Sucrose synthase and enolase expression in actinorhizal nodules ofAlnus glutinosa: comparison with legume nodules

Two different types of nitrogen-fixing root nodules are known — actinorhizal nodules induced byFrankia and legume nodules induced by rhizobia. While legume nodules show a stem-like structure with peripheral vascular bundles, actinorhizal nodule lobes resemble modified lateral roots with a central vascular bundle. To compare carbon metabolism in legume and actinorhizal nodules, sucrose synthase and enolase cDNA clones were isolated from a cDNA library, obtained from actinorhizal nodules ofAlnus glutinosa. The expression of the corresponding genes was markedly enhanced in nodules compared to roots. In situ hybridization showed that, in nodules, both sucrose synthase and enolase were expressed at high levels in the infected cortical cells as well as in the pericycle of the central vascular bundle of a nodule lobe. Legume sucrose synthase expression was studied in indeterminate nodules from pea and determinate nodules fromPhaseolus vulgaris by usingin situ hybridization.     

Sucrose synthase and enolase expression in actinorhizal nodules ofAlnus glutinosa: comparison with legume nodules

Two different types of nitrogen-fixing root nodules are known — actinorhizal nodules induced byFrankia and legume nodules induced by rhizobia. While legume nodules show a stem-like structure with peripheral vascular bundles, actinorhizal nodule lobes resemble modified lateral roots with a central vascular bundle. To compare carbon metabolism in legume and actinorhizal nodules, sucrose synthase and enolase cDNA clones were isolated from a cDNA library, obtained from actinorhizal nodules ofAlnus glutinosa. The expression of the corresponding genes was markedly enhanced in nodules compared to roots. In situ hybridization showed that, in nodules, both sucrose synthase and enolase were expressed at high levels in the infected cortical cells as well as in the pericycle of the central vascular bundle of a nodule lobe. Legume sucrose synthase expression was studied in indeterminate nodules from pea and determinate nodules fromPhaseolus vulgaris by usingin situ hybridization.     

Lignification of cell walls of infected cells in Casuarina glauca nodules that depend on symplastic sugar supply is accompanied by reduction of plasmodesmata number and narrowing of plasmodesmata

The oxygen protection system for the bacterial nitrogen‐fixing enzyme complex nitrogenase in actinorhizal nodules of Casuarina glauca resembles that of legume nodules: infected cells contain large amounts of the oxygen‐binding protein hemoglobin and are surrounded by an oxygen diffusion barrier. However, while in legume nodules infected cells are located in the central tissue, actinorhizal nodules are composed of modified lateral roots with infected cells in the expanded cortex. Since an oxygen diffusion barrier around the entire cortex would also block oxygen access to the central vascular system where it is required to provide energy for transport processes, here each individual infected cell is surrounded with an oxygen diffusion barrier. In order to assess the effect of these oxygen diffusion barriers on oxygen supply for energy production for transport processes, apoplastic and symplastic sugar transport pathways in C. glauca nodules were examined. The results support the idea that sugar transport to and within the nodule cortex relies to a large extent on the less energy‐demanding symplastic mechanism. This is in line with the assumption that oxygen access to the nodule vascular system is substantially restricted. In spite of this dependence on symplastic transport processes to supply sugars to infected cells, plasmodesmal connections between infected cells, and to a lesser degree with uninfected cells, were reduced during the differentiation of infected cells.     

Is the Legume Nodule a Modified Root or Stem or an Organ sui generis?

The legume nodule, which houses nitrogen-fixing rhizobia, is a unique plant organ. Its homology with lateral roots has been inferred by a comparison with other nitrogen-fixing nodules, especially those formed on actinorhizal plants in response to Frankia inoculation or on Parasponia roots following inoculation with Bradyrhizobium species. These nodules are clearly modified lateral roots in terms of their structure and development. However, legume nodules differ from lateral roots and these other nodules in their developmental origin, anatomy, and patterns of gene expression, and, consequently, several other evolutionary derivations, including from stems, wound or defense responses, or the more ancient vesicular-arbuscular mycorrhizal symbiosis, have been postulated for the legume nodule. In this review, we first present a broad view of the legume family showing the diversity of nodulation occurrence and types in the different subfamilies and particularly within the subfamily Papilionoideae. We then define the typological and molecular criteria used to discriminate the basic organs — root, stem, leaf— of the plant. Finally, we discuss the possible origins of the legume nodule in terms of these typological and molecular bases.     

