scholarly journals A Toolbox for Nodule Development Studies in Chickpea: A Hairy-Root Transformation Protocol and an Efficient Laboratory Strain of Mesorhizobium sp.

2019 ◽  
Vol 32 (4) ◽  
pp. 367-378 ◽  
Author(s):  
Drishti Mandal ◽  
Senjuti Sinharoy
Keyword(s):  
Nitrogen Fixation ◽  
Hairy Root ◽  
Root Nodules ◽  
Laboratory Strain ◽  
Nodule Development ◽  
Model Legume ◽  
Transformation Protocol ◽  
Evolutionary Diversification ◽  
Fluorescent Markers ◽  
Root Transformation

A Mesorhizobium sp. produces root nodules in chickpea. Chickpea and model legume Medicago truncatula are members of the inverted repeat–lacking clade (IRLC). The rhizobia, after internalization into the plant cell, are called bacteroids. Nodule-specific cysteine-rich peptides in IRLC legumes guide bacteroids to a terminally differentiated swollen (TDS) form. Bacteroids in chickpea are less TDS than those in Medicago spp. Nodule development in chickpea indicates recent evolutionary diversification and merits further study. A hairy-root transformation protocol and an efficient laboratory strain are prerequisites for performing any genetic study on nodulation. We have standardized a protocol for composite plant generation in chickpea with a transformation frequency above 50%, as shown by fluorescent markers. This protocol also works well in different ecotypes of chickpea. Localization of subcellular markers in these transformed roots is similar to the localization observed in transformed Medicago roots. When checked inside transformed nodules, peroxisomes were concentrated along the periphery of the nodules, while endoplasmic reticulum and Golgi bodies surrounded the symbiosomes. Different Mesorhizobium strains were evaluated for their ability to initiate nodule development and efficiency of nitrogen fixation. Inoculation with different strains resulted in different shapes of TDS bacteroids with variable nitrogen fixation. Our study provides a toolbox to study nodule development in the crop legume chickpea.

scholarly journals A toolbox for nodule development studies in chickpea: a hairy-root transformation protocol and an efficient laboratory strain of Mesorhizobium sp

2018 ◽  
Author(s):  
Drishti Mandal ◽  
Senjuti Sinharoy
Keyword(s):  
Nitrogen Fixation ◽  
Hairy Root ◽  
Root Nodules ◽  
Laboratory Strain ◽  
Nodule Development ◽  
Model Legume ◽  
Transformation Protocol ◽  
Evolutionary Diversification ◽  
Fluorescent Markers ◽  
Root Transformation

AbstractMesorhizobium sp. produces root nodules in chickpea. Chickpea and model legume Medicago truncatula are members of inverted repeat lacking clade (IRLC). The rhizobia after internalization inside plant cell called ‘bacteroid’. Nodule Specific Cysteine-rich (NCR) peptides in IRLC legumes guide bacteroids to a ‘terminally differentiated swollen (TDS)’ form. Bacteroids in chickpea are less TDS than those in Medicago. Nodule development in chickpea indicates recent evolutionary diversification and merits further study. A hairy root transformation protocol and an efficient laboratory strain are prerequisites for performing any genetic study on nodulation. We have standardized a protocol for composite plant generation in chickpea with a transformation frequency above 50%, as shown by fluorescent markers. This protocol also works well in different ecotypes of chickpea. Localization of subcellular markers in these transformed roots is similar to Medicago. When checked inside transformed nodules, peroxisomes were concentrated along the periphery of the nodules, while ER and golgi bodies surrounded the symbiosomes. Different Mesorhizobium strains were evaluated for their ability to initiate nodule development, and efficiency of nitrogen fixation. Inoculation with different strains resulted in different shapes of TDS bacteroids with variable nitrogen fixation. Our study provides a toolbox to study nodule development in the crop legume chickpea.

scholarly journals Gene Silencing by Expression of Hairpin RNA in Lotus japonicus Roots and Root Nodules

