Large-Scale Phenotyping of Root Traits in the Model Legume Lotus japonicus

Phosphoenolpyruvate carboxylases (PEPCs), one form of which in each legume species plays a central role in the carbon metabolism in symbiotic root nodules, are activated through phosphorylation of a conserved residue by a specific protein kinase (PEPC-PK). We characterized the cDNAs for two PEPC isoforms of Lotus japonicus, an amide-translocating legume that forms determinate nodules. One gene encodes a nodule-enhanced form, which is more closely related to the PEPCs in amide-type indeterminate nodules than those in ureide-type determinate nodules. The other gene is expressed in shoots and roots at a low level. Both forms have the putative phosphorylation site, Ser11. We also isolated a cDNA and the corresponding genomic DNA for PEPC-PK of L. japonicus. The recombinant PEPC-PK protein expressed in Escherichia coli phosphorylated recombinant maize C4-form PEPC efficiently in vitro. The level of mRNA for PEPC-PK was high in root nodules, and those in shoots and roots were also significant. In situ hybridization revealed that the expression patterns of the transcripts for PEPC and PEPC-PK were similar in mature root nodules, but were different in emerging nodules. When L. japonicus seedlings were subjected to prolonged darkness and subsequent illumination, the activity of PEPC-PK and the mRNA levels of both PEPC and PEPC-PK in nodules decreased and then recovered, suggesting that they are regulated according to the amounts of photosynthates transported from shoots.

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Αναπρογραμματισμός του μεταβολισμού του Lotus japonicus σε μεταγραφικό, βιοχημικό και μεταβολομικό επίπεδο κατά τη συμβιωτική αζωτοδέσμευση

10.12681/eadd/37826 ◽

2016 ◽

Author(s):

Χρυσάνθη Καλλονιάτη

Keyword(s):

Nitrogen Fixation ◽

Metabolite Profiling ◽

Symbiotic Nitrogen Fixation ◽

Sulfur Metabolism ◽

Lotus Japonicus ◽

Nitrogen Fixing ◽

Model Legume ◽

Whole Plant ◽

Plant Level ◽

Molecular And Biochemical Mechanisms

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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Recent Advances in the Molecular Genetics of The Model Legume Lotus japonicus

Biological Fixation of Nitrogen for Ecology and Sustainable Agriculture ◽

10.1007/978-3-642-59112-9_52 ◽

1997 ◽

pp. 255-258

Author(s):

Leif Schauser ◽

Leszek Boron ◽

Eloisa Pajuelo ◽

Thomas Thykjær ◽

Dorthe Danielsen ◽

...

Keyword(s):

Molecular Genetics ◽

Lotus Japonicus ◽

Model Legume ◽

Recent Advances

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Flavonoids and Isoflavonoids Biosynthesis in the Model Legume Lotus japonicus; Connections to Nitrogen Metabolism and Photorespiration

Plants ◽

10.3390/plants9060774 ◽

2020 ◽

Vol 9 (6) ◽

pp. 774 ◽

Cited By ~ 1

Author(s):

Margarita García-Calderón ◽

Carmen M. Pérez-Delgado ◽

Peter Palove-Balang ◽

Marco Betti ◽

Antonio J. Márquez

Keyword(s):

Abiotic Stress ◽

Nitrogen Metabolism ◽

Lotus Japonicus ◽

Regulatory Proteins ◽

Phenylpropanoid Metabolism ◽

Biological Processes ◽

Model Legume ◽

Different Types ◽

Made In

Phenylpropanoid metabolism represents an important metabolic pathway from which originates a wide number of secondary metabolites derived from phenylalanine or tyrosine, such as flavonoids and isoflavonoids, crucial molecules in plants implicated in a large number of biological processes. Therefore, various types of interconnection exist between different aspects of nitrogen metabolism and the biosynthesis of these compounds. For legumes, flavonoids and isoflavonoids are postulated to play pivotal roles in adaptation to their biological environments, both as defensive compounds (phytoalexins) and as chemical signals in symbiotic nitrogen fixation with rhizobia. In this paper, we summarize the recent progress made in the characterization of flavonoid and isoflavonoid biosynthetic pathways in the model legume Lotus japonicus (Regel) Larsen under different abiotic stress situations, such as drought, the impairment of photorespiration and UV-B irradiation. Emphasis is placed on results obtained using photorespiratory mutants deficient in glutamine synthetase. The results provide different types of evidence showing that an enhancement of isoflavonoid compared to standard flavonol metabolism frequently occurs in Lotus under abiotic stress conditions. The advance produced in the analysis of isoflavonoid regulatory proteins by the use of co-expression networks, particularly MYB transcription factors, is also described. The results obtained in Lotus japonicus plants can be also extrapolated to other cultivated legume species, such as soybean, of extraordinary agronomic importance with a high impact in feeding, oil production and human health.

