Ethylene treatment induces the nuclear accumulation of PDC

We have observed the nuclear localization of PDC complex with the presence of ethylene (Fig. 1). To further confirm that the PDC nuclear translocation is induced by ethylene, we examined the nuclear appearance of E1, E2, and E3 proteins over a time series of ethylene gas treatments. E1 had a basal nuclear distribution in the absence of ethylene, but its nuclear levels were notably elevated by ethylene treatment and were positively correlated with the duration of ethylene treatment while E1 cytoplasmic levels showed gradual decrease as the ethylene treatment prolongs (Fig. 2A and fig. S2, A and B). Neither E2 nor E3 proteins were detected in the nucleus in the absence of ethylene, but these two proteins were accumulated in the nuclear fraction after 4 hours of ethylene treatment, and their levels were also positively correlated with the duration of ethylene treatments (Fig. 2, B and C, and fig. S2, C to F). Similar to cytoplasmic E1, we also observed that cytoplasmic E2 and E3 protein levels decrease in response to ethylene (Fig. 2, B and C, and fig. S2, C to F). Notably, the total E1 and E2 subunit protein levels were not altered by the ethylene treatments, but E3 total protein level showed slight elevation after 12-hour ethylene treatment. We then monitored the nuclear localization of PDC over a time series of ethylene treatments in living cells. In the absence of ethylene, a basal level of E1 was observed in the nucleus, but no nuclear E2 and E3 were observed (Fig. 2, D to F, and fig. S2, G to L). Upon ethylene treatment, E1, E2, and E3 accumulated in the nucleus, and their accumulation was positively associated with the duration of ethylene treatment, the cytoplasmic fraction of PDC remains in the mitochondria even with the ethylene treatment (Fig. 2, D to F, and fig. S2, G to R). This provides an additional piece of cellular evidence that ethylene induces PDC nuclear accumulation.

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Fig. 2.

PDC accumulates in the nucleus in response to ethylene.

(A to C) Fractionation Western blot to examine the subcellular localization of PDC in E1-YFP-HA (A), E2-FLAG-GFP (B), and E3-FLAG-GFP (C) transgenic plants with time series of ethylene gas treatments. PDC E1 was probed with anti-HA, and E2 and E3 were probed with anti-FLAG in total protein extracts, cytoplasmic fractions, and nuclear fractions. PEPC, ATP synthase beta (ATPB), FUM1/2, Cyt C, and histone H3 were used to assess purities of nuclear and cytosolic fractionations and loading controls. Blue number indicates PDC band intensity that normalized to cytoplasmic PEPC signal. Red number indicates PDC band intensity that normalized to histone H3 signal. (D to F) Confocal microscopy images showing the subcellular localization of E1-YFP-HA (D), E2-FLAG-GFP (E), and E3-FLAG-GFP (F) with time series of ethylene gas treatments. 4′,6-Diamidino-2-phenylindole (DAPI) staining labels nuclei. Scale bars, 20 μm.

PDC subunits are required for ethylene response

Given that the interaction between PDC and EIN2-C occurs in the nucleus in response to ethylene, we decided to investigate the functions of PDC in the ethylene response. We first obtained two transferred DNA (T-DNA) insertion lines for E1 (e1-2-2 and e1-2-4) and two for E3 (e3-2-1 and e3-2-2). Reverse transcription–quantitative polymerase chain reaction (RT-qPCR) assays showed that the expression levels of these two genes were drastically reduced in their T-DNA insertion mutants (fig. S3, A to D). No E2 T-DNA homozygous plants were obtained because of homozygous lethality (46). Using CRISPR-Cas9 mutagenesis, we generated two weak alleles of e2 mutants (e2-2 #17 and e2-2 #215) that were variable (fig. S3, E to G). Phenotypical assay showed that the e2 single mutants, but not e1 nor e3 single mutant, displayed a mild ethylene insensitivity both in roots and in hypocotyls (Fig. 3, A to C). We then generated a variety of their higher-order mutants and examined their ethylene responses (fig. S3, E to G). The mild ethylene-insensitive phenotype of the e2 single mutant was significantly enhanced in e1e2 (e1-2-2 e2-2 #17 and e1-2-4 e2-2 #215) and e2e3 (e2-2 e3-2-1 #99 and e2-2 e3-2-2 #67) double mutants and in e1e2e3 triple mutants (e1-2-2 e2-2 e3-2-1 #167 and e1-2-2 e2-2 e3-2-2 #67) (Fig. 3, D to F). No significant difference was observed between the e1e3 (e1-2-2 e3-2-1 and e1-2-2 e3-2-2) double mutants and e1 or e3 single mutants (Fig. 3, A to F). Notably, e1e2e3 triple mutants exhibit mild growth defects including shortened hypocotyls and roots compared to the wild-type Columbia (Col-0) and other pdc mutants when grown in the dark in the absence of 1-aminocyclopropane-1-carboxylic acid (ACC) (Fig. 3, D to F). The triple mutant also displayed reduced root and vegetative growth under light condition (fig. S3, H and I), suggesting that PDC complex potentially influences plant growth and development. The e1e2, e2e3, and e1e2e3 ethylene-insensitive phenotype and the growth defect of e1e2e3 etiolated seedlings were complemented by introducing proE2:gE2-FLAG-GFP into each mutant background, respectively (fig. S3, J and K). These genetic results demonstrated that PDC is involved in the ethylene response, E2 is the most important subunit genetically, and E1 and E3 enhance the function of E2 in the ethylene response.

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Fig. 3.

PDC is involved in the ethylene response.

