PROCEEDINGS OF THE OKLAHOMA ACADEMY OF SCIENCE Volume 56—1976

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THE RESPONSE OF A CYLINDROSPERMUM SPECIES TO DIFFERENT SOURCES OF NITROGEN

Lawrence A. Kapustka* and James R. Rosowski

The Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma, and The School of Life Sciences, University of Nebraska, Lincoln, Nebraska

Cylindrospermum sp. (IUCC 942) was grown for six weeks in a modified Bristol's solution that supplied N in different oxidation states and concentrations. The occurrence and position of heterocysts, the criteria used to identify this genus, were shown to be dependent on the source and concentration of N. Vegetative cell to heterocyst ratios increased as the N concentration increased. No heterocysts were formed in high concentrations of NH₄⁺-N. Intercalary heterocysts were common in young cultures.

C₂H₂ reduction was used to estimate the N₂-fixing capacity of the alga. Cells grown with NH₄⁺-N at concentrations of 40 ppm and greater had no N₂-fixing activity, while cells with NO₃¯-N had activity even at 400 ppm.

The physiological and morphological responses to changes in N indicate the need for additional work on the taxonomy of the Nostocaceae.

The heterocyst, a specialized cell in the trichomes of the Nostocaceae, is distinguished from vegetative cells by its different size, shape, color, and function (1, 2). The position of the heterocyst in the trichome is of taxonomic importance at the generic level (3, 4, 5). The physiological function of the heterocyst has been debated extensively. Several major roles have been suggested. Among these are the regulation of cell division and growth of the trichome and N₂ fixation (1, 2, 6, 7).

The possibility for regulation of the activities of the trichome was supported by the demonstration of cytoplasmic connections between heterocysts and vegetative cells (8, 9). Wolk (10) reported a rather constant number of vegetative cells separating spores and suggested the presence of a gradient of some substance that somehow regulates the differentiation of vegetative cells.

The presence of nitrogenous compounds has been shown to affect the formation of heterocysts. Anabaena cylindrica had fewer heterocysts when grown on NO₃¯-N or NH₄⁺-N than when grown with only atmospheric N₂ (11, 12, 13). Anabaena flos-aquae A-37 responded in the same manner to NO₃¯-N but in the presence of NH₄⁺-N, heterocyst formation was completely inhibited (14). Likewise, Cylindrospermum trichotosporum grown on media with NH₄Cl or (NH₄) ₃PO₄ at concentrations of 3mg/ml did not form heterocysts (15).

The isolation of mutant strains of Cylindrospermum that lack heterocysts has led to the identification of two categories of genetic regulation of heterocyst formation (16). The first category is an inducible-repressible system whereby combined N represses heterocyst formation. The other category is constitutive in that combined N does not affect the development of heterocysts, as in C. majus and Anabaenopis raciborskii (16).

Cylindrospermum sp. (IUCC 942) may be atypical for the genus in that it contains a high frequency of intercalary heterocysts in young cultures. In this paper we describe the pattern of formation of intercalary heterocysts and examine the effect of N supplied in different oxidation states and concentrations with respect to heterocyst formation and N₂ fixation.

Cylindrospermum sp. (IUCC 942) was grown in soil-water medium and in Bristol's medium (17) modified to provide N in the following concentrations and oxidation states: atmospheric N₂; 40, 80, and 400 ppm NO₃¯-N; 40, 80, and 400 ppm NO₃¯-N; 40, 80, and 400 ppm NH₄⁺-N; and 40, 80, and 400 ppm N as NH₄NO₃. The stock culture was examined for N₂-fixing contaminants.

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Aliquots of the stock culture were plated on N-free Bristol's, N-free Burk's minimal salts (18) and modified Line-Loutit (19) media and incubated in aerobic and anaerobic atmospheres. No growth, other than that of Cylindrospermum on Bristol's medium, was observed in any of the conditions.

One hundred ml of medium were placed in each of two 250-ml Erlenmeyer flasks per treatment. Each flask was inoculated with a 2-ml cell suspension from a stock culture that was previously conditioned to grow on N-free medium. The cultures were maintained on a 12-hour photoperiod at approximately 3000 lux illumination at 21 C. After 6 weeks a portion of the algal mass was removed from the medium with a wire transfer loop and placed on a slide for microscopic examination. Twenty-five trichomes on each slide which could be observed the entire length of the filament were selected for the counting of vegetative cells and heterocysts.

