Arabidopsis TIE1 and TIE2 transcriptional repressors dampen cytokinin response during root development.

Cytokinin plays critical roles in root development. Cytokinin signaling depends on activation of key transcription factors known as type B Arabidopsis response regulators (ARRs). However, the mechanisms underlying the finely tuned regulation of type B ARR activity remain unclear. In this study, we demonstrate that the ERF-associated amphiphilic repression (EAR) motif-containing protein TCP interactor containing ear motif protein2 (TIE2) forms a negative feedback loop to finely tune the activity of type B ARRs during root development. Disruption of TIE2 and its close homolog TIE1 causes severely shortened roots. TIE2 interacts with type B ARR1 and represses transcription of ARR1 targets. The cytokinin response is correspondingly enhanced in tie1-1 tie2-1. We further show that ARR1 positively regulates TIE1 and TIE2 by directly binding to their promoters. Our findings demonstrate that TIEs play key roles in controlling plant development and reveal an important negative feedback regulation mechanism for cytokinin signaling.

INTRODUCTION

The plant root system is essential for plant survival because of its critical role in absorption of nutrients and water from the soil and provision of anchorage. Thus, the developmental plasticity of roots is essential for adaptation of sessile plants to diverse soil conditions (). In Arabidopsis, the mature root is longitudinally divided into the stem cell niche (SCN), the proliferation zone (PZ), the transition zone (TZ), the elongation zone (EZ), and the differentiation zone (DZ) (). When the roots grow, the stem cells surrounding the quiescent center (QC) in the SCN asymmetrically divide to generate stem cell daughters. The daughter cells further divide several times in the PZ and then transit to the EZ via the TZ. Cells in the EZ elongate longitudinally and enter the DZ for differentiation (). The plasticity of root growth and development is determined by coordinated cell division, elongation, and differentiation in the PZ, TZ, EZ, and DZ, which are tightly regulated by various internal hormones and external conditions ().

The phytohormone cytokinin is one of the most important hormones governing root development (). Genetic and mutant analysis indicates that cytokinin negatively regulates the size of the root meristem by repressing cell division in the PZ and promoting cell differentiation in the TZ (–). Disruption of genes in cytokinin biosynthesis or signaling leads to a larger root meristem (). In the EZ, cytokinin inhibits cell elongation by altering the direction of microtubule disposition (). Cytokinin applications result in shorter roots (), while the disruption of type B Arabidopsis response regulators (ARRs), which play key roles in cytokinin signaling, produces longer roots in arr1, arr1 arr12, and arr1 arr10 arr12 mutants (, ). In the DZ, cytokinin positively regulates root hair development. Application of cytokinin increases the density of root hairs (). In addition, cytokinin represses the emergence of lateral roots and responses to environmental conditions (, ). These findings suggest that cytokinin regulates root morphology by participating in almost all aspects of root development, and thus, tight regulation of cytokinin signal output is pivotal for root developmental plasticity.

Cytokinin is perceived by a group of Arabidopsis histidine kinase (AHK) receptors and triggers a multistep phosphorelay, in which AHKs are first autophosphorylated and the phosphate is lastly relayed to a class of ARR transcription factors via Arabidopsis histidine protein 1 to 5 (AHP1 to AHP5) to elicit the cytokinin response (, ). Eleven type B, 10 type A, and 2 type C ARRs comprise the Arabidopsis ARR family. The type B ARRs contain an N-terminal receiver domain and a C-terminal MYB-like DNA binding domain, whereas type A ARRs lack a DNA binding domain (, ). Phosphorylation of a conserved aspartate (Asp) in the receiver domain by cytokinin leads to the activation of type B ARRs (). Type B ARRs mediate major cytokinin-dependent transcriptional outputs and directly regulate thousands of genes (–). Tight regulation of the activity of type B ARRs is very important for fine-tuning of the cytokinin response at appropriate spatiotemporal scales and, thus, for plant developmental plasticity (–). To accurately tune cytokinin outputs, some genes, including cytokinin oxidases (CKX), AHK4, AHP6, and type A ARRs are up-regulated by cytokinin and, in turn, dampen cytokinin signaling in negative feedback loops (). CKXs decrease the level of cytokinin by mediating cytokinin degradation (). AHK4 acts as a phosphatase and dephosphorylates AHPs in the absence of cytokinin (). AHP6 has no conserved phosphorylation site and competes with other AHPs for interaction with AHK receptors, while type A ARRs may compete with type B ARRs for the phosphate transferred by AHPs (). In addition, KISS ME DEADLY (KMD) family genes encoding F-box proteins negatively regulate the cytokinin response by forming S-phase kinase-associated protein1/Cullin/F-box protein E3 ubiquitin ligase complexes to mediate several type B ARR proteins for degradation via the 26S proteasome (). However, the molecular mechanisms by which plants finely control cytokinin outputs in various developmental contexts remain to be elucidated.

The ethylene response factor (ERF)–associated amphiphilic repression (EAR) motif has been identified as a plant-specific repression domain with a conserved short sequence LXLXL (L indicates leucine, and X represents any amino acid) (). More than 200 transcriptional regulators in the Arabidopsis genome have been found to contain the EAR motif (). Many of them have been identified to be essential for different biological processes (). For example, EAR motif–containing proteins are repeatedly used by plants to control hormone signaling pathways. Auxin/indole-3-acetic acid (AUX/IAA), novel interactor of jaz (NINJA), suppressor of more axillary growth2 1-like (SMXL), ABI five binding protein (AFP), and brassinazole-resistant 1 (BZR1) proteins are well-known EAR motif–containing transcriptional repressors that play pivotal roles in the auxin (), jasmonic acid (JA) (), strigolactone (SL) (), abscisic acid (ABA) (), and brassinosteroid (BR) pathways (), respectively. It has been proposed that these different EAR motif–containing proteins recruit topless/topless-related (TPL/TPR) co-repressors and histone deacetylases (HDAs) to cause local chromatin condensation and, thus, the repression of key transcription factors in hormone signaling pathways (). Although cytokinin causes dynamic changes in chromatin condensation for which type B ARRs are necessary (), no EAR motif–containing transcriptional repressors have yet been found to regulate the activity of type B ARR transcription factors in the cytokinin signaling pathway.

We previously identified TCP interactor containing EAR motif protein 1 (TIE1) by analyzing a T-DNA activation tagging curly leaf mutant tie1-D (). TIE1 contains a C-terminal EAR motif that recruits TPL/TPRs to repress the activity of teosinte branched1/cycloidea/proliferating cell factor (TCP) family transcription factors during leaf development and shoot branching (, ). We further demonstrated that TIE1 is regulated by degradation via the 26S proteasome, and we showed that the degradation of TIE1 is mediated by TIE1-associated ring-type E3 ligase1 (TEAR1) (). TIE1 has three close homologs in the Arabidopsis genome: TIE2, TIE3, and TIE4. They all contain EAR motifs at the C terminus and interact with TPL/TPR co-repressors (). Overexpression of TIE2, TIE3, or TIE4 causes curly leaves resembling those observed in TIE1 overexpression mutant tie1-D (), suggesting that these four proteins may have similar biochemical functions. Among these four TIEs, TIE2 is predominantly expressed in roots (). However, the roles of TIE2 and other TIEs during root development are still unknown.

