Organization of protein complexes under photomorphogenic UV-B in Arabidopsis

Abstract

Low-fluence and long-wavelength UV-B light promotes photomorphogenic development in Arabidopsis. CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) is a positive regulator in this pathway while it is a negative regulator of the traditional photomorphogenesis triggered by far-red and visible light. We have recently reported the mechanism by which the switch of COP1 function is accomplished in distinct light contexts. In response to photomorphogenic UV-B, the photoactivated UV RESISTANCE LOCUS 8 (UVR8) associates with the COP1- SUPRESSOR OF PHYA (SPA) core complexes, resulting in the physical and functional disassociation of COP1-SPA from the CULLIN4-DAMAGED DNA BINDING PROTEIN 1 (CUL4-DDB1) E3 scaffold. These UV-B dependent UVR8-COP1-SPA complexes promote the stability and activity of ELONGATED HYPOCOTYL 5 (HY5), and eventually cause COP1 to switch from repressing to promoting photomorphogenesis. In addition, it is possible that CUL4-DDB1 might simultaneously recruit alternative DDB1 BINDING WD40 (DWD) proteins to repress this UV-B-specific signaling. Further investigation is required, however, to verify this hypothesis. Overall, the coordinated organization of various protein complexes facilitates an efficient and balanced UV-B signaling.

Keywords: :

• UV-B
• protein complex
• light signaling

This article refers to:

To cope with the changing light environment, higher plants have evolved the capacity to sense and interpret diverse light signals, in order to optimize their growth. UV-B light (280—315 nm) comprises a minor proportion of sunlight that reaches the earth surface, and is processed by plants not only as a damaging stimulus, but also as an informational signal. Specifically, high-fluence and short-wavelength UV-B light induces stress-related physiological responses, while low-fluence and long-wavelength UV-B light promotes photomorphogenic development.¹

UV RESISTANCE LOCUS 8 (UVR8) is a UV-B light receptor that has been recently identified in Arabidopsis thaliana. Upon irradiation with photomorphogenic UV-B, the dark-state dimeric UVR8 perceives UV-B light via its own tryptophan residues, which acts as internal chromophores, and monomerizes by disrupting the critical intermolecular bonds shaped by its own arginine residues.^2,3 The lit-state monomeric UVR8 then rapidly interacts with CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) in order to trigger downstream signaling.^4,5

COP1 is an evolutionally conserved and multifunctional RING E3 ubiquitin ligase. It was originally identified by screening Arabidopsis seedlings that exhibit constitutive photomorphogenesis in darkness.⁶ Molecular and biochemical studies have revealed that COP1 functions in COP1-SUPRESSOR OF PHYA (SPA) complexes.⁷ They serve as substrate receptors for the CULLIN4- DAMAGED DNA BINDING PROTEIN 1 (CUL4-DDB1) E3 apparatus, to target photomorphogenesis promoting transcription factors including ELONGATED HYPOCOTYL 5 (HY5) for the ubiquitin-proteasome system (UPS)-mediated degradation.⁸ Thus, COP1 is a central repressor of the traditional photomorphogenesis triggered by far-red and visible light.⁹

Intriguingly, COP1 has also been shown to be a positive regulator of UV-B-induced photomorphogenesis,^4,10 and to no longer act antagonistically against HY5 under UV-B.^11,12 The loss-of-function mutation of either COP1 or HY5 suffers from abolished transcriptomic responses to photomorphogenic UV-B, and consequently results in defective UV-B-induced photomorphogenesis and UV-B acclimation.¹⁰ In addition to these discoveries, several additional observations listed below prompted us to investigate the mechanism of COP1’s role in this UV-B specific signaling, particularly with regard to the changes induced in the relevance interaction between COP1 and HY5. Both COP1 and HY5 show nuclear localization under UV-B.¹⁰ COP1 is required for the UV-B-mediated HY5 induction,⁴ and HY5 activates COP1 in a positive feedback by targeting the COP1 promoter.¹²

We found that under photomorphogenic UV-B, monomerized UVR8 sequestered COP1 from DDB1, which in turn caused COP1-SPA to disassociate from CUL4-DDB1. In agreement with this physical segregation, COP1-SPA also displayed functional disassociation from CUL4-DDB1, and CUL4 was identified as a repressor of UV-B-induced photomorphogenesis.¹¹

