Levels Of Nitrogenase Activity Research Articles

Organelle-dependent polyprotein designs enable stoichiometric expression of nitrogen fixation components targeted to mitochondria

Introducing nitrogen fixation (nif ) genes into eukaryotic genomes and targeting Nif components to mitochondria or chloroplasts is a promising strategy for engineering nitrogen-fixing plants. A prerequisite for achieving nitrogen fixation in crops is stable and stoichiometric expression of each component in organelles. Previously, we designed a polyprotein-based nitrogenase system depending on Tobacco Etch Virus protease (TEVp) to release functional Nif components from five polyproteins. Although this system satisfies the demand for specific expression ratios of Nif components in Escherichia coli, we encountered issues with TEVp cleavage of polyproteins targeted to yeast mitochondria. To overcome this obstacle, a version of the Nif polyprotein system was constructed by replacing TEVp cleavage sites with minimal peptide sequences, identified by knowledge-based engineering, that are susceptible to cleavage by the endogenous mitochondrial-processing peptidase. This replacement not only further reduces the number of genes required, but also prevents potential precleavage of polyproteins outside the target organelle. This version of the polyprotein-based nitrogenase system achieved levels of nitrogenase activity in E. coli, comparable to those observed with the TEVp-based polyprotein nitrogenase system. When applied to yeast mitochondria, stable and balanced expression of Nif components was realized. This strategy has potential advantages, not only for transferring nitrogen fixation to eukaryotic cells, but also for the engineering of other metabolic pathways that require mitochondrial compartmentalization.

The Effect of In Vitro Coinoculation on the Physiological Parameters of White Lupine Plants (Lupinus albus L.)

The aim of the study was to select microbiological inoculants for a specific plant species, i.e., white lupine (Lupinus albus L.), to increase the efficiency of the diazotroph process. The research involved an in vitro assessment of interactions between the symbiotic bacteria (Bradyrhizobium sp. isolated from Nitragina and Nitroflora commercial preparations dedicated to white lupine) and selected endophytes (Pseudomonas fluorescens or Bacillus subtilis) used for seed coinoculation. In addition, selected morphological traits of plants (the weight and length of aboveground and belowground parts) were examined after the inoculation/coinoculation. The degree of root colonisation by selected endophytes used as individual inoculants and in combination with bacteria of the Bradyrhizobium genus was determined. The diazotrophic parameters were also investigated (nitrogenase activity, the number, and weight of nodules). The results showed no antagonistic interactions have been demonstrated between bacterial strains of the genus Bradyrhizobium sp. isolated from Nitragina and Nitroflora, and the endophytes Pseudomonas fluorescens or Bacillus subtilis used for the study. The applied coinoculation in vitro had a stimulating effect on the weight of the stems and roots of white lupine causing an average increase of 13% and 28%, respectively. The level of nitrogenase activity in the coinoculation variants increased from 3.5 nMC2H4 plant−1 h−1 to an average of 32.34 nMC2H4 plant−1 h−1.

The nodD1 Gene of Sinorhizobium fredii HH103 Restores Nodulation Capacity on Bean in a Rhizobium tropici CIAT 899 nodD1/nodD2 Mutant, but the Secondary Symbiotic Regulators nolR, nodD2 or syrM Prevent HH103 to Nodulate with This Legume.

Rhizobial NodD proteins and appropriate flavonoids induce rhizobial nodulation gene expression. In this study, we show that the nodD1 gene of Sinorhizobium fredii HH103, but not the nodD2 gene, can restore the nodulation capacity of a double nodD1/nodD2 mutant of Rhizobium tropici CIAT 899 in bean plants (Phaseolus vulgaris). S. fredii HH103 only induces pseudonodules in beans. We have also studied whether the mutation of different symbiotic regulatory genes may affect the symbiotic interaction of HH103 with beans: ttsI (the positive regulator of the symbiotic type 3 protein secretion system), and nodD2, nolR and syrM (all of them controlling the level of Nod factor production). Inactivation of either nodD2, nolR or syrM, but not that of ttsI, affected positively the symbiotic behavior of HH103 with beans, leading to the formation of colonized nodules. Acetylene reduction assays showed certain levels of nitrogenase activity that were higher in the case of the nodD2 and nolR mutants. Similar results have been previously obtained by our group with the model legume Lotus japonicus. Hence, the results obtained in the present work confirm that repression of Nod factor production, provided by either NodD2, NolR or SyrM, prevents HH103 to effectively nodulate several putative host plants.