In this study, we generated a tie1-1 tie2-1 double mutant and observed that tie1-1 tie2-1 produced obviously shorter roots, indicating that TIE1 and TIE2 play an important role in root elongation. TIE1 and TIE2 are predominantly expressed in the EZ and DZ and specifically control the length of cells in the EZ and DZ. We show that the N terminus of TIE2 interacts with several type B ARR transcription factors, including ARR1 and ARR2, and the C-terminal EAR motif is required for its repression activity. The expression of an ARR1 or ARR2 fusion protein with the C terminus of TIE2 rescues the shorter roots of tie1-1 tie2-1. We further demonstrate that ARR1 directly up-regulates TIE1 and TIE2 by binding to their promoter regions. Therefore, our findings elucidate the important roles of TIE1 and TIE2 in the control of root developmental plasticity and establish an unknown negative feedback loop in cytokinin signaling during root development. In this loop, cytokinin activates type B ARRs to directly promote transcription of TIE1 and TIE2, and the increased abundance of TIE1 and TIE2, in turn, suppresses cytokinin signaling by inhibiting the activity of type B ARRs.

RESULTS

The double mutant tie1-1 tie2-1 produced short roots

TIE2 is a homolog of TIE1 that acts as a transcriptional repressor. In comparison with TIE1, which is expressed in both roots and shoots, TIE2 is predominantly expressed in roots (). To reveal the possible roles of TIE2 in root development, we first generated a tie2-1 mutant with a 7–base pair (bp) deletion in the first exon of the TIE2 gene using CRISPR-Cas9 technology (fig. S1B). The roots of tie2-1 had no obvious phenotype when compared to those of wild-type plants (fig. S1, D and E). To overcome potential functional redundancy, we identified a new tie1-1 (GABI_372F04) mutant, in which T-DNA was inserted into the first exon of TIE1 (fig. S1A). The T-DNA insert disrupted the function of TIE1 in tie1-1 (fig. S1C). The tie1-1 mutant also displayed roots that were comparable to those of the wild-type control (fig. S1, D and E). Next, we crossed tie2-1 and tie1-1 to generate the tie1-1 tie2-1 double mutant. We observed that tie1-1 tie2-1 displayed severely shorter roots and produced obviously fewer lateral roots (Fig. 1, A to D). To confirm that the root defects in tie1-1 tie2-1 were caused by disruption of TIE1 or TIE2, we generated TIE1pro-TIE1-GFP (green fluorescent protein) and TIE2pro-TIE2-GFP constructs, in which the sequence encoding GFP at the C-terminal end of TIE1 or TIE2 was driven by their own promoters, respectively. Either TIE1pro-TIE1-GFP or TIE2pro-TIE2-GFP transformation completely rescued the shorter roots of tie1-1 tie2-1 (Fig. 1E and fig. S2). We next investigate the root length of an activation tagging mutant tie1-D in which TIE1 gene is overexpressed (). The results showed that tie1-D produced longer roots and exhibited a root phenotype opposite to tie1-1 tie2-1 (fig. S3, A to D). These results indicate that TIEs play key roles in root elongation.

Fig. 1.

The tie1-1 tie2-1 double mutant displayed severely shorter roots and fewer lateral roots.

(A) Five-day-old wild type and tie1-1 tie2-1. (B) Fourteen-day-old wild type and tie1-1 tie2-1. (C) The length of roots from wild type and tie1-1 tie2-1. Data are means ± SD (n = 21) of three independent experiments. (D) The density of lateral roots from wild type and tie1-1 tie2-1. Data are means ± SD (n = 34 for wild type and n = 67 for tie1-1 tie2-1) of three independent experiments. (E) Complementation of tie1-1 tie2-1 by transforming TIE1pro-TIE1-GFP or TIE2pro-TIE2-GFP. (F and G) Observation of the root meristem, EZ, and DZ of the longitudinal roots from wild type and tie1-1 tie2-1. (H) Propidium iodide (PI) staining of roots shows the elongation of cells in the EZ and the DZ of roots from wild type (top) and tie1-1 tie2-1 (bottom). (I) The cell number of the root meristems from wild type and tie1-1 tie2-1. Data are means ± SD (n = 17) of three independent experiments. (J and K) CyclinB1;1:GUS staining of the root meristem in the wild-type or tie1-1 tie2-1 background. (L) Statistical analysis of the length of GUS staining regions in the wild-type or tie1-1 tie2-1 roots shown in (J and K). Data are means ± SD (n = 22) of three independent experiments. (M) The length of cells in the EZ and DZ of roots from 4- and 5-day-old wild type and tie1-1 tie2-1. Data are means ± SD (n = 38) of three independent experiments. Scale bar, 1 mm (A, B, and E) and 25 μm (F to H, J, and K). Student’s t test was used for significance testing, ***P < 0.001 in (C, D, I, L, and M).

To comprehensively assess the root phenotypes of tie1-1 tie2-1, we first performed starch staining with Lugol’s solution (), which showed that the distribution of starch granules and the columella cell differentiation in the roots of tie1-1 tie2-1 had no obvious differences from that of wild-type plants (fig. S4, A and B). We then introduced the QC marker WUSCHEL-related homeobox 5 (WOX5)::GFP into tie1-1 tie2-1 (fig. S4, C and D) (). GFP fluorescence was clearly observed in the QC of tie1-1 tie2-1 and wild-type controls. These results indicate that the QC identity and the columella cell differentiation have no obvious defects in the roots of tie1-1 tie2-1. We next investigated the size of the root meristem, as indicated by the number of cells in a cortex file from the QC to the first elongated cell. The statistical analysis showed that the size of the root meristem in the roots of tie1-1 tie2-1 was also not substantially different from that of the wild-type controls (Fig. 1, F, G, and I). We confirmed that the root meristem was not affected by disruption of TIE1 and TIE2 in tie1-1 tie2-1 by introducing Cyclin B1;1:GUS (β-glucuronidase) into tie1-1 tie2-1 and GUS staining analysis (Fig. 1, J to L). We then observed the cells in the EZ and DZ of tie1-1 tie2-1. The results showed that cell elongation in the EZ and DZ was severely affected in tie1-1 tie2-1 (Fig. 1, H and M), and the cells were significantly shorter than those of the wild-type control (Fig. 1M). These results indicate that TIE1 and TIE2 control root length by promoting cell elongation in the EZ and thus increasing the cell length in the DZ.

