HISTORY


The first writings, at least in the western canon, to recognize diurnal rhythms come from the fourth century BC. Androsthenes described the observation of daily leaf movements of the tamarind tree, Tamarindus indicus, that were observed on the island of Tylos (now Bahrein) in the Persian Gulf during the marches of Alexander the Great (Bretzl, 1903). There was no suggestion that the endogenous origin of these rhythms was suspected at the time, and it took more than two millennia for this to be experimentally tested. The scientific literature on circadian rhythms began in 1729 when the French astronomer de Mairan reported that the daily leaf movements of the sensitive heliotrope plant (probably Mimosa pudica) persisted in constant darkness, demonstrating their endogenous origin (de Mairan, 1729). Presciently, de Mairan suggested that these rhythms were related to the sleep rhythms of bedridden humans. It took 30 years before de Mairan's observations were independently repeated (Hill, 1757; Duhamel duMonceau, 1759; Zinn, 1759). These studies excluded temperature variation as a possible zeitgeber driving the leaf movement rhythms.

Nearly a century passed before period length of these leaf movements was accurately measured and it was realized that these rhythms were only ∼24 h, making the rhythms circadian and suggesting that these rhythms were endogenous and not simply responses to environmental time cues. de Candolle (1832) determined that the free running period of M. pudica was 22 to 23 h, discernably shorter than 24 h. He further showed that the rhythm could be inverted by reversing the alternation of light and dark. A number of authors repeated and expanded these observations through the 19th and early 20th centuries, in each case exploiting plant leaf movements , the only known circadian rhythm (for a more complete historical account, see Bünning, 1960; Cumming and Wagner, 1968). As an aside, animal circadian rhythms were not scientifically described until much later, with pigment rhythms in arthropods (Kiesel, 1894) and daily activity in rats (Richter, 1922) being among the first in the literature.



Not surprisingly, that these circadian rhythms in leaf movements were truly endogenous was disputed. Pfeffer (1873), for example, suspected that light leaking into the darkrooms (and wine cellars and caves) employed in these studies foiled the attempts to provide constant conditions and invalidated the claims that these rhythms had endogenous origins. However, the critics ultimately were persuaded by the accumulating mass of evidence. Pfeffer himself extensively studied leaf movements and provided many examples of the free-running periods of leaf movement rhythms differing from 24 h (Pfeffer, 1915). That the rhythms were circadian and not exactly 24 h was an extremely important point because it was the best evidence, until experiments on the fungusNeurospora crassa were conducted in space (Sulzman et al., 1984), that these rhythms were truly endogenous and not driven by some subtle and undetected geophysical cue associated with the rotation of the earth on its axis.

The third key criterion of circadian rhythms is temperature compensation, and it took much longer for this attribute to become appreciated. The rationale for examining temperature dependence of the period length emerged from the expectation that the clock mechanism was based on alternating chemical processes. Thus, it was anticipated that, like chemical processes, the clock should exhibit marked temperature dependence. The rate of a typical chemical reaction doubles with a 10° increase in temperature (Q10 = 2). However, the period of leaf movement in P. coccineus exhibited a Q10 of only 1.2 (Bünning, 1931). By the 1960s, this observation had been extended to many other plants as well as to animals (Sweeney and Hastings, 1960). That the clocks were not temperature independent but, instead, exhibited less than expected temperature dependence strongly supported the concept of a temperature compensation mechanism that was imperfect. Consistent with this view was the observation of Q10 values of <1.0: an imperfect compensation mechanism could lengthen the period either insufficiently or too greatly at higher temperatures or, conversely, shorten the period too little or too much at lower temperatures.

As early as 1880, Charles and Francis Darwin suggested the heritability of circadian rhythms (Darwin and Darwin, 1880), as opposed to the imprinting of a 24-h period by exposure to diurnal cycles during development. This was initially explored in the 1930s by two strategies. In one, plants or animals were raised in constant conditions for multiple generations. One of the most grueling among such studies demonstrated the retention of stable rhythms among fruit flies reared in constant conditions for 700 generations (reviewed in Johnson, 2005). In a second strategy, seedlings or animals were exposed to cycles that differed from 24 h in an effort to imprint novel periods; such studies could sometimes impose the novel period length during the novel cycles, but upon release into continuous conditions, the endogenous circadian period was restored (Bünning, 1973). The inheritance of period length among progeny from crosses of parents with distinct period lengths was first reported in Phaseolus; hybrids had period length intermediates between those of the parents (Bünning, 1932, 1935).