(A) Photographs of pdc single mutants and Col-0 seedlings grown on MS medium containing 0, 1, 2, and 5 μM ACC. (B and C) Measurements of hypocotyl lengths (B) and root lengths (C) of indicated mutants. (D) Representative higher-order PDC mutants and Col-0 grown on MS medium containing 0, 1, 2, and 5 μM ACC. (E and F) Measurements of hypocotyl lengths and root lengths from indicated plants. In (B), (C), (E), and (F), each value is means ± SD of at least 30 seedlings, and different letters indicate significant differences between genotypes [P ≤ 0.05, calculated by a one-way analysis of variance (ANOVA) test followed by Tukey’s post hoc test for multiple comparisons]. (G and H) Scatterplots comparing the log₂FC of ethylene up-regulated genes in Col-0 with that in e1-2-2 e2-2 #17 (G) or in e2-2 e3-2-2 #67 (H) in mRNA-seq. Each dot represents a DEG that its transcription level is significantly elevated by ethylene in Col-0 (log₂FC > 1, P adjusted < 0.05). In (G) and (H), red dots correspond to DEGs in the elevated group, gray dots to DEGs in the unchanged group, and green dots to DEGs in the compromised group as described in Results. (I) Violin plot of log₂FC distributions of ethylene up-regulated genes in the indicated plants. P values were determined by a two-tailed t test. (J) Venn diagram showing the ethylene up-regulation compromised genes in e1-2-2 e2-2 #17 and in e2-2 e3-2-2 #67. (K) Gene ontology (GO) analysis of transcriptionally co-compromised genes in both e1-2-2 e2-2 #17 and in e2-2 e3-2-2 #67. Top 15 GO categories ranked by P values were plotted. Dot size indicates the gene percentage from each GO category of all co-compromised genes (gene ratio %); dot colors indicate the P value of the GO categories.

To further understand the functions of E1, E2, and E3 in the ethylene response, we generated their gain-of-function mutants and selected two individual transgenic lines of each with similar protein expression levels for further ethylene response analysis (fig. S4, A to C). Compared to Col-0, all the plants that overexpressed PDC E1, E2, and E3 subunits had enhanced ethylene-responsive phenotype in the presence of ACC, but the phenotypes of E2ox and E3ox did not differ from Col-0 in the absence of ACC (fig. S4D). This showed that the overexpression of PDC E2 and E3 alone was not sufficient to trigger ethylene response in the absence of the hormone when they were not accumulated in the nucleus. PDC E1ox displayed a mild ethylene hypersensitivity in the absence of ACC compared to Col-0, which is in line with the observation that there was a basal E1 in the nucleus without ethylene treatment (fig. S4D). Next, we expressed E1 and E2 fused with nuclear localization signal (NLS) peptide derived from EIN2-C (E1-NLS-GFP and E2-NLS-GFP) in Col-0 (fig. S4E). E1–NLS–green fluorescent protein (GFP) and E2-NLS-GFP fusion proteins could be localized to the nucleus in the absence of ethylene; the E1-NLS-GFP and E2-NLS-GFP plants displayed a clear ethylene hypersensitivity phenotype even in the absence of ethylene (fig. S4, F and G). Together, these data demonstrate that the nuclear localization of PDC can trigger the ethylene response.

To evaluate the function of PDC in ethylene response at a molecular level, we performed RNA sequencing (RNA-seq) using e1-2-2 e2-2 #17 and e2-2 e3-2-2 #67 double mutants treated with or without 4 hours of ethylene gas (fig. S5). We found that about 50% genes up-regulated by ethylene in Col-0 were not differentially expressed in either of the two mutants in response to ethylene (fig. S6, A and B). For further statistical quantification, we plotted the log₂ fold change (log₂FC) of each gene up-regulated by ethylene in Col-0 against that in each double mutant. These genes were then divided into three groups: elevated group (log₂FC in mutants is 30% greater than in Col-0, plotted as red dots), unchanged group (log₂FC in mutants is within 30% of that in Col-0, plotted as gray dots), and compromised group (log₂FC in mutants is 30% less than that in Col-0, plotted as green dots). We found that the elevation of more than half of ethylene up-regulated genes in Col-0 was significantly compromised in both double mutants (Fig. 3, G to I). Most of the compromised genes identified from e1-2-2 e2-2 #17 seedlings were also compromised in the e2-2 e3-2-2 #67 seedlings (Fig. 3J), and the log₂FC profile showed a high similarity (Fig. 3I and fig. S6C). Further gene ontology analysis using those up-regulation compromised genes showed that the ethylene signaling genes were overrepresented (Fig. 3K and fig. S6D). Together, these data provide genetic and molecular evidence that E1, E2, and E3 function coordinately to regulate ethylene response.

PDC regulates ethylene-dependent elevation of histone acetylation at H3K14 and H3K23

