Three PRR proteins interact with ARF10 and ARF16 to enhance ABI5 activation and auxin-stimulated ABA responses during Arabidopsis seed germination.

The circadian clock and auxin regulate abscisic acid (ABA)-induced delayed seed germination in Arabidopsis thaliana. However, the molecular mechanisms underlying the crosstalk between circadian clock and auxin in modulating ABA signaling remain obscure. Here, we found that the central circadian clock proteins PSEUDO-RESPONSE REGULATOR5 (PRR5), PRR7, and PRR9 are crucial for auxin-increased ABA responses. Disrupting PRR5, PRR7, and PRR9 simultaneously conferred germinating seeds substantially less sensitive to combined auxin and ABA treatment, whereas PRR5 overexpression partially restores the ABA-hyposensitive phenotype of the auxin signaling-defective mutants (i.e., axr3-1). PRR5, PRR7, and PRR9 interact with the auxin-dependent transcription factors AUXIN RESPONSE FACTOR10 (ARF10) and ARF16. Consistently, PRR5, PRR7, and PRR9 cooperate with ARF10/ARF16 to promote auxin-enhanced ABA responses. Importantly, PRR5 and ARF16 synergistically stimulate the transcriptional activation by ABSCISIC ACID INSENSITIVE5 (ABI5, a master activator of ABA signaling) and the enrichment of ABI5 at the promoters of downstream targets. Moreover, PRR5 and ARF16 coordinately enhance the ABA-induced accumulation of ABI5 in germinating seeds. Collectively, our results demonstrate that the auxin stimulation on ABA signaling requires PRR5, PRR7, and PRR9, and explain the mechanism by which PRR proteins synergize with ARF10/ARF16 to activate ABI5 and integrate the circadian clock, auxin, and ABA pathways.