Clock Classics: It all started with the plants

This is a repost of a May 29, 2008 post:

I was wondering what to do about the Classic Papers Chellenge. The deadline is May 31st, and I am so busy (not to mention visiting my dentist twice week which incapacitates me for the day, pretty much), so I decided to go back to the very beginning because I already wrote about it before and could just cannibalize my old posts: this one about the history of chronobiology with an emphasis on Darwin's work, and this one about Linnaeus' floral clock and the science that came before and immediately after it.

In the old days, when people communed with nature more closely, the fact that plants and animals did different things at different times of day or year did not raise any eyebrows. That's just how the world works - you sleep at night and work during the day, and so do (or in reverse) many other organisms. Nothing exciting there, is it? Nobody that we know of ever wondered how and why this happens - it just does. Thus, for many centuries, all we got are short snippets of observations without any thoughts about causes:

"Aristotle [noted] that the ovaries of sea-urchins acquire greater size than usual at the time of the full moon."(Cloudsley-Thompson 1980,p.5.)

"Androsthenes reported that the tamarind tree..., opened its leaves during the day and closed them at night."(Moore-Ede et al. 1982,p.5.)

"Cicero mentioned that the flesh of oysters waxed and waned with the Moon, an observation confirmed later by Pliny."(Campbell 1988, Coveney and Highfield 1990)

"...Hippocrates had advised his associates that regularity was a sign of health, and that irregular body functions or habits promoted an unsalutory condition. He counseled them to pay close attention to fluctuations in their symptoms, to look at both good and bad days in their patients and healthy people."(Luce 1971,p.8.)

"Herophilus of Alexandria is said to have measured biological periodicity by timing the human pulse with the aid of a water clock."(Cloudsley-Thompson 1980, p.5.)

"Early Greek therapies involved cycles of treatment, known as metasyncrasis....Caelius Aurelianus on Chronic and Acute Diseases...describes these treatments.. ."(Luce 1971, p.8.)

"Nobody seems to have noticed any biological rhythmicities throughout the Middle Ages. The lone exception was Albertus Magnus who wrote about the sleep movements of plants in the thirteenth century" (Bennet 1974).

The first person to ask the question - and perform the very first experiment in the field of Chronobiology - was Jean-Jacques d'Ortous de Mairan, a French astronomer. What did he do?

In 1729, intrigued by the daily opening and closing of the leaves of a heliotrope plant (the phenomenon of 'sleep in plants' was well known due to Linneaus), de Mairan decided to test whether this biological "behavior" was simply a response to the sun. He took a plant (most likely Mimosa pudica but we do not know for sure as Linnean taxonomy came about a decade later) and placed it in a dark closet. He then observed it and noted that, without having access to the information about sunlight, the plant still raised its leaves during the day and let them droop down during the night.

However, De Mairan was an astronomer busy with other questions:

"....about the aurora borealis, and the relation of a prism's rainbow colors to the musical scale, and the diurnal rotation of the earth, and the satellites of Venus, and the total eclipse of the sun that had occurred in 1706. He would waste no time writing to the Academy about the sleep of a plant!"(Ward 1971,p.43.)

He did not wanted to waste his time writing and publishing a paper on a mere plant. So his experiment was reported by his friend Marchant. It was not unusual at that time for one person to report someone else's findings. Marchand published it in the Proceedings of the Royal Academy of Paris as he was a member, and the official citation is: De Mairan, J.J.O. 1729. Observation Botanique, Histoire de l'Academie Royale des Sciences, Paris, p.35.

In the paper Marchant wrote:

"It is well known that the most sensitive of the heliotropes turns its leaves and branches in the direction of the greatest light intensity. This property is common to many other plants, but the heliothrope is peculiar in that it is sensitive to the sun (or time of day) in another way: the leaves and stems fold up when the sun goes down, in just the same way as when touches or agutates the plant.

But M. de Mairan observed that this phenomenon was not restricted to the sunset or to the open air; it is only a little less marked when one maintains the plant continually enclosed in a dark place - it opens very appreciably during the day, and at evening folds up again for the night. This experiment was carried out towards the end of one summer, and well duplicated. The sensitive plant sense the sun without being exposed to it in any way, and is reminiscent of that delicate perception by which invalids in their beds can tell the difference between day and night. (Ward 1971)"

Marchant and de Mairan were quite careful about not automatically assuming that the capacity for time measurement resides within the plant. They could not exclude other potential factors: temperature cycles, or light leaks, or changes in other meteorological parameters. Also, the paper, being just a page long (a "short communication"), does not provide detailed "materials and methods" so we do not know if "well repeated" experiments meant that this was done a few times for a day or two, or if the same plants were monitored over many days. We also do not know how, as well as how often and when, did de Mairan check on the plants. He certainly missed that the plants opened up their leaves a little earlier each day - a freerunning rhythm with a period slightly shorter than 24 hours - a dead giveaway that the rhythm is endogenous.

