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Regulated proteolysis in light-related signaling pathways
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Regulated proteolysis in light-related signaling pathways Rossana Henriques1, In-Cheol Jang1 and Nam-Hai Chua Post-translational modification is an important mechanism to determine protein levels and/or activities in cells. The process of conjugation of ubiquitin units to particular proteins, ubiquitination, usually leads to proteasomal degradation. During the past several years considerable work has been done to reveal the role of ubiquitination in the regulation of plant signaling and development. This article focuses on recent advances made on the study of ubiquitin-mediated proteolysis of several light-related signaling pathways, such as photomorphogenesis, circadian clock function, and photoperiodic flowering.
sequential steps. This is done by the concerted action of E1, E2 and E3 enzymes. E1 is the Ub activating enzyme, E2 the Ub conjugating enzyme and the Ub ligase which confers substrate specificity is known as E3. Proteins tagged with four or more Ub’s with K48 linkages are usually degraded by 26S proteasomes [3].
Address Laboratory of Plant Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA
E3 ligases in Arabidopsis
Corresponding author: Chua, Nam-Hai ([email protected]) 1 These authors contributed equally to this work.
Current Opinion in Plant Biology 2009, 12:49–56 This review comes from a themed issue on Growth and Development Edited by Charles S. Gasser and Caroline Dean Available online 10th December 2008 1369-5266/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2008.10.009
Introduction Research on plant signaling pathways has progressed from investigations on transcriptional control to post-transcriptional regulation. Prominent among post-transcriptional regulatory events is the control of the half-life of signal receptors and signaling components through ubiquitination and proteolysis. This regulatory strategy has been shown to initiate or terminate signaling and to determine the strength and duration of signal flux. Several previous reviews [1–5] have extensively discussed how ubiquitination/proteolysis regulates hormone signaling pathways, circadian clock function, flowering time and light signal transduction. Because of space limitation we have restricted this review to recent results on light-related signaling pathways.
Ubiquitination Most cellular proteins are degraded via the ubiquitination/proteasome pathway. Ubiquitination involves the attachment of several units of a small protein, ubiquitin (Ub) to specific lysine residues of a substrate protein in www.sciencedirect.com
On the other hand, proteins modified by monoubiquitin or ubiquitin chains with linkages other than K48 are not destined for destruction but rather they are directly implicated in various signaling events.
E3s include a wide family of proteins/protein complexes divided in two groups: (1) single unit E3 ligases that possess a HECT domain [an active site first found at the C-terminus of the human E3 (E6-AP)] and bind directly to an activated Ub and (2) RING/U-box E3s, which rely on E2 for transfer of Ub to the substrate. RING E3s can be single units or multi-subunit protein complexes [2,3]. In Arabidopsis, E3s ligases constitute the largest group in the ubiquitin/proteasome pathway. So far seven genes (UPL1-7; Ubiquitin Protein Ligase) encoding HECT E3s have been identified [6]. RING E3 ligases constitute the majority of the known E3s, since more than 500 genes have been identified [2,3]. Single unit RING E3s, such as COP1 (CONSTITUTIVE PHOTOMORPHOGENESIS1) and SINAT5 (SEVEN IN ABSENTIA IN ARABIDOPSIS THALIANA 5), possess both the substrate and E2 binding motifs. Multi-subunit E3s divide the two functional domains into separate proteins. This is the case for the SCF (SKP1-CUL1-F-box), APC (Anaphase Promoting Complex) and CUL3-BTB complexes (Broadcomplex, Tramtrack, Bric-a-Brac) [2,3,5,7]. The SCF E3 ligases contain four subunits: SKP1 (in plants ASK for Arabidopsis SKP1), cullin, F-box and RBX1 (Ring-Box 1). The cullin subunit functions as a scaffolding protein binding both SKP1 and RBX1. The SKP1 in turn binds to an F-box protein which determines the substrate specificity of the SCF complex. The Arabidopsis genome contains genes encoding 21 ASKs, 5 cullins, 2 RBX1 proteins and more than 700 F-box proteins. This large number of subunit proteins predicts an innumerable number of possible combinations of SCF complexes targeting a wide variety of possible targets [2–4,7,8].
Light signaling In addition to providing energy for photosynthesis, light also signals vegetative and reproductive development. Current Opinion in Plant Biology 2009, 12:49–56
50 Growth and Development
Plants perceive wavelengths, intensities, direction and duration of light signals through a number of photoreceptors: phytochromes (phy) that detect red (R) and far-red (FR) light and cryptochromes (cry) and phototropins that function as blue light (B) photoreceptors [5]. In Arabidopsis there are five phytochromes: phyA is the major regulator of FR signaling, phyB mediates RL responses whereas the other phytochromes have a redundant role. Upon activation by light, phytochrome dimers translocate to the nucleus where they initiate specific signaling cascades [9,10]. Downstream of phytochromes, several transcription factors (TFs) act as positive or negative regulators of photomorphogenesis. In FR signaling, the positive regulators HYPOCOTYL5 (HY5) and HY5 HOMOLOG (HYH) are degraded in darkness but become stabilized in the light to promote photomorphogenesis [11,12]. Two other positive factors, LONG AFTER FAR-RED LIGHT1 (LAF1) and LONG HYPOCOTYL IN FAR-RED1 (HFR1) are degraded in the light as part of the signal attenuation mechanism [13–17]. Negative regulators such as PHYTOCHROME INTERACTING FACTOR1, 3, 4, 5 (PIF1, PIF3, PIF4, PIF5) are stable in darkness but rapidly degraded upon light exposure [18–20]. Although the stability of these factors is regulated in an opposing manner in darkness and light, all proteins are degraded by 26S proteasomes. A major repressor of photomorphogenesis in darkness is the RING protein CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1). As a nuclear protein in darkness, COP1 presumably becomes depleted from the nucleus upon prolonged light exposure [21] to allow the accumulation of positive regulators. This RING protein possesses auto-ubiquitination activity in vitro and in vivo [16] and can ubiquitinate LAF1, HY5, and HFR1 through direct interaction [13,16,17,22]. The importance of COP1-mediated protein degradation in light signaling renders this step the target of multiple regulations. One mechanism is to directly modulate COP1 E3 activity with regulatory factors. All four SUPPRESSOR OF PHYTOCHROME A (SPA) proteins function in concert with COP1 to suppress photomorphogenesis in dark-grown seedlings [23]. Consistent with this, the SPA1 coiled–coil domain promotes LAF1 ubiquitination by COP1 at low COP1 concentrations [16]. On the other hand, full-length SPA1 inhibits COP1 E3 ligase activity towards HY5 [22]. Phosphorylation status of receptors and signaling factors is known to affect their subsequent ubiquitination and proteasomal degradation. Degradation of the photoreceptor, phyA, is mediated by COP1 as part of the signal termination process [24]. Recently, Saijo et al. [25] reported that SPA1 enhances COP1 E3 ligase activity Current Opinion in Plant Biology 2009, 12:49–56
