High-throughput Identification of Novel Heat Tolerance Genes via Genome-wide Pooled Mutant Screens in the Model Green AlgaChlamydomonas reinhardtii

Abstract

High temperatures adversely affect crop and algal yields. Photosynthetic cells show varying physiological responses to different heat intensities. The specificity of genes required for thermotolerance at different temperatures in photosynthetic cells remains elusive. Additionally, organic carbon sources are present in nature for both plants and algae, but it is unclear how environmental availability of carbon sources interface with heat responses. We utilized the insertional mutant library of the unicellular, eukaryotic green alga Chlamydomonas reinhardtii (CLiP) to perform genome-wide, quantitative, pooled screens under moderate (35oC) or acute (40oC) high temperatures with or without organic carbon. We screened for heat sensitive mutants based on growth rate quantification and identified putative heat tolerance genes (HTGs). By triangulating HTGs with heat-induced transcripts/proteins in wildtype cultures and MapMan functional annotation data, we present a high-confidence list of 933 Chlamydomonas genes with putative roles in heat tolerance. These include genes with known roles in thermotolerance and those with little or no functional annotation, suggesting novel players in thermotolerance. About 50% of these high-confidence HTGs in Chlamydomonas have orthologs in green lineage model organisms, including crop species. Arabidopsis thaliana mutants deficient in the ortholog of a Chlamydomonas HTG were also heat sensitive. This work expands our knowledge of heat responses in photosynthetic cells and provides engineering targets to improve thermotolerance in algae and crops.