Effect of Light Quality and Light Intensity and Various Sugars on the Sexual Expression and Some Observations on the Red Pigment in Equisetum Gametophytes

The purposes of this study were to explore what effect light quality and light intensity and various sugars have on the sexual expression of Eguisetum gametophytes and to determine some characteristics of the red pigment in Eguisetum gametophytes. Light quality and light intensity were employed to seek the presence of a morphogenetic factor involved in the sexual expression of Eguisetum gametophytes. The gametophytes were grown in mass culture in petri dishes and singly in test tubes under red light and white light or under high light intensity and low light intensity. Light quality was employed when the Eguisetum species was Eguisetum hyemale. When the species was Eguisetum arvense, light intensity was employed. A higher percentage of antheridial gametophytes in mass culture in petri dishes than singly in test tubes under either red light or high light intensity would indicate an interaction among the gametophytes due to a diffusible substance and thus would indicate that light quality or light intensity was probably involved in a mechanism which activated a morphogenetic factor determining the sex of Eguisetum gametophytes. In mass culture in petri dishes, the morphogenetic factor activated by either red light or high light intensity would influence the sexual expression of other gametophytes in the same petri dish; whereas, singly in test tubes the gametophytes are isolated from each other so that any morphogenetic factor that is produced could not influence the sexual expression of the gametophytes. The factor would probably be some sort of diffusible substance comparable to the antheridogens of ferns. The results were ambiguous. Mannitol, sucrose and glucose we~e added separately to Bold's basal medium to determine what effect these sugars have on the sexual expression of Eguisetwn gametophytes. The results were ambiguous. The red pigment often associated with Eguisetum antheridial production was isolated by column chromatography and some characteristics were determined using visible light spectrophotometry. It is definitely not rhodoxanthin, as reported, but could not be identified other than to be a carotenoid. This study showed that Eguisetum gametophytes are a difficult system with which to experiment. It seems the sensitivity of Eguisetwn spores to light quality and light intensity can vary.

INTRODUCTION Eguisetum, a vascular cryptogam, has a life cycle of two alternating generations, the sporophyte generation and the gametophyte generation.
The sporophyte generation bears the strobili that shed the spores. The gametophyte generation, the sexual phase, bears the gametangia (archegonia and antheridia) which form the gametes (egg and sperm).
Uncertainty over the nature of sexuality in Eguisetum gametophytes is seen in the morphology textbooks. Some authors (Bold, 1957;Campbell, 1913) have considered them to be unisexual. Other authors (Eames, 1936;Foster and Gifford, 1957;Haupt, 1953) have considered them bisexual. Hauke (1967) believes that Eguisetum gametophytes have the potential to be bisexual, but most gametophytes are unisexual and those that are bisexual are postmature and were once unisexual. There is only one species of Eguisetum which has bisexual gametophytes from the beginning (Hauke, 1963 and. Factors other than genetic seem to play an important role in the sexual expression in Eguiseturn gametophytes. Several authors (Campbell, 1913;Schratz, 1928;Walker, 1931;Eames, 1936;Williams, 1938;Haupt, 1953;Scott and Ingold, 1955;and Doyle, 1970) have reported crowded and unfavorable conditions increase antheridial formation in Eguisetum gametophytes. Factors such as nutritional deficiencies, hormonelike substances, and the accumulation of waste products may influence sexual expression in Eguiseturn gametophytes.
It may be that a hormonelike substance may control sexual expression in Equisetum gametophytes. Already antheridogens that control the forma-tion of antheridia have been found in fern gametophytes (D8pp, 1950;NHf, 1956NHf, , 1959NHf, , 1960NHf, and 1961Voeller, 1964). Because fern gametophytes are much like those of Eguisetum in that they are both photosynthetic, dorsiventral and terrestrial, substances comparable to those in ferns may be operating in Equisetum.
Recently Hauke (1971) has shown that light quality affected sexual expression in Eguisetum hyemale gametophytes and that light intensity affected the sexual expression in Eguisetum arvense gametophytes. One purpose of this study was to further explore the effect of light quality on sexual expression in Eguisetum hyemale gametophytes and of light intensity on Eguisetum arvense gametophytes. Hauke (1971) has shown that sucrose, a sugar, also affects the sexual expression in both of these species of Eguisetum. Another purpose of this study was to determine what effect sugars have on these species of Eguisetum gametophytes. Glucose, mannitol and sucrose were the sugars chosen for study. If the effect of these three sugars were the same, the effect would be assumed to be osmotic. The effect would be considered nutritional if mannitol showed no effect and the other sugars did. If only one of the sugars produced an effect, this would indicate some special morphogenetic effect.
A red pigment has been observed in Eguisetum gametophytes (Eames, 1936;Chatterjee and Ram, 1968). The red pigment is seen under the microscope in chromoplasts in cells adjacent to antheridia, so that it seems the red pigment is related to the sexual expression of Eguisetum gametophytes. The final purpose of this study was to determine some characteristics of this red pigment.

