Identifying abnormalities in symbiotic development between Trifolium spp. and Rhizobium leguminosarum bv. trifolii leading to sub-optimal and ineffective nodule phenotypes

Background and Aims

Legumes overcome nitrogen limitations by entering into a mutualistic symbiosis with N₂-fixing bacteria (rhizobia). Fully compatible associations (effective) between Trifolium spp. and Rhizobium leguminosarum bv. trifolii result from successful recognition of symbiotic partners in the rhizosphere, root hair infection and the formation of nodules where N₂-fixing bacteroids reside. Poorly compatible associations can result in root nodule formation with minimal (sub-optimal) or no (ineffective) N₂-fixation. Despite the abundance and persistence of strains in agricultural soils which are poorly compatible with the commercially grown clover species, little is known of how and why they fail symbiotically. The aims of this research were to determine the morphological aberrations occurring in sub-optimal and ineffective clover nodules and to determine whether reduced bacteroid numbers or reduced N₂-fixing activity is the main cause for the Sub-optimal phenotype.

Methods

Symbiotic effectiveness of four Trifolium hosts with each of four R. leguminosarum bv. trifolii strains was assessed by analysis of plant yields and nitrogen content; nodule yields, abundance, morphology and internal structure; and bacteroid cytology, quantity and activity.

Key Results

Effective nodules (Nodule Function 83–100 %) contained four developmental zones and N₂-fixing bacteroids. In contrast, Sub-optimal nodules of the same age (Nodule Function 24–57 %) carried prematurely senescing bacteroids and a small bacteroid pool resulting in reduced shoot N. Ineffective-differentiated nodules carried bacteroids aborted at stage 2 or 3 in differentiation. In contrast, bacteroids were not observed in Ineffective-vegetative nodules despite the presence of bacteria within infection threads.

Conclusions

Three major responses to N₂-fixation incompatibility between Trifolium spp. and R. l. trifolii strains were found: failed bacterial endocytosis from infection threads into plant cortical cells, bacteroid differentiation aborted prematurely, and a reduced pool of functional bacteroids which underwent premature senescence. We discuss possible underlying genetic causes of these developmental abnormalities and consider impacts on N₂-fixation of clovers.     

Comparison of nucleic acid content in populations of free-living and symbiotic Rhizobium meliloti by flow microfluorometry.

Populations of symbiotic Rhizobium meliloti extracted from alfalfa nodules were shown by flow microfluorometry to contain a significant number of bacteroids with higher nucleic acid content than the free-living rhizobia. Bacteroids with lower nucleic acid content than the free-living bacteria were not detected in significant quantities in these populations. These results indicate that the incapability of bacteroids to reestablish growth in nutrient media may not be caused by a decrease in nucleic acid content of the symbiotic rhizobia.     

Acetylcholine promotes the emergence and elongation of lateral roots of Raphanus sativus

Radish (Raphanus sativus L.) was grown on four layers of paper towel moistened with distilled water with and without acetylcholine (ACh) for five days in the dark after sowing. ACh at 1 nM promoted the growth (emergence and elongation) of lateral roots of radish plants, but had no effect on the stems and main roots. Moreover, ACh enhanced the dry weight of roots [main (primary) + lateral roots]. Neostigmine, an inhibitor of acetylcholinesterase (AChE) also promoted the emergence and elongation of lateral roots, and atropine, a competitive inhibitor of ACh receptor, suppressed the emergence and elongation. ACh promoted the activities of glyceraldehyde-3-phosephate dehydrogenase (G-3-PD), nicotinamide adenine dinucleotide-specific isocitrate dehydrogenase (NAD-ICDH), succinate dehydrogenase (SDH) and cytochrome-c oxidase (Cyt-c OD) in seedlings. Moreover, ACh suppressed the activity of AChE and increased the amount of proteins and pyridine nucleotides (NAD and NADH) in the roots of the seedlings. It also increased the activities of NAD-forming enzymes [NAD synthetase and ATP-nicotinamide mononucleotide (ATP-NMN) adenyltransferase], and enhanced the amount of DNA in the roots of the seedlings. The relationship between ACh and the emergence and growth of lateral roots was discussed from a biochemical viewpoint.Key words: acetylcholine, acetylcholine esterase, DNA, lateral root, NAD, NAD-forming enzyme, protein, Raphanus sativus     

The Rhizobium--legume symbiosis.

