Stoichiometry changes to photosynthetic structures in algae in response to environmental stimuli and genetic engineering
EMSL Project ID
60580
Abstract
Photosynthesis is essential for all life on Earth, with the photosynthetic electron transfer chain and the Calvin-Benson cycle being the key molecular machineries assimilating inorganic carbon with the energy of sunlight. In response to changes in their immediate environment, especially with regards to light and CO2 availability, plants and algae employ several short-term acclimation strategies (e.g. NPQ, CCM) to maximize photosynthetic yield and minimize potential damage to the cell. Long-term acclimation to nutritional and environmental challenges additionally involves inducing conserved transcriptionally-encoded programs, modulating protein and metabolite pools for improved carbon capture and light utilization. The immediate goal of this project is to define the compositional dynamics in the photosynthetic apparatus in response to changes in environmental and nutritional resources with the long term goal of building the infrastructure for understanding adjustments of the photosynthetic apparatus in genetically engineered algae, which is a pre-requisite for efficient bioproduct and biofuel production. We propose to analyze the acclimation of the photosynthetic apparatus in algae to changes in light intensity and quality, nutrient deficiencies (N, S, Zn, Fe) and in specific mutant strains defective for Chlorophyll biosynthesis in the dark, allowing to study the process of de-etiolation in algae, with quantitative output (absolute abundances and stoichiometries). For this purpose, we assembled a set of peptides from proteins involved in photosynthetic light capture and utilization that can be identified in mass spectrometry experiments. It includes, two peptides each, of the core photosynthetic complexes, the mobile electron carriers, known proteins associated with the photosystems, proteins involved in their maturation, chloroplast transcriptional and translational control, Calvin-Benson cycle enzymes and maturation factors, proteins facilitating non-photochemical quenching (NPQ), state transitions, cyclic electron flow, carbon-concentrating mechanisms (CCM), alternative oxidases, cofactor biosynthesis and assembly, alternative electron acceptors, biomarkers of starch metabolism and lipid bodies. We will use this information to generate a full set of isotopically labeled peptides to directly and absolutely quantify ~300 proteins using multiple reaction monitoring (MRM/SRM) in two eukaryotic green algae, the model Chlamydomonas reinhardtii and the biotechnologically relevant Auxenochlorella protothecoides. For this exploratory project, we will work with a subset of these proteins, ~ 100 polypeptides, from which at least 5 unique peptides can be captured on mass spectrometry pipelines suitable for MRM. In this exploratory project, we will establish the determination of the composition / stoichiometries of the photosynthetic complexes. Using this workflow, we will utilize absolute protein quantitation with MRM/SRM proteomics of a larger set of proteins involved in photosynthetic energy production and carbon capture, to uncover the full picture of acclimation of algae to changes in light, nutrient supply, and during the process of greening.
Project Details
Project type
Exploratory Research
Start Date
2022-12-01
End Date
N/A
Status
Active
Released Data Link
Team
Principal Investigator
Co-Investigator(s)
Team Members