TIE1 and TIE2 have overlapping expression in the EZ and DZ

To elucidate the spatiotemporal expression pattern of TIE1 and TIE2 during root development, we generated TIE1pro-GUS and TIE2pro-GUS constructs, in which the GUS gene was driven by a 2790-bp-long TIE1 promoter or a 5104-bp-long TIE2 promoter. The roots of more than 30 independent TIE1pro-GUS or TIE2pro-GUS transgenic lines displayed similar expression patterns. We subjected one stable TIE1pro-GUS line and one stable TIE2pro-GUS line to detailed analysis. In 1-day-old germinated seeds, TIE1 was found to be expressed in the cotyledons, while no expression of TIE1 was observed in the protruded root tips (Fig. 2A). TIE1 started to be expressed in the proximal part of the root, but not in the root tip, of 1.5-day-old TIE1pro-GUS plants (Fig. 2B). The expression pattern was maintained as plants grew, with no expression in the root tip (Fig. 2, C to G). Strong GUS staining was found in the roots, excluding the root tips, in the 2-, 3-, 4-, 5-, and 9-day-old TIE1pro-GUS seedlings (Fig. 2, C to G). GUS staining was also observed at the initial sites of lateral roots (Fig. 2G). The young lateral roots had a similar expression pattern, with GUS staining in the proximal parts but not in the lateral root tips (Fig. 2G). The expression pattern shown by TIE1pro-GUS in the vasculature and root tip is consistent with the results that we reported previously (). In comparison with TIE1, no GUS staining was observed in the root of 1-day-old germinated seed from TIE2pro-GUS plant (Fig. 2H). TIE2 was clearly expressed in the roots of the 2-, 3-, 4-, 5-, and 9-day-old seedlings (Fig. 2, I to M). A small region neighboring the root tip never showed GUS staining in the roots (Fig. 2, I to M). As the plants grew, the GUS staining faded in the root region near the hypocotyl (Fig. 2, J to M). The expression of TIE2 was also found at the initial sites of lateral roots (Fig. 2M). The expression pattern of the young lateral roots was like that of the main roots (Fig. 2M). To further determine the expression patterns of TIE1 and TIE2 in the root tip region, we observed the GFP fluorescence of TIE1pro-TIE1-GFP and TIE2pro-TIE2-GFP, which complemented the shorter root phenotype of tie1-1 tie2-1. Clear GFP fluorescence was observed in the TZ and DZ, but not in the root meristem, in TIE1pro-TIE1-GFP roots (Fig. 2, N to Q). In TIE2pro-TIE2-GFP roots, GFP fluorescence was found in the TZ, the DZ, columella cells, and lateral root cap (Fig. 2, R to U). These results suggest that both TIE1 and TIE2 are expressed in specific root domains, and the expression patterns of TIE1 and TIE2 overlap in the EZ and DZ, corresponding to the shorter cells in the EZ and DZ in the roots of tie1-1 tie2-1.

Fig. 2.

The expression patterns of TIE1 and TIE2 in the roots.

(A to G) GUS staining of the stable TIE1pro-GUS transgenic line. (A) A 1-day-old seedling. (B) A 1.5-day-old seedling. (C) A 2-day-old seedling. (D) A 3-day-old seedling. (E) A 4-day-old seedling. (F) A 5-day-old seedling. (G) A 9-day-old seedling. The close-up view in (D) shows staining of the root tip. The close-up view in (G) shows GUS staining in the lateral roots. (H to M) GUS staining of the stable TIE2pro-GUS transgenic line. (H) A 1-day-old seedling. (I) A 2-day-old seedling. (J) A 3-day-old seedling. (K) A 4-day-old seedling. (L) A 5-day-old seedling. (M) A 9-day-old seedling. The close-up view in (K) shows staining of the root tip. The close-up view in (M) shows GUS staining in the lateral roots. (N to Q) GFP fluorescence of 3-, 4-, 5-, and 7-day-old TIE1pro-TIE1-GFP roots. (R to U) GFP fluorescence of 3-, 4-, 5-, and 7-day-old TIE2pro-TIE2-GFP roots. Scale bars, 0.1 mm [(A, B, H, and N to U) and insets of (D, G, K, and M)] and 1 mm (C to G and I to M).

TIE2 is a transcriptional repressor

TIE1 was previously demonstrated to be a transcriptional repressor. Overexpression of TIE2 leads to curly leaves resembling those of TIE1 activation tagging mutant tie1-D, suggesting that TIE2 may also encode a transcriptional repressor (). To confirm that TIE2 can function as a transcriptional repressor in nuclei, we first observed the fluorescence of TIE2pro-TIE2-GFP, which rescued the shorter root phenotype of tie1-1 tie2-1. The GFP fluorescence of TIE2-GFP was distributed in the cytoplasm and nuclei, but the signals were clearer and stronger in nuclei (Fig. 3, A to C, and fig. S5). We next generated the reporter 35S-UAS-LUC, in which the Luciferase (LUC) gene was driven by a synthetic promoter containing a cauliflower mosaic virus (CaMV) 35S promoter fusion with six upstream activating sequences (UAS) bound by GAL4, and the reporter 35S-REN-35S-UAS-LUC, in which the Renilla Luciferase (REN) gene was driven by a CaMV 35S promoter, and used it as an internal expression control (Fig. 3D). We generated the effector 35S-G4BD-TIE2 in which a CaMV 35S promoter was used to drive TIE2 fusion to the sequence encoding the GAL4 DNA binding domain (G4BD). We also generated the control vector 35S-G4BD or 35S-G4BD-TIE2mEAR in which a CaMV 35S promoter was used to drive G4BD or G4BD fusion with TIE2mEAR encoding mutated TIE2 with the three mutations (the conserved L was mutated to S) in the EAR motif. We cotransformed 35S-UAS-LUC with 35S-G4BD-TIE2, the negative control 35S-G4BD, 35S-G4BD-TIE2mEAR, or the positive control 35S-G4BD-TIE1 into tobacco leaves (Fig. 3, E and F). The results showed that LUC reporter expression was higher in leaves transformed with the combination with 35S-G4BD, as shown by the stronger fluorescence, while the expression of LUC was repressed in the leaves transformed with the combination with 35S-G4BD-TIE2 or 35S-G4BD-TIE1, suggesting that TIE2 and TIE1 had transcriptional repression activity. However, repression of the LUC reporter was released by transforming with the combination with 35S-G4BD-TIE2mEAR, indicating that the EAR motif was required for the repression activity of TIE2 (Fig. 3, E and F). We then cotransformed the 35S-REN-35S-UAS-LUC reporter with 35S-G4BD, 35S-G4BD-TIE2, or 35S-G4BD-TIE2mEAR into Arabidopsis protoplasts. The quantitative analysis showed that TIE2 repressed the reporter LUC, and the EAR motif was required for the repression (Fig. 3G). These results indicate that TIE2 is an EAR motif–containing regulator with transcriptional repression activity.

Fig. 3.

TIE2 is a nuclear transcriptional repressor.

(A to C) The subcellular localization of TIE2 is shown by the GFP fluorescence in the cells of roots from TIE2pro-TIE2-GFP plants, which rescued the short root phenotype of tie1-1 tie2-1. TIE2 was predominantly localized to nuclei. (A) GFP fluorescence. (B) DAPI (4′,6-diamidino-2-phenylindole) staining. (C) The merged picture of (A) and (B). Scale bar, 10 μm. (D) A schematic representation of the constructs used in (E to G). (E and F) TIE2 displayed transcriptional repression activity similar to that of TIE1, and the EAR motif was required for the repression activity. (E) Bright-field image and (F) fluorescence of tobacco leaves cotransformed with the different combinations of constructs shown in (D). (G) The protoplast transient expression system indicated that TIE2 acts as a transcriptional repressor. 35S-REN-35S-UAS-LUC was transformed into Arabidopsis protoplasts with 35S-G4BD, 35S-G4BD-TIE2, or 35S-G4BD-TIE2mEAR, respectively, and the LUC/REN ratio was measured. The data are means (± SD) of three replicates. Student’s t test was used for significance testing, *P < 0.05.

