Plant material and growth conditions

Arabidopsis thaliana accession Col-0 was used as wild-type control in this study. The T-DNA mutant line (cti1-2; N599125) was ordered from ABRC (Arabidopsis Biological Resource Center). Characterization of the wri1-3 and wri1-4 T-DNA insertion lines and construction of the Pro35Sdual:WRI1 lines (T63 and T81) were previously reported²⁸. All plants were grown in growth chamber at 22 °C. Light intensity was 82–115 (98 on average) μmol m⁻² s⁻¹ and humidity was 50%. The long-day (LD) photoperiod corresponded to an alternate 16 h light/8 h dark regime. The short-day (SD) photoperiod corresponded to an alternate 12 h light/12 h dark regime.

Constructs preparation and stable transformation of Arabidopsis

The primers used for construct preparation are listed in Supplementary Table 3.

We designed single-guide RNAs (sgRNA) for CRISPR/Cas9 using the online CRISPR-P 2.0 software⁴⁹. We annealed the oligo pairs to generate double-stranded DNA and cloned the sgRNAs into the BbsI site of the gateway compatible entry vector U6-sgRNA. We then introduced the U6-sgRNA cassette into a modified pCambia1300 binary vector, in which hspCas9 expression is driven by the YAO promoter⁵⁰.

To construct ProCTI1:CTI1:uidA, ProCTI2:CTI2:uidA, and ProCTI3:CTI3:uidA transgenes, gene fragments extending from up to 2 kb upstream of the ATG of the gene to the last coding codon were amplified with the Pfu Ultra DNA polymerase (Stratagene) from Col-0 genomic DNA. The PCR products were introduced by BP recombination into the pDONR207 entry vector (Invitrogen) and transferred into the destination vector pBI101-R1R2-GUS⁵¹ by LR recombination. The resulting binary vector was electroporated into Agrobacterium tumefaciens C58C strain and used for transformation⁵¹. Depending on the construct considered, between 17 and 19 independent transgenic lines were analyzed. To construct the ProCTI1:uidA transgene, region -994 to -1 bp relative to the CTI1 translational start codon was introduced by BP recombination into the pDONR207 entry vector as described above, and transferred into the destination vector pBI101-R1R2-GUS by LR recombination. Construction of the ProBCCP2:uidA transgene²⁸, just like that of the ProODD:uidA transgene⁵² followed a similar procedure.

To construct ProAT2S2:CTI transgenes, CTI cDNAs were amplified from a mixture of seed cDNA (Col-0 accession), cloned into the pDONR207 as described above, and finally transferred to the binary vector ProAT2S2-R1R2-HYGRO⁵³ by LR recombination. The corresponding binary vectors were used for agroinfiltration of cti1-2 flower buds. T2 seeds were subjected to segregation analyses for hygromycin resistance and lines segregating 3:1 were selected (heterozygous lines, one insertion locus). T2 lines were then grown in a greenhouse and their progeny (T3 seeds) was subjected to segregation analyses. Lines producing 100% resistant plantlets were selected (homozygous lines, single insertion locus) and used for further characterization.

To construct Pro35S:CTI1:YFP, Pro35S:CTI2:YFP, Pro35S:CTI3:YFP, and Pro35S:α-CT:YFP transgenes for protoplast transient transformation, CTI and α-CT coding sequences (CDSs) without STOP codons were amplified from a mixture of cDNA (Col-0 accession), cloned into the pENTR/D-TOPO, and transferred into the pAM-PAT-35SS::YFP:GW vector⁵⁴ by LR recombination.

To construct Pro35S:CTI1:GFP, Pro35S:α-CT:RFP, and Pro35S:TGD2:RFP transgenes, CDSs were cloned into the pENTR/D-TOPO vector as above, before being transferred into destination vectors pGWB605 (GFP) and pGWB654 (RFP) by LR recombination⁵⁵.

To construct BiFC and split-luciferase vectors, CDSs previously cloned into the pENTR/D-TOPO were transferred into pAM-PAT-35SS::cYFP/nYFP:GW, and pEarley-nLUC/cLUC vectors by LR recombination.

To purify the coiled-coil domains of the CTIs and to perform yeast two-hybrid assays, the CDSs corresponding to CTI coiled-coil domains were amplified by PCR, cloned into the pENTR/D-TOPO entry vector and transferred into destination vectors pGADT7-GW by LR recombination.

