Ineffective Root Nodules Research Articles

Pursue the truth, devote to education: Chen Hua-Kui, a respectable educator and pioneer of China's soil microbiology.

Pursue the truth, devote to education: Chen Hua-Kui, a respectable educator and pioneer of China's soil microbiology.

Morphogenesis of root nodules in white clover. II. The effect of mutation in genes nod IJ of the microsymbiont upon the nodule structure

Morphogenesis of ineffective root nodules initiated on the roots of white clover 'Astra' by the <em>Rhizobium leguminosarum</em> biovar. <em>trifolii</em> strains ANU261 (Tn5 insertion in nod 1 gene) and ANU262 (Tn5 insertion in nod J gene) was investigated. Following changes were observed, as compared to the wild-type nodulation: the exaggerated, not delayed reaction of root hairs; the delay in nodulation with the number of nodules the same as in plants inoculated with a wild strain; the formation and organization of the nodule primordium not changed in comparison with the wild-type nodules; infection threads abnormally branched and diffusing with bacteria deprived of light zone and enriched with storage material; infected cells of bacteroidal tissue abnormally strongly osmiophilic and only slightly vacuolated; symbiosomes with very narrowed peribacteroidal space, subject to premature degradation; abnormal accumulation of starch in the nodule tissues; nodule development blocked at the stage of laterally situated meristem and single nodule bundle; inhibition of divisions in the meristem and vacuolation of its cells; the appearance of single cells with colonies of saprophytic rhizobia embedded in the fibrillar matrix in the old, degraded regions of the bacteroidal tissue.

A ClpB Chaperone Knockout Mutant of Mesorhizobium ciceri Shows a Delay in the Root Nodulation of Chickpea Plants

Several molecular chaperones are known to be involved in bacteria stress response. To investigate the role of chaperone ClpB in rhizobia stress tolerance as well as in the rhizobia-plant symbiosis process, the clpB gene from a chickpea microsymbiont, strain Mesorhizobium ciceri LMS-1, was identified and a knockout mutant was obtained. The ClpB knockout mutant was tested to several abiotic stresses, showing that it was unable to grow after a heat shock and it was more sensitive to acid shock than the wild-type strain. A plant-growth assay performed to evaluate the symbiotic performance of the clpB mutant showed a higher proportion of ineffective root nodules obtained with the mutant than with the wild-type strain. Nodulation kinetics analysis showed a 6- to 8-day delay in nodule appearance in plants inoculated with the ΔclpB mutant. Analysis of nodC gene expression showed lower levels of transcript in the ΔclpB mutant strain. Analysis of histological sections of nodules formed by the clpB mutant showed that most of the nodules presented a low number of bacteroids. No differences in the root infection abilities of green fluorescent protein-tagged clpB mutant and wild-type strains were detected. To our knowledge, this is the first study that presents evidence of the involvement of the chaperone ClpB from rhizobia in the symbiotic nodulation process.

Brief communication. A dominant, host plant mutation conferring ineffective nodulation in the chickpea-Rhizobium symbiosis

Chickpea (Cicer arietinum L.) mutant PM638, a derivative of wild-type, desi chickpea line ICC 640, forms yellowish, ineffective root nodules and differs phenotypically from previously described chickpea nodulation mutants. To establish the mode of inheritance of the mutant nodule trait, and to eliminate independent chlorophyll and sterility mutations carried by the mutant, PM638 was crossed with ICC 640 and an F5 generation mutant was reciprocally backcrossed to ICC 640. Segregation analysis indicated that the mutant phenotype was conferred by a single, dominant nuclear gene mutation. In keeping with established nomenclature for chickpea nodulation genes, the mutant allele is tentatively designated Rn7.

