Photoactivated CRY1 and phyB Interact Directly with AUX/IAA Proteins to Inhibit Auxin Signaling in Arabidopsis

Abstract

Light is a key environmental cue that inhibits hypocotyl cell elongation through the blue and red/far-red light photoreceptors cryptochrome- and phytochrome-mediated pathways in Arabidopsis. In contrast, as a pivotal endogenous phytohormone auxin promotes hypocotyl elongation through the auxin receptors TIR1/AFBs-mediated degradation of AUX/IAA proteins (AUX/IAAs). However, the molecular mechanisms underlying the antagonistic interaction of light and auxin signaling remain unclear. Here, we report that light inhibits auxin signaling through stabilization of AUX/IAAs by blue and red light-dependent interactions of cryptochrome 1 (CRY1) and phytochrome B with AUX/IAAs, respectively. Blue light-triggered interactions of CRY1 with AUX/IAAs inhibit the associations of TIR1 with AUX/IAAs, leading to the repression of auxin-induced degradation of these proteins. Our results indicate that photoreceptors share AUX/IAAs with auxin receptors as the same direct downstream signaling components. We propose that antagonistic regulation of AUX/IAA protein stability by photoreceptors and auxin receptors allows plants to balance light and auxin signals to optimize their growth.

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Introduction

Light, as both energy source and informational signal, profoundly influences plant growth and development during the whole life span from seed germination to flowering (Fankhauser and Chory, 1997Deng and Quail, 1999). Plant senses light through multiple photoreceptors including blue/UV-A light receptors cryptochromes (CRY) and phototropins, UV-B receptor UVR8, and red and far-red light receptors phytochromes (PHY) (Cashmore et al., 1999Briggs and Christie, 2002Quail, 2002Rizzini et al., 2011). Cryptochromes and phytochromes are known to regulate photomorphogenesis, stomatal development, circadian rhythm, and flowering in Arabidopsis (Guo et al., 1998Somers et al., 1998Cashmore et al., 1999Yang et al., 2000Quail, 2002Kang et al., 2009). Cryptochromes exist not only in plants but also in a variety of other organisms from bacterium to human. In Arabidopsis, there are two homologous cryptochromes, CRY1 and CRY2. Genetic analyses show that CRY1 plays a major role in mediating blue light inhibition of hypocotyl elongation, whereas CRY2 acts to inhibit hypocotyl growth mainly under low intensity of blue light (Ahmad and Cashmore, 1993Lin et al., 1998). Consistent with their functions, CRY1 protein is stable but CRY2 is labile upon high intensity of blue light irradiation (Lin et al., 1998). However, CRY2 is the major blue light photoreceptor to promote floral initiation under a long-day photoperiod (Guo et al., 1998). It is known that cryptochrome serves as photoreceptor to entrain the circadian clock in Drosophila or an integral component of the circadian clock in mammals (Emery et al., 1998Kume et al., 1999). In migratory butterflies and birds, cryptochrome is responsible for sensing the Earth’s magnetic field and providing precise navigation during their long-distance migration (Gegear et al., 2010).

Cryptochromes are flavoproteins that show sequence similarity to photolyases, a rare class of flavoproteins that mediate repair of UV-damaged DNA (Sancar, 1994). They are structurally divided into N-terminal photolyase homology region and C-terminal extension domain, which is absent in photolyases. Arabidopsis CRY1 is localized in both cytoplasm and nucleus, whereas CRY2 is exclusively localized in the nucleus (Guo et al., 1999Wu and Spalding, 2007). It has been shown that the C-terminal domain of Arabidopsis CRY1 and CRY2 (CCT1/CCE1 and CCT2/CCE2) mediates blue light signaling by interactions with COP1 (Yang et al., 2000Yang et al., 2001Wang et al., 2001), a RING-finger E3 ubiquitin ligase (Deng et al., 1992) that interacts with and targets the degradation of a set of transcription factors, such as HY5 and CONSTANS (CO) (Osterlund et al., 2000Jang et al., 2008Liu et al., 2008), to regulate photomorphogenesis and flowering. Moreover, CRY1 and CRY2 also interact with the COP1 enhancer, SPA1 (Lian et al., 2011Liu et al., 2011Zuo et al., 2011), which interacts with COP1 to enhance its E3 ligase activity (Seo et al., 2003). The consequence of interactions of CRY1/CRY2 with COP1/SPA1 is disruption of the COP1–SPA1 core complex, thus promoting HY5/CO accumulation. The N-terminal domain of CRY1 and CRY2 (CNT1 and CNT2) mediate CRYs dimerization (Sang et al., 2005Yu et al., 2007), and CRY2 dimerization is inhibited by BIC1 (Wang et al., 2016). It has been demonstrated that overexpression of CNT1 in cry1 mutant confers a blue light-dependent shortened hypocotyl phenotype and that this phenopyte is not related to HY5 protein accumulation, implying that CNT1 is involved in mediating CRY1 signaling independent of the CRY1 C terminus (He et al., 2015). However, the underlying mechanism is not well understood.