A possible role for phenyl acetic acid (PAA) on Alnus glutinosa nodulation by Frankia

Phenylacetic hopanetetrol is a Frankia specific lipid present in vesicles. Phenylacetic acid (PAA) is known as an auxinomimetic, exhibiting the same effect on plant growth as indole acetic acid (IAA). We hypothesize that PAA, only bound by an ester link to the hopanetetrol basic unit, would be easily released and could thus play a role in nodule formation. HPLC and mass spectrometry analysis allowed us to show that 2 Alnus- (ACoN24d and ACN14a) and 2 Elaeagnus-infective strains (EaI1 and EaI3) released PAA into the culture medium, at concentrations of about 10^–5 to 10^–6 M, whereas IAA was not detected. Furthermore, exogenous PAA added to axenically-grown Alnus glutinosa roots at a concentration of 5×10^–5 M, resulted in the formation of thick, short lateral roots which resembled actinorhizal nodules. phenylalanine ammonia lyase (PAL) and chalcone syntase (CHS) induction by incompatible and compatible Frankia strains in A. glutinosa roots and the different contents in salicylic acid precursors (cinnamic acid and benzoic acid) observed between nodules and roots support the idea that PAA would be produced in nodules to the detriment of salicylic acid production. These results provide evidence that in actinorhizal root nodules, phenylpropanoid metabolism may play a multiple role in symbiotic interactions including the limitation of the induction of the systematic acquired resistance (SAR) by the plant.     

Purification of Hemoglobin from the Actinorhizal Root Nodules of Myrica gale L

Hemoglobins are generally absent or present in low concentrations in the nodules of actinorhizal plants. An exception is Casuarina, where a hemoglobin occurs at relatively high concentration. However, this plant is unique in that Frankia, the microsymbiont, lacks the vesicles that are normally the site of nitrogen fixation. The present paper shows that a hemoglobin also occurs at high concentrations in Myrica gale L., an actinorhizal plant in which Frankia does form vesicles. Hemoglobin was extracted from root nodules under anaerobic conditions using a buffer containing CO, detergent, and a reducing agent. Carboxyhemoglobin was purified using gel filtration followed by aerobic ion-exchange chromatography. The optical absorption spectra of the oxy-, deoxy-, and carboxyhemoglobins were similar to those of other hemoglobins. The molecular mass of the native hemoglobin estimated by gel filtration was 38,500 D. The molecular mass of the subunits estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 16,200 D, consistent with the mass of other hemoglobin subunits. Thus, the native hemoglobin is probably a dimer.     

Endophyte sporulation in root nodules of actinorhizal plants

All strains of isolated Frankia possess the genetic capacity to form sporangia since, when grown in vitro, they usually sporulate freely, depending on the physical and chemical environment in which they are cultured. Endophytic sporulation involving Frankia differentiation of sporangia within root nodules has been described in only 16 host species in 9 genera within six families of actinorhizal plants. From studies published to date, endophytic sporulation cannot be correlated with specific environmental conditions surrounding the host plants. Based on the literature and on previously unpublished observations from field and greenhouse studies, an account is given of the occurrence of sporulation in actinorhizal plants with emphasis on Alnus, Casuarina, Comptonia, Elaeagnus and Myrica . The possible role of the host plant in controlling Frankia sporulation as contrasted to the control exerted by the genetic constitution of the microbial symbiont is explored.     

OXYGEN CONCENTRATION WITHIN THE NITROGEN-FIXING ROOT NODULES OF MYRICA GALE L.

Microelectrodes were used to study the oxygen concentration within Myrica gale L. nodules. Low oxygen concentrations were found only in the region of the mature, nitrogen-fixing endophyte, and appeared to correspond to clusters of infected host cells. The oxygen concentration in the remainder of the nodule was much higher. Interconnected intercellular air spaces were demonstrated by infiltration with India ink. Infiltration of the spaces with water greatly reduced oxygen concentration throughout the nodule, indicating that they function in supplying oxygen to the infected cells and remainder of the nodule. These results differ from those found previously for soybean nodules and provide evidence that legume and actinorhizal nodules have different mechanisms for protecting nitrogenase from oxygen.     

