Nodule-specific polypeptides from effective alfalfa root nodules and from ineffective nodules lacking nitrogenase

In addition to leghemoglobin, at least nine nodule-specific polypeptides from the alfalfa (Medicago sativa L.)-Rhizobium meliloti symbiosis were identified by immune assay. Some of these polypeptides may be subunits of larger proteins but none appeared to be subunits of the same multimeric protein. All nine of the nodule-specific polypeptides were localized to within the plant cytosol; they were not found in extracts of bacteroids or in the peribacteroid space. At least one of these nodule-specific polypeptides was found to be antigenically related to nodule-specific polypeptides in pea and/or soybean. Ineffective nodules elicited by R. meliloti strains containing mutations in four different genes required for nitrogenase synthesis contained reduced concentrations of leghemoglobin and of several of the nodule-specific polypeptides. Other nodule-specific polypeptides were unaltered or actually enriched in the ineffective nodules. Many of the differences between the ineffective and effective nodules were apparent in nodules harvested shortly after the nodules became visible. These differences were greatly amplified in older nodules. When the four ineffective nodule types were compared to one another, there were clear quantitative differences in the concentrations of several of the nodule-specific polypeptides. These differences suggest that lack of a functional nitrogenase does not have a single direct effect on nodule development.     

Turnover of nitrogenase and leghemoglobin in root nodules of Pisum sativum

Turnover rates of the two nitrogenase components and leghemoglobin in root nodules of pea plants nodulated with Rhizobium leguminosarum were determined with three different methods: 1, Kinetics of 35S incorporation into protein; 2, pulse-chase experiments; 3, chloramphenicol inhibition of bacteroid protein synthesis. Methods 1 and 3 revealed that the turnover rates of the two nitrogenase components and leghemoglobin are identical to the average rate of bacteroid and plant nodule protein turnover. The t1/2 times of component I and II and leghemoglobin were about 2 days. Pulse-chase experiments with 35SO(2-)4 appeared to be rather unsuitable for determination of turnover rates in pea root nodules.     

Acetylene reduction by effective and ineffective clover and soybean root nodules

Summary Acetylene was reduced to ethylene by effective white clover nodules and by fully and partially effective intact nodules, nodule homogenates, and bacteroids of soybeans. Succinate and several amino acids markedly stimulated the reduction by effective soybean bacteroids, but the stimulation was slight with partially effective bacteroids. Acetylene metabolism by effective soybean bacteroids was also enhanced by excretions of in vitro-grown Rhizobium japonicum, excretions of bacteria derived from effective and ineffective nodules, and the soluble fraction from these nodules. Inhibitors of nitrogen fixation were not found in ineffective nodules. Ineffective soybean and white clover nodules and homogenates or isolated bacteria from ineffective soybean nodules did not reduce acetylene. Additions of succinate, amino acids, the soluble fraction of effective nodules, or excretions of effective bacteroids or of in vitro-grown cells of an effective R. japonicum strain did not promote nitrogen fixation by bacterial cells obtained from ineffective soybean nodules.     

Carbohydrate and organic acid composition of effective and ineffective root nodules of Phaseolus vulgaris

Dicarboxylic acid transport mutants of Rhizobium species are usually deficient in their ability to fix atmospheric dinitrogen. We report here a study comparing the physiology of root nodules on Phaseolus vulgaris L. cv. Goldie induced by an effective strain of Rhizobium leguminosarum biovar phaseoli and a C₄-dicarboxylic acid utilization mutant. The mutant, while able to form nodules, was ineffective in N₂ fixation. Carbohydrates and organic acids of roots and nodules formed by the 2 strains were monitored at 3-day intervals from 13 to 34 days after inoculation. Both carbohydrates and organic acids accumulated in ineffective nodules in comparison with the effective nodules. The concentration of malic acid was tenfold higher in ineffective nodules than in effective nodules. Other organic acids, i.e., lactate, malonate, ascorbate and gluconate, were also detected. Lactate and ascorbate were the only other organic acids accumulating in ineffective nodules. The most prevalent carbohydrates found in both types of nodules were sucrose, glucose and fructose. Myo-inositol was the only cyclitol detected in both types of nodules. Carbohydrates and organic acids were present in lower concentration in roots than in nodules, except for lactate. These compounds were not consistently detected in higher concentration in roots from plants inoculated with the mutant strain, as was the case in nodules.     

