The role of phosphorus in nitrogen fixation by young pea plants (Pisum sativum)

The influence of P on N₂ fixation and dry matter production of young pea ( Pisum sativum L. cv. Bodil) plants grown in a soil-sand mixture was investigated in growth cabinet experiments. Nodule dry weight, specific C₂H₂ reduction and P concentration in shoots responded to P addition before any growth response could be observed. The P concentration in nodules responded only slightly to P addition. A supply of P to P-deficient plants increased both the nodule dry weight, specific C₂H₂ reduction and P concentration in shoots relatively faster than it increased shoot dry weight and P concentration in nodules. Combined N applied to plants when N₂ fixation had commenced, increased shoot dry weight only at the highest P levels. This indicates that the smaller plant growth at the low P levels did not result from N deficiency. The reduced nodulation and N₂ fixation in P-deficient plants seem to be caused by impaired shoot metabolism and not by a direct effect of P deficiency of the nodules.     

Genetic diversity of root-nodulating bacteria isolated from pea (Pisum sativum) in subtropical regions of China

Diversity of 42 isolates from effective nodules of Pisum sativum in different geographical regions of China were studied using 16S rRNA gene RFLP patterns, 16S rRNA sequencing, 16S–23S rRNA inter-genic spacer (IGS) region RFLP patterns and G-C rich random amplified polymorphic DNA (RAPD). The isolates were distributed in two groups on the basis of their 16S rRNA gene RFLP patterns. The 16S rRNA gene sequences of strains from 16S rRNA gene RFLP patterns group I were very closely related (identities higher than 99.5%) to Rhizobium leguminosarum USDA 2370. Group II consisting of WzP3 and WzP15 was closely related to Rhizobium etli CFN42. The analysis of the 16S–23S IGS RFLP pat-terns divided the isolates into 18 genotypes and four groups. Group I was clustered with R. legumino-sarum USDA2370. Group II consisted of YcP2, YcP3 and CqP7. The strains of group III were distributed abroad. Group IV consisted of WzP3, WzP15 and R. etli CFN42. RAPD divided the isolates into nine clusters in which group IV only consisted of YcP2 and the strains of group V and IX were from Wenzhou and Xiantao, respectively. This assay demonstrated the geographical effect on genetic diversity of pea rhizobia.     

Characteristics of nitrate uptake into seedlings of pea (Pisum sativum L. cv. Feltham First). Changes in net NO−3 uptake following inoculation with Rhizobium and growth in low nitrate concentrations

Abstract Nitrate uptake into intact pea seedlings (Pisum sativum L. cv. Feltham First) grown in hydroponic culture has been investigated. Following inoculation with Rhizobium leguminosarum a twofold increase in net nitrate uptake was observed. Changes in morphological characteristics following inoculation were found to decrease the effective area available for absorption. There was a two-fold decrease in net nitrate uptake into intact seedlings grown in the presence of N compared with N free media. In the former case net nitrate uptake appeared to stall at regular intervals. In both cases only the initial rates of nitrate uptake were found to be responsive to the external nitrate concentration. The results are discussed in terms of current models for the regulation of NO^?₃ uptake by higher plants.     

Cadaverine formation by specific lysine decarboxylation in Pisum sativum seedlings

Cadaverine was found to be formed in Pisum sativum seedlings via a specific lysine decarboxylation pathway as revealed by specific inhibitor studies. Lysine decarboxylation activity was recorded in the meristems and non-meristematic tissue of the shoots and the roots. In the shoot elongation zone, the specific activity was double that in the other tissues and cadaverine level was 90-fold higher. The results presented in this study suggest possible regulatory control by polyamines of lysine decarboxylase activity in Pisum sativum seedlings.Abbreviations LDC lysine decarboxylase - -DFMO DL--difluoromethyl ornithine - -DFMA DL--difluoromethyl arginine - PLP pyridoxal phosphate - DTT dithiothreitol This paper is Contribution No. 1380-E, 1985 series, from the Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel     

1,3-beta-D-glucanases from Pisum sativum seedlings. I. Isolation and purification.

Two buffer-soluble endo-1,3-beta-D-glucanases (EC 3.2.1.6) have been purified to within 1% of electrophoretic homogeneity from etiolated Pisum sativum stem tissues. Purified glucanase I and II differ in physical properties, such as electrophoretic mobility in sodium dodecyl sulfate polyacrylamide gels (Mr values were 22 000 and 37 000, respectively) and isoelectric focusing, (pI values were 5.4 and 6.8, respectively). Although the enzymes have similar pH optima (5.5--6.0), Km values for various substrates (0.6--7.4 mg/ml) and thermal inactivation profiles, they are localized in different tissues and they differ markedly in the rates with which they attack the internal linkages of long- vs. short-chain substrates. Glucanase I is concentrated in apical regions of the stem and is most effectively assayed reductometrically (as laminarinase), while glucanase II is localized in mature regions and is relatively more active in viscometric assays (as carboxymethyl-pachymanase).     

