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

Biological rhythms are ubiquitous. They can be generated by circadian oscillators, which produce daily rhythms in physiology and behavior, as well as by developmental oscillators such as the segmentation clock, which periodically produces modular developmental units. Here, we show that the circadian clock controls the timing of late-stage floret development, or anthesis, in domesticated sunflowers. In these plants, up to thousands of individual florets are 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. Delays in the phase of floral anthesis delay morning visits by pollinators, while disruption of circadian rhythms in floral organ development causes loss of pseudowhorl formation and large reductions in pollinator visits. We therefore 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.

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.

A TILLING by sequencing approach to identify induced mutations in sunflower genes.

The Targeting Induced Local Lesions in Genomes (TILLING) technology is a reverse genetic strategy broadly applicable to every kind of genome and represents an attractive tool for functional genomic and agronomic applications. It consists of chemical random mutagenesis followed by high-throughput screening of point mutations in targeted genomic regions. Although multiple methods for mutation discovery in amplicons have been described, next-generation sequencing (NGS) is the tool of choice for mutation detection because it quickly allows for the analysis of a large number of amplicons. The aim of the present work was to screen a previously generated sunflower TILLING population and identify alterations in genes involved in several important and complex physiological processes. Twenty-one candidate sunflower genes were chosen as targets for the screening. The TILLING by sequencing strategy allowed us to identify multiple mutations in selected genes and we subsequently validated 16 mutations in 11 different genes through Sanger sequencing. In addition to addressing challenges posed by outcrossing, our detection and validation of mutations in multiple regulatory loci highlights the importance of this sunflower population as a genetic resource.