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

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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Transcriptomic Analysis of Sinorhizobium meliloti and Medicago truncatula Symbiosis Using Nitrogen Fixation–Deficient Nodules

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-12-14-0407-r ◽

2015 ◽

Vol 28 (8) ◽

pp. 856-868 ◽

Cited By ~ 15

Author(s):

Claus Lang ◽

Sharon R. Long

Keyword(s):

Gene Expression ◽

Nitrogen Fixation ◽

Bacterial Infection ◽

Medicago Truncatula ◽

Sinorhizobium Meliloti ◽

Cell Envelope ◽

Root Nodules ◽

Bacterial Cells ◽

Nitrogen Fixing ◽

Bacterial Genes

The bacterium Sinorhizobium meliloti interacts symbiotically with legume plant hosts such as Medicago truncatula to form nitrogen-fixing root nodules. During symbiosis, plant and bacterial cells differentiate in a coordinated manner, resulting in specialized plant cells that contain nitrogen-fixing bacteroids. Both plant and bacterial genes are required at each developmental stage of symbiosis. We analyzed gene expression in nodules formed by wild-type bacteria on six plant mutants with defects in nitrogen fixation. We observed differential expression of 482 S. meliloti genes with functions in cell envelope homeostasis, cell division, stress response, energy metabolism, and nitrogen fixation. We simultaneously analyzed gene expression in M. truncatula and observed differential regulation of host processes that may trigger bacteroid differentiation and control bacterial infection. Our analyses of developmentally arrested plant mutants indicate that plants use distinct means to control bacterial infection during early and late symbiotic stages.

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cDNA Macroarray Analysis of Gene Expression in Ineffective Nodules Induced on the Lotus japonicus sen1 Mutant

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2004.17.11.1223 ◽

2004 ◽

Vol 17 (11) ◽

pp. 1223-1233 ◽

Cited By ~ 20

Author(s):

Norio Suganuma ◽

Atsuko Yamamoto ◽

Ai Itou ◽

Tsuneo Hakoyama ◽

Mari Banba ◽

...

Keyword(s):

Gene Expression ◽

Nitrogen Fixation ◽

Nitrogenase Activity ◽

Cysteine Proteinase ◽

Lotus Japonicus ◽

Vascular Bundles ◽

Cdna Macroarray ◽

Plant Genes ◽

Macroarray Analysis ◽

Mutant Forms

The Lotus japonicus sen1 mutant forms ineffective nodules in which development is arrested at the stage of bacterial differentiation into nitrogen-fixing bacteroids. Here, we used cDNA macroarray systems to compare gene expression in ineffective nodules induced on the sen1 mutant with gene expression in wild-type nodules, in order to identify the host plant genes that are involved in nitrogen fixation. Macroarray analysis coupled with Northern blot analysis revealed that the expression of 18 genes was significantly enhanced in ineffective sen1 nodules, whereas the expression of 30 genes was repressed. Many of the enhanced genes encoded hydrolase enzymes, such as cysteine proteinase and asparaginase, that might function in the early senescence of sen1 nodules. By contrast, the repressed genes encoded nodulins, enzymes that are involved in carbon and nitrogen metabolism, membrane transporters, enzymes involved in phytohormone metabolism and secondary metabolism, and regulatory proteins. These proteins might have a role in the establishment of nitrogen fixation. In addition, we discovered two novel genes that encoded glutamate-rich proteins and were localized in the vascular bundles of the nodules. The expression of these genes was repressed in the ineffective nodules, which had lower levels of nitrogenase activity.

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Nitrate restricts nodule organogenesis through inhibition of cytokinin biosynthesis in Lotus japonicus

Nature Communications ◽

10.1038/s41467-021-26820-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Jieshun Lin ◽

Yuda Purwana Roswanjaya ◽

Wouter Kohlen ◽

Jens Stougaard ◽

Dugald Reid

Keyword(s):

Nitrogen Fixation ◽

Developmental Process ◽

Root Zone ◽

Root Nodules ◽

Lotus Japonicus ◽

Nodule Development ◽

Nodule Formation ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Cytokinin Biosynthesis

