Statistics
Statistical methods were not used to predetermine the sample size. The experiments were not randomized, and the investigators were not blinded to allocation during experimental procedures and data assessment.
Algal Material
The strains used in this study included Chlamydomonas phot (defective in PHOT; gene ID: Cre03.g199000) and phot-C1 (phot strain complemented with WT PHOT gene), as well as their background strain CC-125, which have been previously described25. Additionally, Chlamydomonas acry24 (defective in animal-type cryptochrome, aka aCRY; gene ID: Cre06.g278251), pcry (defective in plant-type cryptochrome, aka pCRY; gene ID: Cre06.g295200), and acrypcry (defective in both animal-type and plant-type cryptochrome) were generated through CRISPR-CAS9 provided by following the protocol described in ref. 24. The pmsk1 (defective in phototropin-mediated signaling kinase 1, aka PMSK1; gene ID: Cre16.g659400) and photpmsk1 (defective in both PHOT and PMSK1) mutants were generated using insertional CRISPR-Cas9 RNP method described by Kim et al.41 with a few modifications. The target sgRNA sequence of PMSK1 was designed by Cas-Designer (http://www.rgenome.net/cas-designer) and selected considering the recommendation guideline. To induce early termination of translation, the sgRNA targets were selected in exon 2 (Supplementary Data 1). To form an RNP complex in vitro, 100 μg of purified Cas9 protein (Cas9 expression plasmid: Plasmid #62934, addgene, US) and 70 μg of sgRNA synthesized by using GeneArt™ Precision gRNA Synthesis Kit (ThermoFisher, US), were mixed gently. For efficient and fast screening, 0.5 μg of paromomycin-resistance gene cassette was co-transformed with RNP complex. The Chlamydomonas cell wall was permeabilized by treatment of Max Efficiency buffer (ThermoFisher, US) following the manufacturer’s protocol. The Chlamydomonas transformation was performed in the 4 mm gap electroporating cuvette by electroporation with the specific parameter (600 V, 50 μF, 200 Ω). One day after transformation, cells were plated on TAP medium containing 1.5% agar and paromomycin (25 μg/ml). Once colonies appear after transformation, genomic DNA PCR and Sanger sequencing were performed to validate knockout events.
To prepare transgenic lines with a knockdown of GAP1, pChlamiRNA3int-GAP1 was transformed into the phot strain. Generation of amiRNA plasmids was performed according to42. The oligonucleotides designed for targeting GAP1 (Data S1) using the WEB MicroRNA Designer platform (WMD3: http://wmd3.weigelworld.org/cgi-bin/webapp.cgi. Ossowski Stephan, Fitz Joffrey, Schwab Rebecca, Riester Markus and Weigel Detlef, personal communication) were annealed and ligated into pChlamiRNA3int (SpeI digested) to create pChlamiRNA3int-gap1. Transformed cells were selected and further checked by RT-qPCR.
For the preparation of transgenic lines overexpressing GAP1(Glyceraldehyde 3-phosphate dehydrogenase, aka GAP1; gene ID: Cre12.g485150) or different versions of PMSK1, the genomic sequences of GAP1 and PMSK1 were PCR amplified from genomic DNA of Chlamydomonas CC-125 and cloned into pLM005 in-frame with a C-terminal Venus-3Xflag using Gibson Assembly43, and then transformed into WT or phot strains. The primers used for different gene amplification and point mutations are described in Supplementary Data 1. All Chlamydomonas reinhardtii strains used in this study are listed in Supplementary Data 4.
Chlamydomonas reinhardtii cultivation
All Chlamydomonas strains were maintained on solid Tris-acetate-phosphate (TAP)44 agar plates with or without appropriate antibiotic at 22 °C and 5 µmol photons m−2 s−1. Prior to the start of the experiments, cells were cultured in 50 mL TAP medium in 250 ml Erlenmeyer flasks at 23 °C, 120 rpm/min and 15 µmol photons m−2 s−1. The experiments were conducted in Sueoka’s high salt medium (HSM)45 at an initial cell density of 1 million cells/ml at 50 µmol photons m−2 s−1 unless otherwise stated.
For continuous light experiments, the cells were transferred to HSM medium and grew at 23 °C, 120 rpm/min, and 50 µmol photons m−2 s−1.
