Arabidopsis synchronizes jasmonate-mediated defense with insect circadian behavior

Abstract

Diverse life forms have evolved internal clocks enabling them to monitor time and thereby anticipate the daily environmental changes caused by Earth's rotation. The plant circadian clock regulates expression of about one-third of the Arabidopsis genome, yet the physiological relevance of this regulation is not fully understood. Here we show that the circadian clock, acting with hormone signals, provides selective advantage to plants through anticipation of and enhanced defense against herbivory. We found that cabbage loopers (Trichoplusia ni) display rhythmic feeding behavior that is sustained under constant conditions, and plants entrained in light/dark cycles coincident with the entrainment of the T. ni suffer only moderate tissue loss due to herbivory. In contrast, plants entrained out-of-phase relative to the insects are significantly more susceptible to attack. The in-phase entrainment advantage is lost in plants with arrhythmic clocks or deficient in jasmonate hormone; thus, both the circadian clock and jasmonates are required. Circadian jasmonate accumulation occurs in a phase pattern consistent with preparation for the onset of peak circadian insect feeding behavior, providing evidence for the underlying mechanism of clock-enhanced herbivory resistance. Furthermore, we find that salicylate, a hormone involved in biotrophic defense that often acts antagonistically to jasmonates, accumulates in opposite phase to jasmonates. Our results demonstrate that the plant circadian clock provides a strong physiological advantage by performing a critical role in Arabidopsis defense.

Fig. 1.

Arabidopsis is more resistant to herbivory when entrained in-phase rather than out-of-phase with T. ni looper entrainment. (A) Light/dark cycle entrainment scheme and experimental protocol. Rectangles symbolize 12-h periods of light (open), darkness (filled), darkness representing subjective day (light gray), and darkness representing subjective night (dark gray). The two arrows represent timing of T. ni looper addition and looper removal, respectively. Double vertical bars symbolize the shift from light/dark cycles to constant darkness. (B) Photographs of representative plant tissue remaining from plants entrained in-phase and out-of-phase with looper entrainment after 72 h of plant–T. ni coincubation. (C) Area of plant tissue remaining from plants entrained in-phase (white bars) and out-of-phase (filled bars) with T. ni entrainment after 72 h of incubation without (control) or with T. ni loopers (looper). Mean area ± SE; n = 6; *P < 0.0002; two-tailed paired t test. (D) Looper wet weights. Mean ± SE; n = 15; *P < 0.05; two-tailed paired t test. (E) Representative loopers at 72 h postcoincubation. (Scale bar, 0.5 mm.)

Fig. 2.

T. ni feeding is circadian-regulated, with enhanced eating during subjective day. T. ni entrained in 12-h light/dark cycles were provided food under (A) light/dark or (B) constant dark conditions. Diet weight lost as a result of looper feeding after each 4-h interval is graphed. Graphed values were determined by calculating the difference of diet weight before and after incubation with loopers and, to account for evaporative weight loss, subtracting the weight difference of comparable diet samples before and after incubation without loopers for the same 4-h interval under similar conditions. Fresh diet was given every 4 h. Mean ± SE; n = 3.

Fig. 3.

Arrhythmic Arabidopsis plants lack enhanced herbivory resistance when entrained in-phase with T. ni loopers. (A) Photographs of representative plant tissue remaining from CCA1-OX and lux2 entrained in-phase and out-of-phase with T. ni entrainment after 72 h of plant–T. ni coincubation. (B) Area of plant tissue remaining from plants entrained in-phase (white bars) and out-of-phase (filled bars) with T. ni entrainment after 72 h of incubation without (control) or with T. ni (looper). Mean area ± SE; n = 6; P < 0.8; two-tailed paired t test. (C) Wet weights of T. ni fed on in-phase (open bars) or out-of-phase (filled bars) plants. Mean ± SE; n = 15; P < 0.05; two-tailed paired t test. (D) Representative T. ni loopers at 72 h postcoincubation. (Scale bars, 0.5 mm.)

Fig. 4.

Jasmonates are required for enhanced herbivory resistance when entrained in-phase with T. ni loopers, and jasmonates and salicylate accumulation patterns show circadian rhythms with opposite phasing. (A) Photographs of representative plant tissue remaining from gl-1, aos, and jar1 entrained in-phase and out-of-phase with T. ni entrainment after 72 h of plant–T. ni coincubation. (B) Area of plant tissue remaining from plants entrained in-phase (white bars) and out-of-phase (filled bars) with T. ni entrainment after 72 h of incubation without (control) or with T. ni (looper). Mean area ± SE; n = 6; *P < 0.0002; two-tailed paired t test. (C) Wet weights of T. ni fed on in-phase (open bars) or out-of-phase (filled bars) plants. Mean ± SE; n = 15; *P < 0.05; two-tailed paired t test. (D) Representative T. ni loopers at 72 h postcoincubation. (Scale bars, 0.5 mm.) (E) Jasmonate and salicylate accumulation patterns are circadian-regulated with opposite phasing. Jasmonates peak in the middle of subjective day (black line) and salicylates (gray line) peak in the middle of subjective night. Mean ± SE; n = 3. fw, fresh weight.