New Results

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

Ningning Zhang, Ming Xia, Catherine E. Bailey, Erin M. Mattoon, Ru Zhang
doi: https://doi.org/10.1101/2022.12.04.519034

Abstract

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.

Highlight We revealed the overlooked, dynamic effects of moderate high temperature in algae with different carbon availabilities and demonstrated the importance of carbon metabolism in thermotolerance of photosynthetic cells.

Introduction

Many biological processes in photosynthetic cells are sensitive to high temperatures (Mittler et al., 2012; Schroda et al., 2015; Janni et al., 2020), including but not limited to photosynthesis (Sharkey and Zhang, 2010; Zhang et al., 2022a), cell cycle (Hemme et al., 2014; Zachleder et al., 2019), membrane fluidity and Ca2+ channels (Saidi et al., 2010; Gao et al., 2012), DNA/RNA integrity (Su et al., 2018; Han et al., 2021), and protein stability (Rütgers et al., 2017). Global warming increases the intensity, duration, and frequency of high temperatures in the field, reducing plant growth and food/biofuel production (Janni et al., 2020). Nine of the ten hottest years on record occurred in the last 10 years from 2010 to 2021 due to human activities and the accumulation of greenhouse gases (NASA, Goddard Institute for Space Studies). A recent model revealed high temperature as the primary driver for yield loss in crops from 1981-2017 in US (Ortiz-Bobea et al., 2019). Considering the increasing global temperature and human population, it is imperative to improve plant thermotolerance and understand how photosynthetic cells respond to high temperatures.

High temperatures in the field have different intensities and durations. For many land plants or algae grown in moderate-temperature regions, moderate high temperatures refer to heat slightly above the optimal temperature for plant/algal growth (at or around 35°C), while acute high temperatures refer to heat at or above 40°C (Zhang et al., 2022a). Moderate high temperatures are often long-lasting and frequent in nature with mild effects on photosynthetic cells, while acute high temperatures are often short-term but damaging. Most previous heat-stress experiments in plants used acute high temperatures at or above 40°C (Balfagón et al., 2019; Kim et al., 2020; Ji et al., 2021), likely due to the rapid onset and easily quantifiable phenotypes as compared to moderate high temperatures. Although the impact of moderate high temperatures in photosynthetic cells can be difficult to investigate due to comparatively mild phenotypes, moderate high temperatures are physiologically relevant stresses in field conditions and their frequent and long-lasting features could have significant impacts on agricultural yield (Delorge et al., 2014; Anderson et al., 2021). Global warming can further increase the frequency and duration of moderate high temperatures in nature. However, the effects of moderate high temperatures in photosynthetic cells are underexplored and often overlooked.

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas throughout) is a superior model to study heat responses in photosynthetic cells (Schroda et al., 2015). Chlamydomonas can grow in light under photoautotrophic conditions using photosynthesis and its photosynthetic structures are very similar to land plants (Minagawa and Tokutsu, 2015), presenting an excellent model to study heat effects on photosynthesis. Chlamydomonas can also grow with supplied organic carbon source (acetate) in light (mixotrophic) or dark (heterotrophic) conditions (Sasso et al., 2018), providing a platform to study heat responses under different light/carbon conditions. Additionally, it has a haploid, sequenced, well-annotated, small genome (111 Mb, 17,741 protein-encoding genes) with simpler gene families and fewer gene duplications than land plants (Merchant et al., 2007; Karpowicz et al., 2011). Many land plants, like Arabidopsis thaliana, have 21 or more copies of heat shock transcription factors (HSFs, the master regulators of heat responses) (Guo et al., 2016). However, Chlamydomonas only has two HSFs, making it relatively easy to dissect the transcriptional regulation of heat responses in photosynthetic cells (Schulz-Raffelt et al., 2007). Abundant transcriptome and proteome datasets under different conditions or from different cellular compartments are available in Chlamydomonas, facilitating systems-wide analysis and gene function prediction (Terashima et al., 2011; Hemme et al., 2014; Romero-Campero et al., 2016; Salomé and Merchant, 2021; Zhang et al., 2022a). Furthermore, several well-established gene editing and cloning tools are available in Chlamydomonas (Shimogawara et al., 1998; Greiner et al., 2017; Crozet et al., 2018; Wang et al., 2019; Dhokane et al., 2020; Emrich-Mills et al., 2021). 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).

Algae have great potential for production of biofuels and bioproducts (Scranton et al., 2015; Mathimani and Pugazhendhi, 2019). However, how algal cells respond to high temperatures is under-investigated as compared to land plants (Schroda et al., 2015). Outdoor algal ponds frequently experience moderate high temperatures around 35°C during summer time (Mata et al., 2010), but the effects of moderate high temperatures on algal growth have been overlooked. Previously published algal heat treatments were often conducted in flasks with incubation in pre-warmed water baths at or above 42°C with sharp temperature switches and without control of nutrients (Hemme et al., 2014; Rütgers et al., 2017)). While previous research was highly valuable to understand algal heat responses, high temperatures in the field or outdoor ponds often increase gradually. Heating speeds affect heat responses in plants (Mittler et al., 2012) and rapid increases to high temperatures largely reduce algal viability (Zhang et al., 2022a). Acute high temperatures at or above 40°C inhibit algal cell division (Mühlhaus et al., 2011b; Hemme et al., 2014; Zachleder et al., 2019; Ivanov et al., 2021/5), while moderate high temperature at 35°C only transiently inhibits cell division during the first 4-8 hour (h) heat (Zhang et al., 2022a). Thus, conducting long-term experiments at moderate high temperatures in flasks can result in overgrown cultures, nutrient depletion, and light limitation, complicating data interpretation.

Consequently, it is advantageous to investigate algal heat responses under highly- controlled conditions in photobioreactors (PBRs) (Zhang et al., 2022a; 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. Utilization of PBRs for algal cultivation and heat treatments can largely reduce compounding effects during high temperature treatments and improve our understanding of algal heat responses.

