Antiphase light and temperature cycles affect PHYTOCHROME B-controlled ethylene sensitivity and biosynthesis, limiting leaf movement and growth of Arabidopsis

Abstract

In the natural environment, days are generally warmer than the night, resulting in a positive day/night temperature difference (+DIF). Plants have adapted to these conditions, and when exposed to antiphase light and temperature cycles (cold photoperiod/warm night [-DIF]), most species exhibit reduced elongation growth. To study the physiological mechanism of how light and temperature cycles affect plant growth, we used infrared imaging to dissect growth dynamics under +DIF and -DIF in the model plant Arabidopsis (Arabidopsis thaliana). We found that -DIF altered leaf growth patterns, decreasing the amplitude and delaying the phase of leaf movement. Ethylene application restored leaf growth in -DIF conditions, and constitutive ethylene signaling mutants maintain robust leaf movement amplitudes under -DIF, indicating that ethylene signaling becomes limiting under these conditions. In response to -DIF, the phase of ethylene emission advanced 2 h, but total ethylene emission was not reduced. However, expression analysis on members of the 1-aminocyclopropane-1-carboxylic acid (ACC) synthase ethylene biosynthesis gene family showed that ACS2 activity is specifically suppressed in the petiole region under -DIF conditions. Indeed, petioles of plants under -DIF had reduced ACC content, and application of ACC to the petiole restored leaf growth patterns. Moreover, acs2 mutants displayed reduced leaf movement under +DIF, similar to wild-type plants under -DIF. In addition, we demonstrate that the photoreceptor PHYTOCHROME B restricts ethylene biosynthesis and constrains the -DIF-induced phase shift in rhythmic growth. Our findings provide a mechanistic insight into how fluctuating temperature cycles regulate plant growth.

Figure 1.

Rhythmic growth and movement of leaves under control and −DIF conditions. A, Two 36-d-old Arabidopsis (Col-0) rosette plants exposed to −DIF (top) or control (+DIF; bottom) conditions and photographed at the end of the fourth photoperiod. B, Projected oscillations for leaves developing 7 d under control (solid line) or −DIF (dashed line) conditions. se values are depicted per 20-min time point as shading; n = 8 leaves. Gray areas indicate the dark period. C, Closeup of B for day 4, depicting an example of amplitude, phase, and phase shift for control leaves compared with −DIF. Error bars represent se; n = 8. D, Average amplitudes of days 2 to 6 calculated from the projected oscillations in B for leaves developing under control (black bar) or −DIF (gray bar) conditions. Error bars represent se; n = 8. E, The phase of oscillations for leaves developing under −DIF conditions (dashed line, white circles) shifts compared with the control (solid line, black triangles). Error bars represent se; n = 8. [See online article for color version of this figure.]

Figure 2.

Ethylene signaling controls amplitude and phase of diurnal leaf movement. A, Average amplitudes (days 2–6) calculated from the projected oscillations (in B and C) for leaves treated with mock (gray bar) or 50 µm STS (black bar) and for Col-0 (light gray bar), etr1-1 (black bar), and ein2-1 (dark gray bar) developing under control conditions. Error bars represent se; n = 8. B, Projected oscillations of Col-0 leaves under control conditions treated with 50 µm STS (solid line) compared with mock (dashed line). Error bars represent se; n = 8. Gray areas indicate the dark period. C, Projected oscillations for leaves of etr1-1 (solid black line) and ein2-1 (solid gray line) compared with the Col-0 wild type (black dashed line) under control conditions. Error bars represent se; n = 8. Gray areas indicate the dark period. D, Phase shifts of leaf movements between control and −DIF conditions for leaves treated with mock or 50 µm STS and for Col-0, ein2-1, and etr1-1. Significant phase shifts (P < 0.05) are indicated with arrows, and nonsignificant shifts are indicated with bars. Each arrow depicts the direction and strength of the shift in phase: the start of the arrow indicates average phase during days 2 to 6 under control conditions, and the arrowhead indicates the average phase for −DIF (days 2–6). Error bars represent se; n = 8.

Figure 3.

