Illuminating Progress in Phytochrome-Mediated Light Signaling Pathways
2015; Elsevier BV; Volume: 20; Issue: 10 Linguagem: Inglês
10.1016/j.tplants.2015.06.010
ISSN1878-4372
AutoresXiaosa Xu, Inyup Paik, Ling Zhu, Enamul Huq,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoTwo classes of repressors called COP/DET/FUS complex and PIFs synergistically repress photomorphogenesis in darkness. Light signals perceived by phytochromes inhibit these repressors to promote photomorphogenesis. CUL3LRB induces polyubiquitylation and subsequent co-degradation of PIF3 and PHYB through the 26S proteasome pathway. CUL4COP1–SPA E3 ligase promotes rapid light-induced degradation of PIF1 to promote photomorphogenesis. Phytochromes directly interact with SPA1 and reorganize the COP1–SPA interaction to inhibit COP1 activity. Light signals regulate a plethora of plant responses throughout their life cycle, especially the red and far-red regions of the light spectrum perceived by the phytochrome family of photoreceptors. However, the mechanisms by which phytochromes regulate gene expression and downstream responses remain elusive. Several recent studies have unraveled the details on how phytochromes regulate photomorphogenesis. These include the identification of E3 ligases that degrade PHYTOCHROME INTERACTING FACTOR (PIF) proteins, key negative regulators, in response to light, a better view of how phytochromes inhibit another key negative regulator, CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), and an understanding of why plants evolved multiple negative regulators to repress photomorphogenesis in darkness. These advances will surely fuel future research on many unanswered questions that have intrigued plant photobiologists for decades. Light signals regulate a plethora of plant responses throughout their life cycle, especially the red and far-red regions of the light spectrum perceived by the phytochrome family of photoreceptors. However, the mechanisms by which phytochromes regulate gene expression and downstream responses remain elusive. Several recent studies have unraveled the details on how phytochromes regulate photomorphogenesis. These include the identification of E3 ligases that degrade PHYTOCHROME INTERACTING FACTOR (PIF) proteins, key negative regulators, in response to light, a better view of how phytochromes inhibit another key negative regulator, CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), and an understanding of why plants evolved multiple negative regulators to repress photomorphogenesis in darkness. These advances will surely fuel future research on many unanswered questions that have intrigued plant photobiologists for decades. Light is an essential commodity for photosynthetic energy production as well as an environmental cue for increasing awareness and fitness to the surrounding conditions. Plants employ two contrasting developmental programs to succeed in ambient light conditions: skotomorphogenesis and photomorphogenesis (Figure 1). Skotomorphogenesis is characterized by elongated hypocotyl, closed cotyledon, and an apical hook to allow young seedlings to grow rapidly in darkness using the reserve energy present in the seed. By contrast, photomorphogenesis is the process where light signals inhibit the rapid elongation of hypocotyl, expand the cotyledons, and promote greening to allow the seedling body to adjust for optimal light-harvesting capacity and autotrophic growth. To promote photomorphogenesis and actively suppress skotomorphogenic development, plants have evolved multiple photoreceptors to track a wide spectrum of light wavelengths in a local environment. These include the UVB-RESISTANCE 8 (UVR8 for UV-B light), cryptochromes (CRY), phototropins, and ZEITLUPE/FLAVIN-BINDING, KELCH REPEAT, F BOX 1/LOV KELCH PROTEIN 2 family of photoreceptors (ZTL/FKF1/LKP2) (for UV-A/blue light) and phytochromes (phy for red/far-red light) [1Galvão V.C. Fankhauser C. Sensing the light environment in plants: photoreceptors and early signaling steps.Curr. Opin. Neurobiol. 2015; 34: 46-53Crossref PubMed Scopus (240) Google Scholar]. This review will focus on the phytochrome family of photoreceptors that are encoded by five genes in Arabidopsis thaliana (PHYA–PHYE) [2Mathews S. Sharrock R.A. Phytochrome gene diversity.Plant Cell Environ. 1997; 20: 666-671Crossref Scopus (256) Google Scholar]. Phytochromes perceive the ambient red (R) and far-red (FR) light signals in the environment and promote gradual progression to photomorphogenic development by orchestrating an elaborate signaling mechanisms [3Jiao Y. et al.Light-regulated transcriptional networks in higher plants.Nat. Rev. Genet. 