) have been further characterized, which exhibit different spatiotemporal expression patterns during nodule development. Lj αCA2 was expressed in the central infection zone of the mature nodule, including both infected and uninfected cells. Lj αCA6 was restricted to the vascular bundle of the root and nodule. As for Lj βCA1 , it was expressed in most cell types of nodule primordia but only in peripheral cortical cells and uninfected cells of the mature nodule. Using CRISPR/Cas9 technology, the knockout of Lj βCA1 or both LjαCA2 and its homolog, LjαCA1 , did not result in abnormal symbiotic phenotype compared with the wild-type plants, suggesting that LjβCA1 or LjαCA1/2 are not essential for the nitrogen fixation under normal symbiotic conditions. Nevertheless, the nodule-enhanced expression patterns and the diverse distributions in different types of cells imply their potential functions during root nodule symbiosis, such as CO 2 fixation, N assimilation, and pH regulation, which await further investigations.

The lotus produces flower buds at each node, yet most of them are aborted because of unfavorable environmental changes and the mechanism remains unclear. In this work, we proposed a potential novel pathway for ABA-mediated flower timing control in the lotus, which was explored by combining molecular, genetic, transcriptomic, biochemical, and pharmacologic approaches. We found that the aborting flower buds experienced extensive programmed cell death (PCD). The hormonal changes between the normal and aborting flower buds were dominated by abscisic acid (ABA). Seedlings treated with increasing concentrations of ABA exhibited a differential alleviating effect on flower bud abortion, with a maximal response at 80 μM. Transcriptome analysis further confirmed the changes of ABA content and the occurrence of PCD, and indicated the importance of PCD-related SNF1-related protein kinase 1 (NnSnRK1). The NnSnRK1-silenced lotus seedlings showed stronger flowering ability, with their flower:leaf ratio increased by 40%. When seedlings were treated with ABA, the expression level and protein kinase activity of NnSnRK1 significantly decreased. The phenotype of NnSnRK1-silenced seedlings could also be enhanced by ABA treatment and reversed by tungstate treatment. These results suggested that the decline of ABA content in lotus flower buds released its repression of NnSnRK1, which then initiated flower bud abortion.

Plant hormones have been identified to be versatile signaling molecules essential for plant growth, development, and stress response. Their content levels vary depending on the species, and they also change in response to any external stimuli. Thus, simultaneous quantification of multiple plant hormones is required to understand plant physiology. Sensitive and quantitative analysis using liquid chromatography-linked mass spectrometry (LC-MS/MS) has been used in detecting plant hormones; however, quantification without stable isotopes is yet to be established. In this study, we quantified seven representative plant hormones of Lotus japonicus, which is a model legume for standard addition method. Accurate masses for monoisotopic ions of seven phytohormones were determined for high-resolution mass spectrometry (HR-MS). Selected ion monitoring (SIM) mode based on accurate masses was used in detecting phytohormones in the roots, stems, and leaves. Evaluation of matrix effects showed ion suppression ranging from 10.2% to 87.3%. Both stable isotope dilution and standard addition methods were able to detect plant hormones in the roots, stems, and leaves, with no significant differences in using both approaches and thus a standard addition method can be used to quantify phytohormones in L. japonicus. The method will be effective, especially when stable isotopes are not available to correct for matrix effects., Competing Interests: The authors have declared that no competing interests exist.

Lotus ( Nelumbo nucifera ) seeds are widely consumed as functional food or herbal medicine, of which cotyledon (CL) is the main edible part, and lotus plumule (LP) is commonly utilized in traditional Chinese medicine. However, few studies have been conducted to investigate the chemical components of CL and LP in dry lotus seeds, not to mention the comparison between wild and domesticated varieties. In this study, a widely targeted metabolomics approach based on Ultra Performance Liquid Chromatography-electrospray ionization-Tandem mass spectrometry (UPLC-ESI-MS/MS) was utilized to analyze the metabolites in CL and LP of China Antique ("CA", a wild variety) and Jianxuan-17 ("JX", a popular cultivar). A total of 402 metabolites were identified, which included flavonoids (23.08% to 27.84%), amino acids and derivatives (14.18-16.57%), phenolic acids (11.49-12.63%), and lipids (9.14-10.95%). These metabolites were classified into ten clusters based on their organ or cultivar-specific characters. Most of these metabolites were more abundant in LP than in CL for both varieties, except for metabolites belonging to organic acids and lipids. The analysis of differentially accumulated metabolites (DAMs) demonstrated that more than 25% of metabolites detected in our study were DAMs in CL and LP comparing "JX" with "CA", most of which were less abundant in "JX", including 35 flavonoids in LP, 23 amino acids and derivatives in CL, 7 alkaloids in CL, and 10 nucleotides and derivatives in LP, whereas all of 11 differentially accumulated lipids in LP were more abundant in "JX". Together with the fact that the seed yield of "JX" is much higher than that of "CA", these results indicated that abundant metabolites, especially the functional secondary metabolites (mainly flavonoids and alkaloids), were lost during the process of breeding selection.