METHODS AND MATERIALS
Spores ~ inoculation of cultures. Eguisetum gametophytes were grown from spores on Bold's basal medium agar (BBMA) prepared f rom stock solutions (Bold, 1967 Cheesecloth was used to regulate light intensity but did not affect 1 ight quality. Two layers of cellophane "K" 210 FC Red from DuPont were The gametophytes were allowed to grow about six weeks before they were harvested to determine the percentage of antheridial gametophytes that formed. The percentage of archegonial gametophytes formed was not determined because of the difficulty in observing archegonia in Eguise-.E!fil gametophytes. Antheridial gametophytes were however identified without difficulty by observing antheridia under a dissecting microscope.
Whenever there was any doubt, the gametophyte was observed under a microscope using lOOX magnification to eliminate any uncertainty.
Those gametophytes that were not identified as antheridial gametophytes were considered either archegonial or neutral. containing the red pigment was treated in exactly the same manner as the original acetone homogenate and the resulting residue was taken up again in 3 ml of light petroleum ether and chromatogrammed as before, but using a column of aluminum oxide (activity of 2). The red band was eluted and the eluate was treated again in exactly the same manner as the original acetone homogenate and the dried purified residue was taken up again in 3 ml of light petroleum ether. An absorption curve was determined between 300 nm and 700 nm using a Cary recording model 15 spectrophotometer. This procedure is an adaptation from that of Jagels (1970) and Foppen (1969 Table 3. The first set of experiments showed that sucrose and glucose had no effect on the formation of antheridial gametophytes but that mannitol stimulated antheridial production. In the second set of experiments the results showed mannitol inhibited antheridial gametophyte development, but glucose had no effect.
The results from the third set of experiments showed glucose and sucrose increased the formation of antheridial gametophytes, but mannitol had no effect. The fourth set of experiments showed that glucose had no significant effect on the formation of antheridial gametophytes. In the fifth set of experiments the results showed that glucose, sucrose and mannitol all increased antheridial gametophyte formation. Finally the results from the last set of experiments showed that mannitol inhibited the formation of antheridial gametophytes, but glucose had no effect. It must be noted that contamination might have been a factor in these results,    and sucrose showed no effect on the sexual expression of Eguisetum hyemale gametophytes under 1,500 ergs/cm 2 /sec but did under 5,000 ergs/cm 2 /sec.
The results from experiments using light intensities of 15,000 and 20,000 2 ergs/cm /sec were not interpreted because the spores used in these experiments were either stored at -10 C in glycerol or obtained outside in freezing temperatures during the winter which might have affected the physiology of the spores.