PDC catalyzes the synthesis of acetyl CoA, the substrate of histone acetylation. Given that the acetylation of histone at H3K14 and H3K23 is induced by ethylene, we compared their global levels in Col-0 and in different pdc mutants. The ethylene-induced global elevation of H3K14ac and H3K23ac in Col-0 was still detected in e1-2-2 e2-2 #17 but with lower levels; whereas ethylene-induced global elevation was not detected in e2-2 e3-2-2 #67 nor in e1-2-2 e2-2 e3-2-2 #67 (Fig. 4A and fig. S7, A to C). In contrast, the H3K14ac and H3K23ac levels were elevated in the plants that overexpress E2 with ethylene treatment and in the E2-NLS-GFP transgenic plants even without ethylene treatment (Fig. 4B and fig. S7, D to F). We then conducted H3K14ac and H3K23ac ChIP-seq in e1-2-2 e2-2 #17 (fig. S8, A to D). H3K14ac and H3K23ac levels were reduced in e1-2-2 e2-2 #17 compared to that in Col-0 over genes that were transcriptionally compromised in both e1-2-2 e2-2 #17 and e2-2 e3-2-2 #67 plants, and the ethylene-induced elevation was notably impaired (Fig. 4, C and D, and fig. S8, E to H). When we evaluated the ChIP signals within the first 500 bp downstream of transcription start sites as histone acetylation is associated with active transcription, an even more significant reduction in H3K14ac and H3K23ac enrichment was detected in e1-2-2 e2-2 #17 both with and without ethylene treatments when compared to those in Col-0 (Fig. 4, E and F). To further investigate the regulation of PDC on histone acetylation in ethylene-activated genes, we examined the H3K14ac and H3K23ac levels in 250 randomly selected, non-ethylene–regulated genes. These genes were chosen to match the number of co-compromised differentially expressed genes (DEGs) analyzed in Fig. 4 (C to F) and had expression levels within the 25th to 75th percentiles of the expression range of co-compromised DEGs in Col-0 without ethylene treatment (fig. S8I). No association between changes in H3K14ac or H3K23ac levels and the expression of these random genes was detected (fig. S8, J to M). This further confirms that PDC regulates H3K14ac and H3K23ac specifically in ethylene-activated genes in response to ethylene. We then conducted H3K14ac and H3K23ac ChIP-qPCR assays in the selected targets which show compromised transcriptional response to ethylene and reduced histone acetylation at H3K14 and H3K23 in pdc mutants (figs. S6D and S8, E to H), and the results further validated the ChIP-seq data (fig. S8, N and O). Given that e2-2 e3-2-2 #67 and e1-2-2 e2-2 e3-2-2 #67 have a similar ethylene-responsive phenotype as e1-2-2 e2-2 #17, we also conducted ChIP-qPCR assays in those two mutants, and we observed that ethylene-induced elevations of H3K14ac and H3K23ac enrichments were also impaired in e2-2 e3-2-2 #67 and e1-2-2 e2-2 e3-2-2 #67 as in e1-2-2 e2-2 #17 (fig. S8, N and O). In contrast, we found that in the absence of ethylene, the basal levels of H3K14ac and H3K23ac in E2-NLS are elevated when compared to that in Col-0 and also higher than that in E2ox, indicating that the nuclear-localized E2 is able to induce histone acetylation at ethylene-responsive target genes (fig. S8, P and Q). Together, these results suggest that PDC regulates the histone acetylation H3K14ac and H3K23ac in response to ethylene.

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Fig. 4.

PDC is involved in the ethylene-induced histone acetylation.

(A) Western blot analysis of total histone acetylation of H3K14 and H3K23 in response to ethylene in different genetic backgrounds as indicated in the figure. Anti-H3 Western blot served as a loading control. (B) H3K14ac and H3K23ac levels in Col-0, E2ox, and E2-NLS-GFP treated with air or 4 hours of ethylene gas. Histone H3 served as a loading control. Red number indicates the quantification of H3K14ac and H3K23ac Western blot signal intensity normalized with that of histone H3. (C and D) Heatmaps of H3K14ac (C) and H3K23ac (D) ChIP-seq signal (log₂ ChIP signal) from the genes that their ethylene-induced expressions are co-compromised in e1-2-2 e2-2 #17 and e2-2 e3-2-2 #67. TSS, transcription start site; TTS, transcription termination site. (E and F) Violin plots illustrating log₂ normalized H3K14ac ChIP signal (E) and H3K23ac ChIP signal (F) per bin (bin size = 1) from 500 bp downstream of TSS in the genes that were co-compromised in two pdc double mutants (Fig. 3J) in the indicated genotypes and conditions. P values were calculated by a two-tailed t test.

Enzymatically active nuclear PDC regulates acetyl CoA production in the nucleus

Because the biological function of PDC is to generate acetyl CoA, we hypothesized that the reduced histone acetylation level in the plants that lack PDC subunits results from the decreased acetyl CoA in the nucleus. We first measured the total acetyl CoA and nuclear acetyl CoA concentrations in Col-0 and in the e1-2-2 e2-2 e3-2-2 #67 triple mutant treated with 4 hours of ethylene gas by LC-MS, and the levels of total acetyl CoA and nuclear acetyl CoA were significantly decreased in the pdc triple mutant compared to levels in Col-0 while the concentrations of PDC-irrelevant CoA species malonyl CoA remain unchanged (Fig. 5, A and B, and fig. S9A). We then decided to examine whether the nuclear PDC was functional to synthesize acetyl CoA. It has been shown that dephosphorylated E1 is required for the PDC activity (44, 47); therefore, we first examined the phosphorylation status of E1 in the nucleus in response to ethylene. Western blot assay by anti-pSer antibody showed that most E1 was in a dephosphorylated state in the nucleus after ethylene treatment (Fig. 5C). Phos-tag electrophoresis assay confirmed this result (Fig. 5D). Next, we conducted LC–tandem MS (MS/MS) analysis of E1 phosphorylation. We detected phosphorylation on Ser²⁹² (fig. S9B), a residue that is evolutionary conserved with the reported inhibitory phosphorylation site in human E1 (fig. S9C) (47). Further quantification of E1 phosphopeptides by MS showed that the ratio of phosphorylated E1 to nonphosphorylated E1 in the nucleus was significantly lower than that in the cytosol after 4 hours of ethylene treatment (Fig. 5E), suggesting that the dephosphorylated E1 is the main species in the nucleus to function in the presence of ethylene. Next, we assessed E1 activities in the nucleus using a PDH activity assay. E1 activity was detected in the nucleus in Col-0, and its activity was elevated by the ethylene treatment (Fig. 5F). However, the ethylene-induced E1 activity was not detected in e1-2-2 e2-2 #17 or in e1-2-2 e2-2 e3-2-2 #67 (Fig. 5F and fig. S9D). To further directly monitor PDC acetyl CoA biosynthesis enzyme activity in the nucleus in response to ethylene, we used a ¹³C isotopic tracing experiment. By feeding 2,3-¹³C₂ pyruvate to the nuclear extracts and followed by the LC-MS/MS, we are able to measure 1,2-¹³C₂–labeled acetyl CoA (1,2-¹³C₂ acetyl CoA) that will be converted from 2,3-¹³C₂ pyruvate by PDC complex (Fig. 5G and fig. S9E). This experiment assesses the nuclear PDC activity since PDC is the only enzyme that synthesizes acetyl CoA from pyruvate. As shown in Fig. 5H, we detected 1,2-¹³C₂ acetyl CoA in the nuclear extracts of Col-0 treated with 4 hours of ethylene gas, and the 1,2-¹³C₂ acetyl CoA levels were significantly increased in the presence of ethylene (Fig. 5H and fig. S9F). This suggests that functional PDC complex is accumulated in the nucleus in response to ethylene in Col-0. Further comparison showed no significant differences in the levels of 1,2-¹³C₂ acetyl CoA in the nuclei of Col-0 compared to that of e1-2-2 e2-2 #17 or of e1-2-2 e2-2 e3-2-2 #67 in the absence of ethylene (Fig. 5H and fig. S9F). However, the ethylene-induced elevation of 1,2-¹³C₂ acetyl CoA detected from Col-0 nucleus was significantly reduced from the nucleus of e1-2-2 e2-2 #17 or e1-2-2 e2-2 e3-2-2 #67 (Fig. 5H), providing further biochemical evidence that functional PDC is translocated into the nucleus in response to ethylene to synthesize acetyl CoA.

