Characteristics of CO2 output patterns of axenic cultures of Lemna perpusilla are being sought that would serve as biochemically definable indicators of the crucial photoperiodic events during inductive light/dark schedules, using cycles with skeleton main light periods and overall periodicities of 24 hr. As the length of the main light period is increased over the range [4][5][6][7][8][9][10] Carbon dioxide output was measured as described earlier, except that 50 ml of medium in 125-ml Erlenmeyer flasks was used instead of 100 ml in bottles (6). After 4-6 days under the conditions being tested, times of maximal and minimal output were estimated to the nearest 0.5 hr by counting the points (at 0.5-hr intervals) on the strip chart records. Maximal or minimal plateaus lasting 3 hr or less are assigned to the time of their midpoints; those lasting longer, to the time of their termination. On both nitrate and ammonium media, the maximum referred to is the second of the two daily peaks observable under schedules with main light periods shorter than about 8 hr and the only one remaining with longer light periods (6). In order to minimize temperature changes and direct light effects on CO2 flux, main light periods in all experiments were substituted by skeleton photoperiods in which 3 hr or less of darkness separated successive brief light exposures. Thus, to simulate main light periods of various lengths, 0.25-hr light exposures were given, starting at the indicated times, with hr 0.0 taken as the start-4 hr: 0, 1.75, 2.75, 3.75; 6 hr: 0, 1.75, 3.75, 5.75; 8 hr: 0, 3.25, 6.5, 7.75; and 10 hr: 0, 3.25, 6.5, 9.75. Light in all experiments was red, at an incidence in the range of 10-35 ,uW X cm2, obtained by screening cool white fluorescent tubes with Rohm and Haas Plexiglas, color red 2444 (11). No net CO2 uptake is observed at any time under these conditions, and the new fronds produced in them are yellow-white.
RESULTSGrowth in all the media described is excellent. The rate of flowering is not the same in all, but experiments summarized elsewhere (12) show no effects of nitrogen source on the critical daylength, as estimated from the relative response to schedules with skeleton main light periods equivalent to 10, 12, 14, or 16 hr. A survey of CO2 output patterns entrained under the same set of conditions (12) indicated that the time between the daily maximum on N03-medium and the nearest light exposure decreases sharply with increasing daylength in the range used, as does flowering (9). Parallel results were obtained on NH4+ medium, while no feature of CO2 output on aspartate showed any apparent relation to photoperiodic response. A more rigorous test of the N03-maximum as a photoperiodic indicator thus seemed in order.501