EMBO/soda-nel huggingface dataset:
Tyr-Asp inhibition of glyceraldehyde 3-phosphate dehydrogenase affects plant redox metabolism. (2021)
Figure 3

Figure 3. Tyr-Asp improvement of Arabidopsis stress tolerance is associated with the GAPC1/2 inhibition.

(A-C) Fresh weight measurements of Arabidopsis wild-type and gapc1/gapc2 double k.o. plants subjected to the oxidative stress (e.g. catechin) and Tyr-Asp treatments. Each treatment was compared to its respective control, corresponding to plants grown in one 24-well plate (represented by the adjoined bars).

(D-F) Representative Arabidopsis wild type and gapc1/gapc2 double k.o. seedlings from the different treatments. All plants were 10-day-old at the stress onset. The seedlings were exposed to catechin for three days.

Data information: Data are mean ± SEM of n=10-12 (seedlings). Unpaired two-tailed Student t-test was performed to compare treatments with control. Scale bar: 10 cm. Data from an independent experiment are shown in (Appendix Figure 7).
Tyr-Asp inhibition of glyceraldehyde 3-phosphate dehydrogenase affects plant redox metabolism. (2021)
Figure 6

Figure 6. AthPEPCK1 activity is inhibited by dipeptides.

(A-B) Recombinant AthPEPCK1 activity was measured at increasing concentrations of six different H-P dipeptides (A) or Tyr-Asp (B). I0.5 values are indicated on each graph. Measurements were performed in the decarboxylation direction, as described in the Methods section. Data were adjusted to a modified Hill equation. The value of 1 corresponds to the activity of 4.3 ± 0.2 U mg-1. Data are mean ± SEM of 4 independent measurements.

(C) PEPCK activity was measured in a protein lysate prepared from 3-week-old Arabidopsis leaves in absence (control, C) or presence of 500 µM of the different dipeptides. Measurements were performed in the carboxylation direction, as described in the Methods section. Data are mean ± SE of 4 independent lysate preparations. Unpaired two-tailed Student t-tests were performed to compare dipeptide treatments with the control.
HDAC6 regulates cellular viral RNA sensing by deacetylation of RIG-I (2015)
Figure 1

Figure 1. HDAC6-/- mice are susceptible to lethal RNA virus infection and show decreased cytokine production.

A. The survival of age- and sex-matched HDAC6+/+ mice and HDAC6-/- mice was monitored for 9 days after intravenous VSV-Indiana infection (2 × 108 pfu/mouse; n = 12 per group; log-rank test).

B. Determination of the viral load in organs by standard plaque assay. HDAC6+/+ and HDAC6-/- mice were intravenously infected with VSV-Indiana, and the brain and spleen were collected at 5 dpi (2 × 108 pfu/mouse; n = 6 per group; Mann-Whitney test).

C. Determination of viral loads in organs by Q-PCR of VSV viral transcripts. The brain and spleen were collected at 5 dpi after HDAC6+/+and HDAC6-/- mice were intravenously infected with VSV-Indiana (2 × 108 pfu/mouse; n = 9 per group; Mann-Whitney test).

D. The viral load in the serum was assessed by standard plaque assay. HDAC6+/+ and HDAC6-/- mice were intravenously infected with VSV-GFP. Serum was collected at the indicated time points and analyzed (4 × 108 pfu/mouse; n = 3 per group; Student’s t-test).

E. ELISA of IFN-β (left) and IL-6 (right) in the serum of mice described in (d) (Student’s t-test).

F. ELISA of serum IFN-β (left) and IL-6 (right) after poly(I:C) injection. HDAC6+/+ and HDAC6-/- mice were intravenously injected with poly(I:C). Serum was collected at the indicated time points and analyzed (200 μg/mouse; n = 3 per group; Student’s t-test).