The influence of seed-dressings on the formation and functioning of symbiotic soybean systems with fungicide-resistant rhizobia

. The application of bacterial fertilizers, prepared on the basis of active, competitive strains of nitrogen-fixing microorganisms and fungicidal dressings for pre-sowing seed treatment in soybean cultivation technologies is characterized by high biological and economic efficiency. Therefore, it is important to find such combination of chemicals and microbial preparations, for the complex use of which will be maintained their main purpose. The aim of the work was to investigate the formation and functioning of symbiotic soybean systems with fungicide-resistant nodule bacteria Bradyrhizobium japonicum PC09 and B144 under the influence of Standak Top and Fever seed dressings. Differences in the effect of chemical plant protection products on the formation of symbiotic systems of soybean variety Almaz and nitrogen-fixing microorganisms have been established. A decrease by 9.2‒24.1 % in the number of nodules formed on the roots during the soybean growing season with the combined use of Fever with rhizobia bacterization of PC09 strain was revealed. At inoculation the seed with Tn5- mutant B144, use of the same preparation, the decrease of this indicator compared to control plants was 6.5‒32.8 %. An increase in the weight of root nodules by 10.3‒36 % was found under the effect of Standak Top and bacterization by both strains of rhizobia.There was a decrease in the intensity of N2 assimilation compared to control plants by 24.2 and 42.3 % in the stage of three true leaves with the combined use of Fever with inoculation and a gradual decrease of the toxic effect of pesticides on the functioning of symbiotic systems in the budding-flowering stage. At shared treatment of seeds with rhizobia strains B144 and PC09 and Standak Top revealed an increase in nitrogen fixation intensity by 6.2‒25 % in the stage of three true leaves, 12.5‒58 % in the stage of budding-beginning of flowering and by 28.1‒42.3 % in the stage of bean formation. Maintaining high level of nitrogenase activity of legume-rhizobial symbiosis, formed with the participation of pesticide-resistant nodule bacteria with using of seed dressings, will provide plants with available forms of nitrogen, protect sown seeds from soil and seed infections and create conditions for grain production.

Using synthetic biology to overcome barriers to stable expression of nitrogenase in eukaryotic organelles

Engineering biological nitrogen fixation in eukaryotic cells by direct introduction of nif genes requires elegant synthetic biology approaches to ensure that components required for the biosynthesis of active nitrogenase are stable and expressed in the appropriate stoichiometry. Previously, the NifD subunits of nitrogenase MoFe protein from Azotobacter vinelandii and Klebsiella oxytoca were found to be unstable in yeast and plant mitochondria, respectively, presenting a bottleneck to the assembly of active MoFe protein in eukaryotic cells. In this study, we have delineated the region and subsequently a key residue, NifD-R98, from K. oxytoca that confers susceptibility to protease-mediated degradation in mitochondria. The effect observed is pervasive, as R98 is conserved among all NifD proteins analyzed. NifD proteins from four representative diazotrophs, but not their R98 variants, were observed to be unstable in yeast mitochondria. Furthermore, by reconstituting mitochondrial-processing peptidases (MPPs) from yeast, Oryza sativa, Nicotiana tabacum, and Arabidopsis thaliana in Escherichia coli, we demonstrated that MPPs are responsible for cleavage of NifD. These results indicate a pervasive effect on the stability of NifD proteins in mitochondria resulting from cleavage by MPPs. NifD-R98 variants that retained high levels of nitrogenase activity were obtained, with the potential to stably target active MoFe protein to mitochondria. This reconstitution approach could help preevaluate the stability of Nif proteins for plant expression and paves the way for engineering active nitrogenase in plant organelles.

