Most organisms, from plants to insects and humans, anticipate the rise and set of the sun through an internal biological timekeeper, called the circadian clock. Plants like the common sunflower use this clock to open their flowers at dawn in time for the arrival of pollinating insects.

Sunflowers are composed of many individual flowers or florets, which are arranged in spirals around a centre following an age gradient: the oldest flowers are on the outside and youngest flowers on the inside. Each day, a ring of florets of different developmental ages coordinates their opening in a specific pattern over the day. For example, petals open at dawn, pollen is presented in the morning, and stigmas, the female organs that receive pollen, unfold in the afternoon. This pattern of flowering, or floret maturation, is repeated every day for five to ten days, creating daily rhythms of flowering across the sunflower head. Previously, it was unclear how florets within each flowering ring synchronize their flowering patterns to precise times during the day.

To find out more, Marshall et al. analysed time-lapse videos of sunflowers that were exposed to different day length and temperature conditions. Sunflowers opened a new floret ring every 24 hours, regardless of the length of the day. In all three day-length scenarios (short, middle, long), the development of the florets remained highly coordinated. Even flowers kept in the dark for up to four days were able to maintain the same daily growth rhythms. This persistence of daily rhythms in the absence of environmental cues suggests that the circadian clock regulates the genetic pathways that cause sunflowers to flower. However, when sunflowers whose circadian rhythms were delayed relative to the sun were placed out in a field, the sunflowers flowered later and thus attracted less pollinators.

Marshall et al. show that the circadian clock is important for regulating flowering patterns in sunflowers to ensure their successful pollination. A better understanding of the interplay between pollinators, flowering plants and their environment will provide more insight into how climate change may affect pollination efficiency. By identifying the genes and pathways underlying flowering patterns, it may be possible to develop breeds that flower at the optimal times of day to promote pollination. This could help mitigate the effects of climate change and declining populations of pollinators.

From the earliest stages of cellular differentiation to reproductive maturity, spatio-temporal regulation of developmental transitions plays an important role in biological success. In some cases, internal temporal rhythms are used to spatially organize a body plan. For example, in vertebrate embryos a molecular ‘segmentation clock’ acts at regular intervals to divide the developing body axis into discrete blocks of cells that later differentiate into individual somites (Richmond and Oates, 2012). In plants, the regular spacing of lateral roots along the length of the primary root axis is controlled by a ‘root clock’ that generates a temporally oscillating developmental signal (Xuan et al., 2020). Thus in both plants and animals, internal body clocks can generate serially repeated organs. These types of clocks are distinct from the circadian oscillator, the biological clock found in most eukaryotes and some prokaryotes (Mosier and Hurley, 2021). Circadian clocks coordinate development on daily and seasonal time scales. In both plants and animals, the circadian clock can integrate environmental cues such as day length and temperature to time the transition to reproductive stages to the appropriate season (Ikegami and Yoshimura, 2012; Inoue et al., 2018). On a diel scale, the circadian clock can coordinate developmental processes with environmental transitions associated with the earth’s rotation, such as leaf movement in plants, eclosion in Drosophila, and conidiation in Neurospora (Brady et al., 2021; Larrondo and Canessa, 2018; McClung, 2019). However, a role for the circadian clock in the spatial patterning of development has not previously been reported.

Circadian clocks in eukaryotes are cell-autonomous systems made up of transcriptional-translational feedback loops that create rhythms of gene expression to regulate biological timing. Circadian rhythms, biological processes regulated by the circadian clock, meet several fundamental criteria (Rensing and Ruoff, 2002). First, they are stably entrained to environmental cues. Second, they persist in free-running environmental conditions. Third, circadian rhythms are temperature compensated such that they maintain a uniform period over a range of ambient temperatures, in contrast to the general rule that the rate of enzymatic processes changes with temperature. In addition, the circadian clock gates certain biological processes so that responsiveness to environmental cues is time-of-day dependent. For example, studies in humans show the timing of drug treatment can be critical to creating an effective therapeutic response (Ruan et al., 2021). A functioning circadian clock whose biological rhythms match that of the environment provides a fitness advantage for many organisms, including plants (Dodd et al., 2005), animals (Miller et al., 2004; Emerson et al., 2008; Spoelstra et al., 2016), bacteria (Ouyang et al., 1998), and fungi (Koritala et al., 2020).

