Two seasons’ study on nifH gene expression and nitrogen fixation by diazotrophic endophytes in sugarcane (Saccharum spp. hybrids): expression of nifH genes similar to those of rhizobia

2010 ◽  
Vol 338 (1-2) ◽  
pp. 435-449 ◽  
Author(s):  
Nisarat Thaweenut ◽  
Yusuke Hachisuka ◽  
Shotaro Ando ◽  
Shuichi Yanagisawa ◽  
Tadakatsu Yoneyama
Keyword(s):  
Gene Expression ◽  
Nitrogen Fixation ◽  
Nifh Gene ◽  
Saccharum Spp ◽  
Diazotrophic Endophytes ◽  
Nifh Genes

scholarly journals Seasonal dynamics of nitrogen fixation and the diazotroph community in the temperate coastal region of the northwestern North Pacific

2015 ◽  
Vol 12 (1) ◽  
pp. 865-889
Author(s):  
T. Shiozaki ◽  
T. Nagata ◽  
M. Ijichi ◽  
K. Furuya
Keyword(s):  
Nitrogen Fixation ◽  
North Pacific ◽  
Gene Diversity ◽  
Nifh Gene ◽  
Anaerobic Bacteria ◽  
Coastal Region ◽  
Euphotic Zone ◽  
Early Summer ◽  
Subsurface Layers ◽  
Nifh Genes

Abstract. Nitrogen fixation in temperate oceans is a potentially important, but poorly understood process that may influence the marine nitrogen budget. This study determined seasonal variations in nitrogen fixation and nifH gene diversity within the euphotic zone in the temperate coastal region of the northwestern North Pacific. Nitrogen fixation as high as 13.6 nmolN L−1 d−1 was measured from early summer to fall when the surface temperature exceeded 14.2 °C and the surface nitrate concentration was low (≤ 0.30 μM), although we also detected nitrogen fixation in subsurface layers (42–62 m) where nitrate concentrations were high (> 1 μM). During periods with high nitrogen fixation, the nifH sequences of UCYN-A were recovered, suggesting that these groups played a key role in nitrogen fixation. The nifH genes were also recovered in spring and winter when nitrogen fixation was undetectable. These genes consisted of many sequences affiliated with Cluster III diazotrophs (putative anaerobic bacteria), which hitherto have rarely been reported to be abundant in surface diazotroph communities in marine environments.

scholarly journals mRNA Extraction and Reverse Transcription-PCR Protocol for Detection of nifH Gene Expression by Azotobacter vinelandii in Soil

2003 ◽  
Vol 69 (4) ◽  
pp. 1928-1935 ◽  
Author(s):  
Helmut Bürgmann ◽  
Franco Widmer ◽  
William V. Sigler ◽  
Josef Zeyer
Keyword(s):  
Gene Expression ◽  
Nitrogen Fixation ◽  
Reverse Transcription ◽  
Liquid Culture ◽  
Azotobacter Vinelandii ◽  
Nifh Gene ◽  
Rna Extraction ◽  
Soil Microbial Communities ◽  
Nitrogen Fixing ◽  
Reverse Transcription Pcr

ABSTRACT The study of free-living nitrogen-fixing organisms in bulk soil is hampered by the great diversity of soil microbial communities and the difficulty of relating nitrogen fixation activities to individual members of the diazotroph populations. We developed a molecular method that allows analysis of nifH mRNA expression in soil in parallel with determinations of nitrogen-fixing activity and bacterial growth. In this study, Azotobacter vinelandii growing in sterile soil and liquid culture served as a model system for nifH expression, in which sucrose served as the carbon source and provided nitrogen-limited conditions, while amendments of NH4NO3 were used to suppress nitrogen fixation. Soil RNA extraction was performed with a new optimized direct extraction protocol that yielded nondegraded total RNA. The RNA extracts were of high purity, free of DNA contamination, and allowed highly sensitive and specific detection of nifH mRNA by a reverse transcription-PCR. The level of nifH gene expression was estimated by PCR amplification of reverse-transcribed nifH mRNA fragments with A. vinelandii-specific nifH primers. This new approach revealed that nifH gene expression was positively correlated with bulk nitrogen fixation activity in soil (r 2 = 0.72) and in liquid culture (r 2 = 0.84) and therefore is a powerful tool for studying specific regulation of gene expression directly in the soil environment.