Endophytic Microbes from Diverse Wheat Genotypes and Their Potential Biotechnological Applications in Plant Growth Promotion and Nutrient Uptake

Endophytic microbes residing inside the tissues of plants play a significant role to enhance the growth and health of plants by different plant growth-promoting mechanisms. In the present investigation, N2-fixing endophytic bacteria were isolated and characterized by plant growth. A total of one hundred fifty-nine endophytic bacteria were isolated from surface-sterilized roots and stem of different genotypes of wheat growing in the Divine Valley of Baru Sahib, Himachal Pradesh. The isolated bacterial endophytes were screened in vitro for plant growth-promoting attributes. Out of one hundred fifty-nine, thirteen endophytic bacteria were selected based on multifarious plant growth-promoting attributes. Among plant growth-promoting activities, hydrogen cyanide producers (19%) were higher when compared to siderophores producers (16%) and P-solubilizers (16%), ammonia producers (14%), K-solubilizers (14%), IAA producers (12%), Zn-solubilizers (5%), N2-fixers (2%) and biocontrol (2%). One of the isolates EU-B2RT.R1 demonstrated that a significant level of nitrogenase activity, P-solubilization and IAA production was identified as Acinetobacter guillouiae EU-B2RT.R1 based on 16S rRNA gene sequencing and BLAST analysis. Acinetobacter guillouiae EU-B2RT.R1, exhibiting multifarious beneficial traits, is further evaluated for plant growth promotion of wheat cultivar PBW 343+Lr24+GPC in pot experiment under greenhouse conditions. The Acinetobacter guillouiae EU-B2RT.R1 with multifarious plant growth-promoting activity has emerged as one of the efficient biofertilizers that need to be explored for sustainable agriculture.

Artificial activation of nif gene expression in nodule bacteria Ex Planta

Background. Rhizobia are the most effective nitrogen-fixing organisms that can fix nitrogen only in symbiosis with leguminous plants. The general transcriptional activator of nitrogen fixation genes in diazotrophic bacteria is NifA. In this work, the possibility of modifying the regulation of nitrogen fixation in the nodule bacteria Mesorhizobium, Ensifer and Rhizobium was studied by introducing an additional copy of the nifA gene into the bacterial genomes during the regulation of induced bacterial promoters.
 Materials and methods. A series of expression genetic constructs with NifA genes of nodule bacteria strains under the control of an inducible promoter Pm were created. The resulting constructs were transformed into strains of nodule bacteria. The obtained recombinant strains were investigated for the appearance of their nitrogen-fixing activity in the free-living state.
 Results. It was shown that the expression of nifA in recombinant cells of all three genera of bacteria leads to the appearance of insignificant nitrogenase activity. At the same time, the level of nitrogenase activity does not have a correlation with the level of expression of the introduced nifA gene, which, most likely, is a consequence of the multilevel regulation of nitrogen fixation.
 Conclusion. The possibility of artificial activation of nitrogenase activity in nodule bacteria in the free-living state by introducing the NifA regulatory protein gene into bacteria was shown.

MiR172 Regulates Soybean Nodulation

Micro-RNAs (miRNAs) play a pivotal role in the control of gene expression and regulate plant developmental processes. miRNA 172 (miR172) is a conserved miRNA in plants reported to control the expression of genes involved in developmental phase transition, floral organ identity, and flowering time. However, the specific role of miR172 in legume nodulation is undefined. Ectopic expression of soybean miR172 resulted in an increase in nodule numbers in transgenic roots and an increase in the expression of both symbiotic leghemoglobin and nonsymbiotic hemoglobin. These nodules showed higher levels of nitrogenase activity. Further analysis revealed a complex regulatory circuit in which miR156 regulates miR172 expression and controls the level of an AP2 transcription factor. The latter, either directly or indirectly, controls the expression of nonsymbiotic hemoglobin, which is essential for regulating the levels of nodulation.