One important way the circadian clock promotes fitness is by controlling timing of reproductive development, for example, by allowing organisms to distinguish between the long and short days of different seasons. In plants, the circadian system mediates the effects of day length during the transition from vegetative to reproductive growth, usually referred to as flowering time (Imaizumi and Kay, 2006). The circadian clock also regulates daily aspects of floral development, for example, coordinating daily rhythms in scent emission and floral opening and closing with pollinator activity (Fenske et al., 2015; Inoue et al., 2018; Muroya et al., 2021; Yon et al., 2017). In addition, the circadian clock is implicated in the uniform eastward orientation observed in mature sunflower plants; this eastward orientation was shown to enhance both male and female reproductive fitness (Atamian et al., 2016; Creux et al., 2021).

Domesticated sunflower is an excellent model system to study the temporal regulation of floral development. Sunflowers are members of the Asteraceae, a highly successful family comprised of about 30,000 distinct species that are widely dispersed around the world (Funk et al., 2009). Asteraceae are characterized by the arrangement of individual florets together in a complex, compressed inflorescence called the capitulum. Individual florets are arranged on the capitulum disk in spiral patterns, a classic type of phyllotaxis observed in many plants (Figure 1A and B). These phyllotactic spirals, called parastichies, are generated by the precise specification of floral primordia, first at the outer rim and then moving into the center of the capitulum disk (Zhang et al., 2021). In domesticated sunflower, specification of the hundreds or even thousands of floret primordia found on a single capitulum takes approximately 10–14 days (Marc and Palmer, 1981; Palmer and Steer, 1985), followed approximately 20 days later by the onset of anthesis, or floral opening, of the first florets (Lindström and Hernández, 2015). Anthesis of all florets on a capitulum head typically occurs over 5–10 days (Seiler, 2015).

Figure 1

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Intriguingly, the timing of anthesis does not follow the gradual and continuous pattern of development seen during specification of floret primordia on the developing capitulum (Marc and Palmer, 1981; Zhang et al., 2021). Instead, discrete developmental boundaries are observed with obvious rings of florets at distinct stages of anthesis (Figure 1E and F). Each ring, or pseudowhorl, undergoes anthesis on successive days. While previous studies examined the effects of hormones and environmental factors on the timing of development of florets within a pseudowhorl (Baroncelli et al., 1990; Lobello et al., 2000), the mechanisms governing the conversion of the continuous spiral pattern of development seen early in floral development (Figure 1A) to the ring-like patterns seen during anthesis (Figure 1E) have not been explored.

Here, we investigate the timing of floral organ development in various environmental conditions. We find that temperature-compensated daily rhythms in development of both male and female floral organs, and the formation of pseudowhorls, persist when flowers are maintained in constant environmental conditions in the absence of light. However, both daily rhythms in development and the formation of pseudowhorls are lost in constant light: late-stage floret maturation occurs continuously along parastichies, with negative impacts on pollinator visits. Importantly, the effects of light on floret development are time-of-day-dependent. Together, these data indicate that late-stage floret development is regulated by the circadian clock. Field experiments reveal that the timing of anthesis affects visits by pollinating insects, thus linking circadian control of floral maturation with plant reproductive success. We propose that pseudowhorls are produced in response to an external coincidence mechanism, in which developmentally competent florets undergo reproductive transitions only when they receive environmental cues at the appropriate time of day. Our data suggest a role for the circadian clock in the spatial as well as temporal control of sunflower development.

Many aspects of floral physiology, including flower opening and scent production, vary across a day (Bloch et al., 2017). In a few cases, such as petal opening and closing in Kalanchoë blössfedliana and flower movements in Nicotiana attenuata, the circadian clock has been shown to regulate these diel rhythms (Bünsow, 1953; Yon et al., 2016). Here, we show that daily rhythms in the development of floral organs in sunflower persist in constant environmental conditions, are temperature-compensated, and exhibit a time-of-day specific response to environmental cues (Figure 3A and D; Figure 4; Figure 5). We therefore conclude that the circadian clock controls floral development in sunflower to ensure release of pollen soon after dawn and the emergence of styles and stigmas late in the day. Although style elongation is largely complete near the end of the first day of floret anthesis (Baroncelli et al., 1990; Figure 2A), stigmas do not become receptive to pollen until the following morning (Putt, 1940; Neff and Simpson, 1990). This temporal separation between release of pollen and stigma receptivity is thought to reduce self-pollination and is prevalent throughout the Asteraceae family (Jeffrey, 2009).

