Showing papers by "Kerstin Huss-Danell published in 1991"

Influence of host (Alnus and Myrica) genotype on infectivity, N2 fixation, spore formation and hydrogenase activity in Frankia

Abstract: summary Nine species of Alnus, two of them represented by several provenances, and Myrica gale were inoculated with a ‘local source’ of Frankia. The inoculum is an effective (N2-fixing), spore-Forming type of Frankia that lacks H, uptake (hydrogenase) activity in symbiosis with A. incana. Despite their wide geographical origin (Europe, China, North America) and their various taxonomie grouping within the genus, all Alnus species/provenances formed Infixing nodules. In all studied nodules Frankia had the phenotype Spore+. None of the studied symbioses had hydrogenase activity. The Frankia inoculum was infective also on M. gale, and gave effective nodules without hydrogenase activity. Unlike some other spore-forming types of symbiotic Frankia, all studied symbiotic phenotypes of the ‘local source’ of Frankia were thus stable characters not influenced by the various host genotypes included in the study.

Nitrogenase Activity and Amounts of Nitrogenase Proteins in a Frankia-Alnus incana Symbiosis Subjected to Darkness.

Abstract: Effects of prolonged darkness on nitrogenase activity in vivo, nitrogenase activity in vitro, and the amounts of nitrogenase proteins were studied in symbiotic Frankia. Plants of Alnus incana (L.) Moench in symbiosis with a local source of Frankia were grown for 9 to 10 weeks in an 18/6 hour light/darkness cycle. After 12 hours of a light period, the plants were exposed to darkness for up to 40 hours. Nitrogenase activity (acetylene reduction activity) of intact plants was measured repeatedly. Frankia vesicle clusters were prepared from the nodules with an anaerobic homogenization and filtration technique and were used for measurements of in vitro nitrogenase activity and for measurements of the amounts of nitrogenase proteins on Western blots. Antisera made against dinitrogenase reductase (Fe-protein) of Rhodospirillum rubrum and against dinitrogenase (MoFe-protein) of Azotobacter vinelandii were used. Western blots were made transparent and nitrogenase proteins were quantified spectrophotometrically. Nitrogenase activity both in vivo and in vitro decreased after about 23 hours of darkness and continued to decrease to about 25% and 16% of initial activity, respectively, after 40 hours. The amount of Fe-protein and MoFe-protein in Frankia of the same plants decreased to 60% and 35%, respectively, after 40 hours of darkness. Loss of nitrogenase activity thus appeared to be largely explained by loss of MoFe-protein.

Response of nitrogenase to altered carbon supply in a Frankia‐Alnus incana symbiosis

Abstract: To study the effect of altered carbon supply on nitrogenase (EC 1.7.99.2), plants of Alnus incana (L.) Moench in symbiosis with the local source of Frankia were exposed to darkness for 2 days, and then returned to normal light/dark conditions. During the dark period nitrogenase activity in vivo (intact plants) and in vitro (Frankia cells supplied with ATP and reductant), measured as acetylene reduction activity, was almost completely lost. Western blots for both the Fe-protein (dinitrogenase reductase) and the MoFe-protein (dinitrogenase) showed that, in particular, the amount of MoFe-protein was strongly reduced during darkness. Protein stained sodium dodecyl sulphate-polyacrylamide gels of Frankia protein showed that the nitrogenase proteins were the only abundant proteins that clearly decreased during darkness. During recovery, studied for 4 days, nitrogenase activity in vivo recovered to the level before dark treatment but was still only half of control activity, Nitrogenase activity in vitro and the amount of MoFe-protein, both expressed per Frankia protein, recovered and reached similar values in previously dark treated plants and in control plants. The rate of recovery was similar to the increase in activity of control plants, suggesting growth of Frankia in addition to synthesis of nitrogenase proteins during the recovery after carbon starvation.

Respiration of Malate and Glutamate in Symbiotic Frankia Prepared from Alnus incana

Abstract: Frankia vesicle clusters were prepared from root nodules of Alnus incana (L.) Moench inoculated either with a local source of Frankia or with Frankia Cpll. The capacity of vesicle clusters to respire was investigated by respirometric and enzymological studies. Simultaneous addition of malate, glutamate, and NAD+ supported respiration in both types of Frankia, though at a smaller rate compared to the substrates NADH or 6-phosphogluconate. The saturating concentrations of malate and glutamate were also much higher than with the other substrates. No respiration was supported by succinate. Activity of the enzymes malate dehydrogenase (EC 1.1.1.37) and glutamate oxaloacetate transaminase (EC 2.6.1.1) was demonstrated in crude extracts from both types of symbiotic Frankia. Their maximum rates were high enough to account for the respiration of malate and glutamate. This respiration was inhibited by mersalylic acid, an inhibitor of the dicarboxylate shuttle in mitochondria, but it was shown that inhibition of respiration could be due to a direct effect on the enzymes. We conclude that respiration of malate and glutamate is most likely mediated by malate dehydrogenase and glutamate oxaloacetate transaminase, but no explicit evidence for or against the presence of a dicarboxylate carrier was found. The utilization of respiratory substrates was largely similar in the two types of Frankia, except for some differences in maximum rates and cofactor dependency.