AbstractLegumes balance nitrogen acquisition from soil nitrate with symbiotic nitrogen fixation. Nitrogen fixation requires establishment of a new organ, which is a cytokinin dependent developmental process in the root. We found cytokinin biosynthesis is a central integrator, balancing nitrate signalling with symbiotic acquired nitrogen. Low nitrate conditions provide a permissive state for induction of cytokinin by symbiotic signalling and thus nodule development. In contrast, high nitrate is inhibitory to cytokinin accumulation and nodule establishment in the root zone susceptible to nodule formation. This reduction of symbiotic cytokinin accumulation was further exacerbated in cytokinin biosynthesis mutants, which display hypersensitivity to nitrate inhibition of nodule development, maturation and nitrogen fixation. Consistent with this, cytokinin application rescues nodulation and nitrogen fixation of biosynthesis mutants in a concentration dependent manner. These inhibitory impacts of nitrate on symbiosis occur in a Nlp1 and Nlp4 dependent manner and contrast with the positive influence of nitrate on cytokinin biosynthesis that occurs in species that do not form symbiotic root nodules. Altogether this shows that legumes, as exemplified by Lotus japonicus, have evolved a different cytokinin response to nitrate compared to non-legumes.

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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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Starch content and activities of starch-metabolizing enzymes in effective and ineffective root nodules of soybean

Canadian Journal of Botany ◽

10.1139/b91-094 ◽

1991 ◽

Vol 69 (4) ◽

pp. 697-701 ◽

Cited By ~ 15

Author(s):

Sharon I. Forrest ◽

Desh Pal S. Verma ◽

Rajinder S. Dhindsa

Keyword(s):

Nitrogen Fixation ◽

Starch Content ◽

Root Nodules ◽

Starch Synthase ◽

Nodule Development ◽

Wild Type ◽

Starch Phosphorylase ◽

High Concentration ◽

Key Words Nitrogen Fixation ◽

Glycine Max L

Starch content and activities of some enzymes of starch metabolism were determined in wild-type, N2-fixing (fix+) nodules and in two non-N2-fixing (fix−) nodules induced by Bradyrhizobium japonicum mutant strains, T5-95 and T8-1, on soybean (Glycine max L.) roots. The T5-95 nodules are similar to wild type in ultrastructure, but the T8-1 nodules are different in that the bacteroids are not released from the infection thread. After initial accumulation to relatively high concentration, starch was depleted during nitrogen fixation in fix+ nodules. However, in fix− nodules, the accumulated starch was not metabolized. The activity of starch-bound starch synthase (EC 2.4.1.21) declined in fix+ nodules but remained high in fix− nodules. The activity of α-amylase (EC 3.2.1.1) was only slightly higher than wild type in T5-95 but was four times higher than wild type in T8-1 nodules. The activity of starch phosphorylase (EC 2.4.1.1) increased in all nodule types from 14 to 21 days postinfection. A positive correlation was observed between the capacity of nodules to fix N2 and their capacity to degrade starch. Collectively, these results support the concept that starch accumulated during early stages of nodule development is metabolized to supply energy for nitrogen fixation and to meet the metabolic demands of bacteroids. Key words: nitrogen fixation, starch content, effective and ineffective nodules, starch synthase, starch phosphorylase, α-amylase.

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Defects in Rhizobial Cyclic Glucan and Lipopolysaccharide Synthesis Alter Legume Gene Expression During Nodule Development

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-21-1-0050 ◽

2008 ◽

Vol 21 (1) ◽

pp. 50-60 ◽

Cited By ~ 18

Author(s):

Alejandra L. D'Antuono ◽

Thomas Ott ◽

Lene Krusell ◽

Vera Voroshilova ◽

Rodolfo A. Ugalde ◽

...

Keyword(s):

Gene Expression ◽

Mutant Strain ◽

Early Recognition ◽

Lotus Japonicus ◽

Cdna Array ◽

Nodule Development ◽

Marker Genes ◽

Wild Type ◽

Mesorhizobium Loti ◽

Expression Of Genes

cDNA array technology was used to compare transcriptome profiles of Lotus japonicus roots inoculated with a Mesorhizobium loti wild-type and two mutant strains affected in cyclic β(1-2) glucan synthesis (cgs) and in lipopolysaccharide synthesis (lpsβ2). Expression of genes associated with the development of a fully functional nodule was significantly affected in plants inoculated with the cgs mutant. Array results also revealed that induction of marker genes for nodule development was delayed when plants were inoculated with the lpsβ2 mutant. Quantitative real-time reverse-transcriptase polymerase chain reaction was used to quantify gene expression of a subset of genes involved in plant defense response, redox metabolism, or genes that encode for nodulins. The majority of the genes analyzed in this study were more highly expressed in roots inoculated with the wild type compared with those inoculated with the cgs mutant strain. Some of the genes exhibited a transient increase in transcript levels during intermediate steps of normal nodule development while others displayed induced expression during the final steps of nodule development. Ineffective nodules induced by the glucan mutant showed higher expression of phenylalanine ammonia lyase than wild-type nodules. Differences in expression pattern of genes involved in early recognition and signaling were observed in plants inoculated with the M. loti mutant strain affected in the synthesis of cyclic glucan.