The rhizobia are soil microorganisms that can interact with leguminous plants to form root nodules within which conditions are favourable for bacterial nitrogen fixation. Legumes allow the development of very large rhizobial populations in the vicinity of their roots. Infections and nodule formation require the specific recognition of host and Rhizobium, probably mediated by plant lectins. Penetration of the host by a compatible Rhizobium species usually provokes host root cell division to form the nodule, and a process of differentiation by both partners then ensues. In most cases the rhizobia alter morphologically to form bacteroids, which are usually larger than the free-living bacteria and have altered cell walls. At all stages during infection, the bacteria are bounded by host cell plasmalemma. The enzyme nitrogenase is synthesized by the bacteria and, if leghaemoglobin is present, nitrogen fixation will occur. Leghaemoglobin is a product of the symbiotic interaction, since the globin is produced by the plant while the haem is synthesized by the bacteria. In the intracellular habitat the bacteria are dependent upon the plant for supplies of energy and the bacteroids, in particular, appear to differentiate so that they are no longer able to utilize the nitrogen that they fix. Regulation of the supply of carbohydrate and the use of the fixed nitrogen thus appear to be largely governed by the host.     

The spliceosomal intron of the rolA gene of agrobacterium rhizogenes is a prokaryotic promoter

Agrobacterium rhizogenes transfers DNA (T-DNA) from its Ri plasmid to plant cells. All T-DNA genes are expressed in plant cells. The rolA gene is the only T-DNA gene that contains an intron in the untranslated leader region of its mRNA. This paper shows that (i) the rolA gene is also transcribed in bacteria; (ii) the 85 bp corresponding to the spliceosomal intron drives prokaryotic gene expression in agrobacteria, in free-living rhizobia and in bacteroids within root nodules; and (iii) promoter activity is abolished by the deletion of 63 bp from its 5' end and is reduced by mutations changing its sequence near the putative -10 region. The expression pattern of a chimeric reporter gene shows that, in free-living bacteria, gene expression takes place during the exponential phase of growth and increases at the onset of the stationary phase. Within root nodules, reporter gene expression occurs in the invasion, nitrogen fixing and senescent zones.     

Three enzymes for trehalose synthesis in Bradyrhizobium cultured bacteria and in bacteroids from soybean nodules

alpha,alpha-Trehalose is a disaccharide accumulated by many microorganisms, including rhizobia, and a common role for trehalose is protection of membrane and protein structure during periods of stress, such as desiccation. Cultured Bradyrhizobium japonicum and B. elkanii were found to have three enzymes for trehalose synthesis: trehalose synthase (TS), maltooligosyltrehalose synthase (MOTS), and trehalose-6-phosphate synthetase. The activity level of the latter enzyme was much higher than those of the other two in cultured bacteria, but the reverse was true in bacteroids from nodules. Although TS was the dominant enzyme in bacteroids, the source of maltose, the substrate for TS, is not clear; i.e., the maltose concentration in nodules was very low and no maltose was formed by bacteroid protein preparations from maltooligosaccharides. Because bacteroid protein preparations contained high trehalase activity, it was imperative to inhibit this enzyme in studies of TS and MOTS in bacteroids. Validamycin A, a commonly used trehalase inhibitor, was found to also inhibit TS and MOTS, and other trehalase inhibitors, such as trehazolin, must be used in studies of these enzymes in nodules. The results of a survey of five other species of rhizobia indicated that most species sampled had only one major mechanism for trehalose synthesis. The presence of three totally independent mechanisms for the synthesis of trehalose by Bradyrhizobium species suggests that this disaccharide is important in the function of this organism both in the free-living state and in symbiosis.     

Three Enzymes for Trehalose Synthesis in Bradyrhizobium Cultured Bacteria and in Bacteroids from Soybean Nodules