Data information: Error bars, mean ± SEM. *p < 0.05, **p < 0.01
HDAC6 regulates cellular viral RNA sensing by deacetylation of RIG-I (2015)
Figure 2

Figure 2. HDAC6 positively regulates the innate antiviral response in bone marrow derived macrophages and peripheral blood mononuclear cells.

A, B. virus replication at 12 and 24 hpi, in HDAC6+/+ and HDAC6-/- BMDMs in response to VSV-GFP (MOI = 10) infection (A) and PR8-GFP (MOI = 5) infection (B).

C. Virus replication in HDAC6+/+ and HDAC6-/- BMDMs in response to HSV-GFP (MOI = 2) infection.

D. Virus replication in HDAC6+/+ and HDAC6-/- PBMCs in response to VSV-GFP (MOI = 10) infection.

E. ELISA of IFN-β (upper), IL-6 (lower) levels in the supernatant of (A) and (B), and in HDAC6+/+ and HDAC6-/- BMDMs treated with poly(I:C) (20 µg/ml) or transfected with 5’ppp-dsRNA (1 µg/ml).

F. Immunoblot analysis of the phosphorylated and inactive forms of IRF3, IKBα, TBK1, RIG-I, MAVS, HDAC6, and β-actin at the indicated times (0, 2, 4, 8, and 16 h) in HDAC6+/+ and HDAC6-/- BMDMs. BMDMs were stimulated with PR8-GFP (MOI = 3).

G. Induction of mRNA for type I IFN, IL-6, and other IFN-related antiviral genes in HDAC6+/+ and HDAC6-/- BMDMs in response to a RIG-I agonist stimulation at 6 h. HDAC6+/+ and HDAC6-/- BMDMs were stimulated with 5’ppp-dsRNA (0.5 µg/ml) for 6 h.

Data information: Data are representative of at least two independent experiments. Error bars, mean ± SD. *p < 0.05, **p < 0.01, ***P < 0.001 (Student’s t test)
HDAC6 regulates cellular viral RNA sensing by deacetylation of RIG-I (2015)
Figure 3

Figure 3. HDAC6 deficiency reduces the innate immune response on mouse macrophage and mouse embryonic fibroblast.

A, B. Fluorescence microscopy at 24 hpi showing green fluorescence absorbance at 12 and 24 hpi, and virus replication at 12 and 24 hpi, in control and HDAC6 knockdown RAW264.7 cells in response to VSV-GFP (MOI = 1) infection (A) and PR8-GFP (MOI = 1) infection (B). bar, 100µm

C. ELISA of IFN-β (upper), IL-6 (lower) levels in the supernatant of (A) and (B), and in control and HDAC6 knockdown RAW264.7 cells treated with poly(I:C) (20 µg/ml) or transfected with 5’ppp-dsRNA (1 µg/ml).

D, E. Fluorescence microscopy at 24 hpi showing green fluorescence absorbance at 12 and 24 hpi, and virus replication at 12 and 24 hpi, in HDAC6+/+ and HDAC6-/- MEFs in response to VSV-GFP (MOI = 1) infection (D) and PR8-GFP (MOI = 1) infection (E). bar, 50µm

F. ELISA of IFN-β (upper), IL-6 (lower) levels in the supernatant of (D) and (E), and in HDAC6+/+ and HDAC6-/- MEFs transfected with poly(I:C) (1 µg/ml).

Data information: Data are representative of at least two independent experiments. Error bars, mean ± SD. *p < 0.05, **p < 0.01, ***P < 0.001 (Student’s t test)
HDAC6 regulates cellular viral RNA sensing by deacetylation of RIG-I (2015)
Figure 4

Figure 4. Deacetylase activity is responsible for the antiviral effect of HDAC6.

A, B. Fluorescence microscopy at 24 hpi showing green fluorescence absorbance at 12 and 24 hpi, and virus replication at 12 and 24 hpi, in vector, HDAC6, or HDAC6-CDM-overexpressing stable RAW264.7 cells in response to VSV-GFP (MOI = 1) infection (A) and PR8-GFP (MOI = 1) infection (B). bar, 100µm.

