RESEARCH | REPORTS PLANT SCIENCE the east during the subjective night and toward the west during the subjective day, with times of maximal inclination corresponding to subjective dawn and dusk. As is true for many types of cir- Circadian regulation of sunflower cadian outputs after withdrawal of environmen- tal signals, rhythmic movements dampened over heliotropism, floral orientation, and time (Fig. 1D). Another way to distinguish rhythms regulated pollinator visits by the circadian clock from those directly in- duced by environmental cues is to maintain organisms in light-dark cycles with total period Hagop S. Atamian,1 Nicky M. Creux,1 Evan A. Brown,2 Austin G. Garner,2 lengths (T-cycles) that differ from 24 hours (14). We Benjamin K. Blackman,2,3 Stacey L. Harmer1* examined heliotropism in a growth chamber with four directional blue LED (light-emitting diode) Young sunflower plants track the Sun from east to west during the day and then reorient lights sequentially turned on and off to mimic the during the night to face east in anticipation of dawn. In contrast, mature plants cease Sun’s daily path. After several days in a 24-hour movement with their flower heads facing east. We show that circadian regulation of T-cycle (16L:8D), plants bent toward the light directional growth pathways accounts for both phenomena and leads to increased source during the day so that “westward” move- vegetative biomass and enhanced pollinator visits to flowers. Solar tracking movements are ment culminated at dusk. Anticipatory “eastward” driven by antiphasic patterns of elongation on the east and west sides of the stem. Genes movement then occurred throughout the dark implicated in control of phototropic growth, but not clock genes, are differentially expressed period (Fig. 1E and fig. S2). Upon transfer to a on the opposite sides of solar tracking stems. Thus, interactions between environmental 30-hour T-cycle (20L:10D), maximal “westward” response pathways and the internal circadian oscillator coordinate physiological processes inclination no longer occurred at the light-dark with predictable changes in the environment to influence growth and reproduction. transition, and directionality of nighttime move- ment was erratic. The return to a 24-hour T-cycle ost plant species display daily rhythms and enhances plant performance in the natural reestablished anticipatory nighttime movement in organ expansion that are regulated environment. that began when lights were turned off (Fig. 1E on August 5, 2016 by complex interactions between light- Sunflower stems exhibit heliotropic movement and fig. S2). The complex patterns in 30-hour M and temperature-sensing pathways and such that their shoot apices shift from facing east T-cycles suggest uncoordinated growth controlled the circadian clock (1). These rhythms at dawn to facing west at dusk as they track the by both environmental response pathways and arise in part because the abundance of growth- Sun’s relative position. Shoot apices then reorient the circadian clock. Thus, the circadian clock related factors such as light signaling compo- at night to face east in anticipation of dawn (Fig. guides solar tracking in sunflowers. nents and hormones (e.g., gibberellins and 1A and movie S1) (12, 13). We disrupted this Because sunflowers lack pulvini, the special- auxins) are regulated by both the circadian clock process in two ways, either by rotating potted ized motor organs that mediate solar tracking and light (2–4). A further layer of regulation is plants every evening so that they faced east at in some species (15), we hypothesized that reg- afforded by circadian gating of plant responsive- nightfall (and thus faced west each morning ulated stem elongation might drive solar track- ness to these stimuli, with maximal sensitivity to after directional nighttime growth) or by tether- ing. We observed a gradual reduction in the light during the day (5) and to gibberellin and ing plant stems to solid supports to limit their amplitude of heliotropic movements as plants auxin at night (6, 7). tracking movements. In multiple trials, we de- reached maturity, correlating with cessation of http://science.sciencemag.org/ Because the direction and amount of solar tected ~10% decreases in both dry biomass and stem elongation (Fig. 2, A and B, and Fig. 3A). irradiation undergo predictable daily changes, leaf area of the manipulated plants relative to To further investigate the involvement of stem light capture might be optimized by links be- controls in both types of experiments (Fig. 1B elongation in heliotropism, we examined solar tween the circadian clock and directional growth and fig. S1; significance assessed using linear tracking and stem growth rates in dwarf2 (dw2) pathways. One such growth pathway is photo- mixed-effect models with treatment as fixed effect, sunflowers, which are deficient in the produc- tropism, in which plants align their photosynthetic trial and leaf number as random effects), demon- tion of gibberellin growth