scholarly journals A reduced fraction of plant N derived from atmospheric N (%Ndfa) and reduced rhizobial nifH gene numbers indicate a lower capacity for nitrogen fixation in nodules of white clover exposed to long-term CO<sub>2</sub> enrichment

2013 ◽  
Vol 10 (12) ◽  
pp. 8269-8281 ◽  
Author(s):  
T. Watanabe ◽  
S. Bowatte ◽  
P. C. D. Newton
Keyword(s):  
Nitrogen Fixation ◽  
Elevated Co2 ◽  
White Clover ◽  
Trifolium Repens ◽  
Nifh Gene ◽  
Shoot Biomass ◽  
N Fixation ◽  
Dna Amount ◽  
Trifolium Repens L ◽  
Nifh Genes

Abstract. Using the δ15N natural abundance method, we found that the fraction of nitrogen derived from atmospheric N (%Ndfa) in field-grown white clover (Trifolium repens L.) plants was significantly lower (72.0% vs. 89.8%, p = 0.047 in a grassland exposed to elevated CO2 for 13 yr using free air carbon dioxide enrichment (FACE). Twelve months later we conducted an experiment to investigate the reasons behind the reduced N fixation. We took cuttings from white clover plants growing in the FACE and established individual plants in a glasshouse using soil from the appropriate ambient or elevated CO2 treatments. The established plants were then transplanted back into their "rings of origin" and sampled over a 6-week period. We used molecular ecological analyses targeting nifH genes and transcripts of rhizobia in symbiosis with white clover (Trifolium repens L.) to understand the potential mechanisms. Shoot biomass was significantly lower in eCO2, but there was no difference in nodule number or mass per plant. The numbers of nifH genes and gene transcripts per nodule were significantly reduced under eCO2, but the ratio of gene to transcript number and the strains of rhizobia present were the same in both treatments. We conclude that the capacity for biological nitrogen fixation was reduced by eCO2 in white clover and was related to the reduced rhizobia numbers in nodules. We discuss the finding of reduced gene number in relation to factors controlling bacteroid DNA amount, which may imply an influence of nitrogen as well as phosphorus.

Biological nitrogen fixation and nifH gene expression in dry beans (Phaseolus vulgaris L.)

2014 ◽  
Vol 94 (2) ◽  
pp. 203-212 ◽  
Author(s):  
Zafrin Akter ◽  
Binod B. Pageni ◽  
Newton Z. Lupwayi ◽  
Parthiba M. Balasubramanian
Keyword(s):  
Gene Expression ◽  
Nitrogen Fixation ◽  
Phaseolus Vulgaris ◽  
Biological Nitrogen Fixation ◽  
Nifh Gene ◽  
Rapid Test ◽  
Dry Beans ◽  
Phaseolus Vulgaris L ◽  
Dry Bean ◽  
Biological Nitrogen

Akter, Z., Pageni, B. B., Lupwayi, N. Z. and Balasubramanian, P. M. 2014. Biological nitrogen fixation and nif H gene expression in dry beans ( Phaseolus vulgaris L.). Can. J. Plant Sci. 94: 203–212. Dry bean (Phaseolus vulgaris L.) is one of the least efficient nitrogen (N2) fixers among legumes (approximately 30 to 50 kg N ha−1). To identify efficient N2 fixers, 22 dry bean genotypes were screened for symbiotic N2-fixation in potted low-N soil in the greenhouse under four treatments: (1) uninoculated, (2) inoculated with commercial Rhizobium leguminosarum bv. phaseoli inoculant Nitrastik-D®, (3) inoculated with commercial Rhizobium inoculant Nodulator®, and (4) fertilizer N added. Expression of nitrogenase reductase (nifH) gene in selected uninoculated genotypes (nodulated by indigenous rhizobia) was quantified by reverse transcription polymerase chain reaction (RT-PCR) to know if expression of this gene was an indicator of N2-fixation. Nitrogen fixation potential varied among the genotypes and between the rhizobia inoculants. Genotypes differed in shoot (5.17 to 8.39 g plant−1) and root (1.71 to 6.44 g plant−1) dry weights, nodule number (0 to 155 nodules plant−1) and mass (0.055 to 0.250 mg plant−1), and N2-fixed (0 to 129 mg N plant−1). Genotypes GH–196, FR 266 and AC Redbond fixed the most N2 (60 to 112 mg N plant−1) with either rhizobial inoculant, and Viva fixed the highest amount (129 mg N plant−1) when inoculated with Nitrastik-D. In contrast, AC Black Diamond, Island, Winchester, AC Polaris, LEF 2RB and SEA 5 had low N2-fixing potential (3 to 23 mg N plant−1) in inoculated treatments. The qRT-PCR analysis indicated that nifH gene expression was 5 fold higher in GH-196 compared to the non-nodulating mutant R99, which further confirmed the high N2-fixation capacity of GH-196. After confirmation in the field, dry bean genotypes with improved N2-fixation potential identified in this study may be used as parents in breeding programs in the development of future cultivars. The nifH gene expression may be used as a rapid test to select dry beans with high N2-fixation potential.

