A Dicarboxylate Transporter, LjALMT4, Mainly Expressed in Nodules of Lotus japonicus

Legume plants can establish symbiosis with soil bacteria called rhizobia to obtain nitrogen as a nutrient directly from atmospheric N2 via symbiotic nitrogen fixation. Legumes and rhizobia form nodules, symbiotic organs in which fixed-nitrogen and photosynthetic products are exchanged between rhizobia and plant cells. The photosynthetic products supplied to rhizobia are thought to be dicarboxylates but little is known about the movement of dicarboxylates in the nodules. In terms of dicarboxylate transporters, an aluminum-activated malate transporter (ALMT) family is a strong candidate responsible for the membrane transport of carboxylates in nodules. Among the seven ALMT genes in the Lotus japonicus genome, only one, LjALMT4, shows a high expression in the nodules. LjALMT4 showed transport activity in a Xenopus oocyte system, with LjALMT4 mediating the efflux of dicarboxylates including malate, succinate, and fumarate, but not tricarboxylates such as citrate. LjALMT4 also mediated the influx of several inorganic anions. Organ-specific gene expression analysis showed LjALMT4 mRNA mainly in the parenchyma cells of nodule vascular bundles. These results suggest that LjALMT4 may not be involved in the direct supply of dicarboxylates to rhizobia in infected cells but is responsible for supplying malate as well as several anions necessary for symbiotic nitrogen fixation, via nodule vasculatures.

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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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Digalactosyldiacylglycerol Synthase Gene MtDGD1 Plays an Essential Role in Nodule Development and Nitrogen Fixation

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-11-18-0322-r ◽

2019 ◽

Vol 32 (9) ◽

pp. 1196-1209

Author(s):

Zaiyong Si ◽

Qianqian Yang ◽

Rongrong Liang ◽

Ling Chen ◽

Dasong Chen ◽

...

Keyword(s):

Nitrogen Fixation ◽

Essential Role ◽

Symbiotic Nitrogen Fixation ◽

Histochemical Staining ◽

Nodule Development ◽

Nodule Number ◽

Nitrogen Fixation Activity ◽

Fixation Activity ◽

Infected Cells ◽

Nodule Symbiosis

Little is known about the genes participating in digalactosyldiacylglycerol (DGDG) synthesis during nodule symbiosis. Here, we identified full-length MtDGD1, a synthase of DGDG, and characterized its effect on symbiotic nitrogen fixation in Medicago truncatula. Immunofluorescence and immunoelectron microscopy showed that MtDGD1 was located on the symbiosome membranes in the infected cells. β-Glucuronidase histochemical staining revealed that MtDGD1 was highly expressed in the infection zone of young nodules as well as in the whole mature nodules. Compared with the control, MtDGD1-RNA interference transgenic plants exhibited significant decreases in nodule number, symbiotic nitrogen fixation activity, and DGDG abundance in the nodules, as well as abnormal nodule and symbiosome development. Overexpression of MtDGD1 resulted in enhancement of nodule number and nitrogen fixation activity. In response to phosphorus starvation, the MtDGD1 expression level was substantially upregulated and the abundance of nonphospholipid DGDG was significantly increased in the roots and nodules, accompanied by corresponding decreases in the abundance of phospholipids such as phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. Overall, our results indicate that DGD1 contributes to effective nodule organogenesis and nitrogen fixation by affecting the synthesis and content of DGDG during symbiosis.

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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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CRISPR/Cas9 knockout of leghemoglobin genes in Lotus japonicus uncovers their synergistic roles in symbiotic nitrogen fixation

New Phytologist ◽

10.1111/nph.16077 ◽

2019 ◽

Vol 224 (2) ◽

pp. 818-832 ◽

Cited By ~ 13

Author(s):

Longlong Wang ◽

Maria Carmen Rubio ◽

Xian Xin ◽

Baoli Zhang ◽

Qiuling Fan ◽

...

Keyword(s):

Nitrogen Fixation ◽

Symbiotic Nitrogen Fixation ◽

Lotus Japonicus

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The SNARE Protein SYP71 Expressed in Vascular Tissues Is Involved in Symbiotic Nitrogen Fixation in Lotus japonicus Nodules

PLANT PHYSIOLOGY ◽

10.1104/pp.112.200782 ◽

2012 ◽

Vol 160 (2) ◽

pp. 897-905 ◽

Cited By ~ 24

Author(s):

Tsuneo Hakoyama ◽

Ryo Oi ◽

Kazuya Hazuma ◽

Eri Suga ◽

Yuka Adachi ◽

...

Keyword(s):

Nitrogen Fixation ◽

Symbiotic Nitrogen Fixation ◽

Lotus Japonicus ◽

Snare Protein ◽

Vascular Tissues

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Induction and Spatial Organization of Polyamine Biosynthesis During Nodule Development in Lotus japonicus

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2004.17.12.1283 ◽

2004 ◽

Vol 17 (12) ◽

pp. 1283-1293 ◽

Cited By ~ 24

Author(s):

Emmanouil Flemetakis ◽

Rodica C. Efrose ◽

Guilhem Desbrosses ◽

Maria Dimou ◽

Costas Delis ◽

...