GTP-binding proteins in etiolated epicotyls of Pisum sativum (Alaska) seedlings

Seven fractions of GTP-binding proteins separated by gel filtration of an extract of epicotyls of Pisum sativum seedlings were partially characterized. Seven fractions of GTP-binding proteins tentatively designated GP1 to GP7 had the capacity to be ADP-ribosylated by pertussis toxin. Pooled fractions of GP2 to GP7 showed Km values 2, 20, 50, 10, 3 and 1 nM, respectively. The binding of [35S]GTP gamma S to GTP-binding proteins was prevented competitively in the presence of 0.1 mM GTP and also prevented in the presence of 0.1 mM ATP. Binding of [35S]GTP gamma S to the proteins produced a decrease in their molecular weights.     

Tyrosine specific sequential labeling of proteins

We report (a) on the synthesis of a long-wavelength fluorescent coumarin containing an allyloxy acetate moiety, (b) the synthesis of two linkers containing an allyloxy acetate and an alkyne or azide function, respectively, and (c) the selective modification human serum albumin by a sequential method involving Pd(II) catalyzed modification of the phenolic side chain of tyrosine residues with an alkyne bearing linker and a subsequent azide–alkyne click reaction with an azide functionalized long-wavelength emitting coumarin dye. The method is likely to be applicable to various kinds of azido-modified fluorophores, and the Pd(II)-catalyzed modification of the tyrosines may also be used to introduce other kinds of tags. With these reagents, tyrosine specific modulation of proteins and peptides becomes possible either directly or in a sequential manner.     

Immunoelectrophoretic analysis of seed proteins from Pisum sativum L.

Summary Soluble proteins of pea seed were investigated by quantitative immunological methods. Vicilin, legumin, pea seed lectin (PEA), 26 albumins and a globulin (B₁) were detected and observed during seed development, germination and under different extraction and fractionation procedures. Vicilin and legumin were found to be immunologically distinctly different. Legumin was found to be comprised of two similar proteins, Legumin species I and II. Vicilin, but no legumin, was detected in the embryonic axis.Three albumins, B₁ and PEA were found to be synthesized after the onset of legumin synthesis.Among the pea lines investigated, one line exhibited distinct differences with respect to the albumins and PEA.Some observations indicate that PEA might interact with other seed proteins of pea.     

Influence of brassinosteroids on initiation of the root gravitropic response in Pisum sativum seedlings

In roots of Pisum sativum seedlings, the average lag-time required for initiation of the gravitropic response was reduced proportionally to the concentration of 24-epibrassinolide (EBL) added to the root solution (range of 10⁻¹³ to 10⁻⁸ M concentrations). A treatment with clotrimazole, a compound inhibiting steroid synthesis, prevents initiation of the gravitropic response. This effect was partly reverted by addition of EBL. From analysis of variability in the populations, it is suggested that BR conditions the root curvature through a gravitropic-induced change in sensitivity to the PGRs regulating cell elongation.     

Nodule-specific host proteins in effective and ineffective root nodules of Pisum sativum

Nodule-specific root proteins – so called nodulins – were identified in root nodules of pea plants by an immunological assay. Nodulin patterns were examined at different stages of nodule development. About 30 nodulins were detectable during development. Some were preferentially synthesized before nitrogen fixation started, whereas the majority were synthesized concomitantly with leghaemoglobin. Some of the nodulins were located within the peribacteroid membrane. Ineffective Rhizobium strains (a natural nod⁺fix^- and a pop ^-fix^-) appeared to be useful in studying the expression of nodulin genes. Synthesis of some nodulins was repressed in ineffective root nodules, indicating that nodulins are essential for the establishment of nitrogen fixation. In both types of ineffective root nodules, leghaemoglobin synthesis was not completely repressed. Low amounts of leghaemoglobin were always detected in young ineffective root nodules whereas in old nodules no leghaemoglobin was present.     

Isolation and partial characterization of phenolate siderophore from Rhizobium leguminosarum IARI 102

Abstract Rhizobium leguminosarum IARI 102 produced 2,3-dihydroxy benzoic acid, a type of phenolate siderophore, under iron-starved conditions. Hydroxamic acids were not detected. Maximum production of siderophore was found at 26 h of growth in a chemically defined medium at 28°C with shaking. Threonine was detected as the amino acid conjugate of the siderophore. Addition of Fe³⁺ to the culture medium increased the growth yield significantly, but depressed the production of the iron chelating compound.     