2007; 8: 217-230Crossref PubMed Scopus (760) Google Scholar, 4Leivar P. Quail P.H. PIFs: pivotal components in a cellular signaling hub.Trends Plant Sci. 2011; 16: 19-28Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar]. These include allosteric conformation change of phytochromes to a biologically active Pfr form from an inactive Pr form followed by nuclear translocation to inhibit two classes of repressors of photomorphogenesis called CONSTITUTIVELY PHOTOMORPHOGENIC/DEETIOLATED/FUSCA (COP/DET/FUS) complex and Phytochrome Interacting Factors (PIFs) (Figure 1) [4Leivar P. Quail P.H. PIFs: pivotal components in a cellular signaling hub.Trends Plant Sci. 2011; 16: 19-28Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar, 5Lau O.S. Deng X.W. The photomorphogenic repressors COP1 and DET1: 20 years later.Trends Plant Sci. 2012; 17: 584-593Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar]. In darkness, these dual repressors are actively promoting skotomorphogenic development by suppressing photomorphogenesis, and therefore inhibition of these repressors allows gradual progression to photomorphogenic development under light. This review will focus on recent progress on the mechanistic understanding of how phytochromes inhibit these repressors. For a detailed review on other aspects of photomorphogenesis, readers are directed to recent reports and reviews [6Jeong J. Choi G. Phytochrome-Interacting factors have both shared and distinct biological roles.Mol. Cells. 2013; 35: 371-380Crossref PubMed Scopus (64) Google Scholar, 7Leivar P. Monte E. PIFs: systems integrators in plant development.Plant Cell. 2014; 26: 56-78Crossref PubMed Scopus (366) Google Scholar, 8Chew Y.H. et al.Mathematical models light up plant signaling.Plant Cell. 2014; 26: 5-20Crossref PubMed Scopus (31) Google Scholar, 9Burgie E.S. Vierstra R.D. Phytochromes: an atomic perspective on photoactivation and signaling.Plant Cell. 2014; 26: 4568-4583Crossref PubMed Scopus (120) Google Scholar, 10Wu S-H. Gene expression regulation in photomorphogenesis from the perspective of the central dogma.Annu. Rev. 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Fankhauser C. bHLH class transcription factors take centre stage in phytochrome signalling.Trends Plant Sci. 2005; 10: 51-54Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar]. There are seven PIFs in Arabidopsis that function in a partially-differential to a largely-overlapping manner to regulate gene expression and ultimately photomorphogenesis [4Leivar P. Quail P.H. PIFs: pivotal components in a cellular signaling hub.Trends Plant Sci. 2011; 16: 19-28Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar, 16Zhang Y. et al.A quartet of PIF bHLH factors provides a transcriptionally centered signaling hub that regulates seedling morphogenesis through differential expression-patterning of shared target genes in Arabidopsis.PLoS Genet. 2013; 9: e1003244Crossref PubMed Scopus (264) Google Scholar, 17Pfeiffer A. et al.Combinatorial complexity in a transcriptionally-centered signaling hub in Arabidopsis.Mol. Plant. 2014; 7: 1598-1618Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 18Shin J. et al.Phytochromes promote seedling light responses by inhibiting four negatively-acting phytochrome-interacting factors.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 7660-7665Crossref PubMed Scopus (333) Google Scholar, 19Leivar P. et al.Multiple phytochrome-interacting bHLH transcription factors repress premature seedling photomorphogenesis in darkness.Curr. Biol. 2008; 18: 1815-1823Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar]. All PIFs interact with the Pfr forms of phytochromes with differential affinities [4Leivar P. Quail P.H. PIFs: pivotal components in a cellular signaling hub.Trends Plant Sci. 2011; 16: 19-28Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar, 20Castillon A. et al.Phytochrome interacting factors: central players in phytochrome-mediated light signaling networks.Trends Plant Sci. 2007; 12: 514-521Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar]. Phytochromes interact with PIFs through the APB (active phytochrome binding) or APA (active phytochrome A binding) domains present at the N termini of PIFs. Conversely, PIFs displayed higher affinity for the N terminus of phytochromes [21Shen H. et al.Light-induced phosphorylation and degradation of the negative regulator PHYTOCHROME INTERACTING FACTOR 1 depends upon its direct physical interactions with photoactivated phytochromes.Plant Cell. 2008; 20: 1586-1602Crossref PubMed Scopus (208) Google Scholar, 22Zhu Y. et al.Phytochrome B binds with greater apparent affinity than phytochrome A to the basic helix-loop-helix factor PIF3 in a reaction requiring the PAS domain of PIF3.Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 13419-13424Crossref PubMed Scopus (111) Google Scholar]. Direct physical interaction of PIFs with phytochromes leads to the light-induced phosphorylation followed by ubiquitylation and subsequent degradation of PIFs by the ubiquitin/26S proteasome system (UPS). In addition, light-induced phosphorylation is necessary for degradation of PIF3 [23Ni W. et al.Multisite light-induced phosphorylation of the transcription factor PIF3 is necessary for both its rapid degradation and concomitant negative feedback modulation of photoreceptor phyB levels in Arabidopsis.Plant Cell. 2013; 25: 2679-2698Crossref PubMed Scopus (98) Google Scholar]. The degradation kinetics of PIFs under different light qualities/quantities and early post-translational modifications have been extensively investigated [4Leivar P. Quail P.H. PIFs: pivotal components in a cellular signaling hub.Trends Plant Sci. 2011; 16: 19-28Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar]. A putative polyubiquitin binding factor called HEMERA is also necessary for degradation of PIF1 and PIF3 under prolonged light [24Chen M. et al.Arabidopsis HEMERA/pTAC12 initiates photomorphogenesis by phytochromes.Cell. 2010; 141: 1230-1240Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 25Galvao R.M. et al.Photoactivated phytochromes interact with HEMERA and promote its accumulation to establish photomorphogenesis in Arabidopsis.Genes Dev. 2012; 26: 1851-1863Crossref PubMed Scopus (64) Google Scholar]. However, the kinases that phosphorylate PIFs and the E3 ligases that ubiquitylate PIFs in response to light are under intense investigation. Two recent reports described the identification of E3 ligases for PIF degradation [26Ni W. et al.A mutually assured destruction mechanism attenuates light signaling in Arabidopsis.Science. 2014; 344: 1160-1164Crossref PubMed Scopus (183) Google Scholar, 27Zhu L. et al.CUL4 forms an E3 ligase with COP1 and SPA to promote light-induced degradation of PIF1.Nat. Commun. 2015; 6: 7245Crossref PubMed Scopus (82) Google Scholar]. These studies highlight the complex mechanism of how PIFs are regulated to fine-tune photomorphogenesis. A recent study described a CULLIN 3 (CUL3)-based E3 ligase for PIF3 degradation (Figure 2, right). The substrate adaptor component for this ligase is LRB (light response BTB) proteins [26Ni W. et al.A mutually assured destruction mechanism attenuates light signaling in Arabidopsis.Science. 2014; 344: 1160-1164Crossref PubMed Scopus (183) Google Scholar]. LRBs belong to the BTB family (Bric-a-Brack/Tramtrack/Broad) and display strong affinity for the phosphorylated form of PIF3, which is consistent with the light-induced phosphorylation and subsequent degradation of PIFs. In addition, CUL3LRB can catalyze ubiquitylation of a phosphomimic form of PIF3 in vitro. Interestingly, LRBs recruit both PIF3 and phyB in the CUL3LRB complex for polyubiquitylation and subsequent co-degradation by the 26S proteasome pathway. Because LRBs interact with each other to dimerize, it is possible that the PIF3–phyB bimolecular tetramer is recognized by two CUL3LRB complexes for light-induced ubiquitylation [28Zhu L. Huq E. Suicidal co-degradation of the Phytochrome Interacting Factor 3 and phytochrome B in response to light.Mol. Plant. 2014; 7: 1709-1711Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar, 29Christians M.J. et al.The light-response BTB1 and BTB2 proteins assemble nuclear ubiquitin ligases that modify phytochrome B and D signaling in Arabidopsis.Plant Physiol. 2012; 160: 118-134Crossref PubMed Scopus (39) Google Scholar]. This study also highlights the importance of receptor desensitization in many eukaryotic systems where the receptor is activated to transmit the incoming signal and then the receptor is either degraded or endocytosed to inactivate. This prevents the signaling pathways from overactivation under prolonged incoming signals [30Avraham R. Yarden Y. Feedback regulation of EGFR signalling: decision making by early and delayed loops.Nat. Rev. Mol. Cell Biol. 2011; 12: 104-117Crossref PubMed Scopus (492) Google Scholar]. However, the drawback of this study is the lack of any biological significance of PIF3 degradation. lrb double and triple mutants do display photomorphogenic phenotypes; however, these phenotypes are not consistent with PIF3 degradation, but are consistent with phyB degradation. In fact, LRBs have previously been shown to regulate phyB and phyD levels in response to light [29Christians M.J. et al.The light-response BTB1 and BTB2 proteins assemble nuclear ubiquitin ligases that modify phytochrome B and D signaling in Arabidopsis.Plant Physiol. 2012; 160: 118-134Crossref PubMed Scopus (39) Google Scholar]. Not surprisingly, lrb double and triple mutants are strongly hypersensitive to light as they have higher level of phyB and phyD under light. LRBs interact with phyB both in vitro and in vivo [26Ni W. et al.A mutually assured destruction mechanism attenuates light signaling in Arabidopsis.Science. 2014; 344: 1160-1164Crossref PubMed Scopus (183) Google Scholar]. However, it is not clear whether LRBs have higher affinity for the phosphorylated form of phyB than the unphosphorylated form of phyB, although phyB is known to be phosphorylated [31Medzihradszky M. et al.Phosphorylation of phytochrome B inhibits light-induced signaling via accelerated dark reversion in Arabidopsis.Plant Cell. 2013; 25: 535-544Crossref PubMed Scopus (85) Google Scholar]. Therefore, at the molecular level, LRBs function as bona fide E3 ligases for PIF3. However, the morphological phenotypes suggest that the main function of LRBs is to degrade phyB/D for receptor desensitization under prolonged light conditions, and the role of PIF3 is to enhance this process by helping the LRBs to recruit phyB/D. Because LRBs preferentially bind to the phosphorylated form of PIF3, and PIF3 binds to phyB/D, PIF3 might function as a cofactor for LRBs for enhanced substrate recruitment in this process. However, PIF3 is also degraded in this process without having any biological consequence for plants. Thus, it is likely that additional E3 ligase(s) are necessary for PIF3 and other PIFs degradation in response to light. In line with this prediction, a recent study described a well-established CUL4-based E3 ligase for PIF1 degradation in response to light [27Zhu L. et al.CUL4 forms an E3 ligase with COP1 and SPA to promote light-induced degradation of PIF1.Nat. Commun. 