Electron transferring dyes in the nitrate reductase reaction: non-toxic alternatives to methyl viologen.

A range of electron-transferring agents (dyes) were screened for activity with the oxido-reductase, nitrate reductase, from either Pisum sativum or Aspergillus. Reducing equivalents could be transferred efficiently to both enzymes by methyl viologen. Pisum enzyme could also be effectively reduced by reduced Patent blue (food colouring E 131), curcumin (food colouring E 100) or, at lesser rates by Azure A. Aspergillus enzyme could be reduced at only low rates by curcumin, Patent blue and Bromophenol blue.

Perception of Rhizobium nodulation factors by tomato cells and inactivation by root chitinases.

The bacterial genera Rhizobium and Bradyrhizobium, nitrogen-fixing symbionts of legumes, secrete specific lipo-chitooligosaccharides that induce the formation of nodules on their host plants. When preparations of such nodulation-inducing factors (Nod factors) were added to suspension-cultured tomato cells, a rapid and transient alkalinization of the culture medium occurred. Lipo-oligosaccharide preparations from Rhizobium or Bradyrhizobium treated with flavonoids, known inducers of Nod factor synthesis, were up to 100 times more potent in inducing alkalinization than the ones from untreated bacteria. The activity was absent from preparations of the mutant strain Rhizobium sp. NGR234 delta nodABC, unable to produce any Nod factors. Preparations of Nod factors from various bacteria as well as individual, highly purified Nod factors from Rhizobium sp. NGR(pA28) induced alkalinization in the tomato cell cultures at nanomolar concentrations. This demonstrates that Nod factors can be perceived by tomato, a nonhost of rhizobia. Using the alkalinization response as a sensitive bioassay, Nod factors were found to be inactivated by plant chitinases. Root chitinases purified from different legumes differed in their potential to inactivate differently substituted Nod factors produced by Rhizobium sp. NGR(pA28). This indicates that the specificity of the bacterium-host plant interaction may be due, at least in part, to differential inactivation of Nod factors by root chitinases.

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.

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".

Peribacteroid membrane nodulin gene induction by Bradyrhizobium japonicum mutants.

Seventeen translation products from Glycine max root mRNA precipitated with antiserum prepared against a peribacteroid membrane preparation from effective root nodules. Messenger RNA from fix (+) nodules coded for these 17 products plus 7 other nodule-specific polypeptides which bound to the antiserum. Of these 7 nodulins only 4 were present when nodules were infected with Bradyrhizobium japonicum 110 rif 15 2960, which induces the plant to produce 'empty' peribacteroid membranes. In nodules infected with B. japonicum strains inducing either very short-lived or defective peribacteroid membrane, only 5 or 6, respectively, of these nodulins could be detected.From these results we hypothesize that the microsymbiont is responsible for the production of at least 4 different signals leading to peribacteriod membrane formation by the plant.

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.

Choline kinase II is present only in nodules that synthesize stable peribacteroid membranes.

Host-cell cytoplasm from soybean plants infected with the peribacteroid membrane (PBM)-building Rhizobium japonicum strain 61-A-101 (effective, N(2)-fixing) had much higher choline kinase activity than cytoplasm from either uninfected tissue or tissue infected with the non-PBM-building (ineffective, non-N(2)-fixing) strain 61-A-24. Ion-exchange chromatography showed that both types of nodule and root tissue possessed constitutive choline kinase I activity that had a K(m) for choline of approximately 150 muM. The nodules of the effective symbiosis had another activity, choline kinase II (K(m) = 81 muM). Nondenaturing and NaDodSO(4) electrophoresis revealed no multimeric subunit structure of the two enzyme forms but did show the molecular sizes for choline kinase I, 58-59 kDa, and choline kinase II, 60 kDa. Choline kinase I and II and pI values of 8.1 and 8.5, respectively, and two-dimensional gel electrophoresis of whole cytoplasm from control and infected tissue showed a spot corresponding to choline kinase II only in the case of the effective symbiosis, whereas both tissue types had spots corresponding to choline kinase I. Choline kinase II is presumed to be encoded by the plant as neither free-living nor symbiotic (bacteroid) forms of the prokaryote showed any choline kinase activity.

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.

Analysis of glycoconjugate saccharides in organelles isolated from castor bean endosperm.

