Auxin controls seed dormancy through stimulation of abscisic acid signaling by inducing ARF-mediated ABI3 activation in Arabidopsis Xiaodong Liua, Hong Zhanga, Yang Zhaob, Zhengyan Fenga, Qun Lia, Hong-Quan Yangc, Sheng Luand, Jianming Lie, and Zu-Hua Hea,1 aNational Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology and bShanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China; cCollege of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China; dDepartment of Plant and Microbial Biology, University of California, Berkeley, CA 94720; and eDepartment of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109-1048 Edited* by Jian-Kang Zhu, Purdue University, West Lafayette, IN, and approved August 5, 2013 (received for review March 11, 2013) The transition from dormancy to germination in seeds is a key (TIR1)/Additional F box protein (AFB)-Aux/indole-3-acetic acid physiological process during the lifecycle of plants. Abscisic acid (IAA) –AUXIN RESPONSE FACTOR (ARF) signaling system (ABA) is the sole plant hormone known to maintain seed dormancy; (7, 8). Recent studies have also suggested the potential in- it acts through a gene expression network involving the transcrip- volvement of auxin in seed dormancy maintenance. For example, tion factor ABSCISIC ACID INSENSITIVE 3 (ABI3). However, whether exogenous application of auxin enhanced the inhibition of seed Arabidopsis other phytohormone pathways function in the maintenance of germination by ABA in (9, 10) and also delayed seed seed dormancy in response to environmental and internal signals germination of wheat (11, 12). However, whether auxin is di- remains an important question. Here, we show that the plant rectly required for seed dormancy is unclear, and the underlying growth hormone auxin, which acts as a versatile trigger in many mechanism of auxin function in seed dormancy remains un- developmental processes, also plays a critical role in seed dor- known. During our previous studies on auxin-related growth and defense (13, 14), we found that Arabidopsis auxin mutants dis- mancy in Arabidopsis. We show that disruptions in auxin signaling played either accelerated or inhibited germination of freshly in MIR160-overexpressing plants, auxin receptor mutants, or auxin harvested seeds, suggesting an active role of auxin in seed dor- biosynthesis mutants dramatically release seed dormancy, whereas mancy. In the current study, through extensive analysis of the increases in auxin signaling or biosynthesis greatly enhance seed auxin and ABA pathways and their roles in seed dormancy, we dormancy. Auxin action in seed dormancy requires the ABA signaling show that auxin is required for seed dormancy and ABA in- pathway (and vice versa), indicating that the roles of auxin and ABA hibition of seed germination. We have elucidated a molecular link in seed dormancy are interdependent. Furthermore, we show that through which auxin activates ABA signaling to inhibit seed auxin acts upstream of the major regulator of seed dormancy, germination. ABI3, by recruiting the auxin response factors AUXIN RESPONSE FACTOR 10 and AUXIN RESPONSE FACTOR 16 to control the ex- Results pression of ABI3 during seed germination. Our study, thus, uncovers Seed Dormancy Depends On Auxin Levels. During our studies of a previously unrecognized regulatory factor of seed dormancy and auxin mutants, we found that freshly harvested seeds from un- a coordinating network of auxin and ABA signaling in this im- opened siliques (termed fresh wet seeds hereinafter) of trans- portant process. genic plants overexpressing MIR160 (35S:MIR160) (15) displayed greatly decreased dormancy compared with wild-type (WT) hormones | interaction | preharvest sprouting | agriculture | control based on cotyledon greening (Fig. S1 A and B) and evolutionary mechanism radicle protrusion through the seed coat (Fig. S1 C and D). Because MIR160 down-regulates the expression of three tran- PLANT BIOLOGY scription factors, ARF10, ARF16, and ARF17, in auxin signaling eed plants must be equipped with mechanisms to maintain (10, 15, 16), this observation suggested that the intrinsic auxin Sthe dormancy of freshly matured seeds until the proper signal might play an important role in seed dormancy. This season for propagation. The transition of the seed from dor- mancy to germination is a critical step in the lifecycle of plants. Significance Dormancy is crucial to the survival of plant species, because it ensures that seed germination will occur only when environ- mental conditions are optimal for growth. Seed dormancy is also Seed dormancy is a critical step in the lifecycle of plants, and it important for agriculture, because defective seed dormancy is crucial to the survival of plant species; this process is also causes preharvest sprouting when humid conditions persist important for agricultural practice to prevent preharvest sprout- before harvest. ing when humid conditions persist before harvest. This study It has long been known that the relative levels of plant hor- uncovers a previously unrecognized action of auxin in main- mones control seed dormancy and germination. Gibberellins taining seed dormancy through AUXIN RESPONSE FACTOR 10/ (GAs) break seed dormancy and promote germination (1, 2), 16-mediated expression of ABSCISIC ACID INSENSITIVE 3, a key and several other hormones, including brassinosteroids, ethylene, regulator in the abscisic acid-mediated seed dormancy. and cytokinin, have also been shown to promote seed germina- tion (3, 4). However, abscisic acid (ABA) is the only hormone Author contributions: X.L., S.L., J.L., and Z.