Arbuscular mycorrhiza is an ancient symbiosis between most land plants and fungi of the Glomeromycotina, in which the fungi provide mineral nutrients to the plant in exchange for photosynthetically fixed organic carbon. Strigolactones are important signals promoting this symbiosis, as they are exuded by plant roots into the rhizosphere to stimulate activity of the fungi. In addition, the plant karrikin signaling pathway is required for root colonization. Understanding the molecular mechanisms underpinning root colonization by AM fungi, requires the use of plant mutants as well as treatments with different environmental conditions or signaling compounds in standardized cocultivation systems to allow for reproducible root colonization phenotypes. Here we describe how we set up and quantify arbuscular mycorrhiza in the model plants Lotus japonicus and Brachypodium distachyon under controlled conditions. We illustrate a setup for open pot culture as well as for closed plant tissue culture (PTC) containers, for plant-fungal cocultivation in sterile conditions. Furthermore, we explain how to harvest, store, stain, and image AM roots for phenotyping and quantification of different AM structures.

In legumes, rhizobia attach to root hair tips and secrete nodulation factor to activate rhizobial infection and nodule organogenesis. Endosymbiotic rhizobia enter nodule primordia via a specialized transcellular compartment known as the infection thread (IT). The IT elongates by polar tip growth, following the path of the migrating nucleus along and within the root hair cell. Rho-family ROP GTPases are known to regulate the polarized growth of cells, but their role in regulating polarized IT growth is poorly understood. Here, we show that LjSPK1, a DOCK family guanine nucleotide exchange factor (GEF), interacts with three type I ROP GTPases. Genetic analyses showed that these three ROP GTPases are involved in root hair development, but only LjROP6 is required for IT formation after rhizobia inoculation. Misdirected ITs formed in the root hairs of Ljspk1 and Ljrop6 mutants. We show that LjSPK1 functions as a GEF that activates LjROP6. LjROP6 enhanced the plasma membrane localization LjSPK1 in Nicotiana benthamiana leaf cells and Lotus japonicus root hairs, and LjSPK1 and LjROP6 interact at the plasma membrane. Taken together, these results shed light on how the LjROP6-LjSPK1 module mediates the polarized growth of ITs in L. japonicus ., (© 2020 American Society of Plant Biologists. All rights reserved.)

SNAREs (soluble N-ethyl maleimide-sensitive factor attachment protein receptors) mediate membrane fusion of vesicle transport in eukaryotic cells. LjSYP132s are the members of Qa-SNAREs in Lotus japonicus. Two isoforms, LjSYP132a and LjSYP132b, are generated by alternative splicing. Immunoblot analysis detected strong expression of LjSYP132s in infected root nodules and seeds by posttranscriptional modification. In either LjSYP132a or LjSYP132b silenced roots (RNAi-LjSYP132a, RNAi-LjSYP132b), the infection thread (IT) was not elongated, suggesting that both LjSYP132a and LjSYP132b have a role in IT progression. The results were consistent with the data of qRT-PCR showing that both genes were expressed at the early stage of infection. However, during the nodulation, only LjSYP132a was induced. LjSYP132s protein was observed in the Mesorhizobium loti-inoculated roots of mutants, nfr1, castor and pollux, suggesting that LjSYP132s can be induced without Nod factor signaling. Accumulation of LjSYP132s in the peribacteroid membrane suggests the function of not only IT formation but also nutrient transport. In contrast, qRT-PCR showed that LjSYP132b was expressed in the seeds. A stable transgenic plant of LjSYP132b, R132b, was produced by RNAi silencing. In the R132b plants, small pods with a few seeds and abnormal tip growth of the pollen tubes were observed, suggesting that LjSYP132b has a role in pollen tube growth and nutrient transport in the plasma membrane of seeds., (© The Author(s) 2020. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The mitogen-activated protein kinase (MAPK) LjMPK6 is a phosphorylation target of SIP2, a MAPK kinase that interacts with SymRK (symbiosis receptor-like kinase) for regulation of legume-rhizobia symbiosis. Both LjMPK6 and SIP2 are required for nodulation in Lotus japonicus . However, the dephosphorylation of LjMPK6 and its regulatory components in nodule development remains unexplored. By yeast two-hybrid screening, we identified a type 2C protein phosphatase, LjPP2C, that specifically interacts with and dephosphorylates LjMPK6 in vitro. Physiological and biochemical assays further suggested that LjPP2C phosphatase is required for dephosphorylation of LjMPK6 in vivo and for fine-tuning nodule development after rhizobial inoculation. A non-phosphorylatable mutant variant LjMPK6 (T224A Y226F) could mimic LjPP2C functioning in MAPK dephosphorylation required for nodule development in hairy root transformed plants. Collectively, our study demonstrates that interaction with LjPP2C phosphatase is required for dephosphorylation of LjMPK6 to fine tune nodule development in L. japonicus .