To purify the proteins for microscale thermophoresis, the C-terminus of CTI1, CTI3, and α-CT were each fused with a HIS tag, and cloned into pET30a (CTI3 and α-CT) or pDEST17 (CTI1). CTI2 was fused with a GST tag and cloned into the pGEX4T-1 vector.

To construct the Pro35S:CTI1:HA transgene, a HindIII/StuI fragment of the pGWB14 binary vector⁵⁵ containing a 35S promoter-R1-CmR-ccdB-R2-3xHA-NosT sequence was HindIII/SmaI inserted in the pBluescript KS(+) vector (Stratagene), giving the pBKS-35S-R1R2-3xHA vector. CTI1 CDS without STOP codon previously cloned into the pDONR207 (see above) was transferred into the pBKS-35S-R1R2-3xHA vector by LR recombination. To construct the ProCTI1:CTI1:HA transgene, the CTI1 gene was amplified from Col-0 genomic DNA and the 3xHA tag was amplified from the pBKS-35S-R1R2-3xHA containing the Pro35S:CTI1:HA fusion (see above). The two amplified fragments were combined together to serve as overlapping templates for a PCR reaction utilizing primers listed in Supplementary Table 3. The final PCR product was cloned in pDONR207, and finally transferred into the binary vector pBIB-Hyg-GTW⁵¹ by LR recombination. The corresponding binary vector was used for agroinfiltration of cti1-2. Homozygous lines (single insertion locus) were selected as described above.

To construct the Pro35S:TOC33:MYC transgene, the CDS corresponding to TOC33 was amplified by PCR, cloned into the pENTR/D-TOPO entry vector and transferred into destination vectors pGWB617 by LR recombination.

To construct the Pro35Sdual:WRI1⁵¹ and Pro35Sdual:MYB118 transgenes⁵², the corresponding CDSs were amplified by PCR, cloned into the pDONR207 entry vector by BP recombination and transferred into the pMDC32 destination by LR recombination.

RNA isolation and quantitative real-time PCR

For RNA extraction, frozen tissues were ground in liquid nitrogen and total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s instructions. For reverse transcription, RNA was converted into first-strand cDNA using the SuperScript preamplification system for first-strand cDNA synthesis (ThermoFisher) with oligo(dT)₂₂. The real-time quantitative RT-PCR reaction was performed on the LightCycler Instrument (Roche) with the LightCycler-FastStar DNA Master SYBR Green I kit for PCR (Roche) according to the manufacturer’s protocol. Each reaction was performed with 5 µL of 1:50 (v/v) dilution of the first cDNA strands in a total volume of 20 µL. The reaction was incubated at 95 °C for 8 min to activate the hot start recombinant Taq DNA polymerase, followed by 45 cycles of 10 s at 95 °C, 6 s at 55 °C, and 20 s at 72 °C⁵⁶. Specific primer sequences are presented in Supplementary Tables 4, 5. The specificity of the PCR amplification was checked with a heat dissociation protocol (from 65 to 95 °C) following the last cycle of the PCR.

Yeast one-hybrid assay

Construction of the ProCTI1-500:HIS reporter plasmid, like that of the ProBCCP2-180:HIS reporter plasmid²⁹, relied on the amplification by PCR of promoter fragments, that were digested by EcoRI and SacII, and inserted into the pHISi vector between the EcoRI and SacII sites. The plasmids thus obtained were digested with NcoI and integrated into the yeast strain YM4271 at the URA3 locus. Yeast cells presenting the HIS3 reporter gene under the control of the different promoters studied were transformed with pDEST22 using LiAc/SSDNA/PEG method. Yeast cells were grown in YPDA medium and harvested by centrifugation at 3000 × g. For each transformation, around 10⁸ cells were mixed with T Mix (240 μL PEG 3350 50% (w/v), 36 μL 1 M lithium acetate, 50 μL boiled SS-Carrier DNA (2 mg/mL), 34 μL plasmid DNA (0.1–1 μg)). After vigorous mix by vortex, the mixtures were incubated in a 28 °C water bath for 30 min, then in a 42 °C water bath for 25 min. Afterwards, cells were pelleted by centrifugation, washed with sterile water, resuspended in 200 µL sterile water before being plated on selective medium and grown at 30 °C for 3 d.

