Genetic diversity of <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> associated with alfalfa in Chilean volcanic soils and their symbiotic effectiveness under acidic conditions

A study was conducted with the aim of evaluating the genetic diversity of alfalfa rhizobia isolated from volcanic soils in southern Chile and their ability to establish an effective symbiosis with alfalfa. Rhizobial strains isolated from nodules were identified and selected based on PCR analyses and acid tolerance. Symbiotic effectiveness (nodulation and shoot dry weight) of acid-tolerant rhizobia was evaluated in glasshouse experiments under acidic conditions. The results revealed that Sinorhizobium meliloti is the dominant species in alfalfa nodules with a high genetic diversity at strain level grouped in three major clusters. There was a close relationship (r ² = 0.895, P ≤ 0.001, n = 40) between soil pH and the size of rhizobial populations. Representative isolates from major cluster groups showed wide variation in acid tolerance expressed on buffered agar plates (pH 4.5–7.0) and symbiotic effectiveness with alfalfa. One isolate (NS11) appears to be suitable as an inoculant for alfalfa according to its acid tolerance and symbiotic effectiveness at low pH (5.5). The isolation and selection of naturalized S. meliloti strains with high symbiotic effectiveness under acidic conditions is an alternative approach to improving the productivity of alfalfa and for reducing the application of synthetic fertilizers in Chile.     

Nitrogen release from roots of alfalfa and soybean grown in sand culture

An enclosed root chamber containing sterile sand medium was used to study net nitrogen (N) release from actively growing root systems of ‘Saranac’ alfalfa (Medicago sativa L.) and ‘Fiskeby V’ soybean (Glycine max L. Merr.). Plants were inoculated with a rhizobial strain appropriate to each host, irrigated with N-free nutrient solution, and grown either to 85 or to 173 d after germination (alfalfa) or to physiological maturity (soybean). Alfalfa released 4.5% of symbiotically-fixed plant N into the root zone over its growth period; soybean released 10.4% of plant N. Root zone leachates were analyzed for total N and for amino acid and ammonium content. Significant ammonium-N release occurred from the alfalfa but not the soybean root system; little amino-N was released by root systems of either species. Shoot harvest and water deficit caused increased release of N from alfalfa roots. The results provide evidence that alfalfa and soybean released significant proportions of their N into the root zone, and indicate that while substantial ammonium-N was released from alfalfa roots, passive leakage of amino-N was not a primary mechanism for N release from root systems of either species. Cooperative investigation of USDA-ARS and the Minnesota Agric. Exp. Stn. (Scientific J. Series No. 16048). This research was supported in part by a Graduate School Fellowship to LSB from the Univ. of Minnesota.     

Abscisic acid and gibberellin-like substances in roots and root nodules ofGlycine max

Summary The content of endogenous gibberellin (GA)-like substances of roots and root nodules of SOya, and GA production byRhizobium japonicum cultures, were investigated by a combined thin layer chromatographic (TLC)-dwarf pea epicotyl bioassay technique. GAs were more concentrated in root nodules than in the roots, totalling 1.34 and 0.16 nM GA₃ equivalents g⁻¹ dry wt. respectively. GA production byR. japonicum cultures was demonstrated (1.00 nM GA₃ equivalentsl ⁻¹) and comparison of the GA components of plant and bacterial culture medium extracts, suggested that rhizobial GA production may contribute to the nodule GA content. Cis-trans abscisic acid (ABA) was identified in root and nodule extracts by TLC-gas liquid chromatography (GLC), and amounted to 0.18 and 2.21 nM g⁻¹ dry wt. respectively, whereas 0.30 and 4.63 nM ABA equivalents g⁻¹ dry wt. were detected by a TLC-wheat embryo bioassay technique. ABA was not detected in extracts of bacterial cultures.     