The presence of lipid- and protein-bound sugars in the major organelle fractions isolated from germinating castor bean (Ricinus communis L.) endosperm has been established. Microsomes, glyoxysomes and mitochondria were subfractionated into a membrane fraction and a fraction containing peripheral membrane and soluble matrix proteins. The membranes were further subfractionated into monosaccharide lipid, oligosaccharide lipid and lipid-free protein components. The constituent sugars present in the prepared fractions were released and identified by gas-liquid chromatography. While all derived protein fractions contained the N-acetylglucosamine and mannose typically found in the inner core region attached to asparagine residues in many glycoproteins, some differences were noted in the organellar distribution of peripheral sugars such as fucose, arabinose, and xylose.

Glycosylation of exogenous protein by endoplasmic-reticulum membranes from castor-bean (Ricinus communis) endosperm.

Endoplasmic-reticulum membranes isolated from the endosperm tissue of 3-day-old castor-bean (Ricinus communis) seedlings catalysed the enzymic transfer of the sugar moiety from an oligosaccharide--lipid to a chemically unfolded form of ribonuclease A.

Involvement of a lipid-linked intermediate in the transfer of galactose from UDP [(14)C]galactose to exogenous protein in castor bean endosperm homogenates.

A crude organelle preparation from germinating castor bean endosperm catalysed the incorporation of galactose from UDP[(14)C]galactose into chloroform/methanol (2:1)-soluble glactolipids. At least two galactolipids were formed. Most of the [(14)C]galactose was present in a galactolipid synthesized by the microsomal membranes, the remainder was present in a second galactolipid synthesized by other cellular membranes, possibly Golgi-derived. The addition of asialo-agalacto-fetuin reduced incorporation of [(14)C]galactose into the microsomal galactolipid with a concomitant increase in microsomal [(14)C]galactoprotein. Asialo-agalacto-fetuin did not affect galactolipid or galactoprotein synthesis by nonmicrosomal fractions. The results suggest that the endoplasmic reticulum is a major site of protein galactosylation in castor bean endosperm cells, and that galactose transfer from UDP-galactose to protein occurs via a lipid-linked intermediate.

Formation of lipid-linked mono- and oligosaccharides from GDP-mannose by castor bean endosperm homogenates.

A crude organelle preparation from germinating castor bean endosperm catalysed the incorporation of mannose from GDP[(14)C]mannose into acid-labile mannolipids. Solubility and chromatographic properties have identified the most rapidly synthesized products as mannosyl-phosphoryl-polyisoprenol, while the more polar lipid formed was shown to contain oligosaccharide. Little radioactivity from GDP[(14)C]mannose accumulated in insoluble product in the cell-free system, but supplying GDP[(14)C]mannose to intact endosperm tissue has shown that the major incorporation product in vivo is glycoprotein. This product was readily solubilized by either pronase or sodium dodecyl sulphate treatment suggesting it was membrane bound glycoprotein. Incorporation of mannose into mannosyl-phosphoryl-polyisoprenol during the cell-free assay was stimulated by the addition of dolichol monophosphate. This enzymic activity was optimal at pH 7.5 and in the presence of 10 mM Mg(2+). The Km for GDP-mannose was estimated to be 5×10(-7) M. Cellular mannosyl transferase activity changed markedly during early post-germinative growth; from being absent in the dry seed, enzyme activity increased to peak between the second and third days of growth and subsequently declined.

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 Mg(2+) added to maintain ribosome-membrane attachment. Separation of organelles following the incubation of crude particulate fractions with GDP[(14)C]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[(14)C]mannose, [(14)C]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 [(14)C]glycoprotein appears initially in the microsomal fraction and subsequently accumulates in the glyoxysomes. Subfractionation of isolated, [(14)C]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 [(14)C]sugar moiety was associated with several, but not all, constituent polypeptides.

Incorporation of D-[(14)C]galactose into organelle glycoprotein in castor bean endosperm.

Excised casto bean (Ricinus communis L.) endosperm tissue supplied with [(14)C]galactose incorporates radioactivity into particulate cell components. Fractionation of homogenates established that (14)C-labeled trichloroacetic acid-insoluble material was located primarily in the microsomal and glyoxysomal fractions. The capacity of the tissue to incorporate [(14)C]galactose into organelle glycoprotein varied during seedling development, increasing during the first 3 days of germination and subsequently declining. The kinetics of incorporation into the major organelle fractions of 2-day old endosperm tissue showed that the endoplasmic reticulum was immediately labeled whereas a lag period preceded the labeling of glyoxysomes. Sub-fractionation of the isolated organelles established that the greatest proportion of the [(14)C]-galactose labeled glycoprotein was located in the membrane, although a significant incorporation into the matrix protein was also observed.The results indicate that the addition of the carbohydrate moiety to the polypeptide cores occurs in the endoplasmic reticulum during or immediately after their synthesis on membrane-bound ribosomes.