Identification and expression analysis of miRNAs from nitrogen-fixing soybean nodules

miRNAs are ～21 nt non-coding RNAs and play important roles in plant development and response to stress. Symbiotic nitrogen fixation (SNF) is agronomically important for reducing the need of nitrogen fertilizers. The soybean root nodule is the place where SNF takes place. To identify miRNAs that are possibly involved in nitrogen fixation in soybean functional nitrogen-fixing nodules, a small library of RNAs was constructed from the functional nodules harvested 28 days after inoculation with rhizobium. Thirty-two small RNA sequences were identified as belonging to 11 miRNA families. Eight miRNAs are conserved across plant species, twenty are specific to soybean, and the four remaining miRNAs are novel. Expression analysis revealed that miRNAs were differentially expressed in the different tissues. Combinatorial miRNA target prediction identified genes that are involved in multiple biological processes. The results suggest that miRNAs play critical and diverse roles in SNF, nutrient acquisition, and plant development.     

A new approach for the biolistic method: Bombardment of living nitrogen-fixing bacteria into plant tissues

Summary A new utilization of the biolistic gun was developed for the direct introduction of nitrogen-fixing bacteria (Azotobacter vinelandii) into strawberry (Fragaria x ananassa) tissues. This was the first case of using living bacteria as microprojectiles for the bombardment of plant tissues. Bacterial cells, adhered to tungsten particles, were accelerated by a nitrogen-powered device, and delivered into the target leaves and regenerating shoot meristems. The presence of bacteria in the developing strawberry callus tissues and regenerating plants was detected by microscopy, acetylene reduction assay, and selective polymerase chain reaction. Practically, the elaborated method proved to be suitable for the establishment of artificial intereellular, associations between nitrogen-fixing bacteria and higher plants.     

Transfer of a plant chitinase gene into a nitrogen-fixing Azospirillum and study of its expression

Azospirillum is used extensively in rice and other cereal crops as a biofertilizer. There is a substantial opportunity to improve the efficiency of this bacterium through the transfer of genes of agricultural importance from other organisms. Chitinases are antifungal proteins, and expression of chitinase genes in Azospirillum would help to develop strains with potential antifungal activities. So far there are no reports about transfer of plant genes into Azospirillum and their expression. The present study was aimed at expressing an antifungal gene (a rice chitinase) of plant origin in Azospirillum brasilense. A rice chitinase cDNA (RC 7) that codes for a 35 kDa protein was subcloned into a broad host range plasmid pDSK519 under the control of LacZ promoter. The plasmid was mobilized into the nitrogen-fixing bacterium, Azospirillum brasilense strain SP51eFL1, through biparental mating. The conjugation frequency was in the range of 35-40 x 10(-6). The transconjugants grew in nitrogen-free media and fixed gaseous nitrogen in vitro. However, their growth and nitrogen-fixing ability were slightly less than those of the wild-type. Expression of the protein was demonstrated through western blotting of the total cell protein, which detected a 35 kDa band that was immuno-reactive to a barley chitinase antibody. The cell lysates also hydrolyzed various chitin substrates, which resulted in release of free sugars demonstrating the chitinase activity of transconjugants. The expressed protein also had antifungal activity as demonstrated by inhibition of growth of the plant pathogenic fungus, Rhizoctonia solani.     

Nitrogen fixation by cellulolytic communities at aerobic-anaerobic interfaces in straw

Microbial communities involved in both cellulolysis and nitrogen fixation were isolated from decomposing straw. Cellulolytic activity appeared restricted to fungal isolates, predominantly species of Penicillium and Fusarium , growing in the presence of oxygen. Bacteria isolated under anaerobic conditions on nitrogen-free media were identified exclusively as Clostridium butyricum . Enterobacters isolated on media containing fixed nitrogen, particularly Enterobacter cloacae , might also be involved in nitrogen fixation when growing under conditions of restricted aeration. Anaerobic or facultative anaerobic nitrogen-fixing bacteria are thought to be sustained by the products of cellulolytic enzymes from aerobic fungi.     