α,α-Trehalose is a disaccharide accumulated by many microorganisms, including rhizobia, and a common role for trehalose is protection of membrane and protein structure during periods of stress, such as desiccation. Cultured Bradyrhizobium japonicum and B. elkanii were found to have three enzymes for trehalose synthesis: trehalose synthase (TS), maltooligosyltrehalose synthase (MOTS), and trehalose-6-phosphate synthetase. The activity level of the latter enzyme was much higher than those of the other two in cultured bacteria, but the reverse was true in bacteroids from nodules. Although TS was the dominant enzyme in bacteroids, the source of maltose, the substrate for TS, is not clear; i.e., the maltose concentration in nodules was very low and no maltose was formed by bacteroid protein preparations from maltooligosaccharides. Because bacteroid protein preparations contained high trehalase activity, it was imperative to inhibit this enzyme in studies of TS and MOTS in bacteroids. Validamycin A, a commonly used trehalase inhibitor, was found to also inhibit TS and MOTS, and other trehalase inhibitors, such as trehazolin, must be used in studies of these enzymes in nodules. The results of a survey of five other species of rhizobia indicated that most species sampled had only one major mechanism for trehalose synthesis. The presence of three totally independent mechanisms for the synthesis of trehalose by Bradyrhizobium species suggests that this disaccharide is important in the function of this organism both in the free-living state and in symbiosis.     

Endophytic occupation of peanut root nodules by opportunistic Gammaproteobacteria

Several bacterial isolates were recovered from surface-sterilized root nodules of Arachis hypogaea L. (peanut) plants growing in soils from Córdoba, Argentina. The 16S rDNA sequences of seven fast-growing strains were obtained and the phylogenetic analysis showed that these isolates belonged to the Phylum Proteobacteria, Class Gammaproteobacteria, and included Pseudomonas spp., Enterobacter spp., and Klebsiella spp. After storage, these strains became unable to induce nodule formation in Arachis hypogaea L. plants, but they enhanced plant yield. When the isolates were co-inoculated with an infective Bradyrhizobium strain, they were even found colonizing pre-formed nodules. Analysis of symbiotic genes showed that the nifH gene was only detected for the Klebsiella-like isolates and the nodC gene could not be amplified by PCR or be detected by Southern blotting in any of the isolates. The results obtained support the idea that these isolates are opportunistic bacteria able to colonize nodules induced by rhizobia.     

Evolutionary dynamics of rhizopine within spatially structured rhizobium populations

Symbiosis between legumes and nitrogen-fixing bacteria is thought to bring mutual benefit to each participant. However, it is not known how rhizobia benefit from nodulating legume hosts because they fix nitrogen only after becoming bacteroids, which are terminally differentiated cells that cannot reproduce. Because undifferentiated rhizobia in and around the nodule can reproduce, evolution of symbiotic nitrogen fixation may depend upon kin selection. In some hosts, these kin may persist in the nodule as viable, undifferentiated bacteria. In other hosts, no viable rhizobia survive to reproduce after nodule senescence. Bacteroids in these hosts may benefit their free-living kin by enhancing production of plant root exudates. However, unrelated non-mutualists may also benefit from increased plant exudates. Rhizopines, compounds produced by bacteroids in nodules and catabolized only by related free-living rhizobia, may provide a mechanism by which bacteroids can preferentially benefit kin. Despite this apparent advantage, rhizopine genotypes are relatively rare. We constructed a mathematical model to examine how mixing within rhizobium populations influences the evolution of rhizopine genotypes. Our model predicts that the success of rhizopine genotypes is strongly dependent upon the spatial genetic structure of the bacterial population; rhizopine is more likely to dominate well-mixed populations. Further, for a given level of mixing, we find that rhizopine evolves under a positive frequency-dependent process in which stochastic accumulation of rhizopine alleles is necessary for rhizopine establishment. This process leads to increased spatial structure in rhizobium populations, and suggests that rhizopine may expand the conditions under which nitrogen fixation can evolve via kin selection.     

Bacteroid-encoded proteins are secreted into the peribacteroid space by Rhizobium leguminosarum

Bacteroids of Rhizobium leguminosarum in root nodules of Pisum sativum are enclosed by a plant-derived peribacteriod membrane (PBM). The contents of the interstitial peribacteroid space (PBS) between bacteroid membrane and PBM were isolated by a controlled osmotic shock of PBM-enclosed bacteroids and analysed by two-dimensional gel electrophoresis. Silver staining revealed approximately 40 PBS polypeptides. Ex planta ³⁵S-methionine labeling of PBM-enclosed bacteroids revealed that about 90% of the PBS proteins are synthesized by the bacteroid. Approximately 30% of the PBS polypeptides are common between the PBS and the periplasmic space of free-living bacteria; one (38kDa) PBS protein is also excreted by free-living bacteria in the bacterial culture medium. At least four bacteroid-encoded PBS polypeptides were clearly identified as symbiosis-specific.     