In contrast, we showed that COP1 and SPA proteins remained tightly associated with one another regardless of the presence or absence of UV-B. Under photomorphogenic UV-B, UVR8 was observed to associate with the COP1-SPA core to constitute novel complexes. We then verified that SPA proteins, like COP1 and UVR8, played positive regulatory roles in UV-B-induced photomorphogenesis. More importantly, we demonstrated that the UV-B-dependent UVR8-COP1-SPA complexes promoted the stability and activity of HY5, which enabled a functional switch of COP1 from repressing to promoting photomorphogenesis. In YFP-UVR8^W285A transgenic plants, the constitutive UVR8-COP1-SPA complexes were found to continuously facilitate HY5 stability and activity, and to eventually drive the Arabidopsis seedlings to develop constitutive photomorphogenesis in darkness.¹¹ However, how HY5 is stabilized is a subject that awaits further investigation. Our results have indicated that an alternative E3 ubiquitin ligase might be responsible for the turnover of HY5 protein, and UVR8-COP1-SPA complexes might deactivate this E3 ligase. Also, it has been pointed out that the stability and activity of HY5 is modulated by its phosphorylation.¹³ Thus a close examination of the phosphorylation or/and other modifications of HY5 might reveal that how HY5 is regulated post-transcriptionally by photomorphogenic UV-B.

As it does under other light conditions, CUL4 represses photomorphogenesis under UV-B.^11,14 Nevertheless, it remains unknown, regarding the mechanism by which CUL4 plays a repressive role in UV-B-induced photomorphogenesis. It has been extensively studied that DDB1 is an adaptor protein that recruits a group of proteins named DDB1-CUL4 ASSOCIATED FACTOR (DCAF) as substrate receptors for CUL4-based E3 ligase in both animals and plants.^15-19 In Arabidopsis, DCAFs, also designated as DWD proteins, are functionally involved in a wide variety of developmental processes.¹⁹ Therefore, it is reasonable that one or more unknown DWD proteins might join the CUL4-DDB1 E3 scaffold to repress UV-B-induced photomorphogenesis.

Two previously identified negative regulators in this pathway, REPRESSOR OF UV-B PHOTOMORPHOGENESIS 1 (RUP1) and RUP2,²⁰ are potential DWD proteins as each of them possesses one typical WDXR motif that might mediate their direct interaction with DDB1. Furthermore, multiple lines of evidence indicate that CUL4-DDB1 and RUP1/RUP2 might function in concert. For example, a reduction in CUL4 or RUP1/RUP2 function was observed to result in hypersensitivity to photomorphogenic UV-B in Arabidopsis seedlings, which were found to display enhanced induction of UV-B-responsive genes and increased accumulation of HY5 protein, the known UV-B signaling hub.^11,20 Thus, further analyses of the genetic and biochemical interaction between CUL4 and RUP1/RUP2 might assist substantiating this hypothesis. Since RUP1 and RUP2 are known to facilitate the conformational reversion of UVR8,^21,22 it is also of interest and significance to investigate whether or not CUL4 participates in this process.

Taken together, we propose a model for the organization of protein complexes under photomorphogenic UV-B (Fig. 1). Following UVR8-mediated UV-B light perception, monomerized UVR8 directly interacts with COP1 so as to form UVR8-COP1-SPA complexes, which, in turn, promote the stability and activity of HY5. HY5 is then critically involved in downstream transcriptional regulation. On the other hand, when sequestered by UVR8 monomers, COP1-SPA core complexes disassociate from the CUL4-DDB1 E3 apparatus which might then recruit alternative DWD proteins to repress UV-B-induced photomorphogenesis. Overall, these complexes are structurally and functionally coordinated to ensure an efficient and balanced photomorphogenic UV-B signaling.

Figure 1. A model for the organization of protein complexes under photomorphogenic UV-B. Upon UV-B irradiation, UVR8 switches from dimer to monomer, and directly interacts with COP1 to form UVR8-COP1-SPA complexes, which promote the stability and activity of HY5. HY5 positively regulates downstream transcriptional responses so as to facilitate UV-B-induced photomorphogenesis and acclimation. On the other hand, the CUL4-DDB1 E3 apparatus, which releases the COP1-SPA core complexes sequestered by UVR8 monomers, might recruit alternative DWD proteins such as RUP1/RUP2 to repress UV-B-induced photomorphogenesis. Once the UV-B irradiation is removed, RUP1 and RUP2 mediate the redimerization of UVR8. Solid lines indicate direct effects, while dotted lines represent indirect regulation. Question marks denote the hypothesis, which awaits further investigation.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Abigail Coplin for her critical reading and commenting on the manuscript.