2003 ◽  
Vol 16 (8) ◽  
pp. 663-668 ◽  
Author(s):  
Hirotaka Kumagai ◽  
Hiroshi Kouchi
Keyword(s):  
Hairy Root ◽  
Hairy Roots ◽  
Rna Silencing ◽  
Root Nodules ◽  
Lotus Japonicus ◽  
Loss Of Function ◽  
Coding Region ◽  
Hairpin Rna ◽  
Model Legume ◽  
Root Transformation

We investigated the efficacy of self-complementary hairpin RNA (hpRNA) expression to induce RNA silencing in the roots and nodules of model legume Lotus japonicus, using hairy root transformation mediated by Agrobacterium rhizogenes. Transgenic lines that express β-glucuronidase (GUS) by constitutive or nodule-specific promoters were supertransformed by infection of A. rhizogenes harboring constructs for the expression of hpRNAs with sequences complementary to the GUS coding region. GUS activity in more than 60% of the hairy roots was decreased or silenced almost completely. Silencing of the GUS gene was also observed in symbiotic nodules formed on hairy roots in both early and late stages of nodule organogenesis. These results indicate that transient RNA silencing by hairy root transformation provides a powerful tool for loss-of-function analyses of genes that function in roots and root nodules.

scholarly journals Sinorhizobium meliloti Controls Nitric Oxide–Mediated Post-Translational Modification of a Medicago truncatula Nodule Protein

2015 ◽  
Vol 28 (12) ◽  
pp. 1353-1363 ◽  
Author(s):  
Pauline Blanquet ◽  
Liliana Silva ◽  
Olivier Catrice ◽  
Claude Bruand ◽  
Helena Carvalho ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitrogen Fixation ◽  
Medicago Truncatula ◽  
Sinorhizobium Meliloti ◽  
Molecular Mechanisms ◽  
Root Nodules ◽  
Plant Protein ◽  
Post Translational Modification ◽  
Model Legume ◽  
Bacterial Mutants

Nitric oxide (NO) is involved in various plant-microbe interactions. In the symbiosis between soil bacterium Sinorhizobium meliloti and model legume Medicago truncatula, NO is required for an optimal establishment of the interaction but is also a signal for nodule senescence. Little is known about the molecular mechanisms responsible for NO effects in the legume-rhizobium interaction. Here, we investigate the contribution of the bacterial NO response to the modulation of a plant protein post-translational modification in nitrogen-fixing nodules. We made use of different bacterial mutants to finely modulate NO levels inside M. truncatula root nodules and to examine the consequence on tyrosine nitration of the plant glutamine synthetase, a protein responsible for assimilation of the ammonia released by nitrogen fixation. Our results reveal that S. meliloti possesses several proteins that limit inactivation of plant enzyme activity via NO-mediated post-translational modifications. This is the first demonstration that rhizobia can impact the course of nitrogen fixation by modulating the activity of a plant protein.

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

1991 ◽  
Vol 69 (4) ◽  
pp. 697-701 ◽  
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.

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

2009 ◽  
Vol 22 (7) ◽  
pp. 800-808 ◽  
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.

scholarly journals Transcription Profiling of Soybean Nodulation by Bradyrhizobium japonicum

2008 ◽  
Vol 21 (5) ◽  
pp. 631-645 ◽  
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.

scholarly journals Characterization of a Mesorhizobium loti α-Type Carbonic Anhydrase and Its Role in Symbiotic Nitrogen Fixation

2009 ◽  
Vol 191 (8) ◽  
pp. 2593-2600 ◽  
Author(s):  
Chrysanthi Kalloniati ◽  
Daniela Tsikou ◽  
Vasiliki Lampiri ◽  
Mariangela N. Fotelli ◽  
Heinz Rennenberg ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Carbonic Anhydrase ◽  
Mutant Strain ◽  
Symbiotic Nitrogen Fixation ◽  
Plant Traits ◽  
Nitrogenase Activity ◽  
Nodule Development ◽  
Free Living ◽  
Model Legume ◽  
Mesorhizobium Loti

