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Photoperiodism: The consistent use of CONSTANS

Alon Samach and Ayala Gover

Photoperiodic induction of flowering in the long-day plant Arabidopsis is mediated by the circadian regulated CONSTANS gene. New evidence suggests that CONSTANS-like genes have a similar role in shortday induction of flowering of rice and Pharbitis.
Address: Institute of Plant Sciences and Genetics in Agriculture, Faculty of Agriculture, The Hebrew University of Jerusalem P.O. Box 12, Rehovot 76100, Israel. E-mail: samach@agri.huji.ac.il Current Biology 2001, 11:R651R654 0960-9822/01/$ see front matter 2001 Elsevier Science Ltd. All rights reserved.

Genetic background, the age of the plant, plant hormones and environmental signals all have diverse effects on transcript levels of both genes in a way that correlates with flowering [69]. The SOC1 and FT genes are directly activated by the product of the CONSTANS (CO) gene, which mediates the photoperiodic induction of flowering [6]. The CO gene was originally identified because of the late-flowering phenotype of co mutant plants grown in long-day conditions (Figure 1) [10]. Transgenic plants in which CO is overexpressed contain high levels of SOC1 and FT mRNAs, flower very early both in short and long days, and are no longer responsive to daylength [11]. CO encodes a protein with two conserved domains [12,13]: an amino-terminal zinc finger, B-box domain, predicted to mediate proteinprotein interactions; and a carboxy-terminal CCT domain required for nuclear localization and proteinprotein interactions [13,14]. As CO lacks any evident DNA-binding motif, other DNA binding proteins may recruit it to promoters. CO is also involved in the photoperiodic response of the short day rice plant, Oryza sativa [4]. Different rice cultivars show a large degree of genetic variability, from photoperiod insensitivity to a strong photoperiod sensitivity that causes a delay of flowering heading in long days. The heading date is a complex trait controlled by multiple genes known as quantitative trait loci (QTLs). One major QTL, named Hd1, was recently identified as a CO homolog [4]. The Hd1 has both conserved domains and also exhibits sequence similarity with CO and CO-like Arabidopsis proteins in regions between these domains. Comparison of the sequences of hd1 alleles from three different rice cultivars showed that certain alleles that cause decreased photoperiodic sensitivity have insertions or deletions that either truncate the B-box domain or cause the loss of the CCT domain (Figure 1). In the obligatory short-day plant Pharbitis, a CO homolog (PnCO) was identified by differential analysis of induced versus non-induced tissues. When overexpressed in transgenic Arabidopsis plants, PnCO was capable of promoting flowering of co mutants [5]. Short-day plants might have the same downstream flowerpromoting genes FT and SOC1 as long-day plants. In rice, allelic variation in an FT-like gene was recently reported to be the cause of an additional QTL for photoperiod sensitivity Hd3A, [15]. Overexpression of a SOC1 homolog, taken from the long-day mustard plant, was found to cause early flowering in the short-day tobacco variety Maryland Mammoth [9].

In non-equatorial regions, the duration of light hours in a 24 hour day expands and decreases systematically in an annual cycle. Living organisms need to adjust their behavioural programs to such changes in day length, but they also seem to make use of them to initiate developmental programs at a certain time of the year. In many plants, the onset of sexual reproduction flowering is triggered by a change in photoperiod, a phenomenon known as photoperiodism. Short-day plants remain vegetative until they perceive a daylength that is shorter than a certain period, whereas long-day plants flower only when the daylength exceeds a certain limit. Since photoperiodism was discovered by Garner and Allard, intense physiological investigations have provided insight into this process [1]. Most experiments were performed on obligatory plants, which remain vegetative unless they encounter a certain daylength, such as the short-day plant Pharbitis nil. Other plants have less stringent requirements. This is true, for example of the model plant Arabidopsis thaliana in this case, plants grown in longday conditions flower earlier and with fewer leaves than those grown under short days. Molecular genetic studies have identified several genes that participate in the photoperiodic control of flowering of Arabidopsis [2]. Recent findings [35] suggest that these genes play a major role in photoperiodic control of flowering in both long-day and short-day plants. In Arabidopsis, the transition to reproductive development is normally initiated once flower-promoting proteins have accumulated to certain threshold levels. Two such proteins are the products of the FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1; also known as AGL20) genes. Loss-of-function mutations in either of these genes delay flowering, and overexpression of either gene causes very early flowering.