scholarly journals A reduced fraction of plant N derived from atmospheric N (%Ndfa) and reduced rhizobial nifH gene numbers indicate a lower capacity for nitrogen fixation in nodules of white clover exposed to long-term CO<sub>2</sub> enrichment

2013 ◽  
Vol 10 (6) ◽  
pp. 9867-9896 ◽  
Author(s):  
T. Watanabe ◽  
S. Bowatte ◽  
P. C. D. Newton
Keyword(s):  
Nitrogen Fixation ◽  
Elevated Co2 ◽  
White Clover ◽  
Trifolium Repens ◽  
Nifh Gene ◽  
Shoot Biomass ◽  
N Fixation ◽  
Dna Amount ◽  
Trifolium Repens L ◽  
Nifh Genes

Abstract. Using the δ15N natural abundance method, we found that the fraction of nitrogen derived from atmospheric N (%Ndfa) in field grown white clover (Trifolium repens L.) plants was significantly lower (72.0% vs. 89.5%, p = 0.047 in a grassland exposed to elevated CO2 for 13 yr using Free Air Carbon Dioxide Enrichment (FACE). Twelve months later we conducted an experiment to investigate the reasons behind the reduced N fixation. We took cuttings from white clover plants growing in the FACE and established individual plants in a glasshouse using soil from the appropriate ambient or elevated CO2 treatments. The established plants were then transplanted back into their "rings of origin" and sampled over a 6 week period. We used molecular ecological analyses targeting nifH genes and transcripts of rhizobia in symbiosis with white clover (Trifolium repens L.) to understand the potential mechanisms. Shoot biomass was significantly lower in eCO2 but there was no difference in nodule number or mass per plant. The numbers of nifH genes and gene transcripts per nodule were significantly reduced under eCO2 but the ratio of gene to transcript number and the strains of rhizobia present were the same in both treatments. We conclude that the capacity for biological nitrogen fixation was reduced by eCO2 in white clover and was related to the reduced rhizobia numbers in nodules. We discuss the finding of reduced gene number in relation to factors controlling bacteroid DNA amount which may imply an influence of nitrogen as well as phosphorus.

scholarly journals Endophytic Expression of nif Genes of Azoarcus sp. Strain BH72 in Rice Roots

1999 ◽  
Vol 12 (9) ◽  
pp. 813-819 ◽  
Author(s):  
Tanja Egener ◽  
Thomas Hurek ◽  
Barbara Reinhold-Hurek
Keyword(s):  
Gene Expression ◽  
Nitrogen Fixation ◽  
Oryza Sativa ◽  
Nifh Gene ◽  
Immunogold Labeling ◽  
Nif Genes ◽  
Iron Protein ◽  
Rice Roots ◽  
Transcriptional Fusions ◽  
Gene Expression Levels

Transcriptional fusions of gusA and gfp to the nifH gene as well as immunogold labeling with antibodies against the iron protein of nitrogenase revealed high nitrogenase gene expression levels of the endophyte Azoarcus sp. strain BH72 inside infected rice roots (Oryza sativa) in the laboratory. Thus, environmental conditions inside rice roots are permissive for endophytic nitrogen fixation in bacterial microcolonies in the aerenchyma.