Yeast two-hybrid assays

The cDNA library used was prepared by introducing Arabidopsis cDNA prepared from seedlings into the pGATD7-GW vector. The screening assay was conducted according to the Clontech yeast handbook. Briefly, the sequence encoding the coiled-coil domain of α-CT (amino acid 420–769) was cloned into the pGBKT7 vector. This vector was used to transform the AH109 yeast strain. Yeast transformants were selected on synthetic dropout (SD) medium lacking tryptophan. Then, 100 μg of cDNA library were used to transform AH109 cells containing the pGBKT7 vector expressing the coiled-coil domain of α-CT. Cells plated on SD medium lacking leucine, tryptophan, histidine, adenine, were incubated at 30 °C for 4 d and cDNAs of positive clones were amplified by PCR and sequenced using T7 and 3’AD primers (Supplementary Table 6).

For oriented interaction assays, the sequences coding for the coiled-coil domain of CTIs were cloned into the pGADT7 vector as described above. Then, the AH109 strain was cotransformed with the pGADT7 vector expressing CTI coiled-coil domains and the pGBKT7 vector expressing the α-CT coiled-coil domain, before being plated on SD medium lacking leucine and tryptophan. After 3 days at 30 °C, positive clones were transferred to SD medium lacking leucine, histidine, and tryptophan, in the presence of 1 mM 3AT for another 4 d at 30 °C.

Transformation of Arabidopsis protoplasts

Arabidopsis protoplasts were prepared from 4-week-old Col-0 plants⁵⁷. About 40 leaves were cut into strips with a sharp razor blade and then transferred into 20 mL enzyme solution (20 mM MES pH 5.7, 1.5% (w/v) cellulase R10, 0.4% (w/v) macerozyme R10, 0.4 M mannitol, 20 mM KCl and 0.1% BSA) for 3 h at room temperature. After 3 h, the enzyme solution was filtered through 75-μm nylon mesh that was then washed with 20 mL W5 solution (2 mM MES pH 5.7, 154 mM NaCl, 125 mM CaCl₂, 5 mM KCl). The flow-through was centrifuged at 200 × g for 2 min. After removing as much supernatant as possible, the protoplast pellet was resuspended in 1 mL W5 solution and kept on ice for 30 min. The protoplasts were pelleted at 200 × g for 2 min and resuspended in 500 μL MMG solution (4 mM MES pH 5.7, 0.4 M mannitol, 15 mM MgCl₂). Then, 10 μL purified plasmid (10-20 μg) were mixed with 100 μL protoplasts before 110 μL PEG solution, namely 40% (w/v) PEG4000 in ddH₂O containing 0.2 M mannitol and 100 mM CaCl₂, were added. The transfection reaction was mixed by gently tapping the tube and incubated at room temperature for 10 min. The mixture was then diluted in 500 μL W5 solution, mixed by gently rocking the tube to stop the transfection process, and ultimately centrifuged for 2 min at 200 × g. The pellet was resuspended in 1 mL W5 solution and incubated overnight at 23 °C under weak light.

Transient expression in leaves of Nicotiana benthamiana

Agrobacterium tumefaciens strains transformed with the different binary vectors prepared were grown overnight in selective medium. Cells were then pelleted, washed two times and resuspended in injection buffer (50 mM MES pH 5.7, 10 mM MgCl₂). Different strain combinations were coinfiltrated into the leaves of Nicotiana benthamiana in the presence of a vector coding for the P19 protein of tomato bushy stunt virus (TBSV) that prevents the onset of post-transcriptional gene silencing.

Confocal microscopy

Fluorescence was observed by confocal laser microscopy using a Leica TCS SP8 device. Fluorescence were observed at 488–505 nm for GFP, 514–527 nm for YFP, and 558–583 nm for RFP.

Split-luciferase assay

After agrobacterium-mediated infiltration of N. benthamiana leaves, different light/dark treatments were applied and luciferase activity was detected with a CCD camera by applying firefly D-luciferin (Goldbio). For 24-h light recovery assays, after luminescence imaging, the detached leaves were kept in a tray with water to maintain humidity. After 24 h of light recovery, the luciferase activity was detected with the CCD camera again. Ponceau S Staining (1% (w/v) Ponceau S, 20% acetic acid) was used to quantify relative protein contents.

