The occurrence of leghemoglobin protein in the uninfected interstitial cells of soybean root nodules

The distribution of leghemoglobin (Lb) in resin-embedded root nodules of soybean (Glycine max (L.) Merr.) was investigated using immunogold labeling. Using anti-Lb immunoglobulin G and protein A-gold, Lb or its apoprotein was detected both in cells infected by Bradyrhizobium japonicum and in uninfected interstitial cells. Leghemoglobin was present in the cytoplasm, exclusive of the organelles, and in the nuclei of both cell types. In a comparison of the density of labeling in adjacent pairs of infected and uninfected cells, Lb was found to be about four times more concentrated in infected cells. This is the first report of Lb in uninfected cells of any legume nodule; it raises the possibility that this important nodule-specific protein may participate in mediating oxygen flow to host plant organelles throughout the infected region of the nodule.Abbreviations BSA bovine serum albumin - IgG immunoglobulin G - kDA kilodalton - Lb leghemoglobin - TBST Tris-buffered saline plus Tween 20     

Sequential expression of two late nodulin genes in the infected cells of alfalfa root nodules.

The Nms-22 and leghemoglobin (Lb) genes are expressed exclusively in the infected cells of alfalfa root nodules. Expression of these two late nodulin genes originated at distinct cellular boundaries within the symbiotic region of the nodule. The Nms-22 gene was expressed in all infected cells, including those just adjacent to the meristematic region. Lb gene expression was induced in older infected cells and was most prominent in the mature region of the nodule. Despite this temporal separation of gene expression, both the Nms-22 and Lb genes were expressed in nodules elicited by bacA mutants in which bacteroid development has been blocked just after release from the infection thread.     

Lupin leghemoglobins during root nodule development

Two yellow lupin leghemoglobins, Lb I and Lb II, were purified to homogeneity using the HPLC technique for final separation. Lb I and Lb II were identified by the N-terminal sequences and their reaction with antibodies against electrophoretically pure leghemoglobin. The third Lb species was detected by the combined method of isoelectrofocusing and PAGE of Lb I. It seems that Lb III represents a posttranslational modification of Lb I. Developmental changes in Lb multiple forms were examined using the Western blotting method. The content of leghemoglobin, first detectable approximately 3 weeks after infection, increased up to 6-7 weeks, and then it remained at the same level until 8-9 weeks after the infection. At the early stages of nodule formation Lb I prevailed over Lb II, while later Lb II became the predominant form. This suggests physiological role of particular forms and precise regulation of the expression of Lb genes.     

Chromosomal arrangement of leghemoglobin genes in soybean.

A cluster of four different leghemoglobin (Lb) genes was isolated from AluI-HaeIII and EcoRI genomic libraries of soybean in a set of overlapping clones which together include 45 kilobases (kb) of contiguous DNA. These four genes, including a pseudogene, are present in the same orientation and are arranged in the order: 5'-Lba-Lbc1-Lb psi-Lbc3-3'. The intergenic regions average 2.5 kb. In addition to this main Lb locus, there are other Lb genes which do not appear to be contiguous to this locus. A sequence probably common to the 3' region of Lb loci was found flanking the Lbc3 gene. The 3' flanking region of the main Lb locus also contains a sequence that appears to be expressed more abundantly in root tissue. Another sequence which is primarily expressed in root and leaf is found 5' to two Lb loci. Overall, the main leghemoglobin locus is similar in structure to the mammalian globin gene loci.     

A Mutation in Vicia faba Results in Ineffective Nodules with Impaired Bacteroid Differentiation and Reduced Synthesis of Late Nodulins