Morphological and Molecular Characteristics of Host-Conditioned Ineffective Root Nodules in Cowpea

In cowpea (Vigna unguiculata [L.] Walp.) a recessive allele, designated cpi, elicits the formation of non-N2-fixing nodules with all bacterial isolates tested. Comparisons of mutant and wild-type nodules demonstrated that the ineffective nodules were anatomically similar to the wild type and contained both infection threads and bacteroids. Ineffective nodules were smaller, however, largely because of the reduced size of the infected cells. Additionally, the number of bacteroids was reduced and senescence occurred prematurely in the infected cells. Grafting studies demonstrated that the defect in nodule development was controlled by the root rather than the shoot. Northern analysis of four nodulin genes indicated that in the ineffective nodules transcript levels of the early nodulin VuENOD2 were initially reduced but were equivalent to wild-type nodules by 21 d. In contrast, transcript levels of the early nodulin VuB were initially similar in both genotypes but as the nodules matured the mRNA levels declined more slowly in the ineffective nodules. The late nodulins leghemoglobin and uricase were expressed in the ineffective nodules but at greatly reduced levels. Thus, the cpi-conditioned defect in nodulation is associated with impaired bacteroid maturation and maintenance, altered nodulin expression, and accelerated senescence.

Resistance to an ineffective Frankia strain type in Alnus glutinosa (L.) Gaertn.

Characteristics of natural resistance to the ineffective Frankia strain type AgI-WD1 were investigated in different progenies of the host species Alnus glutinosa (L.) Gaertn. with the aid of nodulation experiments. This strain type of Frankia was abundant in the soil of an A. glutinosa stand at Voorne, The Netherlands. Soil suspensions from this site produced high numbers of ineffective root nodules on some of the A. glutinosa seedlings used in biotests, but ii significant number of the seedlings did not produce ineffective nodules. Resistance (R) or susceptibility (S) of the seedlings to Frankia strain type AgI-WD1, as expressed by absence or presence of ineffective root nodules, proved to be persistent in cloned plants of each phenotype. Both R and S-seedlings were susceptible to effective Frankia strains in the soil suspensions. Variation in nodulation time was examined among cloned plants of S-seedlings selected for this trait. Average nodulation times of three selected clones were 11, 26 and 39 d, respectively. Relative growth rates of cloned plants showed no relationship with nodulation time. Both R and S phenotypes were present in all seed-lots of A. glutinosa under study, but the frequency of R phenotypes of seed-lots from different locations ranged from 24 to 100%. R-frequencies of seed-lots of nine trees from a small population of A. glutinosa segregated into two distinct frequency classes which suggested genotypic differentiation of the R-trait among these trees. A significant correlation was found between R-frequencies of seedlots from the same trees over successive years.

Proteolysis during Development and Senescence of Effective and Plant Gene-Controlled Ineffective Alfalfa Nodules.

Plant-controlled ineffective root nodules, conditioned by the in1 gene in Medicago sativa L. cv Saranac, undergo premature senescence and have reduced levels of many late nodulins. To ascertain which factors contribute to premature senescence, we have evaluated proteolysis as it occurs throughout the development of ineffective Saranac (in1Sa) and effective Saranac nodules. Cysteine protease activities with acidic pH optimum and enzyme proteins were present in both genotypes. We found that acidic protease activity was low in effective Saranac nodules throughout their development. In contrast, by 2 weeks after inoculation, acid protease activity of in1Sa nodules was severalfold higher than that of Saranac nodules and remained high until the experiment was terminated 8 weeks later. This increase in protease enzyme activity correlated with an increase in protease protein amounts. Increased protease activity and amount in in1Sa nodules was correlated with a decrease in nodule soluble protein. The time at which in1Sa nodules initially showed increased protease activity corresponded to when symbiosis deteriorated. High levels of phosphoenolpyruvate carboxylase (PEPC) protein were expressed in effective nodules by 12 d after inoculation and expression was associated with low proteolytic enzyme activity. In contrast, although PEPC was expressed in in1Sa nodules, PEPC protein was not found 12 d after inoculation and thereafter. Acidic protease from in1Sa nodules could also degrade purified leghemoglobin. These data indicate that premature senescence and low levels of late nodulins in in1Sa nodules can be correlated in part with increased proteolysis.

