Volume 23, Issue 13, Pages 1235-1241 (July 2013) Postharvest Circadian Entrainment Enhances Crop Pest Resistance and Phytochemical Cycling  Danielle Goodspeed, John D. Liu, E. Wassim Chehab, Zhengji Sheng, Marta Francisco, Daniel J. Kliebenstein, Janet Braam  Current Biology  Volume 23, Issue 13, Pages 1235-1241 (July 2013) DOI: 10.1016/j.cub.2013.05.034 Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 1 The Circadian Clock Regulates Antiherbivore Metabolite Accumulation in Arabidopsis, and Postharvest Cabbage Can Be Re-entrained by Light-Dark Cycles to Demonstrate Phase-Dependent Herbivore Resistance and Circadian Accumulation of Glucosinolates (A) Total Arabidopsis glucosinolates accumulate with circadian regulation. Means ± SE; n = 6. See also Figure S1A. (B) Photographs of representative plant tissue remaining from wild-type (Col-0) and myb28myb29 entrained in phase and out of phase with T. ni after 72 hr of plant-T. ni coincubation. See also Figure S1B. (C) Area of plant tissue lost from plants entrained in phase (green bars) and out of phase (red bars) with T. ni entrainment after 72 hr of incubation with T. ni. Mean areas ± SE; n = 6. Phenotypes with different letters were statistically different at p < 0.05 with a one-way ANOVA with Tukey’s honestly significant difference multiple comparison test; those with the same letter designation were statistically nonsignificant for a difference. See also Figure S1B. (D) Photographs of representative cabbage disks entrained in phase or out of phase with T. ni entrainment after 72 hr of cabbage-T. ni coincubation. Tissue dark spots and holes are sites of herbivory damage. (E) Tissue weight loss remaining from cabbage disks entrained in phase and out of phase with T. ni entrainment after 72 hr of coincubation. Mean areas ± SE; n = 6. ∗p < 0.005, unpaired t test. (F) Representative T. ni larvae at 72 hr after coincubation with cabbage. The scale bar represents 0.5 mm. (G) T. ni larvae weight gain. Means ± SE; n = 18. ∗p < 0.005, unpaired t test. (H) Total glucosinolates accumulate with circadian rhythmicity in re-entrained cabbage. Means ± SE; n = 6. See also Figure S1. Current Biology 2013 23, 1235-1241DOI: (10.1016/j.cub.2013.05.034) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 2 The Ability of Postharvest Cabbage to Demonstrate Enhanced Herbivory Resistance after In-Phase Entrainment Is Lost over Time (A) Photographs of representative cabbage disks, maintained in light-dark cycles either in phase or out of phase with T. ni, 72 hr after incubation with T. ni larvae. Entrainment was initiated the day of purchase (0 days) or 3, 6, or 9 days, as indicated, after purchase. Dark spots and holes are sites of herbivory damage. (B) Photographs of representative T. ni larvae at 72 hr after coincubation with cabbage disks described in (A). Scale bars represent 0.5 mm. (C) T. ni larvae weight gain. Means ± SE; n = 18. ∗p < 0.05, two-tailed unpaired t test. Current Biology 2013 23, 1235-1241DOI: (10.1016/j.cub.2013.05.034) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 3 Maintenance of Light-Dark Cycles Enhances T. ni Resistance and Promotes Circadian Accumulation of the Anticancer Glucosinolate 4MSO in B. oleracea (A) Photographs of representative cabbage disks maintained in light-dark cycles in phase with T. ni or maintained in constant light or darkness 72 hr after incubation with T. ni larvae. Dark spots and holes are sites of herbivory damage. (B) Tissue weight loss from cabbage disks described in (A). Mean areas ± SE; n = 6. ∗p < 0.005, unpaired t test. (C) Representative T. ni larvae at 72 hr after coincubation with cabbage disks described in (A). The scale bar represents 0.5 mm. (D) T. ni larvae weight gain. Means ± SE; n = 6. ∗p < 0.005, unpaired t test. (E) Tissue weight loss from cabbage disks that were entrained in light-dark cycles in phase with T. ni or maintained in constant light and then provided together to T. ni larvae, enabling the insects to choose among the different disks. Means ± SE; n = 15. ∗p < 0.05, two-tailed unpaired t test. (F) The anticancer glucosinolate 4-methylsulfinylbutyl (4MSO) accumulates with circadian rhythmicity in cabbage maintained at 22°C under light-dark cycles (green line); under constant light (gray line) or constant darkness (black line), rhythmic accumulation is lost. Means ± SE; n = 3. (G) 4MSO also accumulates with circadian rhythmicity in cabbage maintained at 4°C under light-dark cycles (green line) but not under constant light (gray line) or constant darkness (black line). Means ± SE; n = 3. See also Figure S2. Current Biology 2013 23, 1235-1241DOI: (10.1016/j.cub.2013.05.034) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 4 The Circadian Clock of Diverse Vegetables and Fruits Can Be Re-entrained Postharvest by Light-Dark Cycles and Confers Phase-Dependent Herbivore Resistance (A) Photographs of representative leaf disk (lettuce and spinach) or whole organs (zucchini, sweet potato, carrot, and blueberry) entrained in phase or out of phase with T. ni entrainment after 72 hr of plant tissue-T. ni coincubation. Tissue damage is apparent as a loss of disk integrity in lettuce and spinach, exposure of inner white tissue of zucchini, exposure of inner dark tissue of sweet potato, holes and crevices in carrot, and loss of outer tissue layer in blueberries. (B) Tissue weight loss from plant tissue entrained in phase and out of phase with T. ni entrainment after 72 hr of coincubation. Mean area ± SE; n = 6. ∗p < 0.005, unpaired t test. (C) Representative T. ni larvae at 72 hr after coincubation. Note that T. ni accumulate pigment from their host; for example, zucchini-fed larvae are green, whereas carrot-fed larvae are orange and blueberry-fed larvae are blue. Scale bars represent 0.5 mm. (D) T. ni larvae weight gain. Means ± SE; n = 18. ∗p < 0.005, unpaired t test. Current Biology 2013 23, 1235-1241DOI: (10.1016/j.cub.2013.05.034) Copyright © 2013 Elsevier Ltd Terms and Conditions