Secondary metabolite profiling of the model legume Lotus japonicus during its symbiotic interaction with Mesorhizobium loti

Symbiotic nitrogen fixation in legumes takes place in specialized organs called nodules,which become the main source of assimilated nitrogen for the whole plant. Symbiotic nitro‐gen fixation requires exquisite integration of plant and bacterial metabolism and involvesglobal changes in gene expression and metabolite accumulation in both rhizobia and thehost plant. In order to study the metabolic changes mediated by symbiotic nitrogen fixationon a whole‐plant level, metabolite levels were profiled by gas chromatography–mass spec‐trometry in nodules and non‐symbiotic organs of Lotus japonicus plants uninoculated or in‐oculated with M. loti wt, ΔnifA or ΔnifH fix‐ strains. Furthermore, transcriptomic andbiochemical approaches were combined to study sulfur metabolism in nodules, its link tosymbiotic nitrogen fixation, and the effect of nodules on whole‐plant sulfur partitioning andmetabolism. It is well established that nitrogen and sulfur (S) metabolism are tightly en‐twined and sulfur is required for symbiotic nitrogen fixation, however, little is known aboutthe molecular and biochemical mechanisms governing sulfur uptake and assimilation duringsymbiotic nitrogen fixation. Transcript profiling in Lotus japonicus was combined with quan‐tification of S‐metabolite contents and APR activity in nodules and in non‐symbiotic organsof plants uninoculated or inoculated with M. loti wt, ΔnifA or ΔnifH fix‐ strains. Moreover,sulfate uptake and its distribution into different plant organs were analyzed and 35S‐flux intodifferent S‐pools was monitored. Metabolite profiling revealed that symbiotic nitrogen fixa‐tion results in dramatic changes of many aspects of primary and secondary metabolism innodules which leads to global reprogramming of metabolism of the model legume on awhole‐plant level. Moreover, our data revealed that nitrogen fixing nodules represent athiol‐rich organ. Their high APR activity and 35S‐flux into cysteine and its metabolites in com‐bination with the transcriptional up‐regulation of several genes involved in sulfur assimila‐tion highlight the function of nodules as a new site of sulfur assimilation. The higher thiolcontent observed in non‐symbiotic organs of nitrogen fixing plants in comparison touninoculated plants cannot be attributed to local biosynthesis, indicating that nodules couldserve as a novel source of reduced sulfur for the plant, which triggers whole‐plant repro‐gramming of sulfur metabolism. Interestingly, the changes in metabolite profiling and theenhanced thiol biosynthesis in nodules and their impact on the whole‐plant sulfur, carbonand nitrogen economy are dampened in fix‐ plants, which in most respects metabolically re‐sembled uninoculated plants, indicating a strong interaction between nitrogen fixation andsulfur and carbon metabolism.

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Whole-Genome Sequence of the Nitrogen-Fixing Symbiotic Rhizobium Mesorhizobium loti Strain TONO

Genome Announcements ◽

10.1128/genomea.01016-16 ◽

2016 ◽

Vol 4 (5) ◽

Cited By ~ 5

Author(s):

Yoshikazu Shimoda ◽

Hideki Hirakawa ◽

Shusei Sato ◽

Kazuhiko Saeki ◽

Makoto Hayashi

Keyword(s):

Comparative Genomics ◽

Genome Sequence ◽

Lotus Japonicus ◽

Whole Genome Sequence ◽

Whole Genome ◽

Nitrogen Fixing ◽

Model Legume ◽

Mesorhizobium Loti ◽

Symbiotic Properties

Mesorhizobium loti is the nitrogen-fixing microsymbiont for legumes of the genus Lotus . Here, we report the whole-genome sequence of a Mesorhizobium loti strain, TONO, which is used as a symbiont for the model legume Lotus japonicus . The whole-genome sequence of the strain TONO will be a solid platform for comparative genomics analyses and for the identification of genes responsible for the symbiotic properties of Mesorhizobium species.

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Expression Islands Clustered on the Symbiosis Island of the Mesorhizobium loti Genome

Journal of Bacteriology ◽

10.1128/jb.186.8.2439-2448.2004 ◽

2004 ◽

Vol 186 (8) ◽

pp. 2439-2448 ◽

Cited By ~ 162

Author(s):

Toshiki Uchiumi ◽

Takuji Ohwada ◽

Manabu Itakura ◽

Hisayuki Mitsui ◽

Noriyuki Nukui ◽

...

