Absolute quantification of components of the algal photosynthetic apparatus in response to environment and modification of chlorophyll biosynthesis
EMSL Project ID
60717
Abstract
The operation of the photosynthetic apparatus is influenced by environment, particularly stress situations like poor iron nutrition, carbon or light limitation, and excess excitation energy, which impact net productivity. To facilitate and improve models of photosynthetic productivity in algae, we need reliable measurements of the actual amounts of individual enzymes / complexes in various situations rather than “fold changes”, which have been the focus of previous investigations. Reliable quantification of structural and catalytic subunits of the enzymes / complexes of the light and carbon reactions and of the regulatory factors that determine their abundance will facilitate iterative design-build-test-learn cycles of synthetic biology efforts to improve photosynthetic productivity. The information is also central to building metabolic models aimed at understanding existing bottlenecks or aimed at optimizing sustainable photosynthesis-based bioproduct outputs in engineered algae. We will develop the knowledge base and methods in Chlamydomonas, a microbial reference organism for discovery in photosynthesis. Genetic analyses and transcriptome profiles have revealed dynamic expression of structural and regulatory subunits, cofactor biosynthesis and assembly factors for the photosynthetic apparatus during the diel period, with an impact of environmental stress on the magnitude of expression of individual genes within a complex. Quantifying the corresponding proteins is the next step. One goal is to measure the ratios of each multi-subunit complex in the thylakoid membrane, which speaks to distribution of light energy, operation of photoprotection mechanisms, demand for reductant vs. ATP. Another goal is to measure the stoichiometry of individual subunits within each thylakoid membrane complex, which speaks to the function of that complex. These measurements will enable us to connect environmental and genetic input to functional output. Another objective is to understand changes in chloroplast carbon metabolism in response to energy (light quality and quantity), carbon source (CO2 or acetate) or nutrient (Fe) input. Time course experiments would allow us to deduce the sequence of events that occur during reorganization of the photosynthetic apparatus in response to the environmental, nutrient or genetic inputs. We have compiled a prioritized list of ~ 300 Chlamydomonas proteins of interest for this project: components of the light reactions including cofactor biosynthesis, enzymes of the carbon reactions, proteins of the carbon concentrating mechanism, biomarkers of light and stress response, regulatory proteins and biogenesis factors, and alternative pathways. The actual proteins (~ 150) chosen for analysis will be based on collaboration with EMSL staff to identify suitable peptides and transitions. At least 2 proteins unrelated to photosynthesis will be included to monitor protein recovery between samples. Each sample will be analyzed in parallel for survey proteomics, lipidomics and select metabolites to provide infrastructure and context for the absolute abundances and stoichiometries and for downstream modeling. Preliminary studies to optimize sample preparation and digestion and to survey the proteins of interest are ongoing in an exploratory project. The peptides / transitions are a potential resource for the community as also are the resulting data, which will be shared with the community through publication.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2023-10-01
End Date
N/A
Status
Active
Released Data Link
Team
Principal Investigator
Co-Investigator(s)
Team Members