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Fig. 5.

Nuclear PDC is enzymatically active to produce acetyl CoA.

(A and B) LC-MS detection of acetyl CoA (A) and malonyl CoA (B) in purified nuclei from Col-0 and e1-2-2 e2-2 e3-2-2 #67 etiolated seedlings treated with 4 hours of ethylene gas. Total protein mass was used to normalize metabolite concentration. P values were calculated by a two-tailed t test, and each data point was plotted as a dot in the bar graph. (C) Western blot analysis of the phosphorylation status of E1 using anti-pSer antibody in the cytosolic fraction and nuclear fractions from E1ox seedlings treated with 4 hours of ethylene gas. (D) Phos-tag phosphoprotein mobility shift gel of E1 phosphorylation status in the cytosolic fraction and nuclear fractions of E1ox etiolated seedlings treated with 4 hours of ethylene gas. Black arrow shows nonphosphorylated E1; red arrow indicates phosphorylated E1 protein. PEPC, CytC, and H3 were used to assess purities of nuclear and cytosolic fractionations. (E) The ratios of phosphorylated to nonphosphorylated YHGHpSMSDPGSTYR E1 peptides in the cytosolic versus in the nuclear fractions from E1ox with 4 hours of ethylene gas treatment. Peak area values were collected from three replicates. P value was calculated by t test. (F) Pyruvate dehydrogenase activity assays in the nuclear fraction from Col-0 and indicated pdc mutants treated with 4 hours of ethylene gas. (G) Schematic diagrams to show the conversion of 2,3-¹³C₂ pyruvate to 1,2-¹³C₂ acetyl CoA. (H) Normalized LC-MS/MS measured the concentration of 1,2-¹³C₂ acetyl CoA that converted from 2,3-¹³C₂ pyruvate by using the nuclear extracts from indicated plants with or without 4 hours of ethylene gas treatment. Quantification from four replicates was normalized by total protein input. Different letters represent significant differences between each group calculated by a one-way ANOVA test followed by Tukey’s post hoc test.

Plasmid construction and generation of transgenic Arabidopsis plants

For yeast two-hybrid assays, the full-length coding regions of E1, E2, and E3 were obtained by RT-PCR from Col-0 cDNA and then cloned into pEXPAD502 [activation domain (AD)] and pDBLeu (BD) backbone. For recombinant protein expression, E1 and E2 cDNA was first cloned into pENTR vector and then cloned into pVP13, and E3 cDNA was amplified and inserted into pMAL-pX2 for maltose-binding protein (MBP) fusion. For plant transformation, full-length E1 cDNA was cloned into pENTR vector, and then E1-pENTR was cloned into pEarleyGate 101 vector using recombination reaction between attL and attR sites (LR reaction) to generate E1-YFP-HA. E2 and E3 cDNA was cloned into pCambia1300 to generate E2-FLAG-GFP and E3-FLAG-GFP. To generate PDC-BFP fusion protein, E1, E2, and E3 cDNA was inserted into modified pCambia1300 with replacement of GFP to BFP. All these genes were expressed under the control of the CaMV 35S promoter. The promoter region and genomic sequence of E2 were cloned from Col-0 genomic DNA and integrated into pCambia1300 backbone. These constructs were introduced into Agrobacterium tumefaciens strain GV3101 through electroporation and transformed into Arabidopsis Col-0 ecotype or desired genetic backgrounds using the floral dip method. Positive T1 transformants were selected on MS medium with appropriate antibiotics. Homozygous T3 transgenic seedlings with single T-DNA insertion were used for further analysis. Table S1 contains details of plasmid and primer sequence information.