Recently, we conducted systems-wide analyses in wild-type (WT) Chlamydomonas cultures during and after 24-h heat of 35°C and 40°C in PBRs with constant nutrient supply and light in acetate-containing medium (mixotrophic condition) (Zhang et al., 2022a). Our results showed that 40°C inhibited algal growth while 35°C increased algal growth. The growth inhibition at 40°C could be explained by reduced photosynthesis, impaired respiration, and cell cycle arrest, while these cell parameters had minor changes in algal cultures treated by 35°C. Our proteomics data indicated that several proteins involved in acetate uptake/assimilation, glyoxylate cycle, and gluconeogenesis were up- regulated during 35°C (Zhang et al., 2022a). Chlamydomonas uptakes acetate and feeds it into the glyoxylate cycle and gluconeogenesis for starch biosynthesis (Johnson and Alric, 2012, 2013). The main function of glyoxylate cycle is to allow growth when glucose is unavailable and two-carbon compounds, e.g., acetate, are the organic carbon source; glyoxylate cycle is a shunt of the tricarboxylic acid (TCA) cycle in mitochondria but without CO2 releases to allow for the anabolism of simple carbon compounds in gluconeogenesis, a process to make sugars, namely glucose (Johnson and Alric, 2012; Chew et al., 2019; Walker et al., 2021).

We hypothesized that the increased growth in Chlamydomonas at 35°C with constant acetate supply was attributable to up-regulated acetate metabolisms, including the pathways mentioned above. The majority of published high temperature research in Chlamydomonas has been conducted in acetate-containing medium (Voß et al., 2010; Mühlhaus et al., 2011a; Hemme et al., 2014; Rütgers et al., 2017; Zhang et al., 2022a), but the interface between acetate supply and algal heat responses is understudied. To validate our hypothesis and address these unknown questions, we cultivated the same Chlamydomonas WT strain in PBRs as before but heated the cultures at 35°C or 40°C with and without constant acetate supply for 1 or 4 days. Our results revealed the overlooked effects of moderate high temperature of 35°C on algal growth which can be beneficial or detrimental based on acetate availability. Acute high temperature at 40°C is lethal to algal cultures, even with constant acetate supply. Our research provided insights to understand algal heat responses and help improve thermotolerance in photosynthetic cells.

Materials and methods

Algal cultivation

Chlamydomonas reinhardtii wild-type strain CC-1690 (also called 21gr, mating type plus) (Pröschold et al., 2005; Zhang et al., 2022b; Sager, 1955-7) was purchased from the Chlamydomonas resource center and used in all experiments. Algal cultures were grown in Tris-acetate-phosphate (TAP, with acetate) or Tris-phosphate (TP, without acetate) medium with revised trace elements (Kropat et al., 2011) in 400 mL photobioreactors (PBRs) (Photon System Instruments, FMT 150/400-RB) as described before (Zhang et al., 2022a). Cultures were illuminated with constant 100 μmol photons m−2 s−1 light (red: blue, 1:1 ratio), and agitated by bubbling with filtered air at a flow rate of 1 L min−1. Algal cultures with targeted cell density around 2 x106 cells mL−1 (~4.0 μg mL−1 chlorophyll content) were maintained at 25°C for 4 days with constant nutrient supply through a turbidostatic mode before different temperature treatments. The turbidostatic mode was controlled by OD680 (optical density at 680 nm), which is proportional to chlorophyll contents (μg mL−1) and was monitored once per min automatically. When OD680 increased to a maximum value slightly above the target value (for target cell density) due to algal growth, OD680 signaled the control computer to turn on a turbidostatic pump to add fresh medium to dilute the culture until a minimum OD680 slightly below the target value was reached, then the turbidostatic pump was turned off automatically. Because of the small OD range we used, the PBR cultures had exponential growth between dilution events through the turbidostatic mode with constant nutrient supply (Fig. 1A). The OD680 data during exponential growth phases in between dilution events was log2 transformed, and the relative growth rate was calculated using the slope of log2(OD680). The relative growth rate is an inverse of the doubling time of an algal culture. For the treatments without constant nutrient supply, the turbidostatic pumps were turned off after PBR cultures reached steady growth and at the start of different temperature treatments.

Fig. 1.
Fig. 1. Without constant acetate supply, moderate high temperature of 35°C transiently increased but then decreased Chlamydomonas biomass accumulation.

(A) Before heat treatments, Chlamydomonas cells (CC-1690, also called 21gr, wild-type) were grown in photobioreactors (PBRs) in Tris-acetate-phosphate (TAP) medium (acetate as an organic carbon source) with a light intensity of 100 μmol photons m−2 s−1 and constantly bubbling of air. Algal cultures were maintained turbidostatically within a small range of OD680 which monitors chlorophyll content (μg chlorophyll mL−1 cultures). When PBR cultures grew to the set maximum OD680, pumps were turned on to add fresh medium and dilute the cultures to the set minimal OD680, then pumps were turned off to allow for exponential growth to the maximum set OD680. Figure cited from a supplementary figure of this paper (Zhang et al., 2022a). (B) Experimental outline. The PBR cultures were first maintained at 25°C with constant nutrient supply using the turbidostatic mode as mentioned above. After the cultures reached steady growth (preheat time point), the turbidostatic pumps were turned off and the cultures were grown under the indicated temperature for 24 hours (h) without fresh medium supply (thus no constant acetate supply). Algal cultures were harvested at different time points for the indicated parameters. Each temperature treatment was conducted in an individual PBR with at least 3 replicates. (C) Without constant acetate supply, algal dry biomass increased with 8-h heat but decreased with 24-h heat of 35°C as compared to the constant 25°C. Mean ± SE, n=3-8 biological replicates. (D) Heat at 35°C accelerated acetate consumption. Algal cultures were harvested at different time points and acetate content in algal supernatant was quantified using Acetate Colorimetric Assay Kit. The red dashed line marks the start of heating at 35°C or 40°C. (C, D) Statistical analyses were performed using two-tailed t-test assuming unequal variance; *, p<0.05; **, p<0.01; Not significant, ns; the colors of the asterisks match the heated condition as compared to 25°C at the same time points; #, p<0.05, for the comparisons between 35°C and 40°C.

