Activation of YUCCA5 by the Transcription Factor TCP4 Integrates Developmental and Environmental Signals to Promote Hypocotyl Elongation in Arabidopsis

Abstract

Cell expansion is an essential process in plant morphogenesis and is regulated by the coordinated action of environmental stimuli and endogenous factors, such as the phytohormones auxin and brassinosteroid. Although the biosynthetic pathways that generate these hormones and their downstream signaling mechanisms have been extensively studied, the upstream transcriptional network that modulates their levels and connects their action to cell morphogenesis is less clear. Here, we show that the miR319-regulated TCP (TEOSINTE BRANCHED1, CYCLODEA, PROLIFERATING CELL FACTORS) transcription factors, notably TCP4, directly activate YUCCA5 transcription and integrate the auxin response to a brassinosteroid-dependent molecular circuit that promotes cell elongation in Arabidopsis thaliana hypocotyls. Furthermore, TCP4 modulates the common transcriptional network downstream to auxin-brassinosteroid signaling, which is also triggered by environmental cues, such as light, to promote cell expansion. Our study links TCP function with the hormone response during cell morphogenesis and shows that developmental and environmental signals converge on a common transcriptional network to promote cell elongation.

Figure 1.

The miR319-Targeted TCP Genes Promote Hypocotyl Elongation. (A) and (B) Seven-day-old seedlings (A) and their average hypocotyl lengths (B). n = 10 to 12. (C) Schematic diagram of mTCP4:GR construct. Synonymous mutations (red) were introduced in the miR319 target region on TCP4 ORF to generate a miR319-resistant version of TCP4 (mTCP4) without changing the protein sequence (Palatnik et al., 2003). LB and RB refer to left border and right border of T-DNA, respectively; HPT, hygromycin phosphotransferase; GR, glucocorticoid receptor. (D) Relative transcript levels of TCP4 determined by RT-qPCR analysis of 9-d-old seedlings. Average of three independent biological samples is shown. Transcript levels were normalized to that of PP2A. (E) and (F) Seven-day-old seedlings grown in the absence (M, Mock) or presence of 12 µM DEX (E) and their average hypocotyl length (F). n = 10 to 12. (G) Relative hypocotyl length of DEX-induced seedlings (a ratio of length under inductive condition to that under non-inductive condition). n = 12. (H) A correlation analysis between TCP4 transcript level and relative hypocotyl length in three TCP4 gain-of-function lines. (I) and (J) Scanning electron microscopy images of the hypocotyls of 7-d-old seedlings (I) grown in the presence or absence of 12 µM DEX and their average epidermal cell length (J). The average cell length was based on 120 to 150 cells from four hypocotyls. Representative cells are highlighted in red, green, and blue (I). Bars = 1 mm in (A) and 2 mm in (E). Error bars indicate sd in (B), (D), (F), and (G) and se in (J). Asterisk indicates P < 0.05; unpaired Student’s t test was used to determine significant differences relative to the Col-0 values ([B], [D], and [G]) or the mock values (F). In (H) and (J), 35S;GR, Col-0;GR, and jaw-D;GR denote Pro35S:mTCP4:GR, ProTCP4:mTCP4:GR, and jaw-D;ProTCP4:mTCP4:GR plants, respectively.

Figure 2.

Altered Auxin and BR Responses of TCP4-Induced Hypocotyl Elongation. (A) Seven-day-old jaw-D;ProTCP4:mTCP4:GR seedlings grown without (M, Mock) or with 12 µM DEX in the presence of 2 µM GA₃, 0.5 µM PPZ, 0.4 µM epiBL, or 1 µM PIC (− indicates no chemicals). (B) Average hypocotyl lengths of the seedlings shown in (A) (n = 10 to 15). (C) Relative hypocotyl lengths of 7-d-old seedlings grown in the presence of 12 µM DEX and various concentrations of GA₃. (D) and (E) Eight-day-old Pro35S:mTCP4:GR seedlings (D) and their hypocotyl lengths (E) grown without (M, Mock) or with 12 µM DEX in the presence of 0.5 µM PPZ. (F) Relative hypocotyl lengths of 7-d-old seedlings grown in the presence of 12 µM DEX and various concentrations of epiBL. (G) Relative hypocotyl lengths of 7-d-old Pro35S:mTCP4:GR seedlings grown without (Mock) or with 12 µM DEX and various concentrations of PIC. Bars = 2 mm in (A) and 1 mm in (D). Error bars indicate sd. Asterisk indicates P < 0.05; ns indicates not significant. An unpaired Student’s t test was used to assess significance. In (C) and (F), jaw-D;GR denotes jaw-D;ProTCP4:mTCP4:GR seedlings.