Yeast two-hybrid assay

Yeast two-hybrid assay was performed using the ProQuest Two-Hybrid System (Invitrogen) by polyethylene glycol–mediated transformation following the manufacturer’s instructions. Briefly, the bait and prey plasmids (pAD-E1, pBD-E1, pAD-E2, pBD-E2, pAD-E3, and pBD-E3) were cotransformed into the yeast strain AH109 as the experiment design. The positive transformants were selected on SD/-Trp-Leu (two-dropout) medium. The growth on SD/-Trp-Leu-His (three-dropout) medium supplemented with appropriate concentrations of 3-amino-1,2,4-triazole indicates interaction between corresponding proteins.

In vitro protein purification and pull-down assays

MBP and recombinant MBP-E1, MBP-E2, MBP-E3, and 6XHis-EIN2-C proteins were expressed in the Escherichia coli strain BL21-CodonPlus(DE3)-RIPL (Agilent) with protein induction by 1 mM IPTG (isopropyl-β-d-thiogalactopyranoside) when OD₆₀₀ (optical density at 600 nm) reached 0.5 for 4 hours at 37°C, and then they were purified using amylose resin column (New England Biolabs, for MBP-related proteins purification) or nickel-nitrilotriacetic acid (Ni-NTA) agarose column (Qiagen, for 6XHis-EIN2-C purification) according to the manufacturer’s menu followed by sonication. MBP-tagged PDC proteins or MBP proteins were added and incubated with 6XHis-EIN2-C–bound Ni-NTA resin for 1 hour at 4°C with gentle rocking. After five times washing with pull-down buffer to remove nonspecific binding, Ni-NTA resin beads were collected by brief centrifugation and then resuspended and denatured in 2× protein sample buffer by boiling. Proteins were then resolved by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and detected with corresponding antibodies.

Confocal imaging and MitoTracker Red staining

Confocal images were acquired by a Zeiss LSM710 confocal microscope with a ×40 (water immersion) or ×63 lens (water immersion). For subcellular localization analysis of EIN2 with MitoTracker staining, true leaves of 3-week-old green seedlings were submerged in ½ MS solution (mock), and 10 μM ACC was dissolved in ½ MS solution (ACC treatment) for 4 hours and then vacuum-infiltrated for 30 min with the addition of 200 nM MitoTracker Red (MitoTracker Red CMXRos, Invitrogen, M7512) before imaging. Images of EIN2-YFP/MitoTracker localization analysis were taken from leaf epidermal cells in central areas in the adaxial side of stained true leaves of Arabidopsis seedlings. For other confocal imaging experiments such as the colocalization of EIN2 and PDC as well as the subcellular localization of PDC under time series of ethylene treatment, 3-day-old etiolated seedlings were used after each indicated treatment, and the images were taken from the similar middle regions of hypocotyls. The excitation/emission spectra for fluorescent proteins and fluorophores used in this study are as follows: YFP, 514 nm/520 to 540 nm; GFP, 488 nm/501 to 528 nm; 4′,6-diamidino-2-phenylindole, 405 nm/437 to 476 nm; BFP, 405 nm/410 to 500 nm, and MitoTracker Red, 579 nm/585 to 615 nm.

Nuclear-cytoplasmic fractionation

Three-day-old etiolated seedlings treated with air or ethylene were first snap-frozen and homogenized in liquid nitrogen (LN2) to fine powders. Approximately 2 g of frozen powders was resuspended in 10 ml of lysis buffer [20 mM tris-HCl (pH 7.4), 20 mM KCl, 2 mM EDTA, 2.5 mM MgCl₂, 25% glycerol, 250 mM sucrose, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1× protease inhibitor cocktail] and then filtered through a 40-μm cell strainer (nylon mesh, Fisher brand). A total of 10% of the flow-through was saved as total extract for the following immunoblot analysis, and the rest of the flow-through was then centrifuged at 1500g for 10 min at 4°C. The supernatant was then transferred to a new tube and centrifuged for 10 min at 10,000g at 4°C, and this supernatant was saved as the cytoplasmic fraction. The pellet was washed for five times with 5 ml of nuclear washing buffer/nuclear resuspension buffer 1 [20 mM tris-HCl (pH 7.4), 2.5 mM MgCl₂, 25% glycerol, 0.2% Triton X-100, 1 mM PMSF, and 1× protease inhibitor cocktail]. Then, the pellet was resuspended in 500 μl of the nuclear resuspension buffer 2 [20 mM tris-HCl (pH 7.4), 10 mM MgCl₂, 250 mM sucrose, 0.2% Triton X-100, 1 mM PMSF, and 1× protease inhibitor cocktail] and then carefully overlaid on top of the 500 μl of the nuclear resuspension buffer 3 [20 mM tris-HCl (pH 7.4), 10 mM MgCl₂, 1.7 M sucrose, 0.2% Triton X-100, 1 mM PMSF, and 1× protease inhibitor cocktail] followed by centrifuge at 16,000g for 45 min at 4°C. The supernatant was then carefully removed, and the final nuclear pellet was saved as the nuclear fraction for the following biochemical assays. The efficiency of the cell fractionation was confirmed by anti–phosphoenolpyruvate carboxylase (anti-PEPC; Abcam, ab347793) for the cytoplasmic fraction, anti–cytochrome C (anti-Cyt C; Agrisera, AS08 343A), anti-fumarase 1 + 2 (anti-FUM1/2; Agrisera, AS16 3966), and anti-ATPB (Agrisera, AS16 3976) for mitochondria contamination examination, anti–luminal-binding protein (anti-BiP; Agrisera, AS09 481) for ER contamination examination, and anti–histone 3 (anti-H3; Abcam, ab1791) for the nuclear fraction in the immunoblots.