Several Lotus species are perennial forage legumes which tolerate waterlogging, but knowledge of responses to partial or complete shoot submergence is scant. We evaluated the responses of 15 Lotus accessions to partial and complete shoot submergence and variations in traits associated with tolerance and recovery after de-submergence. Accessions of Lotus tenuis, L. corniculatus, L. pedunculatus and L. japonicus were raised for 43 d and then subjected to aerated root zone (control), deoxygenated stagnant root zone with shoots in air (stagnant), stagnant root zone with partial (75 %) and complete submergence of shoots, for 7 d. The recovery ability from complete submergence was also assessed. We found inter- and intra-specific variations in the stem extension responses (i.e. promoted or restricted compared to controls) depending on water depth. Eight of 15 accessions promoted the stem extension when in partial submergence, while three of those eight (all L. tenuis accessions) had a restricted stem extension when under complete submergence. Two accessions (belonging to L. corniculatus and L. penduculatus species) also promoted the stem extension under complete submergence. The accessions that attained better recovery in terms of leaves produced after de-submergence, were those that had high leaf and root sugar concentration at de-submergence, and high thickness and persistence of gas films on leaves during submergence (all L. tenuis accessions). We conclude that all Lotus accessions were able to tolerate 7 d of partial and complete shoot submergence, despite adopting different stem extension responses., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Lotus japonicus is an important model legume for studying symbiotic nitrogen fixation as well as plant development. A genomic sequence of L. japonicus (MG20) has been available for more than ten years. However, the low quality of the genome limits its application in functional genomic studies. Therefore, it is necessary to assemble high-quality chromosome sequences of L. japonicus using new sequencing technology to facilitate the study of functional genomics. In this report, we used the third-generation sequencing combined with the Illumina HiSeq platform to sequence the genome of L. japonicus (MG20). We obtained 544 Mb of genomic sequence using third-generation assembly. Based on sequence analysis, 357 Mb of repeats, 28,251 genes, 626 tRNAs, 1409 rRNAs, and 1233 pseudogenes were predicted in the genome. A total of 27,991 genes were annotated into databases. Compared to the previously published data, the new genome database contains complete L. japonicus sequences in the proper order and orientation with a contig N50 2.81Mb and an excellent genome coverage, which provides more accurate genome information and more precise assembly for functional genomic study.

Key Message: An AP2 family gene CBX1 is involved in mycorrhizal symbiosis and growth of Lotus japonicus. APETALA 2 (AP2) transcriptional regulator is highly conserved in plants. CBX1 from Lotus japonicus is a member of AP2 family. AMF (Arbuscular mycorrhizal fungi) inoculation experiment demonstrated that expression of CBX1 was significantly induced by AMF. Further promoter analysis showed that the - 764 to - 498 bp region of the CBX1 promoter containing CTTC motif is the AMF responsive region. Functional analysis of cbx1 mutant suggested CBX1 is critical for mycorrhizal symbiosis, especially for arbuscule formation. Moreover, under noncolonized condition, overexpression of CBX1 reduced the root length of L. japonicus but increased the size of root system and shoot length, whereas cbx1 mutant reduced the root size and shoot length, but not effect on root length. In addition, cbx1 altered activity of monolignol biosynthetic gene and increased lignin levels. Collectively, these data indicated that CBX1 is a positive regulator of symbiotic activity and plays roles in the growth of L. japonicus.

RNA-Binding Protein 1 (RBP1) was first identified as a protein partner of the long noncoding RNA (lncRNA) ENOD40 in Medicago truncatula , involved in symbiotic nodule development. RBP1 is localized in nuclear speckles and can be relocalized to the cytoplasm by the interaction with ENOD40 . The two closest homologs to RBP1 in Arabidopsis thaliana were called Nuclear Speckle RNA-binding proteins (NSRs) and characterized as alternative splicing modulators of specific mRNAs. They can recognize in vivo the lncRNA ALTERNATIVE SPLICING COMPETITOR ( ASCO ) among other lncRNAs, regulating lateral root formation. Here, we performed a phylogenetic analysis of NSR/RBP proteins tracking the roots of the family to the Embryophytes. Strikingly, eudicots faced a reductive trend of NSR/RBP proteins in comparison with other groups of flowering plants. In Medicago truncatula and Lotus japonicus , their expression profile during nodulation and in specific regions of the symbiotic nodule was compared to that of the lncRNA ENOD40 , as well as to changes in alternative splicing. This hinted at distinct and specific roles of each member during nodulation, likely modulating the population of alternatively spliced transcripts. Our results establish the basis to guide future exploration of NSR/RBP function in alternative splicing regulation in different developmental contexts along the plant lineage., Competing Interests: The authors declare no conflict of interest.

Lotus leaves floating on water usually experience short-wavelength edge wrinkling that decays toward the center, while the leaves growing above water normally morph into a global bending cone shape with long rippled waves near the edge. Observations suggest that the underlying water (liquid substrate) significantly affects the morphogenesis of leaves. To understand the biophysical mechanism under such phenomena, we develop mathematical models that can effectively account for inhomogeneous differential growth of floating and freestanding leaves to quantitatively predict formation and evolution of their morphology. We find, both theoretically and experimentally, that the short-wavelength buckled configuration is energetically favorable for growing membranes lying on liquid, while the global buckling shape is more preferable for suspended ones. Other influencing factors such as the stem or vein, heterogeneity, and dimension are also investigated. Our results provide a fundamental insight into a variety of plant morphogenesis affected by water foundation and suggest that such surface instabilities can be harnessed for morphology control of biomimetic deployable structures using substrate or edge actuation.