Keyword(s):

Nitrogen Fixation ◽

Symbiotic Nitrogen Fixation ◽

Expression Patterns ◽

Lotus Japonicus ◽

Cyclin D3 ◽

Nodule Development ◽

Cdna Clones ◽

Long Distance ◽

Polyamine Biosynthesis ◽

Transcript Levels

Putrescine and other polyamines are produced by two alternative pathways in plants. One pathway starts with the enzyme arginine decarboxylase (ADC; EC 4.1.1.19), the other with ornithine decarboxylase (ODC; EC 4.1.1.17). Metabolite profiling of nitrogen-fixing Lotus japonicus nodules, using gas chromatography coupled to mass spectrometry, revealed a two- to sixfold increase in putrescine levels in mature nodules compared with other organs. Genes involved in polyamine biosynthesis in L. japonicus nodules were identified by isolating cDNA clones encoding ADC (LjADC1) and ODC (LjODC) from a nodule library. Searches of the public expressed sequence tag databases revealed the presence of a second gene encoding ADC (LjADC2). Real-time reverse-transcription-polymerase chain reaction analysis showed that LjADC1 and LjADC2 were expressed throughout the plant, while LjODC transcripts were detected only in nodules and roots. Induction of LjODC and LjADC gene expression during nodule development preceded symbiotic nitrogen fixation. Transcripts accumulation was maximal at 10 days postinfection, when a 6.5-fold increase in the transcript levels of LjODC was observed in comparison with the uninfected roots, while a twofold increase in the transcript levels of LjADC1 and LjADC2 was detected. At later stages of nodule development, transcripts for ADC drastically declined, while in the case of ODC, transcript accumulation was higher than that in roots until after 21 days postinfection. The expression profile of genes involved in putrescine biosynthesis correlated well with the expression patterns of genes involved in cell division and expansion, including a L. japonicus Cyclin D3 and an α-expansin gene. Spatial localization of LjODC and LjADC1 gene transcripts in developing nodules revealed that both transcripts were expressed in nodule inner cortical cells and in the central tissue. High levels of LjADC1 transcripts were also observed in both nodule and connecting root vascular tissue, suggesting that putrescine and other polyamines may be subject to long-distance transport. Our results indicate that polyamines are primarily involved in physiological and cellular processes involved in nodule development, rather than in processes that support directly symbiotic nitrogen fixation and assimilation.

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Lotus japonicus Gene Ljsbp Is Highly Conserved Among Plants and Animals and Encodes a Homologue to the Mammalian Selenium-Binding Proteins

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2002.15.4.313 ◽

2002 ◽

Vol 15 (4) ◽

pp. 313-322 ◽

Cited By ~ 25

Author(s):

Emmanouil Flemetakis ◽

Adamantia Agalou ◽

Nektarios Kavroulakis ◽

Maria Dimou ◽

Anna Martsikovskaya ◽

...

Keyword(s):

Polyclonal Antibodies ◽

Lotus Japonicus ◽

Root Hairs ◽

Physiological Role ◽

Cdna Clones ◽

Vascular Bundles ◽

Root Tips ◽

Root Epidermis ◽

Infected Cells ◽

Low Levels

We have isolated and characterized a Lotus japonicus gene (Ljsbp) encoding a putative polypeptide with striking homology to the mammalian 56-kDa selenium-binding protein (SBP). cDNA clones homologous to LjSBP were also isolated from soybean, Medicago sativa, and Arabidopsis thaliana. Comparative expression studies in L. japonicus and A. thaliana showed that sbp transcripts are present in various tissues and at different levels. Especially in L. japonicus nodules and seedpods and A. thaliana siliques, sbp expression appears to be developmentally up-regulated. sbp Gene transcripts were localized by in situ hybridization in the infected cells and vascular bundles of young nodules, while in mature nodules, low levels of expression were only detected in the parenchymatous cells. Expression of sbp transcripts in young seedpods and siliques was clearly visible in vascular tissues and embryos, while in embryos, low levels of expression were detected in the root epidermis and the vascular bundles. Polyclonal antibodies raised against a truncated LjSBP recombinant protein recognized a poly-peptide of about 60 kDa in nodule extracts. Immunohistochemical experiments showed that accumulation of LjSBP occurred in root hairs, in the root epidermis above the nodule primordium, in the phloem of the vasculature, and abundantly in the infected cells of young nodules. Irrespective of the presence of rhizobia, expression of SBP was also observed in root tips, where it was confined in the root epidermis and protophloem cells. We hypothesize that LjSBP may have more than one physiological role and can be implicated in controlling the oxidation/reduction status of target proteins, in vesicular Golgi transport, or both.

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