Nitrate-independent expression of plant nitrate reductase in Lotus japonicus root nodules

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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MIR2111-5 locus and shoot-accumulated mature miR2111 systemically enhance nodulation depending on HAR1 in Lotus japonicus

Nature Communications ◽

10.1038/s41467-020-19037-9 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Nao Okuma ◽

Takashi Soyano ◽

Takuya Suzaki ◽

Masayoshi Kawaguchi

Keyword(s):

Root Nodules ◽

Lotus Japonicus ◽

Dependent Manner ◽

Rna Seq ◽

Signaling System ◽

Nodule Number ◽

Knockout Mutants ◽

Mutualistic Relationship ◽

Dose Dependent

Abstract Legumes utilize a shoot-mediated signaling system to maintain a mutualistic relationship with nitrogen-fixing bacteria in root nodules. In Lotus japonicus, shoot-to-root transfer of microRNA miR2111 that targets TOO MUCH LOVE, a nodulation suppressor in roots, has been proposed to explain the mechanism underlying nodulation control from shoots. However, the role of shoot-accumulating miR2111s for the systemic regulation of nodulation was not clearly shown. Here, we find L. japonicus has seven miR2111 loci, including those mapped through RNA-seq. MIR2111-5 expression in leaves is the highest among miR2111 loci and repressed after rhizobial infection depending on a shoot-acting HYPERNODULATION ABERRANT ROOT FORMATION1 (HAR1) receptor. MIR2111-5 knockout mutants show significantly decreased nodule numbers and miR2111 levels. Furthermore, grafting experiments using transformants demonstrate scions with altered miR2111 levels influence nodule numbers in rootstocks in a dose-dependent manner. Therefore, miR2111 accumulation in leaves through MIR2111-5 expression is required for HAR1-dependent systemic optimization of nodule number.

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Phytosulfokine Is Involved in Positive Regulation of Lotus japonicus Nodulation

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-02-15-0032-r ◽

2015 ◽

Vol 28 (8) ◽

pp. 847-855 ◽

Cited By ~ 13

Author(s):

Chao Wang ◽

Haixiang Yu ◽

Zhongming Zhang ◽

Liangliang Yu ◽

Xiaoshu Xu ◽

...

Keyword(s):

Potential Role ◽

Early Stage ◽

Root Nodules ◽

Lotus Japonicus ◽

Positive Regulation ◽

Transgenic Hairy Roots ◽

Exogenous Addition ◽

Nodule Initiation ◽

Spontaneous Nodules

Phytosulfokine (PSK) is a tyrosine-sulfated peptide that is widely distributed in plants, participating in cell proliferation, differentiation, and innate immunity. The potential role of PSK in nodulation in legumes has not been reported. In this work, five PSK precursor genes were identified in Lotus japonicas, designated as LjPSK1 to LjPSK5. Three of them (LjPSK1, LjPSK4, and LjPSK5) were found to be expressed in nitrogen-fixing root nodules. LjPSK1 and LjPSK4 were not induced at the early stage of nodulation. Interestingly, while the expression of LjPSK4 was also found in spontaneous nodules without rhizobial colonization, LjPSK1 was not induced in these pseudo nodules. Promoter-β-glucuronidase analysis revealed that LjPSK1 was highly expressed in enlarged symbiotic cells of nodules. Exogenous addition of 1 μM synthetic PSK peptide resulted in increased nodule numbers per plant. Consistently, the number of mature nodules but not the events of rhizobial infection and nodule initiation was increased by overexpressing LjPSK1 in transgenic hairy roots, in which the expression of jasmonate-responsive genes was found to be repressed. These results suggest that PSK is a new peptide signal that regulates nodulation in legumes, probably through cross-talking with other phytohormones.

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Expression Analysis of PIN Genes in Root Tips and Nodules of Lotus japonicus

International Journal of Molecular Sciences ◽

10.3390/ijms20020235 ◽

2019 ◽

Vol 20 (2) ◽

pp. 235 ◽

Cited By ~ 4

Author(s):

Izabela Sańko-Sawczenko ◽

Dominika Dmitruk ◽

Barbara Łotocka ◽

Elżbieta Różańska ◽

Weronika Czarnocka

Keyword(s):

Root Nodule ◽

Root Nodules ◽

Quantitative Polymerase Chain Reaction ◽

Lotus Japonicus ◽

Nodule Development ◽

Vascular Bundles ◽

Root Tips ◽

Gus Reporter ◽

Gene Assay ◽

Development And Differentiation

Auxins are postulated to be one of the pivotal factors in nodulation. However, their transporters in Lotus japonicus, the model species for the study of the development of determinate-type root nodules, have been scarcely described so far, and thus their role in nodulation has remained unknown. Our research is the first focusing on polar auxin transporters in L. japonicus. We analyzed and compared expression of PINs in 20 days post rhizobial inoculation (dpi) and 54 dpi root nodules of L. japonicus by real-time quantitative polymerase chain reaction (qPCR) along with the histochemical β-glucuronidase (GUS) reporter gene assay in transgenic hairy roots. The results indicate that LjPINs are essential during root nodule development since they are predominantly expressed in the primordia and young, developing nodules. However, along with differentiation, expression levels of several PINs decreased and occurred particularly in the nodule vascular bundles, especially in connection with the root’s stele. Moreover, our study demonstrated the importance of both polar auxin transport and auxin intracellular homeostasis during L. japonicus root nodule development and differentiation.

