Systems-wide Analysis Revealed Shared and Unique Responses to Moderate and Acute High Temperatures in the Green Alga Chlamydomonas reinhardtii

Abstract

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, promoted growth, and increased starch accumulation. Heat at 40°C arrested the cell cycle, inhibited growth, resulting in carbon uptake over usage and increased starch accumulation. Both high temperatures induced photoprotection, while 40°C decreased photosynthetic efficiency, distorted thylakoid/pyrenoid ultrastructure, and affected the carbon concentrating mechanism. We demonstrated increased transcript/protein correlation during both heat treatments, suggesting reduced post-transcriptional regulation during heat may help coordinate heat tolerance activities efficiently. During recovery after both treatments, 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.

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

“…One round of mating in Chlamydomonas only takes about 2–3 weeks, from the mating process to identifiable progenies. Due to its unicellular nature, high throughput phenotyping tools can be applied to characterize progenies of Chlamydomonas efficiently, e.g., whole plate chlorophyll fluorescence imaging [ 89 , 90 , 91 ], growth quantification on plates using ImageJ [ 23 ], and circadian rhythm monitoring using an automated phototaxis machine [ 92 ]. The CLiP mutant library allows for both forward and reserve genetic screens and candidate genes identified by QTL in Chlamydomonas can be validated using available mutants in the CLiP mutant library [ 37 ].…”

“…Chlamydomonas wildtype strain CC-1690 (also called 21gr, mt + ) and CC-5325 (also called CMJ030, identical to CC-4533, mt − , CLiP library background) were ordered from the Chlamydomonas Resource Center (University of Minnesota). Algal cultures of these two strains were maintained in photobioreactors (PBRs) as described before [ 23 ]. The normal growth condition in PBRs was: 25 °C, Tris-acetate-phosphate (TAP) medium, constant 100 μmol photons m −2 s −1 light (50% red: 50% blue), constant bubbling with filtered air (0.04% CO 2 ), and turbidostatic control by monitoring OD 680 to supply fresh medium frequently and maintain cell density around 2 × 10 6 cells mL −1 .…”

“…Chlamydomonas has rich genetic and genomic resources, and its unicellular nature enables high-throughput approaches and functional genomics. Many transcriptomic analyses under various conditions exist in Chlamydomonas [ 11 ], including light stress [ 12 ], nutrient limitation [ 13 , 14 , 15 ], metal deficiency [ 16 , 17 ], day/night cycle [ 18 , 19 ], oxidative stress [ 20 ], and temperature stresses [ 21 , 22 , 23 ]. Additionally, several proteomics datasets for specific cellular structures are available, including chloroplasts [ 24 ], pyrenoids [ 25 , 26 ], mitochondria [ 27 ], and cilia [ 28 , 29 ].…”

“…Its genome has been sequenced multiple times by several laboratories [ 42 , 43 ]. It has also been used for several transcriptome analyses [ 12 , 23 ] and is an often-preferred background strain for photosynthesis- or stress-related experiments [ 25 , 44 , 45 , 46 ]. CC-5325 is relatively new, available to the Chlamydomonas community since 2014 [ 47 ], but has been frequently used because it is the background strain of the Chlamydomonas CLiP mutant Library [ 37 , 38 ].…”

“…Besides CC-1690 and CC-5325, there are also some other commonly used background strains, e.g., CC-124 and CC-125 [ 42 , 43 ]. We chose to compare CC-1690 and CC-5325 because of our interest in photosynthesis and heat responses, the available transcriptomes of CC-1690 in responses to light and high temperatures [ 12 , 23 ], and the genome-wide screens for mutants deficient in photosynthesis and heat responses by using the CLiP mutant library [ 38 , 54 ]. In this work, we focused on specific phenotypes related to photosynthesis, thermotolerance, circadian rhythm of phototaxis, and ciliary features in these two strains.…”