towards phyA, as was the case with LAF1 [16]. Moreover, COP1/SPA1 preferentially interacts with phosphorylated phyA, whereas underphosphorylated phyA associates with its downstream targets, FHY3 and FHY1 [25]. In addition to phyA, cryptochrome 2 (cry2) also undergoes blue-light induced phosphorylation in vivo and the phosphorylated form is preferentially degraded [26]. This degradation depends on COP1 because phosphorylated cry2 accumulates to higher levels in cop1-6 compared to WT. It is not known whether cry2 is a direct target of COP1. Early work showed that the unphosphorylated HY5 is the preferred COP1 substrate whereas the phosphorylated form accumulates [27]. By contrast, HFR1 can be phosphorylated on multiple N-terminal residues by casein kinase II [28] and the phosphorylated form appears to be the COP1 substrate. This result is consistent with the observation that removal of the HFR1 N-terminal region stabilizes the protein [13]. The suggestion that COP1 would preferably target phosphorylated HFR1 in darkness [15] is difficult to reconcile with the observation that light promotes HFR1 phosphorylation [28]. Moreover, both isoforms of HFR1 are found in light and darkness and light induces the accumulation of both species [28]. Because several TFs operating downstream of phyA are known to form heterodimers, association/dissociation of TFs may regulate their binding to E3 ligases and therefore stability. Jang et al. [14] recently reported that the HFR1 (bHLH)/LAF1 (R2R3-MYB) association attenuates their ubiquitination by the COP1 E3 ligase leading to increased accumulation of these factors. Combinatorial interaction amongst TFs occurring under different environmental conditions may be an important mechanism to regulate TF stability and thus signal flux through particular nodes. COP1 is also known to regulate BL responses. Cryptochromes can bind to COP1 [29] and inhibit COP1 E3 activity in a BL-dependent manner, leading to an increased stability of TFs that activate blue light-dependent gene expression. Hong et al. [30] recently identified the Blue Insensitive Trait (BIT1), a R2R3 type MYB transcription factor, as a positive regulator of BL signaling. Since BIT1 degradation is delayed in cop1-4 mutant this factor is likely a COP1 target, although in vitro ubiquitination of BIT1 by COP1 has not yet been shown. Besides the RING E3 ligase COP1, several SCF complexes likely regulate light signaling as well. Two alleles of CUL1, axr6-3 and cul1-6, show hyper- and hyposensitivity to far-red and red light, respectively [31,32] and several F-box proteins have been implicated in light responses [33]. SCF regulation of light signaling, however, is poorly understood, compared to the impressive progress that has been made in the identification of F-box www.sciencedirect.com
Regulated proteolysis in light-related signaling pathways Henriques, Jang and Chua 51
proteins involved in the control of hormone signaling pathways [33,34].
PHYTOCHROME INTERACTING FACTORS – integrators of multiple signals
dimerize or heterodimerize and they accumulate in darkness. PIF1, PIF3 and PIF4 have been shown to bind to the G-box motif (50 CACGTG 30 ) and activate the transcription of genes controlling the elongation response [9,35–39].
Differences in hypocotyl length between etiolated and light-grown seedlings are largely due to difference in cell elongation. A group of factors, called PIFs (PHYTOCHROME-INTERACTING FACTORS), has emerged as important regulators of cell elongation in darkness. PIFs are TFs belonging to subfamily 15 of the helix-loophelix superfamily. First characterized by their binding to primarily phyB, and sometimes phyA, through their active phytochrome binding (APB) motif, PIFs can homo-
In the light, activated phyB in the nucleus is able to interact with the PIFs via their APB motif. So far PIF1, 3, 4, 5, 6 and PIF7 have been identified as phyB interactors [40–42], whereas PIF1 and PIF3 also associate with phyA although their APA motif is less conserved [35,38]. Immediately after RL exposure, PIFs are involved in activating transcription of genes necessary for de-etiolation [18,38,43]. Upon longer RL exposure, however, phyB
Figure 1
(a) Light-dependent proteasomal degradation of PIFs. In the light, activated phyB translocates into the nucleus and binds to PIFs. PIFs are phosphorylated (P) in a phyB-dependent manner and ubiquitinated (Ub) by an unknown E3 ligase before proteasomal degradation. In the dark, however, accumulated PIFs can promote transcription of target genes, resulting in cell elongation. (b) Regulation of PIF proteins in GA signaling. In the absence of GA, DELLA proteins directly interact with PIFs (PIF3 and PIF4) and inhibit their transcription activity. Upon light exposure, PIFs are phophorylated (P) by an yet unidentified kinase (K), ubiquitinated (Ub) by unknown E3 ligases and finally degraded by 26S proteasome. When GA is present, GID1 binds to GA and leads to DELLA degradation by SCFSLY1/GID2. Released PIFs bind to G-box elements to activate transcription of elongation genes. www.sciencedirect.com
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targets the PIFs for destruction by 26S proteasomes (Figure 1a). In FR light, phyA can also target PIF1 and PIF3 for degradation [18,44]. Therefore, except PIF7, all other PIFs are light labile and this is consistent with the decreased transcription of cell elongation genes in the light [42]. As shown for positive regulators of light responses, PIF1, PIF3 and PIF5 are also phosphorylated, probably in a phyB-dependent manner, and phosphorylation presumably precedes their ubiquitination/degradation [40,44,45]. Consistent with this observation, PIF1 can be stabilized by mutations affecting phyA/B binding [44]. Other than the report that COP1 is not involved in the degradation of PIF3 [18], the E3 ligase(s) responsible for PIF ubiquitination remain to be characterized. Among the various plant hormones, gibberellic acid (GA) is known to promote stem elongation via cell expansion. This raises the question whether PIFs are also involved in this process, and if so, how GA and light signaling pathways interact. Recently, it was shown that both PIF3 and PIF4 transcriptional activities are repressed by DELLA proteins, but degradation of the latter via the SCFSLY1/GID2 E3 ligase is triggered by GA [36,37] (Figure 1b). This mechanism explains very nicely how GA promotes cell elongation through the PIFs. Another member of the PIF family, PIF1, can bind to promoters of genes encoding DELLA proteins [46] and this activity would produce more DELLA proteins which re-repress the GA pathway. Combining the recent results on light and GA signaling, we may assume that upon germination, when GA levels are high and the germinated seedlings are still below the soil surface, the PIF proteins, free from DELLA repression and phyB-dependent degradation, are able to promote hypocotyl growth towards the soil surface. Upon exposure to light, the PIFs are degraded resulting in reduced cell expansion and inhibition of hypocotyl growth. Although phyB is known to regulate the stability of PIFs, the reverse situation may also hold. Immediately after illumination, PIFs (PIF3, 4, 5 and 7) promote de-etiolation, but on longer light exposure, they regulate the levels of available phytochrome, preventing an exaggerated light response [42,43]. PIF levels are regulated, not only by light and GA, but also by the circadian clock. Seedling hypocotyl growth peaks at dawn, and this rhythmic growth depends on both light and the circadian clock [19]. Detailed expression profiling led to the identification of PIF4 and PIF5 as integrators of clock and light signals [19]. The inhibition of PIF4/PIF5 transcription by the clock at dusk is relieved at dawn, allowing accumulation of PIF4/PIF5 proteins which induce transcription of growth-response genes in the dark. Upon light exposure, both PIF4/5 are targeted Current Opinion in Plant Biology 2009, 12:49–56
for degradation by phyB. The diurnal regulation of hypocotyl growth depends on these interconnected mechanisms that affect PIF4/PIF5 transcript (the clock) and protein (light) levels independently [19]. Although the clock does not directly target PIF4-PIF5 proteins, it appropriately constrains their diurnal availability, allowing elongation growth to occur at the more convenient period.
