Identification of transcription factors that regulate ATG8 expression and autophagy in Arabidopsis

Abstract

Autophagy is a conserved catabolic process in eukaryotes that contributes to cell survival in response to multiple stresses and is important for organism fitness. In Arabidopsis thaliana, the core machinery of autophagy is well defined, but its transcriptional regulation is largely unknown. The ATG8 (autophagy-related 8) protein plays central roles in decorating autophagosomes and binding to specific cargo receptors to recruit cargo to autophagosomes. We propose that the transcriptional control of ATG8 genes is important during the formation of autophagosomes and therefore contributes to survival during stress. Here, we describe a yeast one-hybrid (Y1H) screen for transcription factors (TFs) that regulate ATG8 gene expression in Arabidopsis, using the promoters of 4 ATG8 genes. We identified a total of 225 TFs from 35 families that bind these promoters. The TF-ATG8 promoter interactions revealed a wide array of diverse TF families for different promoters, as well as enrichment for families of TFs that bound to specific fragments. These TFs are not only involved in plant developmental processes but also in the response to environmental stresses. TGA9 (TGACG (TGA) motif-binding protein 9)/AT1G08320 was confirmed as a positive regulator of autophagy. TGA9 overexpression activated autophagy under both control and stress conditions and transcriptionally up-regulated expression of ATG8B, ATG8E and additional ATG genes via binding to their promoters. Our results provide a comprehensive resource of TFs that regulate ATG8 gene expression and lay a foundation for understanding the transcriptional regulation of plant autophagy.Abbreviations: ABRC: Arabidopsis biological resource center; AP2-EREBP: APETALA2/Ethylene-responsive element binding protein; ARF: auxin response factor; ATF4: activating transcription factor 4; ATG: autophagy-related; ChIP: chromatin immunoprecipitation; DAP-seq: DNA affinity purification sequencing; FOXO: forkhead box O; GFP: green fluorescent protein; GO: gene ontologies; HB: homeobox; LD: long-day; LUC: firefly luciferase; MAP1LC3: microtubule associated protein 1 light chain 3; MDC: monodansylcadaverine; 3-MA: 3-methyladenine; OE: overexpressing; PCD: programmed cell death; qPCR: quantitative polymerase chain reaction; REN: renilla luciferase; RT: room temperature; SD: standard deviation; TF: transcription factor; TFEB: transcription factor EB; TGA: TGACG motif; TOR: target of rapamycin; TSS: transcription start site; WT: wild-type; Y1H: yeast one-hybrid.

Keywords: ATG8 promoters; Arabidopsis thaliana; TGA9; autophagy; transcription factors; yeast one-hybrid screen.

Figure 1.

Expression of ATG8 genes under abiotic stress. (a) 7-d-old Arabidopsis seedlings were transferred to ½ MS medium with sucrose (control), without sucrose (-SUC) and without nitrogen (-N) for 3 d, and the transcript level for each ATG8 gene was quantified by real-time qPCR. (b) 7-day-old Arabidopsis seedlings were transferred to ½ MS medium with 0.35 M mannitol or 0.16 M NaCl for 6 h, and the transcript level for each ATG8 gene was quantified by real-time qPCR. (c) Relative expression of ATG8 genes in 4-week-old plants after 5 d dark treatment to cause fixed-carbon starvation. Data were extracted from the published RNA-sequencing dataset GSE93420 [56]. Expression of each gene was normalized to expression in control conditions and represented as the mean of 3 biological replicates, except for fixed-carbon starvation which had 2 biological replicates. Error bars indicate SD. Differences are significant at p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***) by Student’s t test.

Figure 2.