C. ELISA of IFN-β (upper) and IL-6 (lower) levels in the supernatant of (A), (B), and in vector, HDAC6, or HDAC6-CDM-overexpressing stable RAW264.7 cells treated with poly(I:C) (20 μg/ml) or transfected with 5’ppp-dsRNA (1 µg/ml).

D, E. Luciferase assay in 293T cells transfected with an IFN-β luciferase promoter and TK-Renilla together with mutant HDAC6 or HDAC6-CDM (100, 200, 400, or 800 ng), followed by PR8-GFP (MOI = 2) infection (D) or by poly(I:C) (E) (1 μg/ml) transfection for another 12 h.

F, G. Luciferase assay in 293T cells transfected with RIG-I (F), MDA-5 (G), an IFN-β luciferase promoter, and TK-Renilla together with mutant HDAC6 or HDAC6-CDM (100, 200, 400, or 800 ng). 24 h later, IFN-β activity was measured by luciferase reporter assay.

Data information: Data are representative of three independent experiments. Error bars, mean ± SD. *p < 0.05, **p < 0.01, ***P < 0.001 (Student’s t test)
HDAC6 regulates cellular viral RNA sensing by deacetylation of RIG-I (2015)
Figure 5

Figure 5. HDAC6 interacts with and deacetylates RIG-I in response to RNA viral infection.

A. 293T cells were transfected with poly(I:C) and cell lysates were prepared at the indicated times for co-immunoprecipitation analysis of endogenous HDAC6 and RIG-I. Actin was used as the loading control.

B. BMDMs were isolated from HDAC6+/+ and HDAC6-/- mice and were transfected with 5ˊppp-dsRNA. Whole cell lysates were prepared after 8 h of transfection and co-immunoprecipitation analysis of RIG-I and HDAC6 was performed.

C. HeLa cells were transfected with 5ˊppp-dsRNA. Cells were stained with anti-RIG-I (red) and anti-HDAC6 H300 (green) antibodies after 8 h of transfection. Nuclei were stained with DAPI (blue) (bar, 20µm).

D. Fluorescence microscopy, green fluorescence absorbance and virus replication at 24hpi in RIG-I+/+ and RIG-I-/- MEFs. Cells were transfected with si-HDAC6 and si-control for 36 h, followed infection with VSV-GFP (MOI = 1). bar, 100µm.

E. HDAC6+/+, HDAC6-/- MEFs, and HDAC6-/- MEFs reconstituted with HDAC6 wild-type were subjected to immunoprecipitation using an anti-RIG-I antibody and immunoblotted with anti-acetyl-lysine and anti-RIG-I antibodies. Acetyl-tubulin was measured in total lysates to determine HDAC6 deacetylase activity.

F. Whole cell lysates from HDAC6+/+ and HDAC6-/- MEFs were prepared and used in dsRNA pull-down assays. Pull-down samples were analyzed by western blotting with anti-RIG-I, anti-HDAC6, and anti-β-actin antibodies. Intensity of pull-downed RIG-I was quantified.

G. Whole cell lysates from HDAC6-/- MEFs and HDAC6-/- MEFs reconstituted with HDAC6 wild-type and HDAC6-CDM were prepared and used in dsRNA pull-down assays. Samples were analyzed by western blotting with anti-RIG-I and anti-HDAC6 antibodies. Intensity of pull-downed RIG-I was quantified.

Data information: Data are representative of three independent experiments. Error bars, mean ± SD. *p < 0.05, **p < 0.01, ***P < 0.001 (Student’s t test)
HDAC6 regulates cellular viral RNA sensing by deacetylation of RIG-I (2015)
Figure 6

Figure 6. HDAC6-dependent RIG-I deacetylation regulates RIG-I binding to double stranded RNA.