TIE2 interacts with type B ARR1 to repress its transactivation activity

We previously screened an Arabidopsis transcription factor library using the N terminus of TIE1 as bait (). We found that type B ARR11 and some other transcription factors, in addition to TCP transcription factors, interacted with TIE1 (fig. S6A) (). To test whether TIE2 can interact with type B ARRs, we first cloned seven type B ARRs genes: ARR1, ARR2, ARR10, ARR11, ARR12, ARR14, and ARR18. Yeast two-hybrid assays were performed using TIE2 as bait and type B ARRs as prey. The results showed that ARR1, ARR2, and ARR14 interacted strongly with TIE2, while ARR10 and ARR11 interacted weakly with TIE2 (fig. S6B). We next confirmed the interactions between TIE2 and type B ARRs using type B ARRs as the bait and TIE2 as the prey. The results showed that ARR11, ARR14, and ARR18 interacted with TIE2 (fig. S6C). We then used firefly luciferase complementation imaging assays to test whether TIE2 can interact with type B ARRs in planta. Fluorescence was clearly detected following cotransformation of nLUC-TIE2 with cLUC-ARR1, cLUC-ARR2, cLUC-ARR10, cLUC-ARR11, cLUC-ARR12, cLUC-ARR14, and cLUC-ARR18, while the cotransformation of nLUC-TIE2 with cLUC-ARR3 (type A ARR) and the other control cotransformation showed no fluorescence (Fig. 4, A to D, and fig. S6, D to F). The results suggest that TIE2 interacts with these type B ARRs in vivo. Firefly luciferase complementation imaging assays also showed that TIE1 interacted with ARR1 in planta (fig. S6G). To further verify the interactions between TIE2 and ARR1/ARR2, we performed coimmunoprecipitation (co-IP) assays by transiently expressing Myc-tagged TIE2 and Flag-tagged ARR1 or ARR2 in Nicotiana benthamiana or Arabidopsis protoplasts. The results showed that TIE2 pulled down ARR1 and ARR2 in both expression systems (Fig. 4, E and F, and fig. S7). These results indicate that TIE2 interacts with ARR1 and ARR2 in vivo.

Fig. 4.

TIE2 repressed the transactivation activity of ARR1 and ARR2 by directly interacting with them.

(A to D) Firefly luciferase complementation assays showed that TIE2 interacted with ARR1, ARR2, ARR10, and ARR11 in planta. cLUC and cLUC-ARR3 were used as negative controls. (E and F) Co-IP assays confirmed that TIE2 interacted with ARR1 or ARR2 in vivo. (G and H) The truncated protein analysis showed that the N terminus of TIE2 interacted with the glutamine-rich domain of ARR2 protein. (G) A schematic diagram of the truncated fragments of TIE2 and ARR2 used in the yeast two-hybrid assays in (H). (I) TIE2 repressed the transactivation activity of ARR1, and the EAR motif was required for the repression activity of TIE2. The reporter 35S-REN-ARR15pro-LUC and other constructs in the upper schematic diagrams were used in the Arabidopsis protoplast assays shown in the bottom. The data are means (±SD) of three replicates. (J and K) Expression of an artificial protein consisting of ARR1 or ARR2 fused with the C terminus of TIE2 using the promoter of ARR1 or ARR2 rescued the short roots and the cell elongation in the EZ and DZ of tie1-1 tie2-1. Data are means ± SD (n = 40) of three independent experiments. (L and M) Expression of an artificial protein consisting of ARR1 fused with the C terminus of TIE2 using the TIE2 promoter rescued the short roots and the cell elongation in the EZ and DZ of tie1-1 tie2-1. Data are means ± SD (n = 48) of three independent experiments. Scale bar, 1 mm (J and L). Student’s t test was used for significance testing, ***P < 0.001 in (I, K, and M).

To determine the domains involved in the interaction between TIE2 and type B ARRs, we divided TIE2 into the N-terminal domain (TIE2-N, 1 to 125) including the helix region and the C-terminal domain (TIE2-C, 126 to 178) containing the EAR motif (Fig. 4G). ARR2 was divided into three parts, including ARR2-DDK (1 to 144), ARR2-Ac-M (145 to 268), and ARR2-Q (269 to 664; Fig. 4G). Yeast two-hybrid assays showed that the N terminus of TIE2 interacted with the glutamine-rich domain of ARR2 (Fig. 4H).

To investigate the effect of TIE2 interaction with type B ARRs, we generated the reporter 35S-REN-ARR15pro-LUC, in which the promoter of type A ARR15 (known to be directly targeted by type B ARRs) was used to drive the LUC gene, and REN was used as an internal control (Fig. 4I). The reporter was cotransformed with 35S-ARR1, in which the ARR1 genes was driven by a CaMV 35S promoter, leading to clear activation of LUC expression, as expected (Fig. 4I). However, 35S-TIE2, in which TIE2 was driven by a CaMV 35S promoter, repressed the activation of the reporter by ARR1, while 35S-TIE2mEAR (carrying mutations in the EAR motif) did not (Fig. 4I). To determine whether the EAR motif mutations in the TIE2mEAR could affect the interaction between TIE2mEAR and ARR1, we first performed yeast two-hybrid assay and found that TIE2mEAR interacted with both ARR1 and ARR2 as TIE2 (fig. S8A). Co-IP analysis confirmed that TIE2mEAR interacted with ARR1 in vivo using the Arabidopsis transient expression system (fig. S8B), suggesting that the result that TIE2mEAR could not repress the transactivation activity of ARR1 was not due to the compromised interaction between TIE2mEAR and ARR1. We further showed that TIE2 repressed the transactivation activity of ARR2, while mutations in TIE2mEAR significantly decreased the repression activity of TIE2 (fig. S9). These results indicate that the interaction of TIE2 with ARR1 or ARR2 suppresses the transactivation activity of ARR1 or ARR2, and the EAR motif of TIE2 is required for its repression activity.

The results described above indicate that TIE2 interacts with type B ARR transcription factors via its N terminus, whereas its C terminus is involved in repressing their activity. We hypothesized that TIE2 could act as a bridge, using its N terminus to connect with type B ARRs and using its C-terminal EAR motif to link with TPL/TPR co-repressors. Compromised expression of TIE1 and TIE2 could lead to de-repression of type B ARRs in tie1-1 tie2-1, causing excessive cytokinin signaling and shorter roots. To confirm this hypothesis, we artificially fused ARR1 with the C terminus of TIE2 by generating ARR1pro-ARR1-TIE2C, ARR2pro-ARR2-TIE2C, or TIE2pro-ARR1-TIE2C, in which the ARR1-TIE2C or ARR2-TIE2C fusion gene was driven by the ARR1, ARR2, or TIE2 promoter (Fig. 4J). We expected the repressive versions of ARR1-TIE2C and ARR2-TIE2C to compete with endogenous type B ARRs, inhibit their activity, and thus rescue the root elongation defects in tie1-1 tie2-1 (). We transformed ARR1pro-ARR1-TIE2C, ARR2pro-ARR2-TIE2C, or TIE2pro-ARR1-TIE2C into the double mutant tie1-1 tie2-1. As expected, transformation with ARR1pro-ARR1-TIE2C, ARR2pro-ARR2-TIE2C, or TIE2pro-ARR1-TIE2C rescued the short root phenotype and the cell elongation in the EZ and DZ of tie1-1 tie2-1 (Fig. 4, J to M). These results strongly suggest that TIE2 acts as a repressor to suppress the activity of type B ARRs.