The N₂-fixing capacity of this Cylindrospermum species was examined also. Bristol's medium having NH₄⁺-N at 0, 5, 10, 20, 40, 80, and 400 ppm and NO₃¯-N at the same concentrations were used. Two 250-ml Erlenmeyer flasks containing 100 ml of medium received a 2-ml cell suspension as described earlier. After 1 week of growth the cells from each concentration were collected on a filter pad and returned to 5 ml of the medium for determination of N₂ase activity. The C₂H₂-reduction assay of Hardy et al. (20) as modified by Stutz and Bliss (21) was used to estimate the N2-fixing capacity of the cells. The cells were incubated for 24 hr in 0.1 atmosphere C₂H₂. Sampling at various times indicated a linear response through 24 hours. C₂H₄ content was measured with a Becker 409 gas chromatograph equipped with a 3 mm × 3 m column containing Porapak N.

General observations on the morphology of our cultures in soil-water medium have been made over the past four years.

The following features of the life cycle of Cylindrospermum sp. (IUCC 942) were observed in soil-water cultures. Perhaps unusual for Cylindrospermum is our observation that intercalary heterocysts are a regular feature of young cultures. The greatest development of intercalary heterocysts occurs in cultures which are about two weeks old and continues until the cultures are a month old. At that time akinete formation begins next to the terminal heterocyst and the formation of intercalary heterocysts slows down. After 2 months, no intercalary heterocysts can be found, although akinete development continues. We do not know when akinete germination first occurs but we have had excellent germination of akinetes in cultures up to 1½ years old. The germling is a single elongated cell which divides, eventually giving rise to a filament with a single terminal heterocyst. A second terminal heterocyst develops on the opposite end of some filaments but such old cultures appear never to have intercalary heterocysts.

Figure 1 presents the developmental sequence for heterocyst formation in the young cultures used in this study. The filament initially has a single terminal heterocyst. As the filament elongates beyond some critical length, which varies with the different sources and amounts of nitrogen, a second heterocyst develops on the end opposite the initial heterocyst. Further elongation of the filament increases the

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distance between the heterocysts and the vegetative cells in the middle of the filament, resulting in the formation of an intercalary heterocyst. The intercalary heterocyst easily separates from the adjacent cells, resulting in two filaments, one with a single terminal heterocyst and one with two terminal heterocysts. Reddy and Talpasayi (15) described a similar sequence for Cylindrospermum trichotosporum except that no intercalary heterocysts were formed in their cultures.

The response to the various sources of N (Table 1) was quite dramatic. The vegetative cell: heterocyst ratio increased as the amount of combined N in the medium increased. It was not possible to make accurate determinations of cell numbers for the NH₄⁺-N media because the growth habit was greatly affected. Filaments grown in the presence of NH₄⁺-N were coiled and twisted making it impossible to trace the entire length of the filament. Filament length was estimated at about 100 vegetative cells/heterocyst in the 40 and 80 ppm NH₄⁺-N media and about 200 vegetative cells/heterocyst for the 40 and 80 ppm NH₄NO₃ -N.

The most striking morphological response was in the 400 ppm NH₄⁺-N and 400 ppm NH4NO3-N media. In addition to the coiling nature there was an obvious absence of heterocysts. The few heterocysts that were found were probably introduced in the initial transfer. No heterocysts were found in the areas of new growth.

C₂H₂ reduction by the cells grown on NH₄⁺-N (Figure 2) indicates N₂ase activity is completely suppressed at concentrations of 40 ppm NH₄⁺-N and above. Nitrate (Figure 2), however, did not cause this suppression. Even at concentrations of 400 ppm NO₃¯-N there was considerable C₂H₂ reduction. These data indicate that Cylindrospermum sp. 942, unlike C. majus, has the inducible-repressible genetic control of heterocyst formation and N₂ fixation described by Singh (16).

Finally the phenotypic plasticity of heterocystic blue-green algae with respect to the presence of the heterocyst and even the position in the trichome exhibits a wide latitude of responses. Cylindrospermum sp. (IUCC 942), as reported here, forms heterocysts in response to N in the medium, with NH₄⁺-N having the greatest effect. The genus Cylindrospermum is variable, having species without heterocysts (15), with only terminal heterocysts (4, 5), and with intercalary heterocysts. This variability along with the responses to N for Cylindrospermum sp. (IUCC 942) and for Anabaena flos-aquae A-37 (13) indicates the need for a critical examination of Nostocaceae in culture to provide more reliable taxonomic characters.

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