Plant Physiol. (1972) 49, 907-911

Photoperiodic Entrainment Patterns in the CO2 Output of Lemnaperpusilla 6746 and of Several Other Lemnaceae

Received for publication August 23, 1971

WILuAM S. HILLMANDepartment of Biology, Brookhaven National Laboratory, Upton, Newv York 11973

ABSTRACT

The C02 output of Lemna perpusilla 6746 in "skeletonphotoperiods" consisting of alternating 10½/2-hour and 13-hourdark periods separated by ¼4-hour illuminations was recordedunder stable high and low nitrate conditions. The phase rela-tionship finally attained between light schedule and output isthe same regardless of which dark period is given first, butentrainment is more rapid (as is flowering) with an initial 13-hour dark period. In all respects other than bistability-theassumption of two different stable phase relationships de-pending on the initial dark period-both flowering and thecourse of CO2 output conform to Pittendrigh's model derivedfrom Drosophila eclosion rhythms, confirming the view that anendogenous circadian rhythm, or biological clock, underliesthe photoperiodic control of flowering in this plant. Experi-ments with rigorous temperature control show that earlierresults with long light exposures were in part due to tempera-ture changes; in consequence, it is clear that entrainmentpatterns with high nitrate differ even more from those in lownitrate than was previously evident, and not simply by theaddition of a "nitrate peak." Other Lemnaceae tested with a fewsimple light-dark schedules in both types of media show avariety of responses, with no obvious correlation to photo-periodic response type.

The carbon dioxide output of Lemna perpusilla strain 6746reflects the activity of an endogenous circadian rhythm, orbiological clock, that is probably involved in photoperiodictiming. Light-dark schedules with a 24-hr periodicity rapidlyentrain CO2 output, and the pattern of entrainment to a givenschedule differs markedly depending upon the presence or ab-sence of nitrate. At least under some circumstances, entrain-ment is exerted through the photoperiodically-active pigmentphytochrome (5-8). Thus, results with a single plant suggestthat CO2 output entrainment patterns are valuable in analyzingboth photoperiodism and certain aspects of plant nutrition. Theexperiments below provide more definitive answers to somequestions raised in the earlier work and they extend theinvestigation in a preliminary fashion to other plants, includingspecies of various photoperiodic response types.

MATERIALS AND METHODS

All work was done in axenic culture. Stocks of Lemna per-pusilla, strain 6746, were grown under continuous light insupplemented Hutner's medium as previously described (6).

Identical stock conditions were used for the following otherclones: strains 6612 and 7001 of L. perpusilla, and strains 6573and 6625 of L. minor, all from Landolt's (10) collection; strainLpm of L. paucicostata, raised from seed supplied by Dr. S. C.Maheshwari but not identical with the clone used in his work(11); strain Spy of Spirodela polyrrhiza (1, 14) and strain Lmyof L. minor (2, 14). Similar conditions, except with only 10hr of light per day, were used for cultures of L. gibba strainsGI and G3 (9) both obtained originally from Dr. R. Kandeler.The photoperiodic responses of L. perpusilla 6746 and of theL. gibba strains are extensively documented in the literature;L. paucicostata Lpm is almost certainly a short day plant (3,1 1); Landolt's (10) observations suggest that L. perpusilla 7001is a short day plant and that L. perpusilla 6612 and L. minor6573 may be long day plants, while neither photoperiodic re-sponses nor flowering have been reported in L. minor 6625, L.minor Lmy, nor in the Spirodela polyrrhiza.At the start of each experiment stock cultures 2 to 4 weeks

old, depending on the strain, were poured into 150 ml of ex-perimental medium usually in a 250-ml cotton-stopperedreagent bottle (6). Carbon dioxide assays were performed aspreviously described (6-8), either by placing each bottle in acanister (with attached light sources) through which the airstream passed, or by fitting a polyethylene enclosure tightlyover the mouth and exposing the stopper to the monitored airstream in that way. The latter procedure was especially usefulin permitting immersion of most of the bottle in a water bathfor accurate temperature control and it was also valuable inbeing adaptable to various vessel types and sizes. Cultures inthe standard 250-ml reagent bottles lay several layers thick onthe surface of the medium. Such cultures were always shakenat 60 to 90 strokes per minute (6). In several experiments,however, the same amount of medium was used in a 1-literErlenmeyer flask so that the plants barely covered the muchlarger surface available; shaking then proved unnecessary forreliable results.The experimental medium was either double-strength Hut-

ner's medium without nitrogen (7), designated NL, or fullstrength M medium (4) containing 25 mm nitrate ion, supple-mented with 30 fuM EDTA and designated E. Sucrose wasadded to both media at 1 % (29 mM) before autoclaving.

