Biological rhythms are ubiquitous. They can be generated by circadian oscillators, which produce daily rhythms in physiology and behavior, as well as by developmental oscillator such as the segmentation clock, which produces modular developmental units in a periodic fashion. Here, we show that the circadian clock controls the timing of late-stage floret development, or anthesis, in domesticated sunflower. In these plants, what appears to be a single inflorescence consists of up to thousands of individual florets tightly packed onto a capitulum disk. While early floret development occurs continuously across capitula to generate iconic spiral phyllotaxy, during anthesis floret development occurs in discrete ring-like pseudowhorls with up to hundreds of florets undergoing simultaneous maturation. We demonstrate circadian regulation of floral organ growth and show that the effects of light on this process are time-of-day dependent. Disruption of circadian rhythms in floral organ development causes loss of pseudowhorl formation. Thus, we show that the sunflower circadian clock acts in concert with environmental response pathways to tightly synchronize the anthesis of hundreds of florets each day, generating spatial patterns on the developing capitulum disk. This coordinated mass release of floral rewards at predictable times of day likely promotes pollinator visits and plant reproductive success.

Sunflower seeds of HA412 HO (USDA ID: PI 603993) genotype were planted into small pots of soil (Sunshine Mix #1, Sun Gro Horticulture) and germinated with a plastic lid in a PGV36 growth chamber (Conviron, Winnipeg, MB, Canada) at 25°C with 16 hours light (provided by metal halide and incandescent lamps, 300 μmol m^-2 s^-1) and 8 hours darkness per day. Plants were watered with nutrient water containing a N-P-K macronutrient ratio of 2:1:2. Two weeks after sowing, seedlings were transplanted to 2-gallon pots with 1 scoop of Osmocote fertilizer (SMG Brands). Approximately 60 days after sowing, sunflower capitula entering anthesis in their first pseudowhorl were transferred to an PGR15 growth chamber (Conviron, Winnipeg, MB, Canada; 200 μmol m^-2 s^-1 provided by metal halide and incandescent lamps) to image floret development under the indicated environmental conditions. Transfer to experimental conditions occurred at ZT 0. Sunflower stalks and capitula were taped to bamboo stakes (to avoid capitulum moving out of the camera frame). Raspberry Pi NoIR V2 cameras were mounted on Raspberry Pi 3 model B computers (Raspberry Pi, Cambridge, UK); cameras were fitted with LEE 87 75x75mm infrared (IR) filters (Lee Filters, Andover, England). Computers were programmed to take a photo every 15 minutes. Infrared LEDs (Mouser Electronics, El Cajon, CA, USA) were programmed to flash during image capture so that the capitula were visible in the dark without disrupting plant growth. Sunflowers were imaged immediately upon transfer to experimental conditions, and through anthesis of all florets in the capitulum. The 15-minute interval images were analyzed sequentially in a stack on ImageJ (Schneider et al., 2012). For each image, the ovaries, stamens, and styles were scored for a change in size from the previous image. Ovary growth was seen as corolla tube swelling above the immature capitulum surface (Figures 1E–G). Late-stage anther filament elongation was observed starting from when the corolla tube cracked open to reveal the stamen tube until its full extension above the corolla surface (Figures 1D–II, 1G). Late-stage style elongation was observed starting with the visible extrusion of pollen out of the top of the anther tube and ending with the style fully extended (Figures 1D–III, 1G). Organs were classified as growing when >5% of the florets in a pseudowhorl showed a change in length in one time-lapse image relative to the previous one. Growth was measured qualitatively as active or inactive.

Sunflower timelapse scoring

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