Robert B. Mellor

Vesicular-arbuscular mycorrhizas of wild-type soybean and non-nodulating mutants with Glomus mosseae contain symbiosis-specific polypeptides (mycorrhizins), immunologically cross-reactive with nodulins

Wild-type soybean (Glycine max L. Merr. cv. Bragg) plants and two soybean mutants derived from cv. Bragg (nod 49 and nod 139) unable to form nodules with Bradyrhizobium japonicum were compared with regard to their reaction to the vesicular-arbuscular mycorrhizal fungus Glomus mosseae. The roots from wild-type and mutant plants entered equally well into vesicular-arbuscular mycorrhiza symbiosis. Polyadenylated RNA was isolated from nodule-free mycorrhizal and non-mycorrhizal roots of wild-type and mutant plants and translated in vitro. The translation products were subjected to immunoprecipitation using antisera reacting with soluble or membrane-bound nodulins. The antisera did not immunoprecipitate any of the translation products from non-mycorrhizal roots. However, they reacted with specific translation products from mycorrhizal roots of both wild-type and mutant plants: two polypeptides (MWs 135–140 and 18 kDa) were immunoprecipitated with the antiserum against soluble nodulins and three (MWs 21–28 kDa) with the antiserum against membrane-bound nodulins. These results indicate that symbiosis-specific polypeptides, possibly identical with nodulins, are induced in the mycorrhiza and therefore can be termed “mycorrhizins”.

Chitinase and peroxidase in effective (fix+) and ineffective (fix-) soybean nodules

Chitinase and peroxidase, two enzymes thought to be involved in the defense of plants against pathogens, were measured in soybean (Glycine max L. Merr.) roots and in nodules colonized by Bradyrhizobium japonicum strains differing in their symbiotic potential. Activities of both enzymes were higher in nodules than in roots. In “effective”, nitrogen-fixing nodules, colonized by wild-type bacteria, chitinase and peroxidase activities had low levels in the central infected zone and were enhanced primarily in the nodule cortex. An ascorbate-specific peroxidase, possibly involved in radical scavenging, had similarly high activities in the infected zone and in the cortex. “Ineffective” nodules colonized by bacteria unable to fix nitrogen symbiotically showed a similar distribution of chitinase and peroxidase. In another type of “ineffective” nodule, colonized by a B. japonicum strain eliciting a hypersensitive response, activities of both enzymes were enhanced to a similar degree in the infected zone as well as in the cortex. Tissue prints using a direct assay for peroxidase and an antiserum against bean chitinase corroborated these results. The antiserum against bean chitinase cross-reacted with a nodule protein of Mr 32 000; it inhibited most of the chitinase activity in the nodules but barely affected the chitinase in uninfected roots. It is concluded that proteins characteristic of the defense reaction accumulate in the cortex of nodules independently of their ability to fix nitrogen, and in the entire body of hypersensitively reacting nodules.

Soybean Root Response to Symbiotic Infection Glyceollin I Accumulation in an Ineffective Type of Soybean Nodules with an Early Loss of the Peribacteroid Membrane

A glyceollin I accumulation of about 6000 pmol · mg dry weight-, a tenfold increase above control root tissue, was found in one type of nodule from Glycine max which had been infected with a fix- strain (61-A-24) of Rhizobium japonicum. In nodules infected with one other ineffective (fix-) strain of Rhizobium japonicum (RH 31-Marburg) or with two fix+ strains of Rhizobium japonicum (61-A-101 and USD A 110) no increase in glyceollin I concentrations above control values was found at either 20 d or 34 d after infection. Nodules infected with Rhizobium japonicum 61-A-24 are distinguished by an early loss of the peribacteroid membrane in the infected host cell, whilst the bacteroids themselves remain stable.

Lysis of bacterioids in the vicinity of the host cell nucleus in an ineffective (fix-) root nodule of soybean (Glycine max)

In nodules of Glycine max cv. Mandarin infected with a nod +fix- mutant of Rhizobium japonicum (RH 31-Marburg), lysis of bacteroids was observed 20 d after infection, but occurred in the region around the host cell nucleus, where lytic compartments were formed. Bacteroids, and peribacteroid membranes in other parts of the host cell remained stable until senescence (40d after infection). With two other nod+ fix- mutants of R. japonicum either stable bacteroids and peribacteroid membranes were observed throughout the cell (strain 61-A-165) or a rapid degeneration of bacteroids without an apparent lysis (strain USDA 24) occurred. The size distribution of RH 31-Marburg-infected nodules exhibited only two maxima compared with four in wild-type nodules and nodule leghaemoglobin content was found to be reduced to about one half that of the wild type. The RH 31-Marburg-nodule type is discussed in relation to the stability of the bacteroids and the peribacteroid membrane system in soybean.

Partial purification and characterization of trehalase from soybean nodules

Summary Soybean ( Glycine max cv. Maple Arrow) was infected with Bradyrhizobium japonicum (strain 61-A-101) and grown under semi-sterile conditions. Trehalase was extracted from nodules under acidic conditions (pH 3.7) and purified 530-fold by chromatography on concanavalin A-agarose, anion exchange chromatography and gel filtration. Its native molecular weight was close to 54 kDa. Activity staining after electrophoresis yielded only one band. Isoelectric focussing under non-denaturing conditions indicated an acidic isoelectric point (ca. pH 5.2). The enzyme had a broad pH-optimum (pH 3.5-7) and a temperature optimum of 59 °C. The K m value for trehalose was 0.28 mM, close to the data given previously for crude nodule extracts. Trehalase was found to be relatively insensitive to chelators and divalent cations, indicating that it does not require a metal cofactor. Validamycin A, a fungitoxic antibioticum isolated from Streptomyces , competitively inhibited trehalase with a K i value of 2 nM.