Colonization of new habitats is expected to require genetic adaptations to overcome environmental challenges. Here, we use full genome re-sequencing and extensive common garden experiments to investigate demographic and selective processes associated with colonization of Japan by Lotus japonicus over the past ~20,000 years. Based on patterns of genomic variation, we infer the details of the colonization process where L. japonicus gradually spread from subtropical conditions to much colder climates in northern Japan. We identify genomic regions with extreme genetic differentiation between northern and southern subpopulations and perform population structure-corrected association mapping of phenotypic traits measured in a common garden. Comparing the results of these analyses, we find that signatures of extreme subpopulation differentiation overlap strongly with phenotype association signals for overwintering and flowering time traits. Our results provide evidence that these traits were direct targets of selection during colonization and point to associated candidate genes.

The rhizobial type III secretion system secretes effector proteins into host plant cells, which may either promote or inhibit symbiosis with legumes. We herein demonstrated that the type III secretion system of Bradyrhizobium sp. SUTN9-2 obstructed symbiosis with Lotus japonicus Miyakojima, L. japonicus Gifu, and Lotus burttii. A mutant of SUTN9-2 that is unable to secrete effector proteins showed better nodulation and plant growth promotion than wild-type SUTN9-2 when paired with these Lotus spp. We propose that SUTN9-2 is a useful strain for understanding the mechanisms by which effector proteins obstruct symbiosis between Bradyrhizobium and Lotus spp.

LAZY1 family genes play important roles in both shoot and root gravitropism in plants. Here we report a Lotus japonicus mutant that displays negative gravitropic response in primary and lateral roots. Map-based cloning identified the mutant gene LAZY3 as a functional ortholog of the LAZY1 gene. Mutation of the LAZY3 gene reduced rootward polar auxin transport (PAT) in the primary root, which was also insensitive to the PAT inhibitor N-1-naphthylphthalamic acid. Moreover, immunolocalization of enhanced green fluorescent protein-tagged LAZY3 in L. japonicus exhibited polar localization of LAZY3 on the plasma membrane in root stele cells. We therefore suggest that the polar localization of LAZY3 in stele cells might be required for PAT in L. japonicus root. LAZY3 transcripts displayed asymmetric distribution at the root tip within hours of gravistimulation, while overexpression of LAZY3 under a constitutive promoter in lazy3 plants rescued the gravitropic response in roots. These data indicate that root gravitropism depends on the presence of LAZY3 but not on its asymmetric expression in root tips. Expression of other LAZY genes in a lazy3 background did not rescue the growth direction of roots, suggesting that the LAZY3 gene plays a distinct role in root gravitropism in L. japonicus., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Food demands of increasing human population dictate intensification of livestock production, however, environmental stresses could jeopardize producers' efforts. Forage legumes suffer from yield losses and poor nutritional status due to salinity increase of agricultural soils. As tools aimed to reduce negative impacts of biotic or abiotic stresses, proteinase inhibitors (PIs) have been promoted for biotechnological improvements. In order to increase tolerance of Lotus corniculatus L. to salt stress, serine PI, BvSTI, was introduced into this legume using Agrobacterium rhizogenes, with final transformation efficiency of 4.57%. PCR, DNA gel-blot, RT-PCR and in-gel protein activity assays confirmed the presence and activity of BvSTI products in transformed lines. Plants from three selected transgenic lines (21, 73 and 109) showed significant alterations in overall phenotypic appearance, corresponding to differences in BvSTI accumulation. Lines 73 and 109 showed up to 7.3-fold higher number of tillers and massive, up to 5.8-fold heavier roots than in nontransformed controls (NTC). Line 21 was phenotypically similar to NTC, accumulated less BvSTI transcripts and did not exhibit an additional band of recombinant trypsin inhibitor as seen in lines 73 and 109. Exposure of the transgenic lines to NaCl revealed different levels of salt stress susceptibility. The NaCl sensitivity index, based on morphological appearance and chlorophyll concentrations showed that lines 73 and 109 were significantly less affected by salinity than NTC or line 21. High level of BvSTI altered morphology and delayed salt stress related senescence, implicating BvSTI gene as a promising tool for salinity tolerance improvement trials in L. corniculatus., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Legumes develop root nodules in symbiosis with nitrogen-fixing rhizobial bacteria. Rhizobia evoke cell division of differentiated cortical cells into root nodule primordia for accommodating bacterial symbionts. In this study, we show that NODULE INCEPTION (NIN), a transcription factor in Lotus japonicus that is essential for initiating cortical cell divisions during nodulation, regulates the gene ASYMMETRIC LEAVES 2-LIKE 18/LATERAL ORGAN BOUNDARIES DOMAIN 16a ( ASL18 / LBD16a ) . Orthologs of ASL18 / LBD16a in nonlegume plants are required for lateral root development. Coexpression of ASL18a and the CCAAT box-binding protein Nuclear Factor-Y ( NF-Y ) subunits, which are also directly targeted by NIN, partially suppressed the nodulation-defective phenotype of L. japonicus daphne mutants, in which cortical expression of NIN was attenuated. Our results demonstrate that ASL18a and NF-Y together regulate nodule organogenesis. Thus, a lateral root developmental pathway is incorporated downstream of NIN to drive nodule symbiosis., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