A, B. 293T cells were transfected with the indicated expression plasmids. At 1 day post-transfection, whole cell lysates were subjected to dsRNA pull-down assays. Pull-down samples were analyzed by western blotting with an anti-FLAG antibody.

C. A luciferase assay was performed using 293T cells transfected with the IFN-β luciferase promoter and TK-Renilla together with 2 ng of RIG-I wild-type, RIG-I 858Q, RIG-I 858R, RIG-I K909Q, or RIG-I K909R mutant, followed by transfection with poly(I:C) (1 μg/ml) for another 12 h.

D. Fluorescence microscopy, green fluorescence absorbance, and virus replication at 24 hpi in RIG-I-/- MEFs. Cells were transiently transfected with empty vector, RIG-I wild-type, RIG-I K909Q, or RIG-I K909R mutant for 24 h, followed infection with VSV-GFP (MOI = 1). bar, 100µm

E. HDAC6-/- MEFs were transfected with the indicated expression plasmids. At 1 day post-transfection, whole cell lysates were prepared and used in dsRNA pull-down assays. Pull-down samples were analyzed by western blotting with anti-RIG-I and anti-β-actin antibodies.

F. 293T cells were transfected with 2 μg of FLAG-tagged RIG-I and 1, 5, or 10 μg of V5-tagged HDAC6. At 36 h post-transfection, whole cell lysates were prepared for co-immunoprecipitation analysis of V5-tagged HDAC6 and acetylated K909 (on RIG-I wild-type). Samples were analyzed by western blotting with a K909 acetylation-specific antibody.

G. 293T cells were transfected with 2 μg of FLAG-tagged RIG-I. At 24 h post-transfection, cells were infected with VSV-GFP (MOI = 0.1). At 2 and 4 h post-infection, whole cell lysates were prepared for immunoprecipitation analysis of acetylated K909 (on RIG-I wild-type). Samples were analyzed by western blotting with a K909 acetylation-specific antibody. Intensity of ac-909K normalized to Flag-RIG-I was quantified.

H. whole cell lysates were prepared for immunoprecipitation analysis of acetylated K909 (on RIG-I wild-type). Samples were analyzed by western blotting with a K909 acetylation-specific antibody. Intensity of ac-909K normalized to RIG-I was quantified.

Data information: Data are representative of three independent experiments. Error bars, mean ± SD. *p < 0.05, **p < 0.01, ***P < 0.001 (Student’s t test)
Adjustment of the PIF7-HFR1 transcriptional module activity controls plant shade adaptation (2020)
Figure 2

Figure 2. Levels of HFR1 transcript are higher in C. hirsuta than A. thaliana seedlings.

Seedlings of ChWT and AtWT were grown for 3 days in W then either kept under the same conditions or transferred to W+FR (R:FR, 0.02) for the indicated times. Plant material was harvested every 24 h. Transcript abundance of HFR1 and PIF7 was normalized to three reference genes (EF1α, SPC25, and YLS8). Expression values are the means ± SE of three independent biological replicates relative to the data of AtWT grown in continuous W at day 3. Asterisks mark significant differences (2-way ANOVA: ** p-value <0.01, *** p-value <0.001) between ChWT and AtWT when grown under W (black asterisks) or W+FR (red asterisks).
Adjustment of the PIF7-HFR1 transcriptional module activity controls plant shade adaptation (2020)
Figure 3

Figure 3. The activity of ChHFR1 is higher than that of AtHFR1 in A. thaliana seedlings.

A Cartoon of constructs containing ChHFR1 or AtHFR1 under the HFR1 promoter of A. thaliana (pAtHFR1) used to complement hfr1-5 mutant of A. thaliana (At hfr1-5).

B Relative expression of HFR1 in seedlings of AtWT, At hfr1-5, hfr1>pAt:ChHFR1 (in blue) and hfr1>pAt:AtHFR1 (in red) lines grown under W+FR (R:FR, 0.02). Expression values are the means ± SE of three independent biological replicates relative to the data of 7 days old AtWT. Transcript abundance is normalized to UBQ10 levels.

