Moderate high temperature is beneficial or detrimental depending on carbon availability in the green alga Chlamydomonas reinhardtii.

High temperatures impair plant growth and reduce agricultural yields, but the underlying mechanisms remain elusive. The unicellular green alga Chlamydomonas reinhardtii is an excellent 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 and genomics resources. Chlamydomonas grows in light by photosynthesis and with externally supplied acetate as an organic carbon source. Understanding how organic carbon sources affect heat responses is important for the algal industry but remains understudied. We cultivated wild-type Chlamydomonas under highly controlled conditions in photobioreactors at 25 °C (control), 35 °C (moderate high temperature), or 40 °C (acute high temperature) with or without constant acetate supply for 1 or 4 day. Treatment at 35 °C increased algal growth with constant acetate supply but reduced algal growth without sufficient acetate. The overlooked and dynamic effects of 35 °C could be explained by induced acetate uptake and metabolism. Heat treatment at 40 °C for more than 2 day was lethal to algal cultures with or without constant acetate supply. Our findings provide insights to understand algal heat responses and help improve thermotolerance in photosynthetic cells.

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

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.

Improved Spirodela polyrhiza genome and proteomic analyses reveal a conserved chromosomal structure with high abundance of chloroplastic proteins favoring energy production.

Duckweeds are a monophyletic group of rapidly reproducing aquatic monocots in the Lemnaceae family. Given their clonal, exponentially fast reproduction, a key question is whether genome structure is conserved across the species in the absence of meiotic recombination. Here, we studied the genome and proteome of Spirodela polyrhiza, or greater duckweed, which has the largest body plan yet the smallest genome size in the family (1C=150 Mb). Using Oxford Nanopore sequencing combined with Hi-C scaffolding, we generated a highly contiguous, chromosome-scale assembly of S. polyrhiza line Sp7498 (Sp7498_HiC). Both the Sp7498_HiC and Sp9509 genome assemblies reveal large chromosomal misorientations relative to a recent PacBio assembly of Sp7498, highlighting the need for orthogonal long-range scaffolding techniques such as Hi-C and BioNano optical mapping. Shotgun proteomics of Sp7498 verified the expression of ~2250 proteins and revealed a high abundance of proteins involved in photosynthesis and carbohydrate metabolism among other functions. In addition, a strong increase in chloroplast proteins was observed that correlated to chloroplast density. This Sp7498_HiC genome was generated cheaply and quickly with a single Oxford Nanopore MinION flow cell and one Hi-C library in a classroom setting. Combining these data with a mass spectrometry-generated proteome illustrates the utility of duckweed as a model for genomics- and proteomics-based education.

Identification and preliminary characterization of conserved uncharacterized proteins from Chlamydomonas reinhardtii, Arabidopsis thaliana, and Setaria viridis.

The rapid accumulation of sequenced plant genomes in the past decade has outpaced the still difficult problem of genome-wide protein-coding gene annotation. A substantial fraction of protein-coding genes in all plant genomes are poorly annotated or unannotated and remain functionally uncharacterized. We identified unannotated proteins in three model organisms representing distinct branches of the green lineage (Viridiplantae): Arabidopsis thaliana (eudicot), Setaria viridis (monocot), and Chlamydomonas reinhardtii (Chlorophyte alga). Using similarity searching, we identified a subset of unannotated proteins that were conserved between these species and defined them as Deep Green proteins. Bioinformatic, genomic, and structural predictions were performed to begin classifying Deep Green genes and proteins. Compared to whole proteomes for each species, the Deep Green set was enriched for proteins with predicted chloroplast targeting signals predictive of photosynthetic or plastid functions, a result that was consistent with enrichment for daylight phase diurnal expression patterning. Structural predictions using AlphaFold and comparisons to known structures showed that a significant proportion of Deep Green proteins may possess novel folds. Though only available for three organisms, the Deep Green genes and proteins provide a starting resource of high-value targets for further investigation of potentially new protein structures and functions conserved across the green lineage.

Comparative Phenotyping of Two Commonly Used Chlamydomonas reinhardtii Background Strains: CC-1690 (21gr) and CC-5325 (The CLiP Mutant Library Background).