2015; 6: 7245Crossref PubMed Scopus (82) Google Scholar]. In this case, COP1 and SPA proteins act as substrate adaptor components in recruiting preferentially the phosphorylated form of PIF1 in the CUL4COP1–SPA complex for light-induced ubiquitylation and subsequent degradation. The light-induced ubiquitylation followed by degradation of PIF1, but not the light-induced phosphorylation of PIF1, is defective in cop1, spaQ, and cul4cs backgrounds compared to wild type. This rapid degradation of PIF1 is mostly regulated by phyA, and phyA is not degraded under these conditions, suggesting that PIF1 and phyA may not be co-degraded as previously shown for PIF3–phyB co-degradation [26Ni W. et al.A mutually assured destruction mechanism attenuates light signaling in Arabidopsis.Science. 2014; 344: 1160-1164Crossref PubMed Scopus (183) Google Scholar]. In addition, cop1 and spaQ mutants display a strong hyposensitive phenotype in seed germination assays, consistent with the major role of PIF1 in this process. COP1 and SPA proteins as well as PIFs are well-established repressors of photomorphogenesis in the dark [4Leivar P. Quail P.H. PIFs: pivotal components in a cellular signaling hub.Trends Plant Sci. 2011; 16: 19-28Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar, 5Lau O.S. Deng X.W. The photomorphogenic repressors COP1 and DET1: 20 years later.Trends Plant Sci. 2012; 17: 584-593Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar]. Strikingly, these repressors function synergistically to repress photomorphogenesis in the dark (see below) [32Xu X. et al.PHYTOCHROME INTERACTING FACTOR1 enhances the E3 ligase activity of CONSTITUTIVE PHOTOMORPHOGENIC1 to synergistically repress photomorphogenesis in Arabidopsis.Plant Cell. 2014; 26: 1992-2006Crossref PubMed Scopus (61) Google Scholar]. However, in response to light, one group of repressors (COP1/SPA) targets another group (PIFs) to promote their rapid degradation under low light conditions. This allows plants to prevent over repression of photomorphogenesis and gradually transition to photomorphogenic development. However, PIF1 is still degraded under prolonged light conditions in all the above mutants, suggesting additional E3 ligases are necessary for PIF1 and other PIF degradation. In addition, because PIF3 is unstable in cop1 and spa mutants [19Leivar P. et al.Multiple phytochrome-interacting bHLH transcription factors repress premature seedling photomorphogenesis in darkness.Curr. Biol. 2008; 18: 1815-1823Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 33Bauer D. et al.Constitutive photomorphogenesis 1 and multiple photoreceptors control degradation of phytochrome interacting factor 3, a transcription factor required for light signaling in Arabidopsis.Plant Cell. 2004; 16: 1433-1445Crossref PubMed Scopus (341) Google Scholar], it is not clear if CUL4-based E3 ligase also plays a role in the degradation of other PIF proteins. One of the first post-translational modifications in PIFs before their degradation is light-induced phosphorylation [4Leivar P. Quail P.H. PIFs: pivotal components in a cellular signaling hub.Trends Plant Sci. 2011; 16: 19-28Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar]. Light-induced phosphorylation is a prerequisite for degradation of PIFs through the 26S proteasome system. As discussed above, both CUL3- and CUL4-based E3 ligases preferentially recruit the phosphorylated form of PIF3 and PIF1, respectively, to the E3 ligase complex for polyubiquitylation (Figure 2, right). Therefore, an intense search is underway to identify the kinases that phosphorylate PIFs in response to light. The first candidate considered for a PIF kinase was phytochrome itself because phytochrome has been shown to function as a serine/threonine kinase with a histidine kinase ancestry [34Yeh K.C. Lagarias J.C. Eukaryotic phytochromes: light-regulated serine/threonine protein kinases with histidine kinase ancestry.Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 13976-13981Crossref PubMed Scopus (351) Google Scholar]. Direct physical interaction with phytochrome is necessary for the light-induced phosphorylation and degradation of PIFs. Plant phytochrome has been shown to phosphorylate other substrates including PKS1, FHY1, and IAA proteins [35Shen Y.P. et al.Phytochrome A mediates rapid red light-induced phosphorylation of Arabidopsis FAR-RED ELONGATED HYPOCOTYL1 in a Low Fluence Response.Plant Cell. 2009; 21: 494-506Crossref PubMed Scopus (55) Google Scholar, 36Fankhauser C. et al.PKS1, a substrate phosphorylated by phytochrome that modulates light signaling in Arabidopsis.Science. 