Nickel and the metabolism of urea by Lemna paucicostata Hegelm. 6746.

With urea as sole nitrogen source, the addition of 5×10(-5) M nickel sulfate to axenic cultures of Lemna paucicostata 6746 approximately doubles the rate of vegetative growth. Under a standard light-dark schedule, Ni(2+) changes the daily pattern of respiratory CO2 output on urea from one having a single daily peak to one with two daily peaks which resembles that on ammonium or nitrate as sole nitrogen source. It also increases CO2 output by as much as 3-fold on a fresh-weight basis. These data represent the first confirmation in an intact higher plant of proposals, based on enzymology and tissue culture responses, for a role of Ni(2+) in urea metabolism. Further, they indicate the possible existence of two distinct pathways of urea utilization.

Calibrating duckweeds: light, clocks, metabolism, flowering.

The roles of photoperiodism and related light-dependent and hormonal processes in plants are not well understood. Rapid growth, aquatic habit, and adaptability to axenic culture make the Lemnaceae, or duckweeds, excellent material for investigating these topics and others in which highly defined conditions or the presence of organic substances are crucial. As a major example among several that are described, recent work with one species suggests a relation between some features of carbon dioxide flux and the photoperiodic timing mechanism, thus providing a system in which the biochemical basis of the latter may be explored.

A metabolic indicator of photoperiodic timing.

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 4-10 hr, the daily times of maximal output on nitrate or ammonium media shift about 2.5 hr; there is a precisely parallel shift in the time of maximal photoperiodic sensitivity of flowering to a night interruption. These times do not depend on either a "drawn" or a "dusk" signal alone, but are functions of both. The time of maximal daily CO2 output on aspartate medium reflects only the dawn signal, suggesting that the display of some component of CO2 flux coupled to the photoperiodic timing mechanism can be controlled by modifying nitrogen metabolism.

Photoperiodic Entrainment Patterns in the CO(2) Output of Lemna perpusilla 6746 and of Several Other Lemnaceae.

The CO(2) output of Lemna perpusilla 6746 in "skeleton photoperiods" consisting of alternating 10(1/2)-hour and 13-hour dark periods separated by (1/4)-hour illuminations was recorded under stable high and low nitrate conditions. The phase relationship finally attained between light schedule and output is the same regardless of which dark period is given first, but entrainment is more rapid (as is flowering) with an initial 13-hour dark period. In all respects other than bistability-the assumption of two different stable phase relationships depending on the initial dark period-both flowering and the course of CO(2) output conform to Pittendrigh's model derived from Drosophila eclosion rhythms, confirming the view that an endogenous circadian rhythm, or biological clock, underlies the photoperiodic control of flowering in this plant. Experiments with rigorous temperature control show that earlier results with long light exposures were in part due to temperature changes; in consequence, it is clear that entrainment patterns with high nitrate differ even more from those in low nitrate than was previously evident, and not simply by the addition of a "nitrate peak." Other Lemnaceae tested with a few simple light-dark schedules in both types of media show a variety of responses, with no obvious correlation to photoperiodic response type.

Factors Affecting the Water-sensitive Phase of Flowering in the Short Day Plant Lemna perpusilla.

The flowering of Lemna perpusilla strain 6746 is inhibited by daily transfers to water for short periods during a sensitive phase. Supplementing the water with Ca(NO(3))(2) partially reverses the inhibition of flowering while MgSO(4) increases the inhibition. The inhibition by MgSO(4) is overcome by low concentrations of Ca(NO(3))(2). Flower-promoting activity was detected in water and in MgSO(4) solutions that had been incubated with plants under dark but not light conditions. The prevention of this effect by light appears to be photosynthetic rather than to depend on phytochrome. The activity is destroyed by autoclaving but not by brief boiling. This loss of a flower-promoting material may explain the inhibiting effect on flowering by transfers to water.

Entrainment of Lemna CO(2) Output Through Phytochrome.

The entrainability of Lemna perpusilla CO(2) output by periodic 15 minute red (R) and far red (F) illuminations was tested in low nitrate medium. R every 8 hour, symbolized R/R/R, gives a flat output (no entrainment) as does F/F/F. However, R/-/- (R every 24 hour) entrains rapidly, and F/-/- does so as well, in a similar manner. The effects of R/R/- and F/F/- also resemble each other closely. Entrainment by R/F/F or R/R/F is rapid and indifferent to order of presentation, e.g., R/F/F and F/R/F lead to the same steady state. Typical phytochrome reversals occur, e.g., R,F/F/F holds output flat, while F,R/F/F entrains in the manner of R/F/F. Blue (B) light acts like R in schedules such as B/F/F but like F in schedules such as B/R/R. In all schedules studied, the zeitgeber (primary synchronizer) appears to be the sharpest transition from a low to a high level of far red-absorbing phytochrome that occurs with a 24-hr periodicity. Thus in entrainment, and by inference in photoperiodic timing, the level of far red-absorbing phytochrome at any time may be less significant than the succession of levels of which it is a part, a conclusion that implies the existence of a "scanning" mechanism that compares levels of far red-absorbing phytochrome at various times of day.