Ethylene restores growth and movement under −DIF. A, Average amplitudes of days 2 to 6 calculated from the projected oscillations (in B and C) of leaves treated with mock (white bar), increasing concentrations of ethephon (gray to black bars) and in Col-0 (light gray bar), etr1-7 (black bar), and etr1-6 etr2-3 ein4-4 (gray bar) developing under −DIF conditions. Error bars represent se; n = 8. B, Ethephon dose response of projected oscillations for Col-0 leaves developing on plants sprayed with increasing ethephon concentrations (0.25–1 mm; gray to black lines) compared with mock (0.0 mm; dashed line) at the start (t = 0 h) of −DIF treatment. Error bars represent se; n = 8. Gray areas indicate the dark period. C, Projected oscillations of leaves of the constitutive ethylene signaling mutant etr1-7 (black line) compared with the Col-0 wild type (dashed line) under −DIF conditions. Error bars represent se; n = 8. Gray areas indicate the dark period. D, Phase shifts of leaf movements between control and −DIF conditions for leaves treated with mock or 1 mm ethephon and for Col-0, etr1-7, and etr1-6 etr2-3 ein4-4. Significant phase shifts (P < 0.05) are indicated with arrows, and nonsignificant shifts are indicated with bars. Each arrow depicts the direction and strength of the shift in phase: the start of the arrow indicates average phase during days 2 to 6 under control conditions, and the arrowhead indicates the average phase for −DIF (days 2–6). Error bars represent se; n = 8.

Figure 4.

Ethylene biosynthesis in the petiole is reduced under −DIF conditions. A, Diurnal ethylene emissions of Col-0 rosette plants growing under control (solid line, black triangles) or −DIF (dashed line, white circles) conditions. Error bars represent se; n ≥ 10. Gray areas indicate the dark period. B, Typical ACS2 promoter activity pattern obtained using histochemical GUS staining in leaves of equal age developed under control or −DIF conditions on 4-week-old plants. C, Promoter activity analysis of ACS2 using histochemical GUS staining in leaves developed during 10 d of control or −DIF conditions in 4-week-old plants. D, Average amplitudes of acs2-1 and acs2-2 leaves developing under control (black bars) or −DIF (gray bars) conditions compared with the Col-0 wild type, calculated from days 2 to 6 of the projected oscillations (in E). Error bars represent se; n = 8. E, Projected oscillations of acs2-1 (dark blue line) and acs2-2 (light blue line) leaves compared with the Col-0 wild type (black dashed line) under control (top) and −DIF (bottom) conditions. Error bars represent se; n = 8. Gray areas indicate the dark period. F, Phase shifts of leaf movement between control and −DIF conditions for acs2-1, acs2-2, and Col-0 wild-type leaves. A significant phase shift (P < 0.05) is indicated with the arrow, and nonsignificant shifts are indicated with bars. The arrow depicts the direction and strength of the shift in phase: the start of the arrow indicates the average phase during days 2 to 6 under control conditions, and the arrowhead indicates the average phase for −DIF (days 2–6). Error bars represent se; n = 8. G, ACC content of acs2-1 and Col-0 wild-type petioles harvested on the peak of ethylene emission (in A) under −DIF or control conditions. Error bars represent se; n = 8. FW, Fresh weight. H, Average amplitudes of days 2 to 6 calculated from the projected oscillations (Supplemental Fig. S4, F and G) of leaves treated with 0.5% (w/w) ACC (dark bars) or mock (light bars) lanolin paste (approximately 1 mg) on the petiole (solid bars) or the leaf blade (dashed bars) under −DIF conditions. Error bars represent se; n = 8. I, Average phase of days 2 to 6 calculated from the projected oscillations (Supplemental Fig. S4, F and G) of leaves treated with 0.5% (w/w) ACC (dark bars) or mock (light bars) lanolin paste (approximately 1 mg) on the petiole (solid bars) or the blade (dashed bars) under −DIF conditions. Error bars represent se; n = 8.

Figure 5.

