DOI: 10.25338/b8865x
title: Sunflower timelapse scoring

description: 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.
description: 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<sup>-2</sup>
 s<sup>-1</sup>) 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<sup>-2</sup>
 s<sup>-1</sup> 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 &gt;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.