Expression of an Adenylate Cyclase Gene of Anabaena cylindrica in the Cyanobacterium Anabaena sp. Strain PCC 7120

A transformant of Anabaena 7120 was made by introducing a plasmidthat includes an adenylate cyclase gene of Anabaena cylindrica.Expression of this gene was driven by the bacterial tac promoter.Transformants accumulate cAMP 170 fold higher than the concentrationin the parental strain. The transformation resulted in the fragmentationof filaments in both nitrogen-replete and nitrogen-free media.It was suggested that this fragmentation caused the inhibitionof growth under nitrogen-fixing conditions. (Received December 26, 1997; Accepted April 30, 1998)     

Humus bacteria of Norway spruce stands: plant growth promoting properties and birch, red fescue and alder colonizing capacity

We studied the potential of the humus layer of the Norway spruce stands to supply beneficial rhizobacteria to birch (Betula pendula), alder (Alnus incana) and fescue grass (Festuca rubra), representatives of pioneer vegetation after clear-cutting of the coniferous forest. Axenically grown seedlings of these species were inoculated with the acid spruce humus, pH 3.7-5.3. Actinorhizal propagules, capable of nodulating alder, were present in high density (10(3) g(-1)) in humus of long-term limed plots, whereas plots with nitrogen fertilization contained almost none (相似文献   

Discovery of a cutinase-producing Pseudomonas sp. cohabiting with an apparently nitrogen-fixing Corynebacterium sp. in the phyllosphere

A phyllospheric bacterial culture, previously reported to partially replace nitrogen fertilizer (B. R. Patti and A. K. Chandra, Plant Soil 61:419-427, 1981) was found to contain a fluorescent pseudomonas which was identified as Pseudomonas putida and a Corynebacterium sp. The P. putida isolate was found to produce an extracellular cutinase when grown in a medium containing cutin, the polyester structural component of plant cuticle. The Corynebacterium sp. grew on nitrogen-free medium but could not produce cutinase under any induction conditions tested, whereas P. putida could not grow on nitrogen-free medium. When cocultured with the nitrogen-fixing Corynebacterium sp., the P. putida isolate grew in a nitrogen-free medium, suggesting that the former provided fixed N2 for the latter. These results suggest that the two species coexist on the plant surface, with one providing carbon and the other providing reduced nitrogen for their growth. The presence of cutin in the medium induced cutinase production by P. putida. However, unlike the previously studied fungal systems, cutin hydrolysate did not induce cutinase. Thin-layer chromatographic analysis of the products released from labeled apple fruit cutin showed that the extracellular enzyme released all classes of cutin monomers. This enzyme also catalyzed hydrolysis of the model ester substrates, p-nitrophenyl esters of fatty acids, and optimal conditions were determined for a spectrophotometric assay with p-nitrophenyl butyrate as the substrate. It did not hydrolyze triacyl glycerols, indicating that the cutinase activity was not due to a nonspecific lipase. It showed a broad pH optimum between 8.0 and 10.5 with 3H-labeled apple cutin as the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of ammonium nitrogen on fixation of elemental nitrogen inAlnus andMyrica

Summary 1. A substantial formation of nodules occurred on plants ofAlnus glutinosa andMyrica gale grown in water culture with different levels of ammonium nitrogen labelled with N¹⁵ present in the culture solution. The nodules tended to be fewer but larger than on plants in solution free of combined nitrogen.2. The nodules continued to fix atmospheric nitrogen despite the presence of ammonium nitrogen in the rooting medium, though fixation per unit weight of nodule tissues formed was somewhat lower than in nitrogen-free solution. Among other possible reasons this could have been due to a substitution of ammonium nitrogen for elemental nitrogen at the nitrogen-fixing centres of the nodule, but evidently this does not occur to any great extent.3. In Alnus but not in Myrica fixation per plant was considerably enhanced in the presence of a low level of ammonium nitrogen, owing to greater nodule development. At higher ammonium levels, in excess of the plants' requirements, fixation per plant was still of comparable order to that in nitrogen-free solution, but now only represented some 24 to 45 per cent of the total nitrogen accumulated by the plants.4. The results suggest that under field conditions some fixation of atmospheric nitrogen will always be associated with nodules present.     