Y1H screen to identify regulators of ATG8B and ATG8E gene expression. (a) Promoter schematic of ATG8B and ATG8E genes. Red lines are fragments used for the Y1H screen. TSS indicates transcription start site. (b) Experimental design of Y1H assays. The library of TFs and ATG8 gene promoter fragments were introduced into yeast mating strains, diploids selected, and OD₆₀₀ and β-galactosidase reporter activity measured. (c) Venn diagram of the number of TFs identified for ATG8B and ATG8E promoters. (d) Comparisons between ATG8-Y1H data and DAP-seq data. DAP-seq TF binding profiles for 336 TFs in the TF library were used to assess the overlap between ATG8 promoter binding and Y1H targets identified in our screen. Overlap significance is shown as – log10(p-value) as calculated in GeneOverlap by Fisher’s exact test.

Figure 3.

TF-promoter interaction network. The ATG8B and ATG8E promoters are indicated by yellow rectangles; interacting TFs are ovals color-coded based on different families. C2H2: Cys(2)His(2) zinc finger domain family; GeBP: GLABROUS1 enhancer binding protein family; HB: homeobox family; NAC: NAC-domain containing protein family; bZIP: basic leucine-zipper protein family; WRKY: WRKY DNA-binding protein family. The definitions and gene IDs are provided in Excel S1 for all the TFs shown in the figure.

Figure 4.

Enrichment of TF families and validation of Y1H candidates in Arabidopsis protoplasts. (a) Enrichment for TF families that bind to the ATG8B promoter. (b) Enrichment for TF families that bind to the ATG8E promoter. Families shown are statistically significant (p < 0.05), calculated using Fisher’s exact test. (c) Validation of Y1H candidates by transiently co-expressing each TF with an ATG8B promoter-luciferase fusion. (d) Validation of Y1H candidates by transiently co-expressing each TF with an ATG8E promoter-luciferase fusion. Candidates were selected based on TF family enrichment. The definitions and gene IDs are provided in Excel S1 for all the TFs shown in the figure. Dual-luciferase (REN and LUC) activities were monitored for each reaction. Data were normalized to a vector control for each promoter and shown as the average value ± SD of 3 biological replicates. Differences are significant at p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***) by Student’s t test.

Figure 5.

Characterization of the TGA9-ATG8B and TGA9-ATG8E promoter interaction. (a) Schematic diagram of the promoter regions (~2000 bp upstream of start codon) of ATG genes. TGA motifs (TGACG) are indicated by blue triangles, and their directions indicate sense or antisense strands. Red arrows are TSS. P represents primer pairs for ChIP-qPCR. Green boxes indicate fragments used for the Y1H screen. (b) Binding of TGA9 to promoter regions and mutated promoter regions of ATG8B and ATG8E in yeast. Bars represent the fold induction of β-galactosidase activity, shown as the average value ± SD of 3 biological replicates. (c) Binding of TGA9 to the promoter regions of ATG8B and ATG8E in Arabidopsis protoplasts. ChIP assays were performed with 35S::GFP-TGA9 and 35S::GFP-GUS as a control after transient expression in protoplasts. (d) Binding of TGA9 to the promoters of selected ATG genes in plants. ChIP assays were performed with 35S::GFP-TGA9 transgenic seedlings and WT (Col) as a control. For (C) and (D), sonicated chromatin was immunoprecipitated with either anti-GFP or anti-IgG antibody. Immunoprecipitated DNA was quantified by real-time quantitative PCR with primers specific for TGA-binding motifs in the promoters. Primers located at −3000 ~ −4000 bp upstream of the promoters were used as negative controls (pro-Neg) for (C). ACTIN2 is a negative control for (D). Fold induction was normalized to anti-IgG and wild-type controls. Data represent means ± SD from 3 biological replicates. For all panels, differences are significant at p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***) by Student’s t test.

Figure 6.