−DIF reduces the ethylene sensitivity of Arabidopsis. A, Responses of 5-d-old etiolated Col-0 seedlings to various concentrations of ACC. After 48 h of germination under control or −DIF conditions, seedlings were kept in the dark with temperature cycles for 72 h. B, Hypocotyl lengths of 5-d-old Col-0 wild-type (black lines) or ein2-1 (red lines) seedlings in response to various concentrations of ACC after 48 h of germination under control or −DIF conditions. Seedlings were then kept in the dark with temperature cycles for 72 h on various ACC concentrations. Error bars represent se; n ≥ 60. [See online article for color version of this figure.]

Figure 6.

PHYB represses ethylene-dependent leaf movement. A and B, Projected oscillations for phyB9 (solid blue line) and Col-0 (dotted black line) leaves developing under control (A) or −DIF (B) conditions. Error bars represent se; n = 8. Gray areas indicate the dark period. C, Average amplitudes of Col-0 and phyB9 leaves developing under control (black bars) or −DIF (gray bars) conditions, calculated from days 2 to 6 of the projected oscillations (in A and B). Error bars represent se; n = 8. D, Phase shifts of leaf movement between control and −DIF conditions for phyB9 and Col-0 wild-type leaves. Significant phase shifts (P < 0.05) are indicated with arrows. Each arrow depicts the direction and strength of the shift in phase: the start of the arrow indicates average phase during days 2 to 6 under control conditions, and the arrowhead indicates the average phase for −DIF (days 2–6). Error bars represent se; n = 8. E and F, Ethylene emissions of phyB9 mutants (blue diamonds, solid lines) compared with Col-0 wild-type emissions (white circles, dashed lines) under control (E) and −DIF (F) conditions. Error bars represent se; n ≥ 10. Gray areas indicate the dark period. FW, Fresh weight. G, Promoter activity analysis of ACS2 using histochemical GUS staining in phyB9 leaves developed during 10 d of −DIF (top) or control (bottom) conditions on 4-week-old plants. H, Relative hypocotyl lengths of 5-d-old phyB9 (solid blue line) or Col-0 wild-type (dotted black line) seedlings in response to various concentrations of ACC. After 48 h of germination under control (triangles) or −DIF (circles) conditions, seedlings were kept in the dark with temperature cycles for 72 h on various ACC concentrations. Absolute lengths (as depicted in Supplemental Fig. S5D) were normalized for the length at 0 µm ACC (100%). Error bars represent se; n = 60.

Figure 7.

Dissection of leaf movement in the phyB9 ein2-1 double mutant. A, Representative 36-d-old phyB9, ein2-1, and phyB9 ein2-1 rosette plants developing under −DIF conditions at the end of the fourth photoperiod. B, Average amplitudes of phyB9, ein2-1, and phyB9 ein2-1 leaves developing under control (black bars) or −DIF (gray bars) conditions, calculated from days 2 to 6 of the projected oscillations (in C and D). Error bars represent se; n = 8. C and D, Projected leaf oscillations of phyB9, ein2-1, and phyB9 ein2-1 double mutant developing under control (C) or −DIF (D) conditions. Error bars represent se; n = 8. Gray areas indicate the dark period. [See online article for color version of this figure.]

Figure 8.

Amplitudes and phase shifts of leaf movement and ethylene emission between control and −DIF conditions in mutants and treatments used in this study. A, Averaged amplitudes (measured during days 2–6) of leaf movements. Solid bars mark the wild-type response under control conditions, and dashed lines mark the −DIF wild-type response. Error bars represent se; n = 8. B, Phase shifts of leaf movements between control and −DIF conditions compared with the wild-type (WT) response. Significant phase shifts (P < 0.05) are indicated with arrows, and nonsignificant shifts are indicated with bars. Each arrow depicts the direction and strength of the shift in phase: the start of the arrow indicates average phase during days 2 to 6 under control conditions, and the arrowhead indicates the average phase for −DIF (days 2–6). Error bars represent se; n = 8. C and D, Average peak amplitudes of ethylene emission (C) and phase shifts of ethylene emission (D) between control and −DIF conditions for phyB9 mutants and the Col-0 wild-type. Error bars represent se; n ≥ 10.