Inhibition of ornithine decarboxylase activity reduces polyamine levels and growth ofAgrobacterium tumefaciens

Flavins in different compartments of effective nodules fromGlycine max cv Maple Arrow xBradyrhizobium japonicum strains were studied by spectrophotometry and chromatographic techniques. Flavins in the peribacteroid space were riboflavin (80%) and FMN (20%), as identified by TLC and HPLC. Flavin concentrations in the soybean root nodule cytoplasm, in the symbiosome space (PBS) and in the cytosol of bacteroids were monitored between 20 and 40 days post infection (d.p.i.) Between the 20th and 29th d.p.i. an at least four times higher flavin/protein ratio was found in PBS of effective nodules compared with the nodule cytoplasm. Between nitrogenase activity in the free-living state and bacterial flavin accumulation, no correlation could be observed. Flavin accumulation is not restricted to an effective symbiosis, as indicated by the analysis of ineffective nodules with strainB. japonicum RH-31 Marburg. Flavin accumulation is absent in uninfected soybean root tissue and in free-living rhizobia, thus indicating that flavin accumulation is a result of symbiotic interaction. Flavin accumulation is also missing in nodules with a hypersensitive response against the bacteria.     

Identification and Characterization of a Bacteroid-Specific Dehydrogenase Complex in Rhizobium leguminosarum PRE

In membranes of Rhizobium leguminosarum bacteroids isolated from nitrogen-fixing pea root nodules, two different protein complexes with NADH dehydrogenase activity were detected. One of these complexes, with a molecular mass of 110 kilodaltons, was also found in membranes of free-living rhizobia, but the other, with a molecular mass of 550 kilodaltons, appeared to be present only in bacteroids. The bacteroid-specific complex, referred to as DH1, probably consists of at least four different subunits. Using antibodies raised against the separate polypeptides, we found that a 35,000-molecular-weight polypeptide (35K polypeptide) in the DH1 complex is bacteroid specific, while the other proposed subunits were also detectable in cytoplasmic membranes of free-living bacteria. Dehydrogenase complex DH1 is also present in bacteroids of a R. leguminosarum nifA mutant, indicating that the synthesis of the dehydrogenase is not dependent on the gene product of this nif-regulatory gene. A possible involvement of the bacteroid-specific DH1 complex in electron transport to nitrogenase is discussed.     

Nitrate reductase and nitrite reductase activity in free-living cells and bacteroids of Rhizobium loti

Cells of Rhizobium loti strains T1 and U226 cultured in defined medium with glutamate as the only nitrogen source and bacteroids isolated from root nodules of Lotus corniculatus, L. pedunculatus and L. tenuis did not show constitutive (non-nitrate induced) nitrate reductase activity (NRA). In contrast, nitrite reductase activity (NiRA) was present in both free-living cells and bacteroids of either strain T1 or U226. Constitutive NRA and NiRA were detected in the cytosol fraction from nodules of all three symbioses examined. An induced NRA was expressed in bacteroids after a 10 h incubation in the presence of nitrate.     

Purification and characterization of NAD(P)H quinone reductase from the latex of Hevea brasiliensis Müll.-Arg. (Euphorbiaceae)

NAD(P)H quinone reductase [NAD(P)H-QR] present in the latex of Hevea brasiliensis Müll.-Arg. (Euphorbiaceae) was purified to homogeniety from the B-serum fraction obtained by freeze-thawing of the bottom fraction of ultracentrifuged fresh latex. The purification protocol involved acetone fractionation, heat treatment, ion exchange chromatography and affinity chromatography. The M(r) determined by SDS-PAGE for the protein subunit was 21 kDa, and the molecular mass of the native enzyme estimated by gel filtration was 83 kDa, indicating that the native enzyme is a homotetramer. The enzyme showed pH stability over a range of 6 to at least 10 (with an optimum at pH 8) and thermal stability up to 80 degrees C. High NAD(P)H-QR activity (70%) was still retained after 10 h of preincubation at 80 degrees C. A comparable substrate specificity for this enzyme was observed among menadione, p-benzoquinone, juglone, and plumbagin, with only duroquinone generating a lower activity. Positive correlations between latex NAD(P)H-QR activity and rubber yield per tapping [fresh latex (r=0.89, P<0.01), dry rubber (r=0.81, P<0.01)] together with flow time (r=0.85, P<0.01) indicated that enzyme activity could possibly be used as a marker to predict the yield potential of selected clones.     