Co-IP and immunoblot assays

For co-IP between EIN2-C and PDC following nuclear-cytoplasmic fractionation, the cytoplasmic fraction was used directly for co-IP assays. The nuclear fraction was first sonicated for 30 s by Bioruptor at 4°C to release the nuclear content, and the cleared nuclear lysate after centrifuge at 16,000g for 10 min at 4°C was used for co-IP. A 10% input aliquot for each co-IP experiment was saved from the cleared supernatant before the rest of the supernatant was subject to immunoprecipitation. Cytoplasmic EIN2-YFP-HA full-length proteins and nuclear EIN2-C–YFP–HA proteins were immunoprecipitated by Pierce anti-HA magnetic beads (Thermo Fisher Scientific), PDC FLAG-BFP fusion proteins were immunoprecipitated by DYKDDDDK Fab-Trap agarose beads (ChromoTek) for 2 hours at 4°C with gentle rotating, and then beads were washed five times with ice-cold 1× phosphate-buffered saline buffer. The EIN2-C native antibody was prebound with the equilibrated protein G–coated magnetic beads (Dynabeads, Thermo Fisher Scientific) with 1% bovine serum albumin for 3 hours with gentle rotation at 4°C and incubated at 4°C after cleared cell fractionation addition with gentle rocking overnight for co-IP. Dynabeads was magnetically precipitated using a DynaMagnetic rack (Thermo Fisher Scientific) and then washed five times with 0.5 ml of ice-cold 1× PBS buffer to eliminate nonspecific binding. After IP and washing, proteins were then released from beads using 2× Laemmli sample buffer by heating at 85°C for 8 min and subject to SDS-PAGE. For EIN2-related Western blots, protein samples were denatured in 2× Laemmli sample buffer with the addition of 3 M urea at 75°C in water bath for 5 min to avoid protein aggregation and degradation.

For protein immunoblots, proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane (0.2 μm; Bio-Rad) by the wet-tank transfer method and blocked with 5% nonfat milk in TBST (Tris-Buffered Saline with 0.1% Tween 20) for 1 hour at room temperature before the overnight inoculation in primary antibody at 4°C with gentle rotation. The following antibodies and dilutions were used for immunoblotting: anti-HA (BioLegend, #901503), 1:10,000; anti-FLAG (rabbit) (Cell Signaling Technology, #14793), 1:2000; anti-FLAG (mouse) (Sigma-Aldrich, F3165),1:5000; anti-H3 (Abcam, ab1791), 1:10,000; anti-PEPC (Abcam, ab34793), 1:2000; anti-Cyt C (Agrisera, AS08 343A), 1:2000; anti-FUM1/2 (Agrisera, AS16 3966), 1:2000; anti-BiP (Agrisera, AS09 481), 1:2000; and anti–EIN2-C [developed in-house (20)], 1:2000. Goat anti-mouse kappa light chain antibody (Bio-Rad, #105001G) and goat anti-rabbit [immunoglobulin G (H + L)–horseradish peroxidase (HRP) conjugate #1706515, Bio-Rad] were used as secondary antibodies at 1:10,000 dilution. HRP activity was detected by enhanced chemiluminescence (GE Healthcare) according to the manufacturer’s instructions using either a ChemiDoc Imaging System (Bio-Rad) or conventional x-ray films.

BiFC assay

The BiFC assay was performed following previously published methods with minor revisions (54). Briefly, Agrobacterium strain GV3101 carrying plasmids of interest (EIN2-nYFP, E1-cYFP, E2-cYFP, and E3-cYFP), NLS-BFP/pCambia1300 served as nuclear marker, and p19 was cultured overnight at 28°C, centrifuged, and resuspended in the fresh infiltration buffer [10 mM MES/KOH (pH 5.7), 10 mM MgCl₂, and 100 μM acetosyringone]. The different combinations of BiFC pairs, NLS-BFP strain, and p19 strain were evenly mixed to yield a final OD₆₀₀ of 0.4 of each strain. Then, the mixture was kept at 28°C for 1 hour and then infiltrated into Nicotiana benthamiana (tobacco) leaves. The YFP fluorescence from tobacco epidermal cells was observed under confocal microscopy (Zeiss LSM710) after 2 days of infiltration. For ACC treatment, leaf discs of infiltrated tobacco leaves were submerged in ½ MS solution (mock), and 20 μM ACC was dissolved in ½ MS solution for 4 hours before confocal microscopy.

CRISPR-Cas9 mutagenesis

The e2-2 #17 and e2-2 #215 single mutants, e2-2 e3-2-1 #99 double mutant, and e1-2-2 e2-2 e3-2-1 #167 and e1-2-2 e2-2 e3-2-2 #67 triple mutants were generated by the CRISPR-Cas9 system in Col-0, e3-2-1 single mutant, and e1-2-2 e3-2-1 and e1-2-2 e3-2-2 double mutant backgrounds, respectively, by following the previous publication (55, 56), because E2 and E3 are linked in chromosome 3 in proximity. e2-2 e3-2-2 #67 was generated by out-crossing e1-2-2 e2-2 e3-2-2 #67 to Col-0. CRISPR-Cas9 transgene was removed by out-crossing to Col-0, and Cas9-free homozygous mutants were used for genetic and molecular experiments. Briefly, pairs of two guide RNA (gRNA) sequences targeting E2 locus were designed by using CRISPR-PLANT v2 (http://omap.org/crispr2/index.html) online tool. gRNA sequences with high specificity in Arabidopsis genome and high predicted efficiency were chosen for PCR amplification using pDT1T2 vector as template and later incorporated into the binary vector pHEE401E. Agrobacterium-mediated floral dipping method was used to transform the E2 gRNA containing pHEE401E vectors into each corresponding genetic background. To genotype deletion mutations, plant genomic DNAs extracted from hygromycin-positive T1 plants were used to PCR amplify of E2 gRNA-targeted regions and followed by Sanger sequencing to identify the CRISPR-Cas9–directed mutation and to obtain homozygotes of e2 mutations.