We found that elongation of ovaries, anther filaments, and styles are all under circadian regulation, but respond differently to environmental cues. For example, transfer to constant light caused a complete inhibition of anther filament elongation, significantly slowed ovary expansion, but had no significant effect on style elongation (Figure 3—figure supplement 2). In contrast, transfer to constant darkness slowed style elongation considerably but had only modest effects on growth of ovaries and anther filaments (Figure 3; Figure 3—figure supplement 2). The strong inhibition of style elongation in constant darkness may help explain why rhythms in style emergence were less robust than those of ovary and anther growth in this condition (Figure 3). Responses of the different organs to temperature were also distinct: temperature cycles slowed ovary expansion and had no effect on anther filament growth in either constant darkness or light. In contrast, temperature cycles slowed style elongation rates in constant darkness but not constant light (Figure 3—figure supplement 2). These results are consistent with our previous field studies that showed modest changes in ambient temperature affected style but not anther filament elongation (Creux et al., 2021). Overall, the different sensitivities of floral organs to light and temperature suggest they may be regulated by distinct growth regulatory pathways. This is consistent with studies in other plants, which revealed that different hormones regulate the growth of male and female reproductive organs (Chandler, 2011; Marciniak and Przedniczek, 2019).

While the molecular pathways controlling elongation of anther filaments and styles in sunflowers are likely different, we demonstrate that their common regulation by the circadian clock results in the fast and near-synchronous release of pollen a few hours after dawn. Intriguingly, many bee- and butterfly-pollinated Asteraceae species release pollen in the morning (Budumajji and Raju, 2018; Hipólito et al., 2013; Neff and Simpson, 1990; Valentin-Silva et al., 2016) while at least one bat-pollinated member of the family releases pollen in the early night (Amorim et al., 2021). Since a delay in the timing of pollen release relative to dawn reduces pollinator visits (Figure 7E–H) and negatively affects male reproductive success in sunflower (Creux et al., 2021), it is tempting to speculate that clock regulation of late-stage floret development may be widespread in the Asteraceae. Identification of the mechanisms by which the circadian system coordinates the growth of male and female floral organs to promote timely pollen release will be of great interest.

The ecological and evolutionary success of the Asteraceae family, estimated to contain ~10% of angiosperm species, is attributed in part to their compressed inflorescences that act together as single false flowers to attract pollinators (Mandel et al., 2019). In sunflowers, the individual disk floret primordia are first initiated on the outer perimeter of the very large, flat head meristem and subsequent primordia are initiated following a centripetal pattern to generate the characteristic spiral pattern of immature florets seen across the disk (Figure 1A; Figure 8A; Marc and Palmer, 1981; Palmer and Steer, 1985). Recent experimental and modeling studies in gerbera daisies attributed formation of this spiral pattern to short-range interactions between spatially close initia (Zhang et al., 2021). In contrast, our work suggests that the ring-like pattern of pseudowhorls that emerges during anthesis (Figure 1E–G) is generated by the action of the circadian clock on florets across the capitulum disk, with loss of synchronized daily rhythms in anthesis in constant light and temperature leading to loss of pseudowhorl formation (Figure 6A, B and F).

Figure 8

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How do florets of different developmental ages temporally coordinate anthesis to generate pseudowhorls? We propose that a type of external coincidence model is at work, analogous to the mechanisms underlying photoperiodic control of the transition from vegetative to reproductive growth in many plants (Pittendrigh and Minis, 1964; Yanovsky and Kay, 2003). In our experiments, developmentally competent immature florets respond to a period of darkness that falls during the subjective night and is at least 4.5 hr long by undergoing the growth events that lead to anthesis (rapid ovary expansion followed by the rapid onset of anther filament and then style elongation) (Figure 8B). This is conceptually similar to photoperiodic control of flowering in short day plants such as rice, in which light at night inactivates a clock-regulated transcription factor that promotes the transition to flowering (Izawa et al., 2002).