RESULTS

Ambiguous Skeleton Photoperiods and L. perpusilla. Whencultures taken from continuous light are exposed to alternating102-hr and 13-hr dark periods separated by ¼/4-hr light pe-riods, the intensity of flowering is strongly dependent on theinitial dark period (5). Thus, the sequence symbolized l/4(13)-¼/4(10½/2), indicating continuous light followed by 13 hr dark-ness followed by '/4 hr light, results in rapid flowering, whileflowering is much less intense under the sequence symbolized

907

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Plant Physiol. Vol. 49, 1972

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FIG. 1. CO2 output ().t-hourly, arbitrary units) of L. perpusilla 6746 as shown in direct strip-chart recordings. Each recording includes the baseline; baseline points in all charts are synchronous, though they may be imperfectly aligned in the figure. Major vertical divisions represent 4 hrThe experiment was started by pouring cultures into E (high nitrate) or NL (nitrogen-less) medium on the first day and maintaining white or dimred light until 1415 hours (-*). Subsequently, all cultures were in darkness except for two 15-min light exposures each day, starting at either 0045and 1400 or 0315 and 1400 hours (A). Numbers 10.5 or 13 indicate length of first dark period in hr.

V4(10'/2)/4(13), continuous light followed by 10'/2-hr darkness.Parallel experiments with CO, output have also indicated a

difference in manner of entrainment depending on which darkperiod was given first (6), but the data seemed inadequate totest the bistability model proposed by Pittendrigh (13) on thebasis of work with Drosophila eclosion rhythms. Subsequentexperiments showed that these early CO2 data had been ob-tained while the medium was being rapidly depleted of nitrate,a condition that would necessarily make interpretations diffi-cult (7). Thus, further tests of the skeleton photoperiods inquestion were carried out in both types of media after firstshowing that the characteristic effects of each medium on en-

trainment would persist for the 7-day experimental period,at least under the simple schedule of ]1 hr of red light per day(see later).

Typical results are shown in Figure 1, the salient points ofwhich can be summarized as follows. In both media, when the13-hr dark period occurs first the record almost immediatelyassumes that phase relationship to the light schedule evidentat the end of the experiment. If the 10/ b-hr dark period occurs

first, a period of several days ensues during which no clearpattern is present, following which the same phase relationshipis established as in the first case. Both records for the high ni-trate E medium show, by the end of the experiment, minima atthe start of each 13-hr dark period and maxima at the close.With the 13-hr dark period given first (second record from thetop) this relationship is already present in the period May 11

and 12, while in the opposite situation it is not established untilat least May 14. Similarly, by the end of the experiment bothrecords for the low nitrate NL medium show maxima 3 to 5hr after the start of each 13-hr dark period and minima 3 to 6hr after the start of each 10 2-hr dark period, with the relation-ship evident at least 2 days sooner when the 13-hr dark periodoccurs first. Additional experiments in both media showed thatthe phase relationship reached eventually under either schedulecan be produced unambiguously, that is, at the same rapid rateregardless of the length of the initial dark period, by theschedule 1/4(5)1/4(51/4)V4(13). Typical results are shown in

Figure 2: see also Figure 9 in (6).Experiments with Full Temperature Control. Direct meas-

urements in culture bottles within canisters with light sources

showed that a single 15-min light exposure had no effect on

temperature, but 2 hr of the red light used increased it as muchas 3 C. Thus, to determine what proportion of the results todate might represent a confounding of temperature and lighteffects, experiments were performed using culture vessels im-mersed in a rapidly circulating water bath held at 26.5 C, withthe light source outside the bath. Both small culture bottleswith shaking, and larger flasks, unshaken (see "Materials andMethods") were used in these experiments, without differencesbetween the two. Under these conditions, no effect of anylight period on medium temperature was detectable.

