Arabidopsis COP1 acts to repress photomorphogenesis in the absence of light. It was shown that in the dark, COP1 directly interacts with the bZIP transcription factor HY5, a positive regulator of photomorphogenesis, and promotes its proteasome-mediated degradation. Here we identify a novel bZIP protein HYH, as a new target of COP1. We identify a physical and genetic interaction between HYH and COP1 and show that this interaction results in dark-specific degradation of HYH. Genetic analysis indicates that HYH is predominantly involved in blue-light regulation of development and gene expression, and that the function of HYH in part overlaps with that of HY5. The accumulation of HYH protein, not the mRNA, is dependent on the presence of HY5. Our data suggest that HYH and HY5 can, respectively, act as heterodimers and homodimers, thus mediating light-regulated expression of overlapping as well as distinct target genes. We propose that COP1 mediates light control of gene expression through targeted degradation of multiple photomorphogenesis-promoting transcription factors in the nucleus.

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CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) and ELONGATED HYPOCOTYL5 (HY5) are two major regulators of light signaling in plants. Here, we identify SALT TOLERANCE HOMOLOG2 (STH2) as a gene that interacts genetically with both of these key regulators. STH2 encodes a B-box-containing protein that interacts physically with HY5 in yeast and in plant cells. Whereas STH2 is uniformly nuclear by itself, it shows a COP1-dependent localization to speckles when coexpressed with COP1. We identified two independent T-DNA insertion lines in STH2. Both alleles are hyposensitive to blue, red, and far-red light. The sth2 mutant, like hy5, shows an enhanced number of lateral roots and accumulates less anthocyanin. Analysis of double mutants between sth2 and hy5 indicates that STH2 has both HY5-dependent and -independent functions. Furthermore, besides partially suppressing the hypocotyl phenotype of dark-grown cop1 alleles, sth2 also suppresses the reduced number of lateral roots and high anthocyanin levels in light-grown cop1 alleles. Interestingly, we found that STH2 can activate transcription. Transient transfection assays in protoplasts using a LUC reporter driven by the chalcone isomerase promoter show that the B-boxes in STH2 and a functional G-box element in the promoter are required for this activity. In conclusion, we have identified STH2, a B-box protein in Arabidopsis thaliana, as a positive regulator of photomorphogenesis and report that the B-box domain plays a direct role in activating transcription in plants.

B-box containing proteins play an important role in light signaling in plants. Here, we identify LIGHT-REGULATED ZINC FINGER1/SALT TOLERANCE HOMOLOG3 (STH3), a B-box encoding gene that genetically interacts with two key regulators of light signaling, ELONGATED HYPOCOTYL5 (HY5) and CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1). STH3 physically interacts with HY5 in vivo and shows a COP1-dependent localization to nuclear speckles when coexpressed with COP1 in plant cells. A T-DNA insertion mutant, sth3, is hyposensitive to high fluence blue, red, and far-red light and has elongated hypocotyls under short days. Analyses of double mutants between sth3, sth2, and hy5 suggest that they have partially overlapping functions. Interestingly, functional assays in protoplasts suggest that STH3 can activate transcription both independently and together with STH2 through the G-box promoter element. Furthermore, sth3 suppresses the cop1 hypocotyl phenotype in the dark as well as the anthocyanin accumulation in the light. Finally, COP1 ubiquitinates STH3 in vitro, suggesting that STH3 is regulated by COP1. In conclusion, we have identified STH3 as a positive regulator of photomorphogenesis acting in concert with STH2 and HY5, while also being a target of COP1-mediated ubiquitination.

CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) is an E3 ubiquitin ligase that represses photomorphogenesis in the dark. Therefore, proteins interacting with COP1 could be important regulators of light-dependent development. Here, we identify CONSTANS-LIKE3 (COL3) as a novel interaction partner of COP1. A green fluorescent protein-COL3 fusion protein colocalizes with COP1 to nuclear speckles when transiently expressed in plant cells. This localization requires the B-box domains in COL3, indicating a novel function of this domain. A loss-of-function col3 mutant has longer hypocotyls in red light and in short days. Unlike constans, the col3 mutant flowers early and shows a reduced number of lateral branches in short days. The mutant also exhibits reduced formation of lateral roots. The col3 mutation partially suppresses the cop1 and deetiolated1 (det1) mutations in the dark, suggesting that COL3 acts downstream of both of these repressors. However, the col3 mutation exerts opposing effects on cop1 and det1 in terms of lateral roots and anthocyanin accumulation, suggesting that COL3 also has activities that are independent of COP1 and DET1. In conclusion, we have identified COL3 as a positive regulator of photomorphogenesis that acts downstream of COP1 but can promote lateral root development independently of COP1 and also function as a daylength-sensitive regulator of shoot branching.

