Evaluation of active versus passive uptake of metabolites by Rhizobium japonicum bacteroids.

Rhizobium japonicum bacteroids were isolated anaerobically from soybean [Glycine max (L.) Merr] nodules. The bacteroids, which were capable of acetylene reduction and respiration, were used to study the uptake of metabolites by a method which permits correction for nonspecific adsorption of metabolites and estimation of total cell volume. These determinations permit active uptake to be assessed from metabolite accumulation against a concentration gradient. Succinate, malate, alpha-ketoglutarate, and glutamate were absorbed via an active mechanism. Plots of 1/V versus 1/[S] for succinate and malate indicated the presence of two uptake components: a saturable and presumably active or carrier-mediated component and a nonsaturable and presumably passive component. The uptake of glucose, malonate, D-pinitol, myo-inositol, and glucose 6-phosphate was slow and not active.

Metabolism of C-labeled photosynthate and distribution of enzymes of glucose metabolism in soybean nodules.

The metabolism of translocated photosynthate by soybean (Glycine max L. Merr.) nodules was investigated by (14)CO(2)-labeling studies and analysis of nodule enzymes. Plants were exposed to (14)CO(2) for 30 minutes, followed by (12)CO(2) for up to 5 hours. The largest amount of radioactivity in nodules was recovered in neutral sugars at all sampling times. The organic acid fraction of the cytosol was labeled rapidly. Although cyclitols and malonate were found in high concentrations in the nodules, they accumulated less than 10% of the radioactivity in the neutral and acidic fractions, respectively. Phosphate esters were found to contain very low levels of total label, which prohibited analysis of the radioactivity in individual compounds. The whole nodule-labeling patterns suggested the utilization of photosynthate for the generation of organic acids (principally malate) and amino acids (principally glutamate).The radioactivity in bacteroids as a percentage of total nodule label increased slightly with time, while the percentage in the cytosol fraction declined. The labeling patterns for the cytosol were essentially the same as whole nodule-labeling patterns, and they suggest a degradation of carbohydrates for the production of organic acids and amino acids. When it was found that most of the radioactivity in bacteroids was in sugars, the enzymes of glucose metabolism were surveyed. Bacteroids from nodules formed by Rhizobium japonicum strain 110 or strain 138 lacked activity for phosphofructokinase and NADP-dependent 6-phosphogluconate dehydrogenase, key enzymes of glycolysis and the oxidative pentose-phosphate pathways. Enzymes of the glycolytic and pentose phosphate pathways were found in the cytosol fraction.In three experiments, bacteroids contained about 10 to 30% of the total radioactivity in nodules 2 to 5 hours after pulse-labeling of plants, and 60 to 65% of the radioactivity in bacteroids was in the neutral sugar fraction at all sampling times. This strongly suggests some absorption and metabolism of sugars by bacteroids in spite of the lack of key enzymes. Bacteroids did possess enzymes for the formation of hexose phosphates from glucose or fructose. Radioactivity in alpha,alpha-trehalose in bacteroids increased until, after 5 hours, trehalose was a major labeled compound in bacteroids. Thus, trehalose synthesis may be a major fate of sugars entering bacteroids.

Effect of 2-(3,4-Dichlorophenoxy)-triethylamine on the Synthensis of cis-Polyisoprene in Guayule Plants (Parthenium argentatum Gray).

The application of 2-(3,4-dichlorophenoxy)-triethylamine to guayule (Parthenium argentatum Gray var 593) plants results in a 2-fold stimulation of rubber synthesis and a 1.5- to 3-fold increase in mevalonic acid kinase, isopentenyl pyrophosphate isomerase, and rubber transferase. The increase in these enzymic activities accounts in part for the chemical induction of rubber synthesis.

Biosynthesis of Starch in Proplastids of Germinating Ricinus communis Endosperm Tissue.