With water bath temperature control, the effects on L.perputsilla 6746 of 15 min of red light per day, and of the

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ambiguous skeleton photoperiods, were identical to those ob-served with the older methods. With longer light periods, how-ever, the results were not the same as those reported earlier(6, 7). Some records for both media, using 8-hr and 12-hr lightperiods, are presented in Figure 3. The most obvious differencebetween these and the earlier data is in the low nitrate medium.The CO,, output there is no longer an almost perfect reflectionof the light-dark cycle, rising during the light and reaching amaximum shortly after the start of darkness. As a consequence,the difference between high and low nitrate records is now

quite complex, no longer appearing as simply the addition of a"nitrate peak" (7).Entrainment Patterns in Various Strains. Records of CO,,

output of various Lemnaceae strains were taken under a stand-ard red light schedule of ¼/ hr per day, in most instances inboth high and low nitrate media. A few strains were also testedwith other schedules. Only the results with L. perpusilla 6746and L. gibba G3 can be considered definitive. Each of the otherstrains was tested at least twice in experiments lasting usually3 to 6 days.

Figure 4 shows typical results with L. perpusilla 6746 andL. gibba G3 under the standard red schedule. Taking the timeat which the daily light exposure starts as hr 0, L. perpusilla6746 in low nitrate conditions (NL) shows a single maximum atabout hr 10 and a minimum at hr 22 to 24: in high nitratemedium (E) there is a sharp maximum at about hr 3, a broaderone at hr 16 to 17, and minima at hr 1 and hr 6 to 8. These re-sults largely confirm those published previously (7, 8), addingfurther detail cn the difference due to nitrate. As for L. gibba

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FIG. 3. As in Figure 1, except that the experiment started July30, the plants were in unshaken 1-1 flasks in water-baths held at26.5 C, and the daily 8-hr (above) or 12-hr red light periods areshown as brackets of the appropriate length and position.

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upper two recordings, L. gibba G3 in the lower; the experimentstarted on April 26, and the daily 15-min red light exposures areindicated by arrowheads.

Plant Physiol. Vol. 49, 1972 CO2 ENTRAINMENT PATTERNS

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Plant Physiol. Vol. 49, 1972

LgG3

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FIG. 5. As in other figures, except all cultures are L. gibba G3,and the experiment began June 7.

G3, the record in low nitrate conditions is nearly featureless,but with a low, broad maximum at about hr 10, while withhigh nitrate there is a distinct maximum at hr 7 to 8 and a

minimum at hr 22 to 24.Additional results with L. gibba G3 are shown in Figure 5

comparing the effects of the standard red schedule with a simi-lar far red schedule. Again, the records with low nitrate are

nearly flat. With high nitrate, the far red schedule gives a

maximum at hr 3 to 4 with a shoulder about hr 8 and a mini-mum at hr 18 to 20. A comparison between the effects of redand far-red schedules on L. perpusilla 6746 in low nitrate con-

ditions was reported earlier (8); in high nitrate medium, 1X4 hrdaily of far red has effects almost indistinguishable from thesame schedule of red, although the timing of the maxima andminima may perhaps be slightly earlier. Typical records withL. gibba GI, L. perpusilla 6612 and L. mninor 6573 are shownin Figure 6.

DISCUSSION

Skeleton Photoperiods and Pittendrigh's Drosophila Model.The work presented here confirms earlier interpretations rend-ered tentative by changes in the medium (6, 7). Indeed, it isnow obvious that at least the early portions of Figures 7 to 9(6) correspond closely to the present Figure 1 records for Emedium; both are in high nitrate conditions, although theHutner's medium used earlier contained less nitrate than E andthus changed more rapidly.

