Materials and Methods

C. reinhardtii cultivation and transformation

C. reinhardtii strain CC-1690 (wild-type strain; WT) was purchased from the Chlamydomonas Resource Center (The University of Minnesota, USA). Growth of C. reinhardtii was performed under mixotrophic conditions with Tris-acetate-phosphate (TAP) medium (Gorman et Levine, 1965) on agar plates or liquid in 6 well plates, 96 well plates or 125 mL shake flasks under 50±10 µmol photons m−2 s−1 light intensity and a photoperiod of 16h light:8h dark cycles at 21±0.5oC, and 130 rpm agitation for liquid cultures.
Nuclear transformation was carried out by electroporation following GeneArtTM Chlamydomonas Protein Expression Vector protocol (Invitrogen, Life technologies, Thermo Fisher Scientifics) using 0.5 µg of linearized pOpt_mVenus_Paro vector (Laursen et al., 2015) by restriction endonucleases Xba I andKpn I. Transformants were selected on TAP agar plates supplemented with paromomycin (10 mg. L−1) for 5-7 days.

Treatment with histone deacetylase inhibitors

For all assays, C. reinhardtii WT and transformed colonies were initially transferred from agar plates to a liquid preculture in 6 well plates for 4-6 days, under culture conditions mentioned in section 2.1. Inoculums of 105 cells. mL-1 in late exponential phase of growth were used in this study.
For initial experiments, 200 µL of C. reinhardtii cells grown to exponential phase were transferred into a 96-well plate (flat bottom; Corning) and incubated for 24 hours with individual HDACi, 50 µM sirtinol, 2.5 µM SAHA, 100 µM OSS-128167, 1 mM nicotinamide (all from MedChem Express) and 5 mM sodium pyruvate (Fishers Bioreagents). A mix of all inhibitors was also tested. As a negative control, samples were incubated with DMSO.
SAHA analogs and other HDACi such as belinostat, dacinostat, panobinostat, mocetinostat, entinostat, and romidepsin (all purchased from MedChem Express) were also tested. Culture inoculums were transferred in 200 µL of TAP medium in 96 well plates, with 5 µM of each HDACi followed over a 6 days growth period. Other experiments were done to follow the impact of SAHA on growth and fluorescence levels, inoculum was transferred into 50 mL of TAP media in 125 mL shaking flasks and treated with SAHA at final concentrations of 2.5, 5 and 10 µM for 6 to 12 days.

Growth curve, chlorophyll and mVenus fluorescence detection using plate reader

Growth curves were obtained using a Synergy H1 plate reader (Biotek, Agilent), from three biological replicates for each C.reinhardtii strain studied as in (Molino et al., 2022). For SAHA treatments, 250 µL of C. reinhardtii cells from 50 mL cultures were transferred, in triplicate, into 96 well flat bottom microplates (Corning Costar 96-Well, Cell Culture-Treated, Fisher Scientifics). Microalgae growth was done by tracking the optical density at 750 nm (OD750, 750/8 nm). Area scan mode was used to measure the mean fluorescence intensity of mVenus (excitation 500/18 nm, emission 541/18 nm) and chlorophyll (excitation 475/18 nm, emission 650/18 nm). C. reinhardtii WT was used as negative control for mVenus fluorescence. Fluorescence intensity of mVenus was normalized to the OD750.

Flow cytometry

A Cytomics FC500 cytometer equipped with Argon (488 nm) and HeNe (633 nm) lasers were used to measure mVenus emission on the FL1 channel (525/15 nm), and chloroplast fluorescence on the FL4 channel (675/15 nm) (Beckman Coulter Life Sciences). At least 10,000 events were acquired. A homogeneous cloud of intact cells was first gated based on the size (forward scatter, FSC) and the granulosity (side scatter, SSC). Then a daughter gate was selected on the cloud of cells with homogenous chlorophyll autofluorescence. The percentage (%) of mVenus+ cells was measured on this daughter population, with a gate in the FL1 channel that (filter at 525/10 nm) excluded cells with autofluorescence levels similar to the negative controls. Cells were considered mVenus+ when both the % of gated mVenus+ cells (and >0.1%) and mVenus mean fluorescence intensity (MFI) were higher than the values of the wildtype (WT) cells used as a control. Propidium iodide (Thermo Fisher, 7 µM) was used to verify viability (Cheloni et al., 2014) and acquired on the FL3 channel (620/20 nm). To select palmelloid cells, a second gate that included events with a minimum of 2-fold increase in FSC mean intensity was drawn, similarly to (Cheloni and Slaveykova, 2021). In that case, cells were acquired on a Beckman Cytoflex S equipped with violet (405nm), blue (488nm), yellow-green (561 nm) and red (638 nm) lasers. Chlorophyll autofluorescence was detected in the PerCP channel (690/50 nm), mVenus on the FITC (525/40 BP). Statistics were obtained using BD FlowJo version 10 software (BD Biosciences, La Jolla, CA, USA, 2020).

