The Azolla, Anabaena azollae Relationship: II. Localization of Nitrogenase Activity as Assayed by Acetylene Reduction

Anaerobic (microaerophilic) acetylene reduction by Azolla caroliniana Willd. was dependent on light and saturated at approximately 450 foot candles. Maximum rates of acetylene reduction were 60 nmoles/mg chlorophyll minute. However, rates of 25 to 30 nmoles/mg chlorophyll minute were more common.The growth of Azolla for 35 days with nitrate or urea as a nitrogen source decreased the rate of acetylene reduction approximately 30% compared to controls grown on nitrogen. Prolonged growth on nitrate or urea (6-7 months) resulted in a 90% decrease in the rate of acetylene reduction.The inhibition of acetylene reduction by 3 (3,4-dichlorophenol) 1,1-dimethylurea (12 muM) was not pronounced until the Azolla became depleted of the reserves formed during photosynthesis. The interval required for this depletion was dependent upon pretreatment and varied from 2 to more than 12 hours. Oxygen evolution was inhibited 75% in 10 minutes by the same concentration of 3 (3,4-dichlorophenol) 1,1-dimethylurea.The addition of oxygen, 20% volume per volume, resulted in a 30 to 40% decrease in the rate of acetylene reduction and the onsetof 3(3,4-dichlorophenol) 1,1-dimethylurea inhibition was more rapid then under microaerophilic conditions. The aerobic dark reduction of acetylene was from 10 to 30% of the rate of aerobic reduction in the light.Acetylene reduction activity was absent in fronds freed ofthe symbiotic algae and present in isolated Anabaena azollae. This study shows that the alga is the agent of acetylene reduction and suggests that there is considerable transport of metabolites between the fern and the blue-green alga.