Starch content and activities of starch-metabolizing enzymes in effective and ineffective root nodules of soybean

Starch content and activities of some enzymes of starch metabolism were determined in wild-type, N2-fixing (fix+) nodules and in two non-N2-fixing (fix−) nodules induced by Bradyrhizobium japonicum mutant strains, T5-95 and T8-1, on soybean (Glycine max L.) roots. The T5-95 nodules are similar to wild type in ultrastructure, but the T8-1 nodules are different in that the bacteroids are not released from the infection thread. After initial accumulation to relatively high concentration, starch was depleted during nitrogen fixation in fix+ nodules. However, in fix− nodules, the accumulated starch was not metabolized. The activity of starch-bound starch synthase (EC 2.4.1.21) declined in fix+ nodules but remained high in fix− nodules. The activity of α-amylase (EC 3.2.1.1) was only slightly higher than wild type in T5-95 but was four times higher than wild type in T8-1 nodules. The activity of starch phosphorylase (EC 2.4.1.1) increased in all nodule types from 14 to 21 days postinfection. A positive correlation was observed between the capacity of nodules to fix N2 and their capacity to degrade starch. Collectively, these results support the concept that starch accumulated during early stages of nodule development is metabolized to supply energy for nitrogen fixation and to meet the metabolic demands of bacteroids. Key words: nitrogen fixation, starch content, effective and ineffective nodules, starch synthase, starch phosphorylase, α-amylase.

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.

Nodulin Gene Expression in Effective Alfalfa Nodules and in Nodules Arrested at Three Different Stages of Development

Nodulin gene expression was analyzed in effective and ineffective root nodules of alfalfa (Medicago sativa L. cv Iroquois) elicited by three different Rhizobium meliloti mutants: an exoB mutant having defective acidic exopolysaccharide that does not fluoresce on plates containing the fluorescent brightener Calcofluor; fix21, a spontaneous mutant that has defective lipopolysaccharide and is Calcofluor bright; and a Rhizobium mutant resulting from a Tn5 insertion in the nifH gene of the nif operon. The ineffective nodules elicited by these various mutant rhizobia are blocked at different stages of nodule development and have unique phenotypes. A distinctive pattern of nodulin gene expression as determined by in vitro translations of total nodule RNA characterizes each nodule phenotype. Seventeen nodulins are found in effective nodules including five leghemoglobins. Only one nodulin gene is expressed in the bacteria-free nodules elicited by the exoB mutant. Other nodulin genes (leghemoglobin and nine others) are expressed in fix21-induced nodules. The genes for nodule-enhanced glutamine synthetase as well as for all the other nodulins are expressed in nodules induced by the nifH mutant. The expression of genes for the nodulins, including leghemoglobin, is independent of the nitrogen-fixing ability of the nodule and appears to correlate with the differentiation of densely cytoplasmic host cells in the nodule and, to some extent, with bacterial release from infection threads.

Host range differentiation of spore‐positive and spore‐negative strain types of Frankia in stands of Alnus glutinosa and Alnus incana in Finland

Host compatibility of different spore‐positive (Sp+)and spore‐negative (Sp−) strain types of Frankia from alder stands in Finland was studied in Modulation tests with hydrocultures of Alnus glutinosa (L.) Gaertner, A. incana (L.) Moench and A. nitida Endl. Root nodules and soil samples from stands of A. incana (Lammi forest and Hämeenlinna forest) were dominated by Sp + types of Frankia (coded AiSp+ and AiSp+ H. respectively), which caused effective root nodules in test plants of A. incana, but failed to induce nodules in A. nitida. In A. glutinosa Frankia strain types AiSp + and AiSp + H caused small, ineffective root nodules with sporangia (coded Ineff −), which were recognized by the absence or near absence of vesicles in the nodule tissue. Ineffective nodules without sporangia (coded Ineff −) were induced on A. glutinosa with soil samples collected at Lammi swamp. The spore‐negative strain type of Frankia was common in root nodules of A. glutinosa in Finland (Lammi swamp) and caused effective Sp− type root nodules (coded AgSp −) in hydrocultures of A. incana, A. glutinosa and A. nitida. A different Sp + strain type of Frankia. coded AgSp+ Finland, was occasionally found in stands of A. glutinosa. It was clearly distinguished from strain type AiSp + by the ability to produce effective nodules on both A. glutinosa and A. incana. The nodulation capacities of soil and nodule samples were calculated from the nodulation response in hydrocutlure and served as a measure for the population density of infective Frankia particles. Sp + nodules from both strain types had equal and high nodulation capacities with compatible host species. The nodulation capacities of Sp type root nodules from A. glutinosa were consistently low. High frequencies of Frankia AiSp+ and AiSp+ H were found in the soil environment of dominant AiSp + nodule populations on A. incana. The numbers of infective particles of this strain type were insignificant in the soil environment of nearby Sp − nodule populations on A. glutinosa and in the former field at Hämeen‐linna near the Sp+ nodule area in Hämeenlinna forest. Strain type AgSp− had low undulation capacity in the soil environment of both A. incana and A. glutinosa stands, Explanations for the strong associations between Frankia strain types AiSp+ and AiSp − H and A. incana and between strain type AgSp− and A. glutinosa are discussed in the light of host specificity and of some characteristics of population dynamics of both strain types. The possible need to adapt the concept of Frankia strain types Sp + and Sp − to strains with some variation in spore development was stressed by the low potentials of strain type AiSp + H to develop spores in symbioses with hydrocultures of A. incnna.

