The Ancestry and Function of Cytochrome c6A

Cytochrome c6A is a homologue of cytochrome c6 found in eukaryotic green algae and higher plants. However it is thought to perform a different function from cytochrome c6. Two current hypotheses exist for this function: that cytochrome c6A acts as a ‘safety valve’, providing an alternative path for electron flow through the photosynthetic electron transport chain and thus alleviating reactive oxygen species production; that cytochrome c6A has a signalling role where it could sense high light stress and convey this signal to the nucleus to affect gene expression. This study aims to provide insight into the ancestry and function of cytochrome c6A. To gain a clearer understanding of the evolutionary history of cytochrome c6A and cyanobacterial homologues cytochrome c6B and c6C, phylogenetic analysis was performed on peptide sequences of these proteins as found in a wide range of photosynthetic organisms. These data were used to refine the model of the evolutionary history of the cytochrome c6 family. This study also found evidence that cytochrome c6B and c6C are in fact orthologues with a similar function. Chlamydomonas reinhardtii is a model organism for eukaryotic photosynthesis, as its unicellularity provides many advantages over land plants. To determine if cytochrome c6A is involved in high light stress in C. reinhardtii, cytochrome c6A knockout and knockdown mutant lines of C. reinhardtii had been previously produced using CrispR-cpf,1 and are characterised in this study. The mutant lines demonstrated potential growth retardation under high or fluctuating light stress, as well as singlet oxygen stress, which was more noticeable under mixotrophic conditions. Chlorophyll fluorescence analysis of the mutant lines established a link between cytochrome c6A and NPQ. As cytochrome c6A is believed to be located in the thylakoid lumen, and initial NPQ is triggered by a change in luminal pH, circular dichroism was used to determine changes in secondary structure of purified cytochrome c6A over a pH range of 2-7. No significant change in structure was observed, but cytochrome c6A did maintain structural integrity even at pH 2. Finally, the transcriptome of the cytochrome c6A knock out line was compared to the background strain under standard and high light conditions through RNAseq. Many photoprotective, motility and CCM genes were differentially regulated under high light stress, but when cytochrome c6A was knocked out these regulations were diminished. Therefore cytochrome c6A has been proposed as a signalling molecule that functions in CCM, NPQ and motility under photosynthetic stress conditions.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2020

Author dc:creator

• Slater, Barnaby

Advisor dc:contributor.advisor

• Howe, Christopher

• Biochemistry
• Photosynthesis
• Phylogeny
• Transcriptomics
• Cytochrome
• Abiotic stress
• Plant
• algae
• Chlamydomonas reinhardtii
• Molecular genetics
• Circular dichroism
• protein structure
• chlorophyll fluorescence
• CrispR
• retrograde signalling
• photosynthetic electron transfer
• redox

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/a946afca-ac9e-46d7-a11a-df468b54f8de/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

en

Author Identifier

0000-0001-9922-8563

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/307693