High temperature treatments in PBRs

After algal cultures in PBRs reached steady growth with turbidostatic control at 25°C for 4 days, PBR temperatures were increased to moderate or acute high temperatures (35°C or 40°C in different PBRs) for the indicated duration with indicated nutrient conditions. PBR temperatures were changed from 25°C to 35°C or 40°C gradually over the course of 30 min with controlled heating speeds. PBR cultures grown under constant 25°C with the same nutrient status and treatment duration served as controls. Algal cultures were harvested from PBRs at different time points during different treatments for various measurements.

Algal biomass quantification

Algal cultures (90 mL) were harvested from PBRs, centrifuged to remove supernatants, flash frozen in liquid nitrogen, then stored in a −80°C freezer until analysis. Algal cell pellets were freeze dried for 24 h over liquid nitrogen. The remaining algal dry biomass were weighted and quantified.

Acetate quantification assay

Algal cultures were harvested from PBRs (2 mL) and centrifuged to collect 500 μL top clear supernatants to a new tube. The supernatant was stored in a −80°C freezer until analysis. The acetate content in the supernatant was quantified using the Acetate Colorimetric Assay Kit (Sigma, Cat No. MAK086) according to manufacture instructions.

RT-qPCR analysis

PBR cultures of 2 mL were pelleted with Tween-20 (0.005%, v/v) by centrifugation at 11,363 × g and 4°C for 2 min, followed by supernatant removal. The cell pellet was flash frozen in liquid nitrogen and then stored in a −80°C freezer until processing. RNA extraction and RT-qPCR analysis were performed as before with minor modifications (Zhang et al., 2022a). Total RNA was extracted with TRIzol reagent (Thermo Fisher Scientific, Cat No. 15596026), digested on-column with RNase-free DNase (Qiagen, Cat No. 79256), purified by RNeasy mini-column (Qiagen, Cat No. 74106), and quantified with Qubit™ RNA BR Assay Kit, (Life technology, Cat No. Q10210). Total 0.4 μg RNA was reverse transcribed with oligo dT primers using SuperScript® III First-Strand Synthesis System (Life technology, Cat No. 18080-051) following manufacturer instructions. Quantitative real-time PCR (RT-qPCR) analysis was performed using a CFX384 Real-Time System (C 1000 Touch Thermal Cycler, Bio-Rad, Hercules, California) using SensiFAST SYBR No-ROS kit (Bioline, BIO-98020) following this set-up: (1) 95°C (2 min); (2) 40 cycles of 95°C (5 s), 60°C (10 s), and 72°C (15 s); (3) final melt curve at 60°C (5 s), followed by continuous temperature ramping from 60°C to 99°C at a rate of 0.5°C s−1. Melting curves and qPCR products were tested to ensure there were no primer dimers or unspecific PCR products. All RT-qPCR products were sequenced for confirmation. Primers and gene IDs for RT-qPCR were included in Supplementary Table S1. CBLP (β- subunit-like polypeptide, Cre06.g278222), (Schloss, 1990; Xie et al., 2013) and EIF1A (Eukaryotic translation initiation factor 1A, Cre02.g103550) (Strenkert et al., 2019) had stable expression among all time points, and were used as reference genes for RT-qPCR normalization. The relative gene expressions were calculated relative to the pre-heat time point by using the 2−ΔΔCT method (Livak and Schmittgen, 2001; Hellemans et al., 2007; Remans et al., 2014). Three biological replicates were included for each time point and treatment.

Cell imaging using light microscopy

Chlamydomonas cultures harvested at select time points with different temperature treatments were fixed with 0.2% glutaraldehyde (VWR, Cat No. 76177-346). Algal cells were imaged with a Leica DMI6000 B microscope and a 63x (NA1.4) oil-immersion objective.

Chlorophyll quantification

Chlorophyll contents in algal cells were quantified as before (Zhang et al., 2022b,a). PBR cultures of 1 mL were harvested in 1.5-mL tubes with 2.5 μL 2% Tween20 (Sigma, P9416-100ML, to help cell pelleting), and centrifuged at 18,407 g at 4°C. After removing supernatant, cell pellets were stored in a −80°C freezer until quantification. Later, cell pellets were thawed, resuspended in 1 mL of HPLC grade methanol (100%, Sigma, 34860-4L-R), vortexed for 1 min, incubated in the dark at 4°C for 5 min, and centrifuged at 15,000 g at 4°C for 5 min. Supernatant was pipetted out to a two-sided disposable plastic cuvettes (VWR, 97000-586) for chlorophyll (Chl) quantification at 652 and 665 nm in a spectrophotometer (IMPLEN Nonophotometer P300) using the following equations: Chl a + Chl b = 22.12*A652 + 2.71*A665 (in μg mL−1 algal cultures) (Porra et al., 1989). Chlorophyll concentrations were also normalized to cell densities (Chl pg cell−1) or algal cell volume (Chl pg μm −3). Cell density and mean cell volume were measured using a Coulter Counter (Multisizer 3, Beckman Counter, Brea, CA).

Differential expression heatmaps

Our previously published RNA-seq and proteomics data (Zhang et al., 2022a) were used to identify the expression patterns of genes of interest. Heatmaps were generated using the R package pheatmap (version 1.0.12. https://CRAN.R-project.org/package=pheatmap).

Results

To investigate how the availability of organic carbon source affected algal heat responses, we first cultivated WT Chlamydomonas cells (CC-1690, 21gr) in PBRs in Tris-acetate- phosphate (TAP, acetate as carbon source) medium at 25°C with constant nutrient supply through turbidostatic control (providing frequent fresh medium and culture dilution) (Fig. 1A, B). After algal cultures reached steady growth rates in PBRs, the turbidostatic control was turned off and cultures were switched to 35°C, or 40°C, or stayed at 25°C for 24 h without constant acetate supply. Algal cultures were harvested at different time points to analyze cell physiologies, transcripts, and biomass (Fig. 1B). Algal dry biomass quantification showed that cultures treated with 35°C had increased biomass at 8-h heat but decreased biomass at 24-h heat as compared to 25°C (Fig. 1C). Acetate quantification in the supernatant of algal cultures indicated that 35°C-treated cultures had increased acetate consumption and depleted acetate faster than 25°C or 40°C (Fig. 1D).