Leghemoglobin-like sequences in the DNA of four actinorhizal plants

Summary A cloned cDNA partial copy of a soybean leghemoglobin mRNA was used to probe genomic DNA of four species of actinorhizal plants. Southern blot hybridization revealed the presence of sequences with homology to the leghemoglobin probe in DNA from Alnus glutinosa, Casuarina glauca, Ceanothus americanus and Elaeagnus pungens. The hybridization patterns of the restriction fragments revealed some fragment size conservation between the DNA of soybean and the DNA of four actinorhizal plants which are taxonomically unrelated to soybean or to each other. The results presented here indicate that globin gene sequences are much more widely distributed in the plant kingdom than has previously been thought. Furthermore, if sequence conservation is actually as high as the restriction fragment patterns suggest, the evolution of the DNA surrounding the globin sequences has been highly constrained.     

Identification of atypical Frankia strains by fatty acid analysis

Abstract: Ineffective, non-infective actinomycetous isolates obtained from actinorhizal nodules of Coriaria nepalensis and Datisca cannabina were identified as Frankia using whole cell fatty acid analysis. The isolates exhibited fatty-acid patterns very similar to those of confirmed Frankia strains from other host plants ( Alnus, Casuarina, Colletia, Comptonia, Elaeagnus and Hippophae ). All Frankia strains, including Coriaria and Datisca isolates, showed fatty-acid profiles very distinct from those of other actinomycetes used as controls ( Actinomyces, Geodermatophilus, Nocardia, Mycobacterium and Streptomyces ). For the genus Frankia , a characteristic pattern of five fatty acids (15:0; 15:1; 16:0 iso; 17:0 and 17:1) was found. These fatty acids comprised 75% or more of the total content. All Frankia strains could be placed into three subgroups. Coriaria isolates were found in the largest subgroup which contained most Frankia strains from other hosts while ineffective strains from Alnus, Elaeagnus and Datisca were distributed in all three subgroups of Frankia .     

Low genetic diversity among Frankia spp. strains nodulating sympatric populations of actinorhizal species of Rosaceae, Ceanothus (Rhamnaceae) and Datisca glomerata (Datiscaceae) west of the Sierra Nevada (California)

Frankia spp. strains typically induce N2-fixing root nodules on actinorhizal plants. The majority of host plant taxa associated with the uncultured Group 1 Frankia strains, i.e., Ceanothus of the Rhamnaceae, Datisca glomerata (Datiscaceae), and all actinorhizal members of the Rosaceae except Dryas, are found in California. A study was conducted to determine the distribution of Frankia strains among root nodules collected from both sympatric and solitary stands of hosts. Three DNA regions were examined, the 5' end of the 16S rRNA gene, the internal transcribed spacer region between the 16S and 23S rRNA genes, and a portion of the glutamine synthetase gene (glnA). The results suggest that a narrow range of Group 1 Frankia spp. strains dominate in root nodules collected over a large area of California west of the Sierra Nevada crest with no apparent host-specificity. Comparisons with Group 2 Frankia strain diversity from Alnus and Myrica within the study range suggest that the observed low diversity is peculiar to Group 1 Frankia strains only. Factors that may account for the observed lack of genetic variability and host specificity include strain dominance over a large geographical area, current environmental selection, and (or) a past evolutionary bottleneck.     

Physiology and chemical diversity ofFrankia spp. isolated from nodules ofComptonia peregrina (L.) Coult. andCeanothus americanus L.

Summary TwoFrankia spp., isolated from the nodules of the plant hostComptonia peregrina, were found to fall into two previously described physiological groups (A and B). Of five frankia isolates fromCeanothus americanus plants of the same provenance, three belonged to physiological group A and two to a novel group whose final disposition remains to be decided. The diversity in whole cell sugar chemistry, morphology and other growth characteristics of these strains is discussed.     

