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The Azolla-Anabaena azollae association permits the study of a symbiotic relationship between a blue-green alga and a green plant under laboratory conditions. Previous studies on the physiology of the symbiotic association were not well defined and were limited in scope. Various aspects of mineral nutrition, temperature, and light intensity on the growth of the organism have been reported (22). We are not aware of any studies on the metabolic functions of N2 fixation, respiration, and photosynthesis in the individual organisms or their interaction in the symbiotic association.This manuscript is a report of initial studies on the characterization of the Azolla-Anabaena azollae symbiotic relationship. We describe the morphology, a method of freeing the fronds of the symbiotic alga, and isolation procedures devised to fractionate Azolla-Anabaena azollae for metabolic studies. The companion publication deals with aspects of acetylene reduction (nitrogenase activity) (25).

This manuscript reports results of initial studies on acetylene reduction by the symbiotic association. It provides evidence that the symbiotic alga is the agent of acetylene reduction and, if acetylene reduction is a valid assay of the ability to fix N,. the site of N, fixation.MATERIALS AND METHODS Azolla plants, nutrient solutions, growth conditions, isolation of the symbiotic algae, and Chl determinations were as described in thr preceding paper (20).Chemicals DCMU was obtained as Diuron from the E. 1. duPont de Nemours Company. Polyvinylpyrrolidone was obtained from the Sigma Chemical Company. All other chemicals we reagent grade. The gas mixtures were obtained from Matheson Gas Products.Acetylene Nmduction Assays. Three to five fronds were incubated under dciher 1% CO,, 1% acetylene in argon (microaerophilic comlidions) (24) or with oxygen added to 21'% (v/v) (aerobic conditions) in calibrated 25-ml Erlenmeyer flasks fitted with serum caps and containing :5 ml of the desired nutrient solution. The flasks were evacuated and flushed prior to filling to a slightly positive pressure. The flasks were illuminated from the bottom with a bank of General Electric white fluorescent lighs. Screens were used to lower the inteasty for the light intensity curve. The intensity for all other assays was maintained at approximately 750 ft-c. Incubations wee carried out at 23 C.The gas phase was analyzed for ethylene by gas chmnatography using a Hewlett-Packard (Model 700) dual flame gas chromatograph with a hydrogen flame ionization detector and a 3.2 mm, 2-m long column containing 80 to 100 mesh Pora-820 www.plantphysiol.org on May 9, 2018 -Published by Downloaded from

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Light, scanning electron, and transmission electron microscopy were used to follow sporocarp development in Azolla mexicana and the mecbanism by whicb sporocarps become inoculated with symbiotic Anahaena. Packaging of the Anabaena into sporocarps is facilitated by branched epidermal tricbomes, termed sporangial pair bairs. Sporocarps are borne in pairs and arise from tbe ventral lobe initial of tbe first leaf of a lateral branch. Four sporangial bairs are associated witb eacb sporangial pair from tbe onset of tbeir development, Tbese bairs differentiate rapidly and exhibit transfer cell ultrastructure, Tbey protrude into tbe mass of undifferentiated Anabaena filaments wbicb comprise tbe apical colony. Subsequently, tbe sporangial primordia and tbe sporangial bairs witb intertwined Anabaena filaments are displaced ventrally and covered by tbe developing involucre, A reservoir of Anabaena filaments is tbus entrapped between tbe developing sporocarp pair and tbe lower surface of tbe involucre, A cup-sbaped indusium forms around tbe base of eacb sporangium, separating it from tbe sporangial bairs wbile entrapping tbe Anahaena filaments. As development continues, tbe indusia completely encapsulate tbe individual sporangia and engulf tbe Anabaena wbicb is differentiating into akinetes. In mature megasporocarps tbe Anahaena cells are packaged into a cbamber near tbe indusial pore and a cluster of simple tricbomes occur external to tbe indusium around tbeir base.Tbe partner lobes of tbe developing sporangia form modified leaf cavities, Tbese cavities are smaller tban tbose in dorsal lobes not partnered witb sporangia and tbey bave a diminisbed complement of botb simple cavity tricbomes and Anahaena. Tbe Anahaena filaments in tbese modified cavities rapidly begin to differentiate akinetes rather tban beterocysts. Factors wbicb may contribute to akinete formation in tbe sporocarps and modified leaf cavities are discussed.