We suspected the transiently increased and then decreased algal biomass in 35°C- treated cultures may be due to increased acetate uptake/usage followed by acetate starvation without constant acetate supply (Fig. 1). Chlamydomonas uptakes acetate and feeds it into the glyoxylate cycle and gluconeogenesis for starch biosynthesis (Johnson and Alric, 2012, 2013) (Fig. 2A). To verify this hypothesis, we investigated transcripts involved in the glyoxylate and gluconeogenesis cycles (Fig. 2A-E). ICL1 and MAS1 are key enzymes involved in glyoxylate cycle; PCK1 and FBP1 are key enzymes involved in gluconeogenesis pathways (Johnson and Alric, 2012, 2013; Plancke et al., 2014) (Fig. 2A). The relative expression levels of three of these four transcripts (except FBP1) were significantly down-regulated at 24-h of constant 25°C without constant acetate supply as compared to the pre-heat time point (with constant acetate supply), consistent with the acetate-depleting condition (Fig. 2B-E). However, the expression levels of three of these four genes (except for FBP1) were significantly higher in algal cultures treated with 24-h 35°C than 25°C. Although only the relative expression levels of MAS1 at a few limited time points were significantly reduced at 40°C than 25°C, the expression levels of ICL1, MAS1, PCK1 were significantly reduced in algal cultures treated with 40°C than 35°C for almost all time points during the 24-h heating, suggesting reduced glyoxylate cycle and gluconeogenesis pathways in 40°C-treated cultures (Fig. 2B-E). Using our previously published transcriptomes and proteomes from algal cultures with constant acetate supply and turbidostatic control (grown in the same PBRs and acetate-containing medium), we confirmed the largely down-regulated transcripts related to glyoxylate cycle and gluconeogenesis in 40°C-treated cultures and up-regulated proteins related to these pathways in 35°C-treated cultures (Fig. F, G). These results suggested glyoxylate cycle and gluconeogenesis increased under 35°C heat but decreased under 40°C heat.

Fig. 2.
Fig. 2. The dynamic effects of 35°C on algal biomass without constant acetate supply is related to the up-regulation of glyoxylate cycle and gluconeogenesis pathways.

(A) Simplified pathways of acetate uptake/assimilation, glyoxylate cycle, and gluconeogenesis based on this paper (Johnson and Alric, 2013). Key enzymes are in boxes and those with asterisks were used for RT-qPCR analysis in panels B-E. Glyoxylate cycle key enzymes: ICL1, isocitrate lyase; MAS1, malate synthase. Gluconeogenesis key enzymes: PCK1, phosphoenolpyruvate carboxykinase; FBP1, fructose-1,6-bisphosphatase (See all gene IDs and annotations in Supplementary Table S1). PEP, phosphoenolpyruvate. Fru-6-P, fructose-6-phosphate. Fru-1,6-P2, fructose- 1,6-bisphosphate. (B-E) Without constant acetate supply, transcripts related to glyoxylate cycle and gluconeogenesis pathways were up-regulated at the end of 24-h heat of 35°C but down-regulated at 24-h of 25°C. The relative expressions were calculated from RT- qPCR results by normalizing to the reference genes CBLP, EIF1A and pre-heat level. Mean ± SE, n = 3 biological replicates. Statistical analyses were performed with two-tailed t-test assuming unequal variance; *, p<0.05, the colors of the asterisks match the heated condition as compared to 25°C at the same time point; #, p<0.05, for the comparisons between 35°C and 40°C at the same time point; ^, p<0.05, for the comparisons between different time points at 25°C (no constant acetate) with the pre-heat, constant-acetate condition. The algal cultivation and heat treatments were the same as in Fig. 1B. The red dashed line marks the start of heat at 35°C (brown squares) or 40°C (red triangles). The control culture was maintained at 25°C (black circles). All cultures started with TAP medium, then no constant acetate supply starting with the 0-h time point. (F, G) The relative expression level of transcripts and proteins related to glyoxylate cycle and gluconeogenesis pathways during 35°C or 40°C heat treatments with constant acetate supply through turbidostatic mode. Heatmaps were plotted based on the transcriptomes and proteosomes data published in this paper (Zhang et al., 2022a). Only annotated transcripts/proteins with significantly changed expressions in at least one time points were included in the heatmaps. FC, fold-change. Differential expression model output log2FC values were sorted into different expression bins. Heating time points were labeled at the bottom: 0 h, reach high temperature of 35°C or 40°C; 0.5 h, heat at 35°C or 40°C for 0.5 h, similar names for other time points during heat.

Without constant acetate supply, 35°C and 40°C affected algal cell physiologies differently. 35°C-treated algal cells had transiently increased then decreased cell size while 40°C-treated algal cells had steadily increased cell size due to heat-inhibited cell division (Fig. 3). We quantified the cellular parameters before and during heat and compared the results with and without constant acetate supply (Fig. 4). Without constant acetate supply and turbidostatic dilution, cell density increased steadily during 25°C and 35°C but had little change during 40°C (Fig. 4A), consistent with cell cycle arrest under 40°C (Zhang et al., 2022a). The cell density was significantly lower at 35°C than 25°C at 8-h and 24-h time points (Fig. 4A). The mean cell volume was constant during 25°C treatment, transiently increased under 35°C, and was close to 3X pre-heat volume at the end of 40°C heat, in agreement with our microscopic images (Fig. 3). Chlorophyll content per culture volume (mL) had no significant differences between 25°C and 40°C but was significantly higher at 8-h of 35°C than 25°C (Fig. 4C), consistent with the increased biomass at the 8-h of 35°C (Fig. 1C). Chlorophyll content normalized to cell density revealed constant chlorophyll per cell during 25°C, but transiently and constantly increased cellular chlorophyll during 35°C and 40°C, respectively (Fig. 4D). Chlorophyll content normalized to cell volume showed the increased cellular chlorophyll during 40°C and 35°C cannot be completely explained by increased cell volume (Fig. 4E).

Fig. 3.
Fig. 3. Heat at 35°C transiently increased algal cell size.