Figure 3.

TCP4 Upregulates Auxin- and BR-Responsive Genes. (A) Venn diagram of global transcriptome analysis of 9-d-old jaw-D;ProTCP4:mTCP4:GR seedlings treated with 12 µM DEX for 2 or 4 h. (B) Genevestigator database (https://genevestigator.com/gv/) analysis showing that the auxin-related genes that were altered in the microarray of jaw-D;ProTCP4:mTCP4:GR plants largely overlapped with the previously reported transcriptome profiles of IAA-treated and naphthaleneacetic acid (NAA)-treated seedlings. The top 14 correlated transcriptome profiles of IAA/NAA-treated microarrays are shown. The original Genevestigator -generated figure is provided in Supplemental Figure 4. (C) to (F) Relative transcript levels of the indicated genes determined by RT-qPCR analysis in 9-d-old jaw-D;ProTCP4:mTCP4:GR seedlings treated with 12 µM DEX for the given durations ([C] and [E]) or of the indicated genotypes ([D] and [F]). TCP4:VP16 denotes ProTCP4:TCP4:VP16 plants. All transcript levels were first normalized to PP2A/TUB2 transcripts and then the test samples were compared with the respective controls. Error bars indicate sd. Asterisk indicates P < 0.05; ns indicates not significant. An unpaired Student’s t test was used. Averages from three independent biological samples are shown.

Figure 4.

TCP4 Directly Upregulates YUC5. (A) Relative transcript levels determined by RT-qPCR analysis of 9-d-old jaw-D;ProTCP4:mTCP4:GR seedlings treated either with 40 µM CHX alone or with a combination of 40 µM CHX and 20 µM DEX (CHX DEX) for 4 h. (B) RT-qPCR analysis of the relative transcript levels in 9-d-old Pro35S:mTCP4:GR seedlings treated with 40 µM CHX or CHX plus 20 µM DEX for 4 h. Transcript levels were first normalized to PP2A ([A] and [B]) and compared with the level with only CHX treatment (Con). For both (A) and (B), error bars indicate sd. Asterisk indicates P < 0.05; ns indicates not significant. An unpaired Student’s t test was used. Averages from biological triplicates are shown. (C) Schematic representation of the YUC5 genomic region and the four predicted TCP4 binding motifs (BS1 to BS4) in the upstream region (black line) and coding region (gray box) of the locus. Arrow indicates the predicted translation start site. The triangle indicates the T-DNA insertion site in the yuc5 line. (D) Electrophoretic mobility shift assay gel showing retardation of radiolabeled oligonucleotides containing BS1-BS4 (shown in [C]) by recombinant TCP4Δ3-MBP protein. The + and the − symbols show the presence and the absence of the indicated compounds, respectively. A 250-fold (left panel) or 25- to 150-fold (right panel) higher concentration of unlabeled oligonucleotides was used as competitors. Anti-MBP antibody was used for the super shift reactions.

Figure 5.