Microscale thermophoresis (MST)

The coiled-coil domains of CTI1, CTI3, and α-CT fused with HIS tag were purified by capture on nickel agarose resin. The coiled-coil domain of CTI2 fused with a GST tag was purified by capture with glutathione resin. Each protein was expressed in Luria Broth. Expression was induced with 100 μM IPTG when OD₆₀₀ reached 0.6. The cells were pelleted and resuspended with lysis buffer (PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄ and 2 mM KH₂PO₄, pH 7.4). After cell disruption by French press and centrifugation, the proteins were purified by nickel agarose resin (Gold Biotechnology, H-320) or glutathione agarose resin (Gold Biotechnology, G-250). The purified protein samples were concentrated to 500 μL with a Pierce concentrator PES with a 3000 Dalton cutoff (ThermoFisher, 88527). The GST tag was proteolytically separated from CTI2 using thrombin treatment. Proteins were quantified by Bradford assays using bovine gamma globulin as a standard. 100 nM α-CT-His protein was fluorescently labeled using His-tag labeling kit (NanoTemper, MO-L008) according to the user manual. After labeling, 10 μL of labeled α-CT protein was mixed with 10 μL of CTI (serial dilutions in PBS buffer). The microthermophoresis was carried out using 100% LED power and high MST power with a NanoTemper monolith NT.115 instrument. The data were analyzed by MO.Offinity Analysis (X86).

Immunoblotting

For quantification of the htACCase subunit content, proteins were extracted from crude chloroplasts using a protein extraction buffer (50 mM HEPES-KOH pH 7.6, 150 mM NaCl, 1 mM EDTA, 10% (v/v) glycerol, 1 mM 2-mercaptoethanol, 1% (v/v) Triton X-100, 1X complete protease inhibitor cocktail). The crude chloroplasts were isolated from 5 g of 4-week-old Col-0 leaf tissues. The proteins were separated by SDS-PAGE and blotted to a PVDF membrane. The BCCP, α-CT and β-CT subunits of plastidic htACCase were immunologically detected with dedicated antisera¹⁹. A horseradish peroxidase-conjugated secondary antibody was used and the horseradish peroxidase activity was detected by enhanced chemiluminescence (ECL) western blotting substrate (ThermoFisher; Cat. No. 32106). Ponceau S Staining was used as a loading control.

Co-immunoprecipitation

Crude chloroplasts were isolated from 4-week-old Col-0 plants. Around 5 g of fresh leaves were ground in 30 mL ice-cold isolation buffer (50 mM HEPES pH 8.0, 2 mM EDTA, 2.5 mM MgCl₂, 5 mM NaHCO₃, 0.33 M sorbitol, 0.5 % BSA). After filtration of the homogenate through miracloth, the flow-through was centrifuged at 1000 × g for 10 min at 4 °C. The pellet was resuspended in 1 mL of protein extraction buffer containing 50 mM Tris pH 7.5, 150 mM NaCl, 1% Triton-X 100 and 1x protease inhibitor cocktail (Sigma). After a 30-min incubation period on ice, the solution was centrifuged at 20,000 × g for 15 min at 4 °C. The supernatant was decanted before addition of 5 μL of anti-α-CT antibody and subsequent incubation for 4 h with end-to-end rotation at 4 °C. After addition of 25 μL of protein A resin (Genescript; Cat. No. L00210), another 2 h incubation period was implemented. The mixture was finally spun down and washed three times with washing buffer (50 mM Tris pH 7.5, 150 mM NaCl). After washing, the resin was incubated with 100 μL of 1× SDS-PAGE loading buffer, and then heated at 80 °C for 10 min. Proteins were separated by SDS-PAGE and then transferred to PVDF membrane. After transfer, the proteins were detected by immunoblotting with anti-CTI1 or anti-α-CT antibody. A horseradish peroxidase-conjugated secondary antibody was used and the horseradish peroxidase activity was detected by ECL western blotting substrate (ThermoFisher; Cat. No. 32106).

