The CONSTANS flowering complex controls the protective response of photosynthesis in the green alga Chlamydomonas

Abstract

Light is essential for photosynthesis, but the amounts of light that exceed an organism's assimilation capacity can result in oxidative stress and even cell death. Plants and microalgae have developed a photoprotective response mechanism, qE, that dissipates excess light energy as thermal energy. In the green alga Chlamydomonas reinhardtii, qE is regulated by light-inducible photoprotective proteins, but the pathway from light perception to qE is not fully understood. Here, we show that the transcription factors CONSTANS and Nuclear transcription Factor Ys (NF-Ys) form a complex that governs light-dependent photoprotective responses in C. reinhardtii. The qE responses do not occur in CONSTANS or NF-Y mutants. The signal from light perception to the CONSTANS/NF-Ys complex is directly inhibited by the SPA1/COP1-dependent E3 ubiquitin ligase. This negative regulation mediated by the E3 ubiquitin ligase and the CONSTANS/NF-Ys complex is common to photoprotective response in algal photosynthesis and flowering in plants.

Fig. 1

CrCO and NF-YB are crucial for photoprotection. a The bleaching phenotypes of the reference strain (LHCSR1–Luc717) and the DSR mutants visualized in multiwell plates. Representative cell cultures treated with low light (LL; left wells) or high light (HL; right wells). Concentrations of the cultures were adjusted to 1.0 ×  10⁷ cells/mL. b Chlorophyll content per cell after LL (closed bar) or HL (open bar) treatment of the cells shown in a. c Maximum quantum yield of photosystem II (Fv/Fm) during HL treatment. d qE quenching capability during HL treatment. e Immunoblot analysis of 3xFLAG-fused proteins (CrCO–FLAG and NF-YB–FLAG in crco-2/CrCO and nfyb-1/NFYB, respectively), LHCSR1, LHCSR3, and PSBS during HL treatment. ATPB protein levels are shown as the loading control. The experiments were performed three times with different biological samples (n = 3 biological replicates; mean ± S.E.M); a representative experiment is shown in e

Fig. 2

CrCO interacts with NF-Y isoforms to form a transcriptional complex that associates with the promoter regions of the photoprotective genes. a Confocal live-cell imaging of the complemented strains, crco-2/CrCO (labelled as CrCO) and nfyb-1/NFYB (labelled as NF-YB), to visualize the localization of CrCO and NF-YB fused with Venus–3xFLAG in C. reinhardtii cells. Scale bars, 5 µm. b GAL4-based yeast two-hybrid (Y2H) assays. CrCO was fused to the GAL4-activation domain (AD–CrCO), whose ability to form heterodimers was tested by cotransformation of the GAL4-binding domain fused with NF–YB or NF-YC (BD–NF-YB or BD–NF-YC). Autoactivation was tested using empty vectors (AD or BD). The cells were plated on permissive (+His) or selective (−His) media and grown at 30 °C for 40 h after being spotted on the plates. c Chromatin immunoprecipitation (ChIP)-PCR assay of CrCO and NF-YB. Agarose gel electrophoresis showing the strength of the association of CrCO or NF-YB with the promoter regions of the photoprotective genes under different light treatments. ChIP was performed using 2 × 10⁸ cells/mL of crco-2/CrCO (labelled as CrCO) or nfyb-1/NFYB (labelled as NF-YB) cells cross-linked with 0.35% (v/v) formaldehyde after a 1-h light treatment

Fig. 3

UVR8 interacts with COP1 and SPA1 to activate the CrCO-based transcriptional complex under UV irradiation. a Confocal images of UVR8–Venus–3xFLAG proteins in the DSR1–comp15 (uvr8/UVR8) strain. The cells were treated with UV for 30 min. Scale bars, 10 µm. b After 1 h of UV treatment, the cells were harvested and UVR8–Venus–3xFLAG proteins were immunoprecipitated by FLAG (M2) antibody with SURE-beads. The coimmunoprecipitated proteins were identified by LC-MS/MS analysis of the Coomassie Brilliant Blue-stained polypeptide bands obtained via SDS-PAGE separation after in-gel trypsin digestion. M, molecular mass standard. c Interaction profiles among COP1, SPA1, and CrCO visualized with Y2H assays, which were performed using COP1, SPA1, and CrCO fused with the AD and/or BD domains of GAL4. The culture conditions were as described for Fig. 2b. d Immunoblot analysis of LHCSRs, PSBS, and 3xFLAG-fused CrCO to visualize protein accumulation in the wild-type control (WT), crco-2, crco-2/CrCO, spa1, spa1 crco-2, and spa1 crco-2/CrCO strains before and after UV treatment. ATPB proteins were included as the loading controls

Fig. 4

Hypothetical signal transduction pathways and physiological functions of CONSTANS/NF-YB/NF-YC-dependent control of the target genes in C. reinhardtii and A. thaliana. Blue light perception by the photoreceptor CRY2 in A. thaliana deactivates the E3 ubiquitin ligase module^,, whereas UV light perception via conversion of the dimeric form of UVR8 to the monomeric form deactivates this module in C. reinhardtii. Circadian clock-regulated CONSTANS transcripts are translated, and successfully accumulate in the organisms when proteasome- and ubiquitin-dependent degradation is inactivated. The CONSTANS/NF-YB/NF-YC transcriptional module then transcribes FT and photoprotective genes (e.g., LHCSR1, LHCSR3, and PSBS) to stimulate flowering and photoprotection in land plants and the green alga, respectively. The kernel, including SPA1/COP1-dependent E3 ubiquitin ligase, circadian regulation of CONSTANS, and the CONSTANS/NF-YB/NF-YC transcriptional complex, was possibly established in an ancestral green photosynthetic organism