Non-leguminous root nodules in Japan

Summary Among plants native to Japan, nodule formation is confirmed in 14 species and varieties of Alnus, 10 of Elacagnus, 2 of Myrica and 1 of Coriaria, in a number of instances for the first time. Plants of 20 foreign species, in 8 genera, which bear nodules in their native countries, were raised in the nursery in Tokyo; only species of Alnus, Myrica and Ceanothus formed nodules. No nodules were found on native plants ofDryas octopetala var.asiatica. In a trial extending over 12 years evidence was obtained that the growth ofPinus thunbergii was benefited by underplanting withMyrica rubra, a result attributed to nitrogen fixation in the root nodules of the latter species. In the attempted isolation of the endophytes from the nodules of Alnus and other non-legume Angiosperms, although actinomycetes peculiar to the host species were usually obtained from the nodules, none of the isolates induced nodules in re-inoculation tests. Also from Podocarpus nodules actinomycetal and bacterial strains were commonly isolated; re-inoculation tests with these are in progress.     

Overexpression of class 1 plant hemoglobin genes enhances symbiotic nitrogen fixation activity between Mesorhizobium loti and Lotus japonicus

Plant hemoglobins (Hbs) have been divided into three groups: class 1, class 2, and truncated Hbs. The various physiological functions of class 1 Hb include its role as a modulator of nitric oxide (NO) levels in plants. To gain more insight into the functions of class 1 Hbs, we investigated the physical properties of LjHb1 and AfHb1, class 1 Hbs of a model legume Lotus japonicus and an actinorhizal plant Alnus firma , respectively. Spectrophotometric analysis showed that the recombinant form of the LjHb1 and AfHb1 proteins reacted with NO. The localization of LjHb1 expression was correlated with the site of NO production. Overexpression of LjHb1 and AfHb1 by transformed hairy roots caused changes in symbiosis with rhizobia. The number of nodules formed on hairy roots overexpressing LjHb1 or AfHb1 increased compared with that on untransformed hairy roots. Furthermore, nitrogenase activity as acetylene-reduction activity (ARA) of LjHb1- or AfHb1 -overexpressing nodules was higher than that of the vector control nodules. Microscopic observation with a NO-specific fluorescent dye suggested that the NO level in LjHb1 - and AfHb1 -overexpressing nodules was lower than that of control nodules. Exogenous application of a NO scavenger enhanced ARA in L. japonicus nodule s , whereas a NO donor inhibited ARA. These results suggest that the basal level of NO in nodules inhibits nitrogen fixation, and overexpression of class 1 Hbs enhances symbiotic nitrogen fixation activity by removing NO as an inhibitor of nitrogenase.     

Distinct patterns of symbiosis-related gene expression in actinorhizal nodules from different plant families

Phylogenetic analyses suggest that, among the members of the Eurosid I clade, nitrogen-fixing root nodule symbioses developed multiple times independently, four times with rhizobia and four times with the genus Frankia. In order to understand the degree of similarity between symbiotic systems of different phylogenetic subgroups, gene expression patterns were analyzed in root nodules of Datisca glomerata and compared with those in nodules of another actinorhizal plant, Alnus glutinosa, and with the expression patterns of homologous genes in legumes. In parallel, the phylogeny of actinorhizal plants was examined more closely. The results suggest that, although relationships between major groups are difficult to resolve using molecular phylogenetic analysis, the comparison of gene expression patterns can be used to inform evolutionary relationships. In this case, stronger similarities were found between legumes and intracellularly infected actinorhizal plants (Alnus) than between actinorhizal plants of two different phylogenetic subgroups (Alnus/Datisca).     

Isolation and Structures of New Peptide Antibiotics,Cirratiomycin A and B

We measured the concentrations of acetyl-CoA and malonyl-CoA in shoots and roots of corn (Zea mays, L., cv. “Peter Corn”). Acetyl-CoA and malonyl-CoA concentrations were found to be relatively constant in shoots and in roots under a light-dark cycle. Acetyl-CoA concentrations were lower in shoots than in roots, whereas malonyl-CoA concentrations were higher in shoots than in roots.     

