Characterization of Aureochromes in the diatom Phaeodactylum tricornutum

Abstract

Aureochromes are a novel group of blue-light dependent transcription factors restricted to Stramenopiles. Phylogenetic analysis of Aureochromes of the model diatom Phaeodactylum tricornutum revealed four distinct clades of these proteins, allowing molecular and functional studies on the individual Aureochromes in this alga. Isoform-specific antisera were validated successfully for individual quantification of PtAUREO protein levels. Using RT-qPCR, a mainly light-independent circadian rhythm was detected for PtAureo1a and PtAureo1c, a mainly light-dependent circadian rhythm for PtAureo1b, whereas PtAureo2 seemed not to be regulated in a circadian manner. For functional studies, a reproducible protocol for a highly efficient TALEN-based genome editing approach was established in P. tricornutum, yielding genetically homogenous colonies, forgoing the need of re-plating before the screening process. Knockout strains for each PtAUREO isoform have been generated. A lack of PtAUREO1a resulted in an increased xanthophyll cycle pool size and a reduced Chlorophyll a content per cell, similar to a previously characterized RNAi knockdown strain. Furthermore, a reduction in cell size was discovered. Interestingly, non-photochemical quenching (NPQ) was strongly reduced in the knockout strain, whereas it had been increased in the knockdown strains. Mono-allelic knockout strains, which eventually became overexpression strains by upregulation of the functional allele over time, showed an increased NPQ capacity, indicating that this contrasting phenotype may not be due to off-targets effects. Western Blots showed a significant reduction in protein level of the photoprotective Lhcx1 in the knockout strains. A shift of red-light adapted wild type or PtAUREO1a knockout strain to blue light analyzed by RNA-seq showed that short-term adaptation to blue light was almost completely blocked: While over 70% of transcripts were significantly up- or downregulated in the wild type after 10 min exposure to blue light, less than 3% were found to be regulated in the knockout strain. Furthermore, around 25% of the transcriptome was differentially regulated in the red-light condition, providing further evidence for a blue-light independent function of PtAUREO1a. While all other photoreceptors and transcription factors were found to be expressed in the knockout strain, their regulation pattern upon blue-light exposure was found to be disturbed. Additionally, transcriptional induction of the photoprotective Lhcx proteins was found to be repressed. In conclusion, PtAUREO1a seems to be a master switch of the short-term light acclimation and photoprotection pathway and its expression needs to be tightly regulated.