Auxin-Induced Modulation of ETTIN Activity Orchestrates Gene Expression in Arabidopsis

Abstract

The phytohormone auxin governs crucial developmental decisions throughout the plant life cycle. Auxin signaling is effectuated by auxin response factors (ARFs) whose activity is repressed by Aux/IAA proteins under low auxin levels, but relieved from repression when cellular auxin concentrations increase. ARF3/ETTIN (ETT) is a conserved noncanonical Arabidopsis thaliana ARF that adopts an alternative auxin-sensing mode of translating auxin levels into multiple transcriptional outcomes. However, a mechanistic model for how this auxin-dependent modulation of ETT activity regulates gene expression has not yet been elucidated. Here, we take a genome-wide approach to show how ETT controls developmental processes in the Arabidopsis shoot through its auxin-sensing property. Moreover, analysis of direct ETT targets suggests that ETT functions as a central node in coordinating auxin dynamics and plant development and reveals tight feedback regulation at both the transcriptional and protein-interaction levels. Finally, we present an example to demonstrate how auxin sensitivity of ETT-protein interactions can shape the composition of downstream transcriptomes to ensure specific developmental outcomes. These results show that direct effects of auxin on protein factors, such as ETT-TF complexes, comprise an important part of auxin biology and likely contribute to the vast number of biological processes affected by this simple molecule.

Figure 1.

Strategy of the Analyses. (A) and (B) Scanning electron microscopy images of wild-type (A) and ett-3 (B) gynoecium apices. Tissues are false-colored: green, valves; pink, style; red, valve margin; orange, replum; blue, stigma. (C) Schematic representation of strategy adopted to compare the data sets obtained through ChIP-seq and RNA-seq analyses. (D) to (H) In situ hybridization with an ETT antisense probe in developing inflorescences. ETT expression can be observed in inflorescence and floral meristem (D), gynoecium and stamen primordia ([E] and [F]), and in developing ovules ([G] and [H]). FM, floral meristem; FP, floral primordia; Gp, gynoecium primordia, IM, inflorescence meristem; Op, ovule primordia; Ov, ovules; rp, replum; SE, sepal; st, style; StP, stamen primordia; sg, stigma; va, valves; vm, valve margin. Bars = 100 µm.

Figure 2.

ETT Regulates Auxin Dynamics in Developing Gynoecium. (A) Representative raw ChIP-seq peaks (only one replicate shown) for PIN1, PIN3, and ASB1/WEI7 target genes and their expression levels in the ett-3 mutant (on the right). Blue rectangles mark the peak regions. Each gene is represented below with blue bars (exons) and lines (introns). (B) and (C) Confocal images of DR5:GFP in wild type (B) and ett-3 mutant (C) at stage 12 of gynoecium development. (D) and (E) Confocal images of DII-VENUS and mDII-VENUS (insets) in the wild type (D) and ett-3 mutant (E) at stage 11 of gynoecium development. (F) and (G) Confocal analyses of PIN1-GFP fusion protein in wild-type (F) and ett-3 (G) stage-10 gynoecia. (H) and (I) Confocal analyses of PIN3-GFP fusion protein in wild-type (H) and ett-3 (I) stage-10 gynoecia. (J) and (K) GUS staining of pASB1:GUS marker line in wild-type (J) and ett-3 (K) stage-14 gynoecia and anthers (inset). (L) and (M) Confocal analyses of TAA1-GFP fusion protein in wild-type (L) and ett-3 (M) stage-10 gynoecia. st, style; sg, stigma; va, valves; vb, vascular bundle. Bars = 100 µm.

Figure 3.

ETT Regulates a Subset of Target Genes in an Auxin-Dependent Manner. (A) Venn diagram of ChIP-seq ±IAA. Schematic representation of the strategy adopted to isolate ETT targets for which binding by ETT is IAA dependent and GO terms corresponding to the isolated set of targets. (B) Representative raw ChIP-seq peaks (one replicate only) for SKP2A, ARR15, IRX14, AP2, FLS1, YUC4, CRF3, and LFY target genes. Blue rectangles mark the peak regions. Each gene is represented below with blue bars (exons) and lines (introns).

Figure 4.

ETT Is Both a Transcriptional Repressor and Activator. (A) to (D) Schematic representation of subdivision in downregulated (magenta arrow) and upregulated (green arrow) targets for each of the data sets emerging from the comparison of ChIP-seq and RNA-seq: 663 set (A), 579 set (B), 136 set (C), and 77 set (D). (E) Schematic representation of GO term frequency associated with targets belonging to the upregulated and downregulated subgroups in the ett-3 mutant background. On the x axis are 15 GO term clusters of molecular function. On the y axis is the percentage of genes associated with the corresponding GO term cluster for each data set in (A) to (D). Black dots indicate the whole set, magenta dots indicate the subgroups of downregulated genes, and green dots indicate the subgroup of upregulated genes. A-B-C-D letters on the top of each GO cluster represent the data sets listed in (A) to (D). (F) Heat map generated with Genevestigator showing expression of the 77-set and 59-set targets (which combined create the 136-set targets). Each set is divided into upregulated (left) and downregulated (right) targets. Color intensity reflects the expression level.

Figure 5.