Although numerous reports have documented the effect of bacterially-inducedineffectiveness on root nodule structure, function, and plantgene expression, few studies have detailed the effect of theplant genome on similar parameters. In this report effective(N₂-fixing) broadbean {Vicia faba L.) and plant-controlled ineffective(non-N₂-fixing) broadbean recessive for the sym-1 gene werecompared for nodule structure, developmental expression of noduleenzyme activities, enzyme proteins, and mRNAs involved in Nassimilation, leghemoglobin (Lb) synthesis, and acetylene reductionactivity (ARA). During development of effective wild-type nodules,glutamine synthetase (GS), aspartate aminotransferase (AAT),phosphoenolpyruvate carboxylase (PEPC) and NADH-glutamate synthase(GOGAT) activities and enzyme proteins increased coincidentwith nodule ARA. The increases in GS, AAT, and PEPC were associatedwith increased synthesis of mRNAs for these proteins. Synthesisof Lb polypeptides and mRNAs during development of effectivenodules was similar to that of GS, AAT, and PEPC. By contrast,ineffective sym-1 nodules displayed little or no ARA and hadneither the increases in enzyme activities nor enzyme proteinsand mRNAs as seen for effective nodules. The effect of the sym-1gene appeared to occur late in nodule development at eitherthe stage of bacterial release from infection threads or differentiationof bacteria into bacteroids. High in vitro enzyme activities,enzyme polypeptides, and mRNA levels in parental effective noduleswere dependent upon a signal associated with effective bacteroidsthat was lacking in sym-1 nodules. Nodule organogenesis didnot appear to be a signal for the induction of GS, PEPC, AAT,and Lb expression in sym-1 nodules. Key words: Vicia faba, mutation, sym-1 gene, nodules     

Molecular cloning of lupin leghemoglobin cDNA

Poly(A)⁺RNA isolated from root nodules of yellow lupin (Lupinus luteus, var. Ventus) has been used as a template for the construction of a cDNA library. The ds cDNA was synthesized and inserted into the Hind III site of plasmid pBR 322 using synthetic Hind III linkers. Clones containing sequences specific for nodules were selected by differential colony hybridization using³²P-labeled cDNA synthesized either from nodule poly(A)⁺RNA or from poly(A)⁺RNA of uninfected root as probes. Among the recombinant plasmids, the cDNA gene for leghemoglobin was identified. The protein structure derived from its nucleotide sequence was consistent with known amino acid sequence of lupin Lb II. The cloned lupin Lb cDNA hybridized to poly(A)⁺RNA from nodules only, which is in accordance with the general concept, that leghemoglobin is expressed exclusively in nodules.     

Expression of lupin nodulin genes during root nodule development

Seven lupin cDNA clones were used to study the expression of corresponding genes during nodule development by Northern blots analysis. They include six nodulin cDNAs: pLLb (lupin leghemoglobin), pLN 13, pLN 21–27, pLN 281, pLN 50, pLNGS (nodule form of glutamine synthetase GS_n and root form of GS: pGS. The appearance of nodulin mRNAs during lupin nodule development showed that the nodulin sequences analysed represent a group of plant genes involved in the nitrogen fixation process rather than formation of nodule. This is based on the observation that they are activated at the time when the nodule has already been formed, prior to the onset of nitrogenase activity. The products of Lb, nodulin 21–67, the nodulin coded by pLN13 and the nodulin 281 genes appeared between 11 and 13 days after infection, whereas the nodulion coded by pLN50 and the nodule form of GS appeared 18 days after inoculation. Twenty-one days post-infection a dramatic increase in the transciption rates of all nodulin genes is observed. This phenomenon may be related to the onset of nitrogenase activity. The possible mechanism of two-step activation of nodulin genes is discussed.     

cDNA cloning and developmental expression of pea nodulin genes

A cDNA library prepared from pea nodule poly(A)⁺ RNA was screened by differential hybridization with cDNA probes synthesized from root and nodule RNA respectively. From the cDNA clones that hybridized exclusively with the nodule probe five clones, designated pPsNod 6, 10, 11, 13 and 14 and each containing unique sequences, were further characterized together with one leghemoglobin and one root-specific cDNA clone. In vitro translation of RNA selected by the pPsNod clones showed that the corresponding genes encode nodulins with molecular weights ranging from 5 800 to 19 000. During pea root nodule development expression of the five PsNod genes starts more or less concomitantly with the onset of nitrogen fixing activity in the nodules and the time course of appearance and accumulation of the nodulin mRNAs is similar to that of leghemoglobin mRNA. In ineffective pea root nodules expression of the PsNod genes is induced but the final accumulation levels of the mRNAs are markedly reduced to various degrees. The expression of another nodulin gene, designated ENOD2, was followed using a heterologous soybean cDNA clone as probe. In pea root nodules the ENOD2 gene is expressed at least five days before the PsNod and leghemoglobin genes, and in contrast to the PsNod mRNAs the concentration of the ENOD2 mRNA is the same in wild type and fix ^- nodules. The results described suggest that in root nodules several regulatory mechanisms exist which determine the final nodulin mRNA amounts accumulating in the root nodule.     