Accumulation of reactive oxygen species in arbuscular mycorrhizal roots

We investigated the accumulation of reactive oxygen species (ROS) in arbuscular mycorrhizal (AM) roots from Medicago truncatula, Zea mays and Nicotiana tabacum using three independent staining techniques. Colonized root cortical cells and the symbiotic fungal partner were observed to be involved in the production of ROS. Extraradical hyphae and spores from Glomus intraradices accumulated small levels of ROS within their cell wall and produced ROS within the cytoplasm in response to stress. Within AM roots, we observed a certain correlation of arbuscular senescence and H₂O₂ accumulation after staining by diaminobenzidine (DAB) and a more general accumulation of ROS close to fungal structures when using dihydrorhodamine 123 (DHR 123) for staining. According to electron microscopical analysis of AM roots from Z. mays after staining by CeCl₃, intracellular accumulation of H₂O₂ was observed in the plant cytoplasm close to intact and collapsing fungal structures, whereas intercellular H₂O₂ was located on the surface of fungal hyphae. These characteristics of ROS accumulation in AM roots suggest similarities to ROS accumulation during the senescence of legume root nodules.     

Hydrogenase in actinorhizal root nodules and root nodule homogenates.

Hydrogenases were measured in intact actinorhizal root nodules and from disrupted nodules of Alnus glutinosa, Alnus rhombifolia, Alnus rubra, and Myrica pensylvanica. Whole nodules took up H2 in an O2-dependent reaction. Endophyte preparations oxidized H2 through the oxyhydrogen reaction, but rates were enhanced when hydrogen uptake was coupled to artificial electron acceptors. Oxygen inhibited artifical acceptor-dependent H2 uptake. The hydrogenase system from M. pensylvanica had a different pattern of coupling to various electron acceptors than the hydrogenase systems from the alders; only the bayberry system evolved H2 from reduced viologen dyes.     

THE STRUCTURE OF THE ROOT NODULES OF CEANOTHUS SANGUINEUS AND CEANOTHUS VELUTINUS,WITH SPECIAL REFERENCE TO THE ENDOPHYTE

Furman , Thomas E. (Escuela Agrícola Panamericana, Tegucigalpa, Honduras.) The structure of the root nodules of Ceanothus sanguineus and Ceanothus velutinus, with special reference to the endophyte. Amer. Jour. Bot. 46(10): 698–703. Illus. 1959.—Past studies of non-leguminous root nodules have cited the endophytes of these structures as bacteria, filamentous fungi, actinomycetes, and members of the Plasmodiophorales. In this study, nodules of Ceanothus sanguineus and of Ceanothus velutinus were examined by using a variety of fixatives and staining methods as checks against artifacts, as well as histochemical tests and phase-contrast microscopy of living material. It was concluded that the nodules of these species were modified short roots, not homologous with leguminous nodules, inhabited by a filamentous microorganism which resembled most closely members of the described genus Streptomyces. The thallus of the endophyte had a definite, firm wall structure which absorbed ruthenium red stain actively, thus indicating a high pectin content.     

The use of PCR-based typing methods to assess the diversity of Frankia nodule endophytes of the actinorhizal shrub Ceanothus

Our understanding of the actinorhizal symbiosis, in particular of the Frankia-Ceanothus association, has been hampered by the failure to isolate infective strains in pure culture. Recently, the polymerase chain reaction (PCR) has been utilized to amplify regions of the Frankia genome, allowing analysis of the microsymbiont genome without first isolating the microbe in pure culture. Root nodules were collected from six Ceanothus spp. common to the coastal regions of the Santa Monica Mountains of southern California. Individual lobes were surface-sterilized, total DNA was extracted and amplified using prokaryotic-specific primers. To assess the genetic diversity of Frankia endophytes in the population studied, the BOX primer was used to generate genomic fingerprints of prokaryotic nodule inhabitants using rep-PCR. Fingerprint patterns fell into twelve distinct groups indicating the occurrence of genetic diversity of Frankia in the nodules sampled. DNA extracts of individual lobes that gave distinct BOX-PCR fingerprints were also amplified by PCR using primers directed against conserved regions of the 16S ribosomal RNA gene. The nucleotide sequences of the PCR products were determined and aligned with the corresponding region from other taxa for phylogenetic analysis. The sequences from Ceanothus nodules share a common ancestor to that of the Elaeagnus –infective strains.