Initial proliferation of cortical cells in the formation of root nodules in Pisum sativum L.

Summary Root nodule initiation in Pisum sativum begins with cell divisions in the inner cortex at some distance from the advancing infection thread. After penetrating almost the entire cortex, the branches of the thread infiltrate the meristematic area previously initiated in the inner cortical cells. These cells are soon invaded by bacteria released from the infection thread and subsequently differentiate into non-dividing, bacteriod-containing cells. As the initial meristematic centre in the inner cortex is thus lost to bacteroid formation, new meristematic activity is initiated in neighbouring cortical cells. As development proceeds, more cortical layers contribute to the nodule, with the peripheral layer and apical meristem of the nodule not invaded by bacteria.Lateral root primordia are initiated in a region separate from that in which nodules are formed, with the lateral primordia being closer to the root apex. This is interpreted to indicate that the physiological basis for nodule initiation is distinct from that for initiation of lateral roots. The role of a single tetraploid cell in nodule initiation is refuted, as is the existence of incipient meristematic foci in the root. It is suggested that the tetraploid cells in nodule meristems arise from pre-existing endoreduplicated cells, or by the induction of endoreduplication in diploid cortical cells by Rhizobium.     

Alfalfa root nodule phosphoenolpyruvate carboxylase: characterization of the cDNA and expression in effective and plant-controlled ineffective nodules

Phosphoenolpyruvate carboxylase (PEPC) plays a key role in N₂ fixation and ammonia assimilation in legume root nodules. The enzyme can comprise up to 2% of the soluble protein in root nodules. We report here the isolation and characterization of a cDNA encoding the nodule-enhanced form of PEPC. Initially, a 2945 bp partial-length cDNA was selected by screening an effective alfalfa nodule cDNA library with antibodies prepared against root nodule PEPC. The nucleotide sequence encoding the N-terminal region of the protein was obtained by primer-extension cDNA synthesis and PCR amplification. The complete amino acid sequence of alfalfa PEPC was deduced from these cDNA sequences and shown to bear striking similarity to other plant PEPCs. Southern blots of alfalfa genomic DNA indicate that nodule PEPC is a member of a small gene family. During the development of effective root nodules, nodule PEPC activity increases to a level that is 10- to 15-fold greater than that in root and leaf tissue. This increase appears to be the result of increases in amount of enzyme protein and PEPC mRNA. Ineffective nodules have substantially less PEPC mRNA, enzyme protein and activity than do effective nodules. Maximum expression of root nodule PEPC appears to be related to two signals. The first signal is associated with nodule initiation while the second signal is associated with nodule effectiveness. Regulation of root nodule PEPC activity may also involve post-translational processes affecting enzyme activity and/or degradation.     

Abscisic acid inhibition of root nodule initiation in Pisum sativum

Summary The effect of exogenous abscisic acid (ABA) on root nodule formation in Pisum sativum cv. Alaska was examined. ABA supplied to the roots at 1.9×10^-6M reduced the number of nodules/plant 61% without affecting root or shoot growth. The first noticeable inhibition of nodulation occurred at 3.8×10^-7M ABA. ABA at a concentration of 1.9×10^-6M inhibited neither root hair formation nor infection of root hairs by Rhizobium leguminosarum. Similar numbers of infection threads penetrated the cortex in both control and treated plants. ABA concentrations of 3.8×10^-6M had no effect on the doubling time or maximum density of R. leguminosarum in pure cultures. Normal nodule formation involves a polyploid cortical proliferation. This response to rhizobial infection can be imitated by culturing 1-mm pea-root segments on a medium containing 4.7×10^-6M kinetin. Under these conditions a highly significant reduction in the number of polyploid mitoses after 72 h is produced by 3.8×10^-8M ABA. A maximum inhibition of 68% was found with 3.8×10^-6M ABA. A similar range of ABA concentrations also inhibited the cytokinin-induced cell division in soybean callus. It is concluded that ABA reduces the number of root nodules/plant by inhibiting the cortical cell divisions required for root nodule formation.     