Changes in four leghemoglobin components in nodules of hypernodulating soybean (Glycine max [L] Merr.) mutant and its parent in the early nodule developmental stage

Soybean nodules contain four major leghemoglobin (Lb) components, Lba, Lbc₁, Lbc₂ and Lbc₃. A sensitive and selective method for quantitative analysis of the four Lb components was devised with capillary isoelectric focusing (CIEF). The changes in the concentrations of four Lb components in nodules during the initial stages of development were compared between hypernodulating soybean mutant NOD1–3 and its parent cv. Williams. The hydroponically cultivated soybean plants were periodically sampled. All the visible nodules were collected from the roots, and then the four Lb components in the largest nodules were analyzed with the CIEF method. In NOD1–3 Lbs were initially detected at 19 days after sowing (DAS), a few days earlier than in Williams at 22 DAS. The Lbcs (Lbc₁, Lbc₂ and Lbc₃) were the main component at the earliest nodule growth stage, and the relative proportion of Lba increased with nodule growth in both NOD1–3 and Williams. This result is in agreement with previous observation, and the CIEF method is considered to be useful for Lb components analysis to define their function and gene expression.     

Developmental regulation of nodule-specific genes in alfalfa root nodules

We have cloned alfalfa nodule-specific cDNAs that code for leghemoglobin (Lb), glutamine synthetase (GS), and three unidentified nodulins. Hybrid-select translation of nodule RNA followed by 2-D gel electrophoresis showed that the Lb-specific cDNA corresponded to at least four Lb species of 12 kDa. One of the unidentified cDNA clones (N-32/34) corresponded to at least five polypeptides of 32-34 kDa; a second unidentified cDNA clone (N-14) corresponded to an individual polypeptide of 14 kDa. The in vitro translation product(s) of the RNA hybrid selected by the third unidentified cDNA clone (N-22) formed a single band at 22 kDa on a one-dimensional gel. Northern and dot blot analyses of RNA isolated from wild-type nodules and from defective nodules elicited by a variety of Rhizobium meliloti mutants showed that 1) RNAs corresponding to the Lb, nodule-specific GS, and three unidentified nodulins were coordinately expressed during the course of nodule development, and 2) all five nodulins were expressed in Fix- nodules that contained infection threads and bacteroids but were not expressed in nodules that lacked infection threads and intracellular rhizobia.     

Crucial role of (homo)glutathione in nitrogen fixation in Medicago truncatula nodules

Legumes form a symbiotic interaction with bacteria of the Rhizobiaceae family to produce nitrogen-fixing root nodules under nitrogen-limiting conditions. We examined the importance of glutathione (GSH) and homoglutathione (hGSH) during the nitrogen fixation process. Spatial patterns of the expression of the genes involved in the biosynthesis of both thiols were studied using promoter-GUS fusion analysis. Genetic approaches using the nodule nitrogen-fixing zone-specific nodule cysteine rich (NCR001) promoter were employed to determine the importance of (h)GSH in biological nitrogen fixation (BNF). The (h)GSH synthesis genes showed a tissue-specific expression pattern in the nodule. Down-regulation of the γ-glutamylcysteine synthetase (γECS) gene by RNA interference resulted in significantly lower BNF associated with a significant reduction in the expression of the leghemoglobin and thioredoxin S1 genes. Moreover, this lower (h)GSH content was correlated with a reduction in the nodule size. Conversely, γECS overexpression resulted in an elevated GSH content which was correlated with increased BNF and significantly higher expression of the sucrose synthase-1 and leghemoglobin genes. Taken together, these data show that the plant (h)GSH content of the nodule nitrogen-fixing zone modulates the efficiency of the BNF process, demonstrating their important role in the regulation of this process.     