Although the molecular processes underlying light and circadian control of floral organ growth are not currently known, we speculate that they could involve circadian control of transcription coupled with light regulation of protein activity. For example, transcription factors with a nighttime phase of circadian gene expression but that are degraded in the light might promote growth of floral organs. In constant darkness, the activity of these factors would oscillate and drive near-normal anthesis rhythms. In constant light, or if light occurred during the subjective night, the abundance of these proteins would decrease and anthesis would be slow and uncoordinated across the capitulum disk. A similar mechanism has been shown to underlie the circadian and photoperiodic control of hypocotyl elongation in Arabidopsis (Kunihiro et al., 2011; Nozue et al., 2007).

We suggest that local signals may also contribute to the coordination of floret anthesis across the capitulum disk. We found that imposition of dark treatments during the subjective day delayed anthesis by close to 24 hr (Figure 4; Figure 4—figure supplement 1J), at which point normal rhythmic patterns of anthesis resumed after entrainment to the new light conditions. However, pseudowhorls that developed after these delays contained the same number of florets as those found on plants that did not experience delays (Figure 4—figure supplement 2). This suggests that signaling may occur between florets in adjacent pseudowhorls to control the onset of anthesis. This might be a positive cue from post-anthesis florets to promote development of the next pseudowhorl, or a negative cue from pre-anthesis florets to inhibit development of nearby florets (Figure 8B).

Plants are generally considered diurnal organisms and often have more robust circadian rhythms in constant light than in constant darkness (Michael et al., 2008). We were therefore surprised to find robust rhythms of anthesis in conditions of constant temperature and darkness but not constant temperature and light (Figure 3; Figure 6). Similar observations have been made in Neurospora crassa and in many animals, with circadian rhythms observed in constant darkness but not in constant light (Aschoff, 1979; Sargent et al., 1966). An interesting question for further study is whether in constant light molecular rhythms are present in sunflower florets but developmental rhythms are masked, or whether the floral circadian clock is arrhythmic in this condition. The very rapid resetting of the different phases of organ growth to new light conditions (Figure 4—figure supplement 1) suggests the latter may be true.

Another interesting question is why florets of different developmental ages undergo coordinated anthesis to generate pseudowhorls. It was reported that the foraging activity of both honeybees and native bees on sunflower heads is positively correlated with the number of florets undergoing anthesis at any given time, and that seed set is strongly correlated with the number of bees foraging on heads (DeGrandi-Hoffman and Watkins, 2000; Neff and Simpson, 1990). In the case of the highly social honeybees, hive members communicate via a dance language to quickly reallocate the numbers of foragers sent to different food sources based on resource availability (Visscher and Seeley, 1982). Plants treated with constant light so that floret anthesis was not coordinated across the capitulum disk were much less attractive to pollinating insects than were control plants (Figure 7A–D), presumably due to the lack of pollen release and/or the uncoordinated development of florets across the capitulum disk. We speculate that both factors have an effect, and that the coordinated release of floral rewards by the hundreds of florets undergoing anthesis within a pseudowhorl each morning may be a mechanism to enhance attractiveness to pollinators and thus promote reproductive success.

All source data have been uploaded to Dryad under the following accession codes: 10.25338/B8865X (timelapse scoring), 10.25338/B86358 (pollinator visits), 10.25338/B8963G (consensus scoring), 10.25338/B8CW5R (ovary measurements), and 10.25338/B8HP9F (organ growth kinetics).

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Author details

1. Carine M Marshall

   Department of Plant Biology, University of California, Davis, Davis, United States

   Contribution

   Conceptualization, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7178-4664

2. Veronica L Thompson

   Department of Plant Biology, University of California, Davis, Davis, United States

   Contribution

   Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0500-5639

3. Nicky M Creux

   1. Department of Plant Biology, University of California, Davis, Davis, United States
   2. Department of Plant and Soil Sciences, FABI, Innovation Africa, University of Pretoria, Pretoria, South Africa

   Contribution

   Conceptualization, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4179-6995

4. Stacey L Harmer

   Department of Plant Biology, University of California, Davis, Davis, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing - original draft, Writing - review and editing

   For correspondence

   slharmer@ucdavis.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6813-6682

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