Light microscopic images of Chlamydomonas cells without constant acetate supply in medium starting with acetate. Images shown are representative results from at least three biological replicates. The algal cultivation and heat treatments were the same as in Fig. 1B.

Fig. 4.
Fig. 4. Acetate availability affected algal cell physiologies during 35°C or 40°C heat in medium starting with acetate.

(A-E) Cell parameters from algal cultures without constant acetate supply. The algal cultivation and heat treatments were the same as in Fig. 1B. (F-J) Cell parameters from algal cultures with constant acetate supply via turbidostatic mode, data plotted based on the results from this paper (Zhang et al., 2022a). Mean ± SE, n = 3-7 biological replicates. (A-J) Two panels on the same row share the same y axis. The red dashed line marks the start of heat. (K-O) Fold change of the indicated cell parameters from algal cultures without (filled bars) and with (empty bars) constant acetate supply. Statistical analyses were performed with two-tailed t-test assuming unequal variance; *, p<0.05, for the comparisons between 35°C or 40°C with 25°C at the same time point under the same acetate condition, the colors of the asterisks match the heat condition; #, p<0.05, for the comparisons between 35°C and 40°C; ^, p<0.05, for the comparisons between with and without constant acetate supply at the same time point. (D-J) Cell parameters at different time points during constant 25°C had little change, not significantly different from the pre-heat time points (p>0.05).

Based on our previously published data, we summarized cell parameters in algal cultures grown in the same PBRs but with constant acetate supply through turbidostatic control (Zhang et al., 2022a) (Fig. 4F-J). Cell parameters at different time points during 25°C had little or no changes as compared to the pre-heat time points, demonstrating the effectiveness of the turbidostatic control with constant acetate supply. The changes of cell volume and chlorophyll content per cell during 35°C and 40°C had the similar trends with and without constant acetate supply, but the increase of these parameters was larger under 40°C in cultures with constant acetate supply than without (Fig. 4B, D, G, I). This was supported by the fold change of cell parameters by comparing the data with and without constant acetate supply (Fig. 4K-O).

In addition to the cellular parameters mentioned above using time-course harvesting, we next utilized non-disruptive methods to quantify algal growth under different temperatures without constant acetate supply. OD680 (optical density at 680 nm) monitors chlorophyll content per mL culture (Chapman et al., 2015; Xiao et al., 2015; Young et al., 2022). OD750 (optical density at 750 nm) monitors light scattering and is thought to be proportional to cell density (Chioccioli et al., 2014; Young et al., 2022). Our PBRs have OD680 and OD720 settings, but no OD750. However, OD720 serves as a proximity for OD750 for light scattering. Thus, we investigated the dynamic changes of OD680 and OD720 relative to chlorophyll content and cell density before and during heat treatments in medium starting with acetate but without constant acetate supply (Fig. 5). The change of OD680 (Fig. 5A) mimicked the change of chlorophyll content per mL algal culture for all three temperature treatments (Fig. 4C) while the change of OD720 (Fig. 5B) mimicked the change of cell density for 25°C and 35°C but not 40°C (Fig. 4A). Combining all data from different temperature treatments and time points, both OD680 and OD720 were linearly proportional to chlorophyll content per mL algal culture (Fig. 5C, D), but they were much less proportional to cell density, with evident deviations at the high OD range (Fig. 5E, F) and even lower correlations at the low OD range (Fig. 5G, H). Our results showed that OD680 and OD720 can be used to accurately estimate chlorophyll accumulation in algal cultures with different heat treatments, thus they can be used to estimate algal relative growth rates based on chlorophyll accumulation during an exponential growth phase between dilution events with turbidostatic control in PBRs (Zhang et al., 2022a) (See methods for details). The relative growth rates calculated from both OD680 and OD720 yielded similar results. Because OD680 had larger values and higher signal/noise ratios than OD720, we used OD680 to estimate relative growth rates of PBR cultures over several days with turbidostatic controls and constant acetate supply to investigate how 35°C and 40°C affected algal heat responses in long-term (Fig. 6). This research goal could not be achieved without turbidostatic controls because acetate in the TAP medium was fully depleted in 24 h during all three temperature treatments of 25°C, 35°C and 40°C without constant acetate supply (Fig. 1D).

Fig. 5.
Fig. 5. OD680 and OD720 both were linearly proportional to chlorophyll contents under different temperatures and can be used to estimate algal growth rates.

(A, B) OD680 and OD720 readings in algal cultures grown in photobioreactors under different temperatures without constant acetate supply (no turbidostatic control) in medium starting with acetate. The algal cultivation and heat treatments were the same as in Fig. 1B. Lines with the same colors represent biological replicates (n= 4-8) under the same condition. (C-F) Chlorophyll (Chl) contents and cell densities were plotted against OD680 or OD720 readings in algal cultures with different treatments mentioned above. Dashed blue lines are linear trendlines of best fit and R-squared values are displayed. (G, H) The same data as in panel E-F but with smaller ranges for X values.

Fig. 6.
Fig. 6. Moderate and acute high temperatures had distinct effects on algal growth and cell parameters during 4-day heating with constant acetate supply and turbidostatic control.

(A) Chlamydomonas cultures were grown in photobioreactors under turbidostatic conditions at different temperatures in acetate-containing medium. Algal cultures were first acclimated at 25°C for 4 days before the temperature was switched to 35°C or 40°C or stayed at 25°C for 4 days. The red dashed line marks the start of heat at 35°C or 40°C. Relative growth rates were calculated based on the cycling of OD680 caused by the turbidostatic control (see Fig. 1A and method for details). Each temperature treatment had 3 biological replicates in separate PBRs. (B-F) Cell parameters were quantified from algal cultures harvested at different time points with different treatments. Statistical analyses were performed with two-tailed t-test assuming unequal variance by comparing 35°C or 40°C with 25°C at the same time point. *, p<0.05, the colors of the asterisks match the heated condition; #, p<0.05, for the comparisons between 35°C and 40°C. Chlorophyll, Chl.