Cytokinin signaling is significantly enhanced in the tie1-1 tie2-1 double mutant

To provide more evidence showing that TIE1 and TIE2 repress the activity of type B ARRs and regulate the cytokinin response, we carried out RNA sequencing (RNA-seq) using the roots of 7-day-old tie1-1 tie2-1 and wild-type control seedlings. The transcriptome data showed that 1357 differentially expressed genes (DEGs; false discovery rate < 0.05; fold change, >2.0 or <−2.0), including 776 up-regulated genes and 581 down-regulated genes, were identified between tie1-1 tie2-1 and the wild-type controls (fig. S10A and data files S1 and S2). Among these DEGs, 468 genes (34% DEGs) were directly targeted by ARR1, ARR10, or ARR12 (fig. S10B) (). Gene Ontology (GO) analysis showed that genes related to pathways including root hair development, cytokinin-activated signaling pathway, and response to cytokinin were highly enriched in the DEGs (fig. S10C). Venn diagram analysis showed that many DEGs overlapped with the golden list of genes regulated by cytokinin (), as well as 813 common target and 1713 union target genes identified by integrating type B ARR chromatin immunoprecipitation sequencing (ChIP-seq) analysis with cytokinin-regulated gene expression (fig. S10, D to G) (). Among them, many type B ARR direct target genes including type A ARR16, ARR15, ARR3, and ARR7 were significantly up-regulated in tie1-1 tie2-1 (data file S3 to S5). Heatmap analysis suggested that the expression levels of 10 type A ARR genes known to be directly targeted by type B ARRs were increased in tie1-1 tie2-1 (Fig. 5A). Motif analysis showed that the core motifs bound by type B ARR or TCP transcription factors were enriched in the 500-bp promoter region upstream of the start codon ATG from the 776 up-regulated genes (fig. S10, H and I) (, ). To verify the RNA-seq data, we performed quantitative real-time polymerase chain reaction (qRT-PCR) using cDNA from the roots of 3- or 7-day-old seedlings to measure the expression levels of the 10 type A ARR genes directly targeted by type B ARRs. The results indicated that the expression levels of the tested type A ARR genes in the roots of 3- and/or 7-day-old tie1-1 tie2-1 seedlings were significantly higher than those in the roots of corresponding wild-type seedlings (Fig. 5B), consistent with the RNA-seq data.

Fig. 5.

Disruption of TIE1 and TIE2 in tie1-1 tie2-1 enhanced cytokinin signaling.

(A) Heatmap analysis of the FPKM (fragments per kilobase million) of type A ARRs directly regulated by type B ARRs. (B) Real-time PCR confirmation of the expression level of type A ARRs in tie1-1 tie2-1 and wild-type roots. The data are means (±SD) of three replicates. (C) TCSn::NLS-3×GFP marker was introduced in tie1-1 tie2-1. The GFP signal was significantly increased in the EZ and DZ of roots in tie1-1 tie2-1, while the signal in the root meristem of tie1-1 tie2-1 was comparable to that of wild-type control. (D) The Western blot confirmed that the expression of the TCSn::NLS-3×GFP marker was significantly induced in the roots of tie1-1 tie2-1. (E and F) Expression of CKX1 driven by the TIE1 or TIE2 promoter in tie1-1 tie2-1 rescued the short root phenotype and the cell elongation in the EZ and DZ. Data are means ± SD (n = 30) of three independent experiments. (G and H) Disruption of ARR1 and ARR2 rescued the short root phenotype and the cell elongation in EZ and DZ of tie1-1 tie2-1. Data are means ± SD (n = 40) of three independent experiments. (I) The inhibition rate of root length of 7-day-old wild type, tie1-1 tie2-1, and tie1-D treated with 6-BA. Data are means ± SD (n = 20) of three independent experiments. Scale bars, 0.1 mm (C) and 1 mm (E and G). Student’s t test was used for significance testing in (B, F, and H). Two-way analysis of variance (ANOVA) and Bonferroni test were used for statistical analysis in (I). **P < 0.01 and ***P < 0.001 in (B, F, H, and I).

To further demonstrate that disruption of the repressors TIE1 and TIE2 leads to activation of cytokinin signaling in roots, we introduced the TCSn::NLS-3×GFP marker, which indicates cytokinin signaling, into tie1-1 tie2-1 (). Fluorescence observation and Western blotting showed that the GFP protein expression level of tie1-1 tie2-1 was much higher than that of the wild-type controls (Fig. 5, C and D). We further generated the constructs TIE1pro-CKX1-GFP and TIE2pro-CKX1-GFP, in which CKX1 (encoding a cytokinin oxidase involved in cytokinin degradation) was driven by the TIE1 promoter or TIE2 promoter, respectively. We transformed the constructs into tie1-1 tie2-1 to decrease the cytokinin input. The results showed that transformation with either TIE1pro-CKX1-GFP or TIE2pro-CKX1-GFP rescued the shorter root phenotype and the cell elongation in the EZ and DZ of tie1-1 tie2-1 (Fig. 5, E and F). To examine whether the short root phenotype of tie1-1 tie2-1 could be dependent on the function of type B ARRs, we performed genetic interaction between tie1-1 tie2-1 and arr1 arr2. The results showed that the disruption ARR1 or ARR2 in the triple tie1-1 tie2-1 arr1 or tie1-1 tie2-1 arr2 mutants weakly rescued the short root phenotype of tie1-1 tie2-1 (fig. S10, J and K), whereas the quadruple mutant tie1-1 tie2-1 arr1 arr2 produced roots with the length comparable to that of wild-type control (fig. S10, J and K, and Fig. 5G). The cell elongation in the EZ and DZ was also rescued in tie1-1 tie2-1 arr1 arr2 (Fig. 5H). These results demonstrate that the short root phenotype of tie1-1 tie2-1 is caused by enhanced cytokinin signaling through the release of suppression of type B ARRs, and TIEs are strong negative regulators of cytokinin signaling.

To provide more evidences to support that TIEs negatively regulate cytokinin signaling, we tested the expression of type A ARR genes including ARR3, ARR4, ARR7, ARR8, ARR9, and ARR15 in the seedlings of tie1-1 tie2-1, the TIE1 overexpression line tie1-D, and wild-type control with or without the treatment of cytokinin. The results showed that these type A ARR genes were induced more significantly in tie1-1 tie2-1 but less in tie1-D when compared to the induction in wild-type control by cytokinin (fig. S11, A to F), suggesting that tie1-1 tie2-1 was more sensitive to cytokinin, while tie1-D is less sensitive to cytokinin. We next treated wild-type control, tie1-1 tie2-1, and tie1-D with cytokinin in different concentrations for evaluation of root elongation. The inhibition rate of root length by cytokinin is lower in tie1-D but higher in tie1-1 tie2-1, when compared with that in wild-type control (Fig. 5I), again suggesting that tie1-D is resistant to cytokinin, while tie1-1 tie2-1 is more sensitive to cytokinin. We further observed the length of root meristem. The results showed that cytokinin treatment significantly inhibited root meristem. However, the inhibition of root meristem was comparable between wild type and tie1-1 tie2-1 (fig. S11G), suggesting that the sensitivity of root meristem to cytokinin is not changed in tie1-1 tie2-1. We lastly treated pTCSn::NLS-3×GFP in the wild-type and in tie1-1 tie2-1 background with cytokinin. The observation of the GFP fluorescence showed that the increased GFP fluorescence is similar in wild type and tie1-1 tie2-1 in the root meristem region (fig. S11, H to K), consistent with the result that the inhibition of root meristem by cytokinin was similar in wild type and tie1-1 tie2-1 (fig. S11G). However, the GFP fluorescence in tie1-1 tie2-1 was increased more significantly than that in wild-type background in the DZ after cytokinin treatment (fig. S11, L to O). These results further demonstrate that TIE1 and TIE2 promote root elongation by repressing cytokinin signaling.

