The circadian clock controls temporal and spatial patterns of floral development in sunflower

Reviewer #1 (Public Review):

This study demonstrates the role of the circadian clock in spatiotemporal regulation of floral development. The authors nicely illustrated floral development patterns in domesticated sunflower. In particular, during anthesis, discrete developmental zones, namely pseudowhorls, are established, and hundreds of florets simultaneously undergo maturation in each psudowhorl in a circadian-dependent manner. Consistently, the flower development follows key features of the circadian clock, such as temperature compensation and gating of plant response to environmental stimuli. Evolutionary advantages of this regulation will add more merit to this study.

Reviewer #2 (Public Review):

Little is known about how the circadian clock regulates the timing of anthesis. This manuscript shows that the circadian clock regulates the diurnal rhythms in floral development of the sunflower. The authors have developed a new method to characterize the timing of floral development under normal conditions as well as constant dark and light conditions. The results from the treatment of darkness during the subjective night and day suggest that the circadian clock regulates the growth of ovary, stamen, and style differently.

All clock papers claim that the circadian clock regulates the fitness of organisms, however, it is hard to evaluate how the circadian clock affects the fitness of organisms. The timing of pollen release and stigma maturity is directly related to plant fitness. That's why the authors suggest that the circadian clock in sunflowers increases plant fitness by regulating the timing of anthesis.

Although the authors manipulated the light and temperature to examine the role of the circadian clock in floral development, the weakness of this manuscript is that there is no molecular evidence to show how the clock regulates floral development.

Reviewer #3 (Public Review):

The flowering heads of species in the Asteracaeae comprise large number of flowers, and this phenotype is thought to contribute to their reproductive success. The Harmer lab has developed sunflower as an experimental model to investigate the contribution of circadian regulation to the processes of reproduction in the Asteraceae, and this paper presents a new addition to this line of research.

The novelty of the article is that it resolves unanswered questions around the processes that underlie coordinated flowering within the disc structure of the floral capitulum. The authors demonstrate a role for circadian clock in the temporal structuring of this process. They identify a free running rhythm in constant darkness of floral anthesis, and this rhythm has several key characteristics of circadian rhythms. The data collected also indicate that the circadian clock might gate the response of anthesis to darkness.

I like the presentation of an external coincidence model for the interaction of light and circadian cues in the floral developmental program of the capitulum. However, I wonder whether this is the only potential explanation. The data in Fig. 4C look like classical entrainment responses. Are the authors sure that they are not just seeing an entrainment process within the capitulum, combined with a masking effect of continuous light upon the rhythmic phenotype? I encourage the authors to retain speculation about the coincidence model within the discussion – it's so important for future work – but perhaps consider alternative interpretations of the data also.

Author Response

Reviewer #1 (Public Review):

This study demonstrates the role of the circadian clock in spatiotemporal regulation of floral development. The authors nicely illustrated floral development patterns in domesticated sunflower. In particular, during anthesis, discrete developmental zones, namely pseudowhorls, are established, and hundreds of florets simultaneously undergo maturation in each psudowhorl in a circadian-dependent manner. Consistently, the flower development follows key features of the circadian clock, such as temperature compensation and gating of plant response to environmental stimuli. Evolutionary advantages of this regulation will add more merit to this study.

We thank the reviewer for this suggestion. We have performed new experiments (Figures 7 and 7-S1) that demonstrate that delays in anthesis relative to dawn and disruption of pseudowhorl formation both negatively impact pollinator visits to flowers. These findings suggest that circadian and light regulation of floral anthesis may have significant impacts on male reproductive fitness.

Reviewer #2 (Public Review):

Little is known about how the circadian clock regulates the timing of anthesis. This manuscript shows that the circadian clock regulates the diurnal rhythms in floral development of the sunflower. The authors have developed a new method to characterize the timing of floral development under normal conditions as well as constant dark and light conditions. The results from the treatment of darkness during the subjective night and day suggest that the circadian clock regulates the growth of ovary, stamen, and style differently.

All clock papers claim that the circadian clock regulates the fitness of organisms, however, it is hard to evaluate how the circadian clock affects the fitness of organisms. The timing of pollen release and stigma maturity is directly related to plant fitness. That's why the authors suggest that the circadian clock in sunflowers increases plant fitness by regulating the timing of anthesis.

Although the authors manipulated the light and temperature to examine the role of the circadian clock in floral development, the weakness of this manuscript is that there is no molecular evidence to show how the clock regulates floral development.

We acknowledge that this study does not demonstrate the molecular mechanisms by which the circadian clock and environmental sensing pathways regulate floral anthesis in sunflower. However, we feel that our demonstration that the circadian clock is involved in the generation of spatial patterns of development on the sunflower inflorescence disk is in itself novel and significant.

Reviewer #3 (Public Review):

The flowering heads of species in the Asteracaeae comprise large number of flowers, and this phenotype is thought to contribute to their reproductive success. The Harmer lab has developed sunflower as an experimental model to investigate the contribution of circadian regulation to the processes of reproduction in the Asteraceae, and this paper presents a new addition to this line of research.

The novelty of the article is that it resolves unanswered questions around the processes that underlie coordinated flowering within the disc structure of the floral capitulum. The authors demonstrate a role for circadian clock in the temporal structuring of this process. They identify a free running rhythm in constant darkness of floral anthesis, and this rhythm has several key characteristics of circadian rhythms. The data collected also indicate that the circadian clock might gate the response of anthesis to darkness.

I like the presentation of an external coincidence model for the interaction of light and circadian cues in the floral developmental program of the capitulum. However, I wonder whether this is the only potential explanation. The data in Fig. 4C look like classical entrainment responses. Are the authors sure that they are not just seeing an entrainment process within the capitulum, combined with a masking effect of continuous light upon the rhythmic phenotype? I encourage the authors to retain speculation about the coincidence model within the discussion- it's so important for future work- but perhaps consider alternative interpretations of the data also.

We thank the reviewer for their positive comments and overall enthusiasm for the study. We agree that it is entirely plausible that continuous light masks circadian clock-controlled rhythms in floral organ development; in our view, this is a restatement of the external coincidence model. We argue that in developing sunflowers, a circadian clock-regulated process controls elongation of floret organs. Normal development depends upon a dark period of at least 4.5 hours occurring during the subjective night. In constant light conditions, or early in re-entrainment when the dark period occurs during the subjective day, normal development is inhibited. This model is analogous to the photoperiodic control of flowering time in short-day plants, in which light perceived during the subjective night inhibits the floral transition.