Nitrogen-tillage-residue management

Summary NCSWAP (nitrogen and carbon cycling in soil, water and plant) is a simulation model of the soil-crop-water system which integrates water flow dynamics, crop growth, N transformations, tillage and residue effects, soil temperature, and solute transport. A small plot field study was initiated in May of 1980 to determine the effects of N rate (2 or 20 g N/m²), tillage (rototill or no-till), and residue management system (residue return or noresidue) on soil parameters, and maize (Zea mays L.) production.Significant differences due to treatments (N rate, tillage, and residue) were not detected in 1981 for the measured soil-plant parameters including soil moisture, yield, and N uptake. Therefore, two representative treatment combinations (N rates of 2 or 20 g N/m²-tilled-no residue) characterized the field research data. Calculated and observed data sets were compared for several parameters including: (1) soluble NO₃–N, (2) N leaching losses (3) plant total-N and¹⁵N, (4) root growth, (5) soil moisture, and (6) fertilizer efficiency.The objectives of this study were to initiate the validation process of the model NCSWAP, and to illustrate how NCSWAP can be used as a research tool to infer operational characteristics of the N cycle.Contribution of the Soil and Water Management Research Unit, USDA-ARS, and the Department of Soil Science, University of Minnesota, St. Paul, MN 55108. Minn Agric. Exp. Sta., Sci. J., Ser. Paper 13907.Senior Laboratory Technician; Research Chemist, USDA-ARS and Professor; Professor of Soil Microbiology; and Soil Scientist, USDA-ARS and Assistant Professor; all Department of Soil Science, University of Minnesota, respectively. Inquiries about NCSWAP should be sent to J. A. E. Molina.     

Charcoal and shrubs modify soil processes in ponderosa pine forests of western Montana

In split-root systems of alfalfa (Medicago sativa L.), already existing nodules or arbuscular mycorrhizal roots suppress further establishment of symbiosis in other root parts, a phenomenon named autoregulation. Roots treated with rhizobial nodulation signals (Nod factors) induce a similar systemic suppression of symbiosis.In order to test the hypothesis that flavonoids play a role in this systemic suppression, split-root systems of alfalfa plants were inoculated on one side of the split-root system with Sinorhizobium meliloti or Glomus mosseae or were treated with Nod factor. HPLC-analysis of alfalfa root extracts from both sides of the split-root system revealed a persistent local and systemic accumulation pattern of some flavonoids associated with the different treatments. The two flavonoids, formononetin and ononin, could be identified to be similarily altered after rhizobial or mycorrhizal inoculation or when treated with Nod factor.Exogenous application of formononetin and ononin partially restored nodulation and mycorrhization pointing towards the involvement of these two secondary compounds in the autoregulation of both symbioses.     

Spontaneous nodules induce feedback suppression of nodulation in alfalfa

A small subpopulation of alfalfa (Medicago saliva L.) plants grown without fixed nitrogen can develop root nodules in the absence of Rhizobium. Cytological studies showed that these nodules were organized structures with no inter- or intracellular bacteria but with the histological characteristics of a normal indeterminate nodule. Few if any viable bacteria were recovered from the nodules after surface sterilization, and when the nodular content was used to inoculate alfalfa roots no nodulation was observed. These spontaneous nodules were formed mainly on the primary roots in the region susceptible to Rhizobium infection between 4 and 6 d after seed imbibition. Spontaneous nodules appeared as early as 10 d after germination and emerged at a rate comparable to normal nodules. The formation of spontaneous nodules on the primary root suppressed nodulation in lateral roots after inoculation with R. meliloti RCR2011. Excision of spontaneous nodules at inoculation eliminated the suppressive response. Our results indicate that the presence of Rhizobium is not required for nodule organogenesis and the elicitation of feedback regulation of nodule formation in alfalfa.Abbreviation RT root tip This work was supported by an endowment to the Racheff Chair of Excellence of the University of Tennessee, and the Soybean Promotion Board, Haskinsville, Tenn., USA. We are indebted to Noel Gerahty for performing the acetylene-reduction assays, and Dr. E.T. Graham for allowing the use of microscope facilities.     