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Figure 1

suggest that what actually causes the difference between long-day and short-day plants is the interaction of CO or its partners with light via phytochrome.
Arabidopsis
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hd1

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Current Biology

Reproductive development in Arabidopsis is induced (arrow) by long days and repressed (T bar) by short days. In rice, short days induce flowering, while long days repress flowering. Mutations in Arabidopsis CO [12] or in the CO-like Kasalath hd1 allele of rice [4] reduce photoperiodic sensitivity. The Kasalath hd1 allele causes a reduction in flowering time in long days. In Arabidopsis, some alleles of CO cause slightly earlier flowering in short days, although others do not [13]. Complete loss of function alleles will determine whether or not the functional protein is actively repressing flowering in non-inductive conditions. Chromophore biosynthesis mutants in rice (se5) [17] and Arabidopsis (hy1) [12] respond similarly to photoperiod. In both mutants, flowering is slightly faster in long days.

The photoperiodic control of flowering time requires a mechanism that measures the duration of a photoperiod. In 1936, Erwin Bnning first proposed that the circadian clock fulfills such a role. The circadian clock is an endogenous pacemaker that generates rhythms, which form an approximate 24 hour cycle. These rhythms are daily entrained by (or synchronized with) daynight transitions of light and temperature, and can further persist, for a few cycles, after transition to continuous conditions of light or dark. Bnning and others showed that the inhibitory effects of night break treatments on the flowering of short-day plants varied with the time of treatment, showing a rhythmic, circadian (approximately 24 hour) pattern (Figure 2) [1,18]. This suggested that a free-running internal circadian oscillator is creating an output involved in gating the effect of light on flowering. There is evidence that light input into the oscillator is gated as well, allowing reduced sensitivity of the circadian clock to transitory fluctuations in light levels during the night. The light input pathway is thus rhythmically regulated by feedback from the oscillator, creating a time-taker that could create rhythmic input even under constant conditions. Recent studies in Arabidopsis have helped to clarify the role of the EARLY FLOWERING 3 (ELF3) gene in both gating responses [1922]. ELF3 masks light signaling both to the clock and to circadian outputs. The product accumulates in the nucleus and can potentially interact with the light receptor phytochrome B [21]. Expression of ELF3 is clock regulated, and by altering the levels of ELF3 so that expression is greatest at night, the plant is able to restrict the sensitivity of its clock to light signals during the night. When a plant is artificially subjected to constant light conditions, the ELF3 product continues to gate light responsiveness in the late subjective day and early subjective night, allowing sustained high-amplitude rhythms of circadian output. In Arabidopsis, most mutations that perturb circadian clock function result in abnormal responses to photoperiod [23]. Indeed, most clock-associated genes were originally identified as flowering-time mutants. Mutations in ELF3 cause early, photoperiod-insensitive flowering, while transgenic plants in which ELF3 is overexpressed are late flowering in long days [21]. Saurez-lopez and colleagues [3] have provided elegant molecular and genetic evidence that CO mediates the flowering phenotype of clock mutants. CO mRNA levels are under circadian clock regulation, with peak expression occurring 1620 hours from the transition to constant conditions. There is still no evidence that CO protein cycles, although indirect evidence suggests that the protein is

Plants detect changes in light quality through photoreceptors, mainly the red/far-red-absorbing phytochromes and the blue/UV-A-absorbing cryptochromes. Physiological and genetic evidence has shown that both types of photoreceptor have roles in measuring and reacting to changes in photoperiod [16]. For example, in the short-day plant Pharbitis, one long inductive night is required for flowering. A short five-minute interruption of this night with low fluence red light night break can keep the plant vegetative. The effects of mutations have revealed the involvement of specific photoreceptors in the regulation of flowering time [16]. In Arabidopsis, CRYPTOCHROME 2 and PHYTOCHROME A seem to be specifically required for longday promotion of flowering, and PHYTOCHROME B (PHYB) acts as a floral repressor. Interestingly, the reduction or absence of functional phytochromes in chromophore biosynthesis mutants causes the normally short-day rice (se5) [17] and long-day Arabidopsis (hy1) [12] to respond similarly to photoperiod (Figure 1). In both mutants, flowering is slightly faster in long-day conditions. This might