For synchronized experiments, cells were grown in HSM for at least 5 days under a 12 h light/12 h dark cycle under white light or different light qualities (light intensity was set at 50 µmol photons m−2 s−1; temperature was 18 °C in the dark and 23 °C in the light). The light spectrum of the LED lighting system used in this study is the same as previously described46.
Transformation of Chlamydomonas reinhardtii
The transformation was performed by electroporation, which follows the protocol of Zhang et al.47 with minor modification. Cells for transformation were collected at 1–2 h before the end of the light phase in a synchronized (12 h light/12 h dark) culture. For three reactions 11 ng/kb linearized plasmid was mixed with 400 μl of 1.0 × 107 Chlamydomonas reinhardtii cells/ml and electroporated at a volume of 125 ml in a 2-mm-gap electro cuvette using a NEPA21 square-pulse electroporator, using two poring pulses of 250 and 150 V for 8 ms each, and five transfer pulses of 50 ms each starting at 20 V with a “decay rate” of 40% (i.e., successive pulses of 20, 12, 7.2, 4.3, and 2.6 V). Electroporated cells were immediately transferred to a 15 ml centrifugation tube containing 9 ml TAP plus 40 mM sucrose. After overnight dark incubation, cells were collected by centrifugation and spread on TAP agar plates which contain the appropriate antibiotic (20 μg/ml paromomycin or 7.5 μg/ml zeocin or 20 μg/ml hygromycin B). Transformants typically appear after 5–7 days.
The putative antibiotic-resistant transformants were transferred into individual wells of a 96-well, flat-bottom transparent microplate, with each well containing 250 μl of TAP medium. Cultures were grown for 3 days under 15 µmol photons m−2 s−1 light without shaking, refreshed by replacing half of the culture with fresh medium, and allowed to grow for an additional day. Transformants were screened for Venus expression using a fluorescent microplate reader (Tecan Group Ltd, Switzerland), with parameters including Venus (excitation 515/12 nm and emission 550/12 nm) and chlorophyll (excitation 440/9 nm and emission 680/20 nm). The fluorescence signal was normalized to the chlorophyll fluorescence signal, and colonies with a high Venus/chlorophyll value were selected as putative complemented strains. These putative positive transformants were further validated by western blotting and RT-qPCR.
Cell counting
Cell concentration was determined using either a hemocytometer or an automated cell counter (Countess II FL, Thermo Fisher, US). For the hemocytometer method, a 10 µL aliquot was mixed with 5% acetate and then loaded onto the hemocytometer, where cells were counted manually under a light microscope in four 1 mm2 squares. For automated counting, a 10 µL aliquot was similarly mixed with 5% acetate and loaded into a disposable chamber for analysis using the automated cell counter. Both methods were performed in triplicate, and the mean values were subsequently reported.
RNA extractions and RT-qPCR analysis
Total RNA for RNA-seq and RT-qPCR was extracted using RNeasy Mini Kit (Qiagen, Germany) and treated with the RNase-Free DNase Set (Qiagen, Germany). One microgram total RNA was reverse transcribed with oligo dT using Sensifast cDNA Synthesis kit (Meridian Bioscience, US). qPCR reactions were performed and quantitated in a Bio-Rad CFX96 system using SsoAdvanced Universal SYBR Green Supermix (BioRad, US). The CBLP gene48 served as the housekeeping control and relative fold differences were calculated on the basis of the ΔCt method (2−(Ct target gene − Ct CBPL)49,50,51. All primers used for the RT-qPCR analyses were synthesized by ThermoFisher (US) or IDT (Integrated DNA Technologies, Inc. Coralville, Iowa, US) and were presented in Supplementary Data 1.
Analyses of total starch content
The total starch content of samples collected daily was determined using Total Starch Assay Kit (K-TSTA-100A, Megazyme, Ireland) as described in its instruction with modifications. The results were calculated according to the standard curve made with glucose solution after starch digestion. To prepare the samples for glucose determination, 10 mL of the liquid culture was pelleted by centrifugation and resuspended in 40 µL of 80% (v/v) ethanol. Next, 400 µL of cold 1.7 M sodium hydroxide solution was added, and the samples were incubated on ice for 15 min. Following this, 1.6 mL of sodium acetate buffer containing calcium chloride (5 mM) was added and mixed well. Subsequently, 20 µL of α-amylase and 20 µL of amyloglucosidase were added, and the samples were incubated at 50 °C for 30 min. The supernatant was collected by centrifugation at 23,000 × g for 5 min and will be ready for glucose determination.