Degradation of circadian regulators is required for maintenance of circadian rhythms Being the biological oscillator responsible for the integration of plant growth/metabolism to the 24 h day/night cycles, the clock translates environmental signals (input) into appropriate cellular responses that increase fitness and survival rate [47,48]. The circadian clock relies on the generation of robust cyclic rhythms created by negative feedback loops at the core of the oscillator. Recent evidence suggests the existence of a morning and an evening loop [1,49,50]. In the morning oscillator, light and CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1)/ LHY (LATE ELONGATED HYPOCOTYL) promote the accumulation of PRR7 and PRR9, (PSEUDORESPONSE REGULATOR7 and 9), which inhibit CCA1/LHY expression. In the evening loop, GI (GIGANTEA) accumulates and promotes the expression of TOC1 (TIMING OF CAB EXPRESSION1), that represses GI, by an unknown mechanism [1,49,50]. In this model the two oscillators are connected by a number of unknown regulators. Initial studies were mostly based on transcript analysis, but new findings have shown that post-translational regulation is critical for the maintenance of robust circadian oscillations [48]. The ZTL (ZEITLUPE) family of F-box proteins [ZTL; FKF1 (FLAVIN BINDING KELCH FBOX 1), and LKP2 (LOV KELCH PROTEIN 2)] is a major regulator of several circadian proteins. These F-box proteins possess an N-terminal LOV (Light-Oxygen-Voltage dependent) domain that responds to blue light, and several Kelch repeats, important for protein-protein interactions [51,52,53]. ZTL associates with three known components of SCF complexes (ASK1, AtCUL1 and AtRBX1), suggesting that ZTL can assemble into an SCF complex in vivo [51]. Indeed, CUL1 association with ZTL is required for a functional SCFZTL complex [54]. Interestingly, ZTL itself is also unstable and the mechanism mediating this instability remains to be investigated [51,55]. Kim et al. [55] recently found that GI associates with the BL-activated form of ZTL thereby stabilizing the latter. As the dark period progresses, GI dissociates from the complex allowing the SCFZTL E3 ligase to interact with its substrates [55], TOC1 and PRR5 (PSEUDORESPONSE REGULATOR5) [52,53]. It should be noted that ZTL regulation of TOC1 and PRR5 levels www.sciencedirect.com
Regulated proteolysis in light-related signaling pathways Henriques, Jang and Chua 53
Figure 2
Transcription of CO depends on the circadian clock. In LD, the formation of GI/FKF1 complex facilitates association of the F-box protein with CDF1, an inhibitor of CO transcription. Upon CDF1 degradation, CO transcript levels increase and, under these conditions, CO protein also accumulates to induce flowering.
is critical for the generation/maintenance of robust circadian rhythms and proper clock function [52,53]. Although the mechanism of interaction might differ, TOC1 and PRR5 are the only PRR family members directly regulated by ZTL [56]. Furthermore, PRR proteins display a diurnal pattern of phosphorylation which, for TOC1 and PRR5, could facilitate ZTL association and increase their degradation. By contrast, phosphorylation enhances the TOC1/ PRR3 interaction and increases TOC1 stability probably by preventing ZTL binding [56]. Para et al., [57] also reported the formation of a TOC1/PRR3 complex in the vasculature suggesting the existence of a tissue-specific clock mechanism. PRR3, PRR5 and TOC1 may constitute a unit in the central oscillator as their transcript and protein levels are tightly and similarly regulated [56]. Other members of the PRR family, PRR9 and PRR7 were also shown to be targets for proteasomes but the E3 ligases involved remain unidentified [58,59]. GI also plays a role in flowering time control by activating CO (CONSTANS), a gene required for the onset of flowering [60]. Interestingly, GI levels are regulated by the 26S proteasome, leading to GI degradation in the dark [60]. Recently, Sawa et al. [61] showed that GI can associate with the F-box protein FKF1 (a ZTL-family member)
Figure 3
Photoperiodic regulation of COSTANS (CO) stability. There are two possible mechanisms for CO protein degradation under LD: a COP1-dependent mechanism, which occurs late in the day and during the night and/or a phyB-dependent mechanism, which occurs early in the morning and/or in response to red light. One or more E3 ligase (X) might be involved in CO degradation early in the day. CO accumulates in the late afternoon and photoreceptor(s) such as CRY may repress COP1 activity. Under SDs, CO protein is degraded in a COP1/SPAs-dependent manner. The reduced CO protein level is not sufficient to induce FT expression resulting in late flowering. www.sciencedirect.com
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and regulate its activity towards CDF1 (CYCLING DOF FACTOR 1), a negative regulator of CO. The GI/FKF1 interaction occurs via a photo-activated LOV domain similarly to GI/ZTL [55]. In long days (LD), the three proteins CDF1, GI and FKF1 accumulate sequentially. CDF1 is able to bind and repress the CO promoter. GI then interacts with CDF1 and recruits the photo-activated FKF1 to target CDF1 for degradation, releasing CO repression (Figure 2). In LD, the CO protein which is stabilized (see below) induces FT (FLOWERING LOCUS T) to promote flowering. In short days (SD), GI and FKF1 proteins peak at different times and their interaction is diminished, resulting in more stable CDF1 and repression of flowering. These results nicely describe a mechanism by which light and circadian clock affect protein levels that control flowering time.
Conclusions Studies on the role of proteolysis in light-related signaling pathways have advanced from substrate identification to regulatory aspects of ubiquitination and proteolysis. Future efforts should be devoted to the development of in vitro biochemical assays which can recapitulate in vivo events with high fidelity, so that putative gene functions predicted by genetic analysis, can be verified and their mechanism of action elucidated.