Keyword(s):

Acid Metabolism ◽

Symbiotic Nitrogen Fixation ◽

Transcriptional Profiling ◽

Lotus Japonicus ◽

Model Legume ◽

Mesorhizobium Loti ◽

Genome Region ◽

Transcriptional Dynamics ◽

Rhizobial Symbiosis ◽

Highly Expressed Genes

ABSTRACT Rhizobia are symbiotic nitrogen-fixing soil bacteria that are associated with host legumes. The establishment of rhizobial symbiosis requires signal exchanges between partners in microaerobic environments that result in mutualism for the two partners. We developed a macroarray for Mesorhizobium loti MAFF303099, a microsymbiont of the model legume Lotus japonicus, and monitored the transcriptional dynamics of the bacterium during symbiosis, microaerobiosis, and starvation. Global transcriptional profiling demonstrated that the clusters of genes within the symbiosis island (611 kb), a transmissible region distinct from other chromosomal regions, are collectively expressed during symbiosis, whereas genes outside the island are downregulated. This finding implies that the huge symbiosis island functions as clustered expression islands to support symbiotic nitrogen fixation. Interestingly, most transposase genes on the symbiosis island were highly upregulated in bacteroids, as were nif, fix, fdx, and rpoN. The genome region containing the fixNOPQ genes outside the symbiosis island was markedly upregulated as another expression island under both microaerobic and symbiotic conditions. The symbiosis profiling data suggested that there was activation of amino acid metabolism, as well as nif-fix gene expression. In contrast, genes for cell wall synthesis, cell division, DNA replication, and flagella were strongly repressed in differentiated bacteroids. A highly upregulated gene in bacteroids, mlr5932 (encoding 1-aminocyclopropane-1-carboxylate deaminase), was disrupted and was confirmed to be involved in nodulation enhancement, indicating that disruption of highly expressed genes is a useful strategy for exploring novel gene functions in symbiosis.

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Responses of a Model Legume Lotus japonicus to Lipochitin Oligosaccharide Nodulation Factors Purified from Mesorhizobium loti JRL501

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2001.14.7.848 ◽

2001 ◽

Vol 14 (7) ◽

pp. 848-856 ◽

Cited By ~ 52

Author(s):

Shinobu Niwa ◽

Masayoshi Kawaguchi ◽

Haruko Imaizumi-Anraku ◽

Svetlana A. Chechetka ◽

Masumi Ishizaka ◽

...

Keyword(s):

High Performance ◽

Molecular Genetic ◽

Lotus Japonicus ◽

Cytoplasmic Bridge ◽

Cortical Cells ◽

Model Legume ◽

Mesorhizobium Loti ◽

Root Hair Deformation ◽

Early Nodulin ◽

Microbe Interactions

Lotus japonicus has been proposed as a model legume for molecular genetic studies of symbiotic plant-microbe interactions leading to the fixation of atmospheric nitrogen. Lipochitin oligosaccharides (LCOs), or Nod factors, were isolated from the culture of Mesorhizobium loti strain JRL501 (MAFF303099), an efficient microsymbiont of L. japonicus B-129 cv. Gifu. High-performance liquid chromatography and mass spectrometric analyses allowed us to identify at least five different structures of LCOs that were produced by JRL501. The major component was NodMl-V(C18:1, Me, Cb, AcFuc), an N-acetyl-glucosamine pentamer in which the nonreducing residue is N-acylated with a C18:1 acyl moiety, N-methylated, and carries a carbamoyl group and the reducing N-acetyl-glucosamine residue is substituted with 4-O-acetyl-fucose. Additional novel LCO structures bearing fucose instead of acetyl-fucose at the reducing end were identified. Mixtures of these LCOs could elicit abundant root hair deformation on L. japonicus roots at a concentration of 10-7 to 10-9 M. Spot inoculation of a few nanograms of LCOs on L. japonicus roots induced the formation of nodule primordia in which the early nodulin genes, ENOD40 and ENOD2, were expressed in a tissue-specific manner. We also observed the formation of a cytoplasmic bridge (preinfection thread) in the swollen outermost cortical cells. This is the first description of cytoplasmic bridge formation by purified LCOs alone in a legume-forming determinate nodules.

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The Lotus japonicus AFB6 Gene Is Involved in the Auxin Dependent Root Developmental Program

International Journal of Molecular Sciences ◽

10.3390/ijms22168495 ◽

2021 ◽

Vol 22 (16) ◽

pp. 8495

Author(s):

Alessandra Rogato ◽

Vladimir Totev Valkov ◽

Marcin Nadzieja ◽

Jens Stougaard ◽

Maurizio Chiurazzi

Keyword(s):

Lateral Root ◽

Target Genes ◽

Lateral Roots ◽

Lotus Japonicus ◽

Auxin Receptor ◽

Symbiotic Interaction ◽

Mesorhizobium Loti ◽

Preferential Expression ◽

Elongation Process ◽

Primary And Lateral Roots

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 N2-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.