TIE1 and TIE2 are directly up-regulated by type B ARR1

In the auxin signaling pathway, EAR motif–containing transcriptional repressors are encoded by AUX/IAA genes, which are directly up-regulated by the key transcription factors known as auxin response factors (ARFs). AUX/IAA proteins, in turn, repress the activity of ARFs by directly interacting with them, thus forming an elegant negative feedback loop to suppress activated auxin signaling (). The interactions between type B ARRs and EAR motif–containing TIE2 strongly imply that a similar feedback loop could prevent persistent cytokinin signaling. To test this hypothesis, we first treated 7-day-old seedlings with cytokinin for a short period of time (45 min) to test whether cytokinin induced expression of TIE1 and TIE2. The results showed that the expression levels of TIE1, TIE2, and the positive control ARR15 were significantly increased by cytokinin treatment (Fig. 6, A to C). To further determine whether the induction of TIE1 and TIE2 by cytokinin could be a primary response, we treated the seedlings with cytokinin and cycloheximide (CHX; a protein synthesis inhibitor). Following treatment with CHX, TIE1, TIE2, and ARR15 were still induced by cytokinin treatment, suggesting that TIE1 and TIE2 are primary genes regulated by cytokinin signaling as ARR15 (Fig. 6, A to C). We then searched the promoter regions of TIE1 and TIE2 and ARR15 for type B ARR binding sites using AthaMap software (), which identified dozens of potential cis-elements recognized by type B ARRs in the 1000-bp promoter regions upstream of the translation initiation sites of TIE1, TIE2, and the positive control ARR15 (fig. S12). We next searched the published ARR1 ChIP-seq data (, ) and found obvious binding signals in the promoter regions of TIE1, TIE2, and ARR15, which increased in binding intensity following cytokinin treatment (Fig. 6, D to F). ChIP-PCR confirmed that ARR1 was significantly enriched near cis-elements bound by ARR1 in the promoter of TIE2 (Fig. 6G). Electrophoretic mobility shift assay (EMSA) showed that ARR1 directly bound to the cis-elements in the promoter of TIE2 in vitro (Fig. 6H). We further generated the reporters 35S-REN-TIE1pro-LUC and 35S-REN-TIE2pro-LUC, in which the LUC gene was driven by the TIE1 promoter or TIE2 promoter, respectively, and REN was used as an internal control. We cotransformed 35S-ARR1 with either 35S-REN-TIE1pro-LUC, 35S-REN-TIE2pro-LUC, or the positive control 35S-REN-ARR15pro-LUC. The LUC reporter was significantly activated by ARR1 following cotransformation with each of the three tested combinations (Fig. 6, I to K). These results demonstrate that ARR1 promotes the expression of TIE1 and TIE2 by directly binding to their promoters, thus forming an elegant negative feedback loop to finely regulate cytokinin signal output.

Fig. 6.

TIE1 and TIE2 are directly induced by ARR1.

(A to C) TIE1 and TIE2 are rapidly up-regulated by cytokinin treatment. The 7-day-old seedlings were treated with mock, 6-BA, CHX, or 6-BA and CHX for 45 min. The type A ARR15 gene was used as the positive control. The data are means (± SD) of three replicates. (D to F) Analysis of ChIP-seq raw reads previously reported at the loci of the ARR15, TIE1, and TIE2 genes (). The peaks in the graphs indicate the binding of type B ARR1. (G) ChIP-PCR analysis confirmed that ARR1 directly bound to the promoter of TIE2. Top: The schematic diagram shows the positions of cis-elements and primers used in the ChIP-PCR in the upstream region of TIE2. The vertical red lines indicate the potential ARR1-binding site 5′-(A/G)GAT(T/C)-3′. The blue arrow indicates TIE2. TIE2-I: −918 to −914 bp; TIE2-II: −737 to −733 bp; TIE2-III: −35 to −31 bp; TIE2-IV: 164 to 168 bp. The black arrows represent the primers used for ChIP-PCR. Five-day-old wild-type and 35S-GFP-ARR1 seedlings were used. The relative enrichment of the wild-type control was set to 1.0. The ARR7 and TUA4 transposon loci were used as positive or negative controls. The data are means (± SD) of three replicates. (H) EMSA experiments confirm that ARR1 directly binds to the promoter of TIE2 in vitro. (I to K) The transient assays showed that ARR1 directly activated the expression of TIE1 and TIE2 in Arabidopsis protoplasts. The data are means (± SD) of three replicates. Student’s t test was used for significance testing, **P < 0.01 and ***P < 0.001 in (A to C, G, and I to K).

It is reported that cytokinin inhibits root elongation by promoting ethylene biosynthesis (). Our GO analysis showed that genes related to ethylene pathway were highly enriched in the DEGs (fig. S10C). To determine whether TIE1 and TIE2 could affect ethylene biosynthesis or signaling, we first searched the reported ChIP-seq data of ethylene insensitive 3 (EIN3), a key positive regulator of ethylene signaling (), and found that EIN3 may bind to the promoter region of TIE1 but not TIE2 (fig. S13, A and B). To test whether ethylene could induce TIE1 and TIE2 rapidly, we treated wild-type seedlings with the ethylene precursor 1–aminocyclopropane-1-carboxylic acid (ACC) in a short time. TIE1 and TIE2 were not significantly induced by ACC treatment, whereas the positive control ERF1 was significantly up-regulated by ACC treatment (fig. S13, C to E). We then treated wild type, tie1-1 tie2-1, constitutive triple response (ctr1), and EIN3 overexpression line EIN3ox with aminoethoxyvinylglycine (AVG), an inhibitor of ethylene biosynthesis (). As expected, the activated ethylene signaling in ctr1 or EIN3ox caused short root phenotype that was not rescued by AVG treatment. However, the short root phenotype of tie1-1 tie2-1 was almost completely rescued by the treatment with ethylene biosynthesis inhibitor AVG (fig. S14), suggesting that TIE1 and TIE2 do not directly affect ethylene signaling. These results further support that TIE1 and TIE2 repressed type B ARR transcription factors to finely tune cytokinin signaling and, thus, the production of ethylene.