Forward genetic analysis to identify components of circadian clocks began in the 1970s. Although now it seems axiomatic that circadian clocks are composed of the products of genes, just how this might be so was the source of considerable controversy. It was argued that forward genetic efforts would be fruitless because clocks were sufficiently complex to reasonably be expected to exhibit polygenic inheritance (Bünning, 1935) and would not yield easily to standard genetic approaches. However, mutations conferring altered period length were identified and characterized in the fruitfly Drosophila melanogaster (Konopka and Benzer, 1971), the green alga Chlamydomonas reinhardtii (Bruce, 1972), and the filamentous fungus N. crassa (Feldman and Hoyle, 1973). It took more than a decade to clone the first clock gene, the Drosophila period (per) gene (Bargiello and Young, 1984; Zehring et al., 1984), and another 5 years to clone the second, the Neurospora frequency gene (McClung et al., 1989). However, the decade of the 1990s saw rapid progress toward the identification of clock components and the elucidation of oscillator mechanisms central to the circadian clock in a number of organisms, most notably Drosophila, Neurospora, and mice (Dunlap, 1999).

In plants, it was realized that the leaf movement rhythm was only one among many rhythms that included germination, growth, enzyme activity, stomatal movement and gas exchange, photosynthetic activity, flower opening, and fragrance emission (Cumming and Wagner, 1968). However, genetic studies of plant clocks languished after Bünning's first experiments. Two critical discoveries changed this. First, Kloppstech (1985) described a circadian rhythm in pea in the abundance of three nuclear-encoded transcripts encoding the light-harvesting chlorophyll a/b binding protein (LHCB; also called CAB), the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, and an early light-induced protein. This observation was replicated and extended in wheat, where it was shown that the transcription rate for the Cab-1 gene was under circadian control (Nagy et al., 1988). Neither pea nor wheat was particularly suitable for positional gene cloning, but Arabidopsis thaliana was emerging as a powerful system in which to combine forward genetic analysis with molecular gene cloning techniques (Somerville and Koornneef, 2002). It was soon established that the transcription rate and transcript accumulation of Arabidopsis LHCB (Millar and Kay, 1991) and a number of other genes (McClung and Kay, 1994) were also under circadian control.

These initial Arabidopsis experiments were quite labor intensive, as tissues for RNA extraction, RNA gel blotting, and nuclear run-on analyses had to be harvested at frequent intervals over fairly lengthy time courses. Such experiments inevitably became exercises in sleep deprivation for the experimenters and provided considerable disincentive to the recruitment of graduate students into the field. Moreover, forward genetic analysis required a sensitive, reliable, and nondestructive assay that could score the circadian activity of individual seedlings without killing them. The luciferases offered a versatile class of noninvasive reporter genes. Firefly luciferase (LUC) catalyzes the ATP-dependent oxidative decarboxylation of luciferin with the concomitant release of a photon at 560 nm; this light emission can be quantified with luminometers or with sensitive charge-coupled device cameras (Welsh et al., 2005). Millar et al. (1992)demonstrated that a short fragment of the Arabidopsis LHCB1*3 (CAB2) promoter would drive rhythmic transcription and mRNA accumulation of LUC mRNA detectable as rhythmic light emission from individual Arabidopsis seedlings bearing the LHCB:LUC transgene. There was an element of luck in this, as it turned out that the LUC protein itself was quite stable, and bulk LUC protein failed to oscillate in abundance. However, LUC protein loses catalytic activity after only a few enzymatic cycles, with the net result that light production requires de novo LUC synthesis that is limited by transcript abundance. The LUC mRNA is sufficiently unstable that its accumulation tracks the transcription rate, which, when driven by the LHCB promoter, is rhythmic. After this initial demonstration inArabidopsis, LUC use in circadian studies spread to other organisms, includingDrosophila and mammals (Welsh et al., 2005).

The development of the LUC assay system permitted the first screen forArabidopsis clock mutants. Arabidopsis seeds bearing the LHCB:LUC transgene were mutagenized, and M2 seedlings were screened to yield the first plant clock mutant, timing of cab expression1 (toc1-1; Millar et al., 1995b). The LHCB:LUCtransgene also was introduced into various genetic backgrounds to provide a sensitive assay system to test mutants for effects on circadian function (Millar et al., 1995a).