Reduced pyridine nucleotides in Pseudomonas carboxydovorans are formed by reverse electron transfer linked to proton motive force

In cell suspensions of Pseudomonas carboxydovorans pulsed with lithotrophic substrates (CO or H₂) in the presence of oxygen, formation of reduced pyridine nucleotides and of ATP could be demonstrated using the bioluminescent assay. Experiments employing base-acid transition, an uncoupler and inhibitors of ATPase or electron transport enabled us to propose a model for the formation of NAD(P)H in chemolithotrophically growing P. carboxydovorans.The protonophor FCCP (carbonly-p-trifluormethoxyphenylhydrazon) inhibited both, formation of NAD(P)H and of ATP. In the absence of oxygen, a chemical potential imposed by base-acid transition resulted in the formation of NAD(P)H and ATP when electrogenic substrates (CO or H₂) were present. This suggests proton motive force-driven NAD(P)H formation. The proton motive force was generated by oxidation of substrate, and not by ATP hydrolysis, as obvious from NAD(P)H formation during inhibition of ATP synthesis by oligomycin and N,N-dicyclohexylcarbodiimide.That the CO-born electrons are transferred via the ubiquinone 10-cytochrome b region to NADH dehydrogenase functioning in the reverse direction, was indicated by inhibition of NAD(P)H formation by HQNO (2-n-heptyl-4-hydroxyquinoline-N-oxide) and rotenone, and by resistance to antimycin A.We conclude that in P. carboxydovorans, growing with CO or H₂, electrons and a proton motive force, generated by respiration, are required to drive an reverse electron transfer for the formation of reduced pyridine nucleotides.Abbreviations CODH carbon monoxide dehydrogenase - DCCD N,N-dicyclohexylcarbodiimide - FCCP carbonyl-p-trifluormethoxyphenylhydrazon - HQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - pmf proton motive force     

Reproductive capacity of bacteroids in nodules of Trifolium repens L. and Glycine max (L.) Merr.

Reproductive growth of intracellular bacteria from isolated protoplasts in nodules of clover and soybean was directly investigated using a microchamber with visual and video recording. Differentiated bacteriods from clover nodules uniformly failed to reproduce. Such growth as occurred came from undifferentiated rhizobia from within the protoplast or extracellularly in the nodule. Plating investigation gave results in agreement with this conclusion. Osmoprotective media failed to secure the reproduction of differentiated clover bacteroids. Reproductive growth of bacteroids from protoplasts and crushed nodules of soybean was regularly observed in the microchamber and determined as proportionate colony-forming ability (CFA) on laboratory media. The CFA markedly increased with age of nodule and with the addition of nodule or root extract. The promoting effect of such extracts was reduced after heating for 60 min at 100°C, and lost completely after 20 min at 121°C. High osmolarity in the suspending and culture media was detrimental to bacteroid recovery.Abbreviations BMM Bergersen's modified medium - B^+m BMM with additional mannitol - CDB Chlamydomonas dilution buffer - PDB protoplast dilution buffer - PDB PDB without mannitol or sorbitol - RMM Rhizobium minimal medium - R^+m RMM with mannitol instead of sucrose - YMA, YMB yeast mannitol agar and broth, respectively. For details, see Materials and methods     

Introduction of a novel pathway for IAA biosynthesis to rhizobia alters vetch root nodule development

We introduced into Rhizobium leguminosarum bv. viciae LPR1105 a new pathway for the biosynthesis of the auxin, indole-3-acetic acid (IAA), under the control of a stationary phase-activated promoter active both in free-living bacteria and bacteroids. The newly introduced genes are the iaaM gene from Pseudomonas savastanoi and the tms2 gene from Agrobacterium tumefaciens. Free-living bacteria harbouring the promoter-iaaMtms2 construct release into the growth medium 14-fold more IAA than the wild-type parental strain. This IAA overproducing R. l. viciae, the RD20 strain, elicits the development of vetch root nodules containing up to 60-fold more IAA than nodules infected by the wild-type strain LPR1105. Vetch root nodules derived from RD20 are fewer in number per plant, heavier in terms of dry weight and show an enlarged and more active meristem. A significant increase in acetylene reduction activity was measured in nodules elicited in vetch by RD20.     