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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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Transcription Profiling of Soybean Nodulation by Bradyrhizobium japonicum

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-21-5-0631 ◽

2008 ◽

Vol 21 (5) ◽

pp. 631-645 ◽

Cited By ~ 64

Author(s):

Laurent Brechenmacher ◽

Moon-Young Kim ◽

Marisol Benitez ◽

Min Li ◽

Trupti Joshi ◽

...

Keyword(s):

Nitrogen Fixation ◽

Bradyrhizobium Japonicum ◽

Root Nodules ◽

Expression Patterns ◽

Root Hairs ◽

Defense Responses ◽

Nodule Development ◽

Plant Defense Responses ◽

Polymerase Chain ◽

High Level

Legumes interact with nodulating bacteria that convert atmospheric nitrogen into ammonia for plant use. This nitrogen fixation takes place within root nodules that form after infection of root hairs by compatible rhizobia. Using cDNA microarrays, we monitored gene expression in soybean (Glycine max) inoculated with the nodulating bacterium Bradyrhizobium japonicum 4, 8, and 16 days after inoculation, timepoints that coincide with nodule development and the onset of nitrogen fixation. This experiment identified several thousand genes that were differentially expressed in response to B. japonicum inoculation. Expression of 27 genes was analyzed by quantitative reverse transcriptase-polymerase chain reaction, and their expression patterns mimicked the microarray results, confirming integrity of analyses. The microarray results suggest that B. japonicum reduces plant defense responses during nodule development. In addition, the data revealed a high level of regulatory complexity (transcriptional, post-transcriptional, translational, post-translational) that is likely essential for development of the symbiosis and adjustment to an altered nutritional status.

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Minimal gene set from Sinorhizobium (Ensifer) meliloti pSymA required for efficient symbiosis with Medicago

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2018015118 ◽

2020 ◽

Vol 118 (2) ◽

pp. e2018015118

Author(s):

Barney A. Geddes ◽

Jason V. S. Kearsley ◽

Jiarui Huang ◽

Maryam Zamani ◽

Zahed Muhammed ◽

...

Keyword(s):

Root Nodules ◽

Nodule Development ◽

Nodule Formation ◽

Protein Coding ◽

Bacterial Gene ◽

Gene Set ◽

Background Strain ◽

Genetic Components ◽

Sustainable Agricultural Systems ◽

Bacterial Genes

Reduction of N2 gas to ammonia in legume root nodules is a key component of sustainable agricultural systems. Root nodules are the result of a symbiosis between leguminous plants and bacteria called rhizobia. Both symbiotic partners play active roles in establishing successful symbiosis and nitrogen fixation: while root nodule development is mostly controlled by the plant, the rhizobia induce nodule formation, invade, and perform N2 fixation once inside the plant cells. Many bacterial genes involved in the rhizobia–legume symbiosis are known, and there is much interest in engineering the symbiosis to include major nonlegume crops such as corn, wheat, and rice. We sought to identify and combine a minimal bacterial gene complement necessary and sufficient for symbiosis. We analyzed a model rhizobium, Sinorhizobium (Ensifer) meliloti, using a background strain in which the 1.35-Mb symbiotic megaplasmid pSymA was removed. Three regions representing 162 kb of pSymA were sufficient to recover a complete N2-fixing symbiosis with alfalfa, and a targeted assembly of this gene complement achieved high levels of symbiotic N2 fixation. The resulting gene set contained just 58 of 1,290 pSymA protein-coding genes. To generate a platform for future synthetic manipulation, the minimal symbiotic genes were reorganized into three discrete nod, nif, and fix modules. These constructs will facilitate directed studies toward expanding the symbiosis to other plant partners. They also enable forward-type approaches to identifying genetic components that may not be essential for symbiosis, but which modulate the rhizobium’s competitiveness for nodulation and the effectiveness of particular rhizobia–plant symbioses.

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