Analyses of Total Protein and Lipid content
The total protein content of samples collected daily was determined using BCA Protein Assay Kit (ThermoFisher, US) with the standardized protocol. The total lipid content of samples collected daily was determined using sulfo-phospho-vanillin (SPV) method52.
Transmission electron microscopy
Cells were harvested by centrifugation at 700 × g for 5 min, washed two times in 0.1 M PB (phosphate buffer, pH 7.4), and then were fixed in 0.1 M PB containing 2.5% (v/v) glutaraldehyde for 2 h at room temperature and stored overnight at 4 °C. The cells were then washed five times in 0.1 M PB before being fixed by a 1 h incubation on ice in 0.1 M PB containing 2% osmium and 1.5% ferricyanide potassium. After being washed five times with 0.1 M PB, the samples were resuspended in 0.1 M PB containing 0.1% (v/v) tannic acid and incubated for 30 min in the dark at room temperature. The cells were washed five times with 0.1 M PB, dehydrated in ascending sequences of ethanol, infiltrated with an ethanol/Epon resin mixture, and finally embedded in Epon. Ultrathin sections (50–70 nm) were prepared with a diamond knife on a PowerTome ultramicrotome (RMC Boeckeler, US) and collected on 200 μm nickel grids. The ultrathin sections were examined on a Philips CM120 transmission electron microscope operating at 80 kV.
Confocal microscopy
The preparation of samples for confocal microscopy followed the protocol reported by Mackinder et al.53. The confocal microscope used in the study was from the cell-imaging platform at IBS, Grenoble, France. All confocal microscopy images were analyzed using Fiji54.
Immunoblotting
Protein samples of whole cell extracts (5 μg protein) were loaded on 4–20% SDS-PAGE gels (Mini-PROTEAN TGX Precast Protein Gels, Bio-Rad, US) and blotted onto nitrocellulose membranes. Antisera against ATPB (AS05085, 1:10,000, https://www.agrisera.com/en/artiklar/atpb-beta-subunit-of-atp-synthase.html) and FLAG (AS20 4442, 1:5000, https://www.agrisera.com/en/artiklar/dykddddk-binds-to-sigma-flag-polyclonal.html) were from Agrisera (Sweden); antiserum PHOT (LOV1 domain, 1:5000) was previously described (Fig. 2g in ref. 55). ATPB was used as a loading control. The anti-rabbit horseradish peroxidase–conjugated antiserum (Jackson Immuno Research, US) was used for detection at 1:10,000 dilution. The blots were developed with ECL detection reagent, and images of the blots were obtained using ImageQuant 800 (Cytiva, UK). For the densitometric quantification, data were normalized with ATPB.
Phos-tag gel electrophoresis
Double-layer Phos-tag gels with a concentration of 12% (w/v) acrylamide/bisacrylamide 37.5:1 and 65 mM of Phos-Tag (Wako Pure, US) were prepared as in ref. 56, with the exception that Zn(NO3)2 was added equimolarly to the samples to compensate for the absence of EDTA in the lysis buffer. The gels were denatured for 30 min at 37 °C prior to loading. In vitro dephosphorylation involved resuspending a cell pellet in 5 mM of HEPES at pH 7.5, 10 mM of EDTA, and 1% (v/v) TritonX 100. An aliquot containing 10 mg of protein was then subjected to lambda protein phosphatase reaction mix following the manufacturer’s instructions (New England Biolabs, US) for 1 h at 30 °C, in accordance to ref. 13.
Fluorescence-based measurements
Fluorescence-based photosynthetic parameters were measured with a pulse-modulated amplitude fluorimeter (MAXI-IMAGING-PAM, HeinzWaltz GmbH, Germany). Prior to the onset of the measurements, cells were acclimated to darkness for 15 min. Chlorophyll fluorescence was recorded under different intensities of actinic light; starting with measurements in the dark (indicated as D below the x-axis of the graphs), followed by measurements at 21 μmol photons m−2 s−1 (indicated as L1 below the x-axis of the graphs) and 336 μmol photons m−2 s−1 (indicated as L2 below the x-axis of the graphs) and finishing with measurements of fluorescence relaxation in the dark. The effective photochemical quantum yield of photosystem II was calculated as Y(II) = (Fm′ − F)/Fm′; F and Fm′ are the fluorescence yield in steady state light and after a saturating pulse in the actinic light, respectively.
