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).

A new procedure is reported for high-yield isolation of guard cell protoplasts from Viia faba L. Delayed Light emission and P700 content plus absorption and fluorescence emission spectra of these protoplast extracts are reported. It is concluded that both photosystems are present. The presence of photosystem II 12Step 2. The leaf pieces were infitrated under reduced pressure with enzymic digestion medium consisting of 150 ml 0.3 M mannitol, 10 mm sodium ascorbate, 10 mm CaCl2, 4% (w/v) Cellulysin (pH 5.5). Incubation was for 1 h at 30 C in a 1-liter flask in a shaker bath (60 5-cm excursions/min).Step 3. One hundred fifty ml 0.7 M mannitol was added and incubation was continued for 1 h.Step 4. Digestion was interrupted by pouring the material over a 295-,um screen (Nitex) and washing with 0.7 M mannitol containing 10 mm sodium ascorbate. Epidermal strips (coming from both surfaces) and vein nets retained on the screen were transferred to 0.7 M mannitol containing 10 mM sodium ascorbate. Then the epidermal strips were manually separated from the vein nets and transferred to a separate container. The strips had few adhering mesophyll cells, but epidermal cells were intact.In some experiments, mesophyll cells which passed through the screen were carried through steps similar to the remainder of the isolation procedure and used as controls.Step 5. Digestion was continued for I h as in Step 2 except (a) mannitol was increased to 0.7 M, (b) volume was reduced to 100 ml, and (c) shaker-bath speed was reduced to 30 excursions/min.Step 6. The strips were collected and washed as in Step 4. If microscopic examination showed contaminating cells, Steps 5 and 6 were repeated.Step 7. Digestion was continued until guard cell protoplasts were released (about 3 h). These protoplasts were isolated by passage through a cascade of screens (nominally 295, 166, and 20 pm) and collected by centrifugation (lSOOg, 5 min). The protoplasts were washed three times with 0.7 M mannitol containing 10 mM sodium ascorbate and examined for purity.Comments. Isolation of guard-cell protoplasts has been reported (25,32, 43

No abstract

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

No abstract

No abstract

Biomass increase, C and N content, C2H2 reduction, percentage dry weight and chlorophyll a/b ratios were determined for clones of Azolla caroliniana Willd., A.filiculoides Lam., A. mexicana Presl., and A. pinnata R.Br. as a function of nutrient solution, pH, temperature, photoperiod, and light intensity in controlled environment studies. These studies were supplemented by a glasshouse study.

Measurements were made of the 3.7 msec delayed light emission of chloroplasts treated with a variety of agents which affect the rate of electron transport (Hill reaction) or photosynthetic phosphorylation. The presence of the electron acceptors ferricyanide or pyocyanine increased delayed light emission. Inhibitors of electron transport (3-(3,4dichloro-pheny1)-1, -I-dimethylurea or l,lO(ortho)-penanthroline) inhibited delayed light emission. Uncouplers of photosynthetic phosphorylation caused a decrease in delayed light emission. The addition of a phosphate acceptor system inhibited delayed light emission. This inhibition was reversed by inhibitors of the phosphorylation reaction, e.g. Dio-9 or phlorizin. From these results it was concluded that the 3.7 msec delayed light emission probably occurs as a result of back reactions of intermediates in the coupled electron transport and photosynthetic phosphorylation systems. I N T R O D U C T I O N IN 1951 Strehler and Arnold(') observed a delayed luminescence of an alga. This luminescence had a spectrum corresponding to the fluorescence of chlorophyll in v~v o , (~) indicating the formation in the dark of the first excited singlet state via some back reaction.To explain the phenomenon, Arnold and Sherwood(s) proposed a semiconductor type mechanism in which the luminescence resulted from a recombination of electrons and holes within the grana of the chloroplasts, which they had shown to have semiconductor-like properties. Strehler,(4) on the other hand, preferred a mechanism based on the backreactions of discrete chemical intermediates formed in the light.Albrecht et d,@) Goedheer@) and Bertsch(') have demonstrated the interaction of the two photosystems(*) of green plants in the phenomenon of delayed light emission (DLE).? Bertsch and Bishop(Q) have shown that a Scenedesmus mutant which lacks photosystem I1 has a greatly decreased DLE, while a mutant which lacks photosystem I has an increase in the rapid DLE. Clayton and Bertschc'O) reported that a mutant Rhodopseudomoms spheuoides, which lacks reaction centers and is unable to perform any of the usual photochemistry, has its steady state DLE reduced at least 300 fold compared to the wild type, indicating the necessity of an intact photosystem for normal DLE.The objective of this paper is to present some experimental results which relate the rapid (3.7 msec) delayed light emission to back reactions of electron transport components coupled to the phosphorylation system. *Contribution No. 256 of the Charles F. Kettering Research Laboratory, Yellow Springs, Ohio, U.S.A. t Abbreviations : DLE-delayedlight emission; DCMU-3-(3,4-dichlorophenyl)-lt -1-dimethylurea; CCCP-189 m-chlorocarbonyl cyanide phenylhydrazone; tricine-N-Tris (hydroxymethy1)-methyglycine.