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The rhizobial autotransporter determines the symbiotic nitrogen fixation activity of Lotus japonicus in a host-specific manner

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1913349117 ◽

2020 ◽

Vol 117 (3) ◽

pp. 1806-1815 ◽

Cited By ~ 2

Author(s):

Yoshikazu Shimoda ◽

Yuki Nishigaya ◽

Hiroko Yamaya-Ito ◽

Noritoshi Inagaki ◽

Yosuke Umehara ◽

...

Keyword(s):

Nitrogen Fixation ◽

Host Plant ◽

Symbiotic Nitrogen Fixation ◽

Root Nodules ◽

Lotus Japonicus ◽

Dependent Manner ◽

Strain Specificity ◽

Passenger Domain ◽

Aspartic Peptidase ◽

Specific Manner

Leguminous plants establish endosymbiotic associations with rhizobia and form root nodules in which the rhizobia fix atmospheric nitrogen. The host plant and intracellular rhizobia strictly control this symbiotic nitrogen fixation. We recently reported a Lotus japonicus Fix− mutant, apn1 (aspartic peptidase nodule-induced 1), that impairs symbiotic nitrogen fixation. APN1 encodes a nodule-specific aspartic peptidase involved in the Fix− phenotype in a rhizobial strain-specific manner. This host-strain specificity implies that some molecular interactions between host plant APN1 and rhizobial factors are required, although the biological function of APN1 in nodules and the mechanisms governing the interactions are unknown. To clarify how rhizobial factors are involved in strain-specific nitrogen fixation, we explored transposon mutants of Mesorhizobium loti strain TONO, which normally form Fix− nodules on apn1 roots, and identified TONO mutants that formed Fix+ nodules on apn1. The identified causal gene encodes an autotransporter, part of a protein secretion system of Gram-negative bacteria. Expression of the autotransporter gene in M. loti strain MAFF3030399, which normally forms Fix+ nodules on apn1 roots, resulted in Fix− nodules. The autotransporter of TONO functions to secrete a part of its own protein (a passenger domain) into extracellular spaces, and the recombinant APN1 protein cleaved the passenger protein in vitro. The M. loti autotransporter showed the activity to induce the genes involved in nodule senescence in a dose-dependent manner. Therefore, we conclude that the nodule-specific aspartic peptidase, APN1, suppresses negative effects of the rhizobial autotransporter in order to maintain effective symbiotic nitrogen fixation in root nodules.

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A shared gene drives lateral root development and root nodule symbiosis pathways in Lotus

Science ◽

10.1126/science.aax2153 ◽

2019 ◽

Vol 366 (6468) ◽

pp. 1021-1023 ◽

Cited By ~ 20

Author(s):

Takashi Soyano ◽

Yoshikazu Shimoda ◽

Masayoshi Kawaguchi ◽

Makoto Hayashi

Keyword(s):

Root Development ◽

Lateral Root ◽

Root Nodule ◽

Root Nodules ◽

Lotus Japonicus ◽

Cortical Cell ◽

Cortical Cells ◽

Lateral Root Development ◽

Lateral Organ ◽

Nodule Symbiosis

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-LIKE18/LATERAL ORGAN BOUNDARIES DOMAIN16a (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. japonicusdaphne 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.

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Differential Effects of Combined N Sources on Early Steps of the Nod Factor–Dependent Transduction Pathway in Lotus japonicus

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-20-8-0994 ◽

2007 ◽

Vol 20 (8) ◽

pp. 994-1003 ◽

Cited By ~ 50

Author(s):

Ani Barbulova ◽

Alessandra Rogato ◽

Enrica D'Apuzzo ◽

Selim Omrane ◽

Maurizio Chiurazzi

Keyword(s):