Divergent responses of biomass and enzyme activities suggest differential nutrient limitation in stream periphyton

We used in situ nutrient-enrichment experiments at sites in 4 Texas streams spanning a steep nutrient gradient to test the hypothesis that periphyton photoautotrophs and heterotrophs are differentially limited by N and P, respectively. We contrasted the responses of total periphyton biomass (ash-free dry mass [AFDM]), photoautotrophic biomass (chlorophyll a [CHLA]), % CHLA of AFDM [% CHLA]), alkaline phosphatase activity (APA), and nitrogenase activity (NA) to ambient N and P concentrations among sites and among nutrient-diffusing substrata (NDS) (control, N, P, and N+P) within sites. Periphyton biomass (AFDM and CHLA) increased steadily with increasing ambient N and P among sites. Both APA and NA were relatively high on the control treatments at the lowest nutrient site and decreased with increasing ambient N and P until falling to negligible levels at the highest nutrient site. Within sites, P enrichment decreased APA but did not increase % CHLA or CHLA/cm2. P enrichment did increase AFDM/cm2 at one of the low nutrient sites. N enrichment reduced NA and increased % CHLA and CHLA/cm2 without increasing AFDM/cm2. N+P enrichment resulted in the highest % CHLA and CHLA/cm2 at the 3 lowest nutrient sites but did not increase AFDM/cm2. Combined, these findings suggest that photoautotrophs in periphyton communities may be more limited than heterotrophs by N or become more competitive for P under elevated N. The high levels of APA and NA on the controls coupled with minimal difference in AFDM among nutrient treatments at most sites despite increases in % CHLA on N and N+P treatments provide experimental evidence that differential nutrient limitation probably occurs in periphyton communities. Our results support the view that the single-limiting-nutrient paradigm may lead to overly simplistic interpretations of periphyton responses to nutrient enrichment.

Biochar application reduces nodulation but increases nitrogenase activity in clover

Biochar is produced from the pyrolysis of organic materials, and when buried in soil can act as a long term soil carbon (C) store. Evidence suggests that biochar can also increase crop yields, reduce nutrient leaching and increase biological nitrogen fixation in leguminous plants. However, the potential for increasing biological N2 fixation in agroecosystems is poorly understood, with inconsistent reports of root nodulation following biochar application. Therefore, the aim of this study was to determine the effect of biochar application rate and time since application on nodulation and nitrogenase activity in nodules of clover grown in a temperate agricultural soil. We used replicated field plots with three biochar application rates (0, 25 and 50 t ha−1). Three years after biochar amendment, the plots were further split and fresh biochar added at two different rates (25 and 50 t ha−1) resulting in double-loaded reapplications of 25 + 25 and 50 + 50 t ha−1. Three years after biochar application, there was no significant difference in the total number of root nodules between biochar-amended and unamended soil, regardless of the application rate. However, despite clover root nodules being of a similar number and size the level of nitrogenase activity of individual nodules in biochar-amended soil was significantly higher than in unamended soil. Reapplication of biochar resulted in decreased nodulation, although the rate of nitrogenase activity per nodule remained unaffected. In the short term, biochar influences root nodule number and localised N2 fixation per nodule; however, total nitrogenase activity for the whole root system remained unaffected by the application rate of biochar or time since its application. These results emphasise the importance of long-term field studies, with a variety of applications rates for determining the influence of biochar applications on N2-fixing organisms and in providing data that can meaningfully inform agronomic management decisions and climate change mitigation strategies.

Concomitant colonization of nifH positive endophytic Burkholderia sp. in rice (Oryza sativa L.) promotes plant growth

Six diazotrophic bacteria were isolated from surface-sterilized roots of rice variety HUR-36, which is grown with very low or no inputs of nitrogen fertilizer. Out of six bacteria one isolate, RREM25, showed appreciable level of nitrogenase activity, IAA production, and Phosphate solubilization ability, and was further characterized with a view to exploiting its plant growth promoting activity. Based on 16S rRNA gene sequence analysis, this isolate was identified as Burkholderia cepacia. Diazotrophic nature of this particular isolate was confirmed by Western blot analysis of dinitrogenase reductase and amplification of nifH. Microscopic observation confirmed colonization of gfp/gusA-tagged RREM25 in the intercellular spaces of cortical as well as vascular zones of roots. Inoculation of RREM25 to rice plants resulted in significant increase in plant height, dry shoot and root weight, chlorophyll content, nitrogen content and nitrogenase activity. Plant growth promoting features suggest that this endophytic bacterium may be exploited in rice cultivation after a thorough and critical pathogenicity test.