BBX21 (also known as SALT TOLERANCE HOMOLOG 2), a B-box (BBX)-containing protein, has been previously identified as a positive regulator of light signaling; however, the precise role of BBX21 in regulating seedling photomorphogenesis remains largely unclear. In this study, we report that CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) interacts with BBX21 in vivo and is able to ubiquitinate BBX21 in vitro. Thus, BBX21 is targeted for 26S proteasome-mediated degradation in dark-grown Arabidopsis seedlings in a COP1-dependent manner. Moreover, we show that BBX21 binds to the T/G-box in the ELONGATED HYPOCOTYL 5 (HY5) promoter and directly activates HY5 expression in the light. Transgenic seedlings overexpressing BBX21 exhibit dramatically shortened hypocotyls in the light, and this phenotype is dependent on a functional HY5. Taken together, our data suggest a molecular base underlying BBX21-mediated seedling photomorphogenesis, indicating that BBX21 is a pivotal component involved in the COP1-HY5 regulatory hub.P lant seedlings undergo two distinct developmental processes dependent on the presence and absence of light, termed photomorphogenesis and skotomorphogenesis, respectively (1). CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) is a central repressor of seedling photomorphogenesis, and mutants impaired in COP1 display constitutively photomorphogenic phenotypes in darkness (2, 3). COP1 acts as an E3 ubiquitin ligase targeting a subset of substrates for degradation in darkness, including ELONGATED HYPOCOTYL 5 (HY5), HY5 HOMOLOG (HYH), LONG AFTER FAR-RED LIGHT 1 (LAF1), LONG HYPOCOTYL IN FAR RED (HFR1), B-BOX PROTEIN 22/ SALT TOLERANCE HOMOLOG 3 (BBX22/STH3), and PHYTOCHROME INTERACTING FACTOR 3-LIKE1 (PIL1) (4-11). Of these, HY5 is considered a key signal integration point from dark to light transition (10, 12, 13). Its abundance is directly correlated with the extent of seedling photomorphogenic growth, but inversely correlated with the nuclear abundance of COP1 (4, 10, 14). As a b-ZIP type transcription factor, HY5 specifically interacts with the ACGT-containing elements (ACEs) and might directly bind to approximately one-third of the Arabidopsis genes (12, 13). Thus, HY5 ensures proper expression of a large number of downstream regulatory genes, which in turn eventually promotes photomorphogenesis in the light. Recent studies have shown that HY5 expression is regulated by HY5 itself, as well as by HYH and CALMODULIN7 (CAM7) (15,16).A total of 32 B-box (BBX)-containing proteins have been identified in the Arabidopsis genome (17), among which a number of BBX proteins have been shown to be involved in COP1-and HY5-mediated seedling photomorphogenesis (18). BBX4, BBX20, BBX22, BBX24, and BBX25 genetically and physically interact with COP1 and undergo COP1-mediated degradation in the dark (6,(19)(20)(21)(22)(23)(24). In addition, BBX22, BBX24, and BBX25 physically interact with HY5, and, interestingly, BBX22 acts as a coactivator of HY5 action (6), whereas BBX24 and BBX25 repress the transcriptional activating activity of HY5 ...

ELONGATED HYPOCOTYL5 (HY5) is a basic domain/leucine zipper (bZIP) transcription factor, central for the regulation of seedling photomorphogenesis. Here, we identified a B-BOX (BBX)-containing protein, BBX25/SALT TOLERANCE HOMOLOG, as an interacting partner of HY5, which has been previously found to physically interact with CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1). BBX25 physically interacts with HY5 both in vitro and in vivo. By physiological and genetic approaches, we showed that BBX25 is a negative regulator of seedling photomorphogenesis. BBX25 and its homolog BBX24 regulate deetiolation processes and hypocotyl shade avoidance response in an additive manner. Moreover, genetic relationships of bbx25 and bbx24 with hy5 and cop1 revealed that BBX25 and BBX24 additively enhance COP1 and suppress HY5 functions. BBX25 accumulates in a light-dependent manner and undergoes COP1-mediated degradation in dark and light conditions. Furthermore, a protoplast cotransfection assay showed that BBX24 and BBX25 repress BBX22 expression by interfering with HY5 transcriptional activity. As HY5 binds to the BBX22 promoter and promotes its expression, our results identify a direct mechanism through which the expression of BBX22 is regulated. We suggest that BBX25 and BBX24 function as transcriptional corepressors, probably by forming inactive heterodimers with HY5, downregulating BBX22 expression for the fine-tuning of light-mediated seedling development.

Arabidopsis COP1 is a photomorphogenesis repressor capable of directly interacting with the photomorphogenesis-promoting factor HY5. This interaction between HY5 and COP1 results in targeted degradation of HY5 by the 26S proteasome. Here we characterized the WD40 repeat domain-mediated interactions of COP1 with HY5 and two new proteins. Mutational analysis of those interactive partners revealed a conserved motif responsible for the interaction with the WD40 domain. This novel motif, with the core sequence V-P-E/D-f-G (f = hydrophobic residue) in conjunction with an upstream stretch of 4±5 negatively charged residues, interacts with a de®ned surface area of the b-propeller assembly of the COP1 WD40 repeat domain through both hydrophobic and ionic interactions. Several residues in the COP1 WD40 domain that are critical for the interaction with this motif have been revealed. The fact that point mutations either in the COP1 WD40 domain or in the HY5 motif that abolish the interaction between COP1 and HY5 in yeast result in a dramatic reduction of HY5 degradation in transgenic plants validates the biological signi®cance of this de®ned interaction.