A JAZ Protein in Astragalus sinicus Interacts with a Leghemoglobin through the TIFY Domain and Is Involved in Nodule Development and Nitrogen Fixation

Abstract Background: Flavin-containing monooxygenases (FMOs) catalyze the NADPH-dependent N- or S- oxygenation of numerous foreign chemicals, and thus may mediate interactions between microorganisms and their chemical environment. The aim of this study was to investigate the role of FMO in symbiotic nitrogen fixation of Mesorhizobium huakuii and its host plant Astragalus sinicus.Results: A mutation in M. huakuii fmoA gene was generated by homologous recombination. The fmoA mutant grew more slowly than its parental strain, and displayed decreased antioxidative capacity under higher concentration of H2O2 and cumene hydroperoxide (CUOOH), indicating that FmoA plays an important role in response to the peroxides. The fmoA mutant strain displayed no difference of peroxidase activity and glutathione reductase activity, but significantly lower level of glutathione and hydrogen peroxide content than the wild type. Real-time quantitative PCR showed that the fmoA gene expression is significantly up-regulated in three different stages of nodule development. The fmoA mutant was severely impaired in its rhizosphere colonization, and its symbiotic properties in Astragalus sinicus were drastically affected. Transcriptomes in root-nodule bacteroids were analyzed and compared. A total of 1233 genes were differentially expressed, of which 560 were up-regulated and 673 were down-regulated in HKfmoA bacteroids compared with that in 7653R bacteroids. The transcriptomic data allowed us to determine additional target genes, whose differential expression was able to explain the observed the changes of symbiotic phenotype in the mutant-infected nodules.Conclusions: The fmoA gene is essential for antioxidant capacity and symbiotic nitrogen fixation. Furthermore, RNA-Seq based global transcriptomic analysis provided a comprehensive view of M. huakuii fmoA gene involved in nodule senescence and symbiotic nitrogen fixation.

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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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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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Comparative nodulation, nitrogen fixation, and growth of Glycine wightii cv. Cooper and Phaseolus atropurpureus cv. Siratro seedlings

Australian Journal of Agricultural Research ◽

10.1071/ar9720001 ◽

1972 ◽

Vol 23 (1) ◽

pp. 1 ◽

Cited By ~ 8

Author(s):

JR Wilson

Keyword(s):

Nitrogen Fixation ◽

Full Range ◽

Sand Culture ◽

Nodule Development ◽

Nitrogen Accumulation ◽

Active Nitrogen ◽

Plant Weight ◽

Culture Techniques ◽

Whole Plant ◽

Seed Reserves

The growth, nodulation, and nitrogen fixation of Glycine wightii cv. Cooper (Glycine) and Phaseolus atropurpureus cv. Siratro (Siratro) were compared in a glasshouse, sand culture techniques being used in a series of trials encompassing a full range of seasonal environments. Plants were inoculated with the commercial Rhizobium strain (CB756) for both species either at germination or 12 days later when seed reserves were virtually exhausted. In both early and late inoculation treatments, Siratro nodulated and established nitrogen fixation more rapidly than did Glycine. This difference between the species was not directly associated with the marked difference in seed size. When active nitrogen fixation was fully established, the disadvantage shown by Glycine in the early nodulation phase was not evident, and the treatments were similar in the rate of growth and nitrogen accumulation, proportion of nodule weight to whole plant weight, proportion of pigmented nodules, efficiency of nitrogen fixation, and concentration of nitrogen in the whole plant. The relatively slow initial nodule development in Glycine is probably one of the important factors hindering the field establishment of seedlings, but in subsequent growth the symbiosis appears fully effective.

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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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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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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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EVALUATION OF SEVERAL GROWING MEDIA FOR USE IN NITROGEN FIXATION RESEARCH

HortScience ◽

10.21273/hortsci.27.11.1166b ◽

1992 ◽

Vol 27 (11) ◽

pp. 1166b-1166

Author(s):

Stephanie Rademan ◽

Dyremple Marsh

Keyword(s):

Nitrogen Fixation ◽

Seed Coat ◽

Germination Rate ◽

Dry Weight ◽

Kidney Bean ◽

Nodule Development ◽

Growing Media ◽

Shoot Dry Weight ◽

Gm Plants ◽

Wide Range

A wide range of methodologies, ranging from Leonard jar to growth pouch have been used to investigate the nitrogen fixation process in leguminous crops. The effectiveness of most of these research methods have been questioned. Problems encountered vary from difficulty in root separation to water log conditions. This study was undertaken to determine the effect of different growing media on nodule development and harvestability. Black and Red seed coat kidney bean were surface sterilized and inoculated with the Rhizobium phaseoli strain UMR 1899. Seeds were planted in 8.5 cm diameter sterile clay pots containing the respective growing medium. These growing media were sand, Promix GM, Promix BX, and fritted clay. The black seed coat kidney bean had higher germination rate under all media for all dates recorded. Black kidney bean grown in sand and fritted clay had plant heights significantly greater than ones grown in the other media on the third harvest date. Nodule activities as measured by shoot dry weight and nodule number were significantly higher in both beans grown in fritted clay than in other media. Promix GM plants with dry weight of .45g for the black bean and .32g for the red beans were the lowest. Nodule separation from the growing media was easiest when plants were grown in sand, however, this was not significantly different from that of plants grown in fritted clay.

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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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Extended Hopanoid Loss Reduces Bacterial Motility and Surface Attachment and Leads to Heterogeneity in Root Nodule Growth Kinetics in a Bradyrhizobium-Aeschynomene Symbiosis

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-04-19-0111-r ◽

2019 ◽

Vol 32 (10) ◽

pp. 1415-1428

Author(s):

Brittany J. Belin ◽

Elise M. Tookmanian ◽

Jaime de Anda ◽

Gerard C. L. Wong ◽

Dianne K. Newman

Keyword(s):

Nitrogen Fixation ◽

Root Nodule ◽

Developmental Stages ◽

Growth Parameters ◽

Phenotypic Diversity ◽

Quantitative Model ◽

Nodule Development ◽

In Planta ◽

Surface Attachment

Hopanoids are steroid-like bacterial lipids that enhance membrane rigidity and promote bacterial growth under diverse stresses. Hopanoid biosynthesis genes are conserved in nitrogen-fixing plant symbionts, and we previously found that the extended (C35) class of hopanoids in Bradyrhizobium diazoefficiens are required for efficient symbiotic nitrogen fixation in the tropical legume host Aeschynomene afraspera. Here, we demonstrate that the nitrogen-fixation defect conferred by extended hopanoid loss can be fully explained by a reduction in root nodule sizes rather than per-bacteroid nitrogen-fixation levels. Using a single-nodule tracking approach to quantify A. afraspera nodule development, we provide a quantitative model of root nodule development in this host, uncovering both the baseline growth parameters for wild-type nodules and a surprising heterogeneity of extended hopanoid mutant developmental phenotypes. These phenotypes include a delay in root nodule initiation and the presence of a subpopulation of nodules with slow growth rates and low final volumes, which are correlated with reduced motility and surface attachment in vitro and lower bacteroid densities in planta, respectively. This work provides a quantitative reference point for understanding the phenotypic diversity of ineffective symbionts in A. afraspera and identifies specific developmental stages affected by extended hopanoid loss for future mechanistic work.

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