1999; 284: 1539-1541Crossref PubMed Scopus (330) Google Scholar, 37Colón-Carmona A. et al.Aux/IAA proteins are phosphorylated by phytochrome in vitro.Plant Physiol. 2000; 124: 1728-1738Crossref PubMed Scopus (186) Google Scholar]. In addition, bacterial phytochromes function as histidine kinases [38Vierstra R.D. Davis S.J. Bacteriophytochromes: new tools for understanding phytochrome signal transduction.Semin. Cell Dev. Biol. 2000; 11: 511-521Crossref PubMed Scopus (62) Google Scholar, 39Bhoo S-H. et al.Bacteriophytochromes are photochromic histidine kinases using a biliverdin chromophore.Nature. 2001; 414: 776-779Crossref PubMed Scopus (252) Google Scholar]. However, the drawback of all the above studies is that both the Pr and Pfr forms of phytochromes phosphorylated most of the substrates, despite the fact that only Pfr is the biologically active form of phytochrome. In addition, convincing in vivo evidence supporting the role of phytochrome as a kinase is still lacking because no kinase-inactive mutant form of phytochrome has been described that did not rescue the phy mutant phenotypes. Moreover, constitutively nuclear localized phytochromes do not induce photomorphogenesis in the absence of light, suggesting a Pfr-specific signaling mechanism [40Huq E. et al.Nuclear translocation of the photoreceptor phytochrome B is necessary for its biological function in seedling photomorphogenesis.Plant J. 2003; 35: 660-664Crossref PubMed Scopus (105) Google Scholar, 41Matsushita T. et al.Dimers of the N-terminal domain of phytochrome B are functional in the nucleus.Nature. 2003; 424: 571-574Crossref PubMed Scopus (241) Google Scholar, 42Genoud T. et al.FHY1 mediates nuclear import of the light-activated phytochrome A photoreceptor.PLoS Genet. 2008; 4: e1000143Crossref PubMed Scopus (86) Google Scholar]. In addition, a C-terminal single-nucleotide deletion mutant of phyB (phyB-28) expressing a truncated form without the histidine kinase-related domain is still partially functional in vivo, suggesting that the putative kinase domain is not essential for phyB function [43Krall L. Reed J.W. The histidine kinase-related domain participates in phytochrome B function but is dispensable.Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 8169-8174Crossref PubMed Scopus (80) Google Scholar]. However, several recent studies alleviate the concerns raised above. For example, the majority of the biological functions of phytochromes are located within the nucleus [40Huq E. et al.Nuclear translocation of the photoreceptor phytochrome B is necessary for its biological function in seedling photomorphogenesis.Plant J. 2003; 35: 660-664Crossref PubMed Scopus (105) Google Scholar, 41Matsushita T. et al.Dimers of the N-terminal domain of phytochrome B are functional in the nucleus.Nature. 2003; 424: 571-574Crossref PubMed Scopus (241) Google Scholar], although phyA displays roles in the cytosol [44Paik I. et al.Phytochrome regulates translation of mRNA in the cytosol.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 1335-1340Crossref PubMed Scopus (57) Google Scholar, 45Rösler J. et al.Arabidopsis fhl/fhy1 double mutant reveals a distinct cytoplasmic action of phytochrome A.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 10737-10742Crossref PubMed Scopus (88) Google Scholar]. The Pfr forms of all phytochromes translocate into the nucleus in response to light, while the Pr form is mostly in the cytosol [46Fankhauser C. Chen M. Transposing phytochrome into the nucleus.Trends Plant Sci. 2008; 13: 596-601Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar], suggesting a physical separation of the substrates from the kinase. Moreover, phyB has been shown to sequester PIFs in response to light by direct physical interaction, and this sequestration contributes to the biological function of phyB [47Park E. et al.Phytochrome B inhibits binding of phytochrome-interacting factors to their target promoters.Plant J. 2012; 72: 537-546Crossref PubMed Scopus (119) Google Scholar]. Many targeted mutations in the putative kinase domain have been described previously that rescued the phyA mutant phenotypes [48Boylan M. Quail P. Are the phytochromes protein kinases?.Protoplasma. 1996; 195: 12-17Crossref Scopus (27) Google Scholar]. However, these mutants might have rescued phyA mutant phenotypes due to phyA-mediated sequestration of PIFs similar to phyB, although sequestration of PIFs by phyA has not been demonstrated yet. Therefore, the rate-limiting steps might be at two levels: one at the nuclear translocation step of phytochromes to promote physical proximity to the substrates, and the other at the Pfr-specific interaction and phosphorylation of the substrates. The Pr-induced phosphorylation described previously might simply be forced phosphorylation due to the use of non-physiological amounts of the kinase and substrates in the in vitro experiments [34Yeh K.C. Lagarias J.C. Eukaryotic phytochromes: light-regulated serine/threonine protein kinases with histidine kinase ancestry.Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 13976-13981Crossref PubMed Scopus (351) Google Scholar, 36Fankhauser C. et al.PKS1, a substrate phosphorylated by phytochrome that modulates light signaling in Arabidopsis.Science. 1999; 284: 1539-1541Crossref PubMed Scopus (330) Google Scholar, 37Colón-Carmona A. et al.Aux/IAA proteins are phosphorylated by phytochrome in vitro.Plant Physiol. 2000; 124: 1728-1738Crossref PubMed Scopus (186) Google Scholar]. Thus, the hypothesis that kinase activity is one of the major biochemical functions of phytochromes might be worth revisiting. Despite the above inconclusive hypothesis, targeted candidate gene approaches have identified two kinases recently that phosphorylate PIFs directly. These include casein kinase II (CK2) and BRASSINOSTEROID INSENSITIVE 2 (BIN2) (Figure 2, right) [49Bu Q. et al.Phosphorylation by CK2 enhances the rapid light-induced degradation of PIF1.J. Biol. Chem. 2011; 286: 12066-12074Crossref PubMed Scopus (70) Google Scholar, 50Bernardo-García S. et al.BR-dependent phosphorylation modulates PIF4 transcriptional activity and shapes diurnal hypocotyl growth.Genes Dev. 2014; 28: 1681-1694Crossref PubMed Scopus (147) Google Scholar]. CK2 has been shown to phosphorylate seven serine/threonine (S/T) residues present in PIF1 in vitro [49Bu Q. et al.Phosphorylation by CK2 enhances the rapid light-induced degradation of PIF1.J. Biol. Chem. 2011; 286: 12066-12074Crossref PubMed Scopus (70) Google Scholar]. Serine to alanine substitution mutations in six of these sites, especially the three consecutive S residues at the C-terminal end, drastically reduced the degradation of PIF1 in response to light in vivo. However, PIF1 was still phosphorylated in response to light, suggesting that CK2 is not the light-regulated kinase that phosphorylates PIF1 in response to light. BIN2 has been shown to phosphorylate PIF4 in vitro, and this phosphorylation alters the degradation kinetics of PIF4 in response to light and brassinosteroid (BR) [50Bernardo-García S. et al.BR-dependent phosphorylation modulates PIF4 transcriptional activity and shapes diurnal hypocotyl growth.Genes Dev. 2014; 28: 1681-1694Crossref PubMed Scopus (147) Google Scholar]. However, it is still not clear whether CK2 and BIN2 phosphorylate PIFs in a light-dependent manner in vivo. Therefore, the light-regulated kinase that phosphorylates PIFs in response to light is still unknown [51Bu Q. et al.Multiple kinases promote light-induced degradation of PIF1.Plant Signal. Behav. 2011; 6: 1119-1121Crossref PubMed Scopus (16) Google Scholar]. One of the long outstanding questions in light signaling pathways is how COP1 is inactivated by light to promote photomorphogenesis. Photobiological experiments demonstrated that both phytochromes and cryptochromes inactivate COP1 in response to red/far-red and blue light, respectively [52Osterlund M.T. Deng X.W. Multiple photoreceptors mediate the light-induced reduction of GUS-COP1 from Arabidopsis hypocotyl nuclei.Plant J. 1998; 16: 201-208Crossref PubMed Google Scholar]. These photoreceptors employ dual mechanisms for this purpose. Under prolonged light as well as relatively shorter light exposure, COP1 is excluded from the nucleus [53Pacín M. et al.Rapid decline in nuclear COSTITUTIVE PHOTOMORPHOGENESIS1 abundance anticipates the stabilization of its target ELONGATED HYPOCOTYL5 in the light.Plant Physiol. 2014; 164: 1134-1138Crossref PubMed Scopus (61) Google Scholar, 54Subramanian C. et al.The Arabidopsis repressor of light signaling, COP1, is regulated by nuclear exclusion: mutational analysis by bioluminescence resonance energy transfer.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 6798-6802Crossref PubMed Scopus (88) Google Scholar]. However, COP1 is also rapidly inactivated by these photoreceptors to trigger light-induced gene expression and photomorphogenesis. CRY1 and CRY2 have been shown to directly interact with SPA1 to dissociate the COP1–SPA complex in response to blue light [55Liu B. et al.Arabidopsis cryptochrome 1 interacts with SPA1 to suppress COP1 activity in response to blue light.Genes Dev. 2011; 25: 1029-1034Crossref PubMed Scopus (273) Google Scholar, 56Lian H.L. et al.Blue-light-dependent interaction of cryptochrome 1 with SPA1 defines a dynamic signaling mechanism.Genes Dev. 2011; 25: 1023-1028Crossref PubMed Scopus (226) Google Scholar, 57Zuo Z. et al.Blue light-dependent interaction of CRY2 with SPA1 regulates COP1 activity and floral initiation in Arabidopsis.Curr. Biol. 2011; 21: 841-847Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar]. Nevertheless, how phytochromes inactivate COP1 was unknown until recently. Two recent studies showed that phytochromes also directly interact with SPA1 and reorganize the COP1–SPA complex in a light-dependent manner [58Lu X-D. et al.Red-light-dependent interaction of phyB with SPA1 promotes COP1–SPA1 dissociation and photomorphogenic development in Arabidopsis.Mol. Plant. 