Transcriptome analysis

RNA was extracted from e1-2-2 e2-2 #17 and e2-2 e3-2-2 #67 double mutant etiolated seedlings with or without 4 hours of ethylene gas treatment. RNA extraction was conducted according to the manufacturer’s recommendation using the RNeasy Plant Kit (Qiagen) with deoxyribonuclease I digestion and clean up [Qiagen, RNeasy Mini Kit (250)]. For mRNA-seq, libraries were generated according to the instructions from the manufacturer using the NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490) and the NEBNext Ultra II RNA Library Prep Kit for Illumina (E7770) with 1 μg of RNA as input. Indexed libraries were sequenced on the platform of HiSeq2000 (Illumina). Paired-end raw fastq data were evaluated with FastQC, and low-quality reads were removed with Trim Galore (Babraham Institute). The trimmed and filtered reads were then mapped to the Arabidopsis reference genome (TAIR10) with STAR (57). Mapped reads were counted by featureCounts (Subread 2.0.1), and differentially regulated genes were identified using R package DESeq2 (58) with a P adjusted < 0.05 and |log₂(FC)| > 1. The RNA-seq data for Col-0 air and ethylene treatment were obtained from a previous study [Gene Expression Omnibus (GEO) accession code GSE77396] (26) and reanalyzed with the pipeline described above. For PDC-dependent DEG (compromised DEGs) identification, typical ethylene-responsive up-regulated DEGs were selected from Col-0 RNA-seq dataset based on P adjusted < 0.05 and log₂(FC) > 1 and show P adjusted < 0.05 in pdc mutants air versus C₂H₄ DESeq2 comparison. The scatterplots, violin plots, and dot plots were generated by R package ggplot2, and the heatmaps of log₂FC were generated by R package pheatmap. Gene ontology (GO) enrichment analysis for DEGs was performed by agriGO v2 (59).

Histone extraction

Approximately 2 g of ground fine powders of etiolated seedlings was homogenized in 20 ml of nuclear extraction buffer [250 mM sucrose, 60 mM KCl, 15 mM NaCl, 5 mM MgCl₂, 1 mM CaCl₂, 15 mM Pipes (pH 6.8), 0.5% Triton X-100, 1 mM PMSF, 1× protease inhibitor, and 10 mM sodium butyrate as histone deacetylase inhibitor] for 10 min. After filtering through a single layer of Miracloth twice, the flow-through was then centrifuged at 2880g for 20 min at 4°C to collect the nuclei. The crude nuclear pellet was then resuspended in 800 μl of 0.4 M H₂SO₄ by gentle pipetting and was incubated for 2 hours for complete lysis followed by centrifuge at 16,000g at 4°C. Total histone in the supernatant phase was then precipitated by 33% trichloroacetic acid (TCA) for at least 2 hours and then centrifuged at maximum speed for 10 min at 4°C. The histone pellet was washed twice with ice-cold acetone containing 0.1% HCl and once with pure acetone. The air-dried histone pellet was then dissolved in 2× Laemmli buffer containing 2 M urea, denatured by boiling, and subject to immunoblots.

ChIP-seq analysis

Chromatin immunoprecipitation was performed according to previous publication (24). Briefly, 3-day-old etiolated e1-2-2 e2-2 #17 seedlings treated with air or 4-hour ethylene were harvested and cross-linked in 1% formaldehyde, and the chromatin was isolated and sheared by Bioruptor. Anti-H3K14ac (Millipore; 07-353) and anti-H3K23ac (Millipore; 07-355) antibodies together with Dynabeads were added to the sonicated chromatin followed by incubation overnight to precipitate histone-bound DNA fragments. DNA eluted from chromatin-immunoprecipitated was prepared for library construction by using NEBNext Ultra II DNA Library Prep with Sample Purification Beads according to the standard operating procedures and then sequenced using HiSeq2000 (Illumina). Paired-end raw reads were first mapped to the Arabidopsis genome (TAIR10) using Bowtie2 software with default parameters. Duplicate reads were removed using SAMtools. H3K14ac and H3K23ac ChIP-seq data for Col-0 air and ethylene treatment were obtained from a previous study (GEO accession code GSE77396) (26). Two replicate ChIP-seq files were merged for the following analyses according to a previous publication (26). ChIP-seq coverage tracks shown in the genome browser IGV (Integrative Genomics Viewer) were generated using deepTools 3.5.0 (60) bamCoverage function normalized by reads per genomic content (1× RPGC, 1× normalization) and bin size = 1. The ChIP-seq signals from 2 kb upstream transcription start site to 2 kb downstream transcription end site of PDC specifically regulated gene were calculated with bamCompare and computeMatrix (deepTools) and plotted with plotHeatmap and plotProfile in deepTools or ggplot2 in R.

In vivo E1 phosphorylation detection

Three-day-old E1-YFP-HA transgenic etiolated seedlings after 4 hours of ethylene treatment were first subject to the nuclear-cytoplasmic fractionation procedure with the addition of phosphatase inhibitors (20 mM NaF (sodium fluoride), 1 mM Na₂MoO₄, 10 mM Na₃VO₄, 50 mM β-glycerophosphate, and 1× PhosStop cocktail) to preserve phosphorylation modifications. Nuclear pellets were sonicated briefly by Bioruptor to release nuclear proteins and cleared by centrifuge at 10,000g for 10 min at 4°C. Cytoplasmic and nuclear E1 proteins were then immunoprecipitated by GFP-Trap (ChromoTek) for 2 hours at 4°C with gentle rotating, and then GFP-trap beads were washed five times with IP buffer and boiled in 2× protein sample buffer followed by subsequential immunoblotting. Anti-HA was used to assess E1-YFP-HA loading, and anti-pSer (P3430, Sigma-Aldrich) was used to detect phosphorylated E1 in each fractionation. IPed E1 used for LC-MS/MS phosphorylation detection was also purified following the above-mentioned procedure.