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Identification of tissue-specific gene clusters and orthologues of nodulation-related genes in Vigna angularis

Plant Genetic Resources ◽

10.1017/s1479262114000185 ◽

2014 ◽

Vol 12 (S1) ◽

pp. S21-S26 ◽

Cited By ~ 1

Author(s):

Yang Jae Kang ◽

Jayern Lee ◽

Yong Hwan Kim ◽

Suk-Ha Lee

Keyword(s):

De Novo ◽

Transcriptome Assembly ◽

Lotus Japonicus ◽

Gene Clusters ◽

Specific Gene ◽

Vigna Angularis ◽

Model Legume ◽

Tissue Specific ◽

Autoregulation Of Nodulation ◽

Species Specific

Nitrogen fixation in legumes is an important agricultural trait that results from symbiosis between the root and rhizobia. To understand the molecular basis of nodulation, recent research has been focused on the identification of nodulation-related genes by functional analysis using two major model legumes, Medicago truncatula and Lotus japonicus. Thus far, three important processes have been discovered, namely Nod factor (NF) perception, NF signalling and autoregulation of nodulation. Nevertheless, application of the results of these studies is limited for non-model legume crops because a reference genome is unavailable. However, because the cost of whole-transcriptome analysis has dropped dramatically due to the Next generation sequencer (NGS) technology, minor crops for which reference sequences are yet to be constructed can still be studied at the genome level. In this study, we sequenced the leaf and root transcriptomes of Vigna angularis (accession IT213134) and de novo assembled. Our results demonstrate the feasibility of using the transcriptome assembly to effectively identify tissue-specific peptide clusters related to tissue-specific functions and species-specific nodulation-related genes.

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Molecular Analysis of the Pathway for the Synthesis of Thiol Tripeptides in the Model Legume Lotus japonicus

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2003.16.11.1039 ◽

2003 ◽

Vol 16 (11) ◽

pp. 1039-1046 ◽

Cited By ~ 25

Author(s):

Manuel A. Matamoros ◽

Maria R. Clemente ◽

Shusei Sato ◽

Erika Asamizu ◽

Satoshi Tabata ◽

...

Keyword(s):

Lotus Japonicus ◽

Regulatory Elements ◽

Chromosome 1 ◽

Glutathione Synthetase ◽

Promoter Regions ◽

Model Legume ◽

Cis Acting ◽

Glutamylcysteine Synthetase ◽

Exon 1 ◽

Legume Symbiosis

The thiol tripeptides, glutathione (GSH) and homoglu-tathione (hGSH), perform multiple roles in legumes, including protection against toxicity of free radicals and heavy metals. The three genes involved in the synthesis of GSH and hGSH in the model legume, Lotus japonicus, have been fully characterized and appear to be present as single copies in the genome. The γ-glutamylcysteine synthetase (γecs) gene was mapped on the long arm of chromosome 4 (70.0 centimorgans [cM]) and consists of 15 exons, whereas the glutathione synthetase (gshs) and homoglutathione synthetase (hgshs) genes were mapped on the long arm of chromosome 1 (81.3 cM) and found to be arranged in tandem with a separation of approximately 8 kb. Both genes consist of 12 exons of exactly the same size (except exon 1, which is similar). Two types of transcripts were detected for the gshs gene, which putatively encode proteins localized in the plastids and cytosol. Promoter regions contain cis-acting regulatory elements that may be involved in the plant's response to light, hormones, and stress. Determination of transcript levels, enzyme activities, and thiol contents in nodules, roots, and leaves revealed that γecs and hgshs are expressed in all three plant organs, whereas gshs is significantly functional only in nodules. This strongly suggests an important role of GSH in the rhizobia-legume symbiosis.

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Root, Root Hair, and Symbiotic Mutants of the Model Legume Lotus japonicus

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2002.15.1.17 ◽

2002 ◽

Vol 15 (1) ◽

pp. 17-26 ◽

Cited By ~ 113

Author(s):

Masayoshi Kawaguchi ◽

Haruko Imaizumi-Anraku ◽

Hiroyuki Koiwa ◽

Sinobu Niwa ◽

Akira Ikuta ◽

...

Keyword(s):

Root Hair ◽

Phenotypic Variation ◽

Developmental Process ◽

Lotus Japonicus ◽

Root Hairs ◽

Model Legume ◽

Nodule Number ◽

Stable Mutant ◽

Mutant Lines ◽

Hypernodulating Mutants

To gain an overview of plant factors controlling nodule number and organogenesis, an extensive screening using model legume Lotus japonicus was carried out. This screening involved 40,000 M2 seeds, and 32 stable mutant lines were isolated. From these, 16 mutant lines maintaining the phenotypic variation were selected and genetically analyzed. With respect to nodule number, four loci were identified, Ljsym77, Ljsym78, slippery root (slp), and radial organization1 (rdo1). The former two mutants have an increased number of nodules, while the latter two have a decreased number. Ljsym78-1 and Ljsym78-2 are hypernodulating mutants with a branched root system and were found to be allelic to Ljsym16. The phenotype of the Ljsym77 mutant was highly pleiotropic, being deficient in light and gravity responses. The slp mutant was isolated as a low-nodulating mutant lacking root hairs. Concerning nodule organogenesis, nine symbiotic loci were identified, including the two loci alb1 and fen1. Mutants affecting the developmental process of nodule organogenesis were placed in three phenotypic categories: Nod¯ (Ljsym70 to Ljsym73), Hist¯ (alb1-1, alb1-2, and Ljsym79), and Fix¯ (fen1, Ljsym75, and Ljsym81).

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