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Improved procedures for in vitro regeneration and for phenotypic analysis in the model legume Lotus japonicus

Functional Plant Biology ◽

10.1071/fp05015 ◽

2005 ◽

Vol 32 (6) ◽

pp. 529 ◽

Cited By ~ 10

Author(s):

Ani Barbulova ◽

Enrica D'Apuzzo ◽

Alessandra Rogato ◽

Maurizio Chiurazzi

Keyword(s):

In Vitro Regeneration ◽

Lotus Japonicus ◽

Shoot Formation ◽

Direct Somatic Embryogenesis ◽

Phenotypic Analysis ◽

Indirect Organogenesis ◽

Model Legume ◽

Mesorhizobium Loti ◽

Efficient Gene

As a prerequisite for the development of an efficient gene transfer methodology, the possibility of inducing direct somatic embryogenesis in Lotus japonicus (Regel) K. Larsen explants was investigated. Petiole bases, cotyledons, hypocotyls and stem segments were cultivated in the presence of different amounts of benzylaminopurine (BAP) and / or thidiazuron (TDZ). Regeneration was achieved differentially in the different explants and a higher efficiency of shoot formation was obtained with TDZ. By maintaining the same TDZ regime a second cycle of morphogenesis was achieved and the histological analysis of these structures indicated unambiguously their somatic embryogenic nature. Thidiazuron was also tested as an agent to improve the kinetics of shoot formation in a Lotus japonicus transformation–regeneration procedure based on indirect organogenesis. A very significant, highly reproducible, increase in the rate of the shoot formation was observed in independent transformation experiments. We also present an extensive analysis of the feasibility and reproducibility of an in vitro procedure, which can be very useful for the screening of symbiotic phenotypes in transgenic Lotus plants and for the analysis of the cascade of molecular and cytological events occurring soon after Mesorhizobium loti infection.

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A Novel Fix¯ Symbiotic Mutant of Lotus japonicus, Ljsym105, Shows Impaired Development and Premature Deterioration of Nodule Infected Cells and Symbiosomes

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-19-0780 ◽

2006 ◽

Vol 19 (7) ◽

pp. 780-788 ◽

Cited By ~ 17

Author(s):

Md. Shakhawat Hossain ◽

Yosuke Umehara ◽

Hiroshi Kouchi

Keyword(s):

Nitrogen Deficiency ◽

Lotus Japonicus ◽

Nodule Development ◽

Acetylene Reduction Activity ◽

Nitrogen Fixing ◽

Wild Type ◽

Electron Microscopic ◽

Model Legume ◽

Mesorhizobium Loti ◽

Infected Cells

Nitrogen-fixing symbiosis between legume plants and rhizobia is established through complex interactions between two symbiotic partners. To identify the host legume genes that play crucial roles in such interactions, we isolated a novel Fix¯ mutant, Ljsym105, from a model legume Lotus japonicus MG-20. The Ljsym105 plants displayed nitrogen-deficiency symptoms after inoculation with Mesorhizobium loti under nitrogen-free conditions, but their growth recovered when supplied with nitrogen-rich nutrients. Ljsym105 was recessive and monogenic and mapped on the upper portion of chromosome 4. The mutant Ljsym105 formed an increased number of small and pale-pink nodules. Nitrogenase (acetylene reduction) activity per nodule fresh weight was low but retained more than 50% of that of the wild-type nodules. Light and electron microscopic observations revealed that the Ljsym105 nodule infected cells were significantly smaller than those of wild-type plants, contained enlarged symbiosomes with multiple bacteroids, and underwent deterioration of the symbiosomes prematurely as well as disintegration of the whole infected cell cytoplasm. These results indicate that the ineffectiveness of the Ljsym105 nodules is primarily due to impaired growth of infected cells accompanied with the premature senescence induced at relatively early stages of nodule development. These symbiotic phenotypes are discussed in respect to possible functions of the LjSym105 locus in the symbiotic interactions required for establishment of the nitrogen-fixing symbiosis.

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The nodD1 gene of Sinorhizobium fredii HH103 Restores Nodulation Capacity on Bean in a Rhizobium tropici CIAT 899 nodD1/nodD2 Mutant, but the Secondary Symbiotic Regulators nolR, nodD2 or syrM Prevent HH103 to Nodulate with This Legume

Microorganisms ◽

10.3390/microorganisms10010139 ◽

2022 ◽

Vol 10 (1) ◽

pp. 139

Author(s):

Francisco Fuentes-Romero ◽

Pilar Navarro-Gómez ◽

Paula Ayala-García ◽

Isamar Moyano-Bravo ◽

Francisco-Javier López-Baena ◽

...