Phos-tag gel electrophoresis assay

The nuclear-cytoplasmic fractionation using 3-day-old E1-YFP-HA transgenic etiolated seedlings was performed with aforementioned buffers’ recipes but without the addition of EDTA and subjected to Phos-tag electrophoresis analysis following the manufacturer’s instructions. Briefly, the E1 proteins from cytosolic fraction and nuclear fraction were resolved by using 6% SDS-PAGE containing 100 μM MnCl₂ and 50 μM Phos-tag (FUJIFILM Wako). After electrophoresis, the poly-acrylamide gel was washed five times with the transfer buffer (20 mM tris, 150 mM glycine, and 20% methanol) with the addition of EDTA (1 mM as final concentration) for 10 min with gentle shaking and then washed twice in the buffer without EDTA 10 min before wet-tank transfer overnight at 50 V using the transfer buffer containing 0.01% SDS followed by immunoblotting.

LC-MS/MS analysis of E1 phosphorylation

After GFP-Trap purification using nuclear and cytoplasmic fractions of 3-day-old E1-YFP-HA transgenic etiolated seedlings, SDS-PAGE, and CBB staining/distaining, E1-YFP-HA protein bands were excised and subjected to in-gel digestion with trypsin. Peptide mixtures were desalted using C18 ZipTips (Millipore). Data were acquired in the data-dependent acquisition mode. Peptides were analyzed by LC-MS/MS on an Easy LC 1200 UPLC (ultra performance liquid chromatography) liquid chromatography system (Thermo Fisher Scientific) connected to a Q Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific). Peptides were first trapped using trapping column Acclaim PepMap 100 (75 μm by 2 cm, nanoViper 2Pk, C18, 3 μm, 100 A) and then separated using analytical column AUR2-25075C18A, 25CM Aurora Series Emitter Column (25 cm by 75 μm, 1.6 μm C18) (IonOpticks). The flow rate was 300 nl/min, and a 120-min gradient was used. Peptides were eluted by a gradient from 3 to 28% solvent B (80% (v/v) acetonitrile/0.1% (v/v) formic acid) over 100 min and from 28 to 44% solvent B over 20 min, followed by a short wash at 90% solvent B. Precursor scan was from mass/charge ratio (m/z) 375 to 1600 (resolution, 120,000; AGC 3.0E6), and top 20 most intense multiply charged precursors were selected for fragmentation. Peptides were fragmented with higher-energy collision dissociation with normalized collision energy. MS/MS data were converted to peaklist using PAVA (peaklist generator that provides centroid MS2 peaklist) (61), and data were searched using Protein Prospector against the TAIR10 database (https://arabidopsis.org/). The precursor mass tolerance was set to 5 ppm, and the MS/MS2 tolerance was set to 20 ppm. Carbamidomethylcysteine was searched as a constant modification. Variable modifications include protein N-terminal acetylation, peptide N-terminal Gln conversion to pyroglutamate, and Met oxidation. Subsequent searches were performed to find peptides modified by phosphorylation. The search parameters were as above but also allowing for phosphorylation on serine, threonine, and tyrosine. A phosphorylated peptide YHGHS(phophos)MSDPGSTYR and an unphosphorylated form YHGHSMSDPGSTYR were identified. Extracted ion chromatography on each precursor ion was extracted using Skyline and quantified from each sample (62). The phosphorylation/nonphosphorylation ratio is calculated by dividing intensity of phosphorylated peptide to that of unmodified peptide and then compared between nuclear and cytosolic fraction after ethylene treatment. Three biological replicates were performed. The mass spectrometry E1 phosphorylation data have been deposited to the ProteomeXchange Consortium via the Proteomics Identification Database (PRIDE) with the dataset identifier PXD040689.

E1 PDH activity assay

Pyruvate dehydrogenase activity was determined by using the PDH Activity Assay Kit (Sigma-Aldrich, MAK183) per the manufacturer’s protocol with adaptation. In short, clean nuclear pellets after nuclear-cytoplasmic fractionation performed on 3-day-old etiolated seedlings from indicated genetic backgrounds were resuspended in 500 μl of PDH assay buffer and later subject to brief sonication shearing and centrifuge to obtain soluble nuclear proteins. Fifty microliters of the cleared nuclear contents was then used to perform the colorimetric enzymatic assay using the Pyruvate dehydrogenase assay kit. The reaction mix was incubated at 37°C. Absorbance at 450 nm was measured by using the Flex Station 3 Multi-Mode Microplate Reader every 1 min and then converted to the amount of NADH (reduced form of nicotinamide adenine dinucleotide; nanomoles per well) generated based on NADH standard curve. Each experimental group was performed with at least three replicates.

We thank D. Hernandez for lab maintenance. We thank C. Petucci and Metabolomics Core of Cardiovascular Institute at University of Pennsylvania for nuclear acetyl CoA LC-MS quantification and K. S. Browning for nuclear-cytoplasmic fractionation setup. We thank P. Oliphint, A. Webb, and Microscopy and Imaging Facility at The University of Texas at Austin for the support of confocal microscope imaging. E1 phosphorylation MS analysis was supported by the Carnegie endowment fund to the Carnegie mass spectrometry facility, and we thank A. V. Reyes for assistance in MS analysis. We thank S. Briggs and JadeBio Inc. for proteomic services. We also thank Arabidopsis Biological Resource Center for the Arabidopsis T-DNA insertion mutants.