C Elongation response of seedlings of the indicated lines grown for 7 days in continuous W or 2 days in W then transferred for 5 days to W+FR (R:FR, 0.02). The mean hypocotyl length in W (HypW) and W+FR (HypW+FR) of at least four biological replicates was used to calculate HypW+FR-HypW. Error bars represent SE.

D Relative HFR1 protein levels in seedlings of the indicated lines, normalized to actin protein levels, are the means ± SE of three independent biological replicates relative to hfr1>pAt:ChHFR1 line #22 that is taken as 1. Seedlings were grown for 7 days in continuous W (~20 µmol m-2 s-1) after which they were incubated for 3 h in high W (~100 µmol m-2 s-1) and transferred to W+FR (R:FR, 0.06) for 3 h.

Data information: Different letters denote significant differences (one-way ANOVA with Tukey test, p-value <0.05) among means.
Adjustment of the PIF7-HFR1 transcriptional module activity controls plant shade adaptation (2020)
Figure 4

Figure 4. ChHFR1 and AtHFR1 proteins show different stability in shade.

A Expression of HFR1 and protein levels of HFR1-3xHA in seedlings of hfr1>pAt:ChHFR1 (line #22) and hfr1>pAt:AtHFR1 (line #13). Seedlings were grown for 7 days in continuous W (~20 µmol m-2 s-1) after which they were incubated for 3 h in high W (~100 µmol m-2 s-1) and then either kept at high W or transferred to W+FR (R:FR, 0.06) for 3 or 6 h, as indicated in the cartoon at the top. Relative HFR1 transcript levels, normalized to UBQ10, are the means ± SE of three independent biological replicates relative to hfr1>pAt:ChHFR1 #22 grown for 3 h under W+FR. Relative protein levels, normalized to actin, are the means ± SE of three independent biological replicates relative to hfr1>pAt:ChHFR1 #22. Samples were collected at data points marked in the cartoon with asterisks.

B Cartoon of constructs containing ChHFR1 or AtHFR1 under the 35S promoter used for transient expression of transgenes in N. benthamiana leaves.

C Relative HFR1 transcript levels transiently expressed in tobacco leaves, normalized to the GFP, are the means ± SE of three independent biological replicates (left). Relative HFR1 protein levels, normalized to the GFP levels, are the means ± SE of four independent biological replicates (right). In A and C, asterisks mark significant differences (Student t-test: * p-value <0.05, ** p-value <0.01) between the indicated pairs.

D Degradation of ChHFR1 (35S:ChHFR1) and AtHFR1 (35S:AtHFR1) in tobacco leaf discs treated with cycloheximide (CHX, 100 µM) for the indicated times. Tobacco plants were kept under high W (~200 µmol m-2 s-1) for 3 days after agroinfiltration and then leaf circles were treated with W+FR (R:FR, 0.2) and CHX. Relative HFR1 protein levels (ChHFR1, blue bars; AtHFR1, red bars), normalized to the GFP levels, are the means ± SE of four biological replicates relative to data point 0, taken as 1 for each line. Asterisks mark significant differences (2-way ANOVA: * p-value <0.05) between ChHFR1 and AtHFR1 at the same time point.
Adjustment of the PIF7-HFR1 transcriptional module activity controls plant shade adaptation (2020)
Figure 5

Figure 5. AtHFR1 interacts more strongly than ChHFR1 with the WD40 domain of COP1.

A Overview of the COP1 WD40-AtHFR1 complex (PDB ID 6QTV). The COP1 WD40 domain and the AtHFR1 VP peptide are shown in surface representation and colored in blue and orange, respectively. The N-terminus of HFR1 VP peptide, the amino acid of which differs between AtHFR1 and ChHFR1, is highlighted in magenta.

B Table summaries of the microscale thermophoresis binding assay (see Fig EV5). The sequence of the respective synthetic peptides is indicated.