Phosphoproteomics analysis
Protein extraction: C. reinhardtii pellets were resuspended in 2000 µL of lysis buffer (100 mM Tris-HCl, PhosphoSTOP inhibitors, protease inhibitors) and ultrasonicated in the Covaris for 4 min each. Samples were diluted by adding 2000 µL of dilution buffer (100 mM Tris-HCl, 5 mM TCEP, 30 mM chloroacetamide, 1 mM sodium orthovanadate, phosphoSTOP inhibitors, 1 mM magnesium chloride) and 1 µL Benzonase. Lysates were shaken at 25 °C for 1 h. 8 mL of methanol was added to each sample, followed by 3 mL chloroform and 3 mL water, with vortexing after each subsequent addition. Samples were centrifuged for 10 min at 3220 × g and the top layer was removed, leaving the interphase intact. An additional 10 mL of methanol was added and the samples were centrifuged for 20 min at 3220 × g and the supernatant was removed. Protein pellets were allowed to dry at RT and resuspended in 1 mL of digestion buffer (100 mM Tris-HCl, 2 M urea). Proteins were digested with 12 µg of trypsin overnight and cleaned up via C18 SPE cartridges. Samples were resuspended in 250 µL of water and a BCA assay was performed to determine peptide concentration. Ten micrograms of digested protein was taken for global analysis, and 500 µg was used for phosphopeptide enrichment.
Phosphopeptide enrichment: Phosphopeptides were enriched using a ProPac Fe-IMAC column (ThermoFisher, US) on a Shimadzu Prominence HPLC system. Before enrichment, the column was charged with 25 mM FeCl3 in 100 mM acetic acid. Mobile phase A consisted of 30% acetonitrile in water (v/v) with 0.07% trifluoroacetic acid (v/v). Mobile phase B consisted of 0.3% ammonium hydroxide in water (v/v). Tryptic peptides were diluted to 30% acetonitrile and injected on the column at a flow rate of 0.2 ml/min. After 3 min of loading, flow rate was increased to 2 mL/min. Peptides were eluted by rapidly ramping the gradient to 50% B. Fractions containing phosphopeptides were cleaned up with C18 SPE cartridges (Waters, US) and resuspended in 20 µL of LC-MS water prior to mass spectrometry analysis.
LC-MS analysis: Phosphopeptide samples were analyzed using a nanoACQUITY UPLC (Waters, US) coupled to a TripleTOF 5600 mass spectrometer (Sciex, Canada). Mobile phase A consisted of water with 0.1% formic acid and mobile phase B was acetonitrile with 0.1% formic acid. Injections were made to a Symmetry C18 trap column (100 Å, 5 µm, 180 µm × 20 mm; Waters, US) with a flow rate of 5 µL/min for 3 min using 99% A and 1% B. Peptides were then separated on an HSS T3 C18 column (100 Å, 1.8 µm, 75 µm × 250 mm; Waters, US) using a linear gradient of increasing mobile phase B at a flow rate of 300 nL/min. Mobile phase B increased from 5% to 40% in 90 min before ramping to 85% in 5 min, where it was held for 5 min before returning to 5% in 2 min and re-equilibrating for 13 min.
The mass spectrometer was operated in positive polarity mode. MS survey scans were accumulated across an m/z range of 350–1600 in 250 ms optimized at ≥30,000 resolution. For data-dependent acquisition, the mass spectrometer was set to automatically switch between MS and MS/MS experiments for the first 20 features above 150 counts having +2 to +5 charge state. Precursor ions were fragmented using rolling collision energy and accumulated in high sensitivity mode for 85 ms across an m/z range of 100–1800 optimized at ≥30,000 resolution. Dynamic exclusion for precursor m/z was set to 8 s.
