May 30, 1979 - fern Todea barbara grown in sterile culture were measured using an infrared gas analyzer. Sporophytes consisted of single whole plants with.
Plant Physiol. (1980) 65, 584-586 0032-0889/80/65/0584/03/$00.50/0
Photosynthetic Rates of Sporophytes and Gametophytes of the Fern, Todea barbara Received for publication May 30, 1979 and in revised form October 15, 1979
WILLIAM G. HAGAR AND JOHN A. FREEBERG Department of Biology, University of Massachusetts, Boston, Massachusetts 02125 ABSTRACT
paring sporophyte and gametophyte photosynthesis by using cultured sporophytes and gametophytes in sterile tube culture where conditions can be controlled and the two life cycle stages compared under identical conditions. The entire plant is used in both gametophyte and sporophyte gas exchange measurements, thereby making the comparison of the two more physiologically meaningful in the sense that the root system and stem are included as an integral part of the sporophyte plant being measured.
The photosynthetic rates of intact sporophytes or gametophytes of the fern Todea barbara grown in sterile culture were measured using an infrared gas analyzer. Sporophytes consisted of single whole plants with roots and leaves grown in tubes of agar. Gametophytes were grown as several plants covering the surface of the agar. Sporophytes had photosynthetic rates at light saturation of 8.50 microliters CO2 per hour per milligram dry weight and 1,300 microliters CO2 per hour per milligram chlorophyll, whereas rates for gametophytes were lower, 2.36 microliters CO2 per hour per milligram dry weight and 236 microliters CO2 per hour per milligram chlorophyll. 02 inhibited the photosynthetic rates of both plants to a degree typical of C-3 plants. The light-saturated photosynthetic rates of sporophytes increased 42% and gametophytes increased 27% when the 02 concentration was changed from 21 to 1%.
MATERIALS AND METHODS
Fems, in contrast to seed plants, have two independent phases in their life cycle. The conspicuous sporophyte plant alternates with a less obvious gametophyte. Both gametophyte and sporophyte are free-living and capable of photosynthesis. They are, however, very different in morphology and one might expect differences in photosynthetic rate even though both have basically the same genetic information. Gametophytes resemble algae in their thalloid structure. They are essentially a single sheet of cells except for a thickened central cushion bearing sex organs and rhizoids. The cells contain chloroplasts and the enzyme systems necessary for photosynthesis. Virtually every cell in the sheet is in contact with the atmosphere. Sporophytes have well developed leaf structure with stomata and intercellular air spaces, as well as the stem and root system characteristic of higher plants. Comparisons of the photosynthetic rates of sporophytes and gametophytes of the same fern species using intact plants have not previously been made. One reason for this is the vast difference in size between the two and the difficulty of comparing the entire sporophyte with the much smaller gametophyte. Photosynthetic rates of gametophytic tissue at different levels of ploidy (IN, 2N, 4N) have been investigated in a polyploid series of gametophytes (3) and the photosynthetic rate was found not to depend on gene dosage. Comparisons of sporophyte photosynthetic rates using CO2 uptake have been made on a fresh weight basis among detached sporophyte leaves of ferns from various environments in Singapore and Malaysia. Photosynthetic rates of the detached leaves of sun and shade fern sporophytes were found to be similar to those of higher plants (5). Photosynthetic rates of gametophytes and detached sporophytic leaflets of the Hawaiian tree fern, Cibotium glaucum have been examined, but not compared (4). We have overcome some of the attendant difficulties of com-
Cultures of Todea barbara L., originally obtained from R. H. Wetmore, were grown on a medium (7) consisting of 6.25 mm NH4NO3, 1.47 mm KH2PO4, 0.81 mM MgSO4, and 0.51 mm CaCl2, supplemented with 0.033 mm ferric citrate, 1 ml/l trace element solution (8), and 0.8% (w/v) agar (pH 5.5). Sporophytes and gametophytes were separated from mixed cultures, grown for several weeks, and transferred to slightly slanted tubes of fresh medium where they were allowed to grow for at least 2 weeks before measurements were taken. Gametophytes in sterile culture are similar in size and morphology to those in nature. Cultured sporophytes are much smaller than naturally occurring plants and their leaves tend to be juvenile in form. However, they have roots, stems, and leaves in roughly the same proportion as naturally occurring sporophytes. Sporophyte tubes contained one complete plant with roots and six to eight leaves, the largest leaf being 3-4 cm in length. Gametophyte tubes contained several gametophytes which covered the surface of the agar. Cultures were maintained at 22 C and a combination of cool-white fluorescent and incandescent lamps at an intensity of 160 ,uE m-2 s-' was used for illumination on a 12-h on, 12-h off cycle. Cultures were grown in 25-mm tubes in which the agar surface was slanted so that they could be illuminated at the same angle during growth and during the measurement of photosynthesis. Photosynthetic rates were determined by measuring the change in CO2 concentration in a tank air mixture (0.033% CO2, 21% 02, Balance N2) flowing through the cultures before and after exposure of the cultures to light. Six glass culture tubes containing sporophytes or gametophytes were connected in series by glass and Tygon tubing. These culture tubes were arranged in a circle at a slight angle on a styrofoam holder to allow illumination perpendicular to the slanted culture surface. A photocell was placed in the center of the circle to measure light intensity. Water-saturated air was passed through the culture tubes, and the concentration of CO2 was monitored continously using a Beckman IR CO2 analyzer (model 865). Water vapor was measured using an EG&G dew point hygrometer, and CO2 readings were corrected for the amount of water vapor present. Corrections for water vapor were made by subtracting the offset caused by the IR A of water vapor from the CO2 concentration readings. The same changes in CO2 concentration were also found when the water vapor was removed before the gas analyzer using a drying system consisting of a 0.6 1-m glass tube filled with anhydrous CaSO4. In all of these experiments care
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Sporophytes
Sporophytes
01
E
11 u
0 0
C14
- -
-
Gametophytes
~~Gametophytes
100 Quantum Flux (uE m-2
200
700
-1)
FIG. 1. Photosynthetic rates on a mg dry weight basis of sporophyte (0) and gametophyte (0) cultures of T. barbara versus light energy. Experimental points are average values from seven separate comparisons of sporophytes and gametophytes. Average dry weight of sporophyte cultures was 0.122 g, whereas that of the gametophytes was 0.166 g. Error expressed is SE of mean.