During the legume-rhizobium symbiotic interaction, rhizobial invasion of legumes is primarily mediated by a plant-made tubular invagination called an infection thread (IT). Here, we identify a gene in Lotus japonicus encoding a Leu-rich repeat receptor-like kinase (LRR-RLK), RINRK1 ( Rhizobial Infection Receptor-like Kinase1 ), that is induced by Nod factors (NFs) and is involved in IT formation but not nodule organogenesis. A paralog, RINRK2, plays a relatively minor role in infection. RINRK1 is required for full induction of early infection genes, including Nodule Inception ( NIN ), encoding an essential nodulation transcription factor. RINRK1 displayed an infection-specific expression pattern, and NIN bound to the RINRK1 promoter, inducing its expression. RINRK1 was found to be an atypical kinase localized to the plasma membrane and did not require kinase activity for rhizobial infection. We propose RINRK1 is an infection-specific RLK, which may specifically coordinate output from NF signaling or perceive an unknown signal required for rhizobial infection., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Most land plants establish mutualistic interactions with arbuscular mycorrhizal (AM) fungi. Intracellular accommodation of AM fungal symbionts remodels important host traits like root morphology and nutrient acquisition. How mycorrhizal colonization impacts plant microbiota is unclear. To understand the impact of AM symbiosis on fungal microbiota, ten Lotus japonicus mutants impaired at different stages of AM formation were grown in non-sterile natural soil and their root-associated fungal communities were studied. Plant mutants lacking the capacity to form mature arbuscules (arb - ) exhibited limited growth performance associated with altered phosphorus (P) acquisition and reduction-oxidation (redox) processes. Furthermore, arb - plants assembled moderately but consistently different root-associated fungal microbiota, characterized by the depletion of Glomeromycota and the concomitant enrichment of Ascomycota, including Dactylonectria torresensis. Single and co-inoculation experiments showed a strong reduction of root colonization by D. torresensis in the presence of AM fungus Rhizophagus irregularis, particularly in arbuscule-forming plants. Our results suggest that impairment of central symbiotic functions in AM host plants leads to specific changes in root microbiomes and in tripartite interactions between the host plant, AM and non-AM fungi. This lays the foundation for mechanistic studies on microbe-microbe and microbe-host interactions in AM symbiosis of the model L. japonicus., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

The establishment of nitrogen-fixing root nodules in legume-rhizobia symbiosis requires an intricate communication between the host plant and its symbiont. We are, however, limited in our understanding of the symbiosis signaling process. In particular, how membrane-localized receptors of legumes activate signal transduction following perception of rhizobial signaling molecules has mostly remained elusive. To address this, we performed a coimmunoprecipitation-based proteomics screen to identify proteins associated with Nod factor receptor 5 (NFR5) in Lotus japonicus. Out of 51 NFR5-associated proteins, we focused on a receptor-like cytoplasmic kinase (RLCK), which we named NFR5-interacting cytoplasmic kinase 4 (NiCK4). NiCK4 associates with heterologously expressed NFR5 in Nicotiana benthamiana , and directly binds and phosphorylates the cytoplasmic domains of NFR5 and NFR1 in vitro. At the cellular level, Nick4 is coexpressed with Nfr5 in root hairs and nodule cells, and the NiCK4 protein relocates to the nucleus in an NFR5/NFR1-dependent manner upon Nod factor treatment. Phenotyping of retrotransposon insertion mutants revealed that NiCK4 promotes nodule organogenesis. Together, these results suggest that the identified RLCK, NiCK4, acts as a component of the Nod factor signaling pathway downstream of NFR5., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

MicroRNA (miRNA)-regulated gene expression plays an important role in various plant metabolic processes. Although adventitious roots are critical to plant growth in lotus, the role of miRNA in AR formation remains unclear. Expression profiling of miRNAs was carried out during three different developmental stages of ARs in lotus: no induction of AR stage, initial stage of ARs, and maximum number of ARs. These data are referenced with the whole lotus genome as already identified through high-throughput tag-sequencing. 1.3 × 10 7 tags were achieved, of which 11,035,798, 11,436,062, and 12,542,392 clean tags were obtained from each stage, respectively. miRNA analysis revealed that miRNAs were less than 10% among all small RNAs. In total, 310 miRNAs (240 up-regulated and 70 down-regulated miRNAs) exhibited expression changes from the no induction stage to the initial stage. Moreover, expression of 140 miRNAs was increased and that of 123 miRNAs was decreased between the initial and maximum AR stages, mostly by ~ - 4-4-fold. miRNAs involved in metabolic pathways differed between the initial stage/no induction stage and the maximum number stage/initial stage. Several miRNAs in the initial stage/no induction stage were related to plant hormone metabolism and pyruvate and MAPK pathways, while major miRNAs in the maximum number stage/initial stage were involved in carbohydrate metabolism. All differentially expressed miRNAs associated with AR formation from the initial stage to maximum stage were also analyzed. The expression of 16 miRNAs was determined using qRT-PCR. This work provides a general insight into miRNA regulation during AR formation in lotus.