Bioinformatic analysis: Raw data files were imported into Progenesis for peak alignment and quantification. Spectra were searched in Mascot against the C. reinhardtii phytozome database (v6.1) using a precursor/fragment tolerance of 15 ppm/0.1 Da, trypsin specificity, two possible missed cleavages, fixed modification cysteine carbamidomethylation, and variable modifications of methionine oxidation, protein N-term acetylation, and phosphorylation (STY). Identifications were imported back into Progenesis for peak assignment, and statistical analysis was performed using the QuantifyR workflow, which can be found on the Hicks Lab Github (github.com/hickslab/QuantifyR). Phosphoproteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository57 with the dataset identifier PXD045599.
Lipidomics
Glycerolipids were extracted from freeze-dried cell pellets frozen immediately in liquid nitrogen after harvesting. Once freeze-dried, cell pellets were resuspended in 4 mL of boiling ethanol for 5 min to prevent lipid degradation and lipids were extracted according to58 by addition of 2 mL methanol and 8 mL chloroform at room temperature. The mixture was then saturated with argon and stirred for 1 h at room temperature. After filtration through glass wool, cell remains were rinsed with 3 mL chloroform/methanol 2:1, v/v, and 5 mL of NaCl 1% were then added to the filtrate to initiate biphase formation. The chloroform phase was dried under argon before solubilizing the lipid extract in pure chloroform. Total glycerolipids were quantified from their fatty acids: in an aliquot fraction, a known quantity of 15:0 was added and the fatty acids present were transformed as methyl esters (FAME) by a 1-h incubation in 3 mL 2.5% H2SO4 in pure methanol at 100 °C59. The reaction was stopped by addition of 3 mL water and 3 mL hexane. The hexane phase was analyzed by gas chromatography-flame ionization detector (GC-FID) (Perkin Elmer, US) on a BPX70 (SGE; Trajan Scientific and Medical location, Australia) column. FAME were identified by comparison of their retention times with those of standards (Sigma, US) and quantified by the surface peak method using 15:0 for calibration.
The lipid extracts corresponding to 25 nmol of total fatty acids were dissolved in 100 µL of chloroform/methanol [2/1, (v/v)] containing 125 pmol of each internal standard. Internal standards used were PE 18:0-18:0 and DAG 18:0-22:6 from Avanti Polar Lipid and SQDG 16:0–18:0 extracted from spinach thylakoid60 and hydrogenated as described in ref. 61. Lipids were then separated by HPLC and quantified by MS/MS.
The HPLC separation method was adapted from ref. 62. Lipid classes were separated using an Agilent 1200 HPLC system using a 150 × 3 mm (length × internal diameter) 5 µm diol column (Macherey-Nagel, Germany), at 40 °C. The mobile phases consisted of hexane/isopropanol/water/ammonium acetate 1 M, pH5.3 [625/350/24/1, (v/v/v/v)] (A) and isopropanol/water/ammonium acetate 1 M, pH5.3 [850/149/1, (v/v/v)] (B). The injection volume was 20 µL. After 5 min, the percentage of B was increased linearly from 0% to 100% in 30 min and stayed at 100% for 15 min. This elution sequence was followed by a return to 100% A in 5 min and an equilibration for 20 min with 100% A before the next injection, leading to a total runtime of 70 min. The flow rate of the mobile phase was 200 µL/min. The distinct glycerophospholipid classes were eluted successively as a function of the polar head group.
Mass spectrometric analysis was done on a 6470 triple quadrupole mass spectrometer (Agilent, US) equipped with a Jet stream electrospray ion source under following settings: Drying gas heater: 230 °C, Drying gas flow 10 L/min, Sheath gas heater: 200 °C, Sheath gas flow: 10 L/min, Nebulizer pressure: 25 psi, Capillary voltage: ±4000 V, Nozzle voltage ±2000. Nitrogen was used as collision gas. The quadrupoles Q1 and Q3 were operated at widest and unit resolution respectively. DGTS analysis was carried out in positive ion mode by scanning for precursors of m/z 236 at a collision energy (CE) of 55 eV. SQDG analysis was carried out in negative ion mode by scanning for precursors of m/z −225 at a CE of −55 eV. PE, PI, PG, MGDG, and DGDG measurements were performed in positive ion mode by scanning for neutral losses of 141 Da, 277 Da, 189 Da, 179 Da, and 341 Da at CEs of 29 eV, 21 eV, 25 eV, 8 eV, and 11 eV, respectively. Quantification was done by multiple reaction monitoring (MRM) with 30 ms dwell time. DAG and TAG species were identified and quantified by MRM as singly charged ions [M + NH4]+ at a CE of 19 and 26 eV respectively with 30 ms dwell time. Mass spectra were processed by MassHunter Workstation software (Agilent, US) for identification and quantification of lipids. Lipid amounts (pmol) were corrected for response differences between internal standards and endogenous lipids and by comparison with a quality control (QC). QC extract correspond to a known lipid extract from Chlamydomonas cell culture qualified and quantified by TLC and GC-FID as described in ref. 63.