To investigate the effects of acetate on long-term heating, after algal cultures reached steady growth at 25°C in PBRs with turbidostatic control, we conducted all three temperature treatments with continuously turbidostatic control and constant acetate supply for 4 days (Fig. 6). Without heat treatments, algal growth rates and cell parameters stayed constant at 25°C, demonstrating the effectiveness of our turbidostatic control for algal growth (Fig. 6). At 35°C with constant acetate supply, the relative growth rates increased first, but the increase was reduced to a smaller degree after 2-day heating at 35°C and stabilized thereafter (Fig. 6A). At 40°C, the relative growth rates decreased steadily, and the cultures died after 2-day heating at 40°C, suggesting algal cells could not acclimate to long-term constant 40°C, even with constant acetate supply. Cell densities and chlorophyll per cell of 35°C-treated cultures had cycling pattern, with approximately 24-h period, which diminished at the end of 4-day heating (Fig. 6B, E). Cells treated with 40°C had more than 4-fold increase of cell volume as compared to the pre-heat condition, followed by reduced cellular chlorophyll and cell death (Fig. 6C-F). The turbidostatic mode by OD680 tightly controlled the chlorophyll contents in unit of ug per mL culture in PBRs during the treatments of 25°C and 35°C (Fig. 6D), but not 40°C due to the cell cycle arrest and overwhelmingly increased chlorophyll per cell, and eventually cell death under 40°C (Fig. 6B and E).

In algal cultures with constant medium supply via turbidostatic control but without acetate, the relative growth rates decreased at 35°C (Fig. 7A), in contrast to the increased growth rates under 35°C with constant acetate supply (Fig. 7B). This supports our hypothesis that the increased growth during 35°C with constant acetate supply (Fig. 6A) is due to increased carbon (acetate) metabolisms: acetate uptake/assimilation, glyoxylate cycle, and gluconeogenesis (Fig. 2A).

Fig. 7.
Fig. 7. Chlamydomonas cells had reduced growth during 4-day heat of 35°C with constant nutrient supply but no acetate.

(A) Relative growth rates from algal cultures grown in photobioreactors with constant nutrient supply using the turbidostatic mode in photoautotrophic medium (Tris-phosphate, TP, no acetate). Relative growth rates were calculated based on the cycling of OD680 caused by the turbidostatic control (see Fig. 1A and method for details). The red dashed line marks the start of heat at 35°C. Data plots are three biological replicates. (B) Comparison of relative growth rates during 4-day heat of 35°C using the turbidostatic mode with and without acetate. The data with constant acetate was based on the results from this paper (Zhang et al., 2022a). Mean relative growth rates before heat, during the 1st and 4th day of heat were plotted. Mean ± SE, n = 3 biological replicates, each with 3-7 data points. Statistical analyses were performed with two-tailed t-test assuming unequal variance by comparing with pre-heat or between the 1st and 4th day of heat (under brackets); *, p<0.05; **, p<0.01.

Discussion

By performing algal cultivation under highly controlled conditions in PBRs with and without a constant organic carbon source, acetate, we investigated how the availability of organic carbon supply affected the growth of Chlamydomonas under moderate (35°C) and acuate high temperatures (40°C).

Heat of 35°C was beneficial or detrimental depending on carbon availability

Moderate high temperature of 35°C increased algal growth rates with constant acetate supply (Fig. 6A) (Zhang et al., 2022a). Without acetate in photoautotrophic medium, heat of 35°C decreased agal growth rates (Fig. 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. 1C, D). In medium starting with acetate but without turbidostatic control and frequent dilutions (Fig. 1B), other nutrients, e.g., nitrogen may also be reduced or depleted in 24-h of treatments, but our results strongly pointed to the effects of acetate on algal heat responses (Fig. 7), which is the focus of our research.

Chlamydomonas uptakes acetate and feeds it into the glyoxylate cycle and gluconeogenesis for starch biosynthesis and starch is broken down through glycolysis to make cellular energy (Johnson and Alric, 2012, 2013) (Fig. 2A).The dynamic effects of 35°C on algal growth under different acetate conditions can be explained by the 35°C- induced carbon metabolisms, including but not limited to the up-regulation of acetate uptake/assimilation, glyoxylate cycle and gluconeogenesis pathways, and glycolysis (Fig. 1D, 2) (Zhang et al., 2022a). Our previous proteomic data showed significantly up- regulated proteins involved in these carbon metabolism pathways mentioned above (Zhang et al., 2022a). Energy produced from glycolysis can be used for energy-requiring cellular activities to increase thermotolerance (Olas et al., 2021), e.g., production of heat shock proteins and repair pathways related to photosynthesis (Murata and Nishiyama, 2018; Bourgine and Guihur, 2021). 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). Under low oxygen conditions, plants increase activity of pyruvate kinases to produce more ATP (van Dongen et al., 2011). Heat-treated barley leaves utilized glycolysis as an alternative energy source for thermotolerance based on proteomics analysis (Rollins et al., 2013), consistent with our previous proteomic results in Chlamydomonas (Zhang et al., 2022a).

Additionally, acetate may protect photosynthesis from heat-induced photoinhibition. Acetate is proposed to protect photosystem II (PSII) against photoinhibition by replacing the bicarbonate associated to the non-heme iron at the acceptor side of PSII, changing the environment of plastoquinone, and affecting PSII charge recombination (Roach et al., 2013). Chlamydomonas grown in acetate-containing medium produced less singlet O2 (one kind of reactive oxygen species, ROS) than those grown in non-acetate-containing, photoautotrophic medium (Roach et al., 2013). Furthermore, experimental and modeling analysis suggested that acetate promoted cyclic electron flow (CEF) around photosystem I (PSI); reducing equivalents produced during the acetate metabolism reduce plastoquinone pools and increase CEF activity (Johnson and Alric, 2012; Lucker and Kramer, 2013; Chapman et al., 2015). CEF cycles photosynthetic electrons around PSI, producing only ATP but no NADPH and providing extra ATP needed for photosynthesis and other cellular activities (Munekage et al., 2004; Baker et al., 2007). CEF balances the ATP/NADPH ratio, contributes to the generation of transthylakoid proton motive force, and protects both PSI and PSII from photo-oxidative damage (Johnson, 2011; Yamori and Shikanai, 2016). The increased acetate uptake under 35°C (Fig. 1D) was coupled with induced CEF activity measured by P700 oxidation/reduction in Chlamydomonas (Zhang et al., 2022a).

