Different high temperatures adversely affect crop and algal yields with various responses in photosynthetic cells. The list of genes required for thermotolerance remains elusive. Additionally, it is unclear how carbon source availability affects heat responses in plants and algae. We utilized the insertional, indexed, genome‐saturating mutant library of the unicellular, eukaryotic green alga Chlamydomonas reinhardtii to perform genome‐wide, quantitative, pooled screens under moderate (35°C) or acute (40°C) high temperatures with or without organic carbon sources. We identified heat‐sensitive mutants based on quantitative growth rates and identified putative heat tolerance genes (HTGs). By triangulating HTGs with heat‐induced transcripts or proteins in wildtype cultures and MapMan functional annotations, we presented a high/medium‐confidence list of 933 Chlamydomonas genes with putative roles in heat tolerance. Triangulated HTGs include those with known thermotolerance roles and novel genes with little or no functional annotation. About 50% of these high‐confidence HTGs in Chlamydomonas have orthologs in green lineage organisms, including crop species. Arabidopsis thaliana mutants deficient in the ortholog of a high‐confidence 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.

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High temperatures impair plant/algal growth and reduce food/biofuel production, but the underlying mechanisms remain elusive. The unicellular green alga Chlamydomonas reinhardtii is a superior model to study heat responses in photosynthetic cells due to its fast growth rate, many similarities in cellular processes to land plants, simple and sequenced genome, and ample genetic, genomic, and high-throughput tools. The interaction of heat responses with the availability of organic carbon sources is important for the algal biofuel/bioproduct industry but understudied. We cultivated Chlamydomonas wild-type cultures under highly controlled conditions in photobioreactors at control of 25°C, moderate high temperature of 35°C, or acute high temperature of 40°C with and without constant organic carbon supply (acetate) for 1- or 4-days. Our results showed that moderate high temperature was beneficial to algal growth with constant acetate supply but detrimental without it. The overlooked and dynamic effects of 35°C can be explained by induced carbon metabolisms, including acetate uptake/assimilation, glyoxylate cycle, gluconeogenesis pathways, and glycolysis. Acute high temperature at 40°C for more than 2 days was lethal to algal cultures even with constant acetate supply. Our research provides insights to understand algal heat responses and help improve thermotolerance in photosynthetic cells.

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