Isolation of root hairs from seedlings of Pisum sativum. Identification of root hair specific proteins by in situ labeling

A procedure was developed which allows the large-scale isolation of root hairs from seedlings of Pisum sativum . L. cvs. Kleine Rheinländerin and Rosa Krone. The method may yield up to 50 g fresh weight of root hairs per 3.10⁴ seedlings. In a modified form considerable amounts of root hair material may be harvested, even after incubation of the roots in aqueous solutions. Thus, detailed biochemical studies on the root hair system have become feasible.
The occurrence of specific proteins in membrane fractions of P. sativum root hairs was demonstrated as follows: Incubation of root hairs in situ with 3-azidonaphthalene-2,7-disulfonate – a strongly anionic, photoactivated fluorescent marker – followed by gel electrophoresis of membrane fractions showed the presence of root-hair specific proteins which, since the system was intact, suggests that they are on the outer surface of the cells.     

Capacity for fermentation in roots and Rhizobium nodules of Pisum sativum L.

The aim of this work was to compare the capacities for fermentation and synthesis of malate from phosphoenolpyruvate in roots and Rhizobium nodules of Pisum sativum. The nodules and the cortices and apices of roots had similar activities of glycolytic enzymes and enzymes of ethanolic and lactic fermentation when expressed on a protein basis. The activity of phosphoenolpyruvate carboxylase was similar in nodules and apices, and three to four fold lower in cortices. All three tissues had very high activities of malate dehydrogenase, significant activity of NADP-malic enzyme, and no detectable activity of phosphoenolpyruvate carboxykinase. These results do not support the belief that nodules have a substantially greater capacity to convert phosphoenolpyruvate to malate than roots, or that there are major qualitative differences in the pathways of fermentation of nodules and roots.Abbreviation PEP phosphoenolpyruvate     

Reactive oxygen species production and antioxidative defense in pea (Pisum sativum L.) root nodules after short-term aluminum treatment

Pea plants (Pisum sativum L.) were treated with 50???M aluminum chloride at pH 4.5 for 2 or 24?h at room temperature. Following treatment, root nodule Al uptake, the generation of reactive oxygen species (ROS, O ₂ ^?· and H₂O₂), and the activities of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and peroxidase (POX) were investigated. Aluminum accumulation was found chiefly in the apoplast of the nodule cortex, endodermis and meristem, while the formation of peroxide was detected in the nodule cortex, infection threads and bacteroidal tissue. Further, there were increased levels of superoxide in the meristem and bacteroidal tissue. The activity of SOD (EC 1.15.1.1) and POX (EC 1.11.1.7) increased in the Al-treated nodules and the roots of pea plants, whereas CAT (EC 1.11.1.6) activity decreased. The Al absorbed by the nodules induced ROS production. The POX and SOD are important ROS-scavengers in Al-stressed nodules.     

Protein composition of the peribacteriod space in root nodules of Pisum sativum and Vicia faba induced by Rhizobium leguminosarum biovar viciae

Proteins in the peribacteroid space (PBS) between the bacteroid outer membrane and the peribacteroid membrane in root nodules of Pisum sativum and Vicia faba induced by Rhizobium leguminosarum PRE were analysed by two-dimensional (2-D) gel electrophoresis. Most of the detectable proteins were found to migrate to identical positions; however the level of accumulation of some of these appear to be determined by the host plant. When a different R. leguminosarum strain (RB1) was used to inoculate P. sativum , the majority of the isolated PBS proteins were found to migrate in the 2-D gel to identical positions as those of the other two combinations ( R. leguminosarum PRE x P. sativum and R. leguminosarum PRE x V. faba ).     