Stimulation of indoleacetic acid production in a <Emphasis Type="Italic">Rhizobium</Emphasis> isolate of <Emphasis Type="Italic">Vigna mungo</Emphasis> by root nodule phenolic acids

The influence of endogenous root nodules phenolic acids on indoleacetic acid (IAA) production by its symbiont (Rhizobium) was examined. The root nodules contain higher amount of IAA and phenolic acids than non-nodulated roots. Presence of IAA metabolizing enzymes, IAA oxidase, peroxidase, and polyphenol oxidase indicate the metabolism of IAA in the nodules and roots. Three most abundant endogenous root nodule phenolic acids (protocatechuic acid, 4-hydroxybenzaldehyde and p-coumaric acid) have been identified and their effects on IAA production by the symbiont have been studied in l-tryptophan supplemented yeast extract basal medium. Protocatechuic acid (1.5 μg ml⁻¹) showed maximum stimulation (2.15-fold over control) of IAA production in rhizobial culture. These results indicate that the phenolic acids present in the nodule might serve as a stimulator for IAA production by the symbiont (Rhizobium). Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. An erratum to this article can be found at     

Molecular and whole-plant responses to selection for enzyme activity in alfalfa root nodules: evidence for molecular compensation of aspartate aminotransferase expression

Summary The enzyme aspartate aminotransferase (AAT) plays a key role in the assimilation of fixed-N in alfalfa (Medicago sativa L.) root nodules. AAT activity in alfalfa nodules is due to the activity of two dimeric isozymes, AAT-1 and AAT-2, that are products of two distinct genes. Three forms of AAT-2 (AAT-2a, -2b, and-2c) have been identified. It was hypothesized that two alleles occur at the AAT-2 locus, giving rise to the three AAT-2 enzymes. In a prior study bidirectional selection for root nodule AAT and asparagine synthetase (AS) activities on a nodule fresh weight basis in two diverse alfalfa germ plasms resulted in high nodule enzyme activity subpopulations with about 20% more nodule AAT activity than low enzyme activity subpopulations. The objectives of the study presented here were to determine the inheritance of nodule AAT-2 production and to evaluate the effect of bidirectional selection for AAT and AS on AAT-2 allelic frequencies, the relative contributions of AAT-1 and AAT-2 to total nodule activity, nodule enzyme concentration, and correlated traits. Two alleles at the AAT-2 locus were verified by evaluating segregation of isozyme phenotypes among F₁ and S₁ progeny of crosses or selfs. Characterization of subpopulations for responses associated with selection was conducted using immunoprecipitation of in vitro nodule AAT activity, quantification of AAT enzyme protein by ELISA, and AAT activity staining of native isozymes on PAGE. Results indicate that selection for total AAT activity specifically altered the expression of the nodule AAT-2 isozyme. AAT-2 activity was significantly greater in high compared to low activity subpopulations, and high AAT subpopulations from both germ plasms had about 18% more AAT-2 enzyme (on a nodule fresh weight basis). No significant or consistent changes in AAT-2 genotypic frequencies in subpopulations were caused by selection for AAT activity. Since changes in AAT activity were not associated with changes in AAT-2 genotype, selection must have affected a change(s) at another locus (or loci), which indirectly effects the expression of nodule AAT.Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture, and does not imply its approval to the exclusion of other products or vendors that might also be suitable     

The influence of temperature and nitrate on vegetative growth and nitrogen accumulation by nodulated soybeans