DNA extraction and qPCR for transgene relative quantity

Genomic DNA (gDNA) was extracted using the protocol from theChlamydomonas Resource Center (The University of Minnesota, USA) with minor modifications. Two mL of a 6 days C. reinhardtii cell cultures were harvested and centrifuged in 1.7 tubes for 5 min at 4000 g. The cell pellet was resuspended in 500 µL of CTAB-buffer (2% (w/v) CTAB, 100 mM Tris-HCl pH 8.0, 1,4 M NaCl, 20 mM EDTA pH 8.0 and 2% (v/v) 2-mercaptoethanol) and incubated at 65ºC for 1h. The DNA was extracted with 500 µL of chloroform/isoamyl alcohol (24:1). The upper phase was transferred and 0.7 volumes of isopropanol was added for 15 min at 4ºC. The DNA was spin down at 4ºC for 20 min at 12000 rpm. The pellet was washed twice with 1 mL of cold 70% ethanol and centrifuged at 4ºC for 5 min at 12000 rpm. The supernatant was discarded and the pellet was air dry under the hood until the pellet was completely dried. The gDNA was dissolved in 50 µL of TE-buffer (1 mM Tris-HCl pH 8.0 and 0,1 mM EDTA pH 8.0). gDNA yield and quality were determined by measuring the 260/280 ratios using a Nanodrop instrument (Thermo Fisher Scientific).
gDNA samples were subjected to qPCR amplification using Luna® Universal qPCR Master Mix (New England Biolabs). Briefly, 250 ng of gDNA were used as template in 20 μL reactions according to manufacturer instructions. Initial denaturation was 2 min at 95°C followed by 45 cycles of denaturation for 15 seconds at 95°C and extension for 30 seconds at 60°C. Primers used to detect mVenus and histone 3(h3 ) (reference gene) were designed using PrimerQuestTM Tool (Integrated DNA Technologies, IDT) (primer sequences are listed in Table S1). The oligonucleotides were validated by performing a standard curve and through dissociation curves analysis (60-95°C for the melt curves). SYBR Green fluorescence was recorded in the FAM channel of a CFX connect real time system (Bio-Rad). PCR runs were analyzed with CFX Manager software (Bio-Rad). pOpt_mVenus_Paro was used to determine the copy numbers. Dilutions ranging from 5 to 1000 pg of vector were used to perform the standard curve. Plasmid copy number was estimated following this formula:
(pOpt_mVenus (ng) * 6.0221.1023) / (pOpt_mVenus (bp) * 660 *1.109)
(Integrated DNA Technologies, https://www.idtdna.com/pages/education/decoded/ article/calculations-converting-from-nanograms-to-copy-number).
Relative mVenus gene copy numbers for each strain were determined using the equation of the standard curve log(y) = ax + b; where y = plasmid copy number and x= Ct. Each sample copy number was then determined with the formula 10((Ctsample−b)/a), as in (Masroori et al., 2016), normalized to the gDNA weight (ng). Relative mVenus gene detection was also calculated, using h3 as an endogenous control (Veillette et al., 2013). Experiments were performed with three biological replicates.