Root Nodules ◽

Lotus Japonicus ◽

Small Scale ◽

Nodule Formation ◽

Inhibitory Effects ◽

Nod Factor ◽

Mesorhizobium Loti ◽

N Sources ◽

N Starvation ◽

And Function

The development of nitrogen-fixing nodules in legumes is induced by perception of lipochitin-oligosaccharide signals secreted by a bacterial symbiont. Nitrogen (N) starvation is a prerequisite for the formation, development, and function of root nodules, and high levels of combined N in the form of nitrate or ammonium can completely abolish nodule formation. We distinguished between nitrate and ammonium inhibitory effects by identifying when and where these combined N sources interfere with the Nod-factor-induced pathway. Furthermore, we present a small-scale analysis of the expression profile, under different N conditions, of recently identified genes involved in the Nod-factor-induced pathway. In the presence of high levels of nitrate or ammonium, the NIN gene fails to be induced 24 h after the addition of Nod factor compared with plants grown under N-free conditions. This induction is restored in the hypernodulating nitrate-tolerant har1-3 mutant only in the presence of 10 and 20 mM KNO3. These results were confirmed in Lotus plants inoculated with Mesorhizobium loti. NIN plays a key role in the nodule organogenesis program and its downregulation may represent a crucial event in the nitrate-dependent pathway leading to the inhibition of nodule organogenesis.

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Absence of Symbiotic Leghemoglobins Alters Bacteroid and Plant Cell Differentiation During Development of Lotus japonicus Root Nodules

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-22-7-0800 ◽

2009 ◽

Vol 22 (7) ◽

pp. 800-808 ◽

Cited By ~ 36

Author(s):

Thomas Ott ◽

John Sullivan ◽

Euan K. James ◽

Emmanouil Flemetakis ◽

Catrin Günther ◽

...

Keyword(s):

Gene Expression ◽

Nitrogen Fixation ◽

Cell Differentiation ◽

Root Nodules ◽

Lotus Japonicus ◽

Nodule Development ◽

Primary And Secondary Metabolism ◽

Bacterial Differentiation ◽

Plant Genes ◽

Bacterial Genes

During development of legume root nodules, rhizobia and their host plant cells undergo profound differentiation, which is underpinned by massive changes in gene expression in both symbiotic partners. Oxygen concentrations in infected and surrounding uninfected cells drop precipitously during nodule development. To assess what effects this has on plant and bacterial cell differentiation and gene expression, we used a leghemoglobin-RNA-interference (LbRNAi) line of Lotus japonicus, which is devoid of leghemoglobins and has elevated levels of free-oxygen in its nodules. Bacteroids in LbRNAi nodules showed altered ultrastructure indicating changes in bacterial differentiation. Transcript analysis of 189 plant and 192 bacterial genes uncovered many genes in both the plant and bacteria that were differentially regulated during nodulation of LbRNAi plants compared with the wild type (containing Lb and able to fix nitrogen). These included fix and nif genes of the bacteria, which are involved in microaerobic respiration and nitrogen fixation, respectively, and plant genes involved in primary and secondary metabolism. Metabolite analysis revealed decreased levels of many amino acids in nodules of LbRNAi plants, consistent with the defect in symbiotic nitrogen fixation of this line.

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Redifferentiation of bacteria isolated from Lotus japonicus root nodules colonized by Rhizobium sp. NGR234

Journal of Experimental Botany ◽

10.1093/jexbot/52.364.2181 ◽

2001 ◽

Vol 52 (364) ◽

pp. 2181-2186 ◽

Cited By ~ 25

Author(s):

J. Müller ◽

A. Wiemken ◽

T. Boller

Keyword(s):

Root Nodules ◽

Lotus Japonicus ◽

Rhizobium Sp

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A plant chitinase controls cortical infection thread progression and nitrogen-fixing symbiosis

eLife ◽

10.7554/elife.38874 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 9

Author(s):

Anna Malolepszy ◽

Simon Kelly ◽

Kasper Kildegaard Sørensen ◽

Euan Kevin James ◽

Christina Kalisch ◽

...

Keyword(s):

Root Nodules ◽

Lotus Japonicus ◽

Positional Information ◽

Dependent Manner ◽

Nitrogen Fixing ◽

Nod Factors ◽

Nod Factor ◽

Infection Threads ◽

Bacterial Mutants ◽

Multicellular Organisms

Morphogens provide positional information and their concentration is key to the organized development of multicellular organisms. Nitrogen-fixing root nodules are unique organs induced by Nod factor-producing bacteria. Localized production of Nod factors establishes a developmental field within the root where plant cells are reprogrammed to form infection threads and primordia. We found that regulation of Nod factor levels by Lotus japonicus is required for the formation of nitrogen-fixing organs, determining the fate of this induced developmental program. Our analysis of plant and bacterial mutants shows that a host chitinase modulates Nod factor levels possibly in a structure-dependent manner. In Lotus, this is required for maintaining Nod factor signalling in parallel with the elongation of infection threads within the nodule cortex, while root hair infection and primordia formation are not influenced. Our study shows that infected nodules require balanced levels of Nod factors for completing their transition to functional, nitrogen-fixing organs.

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