Effects of pyridine nucleotides on the gating of ATP-sensitive potassium channels in insulin-secreting cells

Summary The single-channel current recording technique has been used to study the influences that the pyridine nucleotides NAD, NADH, NADP and NADPH have on the gating of ATP-sensitive K⁺ channels in an insulin-secreting cell line (RINm5F). The effects of the nucleotides were studied at the intracellular surface using either excised inside-out membrane patches or permeabilized cells. All four pyridine nucleotides were found to evoke similar effects. At low concentrations, 100 m and less, each promoted channel opening whereas high concentrations, 500 m and above, evoked channel closure. The degree of K⁺ channel activation by pyridine nucleotides (low conc.) was found to be similar to that evoked by the same concentrations of ADP or GTP, whereas the degree of K⁺ channel inhibition (high conc.) was less marked than that evoked by the same concentrations of ATP, and never resulted in refreshment of K⁺ channels following removal. The effects of NAD, NADH, NADP and NADPH seemed to interact with those of ATP and ADP. In the presence of 1mm ADP and 4mm ATP, 10 to 100 m concentrations of the pyridine nucleotides could not evoke channel opening, whereas concentrations of 500 m and above were found to evoke channel closure. In the presence of 2mm ATP and 0.5mm ADP, however, 10 to 100 m concentrations of the pyridine nucleotides were able to activate K⁺ channels.     

Symbiosis of Astragalus cicer with its microsymbionts: partial nodC gene sequence, host plant specificity, and root nodule structure

Astragalus cicer (cicer milkvetch) nodule bacteria were investigated for host plant specificity and partial nodC gene sequences, whilst their native host was studied for the microscopic structure of root nodules. The strains under investigation formed nodules not only on the original host but also on Astragalus glycyphyllos, Astragalus sinicus, Lotus corniculatus, and Phaseolus vulgaris. The nodules induced on the cicer milkvetch were classified as indeterminate and characterized by apical, persistent meristem, a large bacteroid region with infected and uninfected cells, and elongated bacteroids singly located inside peribacteroid membranes. By comparison of the partial nodC gene sequences of a representative strain of astragali rhizobia to those contained in the GenBank database, a close symbiotic relationship of A. cicer microsymbionts to Rhizobium sp. (Oxytropis) was found.     

Development of nodules of Glycine max infected with an ineffective strain of Rhizobium japonicum

Bacteroids in ineffective (nitrogenase negative) nodules of Glycine max, infected with Rhizobium japonicum 61-A-24, as compared to those in effective nodules are characterized by reduced specific activities of alanine dehydrogenase to 15%, of 3-hydroxybutyrate dehydrogenase to 50%, and an increase of glutamine synthetase to 400%. In the plant cytoplasm of ineffective nodules, glutamine synthetase activity is reduced to 10–30%, glutamate dehydrogenase to 50–70%, and the aspartate aminotransferase and alanine aminotransferase are enhanced to 120–200%, depending on the age of the nodules. The total pool of soluble amino acids is reduced to 52 mol per g nodule fresh weight, as compared to 186 mol in effective nodules, with a replacement of asparagine (42 mol% of the amino acids) by an unknown amino compound. This compound is absent in nitrogenase, repressed and derepressed, free-living Rhizobium japonicum cells and in the uninfected root tissue. In nitrogenase derepressed, as compared to the repressed free-living cells of Rhizobium japonicum 61-A-101, arginine shows the most obvious change with a reduction to less than one tenth. The ultrastructure of the ineffective nodule is different from the effective organ even in the early stages. The membrane envelopes of the infection vacuoles are decomposing in heavily infected cells within 18 to 20 d after infection. In lightly infected cells very large vacuoles develop with only a few bacteroids inside. No close associations of cristae-rich mitochondria with amyloplasts are observed as in effective nodules. The uninfected cells keep their large starch granules even 40 d after infection. Some poly--hydroxybutyrate accumulation in the bacteroids is observed but only in the early stages, and it is almost absent in old nodules (40 d). At this age the infected cells are obviously compressed by uninfected cells, whereas in effective nodules with nitrogenase activity and leghaemoglobin formation, the infected cells have a much higher osmotic pressure than the neighbouring uninfected cells.Abbreviations PHBA poly--hydroxybutyric acid Prof. Dr. A. Pirson on the occasion of his 70th birthday