Red and far red effects on phenylalanine ammonia-lyase in raphanus and sinapis seedlings do not correlate with phytochrome spectrophotometry.

In seedlings of Raphanus sativus (radish) and Sinapis alba (mustard), irradiation for 6 hours with far red light significantly increases the extractable activity of phenylalanine ammonia-lyase by the end of the light period. A schedule of 10 minutes red light-110 minutes darkness-10 minutes red-110 minutes darkness-10 minutes red-110 minutes darkness has no effect as compared to dark controls. However, the red light program maintains a level of far red-absorbing phytochrome always measurable by in vivo spectrophotometry during the 6-hour experimental period. We conclude that the far red effect on this enzyme and for this specific material cannot be explained solely by formation and maintenance of far red-absorbing phytochrome.

Nitrate and the Course of Lemna perpusilla Carbon Dioxide Output under Daily Photoperiodic Cycles.

The carbon dioxide output of Lemna perpusilla 6746 on modified Hutner's media under light (dark) cycles of 8 (16) hours is entrained in either of two forms depending on the level of nitrate. With nitrate low or absent, the peak occurs shortly after the start of each dark period, and the minimum, shortly after the start of each light period. With high (about 10 mm) nitrate, an additional or sole major peak occurs about 17 hours after the start of each light period; the minimum does not shift. This generalization probably also holds for cycles at least from 3 (21) to 18 (6). Apparent slow or unstable entrainment in some earlier data was undoubtedly a result of the progressive lowering of nitrate in the medium and, thus, of the nitrate peak. Future work under stable conditions, with or without the nitrate peak, should make possible the more accurate testing of models of entrainment and hypotheses concerning photoperiodic timing in this and related systems.

Response of Lemma perpusilla to Periodic Transfer to Distilled Water.

The flowering of Lemma perpusilla grown on half-strength Hutner's medium with sucrose under inductive photo-periods is inhibited in a periodic manner by daily transfers to water for short periods of time. The phase of maximal inhibition of flowering caused by water treatment is about 1 to 2 hours after the time of maximal sensitivity to light pulses. The rhythm of sensitivity to water treatments does not persist under continuous blue light. Supplementing the water with either Ca(NO(3))(2) or K(2)HPO(4) partially reverses the inhibition of flowering, with the first salt being more effective. Supplementation with NH(4)NO(3) or MgSO(4) increases the inhibition. The water effect on flowering is not observed in plants grown on half-strength Hutner's medium without sucrose. The water treatments may act by removing or destroying a crucial precursor for photoperiodic induction, with the other conditions modifying permeability. The system provides a new technique for investigating the mechanism of photoperiodic induction.

Dark Reversion of Phytochrome in Sinapis alba L.

Phytochrome in Sinapis alba L. (white mustard) seedlings undergoes both decay and reversion after an exposure to red light. This is typical of other crucifers and of dicotyledons in general. In the presence of sodium azide, decay is inhibited, and reversion continues at about the same rate as in buffer alone. The reversion has been demonstrated both in cotyledon plus hypocotyl hook and in hypocotyl hook samples alone and is of the same order of magnitude in both. Contrary conclusions in the literature that there is no reversion in Sinapis are based on indirect measurements and are unjustified.

Carbon dioxide output as an index of circadian timing in Lemna photoperiodism.

Previous work on flowering suggested that photoperiodism in Lemna perpusilla 6746 involves an endogenous circadian "clock," but direct evidence requires study of an overt rhythm in the same plant. The CO(2) output rate of axenic cultures supplied with sucrose has been studied in a system using infrared analysis and monitoring four sets of cultures at once. Alternations of (1/4) to 21 hours of dim red light with darkness in 24-hour cycles can entrain the CO(2) output. In darkness following either continuous dim red light or entrainment to a 12(12) light (dark) schedule, the rate oscillates through two maxima and two minima, with a circadian periodicity, before apparently damping. In continuous red light, the rate is linear. The skeleton photoperiodic schedule (1/4)(5(1/2))(1/4)(18), with its two portions highly unequal, rapidly entrains the CO(2) output in a phase relationship which is the same irrespective of which dark period is given first. The schedules (1/4)(13)(1/4)(10(1/2)) and its inverse, however, with two portions more nearly equal in length, differ markedly from each other with respect to manner of entrainment, as they do in their effects on flowering. These and other results strongly support the concept that a circadian clock is an important component of photoperiodism, and they provide a new experimental system in which to study its action.