Extracellular superoxide production associated with secondary root growth following desiccation of Pisum sativum seedlings

The seedling stage is arguably the most vulnerable phase in the plant life cycle, where the young establishing plant is extremely sensitive to environmental stresses such as drought. Here, the production of superoxide (O₂⁻), a molecule involved in stress signaling, was measured in response to desiccation of Pisum sativum L. seedlings. Following desiccation that was sufficient to kill the radicle meristem, viability could be retained by seedlings that grew secondary roots. Upon rehydration, secondary roots formed in a region that had displayed intense extracellular O₂⁻production on desiccation. Treating partially desiccated seedlings with hydrogen peroxide (H₂O₂) prevented viability loss. In summary, reactive oxygen species (ROS) appear to participate in the signaling required for secondary root formation following desiccation stress of P. sativum seedlings.     

Comparison of geographically distant populations of Rhizobium isolated from root nodules of Phaseolus vulgaris

Seventy-two rhizobial strains were isolated from the root nodules of french beans ( Phaseolus vulgaris ). They were sampled from two geographically distant field populations and 18 additional different sites in France. They were characterized by a) plasmid profiles, (b) RFLP analysis of total cellular DNA using various chromosomal and symbiotic gene probes (including nif H from Rhizobium etli bv. phaseoli ) and c) their ability to nodulate a potential alternative host, L. leucocephala. Over half of the isolates were ascribed to Rhizobium leguminosarum bv. phaseoli on the basis of the hybridization analysis, the possession of multiple copies of nif H and their inability to nodulate L. leucocephala. The remaining isolates belonged to 2 groups which were shown to be genomically distinct from R. leguminosarum bv. phaseoli, R. etli bv. phaseoli and R. tropici. Most members of these two groups shared with R. tropici the ability to nodulate L. leucocephala and, for isolates of only one of these groups, the presence of one copy of nif H. Members of each of the 3 taxa were widely distributed in France and circumstantial evidence of pSym transfer between them was shown. R. leguminosarum bv. phaseoli and one of the two novel groups co-occurred within the two geographically distant populations. Individual genotypes were conserved between them. The finding of a third taxon at various other locations indicated additional diversity among rhizobia nodulating beans.     

The stimulating effect of ammonium on nodulation in Pisum sativum L. is not long lived once ammonium supply is discontinued

Although mineral N generally has a negative effect on legume-rhizobia symbioses, experiments in hydroponic culture in our laboratory (Waterer et al., 1992) have shown that low concentrations of NH⁺ ₄ can stimulate nodulation in pea (Pisum sativum L.). The objectives of the current study were to determine the immediate and residual effects of NH⁺ ₄ on nodulation and N₂ fixation in pea in sand culture. Peas (cv. Express) were exposed to 0.0, 0.5, 1.0, and 2.0 mM of ¹⁵N-labelled (NH₄)₂SO₄ for 28 days after inoculation (DAI). From 28 to 56 DAI the plants were grown on a NH⁺ ₄-free nutrient solution. Plants were harvested at 7, 14, 21, 28 and 56 DAI and nitrogenase activity was measured by gas exchange at 28 and 56 DAI. Root, shoot, and nodule dry weight (DW) and total N content were obtained, in addition to nodule counts and ¹⁵N enrichment of plant composites. The 1.0 and 2.0 mM NH⁺ ₄ treatments consistently resulted in higher total plant DW accumulation than the control (0.0 mM NH⁺ ₄). At 28 DAI, plants exposed to 1.0 and 2.0 mM NH⁺ ₄ had 1.8 to 2.8 times more nodules plant^-1, respectively, and plants exposed to 2.0 mM NH⁺ ₄ had 1.7 fold higher specific nodulation (nodule number g^-1 root DW). However, individual nodule DW was greater in control plants, such that there were no differences in nodule DW per plant among treatments. Ammonium treatment resulted in more nitrogen derived from the atmosphere (NDFA) in peas early in the experiment, but by 28 DAI there were no treatment effects on NDFA. Whole plant and nodule specific nitrogenase activity (µmol H₂ g^-1 nodule DW h^-1) was higher in control plants at 28 DAI. However, by 56 DAI, after an additional 4 weeks of NH⁺ ₄-free nutrition, no differences in nitrogenase activity nor whole plant or specific nodulation were detectable. This study indicates that nodulation in pea is stimulated in sand culture while exposed to NH⁺ ₄. However, once NH⁺ ₄ is removed, relative growth rate, nodulation and nitrogenase activity becomes similar to plants that were never exposed to NH⁺ ₄.