C Cartoon of constructs containing ChHFR1, AtHFR1, ChHFR1* and AtHFR1* derivatives under the 35S promoter used for transient expression of transgenes in N. benthamiana leaves.

D Relative HFR1 protein levels, normalized to the GFP levels, are the means ± SE of four independent biological replicates. Asterisks mark significant differences (Student t-test: * p-value <0.05, ** p-value <0.01) between the indicated pairs.
Adjustment of the PIF7-HFR1 transcriptional module activity controls plant shade adaptation (2020)
Figure 6

Figure 6. AtHFR1 interacts with AtPIF7.

A Y2H growth assay showing the interaction between AtHFR1 and AtPIF7. The BD- and the AD- derivative constructs used in the assay are shown on the left side of the panel. SD-LW or SD-HLW refer to the selective medium (plated as drops in dilutions of 1, 1:10 and 1:100) indicative of transformed cells or interaction between the hybrid proteins, respectively. Truncated forms of murine p53 (BD-fused) and SV40 large T-antigen (AD-fused), known to interact, were used as a positive control. Empty vectors (/) were used as negative controls.

B, C Hypocotyl length of seedlings of AtWT, (B) pif7-1, hfr1-5, pif7-1 hfr1-5 (top graph), pif7-2, hfr1-5 and pif7-2 hfr1-5 (bottom graph) mutants, and (C) transgenic 35S:GFP-ΔNt-HFR1 (35S:ΔNt-HFR1), two lines of 35S:PIF7-CFP (35S:PIF7 #1 and #2), and 35S:GFP-ΔNt-HFR1 35S:PIF7-CFP double transgenic (35S:ΔNt-HFR1 x 35S:PIF7 #1 and #2) seedlings grown under different R:FR. Seedlings were grown in W (R:FR > 1.5) for 7 days or for 2 days in W and then transferred to two W+FR treatments (R:FR 0.06 or 0.02) for 5 additional days. Values of hypocotyl length are the means ± SE of three independent biological replicates (at least 10 seedlings per replica).

D Aspect of representative 7-day-old W-grown seedlings shown in C. Scale bar is 1 cm.
Adjustment of the PIF7-HFR1 transcriptional module activity controls plant shade adaptation (2020)
Figure 7

Figure 7. C. hirsuta has an attenuated hypocotyl elongation at warm temperature and delayed dark-induced senescence (DIS).

A In AtWT, PIFs promote hypocotyl elongation as a response to warm temperature (28ºC). High ChHFR1 activity is expected to inhibit this response by repressing PIFs more effectively in ChWT and attenuate hypocotyl elongation at 28ºC.

B Seedlings were grown for 7 days in W at either 22ºC, 2 days at 22ºC then transferred to 28ºC for additional 5 days (22ºC > 28ºC) or for 7 days at 28ºC, as represented in the panel.

C Hypocotyl length of seedlings of (left) AtWT, pifq, pif7-2, ChWT, (middle) 35S:GFP-ΔNt-HFR1 (ΔNtHFR1), hfr1-5 and (right) chfr1-1 and chfr1-2 lines grown at warm temperatures. Hypocotyl lengths are the means ± SE of three biological replicates. Asterisks mark significant differences (Student t-test: * p-value <0.05, ** p-value <0.01) relative to the same genotype grown at 22ºC (left and right graphs, black asterisks), and between the indicated pairs (middle graph, red asterisks).

D In AtWT, PIF-mediated DIS involves a reduction of chlorophyll levels. HFR1 activity might inhibit DIS through repression of PIFs. If PIF activity is attenuated in ChWT, DIS would be delayed in this species compared to AtWT.

E Seedlings were grown for 7 days in W and then transferred to total darkness for several days to induce senescence, as illustrated at the right panel.