Summary Inoculated soybeans [Glycine max (L.) Merrill] were grown in controlled environments to evaluate the relationship between temperature and applied NO₃−N on growth rates, N accumulation, and acetylene reduction activity during the vegetative growth stage. Soybeans were grown at day/night temperatures of 22/18 and 26/22°C in sand culture with daily applications of 21.4 mM (high) and 2.1 mM (low) NO₃−N in a complete nutrient solution for durations of 14, 21, and 42 days after emergence and with an N-free solution. Dry matter and N accumulation were greater at 26/22 than 22/18°C. In general, both increased as the level and duration of applied NO₃−N was increased. These increases were attributable to an abbreviation in the interval between emergence and onset of rapid growth. The presence and assimilation of NO₃−N, even at the high level, did not inhibit development of functional nodules. Neither mass nor acetylene reduction activity of nodules was reduced by high NO₃−N; however, the root mass was increased by NO₃−N more than the nodule mass. There was an interaction between temperature and NO₃−N on specific nodule activity as measured by acetylene reduction. The specific nodule activity was unaffected by NO₃−N at 22/18°C, but at 26/22°C the specific activity was lower in the absence of NO₃−N than when NO₃−N was present. Apparently, rapid early growth at 26/22°C depleted cotyledonary reserves of N before nodules became active and, thereafter, the plants were unable to develop adequate leaf area to support nodule development and functioning. This result has implications in N fertilization of late-planted soybeans. Paper number 6637 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina, 27650. The research was supported in part by a grant from the North Carolina Soybean Producers Association and by USDA-SEA-CR grant 701-15-26.     

Tissue-culture-facilitated production of aneupolyhaploid Thinopyrum ponticum and amphidiploid Hordeum violaceum x H. bogdanii and their uses in phylogenetic studies

Summary An aneupolyhaploid (2n = 36) of the decaploid Thinopyrum ponticum and an amphidiploid (2n = 28) of Hordeum violaceum x Hordeum bogdanii were produced through anther and inflorescence culture, respectively. Meiotic associations in pollen mother cells at metaphase I of these plants were analyzed. The aneupolyhaploid arose by direct embryogenesis from a microspore without passing through a callus phase. The mean pairing frequencies of 2.67 univalents ⁺ 0.54 rod bivalents ⁺ 8.85 ring bivalents ⁺ 2.75 trivalents ⁺ 0.17 chain quadrivalents ⁺ 0.56 ring quadrivalents ⁺ 0.65 pentavalents in the aneupolyhaploid (2n = 36) best fit the 221 model. However, the frequent multivalents (up to five trivalents, or three quadrivalents, or four pentavalents in a cell) indicated that decaploid T. ponticum has five sets of closely related genomes representable by the genome formula J₁ J₁ J₁ J₂ J₂. Colchicine treatment of inflorescence-derived H. violaceum x H. bogdanii regenerants greatly enhanced the rate of chromosome doubling, and completely doubled regenerants could be isolated. The H. violaceum x H. bogdanii amphidiploid had a mean pairing pattern of 12.53 univalents ⁺ 5.57 rod bivalents ⁺ 1.97 ring bivalents ⁺ 0.07 trivalents ⁺ 0.03 hexavalents, indicating the presence of desynaptic gene(s) in the original diploiid hybrid. Therefore, the pairing frequency in that diploid hybrid was an under-estimate of chromosome homology between the parental genomes, and additional diploid hybrids are needed to assess the genome homology between H. violaceum and H. bogdanii. These two contrasting experiments demonstrated that tissue culture techniques are useful in altering the ploidy level to produce plant materials suitable for genome analysis and phylogenetic studies.Cooperative investigation of the USDA-ARS, Forage and Range Research Laboratory, Logan, UT 84322-6300, and the Utah Agricultural Experiment Station, Utah State University, Logan, UT 84322-4810. Approved as journal paper No. 3991     

Distribution of spore-positive and spore-negative nodules in stands ofAlnus glutinosa andAlnus incana in Finland

Summary The distribution of spore positive (Sp⁺) and spore negative (Sp⁻) nodules on the two native alder species (A. incana andA. glutinosa) in Finland was investigated. Nodules were collected throughout the country from different ecosystems (forests, swamps, lake- sea- and riversides, old pastures and fields as well as from alder plantations). OnA. incana Sp⁺ nodules predominated, whereas onA. glutinosa the vast majority of the nodules were of the Sp⁻ type. Sp⁺ nodules onA. glutinosa were found only at sites where the two alder species grew close together. This distribution pattern indicates an association of nodule type with alder species, the reasons for which are discussed. Indications of saprophytic growth in the Sp⁻ strain were also found.     