Acknowledgement Light-related research done in our lab was supported by a grant from NIH GM44640 to NHC.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
Regulation of photoperiodic flowering The transition from vegetative to reproductive growth is controlled by environmental factors such as temperature and day length. CO, which activates the floral regulators FT and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (SOC1), is required for flowering under LD. Whereas CO transcription is regulated by the circadian clock via GI and the SCFFKF1 E3 ligase, CO protein levels are differentially regulated by light quality, being more stable under FR and BL but unstable under RL [62]. Under SD conditions, CO is rapidly degraded by 26S proteasomes in darkness and remains unstable throughout the day [63]. In darkness, cop1-6 mutant expresses increased FT and SOC1 transcript levels [64], suggesting that COP1 might repress flowering by regulating CO levels. Indeed, CONSTANS-LIKE 3 (COL3), a homolog of CO, has been identified as a novel COP1-interacting protein [65]. Recently, CO was shown to interact with COP1 via its C-terminal CCT domain suggesting that it may be a COP1 substrate [66,67]. Since CO protein stability is also under the control of CRY1/CRY2 and phyB, there must be an additional layer of control besides ubiquitination by COP1 [66,67] (Figure 3). SPA1 and its homologs, SPA3 and SPA4, are likely involved in regulating CO abundance as well. SPA1 acts as a negative regulator of phyA-mediated de-etiolation in concert with COP1 [16,22,68] and regulates circadian rhythms and flowering time [69]. The SPA proteins were shown to interact with CO in vitro and in vivo. Moreover, SPA1 interacts with the CCT-domain of CO [70], the binding site of COP1 [67], and CO protein accumulates in the spa1spa3spa4 triple mutant [70]. Together, these results suggest that, COP1 and SPA1 cooperate to negatively regulate CO protein stability, as is the case with LAF1 [16] (Figure 3). It should be emphasized, however, that more than one E3 ligase may mediate CO degradation early in the day and there is some evidence to implicate a phyB-controlled mechanism (Figure 3). Current Opinion in Plant Biology 2009, 12:49–56
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Regulated proteolysis in light-related signaling pathways Henriques, Jang and Chua 55
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MYB transcription factor, to activate blue light-dependent gene expression in Arabidopsis. Plant J 2008, 55:361-371. A MYB transcription factor Blue Insensitive Trait (BIT1) was identified as a positive regulator of blue light (BL) signaling. Binding of BIT1 to COP1 in vivo was confirmed by co-immunoprecipitation and these two proteins also co-localize in nuclear speckles as shown before in [11–13,16]. BIT1 could be a target of COP1 E3 ligase because it is degraded by COP1dependent proteolysis in darkness. 31. Moon J, Zhao Y, Dai X, Zhang W, Gray WM, Huq E, Estelle M: A new CULLIN 1 mutant has altered responses to hormones and light in Arabidopsis. Plant Physiol 2007, 143:684-696. 32. Quint M, Ito H, Zhang W, Gray WM: Characterization of a novel temperature-sensitive allele of the CUL1/AXR6 subunit of SCF ubiquitin-ligases. Plant J 2005, 43:371-383. 33. Lechner E, Achard P, Vansiri A, Potuschak T, Genschik P: F-box proteins everywhere. Curr Opin Plant Biol 2006, 9:631-638. 34. Yu H, Wu J, Xu N, Peng M: Roles of F-box proteins in plant hormone responses. Acta Biochim Biophys Sin 2007, 39:915-922. 35. Castillon A, Shen H, Huq E: Phytochrome Interacting Factors: central players in phytochrome-mediated light signaling networks. Trends Plant Sci 2007, 12:514-521. 36. de Lucas M, Daviere JM, Rodriguez-Falcon M, Pontin M, Iglesias Pedraz JM, Lorrain S, Fankhauser C, Blazquez MA, Titarenko E, Prat S: A molecular framework for light and gibberellin control of cell elongation. Nature 2008, 451:480-484. 37. Feng S, Martinez C, Gusmaroli G, Wang Y, Zhou J, Wang F, Chen L, Yu L, Iglesias-Pedraz JM, Kircher S et al.: Coordinated regulation of Arabidopsis thaliana development by light and gibberellins. Nature 2008, 451:475-479. The authors, as well as de Lucas et al. [36], independently show how PIFs (PIF3 and PIF4, respectively) are targeted by the GA signaling repressors, the DELLA proteins. Both research teams show that the transcriptional activity of these PIFs is inhibited by their interaction with the DELLA proteins. In the presence of GA, DELLA proteins associate with GID1 and are targeted for degradation by an SCFSLY1/GID2 complex. Upon DELLA degradation, PIF proteins can activate the transcription of genes involved in the elongation response. These results provide important evidence for a cross-talk between GA and light signaling in the control of the elongation response, and further highlight the role of the PIF proteins as integrators of different signals. 38. Monte E, Al-Sady B, Leivar P, Quail PH: Out of the dark: how the PIFs are unmasking a dual temporal mechanism of phytochrome signalling. J Exp Bot 2007, 58:3125-3133. 39. Lorrain S, Allen T, Duek PD, Whitelam GC, Fankhauser C: Phytochrome-mediated inhibition of shade avoidance involves degradation of growth-promoting bHLH transcription factors. Plant J 2008, 53:312-323. 40. Al-Sady B, Ni W, Kircher S, Scha¨fer E, Quail PH: Photoactivated Phytochrome induces rapid PIF3 phosphorylation prior to proteasome-mediated degradation. Mol Cell 2006, 23:439-446. 41. Khanna R, Huq E, Kikis EA, Al-Sady B, Lanzatella C, Quail PH: A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic HelixLoop-Helix transcription factors. Plant Cell 2004, 16:3033-3044. 42. Leivar P, Monte E, Al-Sady B, Carle C, Storer A, Alonso J, Ecker J, Quail P: The Arabidopsis Phytochrome-Interacting Factor PIF7, together with PIF3 and PIF4, regulates responses to prolonged Red light by modulating phyB levels. Plant Cell 2008, 20:337-352. 43. Al-Sady B, Kikis EA, Monte E, Quail PH: Mechanistic duality of transcription factor function in phytochrome signalling. Proc Natl Acad Sci USA 2008, 105:2232-2237. 44. Shen H, Zhu L, Castillon A, Majee M, Downie B, Huq E: Lightinduced phosphorylation and degradation of the negative regulator PHYTOCHROME-INTERACTING FACTOR1 from Arabidopsis depend upon its direct physical interactions with photoactivated phytochromes. Plant Cell 2008, 20:1586-1602. 45. Shen Y, Khanna R, Carle CM, Quail PH: Phytochrome induces rapid PIF5 phosphorylation and degradation in response to Red-light activation. Plant Physiol 2007, 145:1043-1051. Current Opinion in Plant Biology 2009, 12:49–56
56 Growth and Development
46. Oh E, Yamaguchi S, Hu J, Yusuke J, Jung B, Paik I, Lee HS, Sun TP, Kamiya Y, Choi G: PIL5, a Phytochrome-Interacting bHLH protein, regulates Gibberellin responsiveness by binding directly to the GAI and RGA promoters in Arabidopsis seeds. Plant Cell 2007, 19:1192-1208. 47. Dodd AN, Salathia N, Hall A, Kevei E, Toth R, Nagy F, Hibberd J, Millar A, Webb AA: Plant circadian clocks increase photosynthesis, growth, survival, and competitive advantage. Science 2005, 309:630-633. 48. Gardner MJ, Hubbard KE, Hotta CT, Dodd AN, Webb AA: How plants tell the time. Biochem J 2006, 397:15-24. 49. Zeilinger M, Farre´ EM, Taylor SR, Kay SA, Doyle FJ III: A novel computational model of the circadian clock in Arabidopsis that incorporates PRR7 and PRR9. Mol Syst Biol 2006, 2:58. 50. Locke JC, Kozma-Bogna´r L, Gould PD, Fehe´r B, Kevei E, Nagy F, Turner MS, Hall A, Millar AJ: Experimental validation of a predicted feedback loop in the multi-oscillator clock of Arabidopsis thaliana. Mol Syst Biol 2006, 2:59. 51. Han L, Mason M, Risseeuw EP, Crosby WL, Somers DE: Formation of an SCFZTL complex is required for proper regulation of circadian timing. Plant J 2004, 40:291-301. 52. Ma´s P, Kim WY, Somers DE, Kay SA: Targeted degradation of TOC1 by ZTL modulates circadian function in Arabidopsis thaliana. Nature 2003, 426:567-570. 53. Kiba T, Henriques R, Sakakibara H, Chua NH: Targeted degradation of PSEUDO-RESPONSE REGULATOR5 by an SCFZTL complex regulates clock function and photomorphogenesis in Arabidopsis thaliana. Plant Cell 2007, 19:2516-2530. 54. Harmon F, Imaizumi T, Gray WM: CUL1 regulates TOC1 protein stability in the Arabidopsis circadian clock. Plant J 2008, 55:568-579. 55. Kim WY, Fujiwara S, Suh SS, Kim J, Kim Y, Han L, David K, Putterill J, Nam HG, Somers DE: ZEITLUPE is a circadian photoreceptor stabilized by GIGANTEA in blue light. Nature 2007, 449:356-360. The authors show that blue light and GI can stabilize the ZTL protein. Activated ZTL can interact with GI via its LOV motif and the resulting ZTL/ GI complex allows ZTL accumulation at the end of the day. In the dark, the GI-ZTL interaction is weakened and ZTL is released to target other circadian proteins. This paper provides a new insight on the circadian function of GI and their results show how the regulation of a F-box protein is critical for proper clock function. 56. Fujiwara S, Wang L, Han L, Suh SS, Salome PA, McClung CR, Somers DE: Post-translational regulation of the Arabidopsis circadian clock through selective proteolysis and phosphorylation of Pseudo-Response Regulator proteins. J Biol Chem 2008, 283:23073-23083.