Salinity is one of the most frequent limiting conditions in pasture production for grazing livestock. Legumes, such as Lotus spp. with high forage quality and capable of adapting to different environments, improves pasture performance in restrictive areas. In order to determine potential cultivars with better forage traits, the current study assess the response to salt stress of L. tenuis, L. corniculatus and a novel L. tenuis x L. corniculatus accession. For this purpose, chlorophyll fluorescence, biomass production, ion accumulation and anthocyanins and proanthocyanidins levels have been evaluated in control and salt-treated plants PSII activity was affected by salt in L. tenuis, but not in L. corniculatus or hybrid plants. Analyzed accessions showed similar values of biomass, Na + and K + levels after salt treatment. Increasing Cl - concentrations were observed in all accessions. However, hybrid plants accumulate Cl - in stems at higher levels than their parental. At the same time, the levels of anthocyanins considerably increased in L. tenuis x L. corniculatus stems. Chloride and anthocyanin accumulation in stems could explain the best performance of hybrid plants after a long saline treatment. Finally, as proanthocyanidins levels were no affected by salt, L. tenuis x L. corniculatus plants maintained adequate levels to be used as ruminant feed. In conclusion, these results suggest that hybrid plants have a high potential to be used as forage on salt-affected lands. High Cl - and anthocyanins accumulation in Lotus spp. stems seems to be a trait associated to salinity tolerance, with the possibility of being used in legume breeding programs., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Petroleum contamination and its remediation via plant-based solutions have got increasing attention by environmental scientists and engineers. In the current study, the physiological and growth responses of two diesel-tolerant plant species (tolerance limit: 1500-2000 mg/kg), Italian ryegrass (Lolium multiflorum) and Birdsfoot trefoil (Lotus corniculatus), have been investigated in vegetable oil- and diesel oil-amended soils. A long-term (147-day) greenhouse pot experiment was conducted to differentiate the main focus of the study: physical and chemical effects of oil (vegetable and diesel) in freshly spiked soils via evaluating the plant performance and hydrocarbon degradation. Moreover, plant performance was evaluated in terms of seed germination, plant shoot biomass, physiological parameters, and root biomass. Addition of both diesel oil and vegetable oil in freshly spiked soils showed deleterious effects on seedling emergence, root/shoot biomass, and chlorophyll content of grass and legume plants. Italian ryegrass showed more sensitivity in terms of germination rate to both vegetable and diesel oil as compared to non-contaminated soils while Birdsfoot trefoil reduced the germination rate only in diesel oil-impacted soils. The results of the current study suggest that both physical and chemical effects of oil pose negative effects of plant growth and root development. This observation may explain the phenomenon of reduced plant growth in aged/weathered contaminated soils during rhizoremediation experiments.

Nitrogen-fixing rhizobia and arbuscular mycorrhizal fungi (AMF) form symbioses with plant roots and these are established by precise regulation of symbiont accommodation within host plant cells. In model legumes such as Lotus japonicus and Medicago truncatula, rhizobia enter into roots through an intracellular invasion system that depends on the formation of a root-hair infection thread (IT). While IT-mediated intracellular rhizobia invasion is thought to be the most evolutionarily derived invasion system, some studies have indicated that a basal intercellular invasion system can replace it when some nodulation-related factors are genetically modified. In addition, intracellular rhizobia accommodation is suggested to have a similar mechanism as AMF accommodation. Nevertheless, our understanding of the underlying genetic mechanisms is incomplete. Here we identify a L. japonicus nodulation-deficient mutant, with a mutation in the LACK OF SYMBIONT ACCOMMODATION (LAN) gene, in which root-hair IT formation is strongly reduced, but intercellular rhizobial invasion eventually results in functional nodule formation. LjLAN encodes a protein that is homologous to Arabidopsis MEDIATOR 2/29/32 possibly acting as a subunit of a Mediator complex, a multiprotein complex required for gene transcription. We also show that LjLAN acts in parallel with a signaling pathway including LjCYCLOPS. In addition, the lan mutation drastically reduces the colonization levels of AMF. Taken together, our data provide a new factor that has a common role in symbiont accommodation process during root nodule and AM symbiosis., Competing Interests: The authors have declared that no competing interests exist.

The NOOT-BOP-COCH-LIKE (NBCL) genes are orthologs of Arabidopsis thaliana BLADE-ON-PETIOLE1/2. The NBCLs are developmental regulators essential for plant shaping, mainly through the regulation of organ boundaries, the promotion of lateral organ differentiation and the acquisition of organ identity. In addition to their roles in leaf, stipule and flower development, NBCLs are required for maintaining the identity of indeterminate nitrogen-fixing nodules with persistent meristems in legumes. In legumes forming determinate nodules, without persistent meristem, the roles of NBCL genes are not known. We thus investigated the role of Lotus japonicus NOOT-BOP-COCH-LIKE1 (LjNBCL1) in determinate nodule identity and studied its functions in aerial organ development using LORE1 insertional mutants and RNA interference-mediated silencing approaches. In Lotus, LjNBCL1 is involved in leaf patterning and participates in the regulation of axillary outgrowth. Wild-type Lotus leaves are composed of five leaflets and possess a pair of nectaries at the leaf axil. Legumes such as pea and Medicago have a pair of stipules, rather than nectaries, at the base of their leaves. In Ljnbcl1, nectary development is abolished, demonstrating that nectaries and stipules share a common evolutionary origin. In addition, ectopic roots arising from nodule vascular meristems and reorganization of the nodule vascular bundle vessels were observed on Ljnbcl1 nodules. This demonstrates that NBCL functions are conserved in both indeterminate and determinate nodules through the maintenance of nodule vascular bundle identity. In contrast to its role in floral patterning described in other plants, LjNBCL1 appears essential for the development of both secondary inflorescence meristem and floral meristem., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