Plant gene expression in effective and ineffective root nodules of alfalfa (Medicago sativa)

Expression of plant genes involved in the symbiosis between alfalfa (Medicago sativa) and Rhizobium meliloti has been studied by comparing root and root nodule mRNA populations. Two-dimensional gel electrophoretic separation of the in vitro translation products of polyA(+) RNA isolated from either roots or effective root nodules has allowed us to identify thirteen nodule-specific translation products, including those corresponding to the leghemoglobins (Lb). These translation products, representing putative nodulin mRNAs, are first detected between 9 and 12 days after inoculation, a result which has been confirmed for Lb mRNA by Northern blotting and hybridization with a Lb cDNA probe. Analysis of three different types of ineffective root nodules arrested in different stages of development has led to the following conclusions. (i) The transcription of eleven nodule-specific genes, including the Lb genes, is independent of nitrogen-fixing activity. (ii) Differentiation of the primary nodule structure does not require the transcription of any of these genes but can be correlated with a dramatic reduction in the level of at least five transcripts present in the root. (iii) There is enhanced expression of certain plant genes in the case of nodules elicited by an Agrobacterium strain carrying the symbiotic plasmid of R. meliloti.

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.

Phytohormones,Rhizobium mutants, and nodulation in legumes. VII. Identification and quantification of cytokinins in effective and ineffective pea root nodules using radioimmunoassay

Radioimmunoassays (RIA), employing antisera raised in rabbits against bovine serum albumin conjugates of zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine, were used to estimate levels of these cytokinins and their corresponding bases in samples of effective (nitrogen-fixing, Fix+), ineffective (nonnitrogen-fixing, Fix−) pea root nodules and uninoculated roots. Assays were done on extracts of nodule tissue, 1–2 g fresh weight, or approximately 10 g fresh weight of root tissue, and high specific activity [3H]zeatin riboside was added during preparation of the extract for use as a recovery marker. Two different purification procedures were employed, each involving several purification steps. High performance liquid chromatography (HPLC) was the final step in both procedures. Fractions from HPLC were analyzed by RIA using the appropriate antiserum. The cytokinins, zeatin, zeatin riboside, dihydrozeatin riboside, isopentenyl adenine, and isopentenyladenosine were detected and quantified in nodule tissue, and similarly, in root tissue (with the exception of zeatin, which we were unable to quantify in root tissue). Cytokinin levels in nodule tissue were higher than those in root tissue. The major cytokinins detected in nodule tissue were zeatin, followed by zeatin riboside and then dihydrozeatin riboside. The levels of zeatin and zeatin riboside estimated in nodules in the present study by RIA were of the same order of magnitude, though tending to be a little higher, than values obtained previously by bioassay. Dihydrozeatin riboside was identified with confidence for the first time in nodule tissue. There was a general decline with age in cytokinin levels in nodules, but no major qualitative change in nodule cytokinins with age. For theRhizobium strains examined, the data did not indicate a clear correlation between nodule cytokinin levels and the effectiveness of nodules in nitrogen fixation.

Detection of Nodule-Specific Polypeptides from Effective and Ineffective Root Nodules of Medicago sativa L.