Electron photomicrographs of endosperm tissue from germinating seed of Ricinus communis L. cv. Hale show proplastids which contain prominent starch grains. The content of starch in endosperm tissue increased from 500 micrograms per seed, in imbibed seed, to 1,100 micrograms per seed in 5-day-old seedlings. The maximum net rate of starch deposition was 1.1 nanomoles glucose incorporated per minute per seed. About 200 micrograms of starch remained in the endosperm 9 days after imbibition. Starch content followed the same developmental pattern as the content of sucrose, free reducing sugars, and other metabolic processes found in this tissue. Two key enzymes of starch synthesis, adenosine diphosphoglucose (ADPG) pyrophosphorylase and ADPG-starch glucosyl transferase (starch synthetase) exhibited maximum activities at 4 and 5 days after germination, respectively. The maximum activity of ADPG pyrophosphorylase was 8.17 nanomoles ADPG formed per minute per seed, whereas starch synthetase exhibited an activity of 125 nanomoles glucose incorporated per minute per seed. These levels of enzyme activity are sufficient to account for the starch synthesis observed. Other enzymes which may be involved in starch synthesis include 3-phosphoglycerate kinase which showed an activity of 8.76 units per seed, triose-P isomerase (2.56 units per seed), fructose-1,6-bisphosphate aldolase (0.99 units per seed), fructose-1,6-bisphosphatase (0.23 units per seed), phosphoglucose isomerase (12.6 units per seed), and phosphoglucomutase (9.72 units per seed). The activities of these enzymes were similar to previously reported values.Starch synthetase was found in association with the fraction containing proplastids isolated from endosperm tissue. Of the total starch synthetase activity in the endosperm, 38% was particulate. Forty-four% of the total particulate activity of starch synthetase placed on sucrose gradients was associated with the band containing proplastids. The proplastids contained 98% of the ribulose 1,5-bisphosphate carboxylase carboxylase activity placed on the gradient.

A rapid one-step method for the isolation of bacteroids from root nodules of soybean plants, utilizing self-generating Percoll gradients.

Bacteroids were isolated from the nodules of soybean plants by means of self-generating Percoll density gradients. The entire procedure can be performed in less than 1 h using an ordinary refrigerated centrifuge and angle head rotor. All of the markers for cytosol and bacteroid fractions behaved in accord with other reports in the literature. Asparaginase, beta-hydroxybutyrate dehydrogenase, and alanine dehydrogenase were all localized in the bacteroid fraction. Invertase, phosphoenolpyruvate carboxylase, and leghaemoglobin were all found in the cytosol fraction. Very little (less than 7%) cross contamination between the fractions was observed. The isolated bacteroids were viable, and based on electron micrographs, were free from contaminating plant material. Since the entire procedure is performed isosmotically, very little damage to the bacteroids is likely to occur. No organic compounds, except Percoll, were added to the isolating media, thus aiding in the analysis of bacteroid and cytosol metabolites.

Ultrastructure and biochemistry of two mutants in Hosta (Liliaceae).

In a survey of several plastid chimeras of the genus Hosta two white mutants with C4--type ultrastructure were noted. Mesophyll plastids from the white tissue have an extensive system of peripheral reticula, characteristic of C4 plants, and macrograna or vesiculated thylakoids, characteristic of photosystem I deficient mutants. Microbodies were either absent or in low quantities in these cells. Bundle sheath plastids from the white sectors had a concentration of all organelles, with plastids of typical C4 bundle sheath ultrastructure and numerous, ornate mitochondria. Green restituted sectors of one of these mutants retained the ability to make peripheral reticulum yet had normal Hosta granal formation. Despite these changes the plants show a carbon fractionation like C3 plants. Possible reasons for these ultrastructural modifications are discussed.

Fractionation of stable carbon isotopes by phosphoenolpyruvate carboxylase from c(4) plants.

The active species of "CO(2)" and the amount of fractionation of stable carbon isotopes have been determined for a partially purified preparation of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) from corn (Zea mays) leaves. The rates of the enzyme reactions, using substrate amounts of HCO(3) (-), CO(2) or CO(2) plus carbonic anhydrase, show that HCO(3) (-) is the active species of "CO(2)" utilized by PEP carboxylase. The K(m) values for CO(2) and HCO(3) (-) are 1.25 mm and 0.11 mm, respectively, which further suggest the preferential utilization of HCO(3) (-) by PEP carboxylase. The amount of fractionation of stable carbon isotopes by PEP carboxylase from an infinite pool of H(12)CO(3) (-) and H(13)CO(3) (-) was -2.03 per thousand. This enzyme fractionation (delta), together with the fractionation associated with absorption of CO(2) into plant cells and the equilibrium fractionation associated with atmospheric CO(2) and dissolved HCO(3) (-) are discussed in relation to the fractionation of stable carbon isotopes of atmospheric CO(2) during photosynthesis in C(4) plants.