was taken to ensure that the flowing CO2 was not limiting the photosynthesis rate at any light intensity. The CO2 concentration in the last tube was never lower than 50 ,il/l below atmospheric (280 1d/1). The flow rate of the air was monitored using a Hastings mass flow meter. All of the monitoring devices were connected in series with the fern cultures, and all measurements were made at the same time. The cultures were illuminated from above using a high intensity quartz iodide heat-free light system (Bodkin Optics). Different light intensities were obtained by using screen attenuators. PAR was measured using a calibrated quantum sensor (Lambda, model Li 185A). Dry weight and Chl content of the cultures were determined at the completion of the photosynthetic measurements. Dry weights were obtained after drying tissue in a drying oven at 70 C to a constant weight. Chl was extracted from gametophytes and entire sporophytes by grinding in 100%o acetone in a mortar. After several extractions, the solution was diluted with water to 80%Yo acetone and centrifuged. The amount of Chl present was determined spectroscopically (2, 6).
RESULTS AND DISCUSSION The photosynthetic rates of sporophytes were always much higher than gametophytes, whether expressed on a dry weight or ChM basis. Light compensation points were between 15 and 40 JAE m-2 s-1 for both sporophytes and gametophytes. Gametophytes reached light saturation at a lower intensity than did the sporophytes and had a much lower maximum photosynthetic rate. Figure 1 is a plot of the photosynthetic rates of sporophytes and gametophytes versus light intensity. Sporophytes had higher photosynthetic rates at all light intensities. Even greater differences are seen in Figure 2 in which photosynthetic rates are plotted on a Chl basis versus light intensity, because the amount of Chl per dry weight was higher in gametophytes (10 ,ug ChM/mg dry weight) than sporophytes (6.53 jig Chl/mg dry weight). Table I is a listing of the photosynthetic and respiratory rates of the fern cultures. Sporophytes had light-saturated photosynthetic rates five times greater than their dark respiration rates. Similar ratios of 5:1 can be calculated from data published by Hew and Wong (5) in their
l00
200
'I
700
Quantum Flux (fIE mn2 s-i FIG. 2. Photosynthetic rates on a mg Chl basis of sporophyte (0) and gametophyte (0) cultures of T. barbara versus light energy. Conditions are the same as in Figure 1.
Table I. Photosynthetic and Respiratory Rates The ug Chl/mg dry wt content was 6.53 ± 0.16 and 10 ± 0.74 in the sporophytes and gametophytes, respectively.
Rates
Sporophytes
Photosynthetic (ul CO2 h-1 mg dry wt-1; 8.49 ± 0.15 corrected for dark respiration) Dark respiration (U1 CO2 h-' mg dry wt-') 1.67 ± 0.18 5.08/1 Photosynthetic/Respiration
Gameto-
phytes 2.36 + 0.29 1.35 ± 0.41
1.75/1
work with sun and shade sporophyte leaves. The ratio for gametophytes in our work was only 1.75:1, indicating a lower photosynthetic capacity. 02 inhibited the photosynthetic rates of both plants to a degree typical of C-3 plants (1). The light-saturated photosynthetic rate of sporophytes increased 42 ± 4% when the 02 concentration was changed from 21 to 1%. Gametophytes showed a similar increase in their light-saturated photosynthetic rate of 27 ± 3% when the 02 concentration was changed from 21 to 1%. Conditions of growth seemed to be important in the extent of high light 02 inhibition of photosynthesis. In preliminary measurements gametophyte cultures grown under higher light intensities had smaller increases in their light-saturated maximum photosynthetic rates when the 02 concentration was changed from 21 to 1%. Ferns offer an opportunity to study photosynthesis in plants that are genetically comparable, but morphologically different. Sporophytes are more effective photosynthetically even though when compared with gametophytes a smaller proportion of the sporophytes' cells are actually working directly in photosynthesis. Whether this difference in photosynthetic effectiveness can be attributed entirely to morphological differences or to differences in enzyme concentrations or activities remains to be considered. LITERATURE CITED
1. ANDREws TJ, GH LoRsume 1978 Photorespiration-still unavoidable? FEBS Lett 90: 1-9 2. ARNON D 1949 Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol 24: 1-15 3. DEMAGGIO AE, DA STETR 1971 Polyploidy and gene dosage effects on chloroplasts of fern gametophytes. Exp Cell Res 67: 287-294 4. FRamo DIV 1975 Adaptation and adjustment of photosynthetic characteristics
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of gametophytes and sporophytes of Hawaiian tree-fern (Cibotium glaucum) grown at different irradiances. Photosynthetica 9: 157-174 5. HEW CS, SC WONG 1974 Photosynthesis and respiration of ferns in relation to their habitat. Am Fern J 64: 40-48 6. MACKiNNEY G 1941 Absorption of light by chlorophyll solutions. J Biol Chem
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140: 315-322 7. MooRE GT 1903 Methods for growing pure cultures of algae. J Appl Microsc Lab Methods 6: 2309-2314 8. NITSCH JP 1951 Growth and development in vitro of excised ovaries. Am J Bot
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