60. David KM, Armbruster U, Tama N, Putterill J: Arabidopsis GIGANTEA protein is post-transcriptionally regulated by light and dark. FEBS Lett 2006, 580:1193-1197. 61. Sawa M, Nusinow DA, Kay SA, Imaizumi T: FKF1 and GIGANTEA complex formation is required for day-length measurement in Arabidopsis. Science 2007, 318:261-265. This work clarifies the molecular mechanism for photoperiod regulation of flowering. The interaction between GI and FKF1 is shown to be critical for CDF1 degradation and the control of flowering. Blue light promotes the interaction of GI and the LOV domain of FKF1, similarly to the ZTL/GI interaction [55]. GI interaction with CDF1 and FKF1, allows FKF1 to target CDF1 for degradation, releasing the inhibition on CO transcription. The circadian regulation of GI and FKF1, as well as light-induced formation of the GI/FKF1 complex, constitute the molecular mechanism for regulation of flowering under the most favorable conditions. 62. Turck F, Fornara F, Coupland G: Regulation and identity of florigen: FLOWERING LOCUS T moves center stage. Annu Rev Plant Biol 2008, 59:573-594. 63. Valverde F, Mouradov A, Soppe W, Ravenscroft D, Samach A, Coupland G: Photoreceptor regulation of CONSTANS protein in photoperiodic flowering. Science 2004, 303:1003-1006. 64. Nakagawa M, Komeda Y: Flowering of Arabidopsis cop1 mutants in darkness. Plant Cell Physiol 2004, 45:398-406. 65. Datta S, Hettiarachchi GH, Deng XW, Holm M: Arabidopsis CONSTANS-LIKE3 is a positive regulator of red light signaling and root growth. Plant Cell 2006, 18:70-84. 66. Jang S, Marchal V, Panigrahi KC, Wenkel S, Soppe W, Deng XW, Valverde F, Coupland G: Arabidopsis COP1 shapes the temporal pattern of CO accumulation conferring a photoperiodic flowering response. EMBO J 2008, 27:1277-1288. The authors showed that transcription of FT, a CO target gene, is increased in cop1 mutants and decreased in COP1-overexpressing plants. Although COP1 is required for CO protein degradation in darkness but not in the morning or in continuous R, these results led the authors to suggest that a second ubiquitin ligase may be responsible for the phyBmediated turnover of CO early in the day and in R. 67. Liu LJ, Zhang YC, Li QH, Sang Y, Mao J, Lian HL, Wang L, Yang HQ: COP1-mediated ubiquitination of CONSTANS is implicated in cryptochrome regulation of flowering in Arabidopsis. Plant Cell 2008, 20:292-306. Analysis of cry1cry2co and cry1cry2cop1 triple mutants showed that CO acts downstream of COP1 and CRY in regulating photoperiodic flowering. Along with Jang et al. [66], the authors showed that COP1 associates with CO in vitro and in vivo. Moreover, the accumulation of CO protein in the presence of a dominant negative COP1 is consistent with the observation that COP1 can ubiquitinate CO in vitro. The authors proposed that cryptochrome might negatively regulate COP1 through CRY/COP1 interaction thereby elevating CO levels in the evening in LD to induce flowering by activating FT transcription.
57. Para A, Farre´ EM, Imaizumi T, Pruneda-Paz JL, Harmon FG, Kay SA: PRR3 is a vascular regulator of TOC1 stability in the Arabidopsis circadian clock. Plant Cell 2007, 19:3462-3473.
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69. Ishikawa M, Kiba T, Chua NH: The Arabidopsis SPA1 gene is required for circadian clock function and photoperiodic flowering. Plant J 2006, 46:736-746.
59. Ito S, Nakamichi N, Kiba T, Yamashino T, Mizuno T: Rhythmic and light-inducible appearance of clock-associated PseudoResponse Regulator protein PRR9 through programmed degradation in the dark in Arabidopsis thaliana. Plant Cell Physiol 2007, 48:1644-1651.
70. Laubinger S, Marchal V, Le Gourrierec J, Wenkel S, Adrian J, Jang S, Kulajta C, Braun H, Coupland G, Hoecker U: Arabidopsis SPA proteins regulate photoperiodic flowering and interact with the floral inducer CONSTANS to regulate its stability. Development 2006, 133:3213-3222.
Current Opinion in Plant Biology 2009, 12:49–56
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Regulated proteolysis in light-related signaling pathways Rossana Henriques1, In-Cheol Jang1 and Nam-Hai Chua Post-translational modification is an important mechanism to determine protein levels and/or activities in cells. The process of conjugation of ubiquitin units to particular proteins, ubiquitination, usually leads to proteasomal degradation. During the past several years considerable work has been done to reveal the role of ubiquitination in the regulation of plant signaling and development. This article focuses on recent advances made on the study of ubiquitin-mediated proteolysis of several light-related signaling pathways, such as photomorphogenesis, circadian clock function, and photoperiodic flowering.
sequential steps. This is done by the concerted action of E1, E2 and E3 enzymes. E1 is the Ub activating enzyme, E2 the Ub conjugating enzyme and the Ub ligase which confers substrate specificity is known as E3. Proteins tagged with four or more Ub’s with K48 linkages are usually degraded by 26S proteasomes [3].
Address Laboratory of Plant Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA
E3 ligases in Arabidopsis
Corresponding author: Chua, Nam-Hai ([email protected]) 1 These authors contributed equally to this work.