2015; 8: 467-478Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 59Sheerin D.J. et al.Light-activated phytochrome A and B interact with members of the spa family to promote photomorphogenesis in Arabidopsis by reorganizing the COP1/SPA Complex.Plant Cell. 2015; 27: 189-201Crossref PubMed Scopus (196) Google Scholar]. This reorganization leads to the separation of the physical contact between COP1 and SPA1, thereby reducing the ability of COP1 to degrade positively-acting transcription factors (e.g., HY5/HFR1/LAF1 and others) (Figure 3, right). The increased abundance of the positively-acting factors promotes photomorphogenesis in response to light. However, it is still not clear whether this separation only affects the SPA1-mediated enhancement of COP1 activity and/or directly inhibits the ability of COP1 to degrade the positively-acting transcription factors. Although these studies demonstrated a mechanism for rapid inactivation of COP1 by phytochromes, there are discrepancies between the two studies. For example, one study showed that the C terminus of SPA1 interacts with the N terminus of phyA [59Sheerin D.J. et al.Light-activated phytochrome A and B interact with members of the spa family to promote photomorphogenesis in Arabidopsis by reorganizing the COP1/SPA Complex.Plant Cell. 2015; 27: 189-201Crossref PubMed Scopus (196) Google Scholar]; while the second study showed the opposite interaction through the N-terminal kinase-like domain of SPA and the C terminus of phyA [58Lu X-D. et al.Red-light-dependent interaction of phyB with SPA1 promotes COP1–SPA1 dissociation and photomorphogenic development in Arabidopsis.Mol. Plant. 2015; 8: 467-478Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar]. However, both groups showed that the full-length SPA1 interacts with the full-length phyB [58Lu X-D. et al.Red-light-dependent interaction of phyB with SPA1 promotes COP1–SPA1 dissociation and photomorphogenic development in Arabidopsis.Mol. Plant. 2015; 8: 467-478Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 59Sheerin D.J. et al.Light-activated phytochrome A and B interact with members of the spa family to promote photomorphogenesis in Arabidopsis by reorganizing the COP1/SPA Complex.Plant Cell. 2015; 27: 189-201Crossref PubMed Scopus (196) Google Scholar]. Surprisingly, CRY1 and CRY2 also interacted with SPA1 through different domains. The cryptochrome C-terminal extension (CCE) domain of CRY1 interacted with the C terminus of SPA1 [55Liu B. et al.Arabidopsis cryptochrome 1 interacts with SPA1 to suppress COP1 activity in response to blue light.Genes Dev. 2011; 25: 1029-1034Crossref PubMed Scopus (273) Google Scholar, 56Lian H.L. et al.Blue-light-dependent interaction of cryptochrome 1 with SPA1 defines a dynamic signaling mechanism.Genes Dev. 2011; 25: 1023-1028Crossref PubMed Scopus (226) Google Scholar]; however, the N-terminal photolyase-related (PHR) domain of CRY2 interacted with the N terminus of SPA1 [57Zuo Z. et al.Blue light-dependent interaction of CRY2 with SPA1 regulates COP1 activity and floral initiation in Arabidopsis.Curr. Biol. 2011; 21: 841-847Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar]. Although these discrepancies need to be resolved in the future, both studies show a mechanism by which phytochromes can rapidly modulate COP1 activity to allow photomorphogenesis to proceed. As discussed above, photomorphogenesis is repressed by two distinct classes of proteins: one (COP/DET/FUS) complex involves ubiquitin-mediated degradation of the positively-acting factors (Figures 1 and 3, left) and the other encodes bHLH transcription factors (PIFs) (Figures 1 and 2, left) [4Leivar P. Quail P.H. PIFs: pivotal components in a cellular signaling hub.Trends Plant Sci. 2011; 16: 19-28Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar, 5Lau O.S. Deng X.W. The photomorphogenic repressors COP1 and DET1: 20 years later.Trends Plant Sci. 2012; 17: 584-593Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar]. However, the relationship between these two groups of repressors was not clear until recently. Why have plants evolved two classes of repressors? Do they function additively or synergistically? Strikingly, a recent study demonstrated that these two groups of proteins function synergistically to repress photomorphogenesis [32Xu X. et al.PHYTOCHROME INTERACTING FACTOR1 enhances the E3 ligase activity of CONSTITUTIVE PHOTOMORPHOGENIC1 to synergistically repress photomorphogenesis in Arabidopsis.Plant Cell. 2014; 26: 1992-2006Crossref PubMed Scopus (61) Google Scholar]. Genetic analysis showed that cop1pif or spa123pif1 combination mutants were more hypersensitive compared to their respective parents (Figure 4A) . Biochemical analyses showed that HY5, the key target of the COP1/SPA complex, is much more abundant in the cop1pif or spa123pif1 combination mutants compared to the parental genotypes (Figure 4A). PIF1 physically interacted