The phytochrome family consists of five members (phyA to phyE) in Arabidopsis, of which phyB and phyA are the best characterized and shown to play a major role in mediating red and far-red light inhibition of hypocotyl elongation, respectively (Quail, 2002Franklin and Quail, 2010). Phytochromes exist in two photoconvertible forms: the red light-absorbing inactive Pr form and the far-red light-absorbing active Pfr form, which are induced by far-red and red light, respectively (Quail, 2002Rockwell et al., 2006). Phytochromes are localized in the cytoplasm in the dark, and upon red light exposure they translocate to the nucleus (Fankhauser and Chen, 2008), where they interact with a group of transcription factors called PHYTOCHROME-INTERACTING FACTORS (PIFs) (Castillon et al., 2007Leivar and Quail, 2011). It is well established that, upon light exposure, PIFs undergo rapid phosphorylation in a phytochrome-dependent manner, which induces their degradation via the 26S proteasome (Castillon et al., 2007Leivar and Quail, 2011). Interestingly, phytochromes also interact with the cryptochrome-interacting proteins such as COP1 and SPA1 (Seo et al., 2004Jang et al., 2010Lu et al., 2015Sheerin et al., 2015); Conversely, cryptochromes also interact with PIFs to mediate responses to canopy-shade low blue light or high temperature (Ma et al., 2016Pedmale et al., 2016).

Auxin is the first phytohormone identified in plants, which imposes multi-faceted influences on plant growth and development, including hypocotyl cell elongation, apical dominance, stomatal development, floral organ development, and fertility (Lincoln et al., 1990Mockaitis and Estelle, 2008Zhang et al., 2014). It has been established that auxin promotes the assembly of its coreceptor complex comprising F-box proteins TIR1/AFBs and transcription regulators AUX/IAA proteins (AUX/IAAs), and subsequent ubiquitination and degradation of AUX/IAAs, thus releasing the inhibitory effects of AUX/IAAs on a family of transcription factors, auxin response factors (ARFs), to activate auxin-responsive gene expression (Kim et al., 1997Gray et al., 2001Dharmasiri et al., 2005Kepinski and Leyser, 2005Mockaitis and Estelle, 2008Calderon Villalobos et al., 2012). There are 29 AUX/IAAs and 23 ARFs in Arabidopsis. AUX/IAAs are small short-lived proteins with four domains (DI to DIV), of which DII mediates the interactions of AUX/IAAs with TIR1/AFBs, and thus is required for auxin-triggered degradation of AUX/IAAs (Kepinski and Leyser, 2005Calderon Villalobos et al., 2012). The DIII/IV-containing C-terminal region of AUX/IAAs mediates their own homo-oligomerization and hetero-oligomerization with ARFs (Kim et al., 1997Ulmasov et al., 1997Han et al., 2014Korasick et al., 2014Nanao et al., 2014). Typical ARF proteins have a conserved N-terminal DNA-binding domain, followed by a non-conserved middle region and a conserved C-terminal dimerization domain (Liscum and Reed, 2002). The DNA-binding domain is responsible for ARF binding to the promoters of target genes (Ulmasov et al., 1999b). It has been shown that the middle region of ARFs directs transcriptional changes, and some ARFs (ARF5/6/7/8/19) with a Q-rich middle region are believed to function as transcriptional activators (Ulmasov et al., 1999a). The C terminus of ARFs is related in amino acid sequence to DIII and DIV of AUX/IAAs, and is essential for hetero-oligomerization with AUX/IAAs.

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