The effects of root decapitation on lateral root formation and cytokinin production in Pisum sativum

Decapitation of the primary roots of Pisum sativum L. resulted in a significant increase in the initiation of lateral root primordia within 12 h of the surgical treatment. Although this increase occurred both in the light and in the dark, lateral root initiation and development was much more rapid in the dark. The formation of lateral root primordia was accompanied by increased levels of endogenous cytokinins suggesting that newly formed root primordia start producing cytokinins very soon after being initiated. The significance of the present results in terms of possible sites of synthesis in vegetative tissue is discussed.     

Immunocytochemical localization of proteins in differentiating tissues of Pisum sativum

Although there may be documented morphological changes during development, it is obvious that important changes in protein content occur in a vascular plant during the several stages of differentiation. In the absence of the latter information, an approach has been established for the localization of antigenic proteins in developing tissues of Pisum sativum. Monoclonal antibodies were raised to proteins extracted from pea internode tissue, and employed for the localization of three proteins in tissue sections. One of the proteins has two polypeptide subunits with molecular weights of 25,000 and 40,000 daltons, and the antibody binds to both of them. The three monoclonal antibodies produce different patterns of cellular localization in the tissue sections, as visualized by indirect immunocytochemical labeling. In another series of analyses, quantitative and qualitative differences in the protein contents of apical shoot tissue and mature internode shoot tissue have been found. These studies were based on the use of Western blots with both polyclonal (rabbit) antibodies and monoclonal (mouse) antibodies.     

Immunocytochemical localization of proteins in differentiating tissues of Pisum sativum

Summary Although there may be documented morphological changes during development, it is obvious that important changes in protein content occur in a vascular plant during the several stages of differentiation. In the absence of the latter information, an approach has been established for the localization of antigenic proteins in developing tissues of Pisum sativum. Monoclonal antibodies were raised to proteins extracted from pea internode tissue, and employed for the localization of three proteins in tissue sections. One of the proteins has two polypeptide subunits with molecular weights of 25,000 and 40,000 daltons, and the antibody binds to both of them. The three monoclonal antibodies produce different patterns of cellular localization in the tissue sections, as visualized by indirect immunocytochemical labeling. In another series of analyses, quantitative and qualitative differences in the protein contents of apical shoot tissue and mature internode shoot tissue have been found. These studies were based on the use of Western blots with both polyclonal (rabbit) antibodies and monoclonal (mouse) antibodies.In honour of Prof. P. van Duijn     

Apyrase,streptavidin-binding proteins,and antimicrobial activity in Pisum sativum

The 49 kD apyrase (EC 3.6.1.5), streptavidin-binding proteins, and antimicrobial activity in the subcellular fractions from different seed parts of Pisum sativum L. var. Alaska were examined. Except cotyledons, all subcellular fractions contained 49 kD apyrase, and a considerable relationship was found between 49 kD apyrase and NTPase activities that increased with increasing time of germination. The bulk of 49 kD apyrase and NTPase activities was found in the nucleus pellets and cytoskeleton-enriched fraction, indicating their physiological importance. At 72 h of germination, all subcellular fractions of primary stems have a greater amount of 49 kD apyrase and NTPase than primary leaves and much more than primary roots and cotyledonary stalks. All seed parts showed antimicrobial activities, and the bulk of inhibition activities was found in the cytoskeleton-enriched and nucleus pellets, which was greater in the primary stems and leaves than in other parts. Current findings reveal that apyrases have important roles in metabolic activities in all parts of the pea plants except cotyledons. Cotyledons contained much streptavidin-binding proteins, which might have different physiological roles than apyrases.