Regulation of photoprotection gene expression in Chlamydomonas by a putative E3 ubiquitin ligase complex and a homolog of CONSTANS

Abstract

Photosynthetic organisms use nonphotochemical quenching (NPQ) mechanisms to dissipate excess absorbed light energy and protect themselves from photooxidation. In the model green alga Chlamydomonas reinhardtii, the capacity for rapidly reversible NPQ (qE) is induced by high light, blue light, and UV light via increased expression of LHCSR and PSBS genes that are necessary for qE. Here, we used a forward genetics approach to identify SPA1 and CUL4, components of a putative green algal E3 ubiquitin ligase complex, as critical factors in a signaling pathway that controls light-regulated expression of the LHCSR and PSBS genes in C. reinhardtii The spa1 and cul4 mutants accumulate increased levels of LHCSR1 and PSBS proteins in high light, and unlike the wild type, they express LHCSR1 and exhibit qE capacity even when grown in low light. The spa1-1 mutation resulted in constitutively high expression of LHCSR and PSBS RNAs in both low light and high light. The qE and gene expression phenotypes of spa1-1 are blocked by mutation of CrCO, a B-box Zn-finger transcription factor that is a homolog of CONSTANS, which controls flowering time in plants. CONSTANS-like cis-regulatory sequences were identified proximal to the qE genes, consistent with CrCO acting as a direct activator of qE gene expression. We conclude that SPA1 and CUL4 are components of a conserved E3 ubiquitin ligase that acts upstream of CrCO, whose regulatory function is wired differently in C. reinhardtii to control qE capacity via cis-regulatory CrCO-binding sites at key photoprotection genes.

Keywords: light harvesting; light signaling; nonphotochemical quenching; photomorphogenesis; photosynthesis.

Fig. 1.

Three suppressors of npq4 have elevated NPQ capacity when grown in LL (60 µmol photons m⁻² s⁻¹) or HL (350 µmol photons m⁻² s⁻¹). NPQ was induced with actinic light of 600 µmol photons m⁻² s⁻¹ (white bar at Top), followed by recovery in the dark with 0.6 µmol photons m⁻² s⁻¹ far-red light (black bar at Top). (A) LL-grown cells. (B) Cells grown in HL for 3 d. Values represent means ± SD (n = 3).

Fig. 2.

Identification of causative polymorphisms in suppressor mutants. (A) General outline of workflow for bulked segregant analysis and genome sequencing to identify causative SNPs in UV-mutagenized npq4 suppressors. (B) Protein domain predictions for CrSPA1 and AtSPA1 and (C) CrCUL4, with locations of suppressor mutations (spa1-1, spa1-2, and cul4-1) marked by asterisks.

Fig. 3.

Identification of CO-like cis-regulatory binding sites upstream of qE genes. Position-specific weight matrix generated de novo for the CO-like binding site identified upstream of the translation start site (ATG) of the LHCSR and PSBS genes. Only the single, statistically strongest matching site for each qE gene is shown in the alignment.

Fig. 4.

Deletion of CrCO results in low NPQ capacity, affects expression of qE genes, and blocks the high NPQ phenotype of spa1-1 in LL. Cultures of WT, spa1-1, crco, and crco spa1-1 were grown in LL (60 µmol photons m⁻² s⁻¹) and exposed to HL (350 µmol photons m⁻² s⁻¹) for 6 h to minimize photoinhibition in the crco strains (SI Appendix, Fig. S7). (A) NPQ of LL-grown cells. (B) NPQ of cells exposed to HL for 6 h. Values in A and B represent means ± SD (n = 3). NPQ was induced with actinic light of 600 µmol photons m⁻² s⁻¹ (white bar at Top), followed by recovery in the dark with 0.6 µmol photons m⁻² s⁻¹ far-red light (black bar at Top). (C) RT-qPCR analysis of RNA expression levels of LHCSR1, LHCSR3.1, LHCSR3.2, PSBS1, and PSBS2 genes. Fold-change values for each RNA were normalized to the expression level in WT LL for that gene. CβLP was used as the internal reference. Values represent means ± SD (n = 3). (D) Immunoblot analysis of protein levels of LHCSR1, LHCSR3, and PSBS. Chloroplast ATP synthase beta-subunit (ATPB) was used as a loading control.

Fig. 5.

Model depicting how the COP1-SPA1 E3 ubiquitin ligase complex in C. reinhardtii controls the light regulation of qE gene expression through the transcriptional regulator CrCO. In low light, ubiquitination of CrCO by the COP1-SPA1 E3 ubiquitin ligase targets CrCO for degradation. The low level of CrCO that persists in low light activates low levels of transcription from the qE genes (LHCSR1, LHCSR3.1, LHCSR3.2, PSBS1, and PSBS2). In high light, a photoreceptor-mediated signal inhibits the COP1-SPA1 E3 ubiquitin ligase by an undefined mechanism, allowing accumulation of CrCO and its binding to the CO-like binding motif and transcriptional activation of the qE gene promoters. In addition, to explain the high-light induction of qE gene expression in the crco and crco spa1-1 mutants (Fig. 4 C and D) we hypothesize the existence of a second transcriptional regulator bound to the qE gene promoters that mediates a SPA1/CrCO-independent but high-light-dependent regulatory pathway. Whether this pathway constitutes a second photoreceptor-mediated pathway, possibly integrated with a chloroplast retrograde signaling pathway, is unknown.