The idea that clocks are endogenous, residing inside organisms, was controversial for a very long time - top botanists of Europe were debating this throughout the 19th century, and the debate lasted well into the 1970s with Frank Brown and a few others desparately inventing more and more complicated mathematical models that could potentially explain how each individual, with its own period, could actually be responding to a celestial cue (blame Skinner and behaviorism for treating all behaviors as reactive, i.e., automatic responses to the cues from the environment).

The early 18th century science did not progress at a speed we are used to today. But the paper was not obscure and forgotten either - it just took some time for others to revisit it. And revisit it they did. In 1758 and 1759 two botanists repeated the experiment: both Zinn and Duhamel de Monceau (Duhamel de Monceau 1758) controlled for both light and temperature and the plants still exhibited the rhythms. They used Mimosa pudica, which suggests to us today that this was the plant originally tested by de Mairan.

Suspecting light-leaks in de Mairan's experiment, Henri-Louis Duhamel du Monceau repeated the same experiment several times (Duhamel du Monceau 1758). At first, he placed the plants inside an old wine cave. It had no air vent through which the light could leak in, and it had a front vault which could serve as a light lock. He observed the regular opening and closing of the leaves for many days (using a candle for observation). He once took a plant out in the late afternoon - which phase-shifted the clock with a light pulse. The plant remained open all night (i.e.., not directly responding to darkness), but then re-entrained to the normal cycle the next day. Still not happy, he placed a plant in a leather trunk, wrapped it in a blanket and placed it in a closet inside the cave - with the same result: the plant leaves opened and closed every day.

So, he was convinced that no light leaks were responsible for the plant behavior. Yet he was still not sure if the temperature in the cave was absolutely constant, so he repeated the experiment in a hothouse where the temperature was constant and quite high, suspecting that perhaps a night chill prompted the leaves to close. He had to conclude: "I have seen this plant close up every evening in the hothouse even though the heat of the stoves had been much increased. One can conclude from these experiments that the movements of the sensitive plant are dependent neither on the light nor on the heat" (Duhamel de Monceau 1758). He did not know it at the time, of course, but he was the first to demonstrate that circadian rhythms are temperature compesated - the period is the same at a broad range of constant temperatures.

The research picked up speed in the 19th century. Augustus Pyramus de Candolle repeated the experiments while making sure not just that the darkness was absolute and the temperature constant, but also that the humidity was constant, thus eliminating another potential cue. He then showed that the period of diurnal movements of Mimosa is very close to 24 hours in constant darkness, but around 22 hours in constant light (using a bank of six lamps). He also managed to reverse day and night by using artificial light to which the plants responded by reversing their rhythms (De Candolle 1832) after the initial few days of "confusion".

Another astronomer, Svante Arrhenius argued that a mysterious cosmic Factor X triggered the movements (Arrhenius 1898). He attributed the rhythms to the "physiological influence of atmospheric electricity". Charles Darwin published an entire book on the Movement of Plants in 1880, arguing that the plant itself generates the daily rhythms (Darwin 1880).

The most famous botanist of the 19th century, Wilhelm Pfeffer, started out favouring the "external hypothesis", arguing that light leaks were the source of external information for de Mairan's and Duhamel's plants (Pfeffer 1880, 1897, 1899). But his own well-designed experiments (as well as those of Darwin) forced him to change his mind later in his career and accept the "internal" source of such rhythmic movements. Unfortunately, Pfeffer published his latter views in an obscure (surprisingly, considering the short and catchy title) German journal Abhandlungen der Mathematisch-Physischen Klasse der Königlich Sächsischen Gesellschaft der Wissenschaften, so most people were (and still are) not aware that he changed his mind on this matter.

In the early 20th century, Erwin Bunning was the first to really thoroughly study circadian rhythms in plants and to link the daily rhythms to seasonality. He and many others at the time mostly studied photoperiodism and vernalization in plants, two phenomena then thought to be closely related (we know better today). For the rest of the century, animal research took over and only recently, with the advent of molecular techniques in Arabidopsis, has the plant chronobiology rejoined the rest of the field.