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

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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Novel Aspects of Symbiotic Nitrogen Fixation Uncovered by Transcript Profiling with cDNA Arrays

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2002.15.5.411 ◽

2002 ◽

Vol 15 (5) ◽

pp. 411-420 ◽

Cited By ~ 95

Author(s):

Gillian Colebatch ◽

Sebastian Kloska ◽

Ben Trevaskis ◽

Susanne Freund ◽

Thomas Altmann ◽

...

Keyword(s):

Nitrogen Fixation ◽

Symbiotic Nitrogen Fixation ◽

Active Oxygen Species ◽

Lotus Japonicus ◽

Transcript Abundance ◽

Defense Responses ◽

Cdna Clones ◽

Regulation Of Transcription ◽

Gene Transcript ◽

Amino Acid Synthesis

An array of 2,304 cDNA clones derived from nitrogen-fixing nodules of Lotus japonicus was produced and used to detect differences in relative gene transcript abundance between nodules and uninfected roots. Transcripts of 83 different genes were found to be more abundant in nodules than in roots. More than 50 of these have never before been identified as nodule-induced in any species. Expression of 36 genes was detected in nodules but not in roots. Several known nodulin genes were included among the nodule-induced genes. Also included were genes involved in sucrose breakdown and glycolysis, CO2 recycling, and amino acid synthesis, processes that are known to be accelerated in nodules compared with roots. Genes involved in membrane transport, hormone metabolism, cell wall and protein synthesis, and signal transduction and regulation of transcription were also induced in nodules. Genes that may subvert normal plant defense responses, including two encoding enzymes involved in detoxification of active oxygen species and one that may prohibit phytoalexin synthesis, were also identified. The data represent a rich source of information for hypothesis building and future exploration of symbiotic nitrogen fixation.

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Sinorhizobium meliloti nfe (Nodulation Formation Efficiency) Genes Exhibit Temporal and Spatial Expression Patterns Similar to Those of Genes Involved in Symbiotic Nitrogen Fixation

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2000.13.6.583 ◽

2000 ◽

Vol 13 (6) ◽

pp. 583-591 ◽

Cited By ~ 24

Author(s):

Fernando M. García-Rodríguez ◽

Nicolás Toro

Keyword(s):

Nitrogen Fixation ◽

Sinorhizobium Meliloti ◽

Symbiotic Nitrogen Fixation ◽

Expression Patterns ◽

Nodule Development ◽

Spatial Expression ◽

Consensus Sequences ◽

Promoter Sequences ◽

Meliloti Strain ◽

Temporal And Spatial

The nfe genes (nfeA, nfeB, and nfeD) are involved in the nodulation efficiency and competitiveness of the Sinorhizobium meliloti strain GR4 on alfalfa roots. The nfeA and nfeB genes are preceded by functional nif consensus sequences and NifA binding motifs. Here, we determined the temporal and spatial expression patterns of the nfe genes in symbiosis with alfalfa. Translational fusions of the nfe promoters with the gusA gene and reverse transcription-polymerase chain reaction analyses indicate that they are expressed and translated within mature nitrogen-fixing nodules and not during early steps of nodule development. Within the nodules the three nfe genes exhibit a spatial expression pattern similar to that of genes involved in symbiotic nitrogen fixation. We show that nfeB and nfeD genes are expressed not only from their own promoters but also from the upstream nfe promoter sequences. Furthermore, with the use of specific antibodies the NfeB and NfeD proteins were detected within the root nodule bac-teroid fraction. Finally, NfeB was inmunolocalized in the bacteroid cell membrane whereas NfeD was detected in the bacteroid cytoplasm.

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Molecular Characterization of Carbonic Anhydrase Genes in Lotus japonicus and Their Potential Roles in Symbiotic Nitrogen Fixation

International Journal of Molecular Sciences ◽

10.3390/ijms22157766 ◽

2021 ◽

Vol 22 (15) ◽

pp. 7766

Author(s):

Longlong Wang ◽

Jianjun Liang ◽

Yu Zhou ◽

Tao Tian ◽

Baoli Zhang ◽

...

Keyword(s):

Nitrogen Fixation ◽

Carbonic Anhydrase ◽

Root Nodule ◽

Higher Plants ◽

Expression Patterns ◽

Lotus Japonicus ◽

Nodule Development ◽

Model Legume ◽

N Assimilation ◽

Mature Nodule

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 (LjαCA2, LjαCA6, and LjβCA1) have been further characterized, which exhibit different spatiotemporal expression patterns during nodule development. LjαCA2 was expressed in the central infection zone of the mature nodule, including both infected and uninfected cells. LjαCA6 was restricted to the vascular bundle of the root and nodule. As for LjβCA1, 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 LjβCA1 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 CO2 fixation, N assimilation, and pH regulation, which await further investigations.

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Chickpea shows genotype-specific nodulation responses across soil nitrogen environment and root disease resistance categories

BMC Plant Biology ◽

10.1186/s12870-021-03102-6 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Krista L. Plett ◽

Sean L. Bithell ◽

Adrian Dando ◽

Jonathan M. Plett

Keyword(s):

Nitrogen Fixation ◽

Disease Resistance ◽

Soil Nitrogen ◽

Cellular Localization ◽

Expression Patterns ◽

Nodule Development ◽

Nodule Formation ◽

Symbiotic Relationship ◽

Plant Genotype ◽

Factors Affecting

Abstract Background The ability of chickpea to obtain sufficient nitrogen via its symbiotic relationship with Mesorhizobium ciceri is of critical importance in supporting growth and grain production. A number of factors can affect this symbiotic relationship including abiotic conditions, plant genotype, and disruptions to host signalling/perception networks. In order to support improved nodule formation in chickpea, we investigated how plant genotype and soil nutrient availability affect chickpea nodule formation and nitrogen fixation. Further, using transcriptomic profiling, we sought to identify gene expression patterns that characterize highly nodulated genotypes. Results A study involving six chickpea varieties demonstrated large genotype by soil nitrogen interaction effects on nodulation and further identified agronomic traits of genotypes (such as shoot weight) associated with high nodulation. We broadened our scope to consider 29 varieties and breeding lines to examine the relationship between soilborne disease resistance and the number of nodules developed and real-time nitrogen fixation. Results of this larger study supported the earlier genotype specific findings, however, disease resistance did not explain differences in nodulation across genotypes. Transcriptional profiling of six chickpea genotypes indicates that genes associated with signalling, N transport and cellular localization, as opposed to genes associated with the classical nodulation pathway, are more likely to predict whether a given genotype will exhibit high levels of nodule formation. Conclusions This research identified a number of key abiotic and genetic factors affecting chickpea nodule development and nitrogen fixation. These findings indicate that an improved understanding of genotype-specific factors affecting chickpea nodule induction and function are key research areas necessary to improving the benefits of rhizobial symbiosis in chickpea.

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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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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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