Protease protection assay

Four-week-old leaves of transgenic plants stably expressing a TOC33-MYC fusion were harvested and 10 g of fresh material were used to isolate crude chloroplasts according to method described above. Chloroplasts were resuspended in 1 ml of isolation buffer and loaded onto a Percoll gradient. The Percoll gradient was obtained by mixing 15 mL of Percoll with 15 mL of 2× isolation buffer, and centrifuging the mixture at 38,700 × g for 30 min at 4 °C. The chloroplasts were centrifuged on the Percoll gradient using a prechilled swinging bucket rotor at 7700 × g for 10 min at 4 °C with no brake. After centrifugation, the upper green band corresponding to broken chloroplasts was removed and discarded. The lower green band was transferred into a new 50-mL centrifuge tube containing 10 mL of isolation buffer. The mixture was spun down at 1500 × g for 5 min at 4 °C, and the pellet resuspended in 1 mL reaction buffer (50 mM HEPES pH 8.0, 0.33 M sorbitol). The proteolytic digestions were set up as follows: mock—150 μL of chloroplasts were mixed with 100 μL of reaction buffer; thermolysin—150 μL of chloroplasts were mixed with 10 μL of thermolysin stock solution (1 mg/mL, freshly prepared in 5 mM CaCl₂ in reaction buffer) and 90 μL of reaction buffer; trypsin—150 μL of chloroplasts were mixed with 10 μL of trypsin stock solution (1 mg/mL, freshly made in reaction buffer) and 90 μL of reaction buffer. All reactions were incubated on ice for 30 min. Each proteolytic reaction was quenched on ice for 5 min as follows: mock—50 μL of reaction buffer was added; thermolysin—50 μL of quench solution (60 mM EDTA in reaction buffer) was added; trypsin—50 μL of trypsin inhibitor solution (1 mg/mL in reaction buffer; Sigma; Cat. No. T6522) was added. SDS-PAGE loading buffer was then added to each reaction. Western blotting was performed as described above using anti-Myc, anti-α-CT or anti-CTI1 antibodies.

Immunolocalization experiments

Coverslips were sterilized in 95% ethanol and dried before coating. They were then incubated for 5 min at 37 °C in a 50 µg/mL poly-L-lysine solution before drying. Arabidopsis protoplasts were fixed for 30 min with 3% (w/v) paraformaldehyde in PBS supplemented with 30.5 g/L glucose and 30.5 g/L mannitol (pH 7.4; PBS-GM) at room temperature. Fixed protoplasts were washed three times with 50 mM ammonium chloride in PBS-GM. The final suspension was plated onto poly-L-lysine-coated coverslips. Protoplasts were allowed to settle for 10 min at room temperature, and were then air dried for 1 h under a culture hood. To permeabilize protoplasts, a 15 min treatment with 0.5% (v/v) Triton X-100 buffered with PBS-GM was carried out. After three washes with PBS-GM, nonspecific binding was blocked with 1% bovine serum albumin in PBS-GM for 1 h. Protoplasts were then incubated overnight at 4 °C with rabbit anti-E37 antibodies¹⁹ (1:500) followed by 1 h incubation at room temperature with rat anti-HA antibodies (1:2000; Roche). Cells were washed for 20 min in PBS-GM, and incubated with goat anti-rabbit immunoglobulin G-Alexa Fluor 568 (1:1000; Molecular Probes) and with goat anti-rat immunoglobulin G-Alexa Fluor 488 (1:500; Molecular Probes) or with goat anti-mouse immunoglobulin G-Alexa Fluor 488 (1:500; Molecular Probes) for 1 h. Finally, cells were washed for 20 min in PBS and mounted using Vestashield mounting solution (Vector Laboratories). Cells were visualized with an inverted spectral confocal laser microscope (LEICA SP2-AOBS). Preparation of embryos excised from maturing seeds was similar to that of protoplasts except for the permeabilization treatment: cell walls were partially digested by incubating embryos in 2% (w/v) driselase (Sigma) in PBS-GM for 45 min, then membranes were permeabilized by a 1 h treatment with 10% (v/v) DMSO and 3% (v/v) NP-40 in PBS-GM.

Lipid and fatty acid analyses

To analyze polar lipids and triacylglycerols, 500 mg of leaves were harvested and stored at −80 °C prior to lipid extraction. Leaf samples were ground in 7.2 mL of precooled chloroform/methanol/formic acid (10:10:0.5, v/v/v) and incubated at -20 °C overnight. The mixture was centrifuged at 7500 × g for 10 min at 4 °C to pellet the cell debris. Lipids of the pellet were reextracted in 2.64 ml of precooled chloroform/methanol/water (10:10:1, v/v/v). Then, 3.6 mL of cooled Hajra solution (2 M KCl and 0.2 M H₃PO₄) were added to the pooled supernatants. After shaking and centrifugation (7500 × g for 10 min at 4 °C), the lower phase containing lipids was collected and evaporated with a stream of N₂. Lipids were resuspended in chloroform/methanol (2:1, v/v) and separated on thin-layer chromatography plates developed with hexane/diethylether/acetic acid (35:15:0.01, v/v/v). Polar lipids and triacylglycerols were visualized under UV light by staining with sprayed primuline. Lipid spots were ultimately collected and analyzed by gas chromatography⁵⁸.