KARAKTERISASI DAN UJI AKTIVITAS BAKTERI RIZOSFER LAHAN ULTISOL SEBAGAI PEMACU PERTUMBUHAN TANAMAN DAN AGENSIA HAYATI CENDAWAN PATOGEN TULAR TANAH SECARA IN VITRO

Characterization and activities assay of rhizosphere bacteria from ultisol land for plant-growth promoting and biocontrol agents of soil-borne fungus pathogens under in vitro test. Although many studies have been conducted to identify the specific traits of the plant growth-promoting and bioprotecting rhizobacteria (PGPBR), they were limited to studying specific PGPBR isolates from ultisol lands. We selected 273 isolates from bulk soil and plant rhizosphere and examined them for a wide array traits that might inhibit the growth of plant pathogens and increase early cucumber growth in ultisol soil. A subsample of 25 isolates, all positively produce chitinase and sellulose enzymes, 18 positively produce protease and 7 were fluorescens on KB medium under UV lighting. All isolates could produce IAA and be able to solubilize phosphor in vitro test, 10 exhibited low level of nitrogenase activity. Futher test showed that out of 25 isolates, 12 inhibited F. oxysporum, P. capsici, R. solani and S. rolfsii in vitro. All isolates increased seed germination, but only 5 isolates significanty increased early cucumber growth in ultisol soil. The results suggest that rhizobacteria be able to produce extracelluler enzymes, siderophore, ACC deasiminase, and IAA or those which are able to solubilize phosphor in vitro may be potential to be uses as biofertilizer and biological control agents in ultisol land.

Effect of Perturbation of ATP Level on the Activity and Regulation of Nitrogenase in Rhodospirillum rubrum

Nitrogenase activity in Rhodospirillum rubrum and in some other photosynthetic bacteria is regulated in part by the availability of light. This regulation is through a posttranslational modification system that is itself regulated by P(II) homologs in the cell. P(II) is one of the most broadly distributed regulatory proteins in nature and directly or indirectly senses nitrogen and carbon signals in the cell. However, its possible role in responding to light availability remains unclear. Because P(II) binds ATP, we tested the hypothesis that removal of light would affect P(II) by changing intracellular ATP levels, and this in turn would affect the regulation of nitrogenase activity. This in vivo test involved a variety of different methods for the measurement of ATP, as well as the deliberate perturbation of intracellular ATP levels by chemical and genetic means. To our surprise, we found fairly normal levels of nitrogenase activity and posttranslational regulation of nitrogenase even under conditions of drastically reduced ATP levels. This indicates that low ATP levels have no more than a modest impact on the P(II)-mediated regulation of NifA activity and on the posttranslational regulation of nitrogenase activity. The relatively high nitrogenase activity also shows that the ATP-dependent electron flux from dinitrogenase reductase to dinitrogenase is also surprisingly insensitive to a depleted ATP level. These in vivo results disprove the simple model of ATP as the key energy signal to P(II) under these conditions. We currently suppose that the ratio of ADP/ATP might be the relevant signal, as suggested by a number of recent in vitro analyses.

Characterization of Novel Plant Growth Promoting Endophytic Bacterium Achromobacter xylosoxidans from Wheat Plant