TCP4 Induces the YUC-Dependent Auxin Response. (A) and (B) GUS reporter analysis of 6-d-old seedlings grown without (Mock) or with 12 µM DEX. jaw-D;GR denotes the jaw-D;ProTCP4:mTCP4:GR genotype. (C) Average hypocotyl length of 7-d-old seedlings grown with or without 12 µM DEX. Error bars indicate sd. (D) GUS analysis of 6-d-old seedlings grown without (Mock) or with 12 µM DEX. (E) Relative transcript levels determined by RT-qPCR analysis in 5-d-old seedlings (transcript abundances are provided in Supplemental Table 3). Mock and DEX indicate the absence and presence of 12 μM DEX, respectively. All transcript levels were first normalized to PP2A transcripts and then the test samples were compared with the respective controls. Error bars indicate sd. Asterisk indicates P < 0.05; ns indicates not significant. An unpaired Student’s t test was used. Averages from three independent biological samples are shown. (F) GUS analysis of representative cotyledon tips from 6-d-old seedlings grown on medium containing the indicated chemicals; 12 µM DEX and 20 µM l-kyn were used. (G) Hypocotyl length analysis of 8-d-old Pro35S:mTCP4:GR seedlings grown on medium containing mock (gray filled circle) or DEX (black circles) with the indicated concentrations of l-kyn. Error bars indicate sd. Bars in (A), (B), and (D) = 1 mm.

Figure 6.

TCP4-Induced Hypocotyl Elongation Requires Auxin Signaling. (A) and (B) Seven-day-old seedlings grown without or with 12 µM DEX (A) and their hypocotyl lengths averaged from 10 to 12 seedlings (B). jaw-D;GR denotes the jaw-D;ProTCP4:mTCP4:GR genotype. Bar in (A) = 2 mm. (C) Relative transcript level of YUC5 determined by RT-qPCR analysis in 10-d-old seedlings of the indicated genotypes grown without (Mock) or with 12 µM DEX. (D) Relative transcript levels of the indicated genes determined by RT-qPCR analysis in 10-d-old seedlings of the indicated genotypes grown without (Mock) or with 12 µM DEX. Averages from three independent biological samples are shown in (C) and (D). Transcript levels were first normalized to PP2A/TUB2 and then the test samples were compared with the respective controls (Mock). Error bars indicate sd; asterisk indicates P < 0.05; an unpaired Student’s t test was used.

Figure 7.

TCP4-Induced Hypocotyl Elongation Is Dependent on BR Signaling. (A) and (B) Seven-day-old seedlings grown without (M) or with 12 µM DEX (A) and their hypocotyl lengths averaged from 12 to 15 seedlings (B). jaw-D;GR denotes the jaw-D;ProTCP4:mTCP4:GR genotype. (C) Relative increase in hypocotyl length upon DEX induction over mock. (D) and (E) Seven-day-old seedlings (D) and their average hypocotyl lengths (E) of the indicated genotypes grown with or without 12 µM DEX in the presence of 250 nM PPZ. n = 10 to 12. (F) Relative transcript level in 5-d-old jaw-D;ProTCP4:mTCP4:GR plants grown on medium supplemented with 12 µM DEX and/or 0.5 mm PPZ. Transcript levels were normalized to PP2A. n = 3. Bars = 2 mm in (A) and 1 mm in (D). Error bars indicate sd; asterisk indicates P < 0.05. An unpaired Student’s t test was used.

Figure 8.

TCP4 Requires PRE Genes for Hypocotyl Elongation. (A) Seven-day-old seedlings grown without (M) or with 12 µM DEX (left) and their hypocotyl lengths averaged from 10 to 12 seedlings (right). jaw-D;GR denotes the jaw-D;ProTCP4:mTCP4:GR genotype. Bar = 2 mm. Error bars indicate sd. (B) Schematics of a proposed pathway of miR319-targeted TCP-mediated regulation of hypocotyl cell elongation. Both the TCP-mediated developmental signal (shade of blue) and PIF-mediated environmental signal (shade of pink) converge on YUC-dependent auxin biosynthesis (shade of violet). TCP4 is enlarged and in bold, since its contribution was investigated in greater detail in this study. The resulting auxin response utilizes the common central circuit ARF-BZR1 (shown in a box) to activate the downstream regulators (such as PRE and SAUR) to promote hypocotyl cell elongation. Red arrows indicate direct transcriptional activation, black arrows indicate the direct/indirect influence of the downstream mechanism, the T-bar indicates transcript degradation, and the double-sided arrow indicates protein-protein interaction.