High‐throughput identification of novel heat tolerance genes via genome‐wide pooled mutant screens in the model green alga Chlamydomonas reinhardtii

Abstract

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.

Moderate High Temperature is Beneficial or Detrimental Depending on Carbon Availability in the Green AlgaChlamydomonas reinhardtii

“…7), confirming the important role of the organic carbon source, acetate, in heat tolerance. The increased heat tolerance to 35°C with acetate is not strain-dependent because the acetate effects were similar in two different algal strains: CC-1690 (this study) and CC-5325 (Mattoon et al , 2022). In medium starting with acetate but without constant acetate supply, algal biomass increased first but then decreased at 35°C compared to 25°C, which can be explained by increased acetate uptake/usage initially followed by acetate depletion/starvation by the end of the 24-h 35°C treatments (Fig.…”

“…Pyruvate kinase catalyzes the final step of glycolysis and it converts phosphoenolpyruvate and one ADP to pyruvate and one ATP (Baud et al , 2007; Wulfert et al , 2020). Chlamydomonas mutants deficient in pyruvate kinase were heat-sensitive under 35°C in acetate-containing medium (Mattoon et al , 2022), supporting the important roles of glycolysis in thermotolerance of 35°C. Although ATP production mainly comes from mitochondrial respiration, the ATP production from glycolysis can be important under stressful conditions where energy availability is limited (van Dongen et al , 2011).…”

“…A genome-saturating, mapped, indexed, Chlamydomonas insertional mutant library is available for reverse/forward genetic screens and functional genomics (Li et al , 2016, 2019). Genome-wide screens for heat-sensitive Chlamydomonas mutants have been conducted to identify novel genes with putative roles in thermotolerance in photosynthetic cells (Fauser et al , 2022; Mattoon et al , 2022).…”

“…Consequently, it is advantageous to investigate algal heat responses under highly- controlled conditions in photobioreactors (PBRs) (Zhang et al , 2022 a ; Mattoon et al , 2022), which have several evident strengths: (1) controlled heating to mimic the heating speed in nature; (2) precise temperature regulation with a sterile temperature probe inside each algal culture; (3) the availability of turbidostatic control based on defined parameters (e.g., chlorophyll contents) to enable frequent culture dilutions using fresh medium; (4) precisely controlled cultivation condition, including temperature, light, nutrient, and air agitation, allowing for reproducible experiments; (5) automatic recording of culture status every minute, e.g. growth conditions, optical densities, enabling quantitative growth rate measurements; (6) being able to simulate nutrient depletion by turning off the turbidostatic control to investigate how nutrient availability affects algal heat responses.…”

Chlamydomonas: Fast tracking from genomics

“…These advances facilitated the construction of a mapped, indexed library (Li et al, 2016; Li, Patena, et al, 2019; Zhang et al, 2014) containing over 60,000 mutants, with each lesion a unique barcode, allowing for reverse genetic analyses. This library has facilitated the identification of novel genes involved in photosynthesis, the CO 2 concentrating mechanism (CCM), and heat stress responses (among others) using a quantitative pooled mutant screening procedure (Fauser et al, 2022; Li, Patena, et al, 2019; Mattoon et al, 2023).…”