scholarly journals Mutation in the ntrR Gene, a Member of the vap Gene Family, Increases the Symbiotic Efficiency of Sinorhizobium meliloti

2001 ◽  
Vol 14 (7) ◽  
pp. 887-894 ◽  
Author(s):  
Boglárka Oláh ◽  
Erno Kiss ◽  
Zoltán Györgypál ◽  
Judit Borzi ◽  
Gyöngyi Cinege ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Pathogenic Bacteria ◽  
Sinorhizobium Meliloti ◽  
Root Nodules ◽  
Nifh Gene ◽  
Dry Weight ◽  
Nodule Occupancy ◽  
Gene Encoding ◽  
Symbiotic Efficiency ◽  
Host Interactions

In specific plant organs, namely the root nodules of alfalfa, fixed nitrogen (ammonia) produced by the symbiotic partner Sinorhizobium meliloti supports the growth of the host plant in nitrogen-depleted environment. Here, we report that a derivative of S. meliloti carrying a mutation in the chromosomal ntrR gene induced nodules with enhanced nitrogen fixation capacity, resulting in an increased dry weight and nitrogen content of alfalfa. The efficient nitrogen fixation is a result of the higher expression level of the nifH gene, encoding one of the subunits of the nitrogenase enzyme, and nifA, the transcriptional regulator of the nif operon. The ntrR gene, controlled negatively by its own product and positively by the symbiotic regulator syrM, is expressed in the same zone of nodules as the nif genes. As a result of the nitrogen-tolerant phenotype of the strain, the beneficial effect of the mutation on efficiency is not abolished in the presence of the exogenous nitrogen source. The ntrR mutant is highly competitive in nodule occupancy compared with the wild-type strain. Sequence analysis of the mutant region revealed a new cluster of genes, termed the “ntrPR operon,” which is highly homologous to a group of vap-related genes of various pathogenic bacteria that are presumably implicated in bacterium-host interactions. On the basis of its favorable properties, the strain is a good candidate for future agricultural utilization.

Endophytic nitrogen fixation – a possible ‘hidden’ source of nitrogen for lodgepole pine trees growing at unreclaimed gravel mining sites

2019 ◽  
Vol 95 (11) ◽  
Author(s):  
Kiran Preet Padda ◽  
Akshit Puri ◽  
Chris Chanway
Keyword(s):  
Nitrogen Fixation ◽  
Lodgepole Pine ◽  
Nifh Gene ◽  
Control Treatment ◽  
Nitrogen Fixing ◽  
Nitrogen Fixing Bacteria ◽  
Mining Sites ◽  
Greenhouse Study ◽  
Gravel Mining ◽  
Pine Trees

ABSTRACT Lodgepole pine (Pinus contorta var. latifolia) trees have been thriving on unreclaimed gravel mining sites in British Columbia, Canada, with tissue nitrogen-content and growth-rate unaffected by extremely low soil nitrogen-levels. This indicates that pine trees could be accessing a hidden nitrogen source to fulfill their nitrogen requirements – possibly via endophytic nitrogen-fixation. Endophytic bacteria originally isolated from native pine trees growing at gravel sites were selected (n = 14) for in vitro nitrogen-fixation assays and a year long greenhouse study to test the overall hypothesis that naturally occurring endophytic nitrogen-fixing bacteria sustain pine tree growth under nitrogen-limited conditions. Each of the 14 bacteria colonized the internal tissues of pine trees in the greenhouse study and fixed significant amounts of nitrogen from atmosphere (23%–53%) after one year as estimated through 15N isotope dilution assay. Bacterial inoculation also significantly enhanced the length (31%–64%) and biomass (100%–311%) of pine seedlings as compared to the non-inoculated control treatment. In addition, presence of the nifH gene was confirmed in all 14 bacteria. Our results support the possibility that pine trees associate with nitrogen-fixing bacteria, capable of endophytic colonization, to survive at unreclaimed gravel mining pits and this association could potentially be utilized for effective reclamation of highly disturbed sites in a sustainable manner.

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