Current Opinion in Plant Biology 2009, 12:49–56 This review comes from a themed issue on Growth and Development Edited by Charles S. Gasser and Caroline Dean Available online 10th December 2008 1369-5266/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2008.10.009
Introduction Research on plant signaling pathways has progressed from investigations on transcriptional control to post-transcriptional regulation. Prominent among post-transcriptional regulatory events is the control of the half-life of signal receptors and signaling components through ubiquitination and proteolysis. This regulatory strategy has been shown to initiate or terminate signaling and to determine the strength and duration of signal flux. Several previous reviews [1–5] have extensively discussed how ubiquitination/proteolysis regulates hormone signaling pathways, circadian clock function, flowering time and light signal transduction. Because of space limitation we have restricted this review to recent results on light-related signaling pathways.
Ubiquitination Most cellular proteins are degraded via the ubiquitination/proteasome pathway. Ubiquitination involves the attachment of several units of a small protein, ubiquitin (Ub) to specific lysine residues of a substrate protein in www.sciencedirect.com
On the other hand, proteins modified by monoubiquitin or ubiquitin chains with linkages other than K48 are not destined for destruction but rather they are directly implicated in various signaling events.
E3s include a wide family of proteins/protein complexes divided in two groups: (1) single unit E3 ligases that possess a HECT domain [an active site first found at the C-terminus of the human E3 (E6-AP)] and bind directly to an activated Ub and (2) RING/U-box E3s, which rely on E2 for transfer of Ub to the substrate. RING E3s can be single units or multi-subunit protein complexes [2,3]. In Arabidopsis, E3s ligases constitute the largest group in the ubiquitin/proteasome pathway. So far seven genes (UPL1-7; Ubiquitin Protein Ligase) encoding HECT E3s have been identified [6]. RING E3 ligases constitute the majority of the known E3s, since more than 500 genes have been identified [2,3]. Single unit RING E3s, such as COP1 (CONSTITUTIVE PHOTOMORPHOGENESIS1) and SINAT5 (SEVEN IN ABSENTIA IN ARABIDOPSIS THALIANA 5), possess both the substrate and E2 binding motifs. Multi-subunit E3s divide the two functional domains into separate proteins. This is the case for the SCF (SKP1-CUL1-F-box), APC (Anaphase Promoting Complex) and CUL3-BTB complexes (Broadcomplex, Tramtrack, Bric-a-Brac) [2,3,5,7]. The SCF E3 ligases contain four subunits: SKP1 (in plants ASK for Arabidopsis SKP1), cullin, F-box and RBX1 (Ring-Box 1). The cullin subunit functions as a scaffolding protein binding both SKP1 and RBX1. The SKP1 in turn binds to an F-box protein which determines the substrate specificity of the SCF complex. The Arabidopsis genome contains genes encoding 21 ASKs, 5 cullins, 2 RBX1 proteins and more than 700 F-box proteins. This large number of subunit proteins predicts an innumerable number of possible combinations of SCF complexes targeting a wide variety of possible targets [2–4,7,8].
Light signaling In addition to providing energy for photosynthesis, light also signals vegetative and reproductive development. Current Opinion in Plant Biology 2009, 12:49–56
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Plants perceive wavelengths, intensities, direction and duration of light signals through a number of photoreceptors: phytochromes (phy) that detect red (R) and far-red (FR) light and cryptochromes (cry) and phototropins that function as blue light (B) photoreceptors [5]. In Arabidopsis there are five phytochromes: phyA is the major regulator of FR signaling, phyB mediates RL responses whereas the other phytochromes have a redundant role. Upon activation by light, phytochrome dimers translocate to the nucleus where they initiate specific signaling cascades [9,10]. Downstream of phytochromes, several transcription factors (TFs) act as positive or negative regulators of photomorphogenesis. In FR signaling, the positive regulators HYPOCOTYL5 (HY5) and HY5 HOMOLOG (HYH) are degraded in darkness but become stabilized in the light to promote photomorphogenesis [11,12]. Two other positive factors, LONG AFTER FAR-RED LIGHT1 (LAF1) and LONG HYPOCOTYL IN FAR-RED1 (HFR1) are degraded in the light as part of the signal attenuation mechanism [13–17]. Negative regulators such as PHYTOCHROME INTERACTING FACTOR1, 3, 4, 5 (PIF1, PIF3, PIF4, PIF5) are stable in darkness but rapidly degraded upon light exposure [18–20]. Although the stability of these factors is regulated in an opposing manner in darkness and light, all proteins are degraded by 26S proteasomes. A major repressor of photomorphogenesis in darkness is the RING protein CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1). As a nuclear protein in darkness, COP1 presumably becomes depleted from the nucleus upon prolonged light exposure [21] to allow the accumulation of positive regulators. This RING protein possesses auto-ubiquitination activity in vitro and in vivo [16] and can ubiquitinate LAF1, HY5, and HFR1 through direct interaction [13,16,17,22]. The importance of COP1-mediated protein degradation in light signaling renders this step the target of multiple regulations. One mechanism is to directly modulate COP1 E3 activity with regulatory factors. All four SUPPRESSOR OF PHYTOCHROME A (SPA) proteins function in concert with COP1 to suppress photomorphogenesis in dark-grown seedlings [23]. Consistent with this, the SPA1 coiled–coil domain promotes LAF1 ubiquitination by COP1 at low COP1 concentrations [16]. On the other hand, full-length SPA1 inhibits COP1 E3 ligase activity towards HY5 [22]. Phosphorylation status of receptors and signaling factors is known to affect their subsequent ubiquitination and proteasomal degradation. Degradation of the photoreceptor, phyA, is mediated by COP1 as part of the signal termination process [24]. Recently, Saijo et al. [25] reported that SPA1 enhances COP1 E3 ligase activity Current Opinion in Plant Biology 2009, 12:49–56
towards phyA, as was the case with LAF1 [16]. Moreover, COP1/SPA1 preferentially interacts with phosphorylated phyA, whereas underphosphorylated phyA associates with its downstream targets, FHY3 and FHY1 [25]. In addition to phyA, cryptochrome 2 (cry2) also undergoes blue-light induced phosphorylation in vivo and the phosphorylated form is preferentially degraded [26]. This degradation depends on COP1 because phosphorylated cry2 accumulates to higher levels in cop1-6 compared to WT. It is not known whether cry2 is a direct target of COP1. Early work showed that the unphosphorylated HY5 is the preferred COP1 substrate whereas the phosphorylated form accumulates [27]. By contrast, HFR1 can be phosphorylated on multiple N-terminal residues by casein kinase II [28] and the phosphorylated form appears to be the COP1 substrate. This result is consistent with the observation that removal of the HFR1 N-terminal region stabilizes the protein [13]. The suggestion that COP1 would preferably target phosphorylated HFR1 in darkness [15] is difficult to reconcile with the observation that light promotes HFR1 phosphorylation [28]. Moreover, both isoforms of HFR1 are found in light and darkness and light induces the accumulation of both species [28]. Because several TFs operating downstream of phyA are known to form heterodimers, association/dissociation of TFs may regulate their binding to E3 ligases and therefore stability. Jang et al. [14] recently reported that the HFR1 (bHLH)/LAF1 (R2R3-MYB) association attenuates their ubiquitination by the COP1 E3 ligase leading to increased accumulation of these factors. Combinatorial interaction amongst TFs occurring under different environmental conditions may be an important mechanism to regulate TF stability and thus signal flux through particular nodes. COP1 is also known to regulate BL responses. Cryptochromes can bind to COP1 [29] and inhibit COP1 E3 activity in a BL-dependent manner, leading to an increased stability of TFs that activate blue light-dependent gene expression. Hong et al. [30] recently identified the Blue Insensitive Trait (BIT1), a R2R3 type MYB transcription factor, as a positive regulator of BL signaling. Since BIT1 degradation is delayed in cop1-4 mutant this factor is likely a COP1 target, although in vitro ubiquitination of BIT1 by COP1 has not yet been shown. Besides the RING E3 ligase COP1, several SCF complexes likely regulate light signaling as well. Two alleles of CUL1, axr6-3 and cul1-6, show hyper- and hyposensitivity to far-red and red light, respectively [31,32] and several F-box proteins have been implicated in light responses [33]. SCF regulation of light signaling, however, is poorly understood, compared to the impressive progress that has been made in the identification of F-box www.sciencedirect.com
Regulated proteolysis in light-related signaling pathways Henriques, Jang and Chua 51
proteins involved in the control of hormone signaling pathways [33,34].