The unicellular green alga Chlamydomonas reinhardtii is an excellent model organism to investigate many essential cellular processes in photosynthetic eukaryotes. Two commonly used background strains of Chlamydomonas are CC-1690 and CC-5325. CC-1690, also called 21gr, has been used for the Chlamydomonas genome project and several transcriptome analyses. CC-5325 is the background strain for the Chlamydomonas Library Project (CLiP). Photosynthetic performance in CC-5325 has not been evaluated in comparison with CC-1690. Additionally, CC-5325 is often considered to be cell-wall deficient, although detailed analysis is missing. The circadian rhythms in CC-5325 are also unclear. To fill these knowledge gaps and facilitate the use of the CLiP mutant library for various screens, we performed phenotypic comparisons between CC-1690 and CC-5325. Our results showed that CC-5325 grew faster heterotrophically in dark and equally well in mixotrophic liquid medium as compared to CC-1690. CC-5325 had lower photosynthetic efficiency and was more heat-sensitive than CC-1690. Furthermore, CC-5325 had an intact cell wall which had comparable integrity to that in CC-1690 but appeared to have reduced thickness. Additionally, CC-5325 could perform phototaxis, but could not maintain a sustained circadian rhythm of phototaxis as CC1690 did. Finally, in comparison to CC-1690, CC-5325 had longer cilia in the medium with acetate but slower swimming speed in the medium without nitrogen and acetate. Our results will be useful for researchers in the Chlamydomonas community to choose suitable background strains for mutant analysis and employ the CLiP mutant library for genome-wide mutant screens under appropriate conditions, especially in the areas of photosynthesis, thermotolerance, cell wall, and circadian rhythms.

Systems-wide analysis revealed shared and unique responses to moderate and acute high temperatures in the green alga Chlamydomonas reinhardtii.

Different intensities of high temperatures affect the growth of photosynthetic cells in nature. To elucidate the underlying mechanisms, we cultivated the unicellular green alga Chlamydomonas reinhardtii under highly controlled photobioreactor conditions and revealed systems-wide shared and unique responses to 24-hour moderate (35°C) and acute (40°C) high temperatures and subsequent recovery at 25°C. We identified previously overlooked unique elements in response to moderate high temperature. Heat at 35°C transiently arrested the cell cycle followed by partial synchronization, up-regulated transcripts/proteins involved in gluconeogenesis/glyoxylate-cycle for carbon uptake and promoted growth. But 40°C disrupted cell division and growth. Both high temperatures induced photoprotection, while 40°C distorted thylakoid/pyrenoid ultrastructure, affected the carbon concentrating mechanism, and decreased photosynthetic efficiency. We demonstrated increased transcript/protein correlation during both heat treatments and hypothesize reduced post-transcriptional regulation during heat may help efficiently coordinate thermotolerance mechanisms. During recovery after both heat treatments, especially 40°C, transcripts/proteins related to DNA synthesis increased while those involved in photosynthetic light reactions decreased. We propose down-regulating photosynthetic light reactions during DNA replication benefits cell cycle resumption by reducing ROS production. Our results provide potential targets to increase thermotolerance in algae and crops.

High light and temperature reduce photosynthetic efficiency through different mechanisms in the C4 model Setaria viridis.

C4 plants frequently experience high light and high temperature conditions in the field, which reduce growth and yield. However, the mechanisms underlying these stress responses in C4 plants have been under-explored, especially the coordination between mesophyll (M) and bundle sheath (BS) cells. We investigated how the C4 model plant Setaria viridis responded to a four-hour high light or high temperature treatment at photosynthetic, transcriptomic, and ultrastructural levels. Although we observed a comparable reduction of photosynthetic efficiency in high light or high temperature treated leaves, detailed analysis of multi-level responses revealed important differences in key pathways and M/BS specificity responding to high light and high temperature. We provide a systematic analysis of high light and high temperature responses in S. viridis, reveal different acclimation strategies to these two stresses in C4 plants, discover unique light/temperature responses in C4 plants in comparison to C3 plants, and identify potential targets to improve abiotic stress tolerance in C4 crops.