ABSTRACT Carbonic anhydrase (CA) (EC 4.2.1.1) is a widespread enzyme catalyzing the reversible hydration of CO2 to bicarbonate, a reaction that participates in many biochemical and physiological processes. Mesorhizobium loti, the microsymbiont of the model legume Lotus japonicus, possesses on the symbiosis island a gene (msi040) encoding an α-type CA homologue, annotated as CAA1. In the present work, the CAA1 open reading frame from M. loti strain R7A was cloned, expressed, and biochemically characterized, and it was proven to be an active α-CA. The biochemical and physiological roles of the CAA1 gene in free-living and symbiotic rhizobia were examined by using an M. loti R7A disruption mutant strain. Our analysis revealed that CAA1 is expressed in both nitrogen-fixing bacteroids and free-living bacteria during growth in batch cultures, where gene expression was induced by increased medium pH. L. japonicus plants inoculated with the CAA1 mutant strain showed no differences in top-plant traits and nutritional status but consistently formed a higher number of nodules exhibiting higher fresh weight, N content, nitrogenase activity, and δ13C abundance. Based on these results, we propose that although CAA1 is not essential for nodule development and symbiotic nitrogen fixation, it may participate in an auxiliary mechanism that buffers the bacteroid periplasm, creating an environment favorable for NH3 protonation, thus facilitating its diffusion and transport to the plant. In addition, changes in the nodule δ13C abundance suggest the recycling of at least part of the HCO3 − produced by CAA1.

scholarly journals A Convenient, Soil-Free Method for the Production of Root Nodules in Soybean to Study the Effects of Exogenous Additives

2018 ◽  
Author(s):  
Swarup Roy Choudhury ◽  
Sarah M. Johns ◽  
Sona Pandey
Keyword(s):  
Nitrogen Fixation ◽  
Biological Nitrogen Fixation ◽  
Root Nodules ◽  
Growth Conditions ◽  
Nodule Development ◽  
Nodule Formation ◽  
Legume Crop ◽  
Simple Protocol ◽  
Biological Nitrogen

Legumes develop root nodules that harbour endosymbiotic bacteria, rhizobia. These rhizobia convert nitrogen to ammonia by biological nitrogen fixation. A thorough understanding of the biological nitrogen fixation in legumes and its regulation is key to develop sustainable agriculture. It is well known that plant hormones affect nodule formation; however, most studies are limited to model legumes due to their suitability for in vitro, plate-based assays. Specifically, it is almost impossible to measure the effects of exogenous hormones or other additives during nodule development in crop legumes such as soybean as they have huge root system in soil. To circumvent this issue, the present research develops suitable media and growth conditions for efficient nodule development under in vitro, soil free conditions in an important legume crop, soybean. Moreover, we also evaluate the effects of all major phytohormones during soybean nodulation under identical conditions. This versatile, inexpensive, scalable and simple protocol provides several advantages over previously established methods. It is extremely time-and resource-efficient, does not require special training or equipment, and produces highly reproducible results. The approach is expandable to other large legumes as well as for other exogenous additives.

scholarly journals The legume-rhizobia symbiosis can be supported on Mars soil simulants

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0259957
Author(s):  
Randall Rainwater ◽  
Arijit Mukherjee
Keyword(s):  
Nitrogen Fixation ◽  
Sinorhizobium Meliloti ◽  
Root Nodules ◽  
Lateral Roots ◽  
Model Legume ◽  
Mars Atmosphere ◽  
Future Studies ◽  
Sinorhizobium Medicae ◽  
Total Plant ◽  
Atmospheric Nitrogen Fixation

