The free amino acid and ammonia pools of Azolla caroliniana were analyzed by quantitative column chromatography on columns capable of separating all of the nitrogenous constituents normally found in physiological fluids. Comparisons were made of plants containing symbiotic algae and grown on nitrogen-free media, plants grown on media containing nitrate, and algae-free plants also grown on nitrate media. The major feature of the data was a very high level of intracellular ammonia found in plants which coutain N2-flxing algal symbionts. In addition to the more usual amino acids, serine and cystathionine were found in the free amino acid pool.The genus Azolla, small aquatic ferns of worldwide distribution, generally contains symbiotic algae resembling Anabaena sp. within their leaf cavities (3). These algae are capable of fixing N2 and consequently can supply all of the nitrogenous nutrient requirements of the fern. The rapid growth of this fern and its ease in handling for laboratory studies make this plant particularly attractive as experimental material for study of plant nutrition in a symbiotic system. Relatively little work had been done with this system until Peters and Mayne (4, 5) recently made some initial characterizations of the relationship and showed the essential nature of the algae for growth of plants on N-free media.To provide a basis for more extensive studies of the N nutrition of this symbiosis, we have recently separated and quantitatively analyzed the components of the free nitrogenous pools in these ferns with and without algal symbionts, and the results are presented in this communication.
RESULTS AND DISCUSSIONTo provide as broad a base as possible for our comparisons, we have analyzed several dozen samples of different plant tissues of different ages. Plants were usually collected and immediately treated with boiling water to avoid deterioration which might alter pool contents or composition, and samples were either analyzed directly or stored frozen for subsequent analysis. In general, we found the pool size and composition to be rather uniform from one age of plant culture to another, provided the plants were grown under similar conditions. Data in Table I summarize the major effect we have noted in the pools and compares ammonia analyses obtained by microdiffusion and colorimetry and the chromatographic method. As expected, ammonia content determined by direct analysis of extracts was higher, probably due to decomposition of amides during analysis. It should be noted that the levels of ammonia found in N2-fixing plants are too high to be accounted for by hydrolysis of glutamine or any other pool constituent during sample preparation or analysis. The pool of intracellular ammonia in N2-fixing plants can rise to as much as five to 10 times that found in plants grown on media containing a N source, and can constitute as much as half of the total pool N. This effect is greatest when one compares algae-containing and algae-free plants. It should be noted that the growth rates of p...