Nitrogen-fixing activity in peat soils from a raised bog

The nitrogenase (acetylene reductase) activity in monolithic and minced peat samples was found to be low, no more than 0.014-0.022 mg N/(kg h). Incorporation of the 15N2 isotope into organic compounds of peat soil was from 2.71-8.13 mg N/kg over 15 days. The nitrogen-fixing activity was the highest in a 10-20 cm layer of soil and much lower in the upper (under green moss) and deeper (20-30 cm) layers. The addition of glucose to soil samples stimulated nitrogen fixation considerably after 18-26 h. The maximum nitrogenase activity (3.5-3.8 mg N/(kg h)) observed after 60-70 h coincided with the peak of respiratory activity. A repeated addition of glucose after its exhaustion increased nitrogenase activity without a lag period to 8.5 mg N/(kg h). Investigation of the effect of environmental factors (temperature, pH, aeration, and light intensity) on potential nitrogen-fixing activity in peat samples revealed that nitrogen fixation could proceed in a wide range of pH values (from 3.0 to 7.5) and temperatures (from 5 to 35 degrees C). The nitrogen-fixing bacteria belonging to different trophic groups were enumerated by using nitrogen-free media with pH values and mineralization levels close to those in situ. In samples of peat soil, diazotrophic methanol-utilizing bacteria prevailed (2.0-2.5 x 10(6) cells/g); the second largest group was facultatively anaerobic bacteria of the family Enterobacteriaceae.     

Development of a direct isolation procedure for free-living diazotrophs under controlled hypoxic conditions

Free-living diazotrophs are diverse and ubiquitous in soil, contributing the nitrogen pool in natural ecosystems. The isolation of nitrogen-fixing microorganisms has relied on semisolid nitrogen-free medium enrichment, followed by multiple subculturing steps. These procedures limit the diversity of recovered isolates. In the current study, we investigated three different isolation strategies for free-living diazotrophs using a soil sample from the Amazon forest. The methods were (i) direct plating on solid nitrogen-free medium under a 2% O(2) concentration, (ii) enrichment in semisolid nitrogen-free medium before plating on solid nitrogen-free medium under 2% O(2), and (iii) enrichment followed by subculturing in the semisolid nitrogen-free medium before plating on nitrogen containing medium under a 21% O(2) concentration. A total of 794 isolates were differentiated by their genomic fingerprinting patterns, and strains with unique profiles were identified on the basis of sequencing of their 16S rRNA gene. Isolates belonged to four bacterial phyla: Proteobacteria, Firmicutes, Actinobacteria, and Bacteriodetes. The novel strategy of combining a solid N-free medium and hypoxic conditions showed an increase of 62.6% in the diversity of diazotrophs in comparison to that obtained by the conventional semisolid medium-based methods. All isolates grew on the nitrogen-free medium under a 2% O(2) concentration, 78% of them showed the presence of the nifH gene, and 39% tested positive for acetylene reduction activity. Our results suggest that direct plating of soil dilutions on nitrogen-free solid medium under a 2% O(2) concentration is a useful strategy for the isolation of the diverse diazotrophic communities.     

Presence of trifoliin A,a rhizobium-binding lectin,in clover root exudate

Trifoliin A, a Rhizobium-binding glycoprotein from white clover, was detected in sterile clover root exudate by a sensitive immunofluorescence assay employing encapsulated cells of Rhizobium trifolii 0403 heat-fixed to microscope slides. Its presence in root exudate was further examined by immunoaffinity chromatography. The binding of trifoliin A to cells was specifically inhibited by the hapten, 2-deoxyglucose. Significantly higher quantities of trifoliin A were detected in root exudate of seedlings grown hydroponically in nitrogen-free medium than in rooting medium containing 15 mM NO, a concentration which completely suppressed root hair infection by the nitrogen-fixing symbiont. The presence of trifoliin A in root exudate may make it possible for recognition processes to occur before the microsymbiont attaches to its plant host.     

Symbiotic incompatibility between two forage species of Hedysarum, grown in Morocco, and their homologous rhizobia

Abstract Symbiotic experiments in the glasshouse demonstrated that two species of sulla, Hedysarum coronarium and Hedysarum flexuosum , grown in Morocco were mutually incompatible in their requirements for effectively nitrogen-fixing strains of rhizobia: nitrogen-fixing Rhizobium strains isolated from H. coronarium nodulated H. flexuosum but did not fix nitrogen, conversely strains from H. flexuosum were ineffective for H. coronarium . The agronic implications are discussed.     