In summary, the difference in effect of the schedules 1/4(13)¼4(10'/2) and 1/4(101 2)'/4(13) is not that they entrain with equal

efficiency but in opposite phase relationships; rather, the firstentrains more rapidly than the second but both produce thesame phase relationship in the end (Fig. 1). To this extent, thataspect of Pittendrigh's model (13) predicting bistability (thefirst condition described) does not hold for these data. In allother respects, however, both the CO2 and flowering data are

completely consistent with the model. In fact, the lack ofbistability in the CO2 records correlates perfectly with the factthat giving the 101/2 hr dark period first does not prevent flow-ering, but simply delays it (4, 6), thus parallelling the delay in

CO2 entrainment. The CO2 data also confirm the model's pre-

diction that flowering is associated with a phase relationshipcharacteristic of an unambiguous schedule that permits flower-ing: in this case ¼/4(5)¼4(5¼V4)¼/4(13).One aspect of the photoperiodic response not covered by

these results is the question of when, in the observed entrain-ment patterns, the light-sensitive phase (Pittendrigh's "induc-tive phase") occurs. The only conclusion possible is a generalone-that the rapid entrainment with an initial 13-hr darkperiod avoids exposing the sensitive phase to light, while withan initial 10½2 hr dark period, the series of partial resettingsrepresented by the essentially flat portion of the CO2 recordsmust also involve several coincidences of light and the sensitivephase.

It is conceivable that skeleton photoperiods only slightlydifferent from those used here, ¼/4(11)/4(12 '2), for example,might show true bistability with respect to both flowering andCO2 output, and thus resemble the Drosophila situation in allrespects. A few exploratory experiments with CO2 output. how-

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C02 ENTRAINMENT PATTERNS

ever, do not encourage this view. Rather, they suggest that theentrainment mechanism in Lemna resolves the ambiguity in-herent in such schedules by finally assuming, in every case, aphase relationship in which the shorter period is interpreted asthe "day", which is what the eventual adoption of the 14(5)-4_(5 ¼4)¼4(13) phase relationship in the present experimentsamounts to. In any case, the congruence of flowering data, CO,records and Pittendrigh's model in all other respects providessome of the most persuasive evidence as yet available, thatthe photoperiodic control of flowering involves an endogenouscircadian timer, a timer very similar to that controlling theDrosophila eclosion rhythm.Long Light Periods, Temperature, and Nitrate. On the basis

of data such as those in Figure 3, the earlier results with longlight periods (6, 7) must be reinterpreted as involving tempera-ture effects as well. Fortunately, most of this work has pro-ceeded by the use of short light exposures. Hence the point islargely a technical one, relevant mainly to future experiments,except for one major area: the interpretation of nitrate effects.The idea that high nitrate simply adds another maximum to anotherwise basically unchanged entrainment pattern (7) is mis-taken, as a comparison of the two media under various sched-ules will indicate (Figs. 1-4). It originated because with theolder methods, long light periods impose a temperature cyclein addition to the light-dark cycle, thereby producing a greatersimilarity between results in the two media than is observed(Fig. 3) with adequate temperature control. Far from causing asimple change, high nitrate seems to affect several phases of theentrained cycle. Thus, further work involving long light pe-riods, of a kind that must be done if questions of "light-on" and"light-off" rhythms (15) are to be studied in this system, willrequire rigorous temperature control as well as the use of vari-ous media.

Entrainment Pafterns in Various Strains. A survey of severalspecies and strains leads to no simple conclusion except thatdifferent ones react differently to the same conditions. Sinceonly the flowering responses of L. perpusilla 6746 and the twoL. gibba strains have been studied in detail, the possibility thatthe type of entrainment pattern might correlate with the photo-periodic response type could not be rigorously tested, but theevidence against it seems strong. L. paucicostata Lpm, for in-stance, almost certainly a short day plant, presents differentpatterns from L. perpusilla 6746, although the Lpm data are

so variable as to preclude any further statement. L. perpusilla7001, which according to Landolt (10) responds similarly to6746, also resembles 6746 in its entrainment pattern in highnitrate medium. However, similar patterns are also shown inhigh nitrate by the presumptive long day plants L. perpusilla6612 and L. minor 6573 (Fig. 6). Thus it seems unlikely thatthe entrainment pattern under a single standard condition willprove to be diagnostic for a particular photoperiodic response,although obviously, as much of the work with strain 6746 hasindicated, detailed entrainment studies certainly bear on photo-periodic timing.