Activation of autophagy by TGA9 under sucrose starvation. (a) Expression of selected ATG genes in 2 TGA9 OE lines and the tga9-3 mutant after sucrose starvation for 3 d. (b) MDC-labeled autophagosomes in roots of seedlings of the indicated genotypes under control or sucrose starvation conditions. White arrows indicate MDC-labeled puncta. Scale bar: 20 μm. (c) Quantified autophagosome numbers per unit area in the seedlings shown in (B) and shown as means ± SD from 3 biological replicates, with 10–30 images per replicate. (d) mCherry-ATG8E-labeled autophagosomes in mesophyll protoplasts from the indicated genotypes with or without TGA9 expression under control or sucrose starvation conditions. White arrows indicate mCherry-ATG8E-labeled autophagosomes. Scale bar: 10 μm. (e) The percentage of protoplasts with 3 or more mCherry-ATG8E-labeled autophagosomes in samples from (D). Bars indicate means ± SD from 3 biological replicates, with 100 protoplasts per sample per replicate. For all panels, different letters indicate significant differences at p < 0.05 by Student’s t test.

Figure 7.

Activation of autophagy by TGA9 under mannitol-induced osmotic stress. (a) Expression of selected ATG genes in 2 TGA9 OE lines and the tga9-3 mutant after osmotic stress for 6 h. (b) MDC-labeled autophagosomes in roots of seedlings of the indicated genotypes under control or osmotic stress conditions. White arrows indicate MDC-labeled puncta. Scale bar: 20 μm. (c) Quantified autophagosome numbers per unit area in the seedlings shown in (B) and shown as means ± SD from 3 biological replicates, with 10–30 images per replicate. (d) mCherry-ATG8E-labeled autophagosomes in mesophyll protoplasts from the indicated genotypes with or without TGA9 expression under control or osmotic stress. White arrows indicate mCherry-ATG8E-labeled autophagosomes. Scale bar: 10 μm. (e) The percentage of protoplasts with 3 or more mCherry-ATG8E-labeled autophagosomes in samples from (D). Bars indicate means ± SD from 3 biological replicates, with 100 protoplasts per sample per replicate. For all panels, different letters indicate significant differences at p < 0.05 by Student’s t test.

Figure 8.

Activation of autophagy by TGA9 overexpression is compromised in atg5 mutant protoplasts or by 3-MA. (a, b) TGA9 was transiently overexpressed in WT and atg5 mutant protoplasts, with co-expression of mCherry-ATG8E, followed by sucrose starvation for 2 d (a) or osmotic stress for 6 h (b). The percentage of protoplasts with 3 or more mCherry-ATG8E-labeled autophagosomes was determined by epifluorescence microscopy. (c) The percentage of protoplasts with 3 or more mCherry-ATG8E-labeled autophagosomes when transiently overexpressing TGA9 in WT with or without 5 mM 3-MA under sucrose starvation for 2 d. (d) The percentage of protoplasts with 3 or more mCherry-ATG8E-labeled autophagosomes when TGA9 expression was induced by β-estradiol in OE438 with or without 5 mM 3-MA under sucrose starvation for 2 d. For A-D, bars indicate means ± SD from 3 biological replicates, with 100 protoplasts per sample per replicate. (e) Quantified MDC-labeled puncta per unit area in seedlings of WT and OE438 with or without 5 mM 3-MA after sucrose starvation for 3 d. Data are shown as means ± SD from 3 biological replicates, with 10–30 images per replicate. For all panels, different letters indicate significant differences at p < 0.05 by Student’s t test.

Figure 9.

Effects of alterations in TGA9 expression on survival after long-term sucrose starvation. (a) Seedling phenotype after long-term sucrose starvation in the dark followed by 7 d recovery in the light. (b) Percentage of surviving seedlings from (A). (c) Seedling phenotype after 0 d, 8 d and 9 d sucrose starvation with or without 2 mM 3-MA in the dark followed by 7 d recovery in light. (d) Percentage of surviving seedlings from (C). For all panels, seedlings remaining green or with new growth emerging are considered as surviving. Data indicate means from 3 biological replicates and error bars indicate SD. Differences are significant at p < 0.05 (*) by Student’s t test.