Legumes (soybeans, peas, lentils, etc.) play important roles in agriculture on Earth because of their food value and their ability to form a mutualistic beneficial association with rhizobia bacteria. In this association, the host plant benefits from atmospheric nitrogen fixation by rhizobia. The presence of nitrogen in the Mars atmosphere offers the possibility to take advantage of this important plant-microbe association. While some studies have shown that Mars soil simulants can support plant growth, none have investigated if these soils can support the legume-rhizobia symbiosis. In this study, we investigated the establishment of the legume-rhizobia symbiosis on different Mars soil simulants (different grades of the Mojave Mars Simulant (MMS)-1: Coarse, Fine, Unsorted, Superfine, and the MMS-2 simulant). We used the model legume, Medicago truncatula, and its symbiotic partners, Sinorhizobium meliloti and Sinorhizobium medicae, in these experiments. Our results show that root nodules could develop on M. truncatula roots when grown on these Mars soil simulants and were comparable to those formed on plants that were grown on sand. We also detected nifH (a reporter gene for nitrogen fixation) expression inside these nodules. Our results indicate that the different Mars soil simulants used in this study can support legume-rhizobia symbiosis. While the average number of lateral roots and nodule numbers were comparable on plants grown on the different soil simulants, total plant mass was higher in plants grown on MMS-2 soil than on MMS-1 soil and its variants. Our results imply that the chemical composition of the simulants is more critical than their grain size for plant mass. Based on these results, we recommend that the MMS-2 Superfine soil simulant is a better fit than the MMS-1 soil and it’s variants for future studies. Our findings can serve as an excellent resource for future studies investigating beneficial plant-microbe associations for sustainable agriculture on Mars.

scholarly journals Expression Map for Genes Involved in Nitrogen and Carbon Metabolism in Alfalfa Root Nodules

1999 ◽  
Vol 12 (6) ◽  
pp. 526-535 ◽  
Author(s):  
Gian B. Trepp ◽  
Stephen J. Temple ◽  
Bruna Bucciarelli ◽  
Li Fang Shi ◽  
Carroll P. Vance
Keyword(s):  
Nitrogen Fixation ◽  
Glutamine Synthetase ◽  
Carbon Metabolism ◽  
Root Nodule ◽  
Root Nodules ◽  
Constitutive Expression ◽  
Nodule Development ◽  
Relative Distribution ◽  
Nitrogen Fixing ◽  
Alfalfa Root

During root nodule development several key genes involved in nitrogen fixation and assimilation exhibit enhanced levels of expression. However, little is known about the temporal and spatial distribution patterns of these transcripts. In a systematic study the transcripts for 13 of the essential enzymes involved in alfalfa (Medicago sativa) root nodule nitrogen and carbon metabolism were localized by in situ hybridization. A serial section approach allowed the construction of a map that reflects the relative distribution of these transcripts. In 33-day-old root nodules, the expression of nifH, NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) and a cytosolic isoform of glutamine synthetase (GS13; GS; EC 6.3.1.2) were localized predominantly in a 5- to 15-cell-wide region in the distal part of the nitrogen-fixing zone. This zone was also the region of high expression for leghemoglobin, a second cytosolic glutamine synthetase isoform (GS100), aspartate aminotransferase-2 (AAT-2; EC 2.6.1.1), asparagine synthetase (AS; 6.3.5.4), phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31), and sucrose synthase (SuSy; EC 2.4.1.13). This suggests that, in 33-day-old alfalfa root nodules, nitrogen fixation is restricted to this 5- to 15-cell-wide area. The continued significant expression of the GS100 subclass of GS and AS in the proximal part of the nitrogen-fixing zone implicates these gene products in nitrogen remobilization. A low constitutive expression of NADH-dependent glutamate dehydrogenase (NADH-GDH; EC 1.4.1.2) was observed throughout the nodule. The transcript distribution map will be used as a navigational tool to assist in developing strategies for the genetic engineering of alfalfa root nodules for enhanced nitrogen assimilation.

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