RNA extraction and RT-qPCR for mRNA relative quantification

Four mL of a 6 days C. reinhardtii grown culture were harvested and centrifuged in 1.7 mL tube for 5 min at 4000 g. Cells were lysed by immersion in liquid nitrogen for 1 min. Total RNA was extracted using InvitrogenTM TRIzolTM reagent (Life technologies, Thermo Fisher Scientifics) according to the manufacturer protocol with minor modifications, including the addition of NaCl (final concentration of 100 mM) in isopropanol to improve nucleic acids precipitation. Samples were further treated with Turbo DNase (Invitrogen™ TURBO DNA-free ™ Kit, Thermo Fisher Scientifics) according to the manufacturer’s instructions. RNA yield and quality were determined by measuring the 260/280 ratios using a Nanodrop instrument.
Samples of 100 ng total RNA were subjected to reverse transcription and qPCR amplification in a single reaction using the Luna® Universal One-Step RT-qPCR Kit Protocol (New England Biolabs). Briefly, 2 μL of RNA were subjected to reverse transcription performed at 55°C for 10 minutes. Initial denaturation was 1 min at 95°C followed by 45 cycles of denaturation for 10 seconds at 95°C and extension for 30 seconds at 60°C. A melt curve analysis was performed from 60-95°C with an increment of 0.5°C each 5 seconds. Primers used for mVenus transcript and h3 as a housekeeping gene were designed using PrimerQuestTM Tool (IDT) (primer sequences are listed in Table S1). SYBR Green fluorescence was recorded in the FAM channel of a CFX connect real time system. PCR runs were analyzed with Bio-Rad CFX Manager version 3.1 software. Relative mRNA expression levels were determined according to the 2(-ΔΔCt)method (Livak et Schmittegen, 2001). Experiments were performed using three technical replicates.

Protein extraction and western-blot

Twenty-five mL of a 6 days C. reinhardtii cell cultures were harvested and centrifuged in 50 mL tube at 4000 g for 10 min at 4°C. The pellets were washed once with ice-cold PBS 1X supplemented with 5 mM sodium butyrate, to retain levels of histone acetylation. Then, pellets were weighed and resuspended with a ratio 0.5 g FW mL-1 in Triton Extraction Buffer (TEB: PBS 1X containing 0.5% Triton X 100 (v/v), 0.02% (w/v) NaN3). PMSF, final concentration 2 mM, and protease inhibitor (32 µL g FW-1) were subsequently added (both from Thermo Fisher Scientific). Sonication was performed 6 times at 35% amplitude, 30 sec on, 30 sec off for 3 min total using FisherbrandTMModel 505 Sonic Dismembrator (Thermo Fisher Scientific). Protein extracts were centrifuged at 14000 g for 30 min at 4°C. Supernatants containing the total soluble protein fractions were kept at -80°C to be used for western blot. Proteins were quantified with the RC DC™ Protein Assay Kit I (Bio-Rad).
To detect mVenus and Histone H3 Lysine 9 acetylated (H3K9ac), 50 µg and 25 µg of total proteins were loaded respectively, in 15% SDS-PAGEs. Purified mVenus protein from colony 21 using GFP trap agarose (Chromotek, Germany) was loaded as a positive control. Proteins were then transferred to the 0.2 µm PVDF membrane (settings: 1 mA constant and 25 V for 30 min). Primary antibodies for mVenus (27 kDa) and H3K9ac (15.4 kDa) detection were purchased from Cedarlane and from Agrisera, respectively. Both antibodies were diluted at 1:1000 in 3% BSA and were incubated overnight at 4°C. Actin (40 kDa) and histone 3 (H3, 15.4 kDa) detection was performed as internal standards of cytosolic and nuclear proteins, respectively, respectively. Blots were incubated with anti-actin-HRP solution, 1:10,000 in BSA 3%, from Abcam (Cambridge) and anti-H3 (1:1,000 in BSA 3%, Agrisera). After three washes with Tris-buffered saline, 0.1% Tween 20 (TBST) solution, blots were incubated for 1 hour in a 1:20,000 dilution, in 5% milk, of Immun-Star Goat Anti-Mouse (GAM)-HRP Conjugate from Bio-Rad to detect mVenus, and Immun-Star Goat Anti-Rabbit (GAR)-HRP Conjugate from Bio-Rad to detect H3K9ac. After three washes of the membrane with TBST solution, protein detection was revealed using Clarity Max Western ECL Substrate-Luminol solution from Bio-Rad. Chemiluminescence detection and Ponceau S stained (Glacial Acetic Acid 5% v/v, Ponceau Red dye 0.1% m/v) of blots were visualized using ChemiDoc Imaging System with Image Lab Touch Software (Bio-Rad) and Image Lab™ Software (Bio-Rad). The molecular weight of the protein corresponding to the detected band was confirmed with the protein marker (Precision Plus Protein Dual Color Standards #1610374).

Statistical analysis

Statistical analyses were performed using GraphPad Prism (Version 9.4.1, GraphPad Software, US). Data are expressed as means ± SD of three biological replicated performed at least twice in independent experiments. Parametric tests (ANOVA and Student’s t test) were used when population followed normal distribution, and non-parametric (ANOVA and Mann–Whitney test) were used when the population could not be assumed to be normally distributed. A p value < 0.05 was considered to be significant.