Induction of Root Nodule Senescence by Combined Nitrogen in Pisum sativum L

Root nodule senescence induced by nitrate and ammonium in Pisum sativum L. was defined by determining nitrogenase activity and leghemoglobin content with the acetylene reduction and pyridine hemochrome assays. Root systems supplied with 100 mm KNO(3) or 100 mm NH(4)Cl exhibited a decrease in nitrogenase activity followed by a decline in leghemoglobin content. Increasing the CO(2) concentration from 0.000320 atm to 0.00120 atm had no effect on the time course of root nodule senescence when 20 mm KNO(3) was supplied to the roots; in vitro nitrate reductase activity was detected in leaves and roots, but not bacteroids. Nitrate appeared in leaves, roots, and the nodule cytosol fraction but not bacteroids when 20 mm KNO(3) was supplied to roots. When nitrate entered through the shoots, however, no root nodule senescence was observed, and no nitrate was detected in root or nodule cytosol fractions although nitrate and nitrate reductase were found in leaves. The results suggest that nitrate does not induce root nodule senescence through competition between nitrate reductase and nitrogenase for products of photosynthesis.     

Effect of pO(2) on the Formation and Status of Leghemoglobin in Nodules of Cowpea and Soybean

Nodulated cowpea (Vigna unguiculata [L.] Walp. cv Vita 3: Bradyrhizobium strain CB756) and soybean (Glycine max [L.] Merr. cv White Eye: Bradyrhizobium strain CB1809) were grown with their root systems maintained in a flowing gas stream containing a range of pO₂ (1-80%, v/v) in N₂ for up to 28 days after planting. At the extremes of sub- and supra-ambient pO₂, the levels of leghemoglobin (Lb) in nodules were reduced. However, neither the proportional composition of Lb component proteins (eight in soybean, three in cowpea) nor their oxidation state was affected by pO₂. Short-term changes in pO₂ (transferring plants grown with sub- or supra-ambient pO₂ in the rhizosphere to air or vice versa) caused a significant decline in Lb content and, in cowpea but not soybean, where pO₂ was increased, a higher percentage of oxidation of Lb. Combining data on changes in Lb level of cowpea nodules grown in sub-ambient pO₂ with those for their structural adaptation to an under supply of O₂ indicated that, despite the nodules having a lower level of Lb, the amount per infected cell was increased by up to twofold and per bacteroid up to fivefold (in those from 1% O₂) compared to those grown in air. Progressive decline in pO₂ resulted in a progressive increase on this basis, indicating a close relationship between Lb content and the adaptation of nodule functioning to external O₂ level.     

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.     

Nodule structure and functioning in Cicer arietinum as affected by nitrate

Chickpea (Cicer arietinum L.) cv. C-235 inoculated with Rhizobium sp. (Cicer) strain cv4Az was raised in sand culture under natural conditions with nitrogen-free nutrient solution. 45-d-old plants were treated with 20 and 50 mM KNO3 and sampling made 2 and 6 d after treatment. KNO3 application induced premature nodule senescence. Light microscopic investigations showed that KNO3 treatments resulted in structural degradation of the central bacteroidal tissue. The mass of green nodules increased by 35 % under these treatments. This was accompanied by a rapid decline in leghemoglobin (Lb) content of the nodules being 51 - 67 % lower than in control. The total soluble nodule proteins showed relatively minor changes under KNO3 treatments thus suggesting preferential degradation of Lb. These changes were associated with a rapid decline in N2-fixing activity. However, the decline in total soluble sugars was relatively minor as compared to acetylene reducing activity, thus indicating that sugar deprivation is not the cause of decreased nitrogen fixation ability. Glutathione reductase and ascorbate peroxidase activity showed a 10 - 20 % decrease in comparison with the control. Accumulation of H2O2 and structural degradation of the nodular tissue are considered to be the factors leading to nodule senescence under nitrate treatments. This revised version was published online in July 2006 with corrections to the Cover Date.     