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Interactions between the circadian clock and photoperiod, and the way they combine to regulate gene expression leading to seasonal behavioral changes, have also intrigued researchers working on mammalian systems. The Siberian hamster responds to short photoperiods by undergoing gonadal regression, molting to a winter pelage, and a decrease in body and lipid mass. In mammals, the central circadian pacemaker is located in the suprachiasmatic nucleus (SCN). Mammals show a form of memory for day length at the behavioral level. This is clear from aftereffects of day length on several aspects of free-running circadian rhythms. For example, the intrinsic rhythmicity of the SCN in constant dark is dependant on prior day length [25]. Pharbitis plants show a similar effect of day length on the free-running circadian rhythm of night breaks [18]. Pharbitis PnCO mRNA levels are also under circadian regulation. In fact, the free-running circadian peaks of Arabidopsis CO and Pharbitis PnCO genes are quite similar, even though the entrainment and constant conditions used in studies were quite different for the two species light in Arabidopsis and dark in Pharbitis (Figure 2). Other COlike genes in Arabidopsis are circadian regulated, yet their peak expression times and overexpression phenotypes suggest that they differ from CO and PnCO in function [26]. Unfortunately, there are still no data available on PnCO expression in normal photoperiods. PnCO mRNA accumulated in the dark, and long periods of light seemed to reduce the CO mRNA level. A night break treatment, although completely delaying flowering, had no clear immediate or longer term effect on PnCO mRNA levels [5]. The exact mechanism by which CO mediates the photoperiodic response in long-day and short-day plants is still not clear. It is not likely that the differences are intrinsic to the CO protein, as Pharbitis PnCO and Arabidopsis CO were found to have similar effects when expressed at a high

PnCO expression

unstable and should therefore follow a similar pattern [3]. How does CO specifically promote flowering in long days? In Arabidopsis, long days do not cause an overall increase in CO mRNA. At certain time points during the day, the levels of CO mRNA are higher in long-day plants than in short-day plants [3]. In fact, the peak of CO expression is broadened as days become longer, an effect common to other circadian regulated genes in plants and mammals [23,24]. As a result, high levels of CO mRNA during the light on period can only be found in long-day plants [3]. If the same pattern exists for the protein, then the activation of flower-promoting genes by CO might require light [3,23]. Probably as a result to loss of gating of light signals, the elf3 mutant shows abnormally high levels of CO mRNA at dawn in both short and long photoperiods, which might cause early flowering [3]. ELF3 mRNA itself seems to be affected in a similar way in the elf3 mutant [20].

Figure 2

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Flowering

24 (b)

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Circadian rhythms in short-day and long-day plants. (a) When darkgrown Pharbitis seedlings are exposed to a 4 h photoperiod followed by a 48 h dark inductive period, night beak interruptions are most efficient (reduction of flowering) at ~15 and ~39 from the beginning of the light period, suggesting a circadian rhythm [18]. (b) Circadian expression of PnCO in Pharbitis under continuous dark shows an maximum at ~1624 h [5]. (c) Circadian expression of CO in Arabidopsis under continuous light shows a similar pattern [3]. White and black bars represent light and dark, respectively. Hatched bars represent subjective nights.

level in Arabidopsis co mutant plants [5]. It remains possible that the differences lie within the regulatory domains of the down-stream FT and SOC1 genes. Important answers will likely come from comparative data on gene expression, post-transcriptional regulation and in situ localization. Further studies on the CO product, its interacting proteins and its possible modification by light will most likely provide additional insights.

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In plants, changes in photoperiod are thought to be perceived by the leaves, which when induced produce an unknown flowering stimulus known as florigen. This systemic signal is thought to move from the induced leaf to the meristem [1,27]. This was nicely demonstrated when a leaf exposed to inductive conditions caused flowering when grafted to a plant grown in non-inductive conditions. Could this grafted, induced leaf introduce a new rhythm into the non-induced plant? Experiments in mammals show that when explanted from the body, the rhythm within the mammalian SCN reflects the day length the whole animal had been exposed to prior to removal [25]. Plants do not have a centrally localized pacemaker, rather they contain a spatial array of autonomous circadian oscillators [28]. Although different organs on a plant can be entrained independently [28] perhaps at least part of the elusive floral stimulus is the transmissible change in rhythmicity introduced by the induced tissue.
Acknowledgements
We would like to thank George Coupland and Claire Prilleux for insightful comments and the sharing of unpublished data.

References
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