PHYTOCHROME INTERACTING FACTORS – integrators of multiple signals
dimerize or heterodimerize and they accumulate in darkness. PIF1, PIF3 and PIF4 have been shown to bind to the G-box motif (50 CACGTG 30 ) and activate the transcription of genes controlling the elongation response [9,35–39].
Differences in hypocotyl length between etiolated and light-grown seedlings are largely due to difference in cell elongation. A group of factors, called PIFs (PHYTOCHROME-INTERACTING FACTORS), has emerged as important regulators of cell elongation in darkness. PIFs are TFs belonging to subfamily 15 of the helix-loophelix superfamily. First characterized by their binding to primarily phyB, and sometimes phyA, through their active phytochrome binding (APB) motif, PIFs can homo-
In the light, activated phyB in the nucleus is able to interact with the PIFs via their APB motif. So far PIF1, 3, 4, 5, 6 and PIF7 have been identified as phyB interactors [40–42], whereas PIF1 and PIF3 also associate with phyA although their APA motif is less conserved [35,38]. Immediately after RL exposure, PIFs are involved in activating transcription of genes necessary for de-etiolation [18,38,43]. Upon longer RL exposure, however, phyB
Figure 1
(a) Light-dependent proteasomal degradation of PIFs. In the light, activated phyB translocates into the nucleus and binds to PIFs. PIFs are phosphorylated (P) in a phyB-dependent manner and ubiquitinated (Ub) by an unknown E3 ligase before proteasomal degradation. In the dark, however, accumulated PIFs can promote transcription of target genes, resulting in cell elongation. (b) Regulation of PIF proteins in GA signaling. In the absence of GA, DELLA proteins directly interact with PIFs (PIF3 and PIF4) and inhibit their transcription activity. Upon light exposure, PIFs are phophorylated (P) by an yet unidentified kinase (K), ubiquitinated (Ub) by unknown E3 ligases and finally degraded by 26S proteasome. When GA is present, GID1 binds to GA and leads to DELLA degradation by SCFSLY1/GID2. Released PIFs bind to G-box elements to activate transcription of elongation genes. www.sciencedirect.com
Current Opinion in Plant Biology 2009, 12:49–56
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targets the PIFs for destruction by 26S proteasomes (Figure 1a). In FR light, phyA can also target PIF1 and PIF3 for degradation [18,44]. Therefore, except PIF7, all other PIFs are light labile and this is consistent with the decreased transcription of cell elongation genes in the light [42]. As shown for positive regulators of light responses, PIF1, PIF3 and PIF5 are also phosphorylated, probably in a phyB-dependent manner, and phosphorylation presumably precedes their ubiquitination/degradation [40,44,45]. Consistent with this observation, PIF1 can be stabilized by mutations affecting phyA/B binding [44]. Other than the report that COP1 is not involved in the degradation of PIF3 [18], the E3 ligase(s) responsible for PIF ubiquitination remain to be characterized. Among the various plant hormones, gibberellic acid (GA) is known to promote stem elongation via cell expansion. This raises the question whether PIFs are also involved in this process, and if so, how GA and light signaling pathways interact. Recently, it was shown that both PIF3 and PIF4 transcriptional activities are repressed by DELLA proteins, but degradation of the latter via the SCFSLY1/GID2 E3 ligase is triggered by GA [36,37] (Figure 1b). This mechanism explains very nicely how GA promotes cell elongation through the PIFs. Another member of the PIF family, PIF1, can bind to promoters of genes encoding DELLA proteins [46] and this activity would produce more DELLA proteins which re-repress the GA pathway. Combining the recent results on light and GA signaling, we may assume that upon germination, when GA levels are high and the germinated seedlings are still below the soil surface, the PIF proteins, free from DELLA repression and phyB-dependent degradation, are able to promote hypocotyl growth towards the soil surface. Upon exposure to light, the PIFs are degraded resulting in reduced cell expansion and inhibition of hypocotyl growth. Although phyB is known to regulate the stability of PIFs, the reverse situation may also hold. Immediately after illumination, PIFs (PIF3, 4, 5 and 7) promote de-etiolation, but on longer light exposure, they regulate the levels of available phytochrome, preventing an exaggerated light response [42,43]. PIF levels are regulated, not only by light and GA, but also by the circadian clock. Seedling hypocotyl growth peaks at dawn, and this rhythmic growth depends on both light and the circadian clock [19]. Detailed expression profiling led to the identification of PIF4 and PIF5 as integrators of clock and light signals [19]. The inhibition of PIF4/PIF5 transcription by the clock at dusk is relieved at dawn, allowing accumulation of PIF4/PIF5 proteins which induce transcription of growth-response genes in the dark. Upon light exposure, both PIF4/5 are targeted Current Opinion in Plant Biology 2009, 12:49–56
for degradation by phyB. The diurnal regulation of hypocotyl growth depends on these interconnected mechanisms that affect PIF4/PIF5 transcript (the clock) and protein (light) levels independently [19]. Although the clock does not directly target PIF4-PIF5 proteins, it appropriately constrains their diurnal availability, allowing elongation growth to occur at the more convenient period.