Nine diazotrophic bacteria were isolated from surface-sterilized roots and culms of wheat variety Malviya-234, which is grown with very low or no inputs of nitrogen fertilizer. Out of the nine bacteria, four showed indole acetic acid (IAA) production, and five were positive for P solubilization. One isolate, WM234C-3, showed appreciable level of nitrogenase activity, IAA production, and P solubilization ability, and was further characterized with a view to exploiting its plant growth promoting activity. Based on 16S rDNA sequence analysis, this isolate was identified as Achromobacter xylosoxidans. Diazotrophic nature of this particular isolate was confirmed by Western blot analysis of dinitrogenase reductase and amplification of nifH. Analysis of the nifH sequence showed close homology with typical diazotrophic bacteria. Endophytic nature and cross-infection ability of WM234C-3 were tested by molecular tagging with gusA fused to a constitutive promoter followed by inoculation onto rice seedlings in axenic conditions. At 21 days after inoculation, the roots showed blue staining, the most intense color being at the emergence of lateral roots and root tips. Microscopic observation confirmed colonization of gus-tagged WM234C-3 in the intercellular spaces of cortical as well as vascular zones of roots. Inoculation of gus-tagged WM234C-3 to rice plants resulted in significant increase in root/shoot length, fresh weight, and chlorophyll a content. Plant growth promoting features coupled with cross-infection ability suggest that this endophytic bacterium may be exploited as agricultural agent for various crops after a thorough and critical pathogenicity test.

The GntR-type regulators GtrA and GtrB affect cell growth and nodulation of Sinorhizobium meliloti

GntR-type transcriptional regulators are involved in the regulation of various biological processes in bacteria, but little is known about their functions in Sinorhizobium meliloti. Here, we identified two GntR-type transcriptional regulator genes, gtrA and gtrB, from S. meliloti strain 1021. Both the gtrA1 mutant and the gtrB1 mutant had lower growth rates and maximal cell yields on rich and minimal media, as well as lower cell motility on swimming plates, than did the wild-type strain. Both mutants were also symbiotically deficient. Alfalfa plants inoculated with wild-type strain 1021 formed pink elongated nodules on primary roots. In contrast, the plants inoculated with the gtrA1 and gtrB1 mutants formed relatively smaller, round, light pink nodules mainly on lateral roots. During the first 3 approximately 4 weeks post-inoculation, the plants inoculated with the gtrA1 and gtrB1 mutants were apparently stunted, with lower levels of nitrogenase activity, but there was a remarkable increase in the number of nodules compared to those inoculated with the wild-type strain. Moreover, the gtrA1 and gtrB1 mutants not only showed delayed nodulation, but also showed markedly reduced nodulation competition. These results demonstrated that both GtrA and GtrB affect cell growth and effective symbiosis of S. meliloti. Our work provides new insight into the functions of GntR-like transcriptional regulators.

Infection, Multiplication and Evaluation of the Nitrogen-Fixing Ability of Herbaspirillum sp. Strain B501gfp1 in Sugarcane Stems Inoculated by the Vacuum Infiltration Method

The present study was conducted with the aim of finding a suitable delivery method for introducing endophytic bacteria into new sugarcane stalks. Stem pieces of cultivars NiF8 and Ni15 were inoculated with Herbaspirillum sp. strain B501gfp1 by the vacuum infiltration method. The inoculated bacteria showed higher levels of nitrogenase activity in cultivar Ni15 than NiF8. The population density immediately after inoculation (10(7) CFU g [fresh weight](-1)) showed that strain B501gfp1 successfully entered the stem tissues of Ni15, where its presence was confirmed by fluorescence microscopic observations. Colonization was observed in the intercellular spaces and between the cell layers. The population of inoculated bacteria remained stable at 5 days after inoculation. The inoculated stems also showed adequately strong acetylene reduction activity compared to uninoculated stems in the absence of indigenous nitrogen-fixing bacteria.

Two Genes Encoding Protein Kinases of the HstK Family Are Involved in Synthesis of the Minor Heterocyst-Specific Glycolipid in the Cyanobacterium Anabaena sp. Strain PCC 7120