ETT Recruitment to the TEC3 Target Gene Locus Requires the Protein Partner RPL. (A) Strategy adopted to search for targets shared by RPL and ETT that are differentially regulated ±IAA. Uncharacterized isolated genes are named TEC. (B) ChIP analyses of pETT:ETT-GFP in untreated (yellow bars), IAA-treated (dark red bars), and ett-3 rpl-2 background (green bars) confirming dynamic changes in binding at the TEC2, TEC3, YIP4B, and SMXL5 loci after IAA treatment. Enrichment is represented as fold enrichment relative to the internal control TEC1 (set to 1). (C) ChIP analyses of pRPL:RPL-GFP marker line and wild type as negative control. Error bars indicate sd. Statistical analyses were conducted with one-way ANOVA (Supplemental File 1). *P value < 0.05; ***P value < 0.001.

Figure 6.

ETT Recruitment to TEC3 Locus by RPL Regulates TEC3 Expression. (A) Expression analyses by real-time PCR of TEC3 in wild-type, ett-3, rpl-2, ett-3 rpl-2, and ETT^C2S untreated (left, samples labeled a/e) and IAA-treated (right, samples labeled a+/e+) inflorescences and the comparison between the corresponding values (table). Error bars indicate sd. Statistical analyses were conducted with one-way ANOVA. (B) to (F) In situ hybridization with a TEC3 antisense probe in wild-type, ett-3, rpl-2, ett-3 rpl-2, and ETT^C2S untreated (upper row) and IAA-treated (lower row) shoot apical meristems. Stars indicate the center of the inflorescence meristems, and primordia (P1-6) are numbered from the youngest (1) to the oldest (6). Images in 3D result from the joining of two consecutive sections. A schematic representation of TEC3 expression is illustrated below each genotype. TEC3 signal intensity is shown with different shades of gray: light gray means low expression, and dark gray means strong expression. Blue circle with “E” represents ETT, and green circle with “R” represents RPL. (G) to (K) TEC3 in situ hybridization in ovule primordia of wild-type, ett-3, rpl-2, ett-3 rpl-2, and ETT^C2S untreated (upper row) and IAA-treated (lower row) inflorescences. Black arrowheads point to ectopic expression of TEC3 expanded to the epidermis of young ett-3 and ett-3 rpl-2 gynoecia. pl, placenta; op, ovule primordia, ov, ovule; se, septum. Bars = 50 µm.

Figure 7.

ETT, RPL, and TEC3 Regulate Phyllotactic Pattern in Arabidopsis. (A) Expression pattern of pETT:ETT-GFP (upper panel) and pRPL:RPL-GFP (lower panel) in the shoot apical meristem. (B) to (H) Analysis of angles at which successive flowers develop in wild-type, ett-3, rpl-2, ett-3 rpl-2, ETT^C2S, tec3-1, and tec3-2 plants. Angle classes are indicated on the x axis subdivided into 12 categories. 1, Angles from 0 to 30; 2, angles from 31 to 60; 3, angles from 61 to 90; 4, angles from 91 to 120; 5, angles from 121 to 150; and so on until class 12, angles from 330 to 360. Below, phenotypes of stems of wild-type, ett-3, rpl-2, ett-3 rpl-2, ETT^C2S, tec3-1, and tec3-2 plants. Bars = 50 µm in (A) and 1 cm in (B) to (H).

Figure 8.

ETT Binds to Intronic Regions of a Set of bHLH Transcription Factors. (A) Representative raw ChIP-seq peaks (one replicate only) for TEC1 and TEC4 to TEC6. Green lines represent the amplicons tested by real-time PCR. (B) ChIP analyses with the pETT:ETT-GFP line confirming binding of ETT at the intronic regions of TEC1 and TEC4 to TEC6. Error bars indicate sd. ***P value < 0.001.

Figure 9.

TEC1 Is a bHLH Transcription Factor Involved in Auxin-Related Developmental Responses. (A) Schematic representation of the position of T-DNA insertion in the TEC1 locus. (B) to (F) Scanning electron microscopy images of wild-type (B), ett-3 (C), tec1 (D), and ett-3 tec1 double mutant ([E] and [F]) gynoecium with excessive production of stigmatic tissue (arrowhead). (G) GUS staining of pTEC1:TEC1-GUS marker line in inflorescence, shoot apical meristem (inset), at the lower (abaxial) side, and throughout a developing primary branch (arrowhead). (H) GUS staining of p35S(TEC1-2Intron):GUS in inflorescence and developing branch. (I) Expression profile of pTEC1:TEC1-GUS in ett-3 mutant background in the gynoecium and at the lower (abaxial) side of a developing primary branch (arrowhead). (J) Yeast-two-hybrid interaction assay between ETT and TEC1 and respective negative control with empty AD and BD vectors. Interactions are tested on selection media lacking Trp, Leu, His, and adenine (-W-L-H-A) and supplemented with 100 µM IAA. (K) to (O) Emergence of extra accessory shoots (marked with an asterisk) in the wild type (K), tec1 (L), ett-3 (M), and ett-3 tec1 (N) and its frequency in percentage (O). Statistical analysis is conducted with one-way ANOVA. *P < 0.01 ms, main stem; pb, primary branch; va, valves; sg, stigma; st, style. Bars = 100 µm in (B) to (F), (G) (meristem), and (I) (gynoecium) and 5 mm in (G) and (I) (branches) and (K) to (N).

Figure 10.

Proposed Model of Action for ETT and Auxin Effects on ETT Dimerization and Transcription. ETT controls expression of target genes either alone (transcriptome A, t’ome A) or in combination with process-specific protein partners (“X”) (t’ome B). At increased auxin levels, dimerization of ETT with a set of partners is affected, thereby releasing ETT from a set of genomic loci, ultimately leading to a different transcriptional outcome (t’ome C). As auxin destabilizes ETT dimerization, it also promotes ETT association with a set of target sequences possibly through interaction of ETT with other partners (“Z”) (t’ome D).