A quarter of a century has passed since Lotus japonicus was proposed as a model legume because of its suitability for molecular genetic studies. Since then, a comprehensive set of genetic resources and tools has been developed, including recombinant inbred lines, a collection of wild accessions, published mutant lines, a large collection of mutant lines tagged with LORE1 insertions, cDNA clones with expressed sequence tag (EST) information, genomic clones with end-sequence information, and a reference genome sequence. Resource centers in Japan and Denmark ensure easy access to data and materials, and the resources have greatly facilitated L. japonicus research, thereby contributing to the molecular understanding of characteristic legume features such as endosymbiosis. Here, we provide detailed instructions for L. japonicus cultivation and describe how to order materials and access data using the resource center websites. The comprehensive overview presented here will make L. japonicus more easily accessible as a model system, especially for research groups new to L. japonicus research. © 2018 by John Wiley & Sons, Inc., (Copyright © 2018 John Wiley & Sons, Inc.)

Main Conclusion: The nitrate transporters, belonging to NPF and NRT2 families, play critical roles in nitrate signaling, root growth and nodule development in legumes. Nitrate plays an essential role during plant development as nutrient and also as signal molecule, in both cases working via the activity of nitrate transporters. To date, few studies on NRT2 or NPF nitrate transporters in legumes have been reported, and most of those concern Lotus japonicus and Medicago truncatula. A molecular characterization led to the identification of 4 putative LjNRT2 and 37 putative LjNPF gene sequences in L. japonicus. In M. truncatula, the NRT2 family is composed of 3 putative members. Using the new genome annotation of M. truncatula (Mt4.0), we identified, for this review, 97 putative MtNPF sequences, including 32 new sequences relative to previous studies. Functional characterization has been published for only two MtNPF genes, encoding nitrate transporters of M. truncatula. Both transporters have a role in root system development via abscisic acid signaling: MtNPF6.8 acts as a nitrate sensor during the cell elongation of the primary root, while MtNPF1.7 contributes to the cellular organization of the root tip and nodule formation. An in silico expression study of MtNPF genes confirmed that NPF genes are expressed in nodules, as previously shown for L. japonicus, suggesting a role for the corresponding proteins in nitrate transport, or signal perception in nodules. This review summarizes our knowledge of legume nitrate transporters and discusses new roles for these proteins based on recent discoveries.

Rhizobial bacteria are known for their capacity to fix nitrogen for legume hosts. However ineffective rhizobial genotypes exist and can trigger the formation of nodules but fix little if any nitrogen for hosts. Legumes must employ mechanisms to minimize exploitation by the ineffective rhizobial genotypes to limit fitness costs and stabilize the symbiosis. Here we address two key questions about these host mechanisms. What stages of the interaction are controlled by the host, and can hosts detect subtle differences in nitrogen fixation? We provide the first explicit evidence for adaptive host control in the interaction between Lotus japonicus and Mesorhizobium loti. In both single inoculation and co-inoculation experiments, less effective rhizobial strains exhibited reduced in planta fitness relative to the wildtype M. loti. We uncovered evidence of host control during nodule formation and during post-infection proliferation of symbionts within nodules. We found a linear relationship between rhizobial fitness and symbiotic effectiveness. Our results suggest that L. japonicus can adaptively modulate the fitness of symbionts as a continuous response to symbiotic nitrogen fixation.

Legume mutants have shown the requirement for receptor-mediated cytokinin signaling in symbiotic nodule organogenesis. While the receptors are central regulators, cytokinin also is accumulated during early phases of symbiotic interaction, but the pathways involved have not yet been fully resolved. To identify the source, timing, and effect of this accumulation, we followed transcript levels of the cytokinin biosynthetic pathway genes in a sliding developmental zone of Lotus japonicus roots. LjIpt2 and LjLog4 were identified as the major contributors to the first cytokinin burst. The genetic dependence and Nod factor responsiveness of these genes confirm that cytokinin biosynthesis is a key target of the common symbiosis pathway. The accumulation of LjIpt2 and LjLog4 transcripts occurs independent of the LjLhk1 receptor during nodulation. Together with the rapid repression of both genes by cytokinin, this indicates that LjIpt2 and LjLog4 contribute to, rather than respond to, the initial cytokinin buildup. Analysis of the cytokinin response using the synthetic cytokinin sensor, TCSn , showed that this response occurs in cortical cells before spreading to the epidermis in L. japonicus While mutant analysis identified redundancy in several biosynthesis families, we found that mutation of LjIpt4 limits nodule numbers. Overexpression of LjIpt3 or LjLog4 alone was insufficient to produce the robust formation of spontaneous nodules. In contrast, overexpressing a complete cytokinin biosynthesis pathway leads to large, often fused spontaneous nodules. These results show the importance of cytokinin biosynthesis in initiating and balancing the requirement for cortical cell activation without uncontrolled cell proliferation., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Many endophytic bacteria exert beneficial effects on their host, but still little is known about the bacteria associated with plants growing in areas heavily polluted by hydrocarbons. The aim of the study was characterization of culturable hydrocarbon-degrading endophytic bacteria associated with Lotus corniculatus L. and Oenothera biennis L. collected in long-term petroleum hydrocarbon-polluted site using culture-dependent and molecular approaches. A total of 26 hydrocarbon-degrading endophytes from these plants were isolated. Phylogenetic analyses classified the isolates into the phyla Proteobacteria and Actinobacteria. The majority of strains belonged to the genera Rhizobium, Pseudomonas, Stenotrophomonas, and Rhodococcus. More than 90% of the isolates could grow on medium with diesel oil, approximately 20% could use n-hexadecane as a sole carbon and energy source. PCR analysis revealed that 40% of the isolates possessed the P450 gene encoding for cytochrome P450-type alkane hydroxylase (CYP153). In in vitro tests, all endophytic strains demonstrated a wide range of plant growth-promoting traits such as production of indole-3-acetic acid, hydrogen cyanide, siderophores, and phosphate solubilization. More than 40% of the bacteria carried the gene encoding for the 1-aminocyclopropane-1-carboxylic acid deaminase (acdS). Our study shows that the diversity of endophytic bacterial communities in tested plants was different. The results revealed also that the investigated plants were colonized by endophytic bacteria possessing plant growth-promoting features and a clear potential to degrade hydrocarbons. The properties of isolated endophytes indicate that they have the high potential to improve phytoremediation of petroleum hydrocarbon-polluted soils.