Degradation of circadian regulators is required for maintenance of circadian rhythms Being the biological oscillator responsible for the integration of plant growth/metabolism to the 24 h day/night cycles, the clock translates environmental signals (input) into appropriate cellular responses that increase fitness and survival rate [47,48]. The circadian clock relies on the generation of robust cyclic rhythms created by negative feedback loops at the core of the oscillator. Recent evidence suggests the existence of a morning and an evening loop [1,49,50]. In the morning oscillator, light and CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1)/ LHY (LATE ELONGATED HYPOCOTYL) promote the accumulation of PRR7 and PRR9, (PSEUDORESPONSE REGULATOR7 and 9), which inhibit CCA1/LHY expression. In the evening loop, GI (GIGANTEA) accumulates and promotes the expression of TOC1 (TIMING OF CAB EXPRESSION1), that represses GI, by an unknown mechanism [1,49,50]. In this model the two oscillators are connected by a number of unknown regulators. Initial studies were mostly based on transcript analysis, but new findings have shown that post-translational regulation is critical for the maintenance of robust circadian oscillations [48]. The ZTL (ZEITLUPE) family of F-box proteins [ZTL; FKF1 (FLAVIN BINDING KELCH FBOX 1), and LKP2 (LOV KELCH PROTEIN 2)] is a major regulator of several circadian proteins. These F-box proteins possess an N-terminal LOV (Light-Oxygen-Voltage dependent) domain that responds to blue light, and several Kelch repeats, important for protein-protein interactions [51,52,53]. ZTL associates with three known components of SCF complexes (ASK1, AtCUL1 and AtRBX1), suggesting that ZTL can assemble into an SCF complex in vivo [51]. Indeed, CUL1 association with ZTL is required for a functional SCFZTL complex [54]. Interestingly, ZTL itself is also unstable and the mechanism mediating this instability remains to be investigated [51,55]. Kim et al. [55] recently found that GI associates with the BL-activated form of ZTL thereby stabilizing the latter. As the dark period progresses, GI dissociates from the complex allowing the SCFZTL E3 ligase to interact with its substrates [55], TOC1 and PRR5 (PSEUDORESPONSE REGULATOR5) [52,53]. It should be noted that ZTL regulation of TOC1 and PRR5 levels www.sciencedirect.com
Regulated proteolysis in light-related signaling pathways Henriques, Jang and Chua 53
Figure 2
Transcription of CO depends on the circadian clock. In LD, the formation of GI/FKF1 complex facilitates association of the F-box protein with CDF1, an inhibitor of CO transcription. Upon CDF1 degradation, CO transcript levels increase and, under these conditions, CO protein also accumulates to induce flowering.
is critical for the generation/maintenance of robust circadian rhythms and proper clock function [52,53]. Although the mechanism of interaction might differ, TOC1 and PRR5 are the only PRR family members directly regulated by ZTL [56]. Furthermore, PRR proteins display a diurnal pattern of phosphorylation which, for TOC1 and PRR5, could facilitate ZTL association and increase their degradation. By contrast, phosphorylation enhances the TOC1/ PRR3 interaction and increases TOC1 stability probably by preventing ZTL binding [56]. Para et al., [57] also reported the formation of a TOC1/PRR3 complex in the vasculature suggesting the existence of a tissue-specific clock mechanism. PRR3, PRR5 and TOC1 may constitute a unit in the central oscillator as their transcript and protein levels are tightly and similarly regulated [56]. Other members of the PRR family, PRR9 and PRR7 were also shown to be targets for proteasomes but the E3 ligases involved remain unidentified [58,59]. GI also plays a role in flowering time control by activating CO (CONSTANS), a gene required for the onset of flowering [60]. Interestingly, GI levels are regulated by the 26S proteasome, leading to GI degradation in the dark [60]. Recently, Sawa et al. [61] showed that GI can associate with the F-box protein FKF1 (a ZTL-family member)
Figure 3
Photoperiodic regulation of COSTANS (CO) stability. There are two possible mechanisms for CO protein degradation under LD: a COP1-dependent mechanism, which occurs late in the day and during the night and/or a phyB-dependent mechanism, which occurs early in the morning and/or in response to red light. One or more E3 ligase (X) might be involved in CO degradation early in the day. CO accumulates in the late afternoon and photoreceptor(s) such as CRY may repress COP1 activity. Under SDs, CO protein is degraded in a COP1/SPAs-dependent manner. The reduced CO protein level is not sufficient to induce FT expression resulting in late flowering. www.sciencedirect.com
Current Opinion in Plant Biology 2009, 12:49–56
54 Growth and Development
and regulate its activity towards CDF1 (CYCLING DOF FACTOR 1), a negative regulator of CO. The GI/FKF1 interaction occurs via a photo-activated LOV domain similarly to GI/ZTL [55]. In long days (LD), the three proteins CDF1, GI and FKF1 accumulate sequentially. CDF1 is able to bind and repress the CO promoter. GI then interacts with CDF1 and recruits the photo-activated FKF1 to target CDF1 for degradation, releasing CO repression (Figure 2). In LD, the CO protein which is stabilized (see below) induces FT (FLOWERING LOCUS T) to promote flowering. In short days (SD), GI and FKF1 proteins peak at different times and their interaction is diminished, resulting in more stable CDF1 and repression of flowering. These results nicely describe a mechanism by which light and circadian clock affect protein levels that control flowering time.
Conclusions Studies on the role of proteolysis in light-related signaling pathways have advanced from substrate identification to regulatory aspects of ubiquitination and proteolysis. Future efforts should be devoted to the development of in vitro biochemical assays which can recapitulate in vivo events with high fidelity, so that putative gene functions predicted by genetic analysis, can be verified and their mechanism of action elucidated.
Acknowledgement Light-related research done in our lab was supported by a grant from NIH GM44640 to NHC.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
Regulation of photoperiodic flowering The transition from vegetative to reproductive growth is controlled by environmental factors such as temperature and day length. CO, which activates the floral regulators FT and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (SOC1), is required for flowering under LD. Whereas CO transcription is regulated by the circadian clock via GI and the SCFFKF1 E3 ligase, CO protein levels are differentially regulated by light quality, being more stable under FR and BL but unstable under RL [62]. Under SD conditions, CO is rapidly degraded by 26S proteasomes in darkness and remains unstable throughout the day [63]. In darkness, cop1-6 mutant expresses increased FT and SOC1 transcript levels [64], suggesting that COP1 might repress flowering by regulating CO levels. Indeed, CONSTANS-LIKE 3 (COL3), a homolog of CO, has been identified as a novel COP1-interacting protein [65]. Recently, CO was shown to interact with COP1 via its C-terminal CCT domain suggesting that it may be a COP1 substrate [66,67]. Since CO protein stability is also under the control of CRY1/CRY2 and phyB, there must be an additional layer of control besides ubiquitination by COP1 [66,67] (Figure 3). SPA1 and its homologs, SPA3 and SPA4, are likely involved in regulating CO abundance as well. SPA1 acts as a negative regulator of phyA-mediated de-etiolation in concert with COP1 [16,22,68] and regulates circadian rhythms and flowering time [69]. The SPA proteins were shown to interact with CO in vitro and in vivo. Moreover, SPA1 interacts with the CCT-domain of CO [70], the binding site of COP1 [67], and CO protein accumulates in the spa1spa3spa4 triple mutant [70]. Together, these results suggest that, COP1 and SPA1 cooperate to negatively regulate CO protein stability, as is the case with LAF1 [16] (Figure 3). It should be emphasized, however, that more than one E3 ligase may mediate CO degradation early in the day and there is some evidence to implicate a phyB-controlled mechanism (Figure 3). Current Opinion in Plant Biology 2009, 12:49–56
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