2
The results under 1,500 ergs/cm /sec suggested the effect of glucose and sucrose is not nutritional because neither sugar showed an effect. 2 The effect of these two sugars under 5,000 ergs/cm /sec could not be determined to be either nutritional or osmotic because the effect of mannitol was inconsistant. In summary light intensity may have interacted with the various sugars or may have no effect, but at any rate the author interpreted the results both ways.
These experiments would have been repeated until a conclusion had been obtained, but there were no longer available viable spores. In repeating these experiments, light intensity should be kept constant and the sugars should be added to the agar medium in equal molarity concentrations, rather than equal weight to volume concentrations. The results of various sugars on sexual expression in Eguisetum arvense gametophytes showed that the effect of mannitol, which produced an effect under high light intensity, is osmotic and not nutritional because mannitol is not metabolizable by many plants, but sucrose, which produced no effect, is. It appears light intensity interacts with this osmotic effect of mannitol because mannitol showed the effect under high light intensity but not under low light intensity. The ef f ect of glucose is at least partly osmotic because glucose is also, like mannitol, a monosaccharide and not like sucrose, a disaccharide, but the effect is only partly osmotic because glucose had a greater effect than mannitol and some other additional factor must be involved in the effect of glucose. Because glucose showed a much greater effect than mannitol, this additional factor may be a direct morphogenetic one.
It is interesting to note that Wollersheim (19S7) observed a higher percentage of archegonial gametopbytes using Eguisetum fluviatile with a medium supplemented with glucose, but the author observed instead a higher percentage of antheridial gametophytes using Equisetum arvense.
It appears a different mechanism is involved in the effect of glucose on these two species of Eguisetum.
These experiments were done only once because there were no longer available viable spores from Eguisetum arvense. In further experiments sugars should be added to the nutrient medium in equal molarity concentrtions rather than equal weight to volume concentrations.
Red pigment isolated f!:2m Eguisetum. The absorption spectrum of the red pigment in light petroleum ether from both Eguisetum gametophytes and Eguisetum sporophytes indicated that the pigment was a carotenoid but not rhodoxanthin. Rhodoxanthin in light petroleum ether has a different absorption spectrum than the red pigment in the same solvent. The red pigment has absorption peaks at 4S2, 47S and SOS nm in light petroleum ether and rhodoxanthin has peaks at 4S6, 487 and S21 nm in the same solvent (Karrer and Jucker, 1950). Lippmaa (1926a, b and c) had reported the red pigment to be rhodoxanthin in a few Eguisetum sporophytes. He had apparently identified and named the pigment rhodoxanthin just by looking at a few drawings by Schimper (189S) of chromoplasts containing this red pigment in Eguisetum telmatia sporophytes, for he gave no indication in his article of having extracted the pigment from Eguisetum.
The red pigment cannot be considered another els-trans form of rhodoxanthin because unlike rhodoxanthin the red pigment was not reduced by NaBH 4 • This red pigment also has a different partition coefficient value than rhodoxanthin in light petroleum and 953 methanol. The partition coefficient value for the red pigment was determined to be 0:100. Quackenbush (1965) has determined the partition value in hexane and 953 methanol to be 55:45 for rhodoxanthin.
The red pigment could not be identified for the author could not find a red pigment in the literature that had an absorption in light petroleum ether similar to the absorption spectrum of this red pigment in the same solvent. It is possible the red pigment had not yet been identified.
Nuclear magnetic resonance spectrum and infrared absorption spectrum would be the next steps to take in identifying the pigment, but because of the scope of the problem, amount of pure pigment required and equipment required, these steps were not taken.
Under the microscope the . red pigment is found in chromoplasts in cells adjacent to the antheridia. It is possible the red pigment is related to the sexual development of Eguisetum antheridial gametophytes.

CONCLUSIONS
This study showed that working with Equisetum gametophytes is difficult. However~ this study did show that glucose promoted an increase in number of antheridial gametophytes in Eguisetum arvense and that the red pigment observed in Eguisetum is not rhodoxanthin as reported in the literature but some other red carotenoid not yet identified.