Plants contain various factors that transiently interact with subunits or intermediates of the thylakoid multiprotein complexes, promoting their stable association and integration. Hence, assembly factors are essential for chloroplast development and the transition from heterotrophic to phototrophic growth. Snowy cotyledon 2 (SCO2) is a DNAJ-like protein involved in thylakoid membrane biogenesis and interacts with the light-harvesting chlorophyll-binding protein LHCB1. In Arabidopsis thaliana, SCO2 function was previously reported to be restricted to cotyledons. Here we show that disruption of SCO2 in Lotus japonicus results not only in paler cotyledons but also in variegated true leaves. Furthermore, smaller and pale-green true leaves can also be observed in A. thaliana sco2 (atsco2) mutants under short-day conditions. In both species, SCO2 is required for proper accumulation of PSII-LHCII complexes. In contrast to other variegated mutants, inhibition of chloroplastic translation strongly affects L. japonicus sco2 mutant development and fails to suppress their variegated phenotype. Moreover, inactivation of the suppressor of variegation AtClpR1 in the atsco2 background results in an additive double-mutant phenotype with variegated true leaves. Taken together, our results indicate that SCO2 plays a distinct role in PSII assembly or repair and constitutes a novel factor involved in leaf variegation., (Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.)

Leghemoglobins transport and deliver O2 to the symbiosomes inside legume nodules and are essential for nitrogen fixation. However, the roles of other hemoglobins (Hbs) in the rhizobia-legume symbiosis are unclear. Several Lotus japonicus mutants affecting LjGlb1-1, a non-symbiotic class 1 Hb, have been used to study the function of this protein in symbiosis. Two TILLING alleles with single amino acid substitutions (A102V and E127K) and a LORE1 null allele with a retrotransposon insertion in the 5'-untranslated region (96642) were selected for phenotyping nodulation. Plants of all three mutant lines showed a decrease in long infection threads and nodules, and an increase in incipient infection threads. About 4h after inoculation, the roots of mutant plants exhibited a greater transient accumulation of nitric oxide (NO) than did the wild-type roots; nevertheless, in vitro NO dioxygenase activities of the wild-type, A102V, and E127K proteins were similar, suggesting that the mutated proteins are not fully functional in vivo The expression of LjGlb1-1, but not of the other class 1 Hb of L. japonicus (LjGlb1-2), was affected during infection of wild-type roots, further supporting a specific role for LjGlb1-1. In conclusion, the LjGlb1-1 mutants reveal that this protein is required during rhizobial infection and regulates NO levels., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

In Papilionoideae legume, Lotus japonicus, the development of dorsal-ventral (DV) asymmetric flowers is mainly controlled by two TB1/CYCLOIDEA/PCF (TCP) genes, SQUARED STANDARD (SQU) and KEELED WINGS IN LOTUS (KEW), which determine dorsal and lateral identities, respectively. However, the molecular basis of how these two highly homologous genes orchestrate their diverse functions remains unclear. Here, we analyzed their expression levels, and investigated the transcriptional activities of SQU and KEW. We demonstrated that SQU possesses both activation and repression activities, while KEW acts only as an activator. They form homo- and heterodimers, and then collaboratively regulate their expression at the transcription level. Furthermore, we identified two types of post-transcriptional modifications, phosphorylation and ATP/GTP binding, both of which could affect their transcriptional activities. Mutations in ATP/GTP binding motifs of SQU and KEW lead to failure of phosphorylation, and transgenic plants bearing the mutant proteins display defective DV asymmetric flower development, indicating that the two conjugate modifications are essential for their diverse functions. Altogether, SQU and KEW activities are precisely modulated at both transcription and post-transcription levels, which might link DV asymmetric flower development to different physiological status and/or signaling pathways., (Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.)