Auxin controls seed dormancy through stimulation of abscisic acid signaling by inducing ARF-mediated ABI3 activation in Arabidopsis

Abstract

The transition from dormancy to germination in seeds is a key physiological process during the lifecycle of plants. Abscisic acid (ABA) is the sole plant hormone known to maintain seed dormancy; it acts through a gene expression network involving the transcription factor ABSCISIC ACID INSENSITIVE 3 (ABI3). However, whether other phytohormone pathways function in the maintenance of seed dormancy in response to environmental and internal signals remains an important question. Here, we show that the plant growth hormone auxin, which acts as a versatile trigger in many developmental processes, also plays a critical role in seed dormancy in Arabidopsis. We show that disruptions in auxin signaling in MIR160-overexpressing plants, auxin receptor mutants, or auxin biosynthesis mutants dramatically release seed dormancy, whereas increases in auxin signaling or biosynthesis greatly enhance seed dormancy. Auxin action in seed dormancy requires the ABA signaling pathway (and vice versa), indicating that the roles of auxin and ABA in seed dormancy are interdependent. Furthermore, we show that auxin acts upstream of the major regulator of seed dormancy, ABI3, by recruiting the auxin response factors AUXIN RESPONSE FACTOR 10 and AUXIN RESPONSE FACTOR 16 to control the expression of ABI3 during seed germination. Our study, thus, uncovers a previously unrecognized regulatory factor of seed dormancy and a coordinating network of auxin and ABA signaling in this important process.

Keywords: agriculture; evolutionary mechanism; hormones; interaction; preharvest sprouting.

Fig. 1.

Seed dormancy is controlled by auxin levels. (A and B) Visualization of seed germination by cotyledon greening in fresh mature siliques of WT Col-0, (A) iaaM-OX, and (B) yuc1yuc6 plants after 7 d on water-saturated filter papers at 22 °C without stratification. (C and D) Radicle protrusion-based quantification of germination rates of nonstratified fresh wet seeds taken from unopened siliques on 1/2 MS containing no or various concentrations of IAA. (C and D) The average (± SD) values are shown.

Fig. 2.

Auxin detection and signaling are required for seed dormancy. (A, C, and E) Visualization of seed germination by cotyledon greening in fresh mature seeds or siliques from (A) the auxin receptor mutant tir1afb3 and the auxin signaling mutants (C) axr2 and (E) arf10arf16 on water-saturated filter papers without stratification. Images were acquired (A and C) 4 and (E) 5 d after incubation at 22 °C. (B, D, and F) Radicle protrusion-based quantification of germination rates of nonstratified fresh wet seeds taken from unopened siliques of the auxin signaling mutants on 1/2 MS. The average rates (± SD) are shown.

Fig. 3.

Auxin is required for ABA-mediated inhibition of seed germination. (A) Germination of WT seeds on 1/2 MS with 2 μM IAA, 1 μM ABA, or both. (B) Germination rates of WT seeds with increasing concentrations of IAA in the presence of 1 μM ABA at 8 d. (C) Seed germination of Col-0 and the auxin receptor mutants on 1/2 MS with 1 μM ABA. The image was acquired after 5 d of incubation. (D) Seed germination rates were determined after 3 d on 1/2 MS medium with or without ABA. (E and F) Seed germination rates of Col-0 and the ARF mutants on 1/2 MS medium with or without ABA at (E) 6 and (F) 8 d. Significant differences were determined by Student t test (*P < 0.05; **P < 0.01). (A, B, and D–F) Quantification of seed germination was based on radicle protrusion through the seed coat.

Fig. 4.

ARF10 and ARF16 are required to maintain ABI3 expression. (A) RNA blotting detection of ABI3 transcript levels in Col-0, mARF10/−, arf10arf16, and iaaM-OX during imbibition of nonstratified fresh dry seeds in the absence of ABA. (B) RNA blotting detection of ABI3 transcript levels in Col-0, mARF10/−, arf10arf16, and iaaM-OX during seed germination in the presence of ABA after 4-d stratification at 4 °C. The germination percentage at each time point assayed by radicle protrusion is indicated under the blots. DS, dry seed.

Fig. 5.

Auxin-mediated control of seed dormancy depends on ABI3. (A and B) Fresh seeds of iaaM-OX, iaaM-OX /abi3-1, and abi3-1 were germinated on 1/2 MS medium with or without stratification. The Col-0/Ler hybrid was used as a control, because iaaM-OX is in the Col-0 background, whereas abi3-1 is in the Ler background. (C and D) Germination of fresh seeds of mARF16 and mARF16/abi3-1 as well as the abi3-1 and Col-0/Ler controls with or without stratification. Seed germination was (A and C) visualized by cotyledon greening and (B and D) quantified by radicle protrusion after 6 d of incubation on 1/2 MS medium at 22 °C. Values are presented as averages (± SDs) with Student t test (**P < 0.01). (E) A proposed model for the effect of the ABA–auxin interaction on the control of seed dormancy/germination. When auxin signaling is inactivated by low auxin level or signaling disruption, ARF10 and ARF16 are inactivated by the Aux/IAA repressors AXR2 and AXR3. ABI3 expression cannot be maintained, and seed dormancy is released. With auxin signaling activation, auxin binds to the auxin receptor TIR1/AFB F-box proteins and promotes the degradation of IAA7/AXR2 and IAA17/AXR3. The degradation releases the activity of ARF10 and ARF16 and maintains the expression of ABI3, which protects seed dormancy and inhibits seed germination. The solid arrows and lines indicate direct regulation, and the dotted arrows indicate indirect regulation. The black box indicates the ABI3 gene region.