hormones (16). In the organs with the direction of incoming light. Pho- strating that solar tracking promotes growth. absence of exogenous gibberellin, dw2 plants totropism was first recognized by Charles Darwin The nighttime reorientation of young sun- have very short stems and no perceptible heliotro- Downloaded from (8) and is mediated by the perception of blue flowers in the absence of any obvious environ- pism (movie S2). Treatment with exogenous light by phototropin photoreceptors that then mental signal suggests involvement of the gibberellin transiently restored normal elonga- trigger asymmetric growth via the auxin signal- circadian system; however, an alternative expla- tion (Fig. 2A) and heliotropism (Fig. 2B) in the ing pathway (9). Heliotropism, or solar tracking, nation would be an hourglass-like timing mech- mutant. Between days 7 and 14 after the last is a more dynamic form of phototropism, with anism. To distinguish between these possibilities, gibberellin application, stem elongation and the aerial portions of the plant following the Sun’s we examined the kinetics of nighttime reorienta- amplitude of solar tracking rhythms coordinately movement throughout the day. Some heliotropic tion near the summer solstice and the fall equi- diminishedby~35%(Fig.2,AandB).Thus, plants such as sunflowers also reorient during nox. The rate of apical movement at night was stem elongation is essential for heliotropism. the night so that their leaves and apices face east higher at midsummer [16 hours light: 8 hours Many plant species show daily rhythms in before sunrise (10, 11). Here we show that he- dark (16L:8D)] than during the longer nights of nondirectional stem and leaf growth (1). We liotropism in the common sunflower, Helianthus autumn (12L:12D), so that in each case plant hypothesized that heliotropism results from dif- annuus, is generated by the coordinate action of apices face fully east just before dawn (Fig. 1C). ferential elongation on opposite sides of stems. light-signaling pathways and the circadian clock We next investigated whether plants continue Indeed, the growth pattern on the east side of rhythmic tracking movements in the absence of solar tracking sunflower stems was different 1Department of Plant Biology, University of California, One directional light cues. Sunflower plants grown from that on the west side (Fig. 2C). Growth Shields Avenue, Davis, CA 95616, USA. 2Department of in pots in the field in 14L:10D conditions were rates on the east side were high during the day Biology, University of Virginia, PO Box 400328, heliotropic (Fig. 1D). When moved to a growth and very low at night, whereas growth rates on 3 Charlottesville, VA 22904, USA. Department of Plant and chamber with constant, fixed overhead lighting, thewestsidewerelowduringthedayandhigher Microbial Biology, University of California, 111 Koshland Hall, Berkeley, CA 94720, USA. these plants maintained their directional growth at night. The higher growth rate on the east *Corresponding author. Email: [email protected] rhythms for several days. Plants reoriented toward versus west side of the stem during the day SCIENCE sciencemag.org 5AUGUST2016• VOL 353 ISSUE 6299 587 RESEARCH | REPORTS enables the shoot apex to move gradually from growth patterns might be similar to the overall chamber with overhead lighting. Consistent with east to west. At night, the higher growth rate growth rhythms of sunflower plants not man- reports in pea and zinnia (17, 18), stem elongation on the west side culminates in the apex facing ifesting heliotropism. We therefore monitored growth rates were higher at night than during east at dawn. We postulated that one of these plants maintained in 16L:8D cycles in a growth the day under these controlled conditions (Fig. 107 16L:8D 100 Dusk Midnight Dawn 12L:12D 95 99 96 East 91 92 West 91 87 88 Angle of curvature (°) 83 83 84 0 4 8 12162024 0 12 0 12 0 12 24 36 48 Time (h) 25 = control 40 = rotated T-24 T-30 T-24 20 ) 2 30 95 15 20 90 10 5 10 85 Leaf area (cm Dry mass/plant (g) 0 0 80 CR CR C R CR CR Angle of curvature (°) leaf #: 7,8 9,10 11,12 13,14 0 24 48 72 96 120 150 180 210 240 270 294 318 342 Time (h) on August 5, 2016 Fig. 1. The circadian clock regulates solar tracking. (A) Nighttime re- ments after transfer from field to continuous light and temperature conditions. orientation of stem and shoot apex. (B) Disruption of solar tracking by In (C) and (D), white areas denote daytime; dark and light gray areas represent daily evening 180° rotation of experimental plants results in a 7.5% reduction night and subjective night, respectively [mean ± SEM, n =3plants].(E)The in biomass (left) and an 11% reduction in leaf area (right) compared with onset of “eastward” movement in a growth chamber equipped with four 360°-rotated control plants [mean ± SEM (error bars), n =80plants,P =0.01 directional lights is consistently phased with lights being turned off in 24-hour (biomass) and 1 × 10–6 (leaf area), mixed effect linear regression models].