Nitrate Effects on Nodule Oxygen Permeability and Leghemoglobin (Nodule Oximetry and Computer Modeling)

Two current hypotheses to explain nitrate inhibition of nodule function both involve decreased O2 supply for respiration in support of N2 fixation. This decrease could result from either (a) decreased O2 permeability (PO) of the nodule cortex, or (b) conversion of leghemoglobin (Lb) to an inactive, nitrosyl form. These hypotheses were tested using alfalfa (Medicago sativa L. cv Weevlchek) and birdsfoot trefoil (Lotus corniculatus L. cv Fergus) plants grown in growth pouches under controlled conditions. Nodulated roots were exposed to 10 mM KNO3 or KCI. Fractional oxygenation of Lb under air (FOLair), relative concentration of functional Lb, apparent PO, and O2-saturated central zone respiration rate were all monitored by nodule oximetry. Apparent PO and FOLair in nitrate-treated nodules decreased to <50% of values for KCI controls within 24 h, but there was no decrease in functional Lb concentration during the first 72 h. In nitrate-treated alfalfa, but not in birdsfoot trefoil, FOLair, apparent PO, and O2-saturated central zone respiration rate decreased during each light period and recovered somewhat during the subsequent dark period. This species difference could be explained by greater reliance on photoreduction of nitrate in alfalfa than in birdsfoot trefoil. Computer simulations extended the experimental results, showing that previously reported decreases in apparent PO of Glycine max nodules with nitrate exposure cannot be explained by hypothetical decreases in the concentration or O2 affinity of Lb.     

Effetto della rimozione dei fiori sulla crescita delle piante e sul contenuto in leghemoglobina dei tubercoli radicali di pisello e fava

Abstract

Observations on vegetative growth and leghemoglobin contents of root nodules of pea and bean plants after flower bud removal. — These studies found their origin in the papers by MATTIROLO (1899) on the effect of the removal of flowers as they formed in bean plants; he observed that deflowering resulted in extraordinary plant growth, stem branching and flower buds formation as well as in a delayed root nodule senescence. In the light of modern knowledge of the leghemoglobin role in symbiotic nitrogen fixation, the aim of the present research was to ascertain any possible relation between flower bud removal and haeme pigment contents in the root nodules. The experiments were carried out during two growing seasons (1966 and 1970) using Vicia faba L. cv. Regina and Pisum sativum L. cv. Senatore during 1966 and cv. Vittoria in 1970. In both control and test plants the seasonal trends of average plant height, fresh and dry weight of vegetative portions, fresh and dry weight of root system, fresh weight of nodules, root nodule leghemoglobin concentration and total leghemoglobin content per plant, were determined. The data obtained are quoted in Table 2 and reported in Figures 1, 2, and 3. The removal of flower buds caused in both species: an increased plant growth, a marked stem branching, a longer blooming period, an increased flower number, an increased root nodule number and a certain delay in root nodule reabsorption. Deflowering did not significantly extend — at least in the species studied — life span (senescence was delayed only of one week). On the basis of these and of other Authors' results, we conclude that deflowering may actually delay senescence; the size of this delay, however, depends on the plant species considered and is fairly negligible both in pea and bean. The different effects of deflowering and of preventing floral induction on life span extension, are discussed, and these facts lead to consider floral induction as the onset of a chain of processes leading annual plants toward senescence in a more or less delayable, but definitive way. After having stressed the generally accepted importance of leghemoglobin concentration as an index of nodule nitrogen fixing ability, a correlation between biomass increase of test plants and number and total weight increase of root nodules, is put in evidence. No correlation between test plant biomass and the leghemoglobin concentration in root nodules, was however observed. Leghemoglobin concentration in root nodules is known to change in connection with various factors depending either on host plants and on Rhizobium strains and also in connection with several environmental conditions. Any prevented flower onset (ROPONEN and VIRTANEN, 1968) and deflowering (our data) however exerted no significant influence. The effects of flower bud removal were therefore the following: increased stem, leaf and root weights and increased root nodule number; no difference between control and test plants was however observed as regards size and leghemoglobin concentration of root nodules and hence probably no difference as regards their nitrogen fixing ability.