Particle density and protein composition of the peribacteroid membrane from soybean root nodules is affected by mutation in the microsymbiont Bradyrhizobium japonicum

Particle frequency of the peribacteroid membrane (PBM) from nodules of Glycine max (L.) Merr. cv. Maple Arrow infected with Bradyrhizobium japonicum 61-A-101 (wild-type strain) was determined by freeze-fracturing to be about 2200·μm-2 in the protoplasmic fracture face and 700·μm-2 in the exoplasmic fracture face. In membranes isolated from nodules infected with the mutant RH 31-Marburg of B. japonicum, the particle frequency was similar in both fracture faces with 1200–1300 particles·μm-2. Analysis of particlesize distribution on peribacteroid membranes showed a loss, especially of particle sizes larger than 11 nm, in the mutant-infected nodules. Two-dimensional gel electrophoresis (isoelectric focussing and sodium dodecyl sulfate-polyacrylamide) showed 27 different polypeptides in the PBM from nodules infected with the wild-type strain, four of which were absent from the PBM of nodules infected with the mutant RH 31-Marburg, which also exhibited one extra small-molecular-weight polypeptide. At least 14 of the 27 polypeptides in the PBM from the wild-type-infected nodule were glycoproteins. In three of these glycoproteins, post-translational modifications were either lacking or different when the membrane was derived from mutant-infected nodules.

A phytohaemagglutinin from the Azolla-anabaena symbiosis

Abstract Proteins recovered from cell-free extracts of the Azolla-Anabaena azollae symbiosis exhibited haemagglutination activity; galactose was the most effective carbohydrate tested in preventing haemagglutination. Extracts of cyanobacteria-free Azolla also caused haemagglutination but extracts of free-living or symbiotic Anabaena azollae did not. Symbiotic Azolla plants grown on NO 3 − showed lower haemagglutination activity than did those grown on N 2 ; activity increased on removal of NO 3 − . The lower activity of the NO 3 − -grown material may be due to NO 3 su− exerting a direct effect on lectin activity/synthesis, or it may act indirectly by inhibiting the development of Anabaena which in turn affects the Azolla lectin. The purified lectin was shown to be composed of 6 sub-units, each of M.W. 21000.

Glycoproteins of the glyoxysomal matrix

The early postgerminative growth of fat-storing seeds is characterized by a period of gluconeogenic activity during which time stored triglycerides are converted to carbohydrate [l] . Many of the enzymes catalysing this conversion are known to be functionally located within single-membrane bound glyoxysomes [2] . These enzymes are regarded as major components of the granular protein matrix of such organelles which is characteristically observed in electron micrographs [3] . An increasing effort is now underway to sub-fractionate and further characterize proteins of the glyoxysomal matrix [4-71. Information regarding the nature and origin of such proteins is relevant to the question of glyoxysome biogenesis. We report here that several glyoxysomal proteins contain bound carbohydrate, and discuss the occurrence of corresponding glycoproteins in the microsomal fraction in relation to the intracellular segregation of glyoxysomal proteins.

Subcellular localization of mannosyl transferase and glycoprotein biosynthesis in castor bean endosperm

Differential and sucrose density gradient centrifugation have shown that the mannosyl transferase present in germinating castor bean endosperm cells which catalyses the synthesis of mannosyl-phosphoryl-polyisoprenol is exclusively located in the endoplasmic reticulum membrane. This intracellular location was confirmed using both ribosome-denuded microsomes isolated in the presence of EDTA and rough-surfaced microsomes isolated in the presence of excess Mg2+ added to maintain ribosome-membrane attachment. Separation of organelles following the incubation of crude particulate fractions with GDP[14C]mannose demonstrated that most of the mannolipid thus formed remained associated with the microsomal fraction. When organelles were isolated from intact tissue which had previously been incubated with GDP[14C]mannose, [14C]glycoprotein was found to be associated with other cellular fractions in addition to the microsomes, in particular the glyoxysomes. The kinetics of radioactive labelling of these organelles suggest that [14C]glycoprotein appears initially in the microsomal fraction and subsequently accumulates in the glyoxysomes. Subfractionation of isolated, [14C]glycoprotein-labelled glyoxysomes established that over 80% of the total radioactivity was present in the membrane, while sodium dodecyl sulphate-polyacrylamide gel electrophoresis of solubilized glyoxysomal membranes showed that the [14C]sugar moiety was associated with several, but not all, constituent polypeptides.

Protease (thermolysin) inhibition activity in the peribacteroid space of Glycine max root nodules

Summary Peribacteroid space components from nodules of soybean cv. Maple Arrow infected with Bradyrhizobium japonicum 61-A-101(nif + , fix + ) were found to exhibit a protease inhibition effect. Proteins from the peribacteroid space could inhibit thermolysin activity up to 90 %. After separation on Sephadex G 75 a protease inhibitor of a molecular weight of 20 kDa was separated from at least two other inhibitory fractions with higher molecular weights. The purification of this peribacteroid space protease inhibitor gave a specific activity of 30–40 units/μg protein. The heat stable thermolysin inhibitor activity in the unfractionated peribacteroid space protein is about three times larger compared to an extract from seed proteins of soybean.