To analyze total fatty acids in leaves or seeds, total lipids were transmethylated into fatty acid methyl esters (FAMEs). FAMEs were analyzed by a Hewlett Packard 6890 gas chromatograph. Leaves from 4-week-old plants were collected and dried by centrivap SpeedVac overnight. Mature seeds were harvested and dried at room temperature for 1 week prior to analysis.

Lipidomics

Lipid extracts were dissolved in chloroform, and appropriate amounts of internal standards were introduced. The lipids were detected by triple quadrupole mass spectrometer (Applied Biosystems API 4000) with an autosampler (LC Mini PAL; CTC Analytics)⁵⁹. Data processing was performed in a Lipidome DB Data Calculation Environment (http://lipidome.bcf.ku.edu:9000/Lipidomics) with normalization to internal standards.

In vivo acetate labeling

In vivo labeling experiments were performed according to previous report⁶⁰. Leaves from 4-week-old plants were cut into strips after measuring the fresh weight. The leaf strips were transferred into 2 mL of reaction buffer (20 mM MES pH 5.5, half MS, 0.01% (v/v) Tween 20) in a 6-well plate. The labeling assays were started by the addition of 1 μCi of ¹⁴C-acetate (PerkinElmer; Cat. No. NEC084H001MC) or 1 μCi of ¹⁴C-acetate (Morevek; Cat. No. MC213). After this, the 6-well plates were incubated on a shaker in the light (40 µmol m⁻² s⁻¹) at room temperature or in the dark. The samples were collected after 40 min of incubation, washed three times with water and total lipids were then extracted in 800 µL of methanol/chloroform/formic acid (20:10:1, v/v/v) with vortexing for 10 seconds⁶¹. The mixture was incubated for 30 min at 22 °C, complemented with 500 µL of 1 M KCl and 0.2 M H₃PO₄, vortexed and centrifuged at 12,000 × g for 30 sec for collection of the chloroform phase containing the lipids. One milliliter of scintillation cocktail was added to this phase before measurement of the incorporated radioactivity (in cpm) with a scintillation counter.

ACCase activity assay

ACCase activity was directly quantified from 4-week-old leaves by incorporation of H¹⁴CO₃ into acid-stable products. Around 5 g of leaf tissues were harvested. Crude chloroplasts were isolated as above, and chloroplast proteins were extracted using 300 μL of extraction buffer (50 mM Tris-HCl pH 7.5, 100 mM KCl, 5 mM MgCl₂, 1 mM DTT, 1% (v/v) Triton X-100, 10% (v/v) glycerol, and 1× plant protease inhibitor mixture from Sigma). After centrifugation at 13,000 × g for 10 min, the supernatants were desalted with spin desalting columns (ThermoFisher; Cat. No. 89882). The insoluble pellets were resuspended in the extraction buffer. Reactions were initiated by mixing 10 μL of reaction buffer (100 mM Tricine pH 8.2, 100 mM KCl, 15 mM ATP, 5 mM MgCl₂, 1 mM DTT, 2.5 mM acetyl-CoA, 50 μM haloxyfop, and 1 μCi ¹⁴C-NaHCO₃ (Moravek; Cat. No. MC208) with 40 μL of protein extract. After 30 min at RT, the reactions were quenched by adding 50 μL of 12 N HCl. The reaction solutions were transferred into scintillation vials with a filter paper at the bottom of the vial. Samples were dried at 80 °C for 1 h, after which 1 mL of scintillation cocktail was added and radioactivity counted by liquid scintillation. Controls without acetyl-CoA were included to determine and subtract nonspecific background rates.

GUS assay

For histochemical detection of GUS activity, tissues were incubated in 0.1 M phosphate buffer, pH 7.2 containing 2 mM 5-bromo-3-indolyl-β-D-glucuronide, 0.1% (v/v) Triton X-100, 10 mM Na₂-EDTA, and 0.2 (Arabidopsis vegetative organs, flowers, and seeds) or 2 mM (Arabidopsis embryos and N. benthamiana leaf disks) each of potassium ferricyanide and potassium ferrocyanide. A vacuum was applied for 1 h before incubating the samples for 4 h (Arabidopsis embryos and N. benthamiana leaf disks) or 15 h (Arabidopsis vegetative organs, flowers, and seeds) at 37 °C in the dark, and chlorophyll was finally removed by room temperature incubation in 70% (v/v) ethanol (vegetative tissues and flowers only).

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.