Summary At least twelve polypeptides specific to the nodule tissue of Medicago sativa (alfalfa) were detected by the immunoblotting procedure. These polypeptides ranged in size from 12 Kd to 120 Kd. Nodules induced by seven different Rhizobium meliloti strains showing various symbiotic phenotypes were analysed by immunoblotting for the presence of nodule-specific polypeptides. All of these mutant-induced nodules contained the majority of the nodule-specific polypeptides but at reduced concentrations compared to the wild type. Nodules which were induced by mutant isolate 0540 and lacked infection threads and bacteroid-filled cells, exhibited elevated levels of a 97 Kd and 120 Kd polypeptide. Mutants 101.45 and 2204 induced increased amounts of the 39 Kd and 120 Kd protein respectively. While strains 938 (defective in succinate utilization) and 3046 ( nod + fix − ) incited a nodule-specific polypeptides pattern similar to that of strain 0540, strains A34 (defective in heme synthesis) and 9-3 ( nod + fix − ) were similar to the wild type. Comparative analysis following in vivo labelling of root and nodule proteins by 35 Smethionine showed some nodule-specific polypeptides similar in molecular weight to those found by immunoblotting.

Expression of nodule-specific uricase in soybean callus tissue is regulated by oxygen

In soybean root nodules the enzyme uricase is expressed concomitantly with nodule development. The initial expression of this protein does not depend on active nitrogen fixation, as demonstrated by analysis of uricase activity in effective and ineffective root nodules. However, the maximal level of uricase activity is determined by the infecting Rhizobium japonicum strain. Sterile root cultures and callus tissue, devoid of the microsymbiont, were incubated at varying oxygen concentrations and analyzed for uricase activity. The specific activity of uricase was increased by lowering the oxygen concentration, with the highest activity obtained around 4-5% oxygen. The increase in uricase activity was due to increased uricase synthesis, as demonstrated by in vivo labelling of callus culture followed by immunoprecipitation with antibodies raised against highly purified nodule uricase.

Expression of plant genes during the development of pea root nodules.

The expression of plant genes involved in the pea-Rhizobium symbiosis was studied by analysing mRNA from root nodules. The RNA was translated in vitro and the translation products were separated by two-dimensional gel electrophoresis. The results show differential expression of nodulin genes during root nodule development. One gene encoding N-40' is expressed at a significant level 5 days before the leghemoglobin genes. Most other nodulin genes are expressed more of less concomitantly with the leghemoglobin genes whereas the N-21 mRNA is only present late during the development. In the development of ineffective root nodules induced by infection with different nod+fix- mutants of R. leguminosarum all nodulin genes are expressed except for the N-21 gene. The results suggest that neither bacteroid development, heme excretion nor nitrogen fixation are essential for the induction of nodulin gene expression in the host plant. Further, it appears that the amount of leghemoglobin in ineffective nodules is regulated at a post-transcriptional level.

Tissue Cultures Derived from Ineffective Root Nodules of Alfalfa

Callus tissue cultures were developed from apical meristem regions of tumor-like ineffective root nodules of alfalfa. Callus growth was a function of tissue source and hormone composition and concentration. Callus derived from ineffective nodules also were shown not to contain Rhizobium meliloti.Glutamate dehydrogenase, glutamine synthetase, glutamate synthase, glutamate oxaloacetate transaminase and phosphoenolpyruvate carboxylase activities were present in callus cultures and in the respective nodule source used for callus induction. The mean specific activity of all enzymes evaluated was higher in callus cultures than in ineffective nodules. Quantitative but not qualitative differences in enzyme activities were evident between ineffective nodules and callus derived from these nodules. Tissue cultures derived from ineffective nodules may provide a model system to evaluate host plant-Rhizobium interactions.

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 nodfix 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.

Morphogenesis of Effective and Ineffective Root Nodules in Phaseolus vulgaris L.

Seedlings of Phaseolus vulgaris, inoculated with an effective strain of Rhizobium phaseoli (CIAT 73) or an ineffective strain (14c), were maintained on an N-free nutrient solution in sterilized sand. Acetylene reduction was measured periodically, and nodules were harvested for light and electron microscopy. In plant host cells infected with CIAT 73, there was an increase in cell volume, nuclear size, and mitochondrial length as bacteria multiplied and became bacteroidal. Golgi activity and rough endoplasmic reticulum (RER) were sparse at nodule maturity, but close associations of amyloplasts and elongated, energized mitochondria were observed. Nitrogen fixation peaked at 5 wk and dropped sharply coincident with flowering. Plant host cells infected with 14c typically contained numerous mitochondria; Golgi bodies and RER were conspicuous throughout nodule development. By the time the bacteria became bacteroidal, plant shoots were chlorotic, and under nitrogen stress the apex had made the transition to flowe...