In most of the cases studied, the level of nitrate has a signifi-cant effect on the entrainment pattern, extending the observa-tion first made on L. perpusilla 6746. One noteworthy point isthat although the entrainment patterns of that strain and ofL. minor 6573 are almost indistinguishable in high nitrate(Fig. 4) they are very different in low nitrate conditions,where 6746 shows a maximum at hr 10 and a minimum at hr22 while 6573 shows a maximum at hr 2 and a minimum at hr10. Results of this kind make it evident that, whatever theprocesses responsible for the variations in CO2 output in suchsystems, they are not always organized in the same manner, atleast in a temporal sense. Perhaps most important, comparativestudies of strains differing in such ways should aid substanti-ally in picking out the enzyme(s), metabolite(s) or cofactor(s)immediately underlying the CO2 oscillations from among themany possible candidates. Whether such studies might also helpidentify the chain of communication between the CO2 oscilla-tion and the cellular "clock"-if indeed it is meaningful tospeak of the clock as a discrete entity and not a property ofthe system-is more problematical, but worth considering.

Acknowledgments-I am indebted to Rosemarie Dearing and Helen Kelly fortechnical assistance, and to Neal Tempel for his invaluable collaboration onC02-measuring techniques.

LITERATURE CITED

1. FURLYA, M. AND K. V. THIMAN-N-. 1964. The biogenesis of anthocyanins. XI.Effects of gibberellic acid in two species of Spirodela. Arch. Biochem.Biophys. 108: 109-116.

2. HILLMAN, W. S. 1955. The action of benzimidazole on Lemna minor. PlantPhysiol. 30: 535-542.

3. HILLMAN, W. S. 1961. Test-tube studies on flowering: experiments with theLemnaceae, or duckweeds. Bull. Torrey Bot. Club 88: 327-336.

4. HILLMAN, W. S. 1961. Experimental control of flowering in Lemna. III. Arelationship between medium composition and the opposite photoperiodicresponses of L. perpusilla 6746 and L. gibba G3. Amer. J. Bot. 48: 413-419.

5. HILLMAN, W. S. 1964. Endogenous circadian rhythms and the response ofLemna perpusilla to skeleton photoperiods. Amer. Natur. 98: 323-328.

6. HILLMAN, W. S. 1970. Carbon dioxide output as an index of circadian timingin Lemna photoperiodism. Plant Physiol. 45: 273-279.

7. HILLMAN, W. S. 1971. Nitrate and the course of Lemna perpusilla carbondioxide output under daily photoperiodic cycles. Plant Physiol. 47: 431-434.

8. HILLMAN, W. S. 1971. Entrainment of Lemna C02 output through phyto-chrome. Plant Physiol. 48: 770-774.

9. KANDELER, R. 1955. Uber die bliutenbildung bei Lemna gibba L. I. Kulturbe-dingungen und tageslangenabhaingigkeit. Z. Bot. 43: 61-71.

IO. LANDOLT, E. 1957. Physiologische und okologische Untersuchtungen an Lemna-ceen. Ber. Schweiz. Bot. Ges. 67: 271-410.

11. MAHESHWARI, S. C. AND S. GUPTA. 1967. Induction of flowering in Lemnapaucicostata, a short-day plant, by chelating agents and iron. Planta 77:95-98.

12. ODA, Y. 1969. The action of skeleton photoperiods on flowering in Lemnaperpusilla. Plant Cell Physiol. 10: 399-409.

13. PITTENaDRICH, C. S. 1966. The circadian oscillation in Drosophila pseudo-obscura pupae: a model for the photoperiodic clock. Z. Pflanzenphysiol.54: 275-307.

14. POs-\ER, H. B. 1967. Aquatic Vascular Plants. In: F. Wilt and N. Wessels,eds., Methods in Developmental Biology. Thomas Y. Crowell Co., NewYork. pp. 301-317.

15. TAKIMOTO, A. AND K. C. HAs-.ER. 1965. Effect of double red light inter-ruptions on the photoperiodic response of Pharbitis nil. Plant Physiol. 40:855-858.

911Plant Physiol. Vol. 49, 1972

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