The quadruple tie1 tie2 tie3 tie4 mutants produce defective seeds and shorter roots

To overcome the possible redundant roles of TIEs and reveal general involvement of TIEs in fine-tuning cytokinin signaling during plant development, we generated tie3-1 null mutant by CRISPR-Cas9 technology and identified a T-DNA insertion mutant SALK_018686 from public database as tie4-1 (Fig. 7, A and B). A 5-bp deletion was found in the first exon of tie3-1 and caused premature termination of TIE3 translation (Fig. 7A), while the T-DNA insert was located at the fourth exon of TIE4 and disrupted the function of TIE4 (Fig. 7, B and C). We introduced tie3-1 and tie4-1 into tie1-1 tie2-1 to generate the quadruple mutant tie1-1 tie2-1 tie3-1 tie4-1 by crossing. We found that most of tie1-1 tie2-1 tie3-1 tie4-1 seeds did not germinate, indicating that TIEs are important for seed viability (Fig. 7, D to F). The few germinated seeds produced seedlings with very short roots or no roots (Fig. 7G). The seedlings frequently produced one cotyledon or cup-shaped cotyledon resembling the phenotypes of tpl-1 mutant (Fig. 7G) (). To confirm that TIEs played redundant roles in seed development, we used CRISPR-Cas9 technology to generate tie1-2 tie2-2 tie3-2 tie4-2 in which the deletions or insertions were detected in the first exon of each TIE gene and disrupted the function of TIEs (fig. S15). The new allele of quadruple mutant tie1-2 tie2-2 tie3-2 tie4-2 also produced a high ratio of defective seeds that did not germinate (Fig. 7, H and I). The few germinated seeds of tie1-2 tie2-2 tie3-2 tie4-2 also produced the seedlings with short roots, one cotyledon, or fused cotyledon (Fig. 7J). Microscope observation showed that the roots from quadruple mutants produced defective root meristems (Fig. 7, K and L). We further analyzed the root meristem of wild type, tie1-1 tie2-1, and tie1-1 tie2-1 tie3-1 tie4-1 using 5-ethynyl-2′-deoxyuridine (EdU) staining. The results showed that the size of root meristem was comparable between wild type and tie1-1 tie2-1 (fig. S16, A and B). However, the root meristem in tie1-1 tie2-1 tie3-1 tie4-1 was severely shortened, and even no root meristem was stained in some lines of tie1-1 tie2-1 tie3-1 tie4-1 (fig. S16, C and D). These data indicate that TIEs are essential for root development and may have a broad role in different developmental processes by tightly regulating cytokinin signaling.

Fig. 7.

The tie1 tie2 tie3 tie4 quadruple mutants produce defective seeds and shorter roots.

(A and B) Schematic diagram of tie3-1 and tie4-1 mutants. (A) A 5-bp deletion in the first exon of TIE3 was created by CRISPR-Cas9 technology in tie3-1. (B) T-DNA was inserted in the fourth exon of TIE4 in tie4-1. (C) The relative expression level of TIE4 in tie1-1 tie2-1 tie3-1 tie4-1. (D and E) The seed germination of wild type (D) and tie1-1 tie2-1 tie3-1 tie4-1 (E). (F) Seed germination analysis of wild type and tie1-1 tie2-1 tie3-1 tie4-1. (G) Six-day-old seedlings of wild type and tie1-1 tie2-1 tie3-1 tie4-1. The right pictures of (G) are the close-up views of tie1-1 tie2-1 tie3-1 tie4-1 seedlings. (H and I) Seed germination of wild type (H) and tie1-2 tie2-2 tie3-2 tie4-2 (I). (J) Six-day-old seedlings of wild type and tie1-2 tie2-2 tie3-2 tie4-2. The inset pictures in (J) are the close-up views of tie1-2 tie2-2 tie3-2 tie4-2 seedlings. (K and L) Differential interference contrast (DIC) observation of roots from tie1-1 tie2-1 tie3-1 tie4-1 (K) and tie1-2 tie2-2 tie3-2 tie4-2 (L). Scale bars, 1 mm (D, E, and G to J), 0.1 mm [inset of (J)], and 50 μm (K and L).

DISCUSSION

Here, we demonstrate that the EAR motif–containing proteins TIE1 and TIE2 act as transcriptional repressors to strongly inhibit the cytokinin response. The double loss-of-function mutant tie1-1 tie2-1 displays an excessive cytokinin response and thus produces shorter roots. TIE1 and TIE2 have an overlapping expression pattern and are predominantly expressed in the root EZ and DZ. We show that TIE2 is a transcriptional regulator with repression activity that represses the transactivation activity of type B ARR1 by directly interacting with it in vivo. We further demonstrate that TIE1 and TIE2 are direct targets of ARR1. ARR1 directly up-regulates the expression of TIE1 and TIE2 in the primary response to cytokinin. Our data indicate that TIE1 and TIE2 are key regulators of root elongation that functions by preventing an excessive cytokinin response in the EZ and DZ of roots and reveal a previously unidentified potent negative feedback regulation loop for cytokinin signaling.

EAR motif–containing transcriptional repressors are key factors in several different hormone signaling pathways (–). It is well established that these repressors use the EAR motif to recruit TPL/TPR co-repressors to key transcription factors and dampen signaling pathways (). For example, AUX/IAAs act as transcriptional repressors via their association with TPL/TPR co-repressors using their EAR motifs (). In the absence of auxin, AUX/IAAs function as adaptor proteins connecting TPL/TPRs with ARFs and thus repress the activity of key transcription factors to prevent transcription of auxin-responsive genes. In the presence of auxin, AUX/IAAs are degraded via the 26S proteasome, and ARFs are released to activate the expression of auxin-responsive genes, including AUX/IAAs. AUX/IAAs, in turn, repress the activity of ARFs in the absence of auxin (). By this mechanism, the auxin response is tightly and elegantly tuned by the release of ARF activity via the degradation of AUX/IAAs in a process dependent on the level of auxin (). Similar molecular mechanisms have been found in the JA and SL signaling pathways (, ). In JA signaling, jasmonate zim-domain (JAZ) transcriptional repressors recruit TPL/TPRs through the EAR motif–containing protein NINJA to suppress the activity of JA-responsive transcription factor MYC proteins (). JA promotes the degradation of JAZ and relieves MYC transcription factors to activate JA-responsive genes (). More recently, the EAR motif–containing SMXL and its rice homolog D53 have been shown to interact with TPL/TPRs (, ). The degradation of SMXL or D53 transcriptional repressors activates the SL signaling pathway (, ). In addition, EAR motif–containing proteins, including AFPs, ERFs, BZR1, and BES1 (BRI1-EMS-suppressor 1), participate in the signaling pathways of ABA, ethylene, and BR hormones (). We demonstrate that the activity of type B ARR transcription factors, which play key roles in cytokinin signaling, is suppressed by the EAR motif–containing proteins TIE2 and TIE1. As we previously demonstrated regarding TIE1, TIE2 has transcriptional repression activity (). Both TIE2 and TIE1 interact with TPL/TPR co-repressors using the C-terminal EAR motif (). Our data indicate that type B ARRs interact with the N-terminal region of TIE2, suggesting that TIE2 acts as a bridge connecting TPL/TPRs with type B ARRs. Disruption of TIE1 and TIE2 in the double mutant tie1-1 tie2-1 leads to an excessive response to cytokinin in the roots. These data suggest that the type B ARR transcription factors–TIE1/TIE2 adaptor proteins–TPL/TPR co-repressors modules are parallel to ARFs-AUX/IAAs-TPL/TPRs. The difference between these two kinds of modules is that cytokinin can activate unphosphorylated type B ARRs in the nuclei to initiate the cytokinin response, whereas auxin promotes the degradation of AUX/IAAs to release the activity of ARFs. We previously showed that TIE1 is an unstable protein, and we demonstrated that TIE1 degradation is mediated by RING-like E3 ligase TEARs (), but we do not know yet whether or how cytokinin mediates the degradation of TIE1 and TIE2 to relieve the activity of type B ARRs. However, it has been reported that the F-box protein KMD mediates the degradation of type B ARRs (), leading to disassembly of the modules.