F Relative chlorophylls levels of (left) AtWT, pifq, ChWT, (middle) ΔNtHFR1, hfr1-5 and (right) chfr1-1 and chfr1-2 lines after DIS was promoted for the indicated time. For each genotype, values are relative to pigment levels at time 0 (7 days in W). Data are the means ± SE of four independent biological replicates.

G Aspect of 4-day old dark-grown seedlings of AtWT, pifq, pif7-2, hfr1-5 and ΔNt-HFR1 (left panel), and AtWT, ChWT and chfr1-1 (right panel).
ARF2-PIF5 interaction controls transcriptional reprogramming in the ABS3-mediated plant senescence pathway (2022)
Figure 1

Figure 1. ARF2 and PIF5 are required for ABS3-mediated senescence.

A, Seedlings of WT, abs3-1D, FD11-55, and arf2-20 before and after 6 d C-deprivation.

B, Seedlings of WT, abs3-1D, FD18-70, and pif5-10 before and after 6 d C-deprivation.

C and D, RT-qPCR analysis of ABS3 expression levels in 7-d-old seedlings of the same genotypes as shown in A (C) and B (D). Fold changes were calculated with respect to the expression level in the WT. two-tailed t test, ****, p<0.0001, n.s., not significant.

E and F, Chlorophyll and total cellular protein content reduction in genotypes shown in A (E) and B (F) after 6 d C-deprivation.

G and H, RT-qPCR analysis of ORE1 and SGR1 expression levels in genotypes shown in A (G) and B (H) before and after 4 d C-deprivation. Fold changes were calculated with respect to the expression level in the WT before C-deprivation.

I and J, Suppressor mutation sites in FD11-55 (I) and FD18-70 (J). In the gene model, boxes and lines represent exons and introns, respectively. 5'- and 3'-untranslated regions (UTRs) are shaded in grey. The arrow head indicates the position of the suppressor mutation site.

In C-H, data are means ± standard deviation (s.d.) of 3 biological replicates.
ARF2-PIF5 interaction controls transcriptional reprogramming in the ABS3-mediated plant senescence pathway (2022)
Figure 2

Figure 2. ARF2 and PIF5 control the transcriptional reprogramming in the ABS3-mediated senescence pathway.

A and B, Top 10 significantly enriched GO terms derived from genes that are up-regulated (A) or down-regulated (B) in abs3-1D seedlings compared with the WT under C-deprivation. Enriched GO terms were ranked based on their false discovery rate (FDR).

C, Heatmap of log2FC values of 2,587 ABS3-regulated genes (abs3-1D vs. WT) under C-deprivation in indicated comparisons.

D, Percentage of ARF2- or PIF5-dependent abs3-1D-regulated genes under C-deprivation. Genes that were not significantly up- or down-regulated by abs3-1D in the arf2-20 (arf2-20 abs3-1D vs. arf2-20) or pif5-10 (pif5-10 abs3-1D vs. pif5-10) background under C-deprivation were defined as ARF2- or PIF5-dependent ABS3-regulated genes, respectively.

E, Significant overlap between ARF2-, PIF5-, and C-deprivation-regulated genes in the abs3-1D background.

F, Heatmap of log2FC values of 1,326 ARF2, PIF5, and C-deprivation co-regulated genes in indicated comparisons. C-deprivation: abs3-1D 4 d C-deprivation vs. abs3-1D 0 d C-deprivation; ARF2: abs3-1D vs. arf2-20 abs3-1D after 4 d C-deprivation; PIF5: abs3-1D vs. abs3-1D pif5-10 after 4 d C-deprivation. Enlarged were expression changes of senescence genes identified in DAVID GO analysis (https://david.ncifcrf.gov/).

G, PIF5 and PIF4 binding motifs were over-represented in the promoter regions of ARF2, PIF5, and C-deprivation co-regulated genes. Enriched motifs were identified with the AME tool in MEME Suite 5.3.3 (https://meme-suite.org/meme/tools/ame) using 1000 bp promoter sequences.