Keyword(s):

Nitrogenase Activity ◽

Lotus Japonicus ◽

Rhizobium Tropici ◽

Sinorhizobium Fredii ◽

Symbiotic Interaction ◽

Model Legume ◽

Nod Factor ◽

Factor Production ◽

Nodulation Capacity ◽

Type 3

Rhizobial NodD proteins and appropriate flavonoids induce rhizobial nodulation gene expression. In this study, we show that the nodD1 gene of Sinorhizobium fredii HH103, but not the nodD2 gene, can restore the nodulation capacity of a double nodD1/nodD2 mutant of Rhizobium tropici CIAT 899 in bean plants (Phaseolus vulgaris). S. fredii HH103 only induces pseudonodules in beans. We have also studied whether the mutation of different symbiotic regulatory genes may affect the symbiotic interaction of HH103 with beans: ttsI (the positive regulator of the symbiotic type 3 protein secretion system), and nodD2, nolR and syrM (all of them controlling the level of Nod factor production). Inactivation of either nodD2, nolR or syrM, but not that of ttsI, affected positively the symbiotic behavior of HH103 with beans, leading to the formation of colonized nodules. Acetylene reduction assays showed certain levels of nitrogenase activity that were higher in the case of the nodD2 and nolR mutants. Similar results have been previously obtained by our group with the model legume Lotus japonicus. Hence, the results obtained in the present work confirm that repression of Nod factor production, provided by either NodD2, NolR or SyrM, prevents HH103 to effectively nodulate several putative host plants.

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Mutants of Lotus japonicus deficient in flavonoid biosynthesis

Journal of Plant Research ◽

10.1007/s10265-021-01258-8 ◽

2021 ◽

Vol 134 (2) ◽

pp. 341-352

Author(s):

Toshio Aoki ◽

Masayoshi Kawaguchi ◽

Haruko Imaizumi-Anraku ◽

Shoichiro Akao ◽

Shin-ichi Ayabe ◽

...

Keyword(s):

Anthocyanin Biosynthesis ◽

Lotus Japonicus ◽

Flavonoid Biosynthesis ◽

Anthocyanin Accumulation ◽

Type B ◽

Wild Type ◽

Ems Mutagenesis ◽

Model Legume ◽

Mesorhizobium Loti ◽

Wild Accessions

AbstractSpatiotemporal features of anthocyanin accumulation in a model legumeLotus japonicus(Regel) K.Larsen were elucidated to develop criteria for the genetic analysis of flavonoid biosynthesis. Artificial mutants and wild accessions, with lower anthocyanin accumulation in the stem than the standard wild type (B-129 ‘Gifu’), were obtained by ethyl methanesulfonate (EMS) mutagenesis and from a collection of wild-grown variants, respectively. The loci responsible for the green stem of the mutants were named asVIRIDICAULIS(VIC). Genetic and chemical analysis identified two loci, namely,VIC1andVIC2, required for the production of both anthocyanins and proanthocyanidins (condensed tannins), and two loci, namely,VIC3andVIC4, required for the steps specific to anthocyanin biosynthesis. A mutation inVIC5significantly reduced the anthocyanin accumulation. These mutants will serve as a useful system for examining the effects of anthocyanins and proanthocyanidins on the interactions with herbivorous pests, pathogenic microorganisms and nitrogen-fixing symbiotic bacteria,Mesorhizobium loti.

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New Nodulation Mutants Responsible for Infection Thread Development in Lotus japonicus

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-19-0801 ◽

2006 ◽

Vol 19 (7) ◽

pp. 801-810 ◽

Cited By ~ 28

Author(s):

Koji Yano ◽

Myra L. Tansengco ◽

Taihei Hio ◽

Kuniko Higashi ◽

Yoshikatsu Murooka ◽

...

Keyword(s):

Developmental Process ◽

Lotus Japonicus ◽

Infection Thread ◽

Nodule Development ◽

Short Root ◽

Symbiotic Interaction ◽

Model Legume ◽

Infection Threads ◽

Nodulation Mutants ◽

Legume Plants

Legume plants develop specialized root organs, the nodules, through a symbiotic interaction with rhizobia. The developmental process of nodulation is triggered by the bacterial microsymbiont but regulated systemically by the host legume plants. Using ethylmethane sulfonate mutagenesis as a tool to identify plant genes involved in symbiotic nodule development, we have isolated and analyzed five nodulation mutants, Ljsym74-3, Ljsym79-2, Ljsym79-3, Ljsym80, and Ljsym82, from the model legume Lotus japonicus. These mutants are defective in developing functional nodules and exhibit nitrogen starvation symptoms after inoculation with Mesorhizobium loti. Detailed observation revealed that infection thread development was aborted in these mutants and the nodules formed were devoid of infected cells. Mapping and complementation tests showed that Ljsym74-3, and Ljsym79-2 and Ljsym79-3, were allelic with reported mutants of L. japonicus, alb1 and crinkle, respectively. The Ljsym82 mutant is unique among the mutants because the infection thread was aborted early in its development. Ljsym74-3 and Ljsym80 were characterized as mutants with thick infection threads in short root hairs. Map-based cloning and molecular characterization of these genes will help us understand the genetic mechanism of infection thread development in L. japonicus.

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