Plants ofAlnus incana (L.) Moench in symbiosis with a local source ofFrankia were exposed to prolonged darkness under controlled climate conditions.Frankia vesicle clusters were prepared from the root nodules, and the condition ofFrankia was measured as respiratory capacity by supplying the preparation with saturating amounts of four different substrates. During darkness, nitrogenase (EC 1.7.99.2) activity decreased in intact plants and in the vesicle-cluster preparations. The respiratory capacity ofFrankia also decreased. After 4 d in darkness most respiration was lost, though all nitrogenase activity was already lost after 3 d. When the dark treatment was ended after 2 d and normal light/dark conditions restored, nitrogenase activity immediately started to recover. The respiratory capacity continued to decrease and no recovery was observed until the third day after the end of the dark treatment. Whole-plant nitrogenase activity slowly increased at a rate similar to the rate of increase observed in untreated plants. Transmission electron micrographs of the root nodules showed that the cytoplasm of infected host cells and the cells ofFrankia were structurally degraded in response to dark treatment, while young vesicles were frequent during recovery. Growth and differentiation ofFrankia cells were apparently important for recovery of the enzyme activities studied.

It is not clear to what extent genetic, environmental and measurement factors are responsible for the commonly reported decline in nitrogenase activity with the onset of pod-filling in grain legumes. We address this question by observing nitrogenase activity and assimilate partitioning throughout the life span of an indeterminate variety of common bean (GN 1140) under controlled-environment and field conditions. Nitrogenase activity per plant was maintained well into pod-filling in GN 1140 under high-light conditions in growth cabinets. In contrast, plants exposed to a gradual reduction in light intensity during early reproductive growth had a decline in nitrogenase activity on a whole plant basis with the onset of pod-filling. However, the decline was due to an inability to maintain nodule growth, rather than a decrease in specific nitrogenase activity. Under field conditions, acetylene reduction assay of root crowns appeared to indicate a rapid decline in nitrogenase activity with the onset of pod-filling in GN 1140. This decline was not correlated with the water status of the soil or the plant. In contrast, acetylene reduction activity of root cores taken from outside the root crown region ('non-crown') and N accumulation by above-ground biomass during pod-filling suggested that whole plant nitrogenase activity was maintained longer than that indicated by root crown assays. We conclude that although the occurrence of a decline in nitrogenase activity with the onset of pod-filling in grain legumes can be genetically determined, in many cases the decline can be the result of growing conditions and improper measurement techniques.

K. 1987, Purity of Frankia preparations from root noduies of Ainus incana. -Physiol. Piant, 71: 489-494.Frankia vesicle clusters were prepared from Alnus incana (L.) Moench root nodules by a homogenization-filtration procedure. The preparation was examined by transmission electron microscopy and computerized picture analysis to quantify contamination from tbe host plant. Spedai attention was paid to piant mitochondria. Mitochondria were only found in 30% of the 50 sections of clusters examined. In sections containing mitochondria the mean number of mitochondria per cluster section was 1.5, The relative volume of all objects found in the vesicle clusters was calculated. More than 98% of the volume of a preparation consisted of Frankia vesicles and hyphae, while only 0,4% of the volume was host plant mitochondria. The frequency of mitochondria in a preparation could be birther decreased by osmotic shock. It is conduded that Frankia veside clusters, prepared from Alnus incana by the homogenization-filtration technique used here, are suffidently pure to be used for studies of Frankia metabolism.

Frankia vesicle clusters were prepared from Alnus incana (L.) Moench root nodules containing a local source of Frankia by an improved homogenization‐filtration procedure. The capacity of the vesicle clusters to metabolize hexoses was investigated by respirometric and enzymological studies. The vesicle clusters could utilize glucose, glucose‐6‐phosphate and 6‐phosphogluconate provided that appropriate cofactors were added to the preparations. The enzymes hexokinase (EC 2.7.1.1), NADP+: glucose‐6‐phosphate dehydrogenase (EC 1.1.1.49) and NAD+;6‐phosphogluconate dehydrogenase (EC 1.1.1.44) were found in cell‐free extracts of the vesicle clusters and kinetic constants for the enzymes were determined. Hexokinase had a lower Km for glucose than for fructose. Extracts from both symbiotic and propionate grown Frankia AvcII also showed activity of these hexose‐degrading enzymes, indicating that their presence is not necessarily dependent on sugars as carbon source. The NAD+‐ dependent 6‐phosphogluconate dehydrogenase was only present in Frankia cells and not in alder root cells, which makes this enzyme a useful Frankia‐specific marker in these symbiotic systems.

VIKMAN, P.-A. 1992. The symbiotic vesicle is a major site for respiration in Frankia from Alnus incana root nodules.Can. J. Microbiol. 38: 779-784.A technique was developed for preparation of Frankia symbiotic vesicles, free of hyphae. The symbiotic vesicles were isolated by isopycnic centrifugation of disrupted Frankia vesicle clusters prepared from root nodules of Alnus incana (L.) Moench. Activities in symbiotic vesicles were compared with activities in intact symbiotic vesicle clusters on a total protein basis. Respiratory capacity was tested with 6-phosphogluconate, malate + glutamate, and NADH as added substrates. With all three substrates, specific respiration was doubled after symbiotic vesicle isolation. Nitrogenase was used as a symbiotic vesicle specific marker and its specific activity increased similarly to respiration. Activities of four respiratory enzymes were assayed on crude cell-free extracts obtained after sonication of symbiotic vesicle preparations. According to the increased specific rates after symbiotic vesicle isolation, NAD +