Distribution of spore-positive and spore-negative nodules ofAlnus incana ssp.rugosa in Maine,USA

The distribution of spore-positive (sp+) and spore-negative (sp−) root nodules ofAlnus incana ssp.rugosa (DuRoi) Clausen (speckled alder) was examined at 29 sites with a wide range of environmental conditions in Maine, USA. These included: pH 3.4 to 7.0, soil texture ranging from coarse gravel to clay to organic soils, elevation from 3 to 591 m and latitude 43 to 47°N. Habitat types included disturbed areas, streamsides, swamps and old fields. Sp (−) nodules were substantially more common, making up 76% of all nodules, whereas only 24% were sp (+). Sp (−) nodules often occurred in pure stands and predominated at disturbed sites with mineral soils at the surface and in old fields and swamps with pH>4.0 Sp (+) nodules were nearly always found in mixture with sp (−) nodules. They occurred primarily at streamside and lakeshore sites where they made up 40% of the nodules and at sites with pH<4.0 regardless of habitat type. It is suggested that sp (−) strains ofFrankia may be maintained at a site by saprophytic growth in soil and thus nodulate newly established hosts, whereas sp (+) strains may be maintained primarily by spore production within nodules and thus depend on extended presence of the host.     

Immunocytological evidence for abnormal symbiosome development in nodules of the pea mutant line Sprint2Fix− (sym31)

Summary Using a series of antibody probes as markers of symbiosome development, we have investigated the impaired development of symbiosomes in nodules formed by the plant mutant line Sprint2Fix⁻ (sym31). In wild-type pea (Pisum sativum L.) nodules, bacteria differentiate into large pleiomorphic, nitrogen-fixing bacteroids and are singly enclosed within a peribacteroid membrane. In thesym31 mutant, several small undifferentiated bacteroids were often enclosed within one peribacteroid membrane, or were found within a vacuole-like compartment. In wild-type nodules, the monoclonal antibody JIM18, which recognizes a plasmalemma glycolipid antigen, bound to the juvenile peribacteroid membrane, and did not recognize the mature peribacteroid membrane. However, in the mutant, the antibody bound to all peribacteroid membranes within the nodule, suggesting that differentiation of the peribacteroid membrane was arrested. Another antibody, MAC266, recognized plant glycoproteins which normally accumulate in symbiosomes at a late stage of nodule development. Binding of this antibody was much reduced within mutant nodules, labelling only a few mature cells. Similarly, MAC301, which normally recognizes a lipopolysaccharide epitope expressed on differentiated bacteroids prior to the induction of nitrogenase, failed to react with rhizobial cell extracts isolated from nodules of thesym31 mutant. On the basis of these developmental markers, the symbiosomes ofsym31 nodules appeared to be blocked at an early stage of development. The distribution of infection structures was also found to be abnormal in the mutant nodules. Models of symbiosome development are presented and discussed in relation to the morphological and developmental lesions observed in thesym31 mutant.     

Symbiotic effectiveness of Rhizobium meliloti at low root temperature

Laboratory, growth chamber and field experiments were conducted to select among 226 isolates of Rhizobium meliloti for the ability to grow, nodulate alfalfa (Medicago sativa L.) and support N₂-dependent plant growth between 9° and 12°C. There was wide variation in the abilities of R. meliloti isolates to grow and form nodules at 10°C. Culture doubling times (t_d) varied from 1 to 155h, and the number of nodules formed on alfalfa in growth pouches in 2 weeks varied from 0 to 3.8 nodules per plant. Nodulation occurred at 9°C, but there was no significant N₂-dependent plant growth at this temperature. However, several isolates of R. meliloti had the ability to nodulate alfalfa and produce N₂-dependent growth at root temperatures between 10° and 12°C root temperature than did 14 other isolates tested. In field experiments, inoculation with strain NRG-34 resulted in greater nodule numbers, nodule weight, proportion of nodules occupied by the inoculant strain and plant weight than did inoculation with a commercial strain (NRG-185). These results permitted selection of a strain with better low-temperature competitive abilities than the currently available commercial strains.     