-H.H. designed research; X.L., H.Z., Y.Z., Z.F., known to induce and maintain seed dormancy. ABA acts Q.L., and Z.-H.H. performed research; X.L. and H.-Q.Y. contributed new reagents/analytic through the PYR/RCAR-PP2C-SnRK2 signaling cascade (5, 6). tools; X.L. and Z.-H.H. analyzed data; and X.L. and Z.-H.H. wrote the paper. A major downstream component of ABA signaling, ABSCISIC The authors declare no conflict of interest. ACID INSENSITIVE 3 (ABI3), has been long recognized as *This Direct Submission article had a prearranged editor. a major regulator of seed dormancy and ABA inhibition of seed Freely available online through the PNAS open access option. germination (2). 1To whom correspondence should be addressed. E-mail: [email protected]. The hormone auxin regulates many aspects of plant growth This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and development through the Transport inhibitor response1 1073/pnas.1304651110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1304651110 PNAS | September 17, 2013 | vol. 110 | no. 38 | 15485–15490 Downloaded by guest on September 27, 2021 hypothesis was supported by our finding that the seed dormancy TIR1/AFB-Mediated Auxin Pathway Is Required for Seed Dormancy. of the auxin-overproducing transgenic line iaaM-OX (17) was The auxin signaling pathway has been well-studied and involves stronger than the seed dormancy of the WT by the cotyledon a cascade mediated by the auxin receptors TIR1/AFB, the AUX/ greening assay (Fig. 1A). Similarly, the radicle protrusion assay IAA transcriptional repressors, and the ARF transcription fac- also revealed that fresh wet seeds of iaaM-OX plants did not tors (8). We tested the role of TIR1/AFB in the control of seed even germinate at 30 d after imbibition, whereas 50% of similar dormancy. First, we assayed the seed dormancy of the auxin seeds from WT plants germinated in 5 d (Fig. 1C). Consistent receptor mutants (19–21). Both the triple tir1afb2afb3 and the with transgenic experiments, exogenous application of IAA ef- quadruple mutant tir1afb1afb2afb3 (22) failed to develop a hy- fectively enhanced, in a dose-dependent manner, the dormancy pocotyl and root meristem (Fig. S3A), probably because of a of fresh wet WT seeds (Fig. 1D). Interestingly, 4-d stratification requirement for auxin in embryogenesis, similar to the auxin at 4 °C nullified the inhibitory effect of exogenous IAA appli- biosynthetic mutant yuc1yuc4yuc10yuc11 (23). The majority of cation (Fig. S1E), indicating that the auxin-mediated mainte- seeds from this mutant did not germinate normally (Fig. S3). nance of seed dormancy could be prevented by stratification Instead, we examined the dormancy of fresh wet seeds harvested at 4 °C. from the tir1afb2 and tir1afb3 double mutants, which germinated We next examined whether endogenous auxin levels also af- normally like the WT seeds. The results showed that fresh wet fect seed dormancy. The YUCCA (YUC) family of flavin mon- seeds from these mutants germinated earlier than WT seeds ooxygenases are key enzymes in auxin biosynthesis (17, 18), and assayed by both cotyledon greening (Fig. 2A and Fig. S4A) and seed dormancy is established during seed maturation (1). radicle protrusion (Fig. 2B). IAA7/AXR2 and IAA17/AXR3 are Therefore, we searched the Arabidopsis microarray database two well-characterized transcriptional repressors of the TIR1/ axr2-1 axr3-1 (http://www.bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi), which revealed AFB-mediated auxin pathway, and the mutants and that YUC1, YUC2, and YUC6 are expressed during seed de- display a constitutive repression of auxin-responsive genes (24, velopment, with peak levels in the later stages of seed de- 25). We observed that fresh wet seeds of the two mutants also C velopment (Fig. S2). Because the yuc1yuc2yuc6 mutant exhibits germinated earlier than fresh wet seeds of the WT (Fig. 2 and D B low fertility (17), we used the yuc1yuc6 double mutant to test and Fig. S4 ). seed dormancy. The fresh wet seeds of auxin-deficient yuc1yuc6 We also examined the effects of downstream auxin signaling double mutant displayed significantly decreased dormancy com- components on seed dormancy. Consistent with the phenotype of 35S:MIR160 transgenic plants, in which ARF10, ARF16, and pared with similar seeds of the WT by both cotyledon greening ARF17 arf10arf16 (Fig.