Effect of plant nutrient supply on nodule effectiveness and rhizobium strain competition for nodulation ofLotus pedunculatus

Summary The effect of nutrient supply on nodule formation and competition between Rhizobium strains for nodulation ofLotus pedunculatus was studied. Limiting plant growth by decreasing the supply of nutrients in an otherwise nitrogen-free medium, increased the size but decreased the number and the nitrogenase activity of nodules formed by a fast-growing strain of Lotus Rhizobium (NZP2037). In contrast decreasing nutrient supply caused only a small decline in the size, number and nitrogenase activity of nodules formed by a slow-growing strain (CC814s). Providing small quantities of NH₄NO₃ (50 to 250 g N) to plants grown with a normal supply of other nutrients stimulated nodule development by both Rhizobium strains and increased the nitrogenase activity of the NZP2037 nodules. Differences in the level of effectiveness (nitrogen-fixing ability) of nodules formed by different Rhizobium strains on plants grown with a normal supply of nutrients were less apparent when the plants were grown with decreased nutrient supply or when the plants were supplied with low levels of inorganic N.Inter-strain competition for nodulation ofL. pedunculatus between the highly effective slow-growing strain CC814s and 7 other fast- and slow-growing strains, showed CC814s to form 42 to 100% of the nodules in all associations. The greater nodulating competitiveness of strain CC814s prevailed despite changes in the nutrient supply to the host plant. A tendency was observed for partially effective Lotus Rhizobium strains to become more competitive in nodule formation when plant growth was supplemented with low levels of inorganic nitrogen.     

Identification of nitrogen-fixing Paenibacillus from different plant rhizospheres and a novel nifH gene detected in the P. stellifer

A total of 534 isolates were selectively obtained from different plant rhizospheres based on their growth on nitrogen-free medium and their resistance to 80 degrees C for 15 min. Of the 534 isolates, 23 isolates had nifH gene and exhibited nitrogenase activities. Based on 16S rDNA sequence, G + C content assay and DNA-DNA hybridization, by the 23 isolates, which were divided into four monophyletic clusters, all belonged to the Paenibacillus genus. NifH gene deduced amino acid alignment analysis revealed that cluster I, including 15 isolates, showed the highest NifH identity with Paenibacillus genus; while cluster II identified as P stellifer by DNA-DNA hybridization was consistent with four uncultured bacterial clones. This study suggested that the nitrogen-fixing Paenibacillus were distributed in various ecosystems and prevalent in different plant rhizospheres. It was the first demonstration that nitrogen fixation existed in P. jamilae and P. stellifer. In eight isolates identified as P. stellfer species, a novel nifH gene was detected in Paenibacillus.     

INITIATION AND DEVELOPMENT OF ROOT NODULES OF CASUARINA (CASUARINACEAE)

Roots of seedlings of the “beefwood” tree, Casuarina cunninghamiana Miq. grown in nitrogen-free nutrient solution were inoculated with a suspension prepared from crashed root nodules taken from mature plants. Marked deformation of root hairs was evident but no infection threads were observed in root hairs. The mode of infection remains undetermined. Root nodules were initiated within three weeks and thereafter numerous upward-growing nodule roots developed from each nodule. Nodules in this symbiotic nitrogen-fixing plant resulted from an infection caused by an unidentified actinomycete-like soil microorganism. Anatomical analysis of nodule formation showed that nodules are the result of repeated endogenous lateral root initiations, one placed upon another in a complexly branched and truncated root system. The endophyte-infected cortical tissues derived from successive root primordia form the swollen nodular mass. Nodule roots develop from nodule lobes after escaping from the initial inhibitory effects of the endophyte. Included is a discussion of the anatomical similarities between nodules of Casuarina which produce nodule roots and those of Alnus which form coralloid nodules usually lacking nodule roots.     

Nitrogen-Fixing Activity in Peat Soils from a Raised Bog

The nitrogenase (acetylene reductase) activity in monolithic and minced peat samples was found to be low, no more than 0.014–0.022 mg N/(kg h). Incorporation of the ¹⁵N₂ isotope into organic compounds of peat soil was 2.71–8.13 mg N/kg over 15 days. The nitrogen-fixing activity was the highest in a 10- to 20-cm layer of soil and much lower in the upper (under green moss) and deeper (20- to 30-cm) layers. The addition of glucose to soil samples stimulated nitrogen fixation considerably after 18–26 h. The maximum nitrogenase activity (3.5–3.8 mg N/(kg h)), observed after 60–70 h, coincided with the peak of respiratory activity. A repeated addition of glucose after its exhaustion increased nitrogenase activity, without a lag period, to 8.5 mg N/(kg h). Investigation of the effect of environmental factors (temperature, pH, aeration, and light intensity) on potential nitrogen-fixing activity in peat samples revealed that nitrogen fixation could proceed in a wide range of pH values (from 3.0 to 7.5) and temperatures (from 5 to 35°C). The nitrogen-fixing bacteria belonging to different trophic groups were enumerated by using nitrogen-free media with pH values and mineralization levels close to those in situ. In samples of peat soil, diazotrophic methanol-utilizing bacteria prevailed (2.0–2.5 × 10⁶ cells/g); the second largest group was facultatively anaerobic bacteria of the family Enterobacteriaceae.     