Appearance of a novel form of plant glutamine synthetase during nodule development in Phaseolus vulgaris L.

The activities of glutamine synthetase (GS), nitrogenase and leghaemoglobin were measured during nodule development in Phaseolus vulgaris infected with wild-type or two non-fixing (Fix^-) mutants of Rhizobium phaseoli. The large increase in GS activity which was observed during nodulation with the wild-type rhizobial strain occurred concomitantly with the detection and increase in activity of nitrogenase and the amount of leghaemoglobin. Moreover, this increase in GS was found to be due entirely to the appearance of a novel form of the enzyme (GS_n1) in the nodule. The activity of the form (GS_n2) similar to the root enzyme (GS_r) remained constant throughout the experiment. In nodules produced by infection with the two mutant strains of Rhizobium phaseoli (JL15 and JL19) only trace amounts of GS_n1 and leghaemoglobin were detected.Abbreviations DEAE-Sephacel diethylaminoethyl-Sephacel - GS glutamine synthetase     

Immunogold localization of nodule-enhanced phosphoenolpyruvate carboxylase in alfalfa

Phosphoenolpyruvate carboxylase (PEPC; EC 4-1-1-31) plays a paramount role in providing carbon for synthesis of malate and aspartate in alfalfa (Medicago sativa L.) root nodules. PEPC protein and activity levels are highly enhanced in N₂-fixing alfalfa nodules. To ascertain the relationship between the cellular location of PEPC and root nodule metabolism, enzyme localization was evaluated by immunogold cytochemistry using alfalfa nodule PEPC antibodies. Gold labelling patterns in effective nodules showed that PEPC is a cytosolic enzyme and is distributed relatively equally in infected and uninfected cells of the nodule symbiotic zone. A high amount of labelling was also observed in pericycle cells of the nodule vascular system. Labelling was also detected within inner cortical cells, but the density was reduced by 60%. When Lotus corniculatus was transformed with a chimeric gene consisting of the 5′-upstream region of the PEPC gene fused to β-glucuronidase (GUS), GUS staining in nodules was consistent with immunogold localization patterns. The occurrence of PEPC in both infected and uninfected cells of the symbiotic zone of effective nodules coupled to the reduced amounts in ineffective nodules suggests a direct role for this enzyme in supporting N₂-fixation. PEPC localization in the uninfected, interstitial cells of the symbiotic zone indicates that these cells may also have a role in nodule carbon metabolism. Moreover, the association of PEPC with the nodule vascular system implies a role for the enzyme in the transport of assimilates to and from the shoot.     

Frankia-Alnus incana symbiosis: Effect of endophyte on nitrogen fixation and biomass production

The efficiency of different FinnishFrankia strains as symbionts onAlnus incana (L.) Moench was evaluated in inoculation experiments by measuring nitrogen fixation and biomass production. Since all available pure cultures ofFrankia are of the Sp⁻ type (sporangia not formed in nodules), but the dominant nodule endophyte ofA. incana in Finland is of the Sp⁺ type (sporangia formed in nodules), crushed nodules of thisFrankia type were included. The Sp⁻ pure cultures, whether originating fromA. incana orA. glutinosa, produced with one exception, similar biomass withA. incana. The highest biomass was produced with an American reference strain fromA. viridis crispa. Using Sp⁺ nodule homogenates fromA. incana as inoculum, the biomass production was only one third of that produced by Sp⁻ pure cultures from the same host. Hence, through selection of the endophyte it is possible to exert a considerable influence on the productivity ofAlnus incana.     