On the other side, acerate is reported to suppress the activity of photosynthesis (Heifetz et al., 2000; Chapman et al., 2015). In the presence of acetate, photosynthesis is less important for algal growth than in photoautotrophic conditions without acetate. Photosynthesis is one of the most heat sensitive functions in plants (Sharkey, 2005; Sharkey and Zhang, 2010) and is also the major source of ROS when photosynthetic activity is compromised by heat (Pospíšil, 2016; Qin-Di et al., 2021; Niemeyer et al., 2021). Although moderate high temperature of 35°C did not affect thylakoid structures or PSII activities significantly, the increased non-photochemical quenching (NPQ, photoprotection pathway) (Rochaix, 2014; Erickson et al., 2015) in 35°C-treated Chlamydomonas cells still suggested non-optimal or compromised photosynthesis at 35°C (Zhang et al., 2022a). The suppression of photosynthetic activities by acetate may alleviate the heat-induced damages to photosynthesis and reduce ROS production at 35°C.

With constant acetate supply, 35°C is beneficial, increasing carbon metabolisms and energy production, thus improving thermotolerance and growth (Fig. 8). If algal cells can be cultivated under sterile conditions with constant organic carbon supply, 35°C could be used to promote algal growth and increase biofuel/bioproduct generation under mixotrophic conditions in light. Culture temperature of 35°C in closed, outdoor algal ponds may not be difficult to reach with natural sunlight heating in the summertime of the moderate climate regions and most time of the tropical regions.

Fig. 8.
Fig. 8. A model to depict how carbon availability impacts the effect of moderate (35°C) and acute high temperatures (40°C) on Chlamydomonas growth.

Heat of 35°C accelerates carbon metabolisms through increased acetate uptake/assimilation, glyoxylate cycle, and gluconeogenesis pathways. With constant acetate supply, 35°C increases algal growth. Without constant acetate supply, 35°C increases algal growth transiently followed by decreased growth and biomass accumulation due to acetate starvation. Heat of 40°C reduces transcripts/proteins related to acetate uptake, glyoxylate cycle, and gluconeogenesis pathways, decreases photosynthesis, reduces respiration, and inhibits cell division based on our previous results (Zhang et al., 2022a). Thus, Chlamydomonas cells cannot survive heat of 40°C for longer than 2-days, even with constant acetate supply.

Without constant acetate supply, 35°C is detrimental. The 35°C-induced up-regulation carbon metabolism seem independent of acetate availability, but rather resulted from a high-temperature effect (Fig. 2). Without constant acetate supply, when acetate was fully depleted after 24-h growth, transcripts involved in glyoxylate cycle and gluconeogenesis pathways were down-regulated in the 25°C control culture but up-regulated in the 35°C- treated culture (Fig. 2B-E). The 35°C treatment elevated carbon metabolism without sufficient carbon input which may deplete cellular carbon reserves. This was not sustainable in the long term and eventually reduced biomass accumulation (Fig. 1C). Such effects of 35°C can particularly compromise the yields of outdoor algal ponds because they usually do not contain carbon sources but frequently experience moderate high temperatures (Mata et al., 2010; El-Sheekh et al., 2019). The up-regulated carbon metabolism under moderate high temperatures may also occur in land plants, especially in sink tissues (e.g., roots) or mixotrophic tissues (e.g., developing green seeds) (Koley et al., 2022). Heat-induced glyoxylate cycle, gluconeogenesis, and glycolysis have been reported in plants (Rollins et al., 2013; Zhang et al., 2013; Aprile et al., 2013). Carbon metabolism and sugar availability were shown to be essential for heat tolerance in Arabidopsis (Olas et al., 2021). Without sufficient carbon supply from source tissues (e.g., leaves), long-term moderate high temperatures may result in significant loss of plant yield (Li et al., 2015; Qin-Di et al., 2021). Arabidopsis seedlings treated with 5-day heat at 35°C had reduced growth and viability (Song et al., 2021).

With constant acetate supply, heat at 35°C transiently arrested the cell cycle, which fully recovered after 8 h at 35°C, based on DNA content analysis and the expression pattern of cell cycle genes (Zhang et al., 2022a). Additionally, our previous results revealed partial culture synchronization induced by 35°C (Zhang et al., 2022a). Our pre-heat PBR cultures were grown asynchronously under constant light and temperature (25°C) with turbidostatic control and frequent medium supply so the circadian rhythm of the cultures should be none or minimal without heat treatments, as evidenced by the steady relative growth rates and cell parameters under constant 25°C (Fig. 6). The cycling pattern of cell densities and chlorophyll per cell during 4-day 35°C heat may be related to the 35°C- induced culture synchronization (Fig. 6B, E). Synchronized cultures at 35°C grew up between two sequential dilution events as evidenced by increased chlorophyll per cell (Fig. 6E). The speed of increase in chlorophyll per cell (or the relative growth rates) was faster at 35°C than 25°C (Fig. 6A). The turbidostatic dilution is controlled by OD680, which is proportional to chlorophyll per mL culture (Fig. 5, 6D). Thus, the trend of chlorophyll per cell was opposite to that of cell density, with the maximum of the chlorophyll per cell overlapping with the minimum of cell density under 35°C (Fig. 6B, E).

The gradually reduced cycling pattern of the chlorophyll per cell and cell density during 4-day of 35°C may be related to the heat effects on circadian clock (Fig. 6B, E). High temperatures impact the circadian clock in plants (Kusakina et al., 2014; Gil and Park, 2019; Mody et al., 2020). Light and temperatures are the two strongest environmental signals that can entrain the plant biological clock (the period of the clock is synchronized to the period of the entraining signal); however, the mechanisms of temperature entrainment is much less understood as compared to light entrainment (Gil and Park, 2019). How temperatures affect circadian clock in algal cells is largely underexplored but an RNA-binding protein CHLAMY1 was reported to be involved in the regulation and temperature entrainment of circadian clock in Chlamydomonas (Iliev et al., 2006; Voytsekh et al., 2008). The gradually diminished cycling pattern of the chlorophyll per cell and cell density was probably because the onset of heat at 35°C provided a signal to entrain the circadian clock of the asynchronized pre-heat cultures, but the entrainment became less effective over time under the constant 35°C which lacked a circadian rhythm of heat treatments.