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 can fix N(2) under oxic conditions, and the activity of nitrogen fixation occurs exclusively in heterocysts, cells differentiated from vegetative cells in response to a limitation of a combined-nitrogen source in the growth medium. At the late stages of heterocyst differentiation, an envelope layer composed of two glycolipids is formed to limit the entry of oxygen so that the oxygen-sensitive nitrogenase can function. The genome of Anabaena sp. strain PCC 7120 possesses a family of 13 genes (the hstK family), all encoding proteins with a putative Ser/Thr kinase domain at their N termini and a His-kinase domain at their C termini. In this study, we showed that the double mutant D4.3 strain, in which two members of this gene family, pkn44 (all1625) and pkn30 (all3691), were both inactivated, failed to fix N(2) in the presence of oxygen (Fox(-)). In an environment without oxygen, a low level of nitrogenase activity was detectable (Fix(+)). Heterocyst development in the mutant D4.3 was delayed by 24 h and arrested at a relatively early stage without the formation of the glycolipid layer (Hgl(-)). Only the minor species of the two heterocyst-specific glycolipids (HGLs) was missing in the mutant. We propose that DevH, a putative transcription factor, coordinates the synthesis of both HGLs, while Pkn44/Pkn30 and the previously characterized PrpJ may represent two distinct regulatory pathways involved in the synthesis of the minor HGL and the major HGL, respectively.

Nitrogen fixation associated with Suillus tomentosus tuberculate ectomycorrhizae on Pinus contorta var. latifolia.

Tuberculate ectomycorrhizae are a unique form of ectomycorrhiza where densely packed clusters of mycorrhizal root tips are enveloped by a thick hyphal sheath to form a tubercle. The functional significance of such a unique structure has not previously been established. The purpose of the present study was to investigate and measure the potential nitrogenase activity associated with Suillus tomentosus/Pinus contorta tuberculate ectomycorrhizae in two stand ages, young and old, and across a range of nitrogen-poor soil conditions. Short roots were compared with other mycorrhizae and non-mycorrhizal secondary roots using tuberculate ectomycorrhizae. Assessment of nitrogenase activity was determined and quantitative measurements were taken on tuberculate ectomycorrhizae in situ in a variety of different circumstances, by using an adaptation of the acetylene reduction assay. Significant nitrogenase activity was measured associated with S. tomentosus/P. contorta tuberculate ectomycorrhizae whereas no nitrogenase activity was measured with non-tuberculate mycorrhizae or secondary roots without mycorrhizae. Average nitrogenase activity ranged from undetectable to 5696.7 nmol C2H4 g(-1) tubercle 24 h(-1). Maximum nitrogenase activity was 25,098.8 nmol C2H4 g(-1) tubercle 24 h(-1). Nitrogenase activity was significantly higher in young stands than in old stands of P. contorta. Season or some covariate also seemed to affect nitrogenase activity and there was suggestion of a site effect. Suillus tomentosus/P. contorta tuberculate ectomycorrhizae are sites of significant nitrogenase activity. The nitrogenase activity measured could be an important contribution to the nitrogen budget of P. contorta stands. Season and stand age affect levels of nitrogenase activity.

Aktivitas Nitrogenase, serapan hara dan pertumbuhan dua varietas kedelai pada kondisi jenuh air dan kering

The objective of this experiment was to study nitrogenase activity, nutrient uptake, and growth of two soybean varieties under saturated and dry soil conditions. The experiment was conducted in the plastic house at IPB Experimental Station, Babakan Sawah, Bogor, from August to November 2001. The experiment used the complete randomized design with four replications. The first factor was variety, consisted of Wilis and Bromo. The second factor was water condition, consisted of continuous saturated, saturated-dry, and dry conditions. Wilis and Bromo were not significantly different in nitrogenase activity; P and K uptake; nodule, stalk, leaf, pod, and seed dry weight. Wilis and Bromo were significantly different in root dry weight and leaf N uptake at 3 weeks after planting. Root dry weight and leaf N uptake of Wilis was lower than those of Bromo.The levels of nitrogenase activity, nutrient uptake, and plant weight under different water conditions were in the following order, from low to high, dry, saturated-dry, continuous. Under continuous saturated condition, nitrogenase activity, N uptake, P uptake, K uptake, nodule, root, stalk, leaf, and pod dry weight were 6800 %, 265 %, 320 %, 288 %, 1386 %, 362 %, 289 %, 265 %, 329 % of those under dry condition, respectively at 9 weeks after planting. Key words: Soybean, saturated culture, dry culture, nitrogenase activity, nutrient uptake

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

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.