Morphogenesis of lucerne root nodules incited by Rhizobium meliloti in the presence of combined nitrogen

Combined light and transmission electron microscopy were used to examine the effect of nitrate on the development of root nodules in lucerne (alfalfa, Medicago sativa L.) following induction by the nitrogen-fixing symbiont, Rhizobium meliloti. The timing of NO ₃ ^- addition was varied in order to study its effect on all of the recognized morphogenetic steps of nodule formation. Roots of plants inoculated in the presence of 18 mM NO ₃ ^- had straight root hairs which were devoid of adherent rhizobia and infection threads, and developed no nodules. However, nodules were formed on roots if 18 mM NO ₃ ^- was added 5 d after inoculation. At this time, the initiation of nodule primordia had already commenced in the root cortex. The histology and ultrastructure of young nodules which had developed for 5 d in the absence of NO ₃ ^- and another 5 d in the presence of 18 mM NO ₃ ^- resembled nodules developing under N-free conditions, except that in the infection threads within the infection zone of the nodule 1) some bacteria tended to loose their normal shape and gain more electron density, indicating premature degradation, and 2) the matrix of the infection threads was abnormally enlarged. In the presence of high NO ₃ ^- levels in the medium, lysis and degeneration of the bacteria released from the infection threads were observed in the infection and bacteroid zones of developing nodules, indicative of premature senescence. On the other hand, the nodule meristems continued to proliferate even after 12 d of exposure of 18 mM NO ₃ ^- . This was the only morphogenetic step of root nodulation which was insensitive to levels of combined nitrogen that completely prevented infection if present at the time of inoculation. These data indicate that all of the recognized steps of root nodule morphogenesis in which the bacteria play a key role are sensitive to the inhibitory effect of combined nitrogen.     

Localization of acid phosphatase activity in the apoplast of pea (Pisum sativum L.) root nodules grown under phosphorus deficiency

ACPase activity was localized in the apoplast of pea root nodules under phosphorus deficiency. Pea plants (Pisum sativum L. cv. Sze ciotygodniowy) where inoculated with Rhizobium leguminosarum bv. viciae 248 and were cultured on nitrogen-free medium with phosphate (−N/+P) or phosphate-deficient (−N/−P) one. In comparison with control nodules, P-deficient nodules showed the increase of ACPase activity in plant cell walls and the infection threads. The increase in bacterial ACPase activity under P-deficiency may reflect higher demand for inorganic phosphorus that is necessary for bacteria multiplication within the infection threads. The increase of ACPase activity in nodule apoplast under P stress may enlarge the availability of phosphate for plant and bacteria.     

Short-term inhibition of legume N2 fixation by nitrate

The hypothesis of NO ₂ ⁻ toxicity as the causative factor of NO ₃ ⁻ inhibition of nitrogenase (N₂ase; EC 1.18.6.1) activity has been evaluated using a short-term exposure (3 d) of several legumes. Treatment of plants with 10 mM NO ₃ ⁻ induced nitrate reductase (NR) from bacteroids (EC 1.7.99.4) and nodule cytosol (EC 1.6.6.1) in most species. Regardless of the levels of both enzymes, significant accumulation of NO ₂ ⁻ did not occur in nodules. Dissection of nodules into cortical and infected regions, and subsequent NO ₂ ⁻ assays in conditions that suppressed enzyme activities, indicated that, in the short-term, bacteroid NR does not generate NO ₂ ⁻ in vivo. This is probably because NO ₃ ⁻ access is restricted to the nodule cortex. Accumulation of NO ₂ ⁻ at levels that are damaging for N₂ase and leghaemoglobin were only observed when a delay occurred between dissection and assaying of nodules. It is concluded that NO ₂ ⁻ is not responsible for the initial NO ₃ ⁻ -induced decline of N₂ase activity, and that toxic amounts of NO ₂ ⁻ only build up in nodules following longer exposures to NO ₃ ⁻ , when this anion is actively reduced by bacteroid and cytosol enzymes.