Proteomics analysis
Proteins from total extracts of three biological replicates of WT and phot Chlamydomonas reinhardtii were solubilized in Laemmli buffer and heated for 10 min at 95 °C. They were then stacked in the top of a 4–12% NuPAGE gel (ThermoFisher, US), stained with Coomassie blue R-250 (Bio-Rad, US) before in-gel digestion using modified trypsin (Promega, US) as previously described64. The resulting peptides were analyzed by online nanoliquid chromatography coupled to MS/MS (Ultimate 3000 RSLCnano and Q-Exactive HF, ThermoFisher, US) using a 180-min gradient. For this purpose, the peptides were sampled on a precolumn (300 μm × 5 mm PepMap C18, ThermoFisher, US) and separated in a 75 μm × 250 mm C18 column (Reprosil-Pur 120 C18-AQ, 1.9 μm, Dr. Maisch, Germany). The MS and MS/MS data were acquired using Xcalibur (V2.8, ThermoFisher, US).
Peptides and proteins were identified by Mascot (V2.8.0, Matrix Science) through concomitant searches against the C. reinhardtii phytozome database (V5.6) (19526 sequences), the mitochondrion and chloroplast protein sequences (downloaded from NCBI, respectively 69 and 8 proteins), and a homemade database containing the sequences of classical contaminant proteins found in proteomic analyses (e.g. human keratins, trypsin). Trypsin/P was chosen as the enzyme and two missed cleavages were allowed. Precursor and fragment mass error tolerances were set at respectively at 10 and 20 ppm. Peptide modifications allowed during the search were: Carbamidomethyl (C, fixed), Acetyl (Protein N-term, variable) and Oxidation (M, variable). The Proline software65 (V2.2.0) was used for the compilation, grouping, and filtering of the results (conservation of rank 1 peptides, peptide length ≥6 amino acids, false discovery rate of peptide-spectrum-match identifications <1%66, and minimum of one specific peptide per identified protein group). MS data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository67 with the dataset identifier PXD046943. Proline was then used to perform a MS1 label-free quantification of the identified protein groups based on razor and specific peptides.
Statistical analysis was performed using the ProStaR software68 based on the quantitative data obtained with the three biological replicates analyzed per condition. Proteins identified in the contaminant database, proteins identified by MS/MS in less than two replicates of one condition, and proteins quantified in less than three replicates of one condition were discarded. After log2 transformation, abundance values were normalized using the variance stabilizing normalization (vsn) method, before missing value imputation (SLSA algorithm for partially observed values in the condition and DetQuantile algorithm for totally absent values in the condition). Statistical testing was conducted with limma, whereby differentially expressed proteins were selected using a log2(Fold Change) cut-off of 1 and a p-value cut-off of 0.00912, allowing to reach a false discovery rate inferior to 1% according to the Benjamini-Hochberg estimator. Proteins found differentially abundant but identified by MS/MS in less than two replicates, and detected in less than four replicates, in the condition in which they were found to be more abundant were invalidated (p-value = 1).
GO enrichment analysis
GO term enrichment was tested for all proteins significantly differential abundant at a false discovery rate below 5% using the Benjamini-Hochberg estimator69. GO annotation of proteins was obtained from phytozome database (v.5.6) and all ancestral GO terms were added to a protein using the R package GO.db. P-values were obtained according to the null hypothesis, that the number of differential abundance proteins bearing a GO term is a random variable whose probability distribution is described by the hypergeometric distribution. The false discovery rate was controlled below 5% using the Benjamini-Hochberg-estimator69. Only GO terms linked to at least 6 measured proteins were tested.
Statistical analysis
Prism (GraphPad Software) was used for statistical analysis and all error bars represent standard deviation. ANOVA tests and t-tests were performed, with the p-values or degree of significance provided in the figures and the legends.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