with COP1, SPA1, and HY5 both in vitro and in vivo. Moreover, PIF1 enhanced the substrate recruitment as well as auto- and trans-ubiquitylation activities of COP1 toward HY5 (Figure 4B). PIFs have been shown to function as transcriptional regulators controlling gene expression in various signaling pathways including those regulated by light [7Leivar P. Monte E. PIFs: systems integrators in plant development.Plant Cell. 2014; 26: 56-78Crossref PubMed Scopus (366) Google Scholar]. However, the above data suggest that PIFs have a pivotal non-transcriptional role in modulating signaling pathways in addition to transcriptional regulation. In fact, the results suggest that PIF1 functions as a cofactor for COP1 in this process. This is consistent with previous reports that PIFs promote COP1-mediated ubiquitylation of type II phytochromes (phyB–E) in vitro [60Jang I-C. et al.Arabidopsis PHYTOCHROME INTERACTING FACTOR proteins promote phytochrome b polyubiquitination by COP1 E3 ligase in the mucleus.Plant Cell. 2010; 22: 2370-2383Crossref PubMed Scopus (174) Google Scholar], in line with the increased level of phyB in higher-order pif mutants in vivo [4Leivar P. Quail P.H. PIFs: pivotal components in a cellular signaling hub.Trends Plant Sci. 2011; 16: 19-28Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar]. In addition, as discussed above, PIF3 also promotes degradation of phyB through CUL3LRB E3 ligase [26Ni W. et al.A mutually assured destruction mechanism attenuates light signaling in Arabidopsis.Science. 2014; 344: 1160-1164Crossref PubMed Scopus (183) Google Scholar]. A B box-containing protein, BBX19, interacts with COP1 and ELF3 and promotes COP1-mediated degradation of ELF3 [61Wang C-Q. et al.The transcriptional regulator BBX19 promotes hypocotyl growth by facilitating COP1-mediated EARLY FLOWERING3 eegradation in Arabidopsis.Plant Cell. 2015; 27: 1128-1139Crossref PubMed Scopus (70) Google Scholar]. 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In addition, COP1 and DET1 directly interact with multiple proteins to regulate various signaling pathways (Figure 4B) [5Lau O.S. Deng X.W. The photomorphogenic repressors COP1 and DET1: 20 years later.Trends Plant Sci. 2012; 17: 584-593Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar]. If PIFs interact with any of the COP1/DET1 substrates, PIFs might also regulate their abundance post-translationally, increasing the potential of synergistic regulation of multiple signaling and developmental pathways (Figure 4B). Thus it appears that the nontranscriptional roles of PIFs play an increasingly important if not equal role as transcriptional regulation by PIFs. The discovery of multiple repressors functioning synergistically to suppress photomorphogenesis suggests that photomorphogenesis is the default pathway for plant development. Skotomorphogenesis is a repressed state of photomorphogenesis. Plants employ multiple layers of negative regulators to achieve a sufficiently repressed state in the dark. Light-activated phytochromes interact with PIFs to induce their phosphorylation by an as yet unknown kinase, and the phosphorylated form is ubiquitylated by various E3 ligases and degraded through the 26S proteasome pathway to initiate photomorphogenesis. The light-induced degradation of PIF1 by the CUL4COP1–SPA E3 ligase initiates gradual progression toward photomorphogenesis. Although we have a much better understanding of how light controls plant development, several key questions still remain unanswered (see Outstanding Questions Box). The answer to these questions awaits future research.Outstanding QuestionsWhat is the light-regulated kinase that phosphorylates PIFs in response to light?HY5, the key positive regulator, is much more abundant in the pifQ mutant in the dark, potentially contributing to the pifQ phenotype in the dark. Because HY5 and PIFs bind to similar DNA sequence elements, are the PIF target genes also direct targets of HY5?Most importantly, what is the biochemical function of phytochromes? Is phytochrome merely a scaffold protein to bring PIFs and the light-regulated kinase together? Alternatively, is phytochrome the light-regulated kinase as previously suggested? What is the light-regulated kinase that phosphorylates PIFs in response to light? HY5, the key positive regulator, is much more abundant in the pifQ mutant in the dark, potentially contributing to the pifQ phenotype in the dark. Because HY5 and PIFs bind to similar DNA sequence elements, are the PIF target genes also direct targets of HY5? Most importantly, what is the biochemical function of phytochromes? Is phytochrome merely a scaffold protein to bring PIFs and the light-regulated kinase together? Alternatively, is phytochrome the light-regulated kinase as previously suggested? We thank members of the laboratory of E.H. for critical reading of this manuscript. This work was supported by grants from the National Science Foundation (IOS-1120946) and the Human Frontier Science Program (RGP0025/2013) to E.H.
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