Isolation and identification of nitrogen-fixing bacilli from plant rhizospheres in Beijing region

AIMS: To isolate and identify nitrogen-fixing bacilli from the plant rhizospheres in Beijing region of China. METHODS AND RESULTS: A total of 29 isolates were selectively obtained from the rhizospheres of wheat, maize, ryegrass and willow based on their growth on nitrogen-free medium and their resistance to 100 degrees C for 10 min. Of the 29 isolates, seven had nifH gene determined by PCR amplification. The seven isolates were found to belong to the genera Bacillus and Paenibacillus based on phenotypic characterization, 16S rDNA sequence, G+C content and DNA-DNA hybridization. Isolates T1 and W5 were identified as Bacillus cereus and Bacillus marisflavi respectively. Isolates G1, C4 and C5 were identified as Bacillus megaterium. Isolate G2 was identified as Paenibacillus polymyxa and isolate T7 as Paenibacillus massiliensis. CONCLUSIONS: This study suggests that nifH gene could be detected in the both genera Bacillus and Paenibacillus. These degenerate primers for nifH gene fragment used in this study were shown to be useful for identifying nitrogen-fixing bacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: It is the first demonstration that nitrogen fixation exists in B. marisflavi and P. massiliensis and the first report of the sequences of the nifH gene from B. megaterium and B. cereus. The nitrogen-fixing bacilli obtained in this study will be used in our future research for investigating the mechanisms of nitrogen fixation in bacilli.     

Interaction between Medicago truncatula lines and Sinorhizobium meliloti strains for symbiotic efficiency and nodule antioxidant activities

The relationships between symbiotic performance and nodular antioxidant enzymes were studied for the associations between three Medicago truncatula lines and three Sinorhizobium meliloti strains. The results showed that the variability in symbiotic efficiency was dependent on the bacterial partner, host plant and their interaction. The contribution of each factor to the total amount of variance differed with the measured parameter. The aerial biomass production and nitrogen-fixing capacity were affected similarly by the three factors, whereas root and nodule biomass and catalase (CAT, E.C. 1.11.1.6), guaiacol peroxidase (POX, E.C. 1.11.1.7) and ascorbate peroxidase (APX, E.C. 1.11.1.11) antioxidant activities were mainly influenced by the M. truncatula line. The nodule number was dependent on the bacterial strain, and superoxide dismutase (SOD, E.C. 1.15.1.1) was dependent mainly on the plant–rhizobium interaction. A highly significant correlation was found between nitrogen-fixing activity, shoot biomass production, total nodule protein content and catalase activity. The other nodular antioxidant enzymes were differentially expressed between associations and showed no clear correlation with symbiotic efficiency.     

NITROGEN FIXATION IN THE BAYBERRY (MYRICA PENSYLVANICA) AND ITS ROLE IN COASTAL SUCCESSION

The nodules on roots of Myrica pensylvanica (bayberry) contain a bacterial endophyte. By using the acetylene reduction technique these plant endophyte associations were shown to be capable of fixing nitrogen. As nodulation was plentiful and fixation vigorous, it is proposed that the success of M. pensylvanica as an early successional plant of dunes and impoverished coastal soils is due in part to the nitrogen-fixing capacity of its nodular association.     

Immunogold localization of hydrogenase in free-living Frankia CpI1

Abstract The free-living Frankia strain CpI1 cultured under nitrogen-fixing and non-nitrogen-fixing conditions was investigated for occurrence of hydrogenase protein by Western blots. Transmission electron microscopy and immunocytological labelling were used to study the distribution of hydrogenase in the Frankia strain.
Western immunoblots revealed that a 72-kDa protein in the Frankia strain CpI1 was immunologically related to the large subunit of a dimeric hydrogenase purified from Alcaligenes latus . Immunolocalization showed that the hydrogenase protein is located both in vesicles and hyphae in Frankia strain CpI1 grown in a nitrogen-free medium. Earlier reports that nitrogenase is localized in the vesicles [1,2], together with this finding, point out a possible role for hydrogenase in increasing relative efficiency of nitrogen fixation. In CpI1 grown in media containing nitrogen (lacking vesicles), the enzyme was evenly distributed in the hyphae. The impact of this result has to be further analysed.