Heat of 40°C was detrimental to algal cells even with constant acetate supply

Unlike the transient effects of moderate high temperature at 35°C, acute high temperatures at or above 40°C inhibit the algal cell cycle (Mühlhaus et al., 2011b; Hemme et al., 2014; Zachleder et al., 2019; Ivanov et al., 2021/5), alter thylakoid membranes, reduce photosynthesis, and damage respiration (Zhang et al., 2022a) (Fig. 8). These cellular damages took place quickly, within 4 h of heat at 40°C with constant acetate supply (Zhang et al., 2022a). However, 40°C-treated cells grew bigger and gained more biomass with constant acetate supply than without in the short-term (24-h heat) (Fig. 4B, G, D, I, L, N), although the constant acetate supply could not prevent ultimate cell death with 40°C-heat longer than 2-days (Fig. 6). The death of Chlamydomonas cultures was also reported at 39°C for 33 h in photoautotrophic medium without acetate (Zachleder et al., 2019; Ivanov et al., 2021/5). Thus, constant heating at 40°C is lethal for Chlamydomonas cells independent of carbon availability.

Transcripts/proteins of many genes involved in carbon metabolism, e.g., acetate uptake/assimilation, glyoxylate cycle and gluconeogenesis pathways, were significantly down-regulated during 24-h heat of 40°C (Fig. 2F, G) (Zhang et al., 2022a). Our previous results showed that (Zhang et al., 2022a): (1) mitochondrial activities, measured by respiration rates, were particularly sensitive to 40°C heat, reduced to 50% of the pre-heat level with 4 h of heat at 40°C; (2) a large fraction of transcripts related to mitochondrial electron transport was down-regulated with just 30 min heat at 40°C. Most of the reducing power from acetate assimilation is used in mitochondrial respiration to produce ATP (Johnson and Alric, 2012, 2013). Acute high temperatures at or around 40°C cause starch over-accumulation (Zachleder et al., 2019; Zhang et al., 2022a; Ivanov et al., 2021/5). Starch accumulation could be an electron sink to alleviate the over-reduced electron transport chain in chloroplasts due to damaged photosynthesis during 40°C treatment (Hemme et al., 2014). The compromised mitochondrial activities and the over-accumulated starch may restrict acetate uptake/assimilation and reduce photosynthesis further during 40°C heat treatment. The acetate uptake was slightly lower in 40°C-treated cultures than 25°C (Fig. 1D), which, together with reduced photosynthesis and respiration (Zhang et al., 2022a), may contribute to the significantly reduced algal dry biomass in cultures treated with 8h-heat of 40°C than 25°C (Fig. 1C). By the end of 24-h treatment without constant nutrient supply, there were comparable amount of dry biomass in cultures treated with 40°C and 25°C (Fig. 1C), which could be explained by the arrested cell cycle and almost eliminated energy/biomass consumption for cell division during 40°C. Such an inefficient system with reduced carbon input and little energy output is not sustainable, eventually killing all the algal cells by the end of 2-day heat of 40°C, even with constant acetate supply (Fig. 6). As compared to the increased growth rates at 35°C with constant acetate supply, the culture death during 2-day heat of 40°C may highlight the importance of active carbon metabolisms in thermotolerance.

In summary, by using highly controlled cultivation systems and the model green alga Chlamydomonas, we revealed how the availability of organic carbon source interacted with different intensities and duration of high temperatures in photosynthetic cells. Our research revealed the overlooked effects of moderate high temperature of 35°C, which can be beneficial with constant carbon supply or detrimental with insufficient carbon supply. Our results also showed that the damaging effects of acute high temperature of 40°C is dominant and independent of carbon availability. Our research not only helps us understand heat responses in photosynthetic cells but also provides insights for high temperature effects on the production of algal biofuel/bioproducts.

Supplementary data

Table S1: Gene IDs for RT-qPCR analysis and transcript/protein heatmaps.

Author contributions

RZ designed the experiments. MX and CB operated and maintained the algal growth in photobioreactors. MX, NZ, and CB harvested algal samples at different time points and quantified biomass. NZ and MX performed acetate quantification assay. NZ performed RT-qPCR analysis and light microscope imaging. MX and CB quantified cell density, cell size, and chlorophyll contents, and recorded OD608/720 readings. MX and CB performed 4-day heating experiments and quantified growth rates as well as cell parameters. EMM generated the heatmaps from the published RNA-seq data. NZ, MX, CB, RZ, and EMM prepared the figures. RZ wrote the manuscript. RZ, EMM, and CB revised the manuscript.

Conflict of interest

No conflict of interest declared.

Funding statement

This work was supported by the start-up funding from the Donald Danforth Plant Science Center (DDPSC) and the DOE Biological & Environmental Research (BER) grant (DE- SC0020400) to RZ. EMM was supported by the William H. Danforth Fellowship in Plant Sciences and the DDPSC start-up funding (to RZ).

Data availability

All data supporting the findings of this study are available within the paper and within its supplementary materials published online.

Acknowledgements

We would like to thank Dr. Michael Schroda for discussing the initial experimental idea under acetate depleting conditions and Dr. Trent Northen for recommending the acetate quantification assay.

Footnotes

  • Ningning Zhang: lucy.nnzhang{at}gmail.com

  • Catherine E. Bailey: cbailey{at}danforthcenter.org

  • Erin M. Mattoon: EMattoon{at}danforthcenter.org

Abbreviations

PBRs
Photobioreactors
TAP
Tris-acetate-phosphate medium
TP
Tris-phosphate medium

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Posted December 07, 2022.
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Moderate High Temperature is Beneficial or Detrimental Depending on Carbon Availability in the Green Alga Chlamydomonas reinhardtii
Ningning Zhang, Ming Xia, Catherine E. Bailey, Erin M. Mattoon, Ru Zhang
bioRxiv 2022.12.04.519034; doi: https://doi.org/10.1101/2022.12.04.519034
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