TIEs also interact with other transcription factors including TCP transcription factors. The quadruple mutants tie1-1 tie2-1 tie3-1 tie4-1 and tie1-2 tie2-2 tie3-2 tie4-2 produce one cotyledon or cup-shaped cotyledon, resembling the phenotype of topless-1 (tpl-1) and overexpression of microRNA319-resistant TCP4 (, ), suggesting that TIEs may act as bridges to connect TCPs and TPL/TPR co-repressors in the control of the cotyledon and leaf development, as well as shoot branching (, , ). Here, disruption of type B ARRs in the quadruple mutant tie1-1 tie2-1 arr1 arr2 or by expressing ARR2pro-ARR2-TIE2C or TIE2pro-ARR1-TIE2C in tie1-1 tie2-1 rescued the short root phenotype, suggesting that TIEs control root elongation mainly by repressing the activity of type B ARR transcription factors. However, in our RNA-seq data using the root tissues as materials, the promoter analysis of up-regulated genes in tie1-1 tie2-1 showed that the core TCP-binding motif was also enriched in addition to the core type B ARR-binding motif, suggesting that TIEs may also regulate other signaling pathways in the control of root development.

Cytokinin plays critical roles in diverse processes in plant development. The tight control of cytokinin signal output is essential for developmental plasticity during adaptation to different environmental conditions (, ). Type B ARRs are key transcription factors that govern the primary cytokinin signal output by regulating the transcriptome in response to cytokinin (, ). Thus, fine regulation of type B ARR transactivation activity is very important for plant growth and development. The activity of type B ARRs is tightly controlled by different mechanisms. For example, type A ARRs are well-known repressors of type B ARRs that function in a negative feedback loop (). In this negative feedback loop, type B ARRs rapidly induce the expression of type A ARRs in the primary cytokinin response, and type A ARRs, in turn, indirectly suppress the activity of type B ARRs, possibly by competing with them for the phosphate relayed from AHP1 to AHP5 (, ). Although type A ARRs were thought to be the most important factors involved in dampening the cytokinin response in a feedback loop until now, the high-order type A ARR sextuple mutant arr3,4,5,6,8,9 does not display significant morphological defects (, ). We show that TIE1 and TIE2 are also up-regulated in the primary cytokinin response. TIE1 and TIE2 suppress the cytokinin response by directly interacting with type B ARRs. Compromised TIE1/TIE2 function in the double mutant tie1-1 tie2-1 causes severely shortened roots, and the quadruple tie1 tie2 tie3 tie4 mutants produced even shorter or no root phenotype. These indicate that the negative feedback loop mediated by TIEs is pivotal for controlling appropriate cytokinin signal output during plant development.

Cytokinin plays a critical role in flexible root growth and development by influencing cell division and differentiation in different root zones (). During embryogenesis, cytokinin participates in establishment of the SCN (). At the postembryogenesis stage, cytokinin does not affect the SCN, but it negatively regulates the size of the root meristem (, ). In this process, ARR1 is activated by the cytokinin signal to promote the expression of SHORT HYPOCOTYL 2 (SHY2) and thus inhibits auxin transport while simultaneously enhancing auxin degradation by increasing the expression of GRETCHEN HAGEN 3.17 (GH3.17), resulting in an auxin minimum in the TZ, which increases the rate of cell differentiation in the root meristem (). On the other hand, cytokinin activates ARR2 that directly promotes the expression of CELL CYCLE SWITCH52 A1 (CCS52A1). CCS52A1 acts as a cell cycle switch to stop cell division by promoting the degradation of mitotic regulators (). It is reported that the compromise of cytokinin signaling leads to an enlarged root meristem by delaying cell endoreplication and the first rapid cell elongation at the boundary between the PZ and TZ. However, at the boundary of the TZ and EZ, the second rapid elongation cell with more than twofold length increase is not related to endoreplication but involves actin reorganization. Cytokinin promotes the onset of actin bundling and the second rapid cell elongation by activating type B ARRs (). The negative effect of cytokinin on the size of the root meristem is independent of its positive effect on ethylene biosynthesis. In contrast, cytokinin profoundly affects root elongation in the EZ and DZ by promoting ethylene biosynthesis (, ). The application of cytokinin or overexpression of the cytokinin biosynthetic gene ISOPENTENYLTRANS-FERASE (IPT) represses root elongation (, ), while disruption of cytokinin signaling key component type B ARR1, ARR10, and ARR12 in loss-of-function arr1 and arr1 arr10 arr12 mutants leads to longer roots (). We found that TIE1 and TIE2 are potent inhibitors of type B ARRs because disruption of TIE1 and TIE2 causes highly increased expression of the cytokinin response marker TCSn::NLS-3×GFP and severely shortened roots caused by the compromised cell elongation in the EZ and DZ. Consistently, the treatment with ethylene biosynthesis inhibitor rescues the short root phenotype, indicating that TIE1 and TIE2 reduce cytokinin sensitivity and thus prevent excessive ethylene biosynthesis to promote cell elongation. However, tie1-1 tie2-1 did not produce any observed abnormality in the root meristem. This effect can be explained by the high redundant function of TIE family proteins. Our genetic data showed that the disruption of TIE1, TIE2, TIE3, and TIE4 in the quadruple tie1 tie2 tie3 tie4 mutants causes severely developmental phenotypes including short root meristem or no roots. It is proposed that cytokinin signaling regulates cell proliferation in the root meristem by mainly controlling rootward auxin transport through the vasculature while regulating cell elongation in the EZ and DZ by mainly controlling shootward auxin transport through the outer cells of the root cap and TZ (). We showed that TIE1 and/or TIE2 was expressed in lateral root cap, epidermis, and vasculature of roots. The fact that the cell elongation in the EZ and DZ, but not cell proliferation, in root meristem was affected in tie1-1 tie2-1 may be due to the partially activated cytokinin response by loss of TIE1 and TIE2. However, disruption of four TIEs leads to a stronger effect on cytokinin signaling, and both the cell proliferation in the root meristem and cell elongation in the EZ and DZ are affected in the quadruple tie1 tie2 tie3 tie4 mutants.

To summarize the proposed working model for the functions of TIE1 and TIE2 in cytokinin signaling (Fig. 8), in the presence of cytokinin, cytokinin is perceived by AHK receptors, causing autophosphorylation of His residue in AHKs. The phosphate is subsequently transferred to the Asp residue in the receiver domains of AHKs and then relayed by AHP1 to AHP5 to type B ARRs. The type B ARRs are activated by phosphorylation and bind to DNA to regulate the downstream genes including TIE genes and type A ARRs. TIEs, in turn, connect type B ARRs with TPL/TPR co-repressors, achieving timely suppression of the cytokinin response in the absence of cytokinin (Fig. 8). Future studies should aim to identify the important roles of TIEs in regulating the sensitivity of the different plant organs or cells in response to cytokinin signal.

Fig. 8.

A working model of TIE1 and TIE2 in the suppression of cytokinin signaling.

In the presence of cytokinin, cytokinin is perceived by AHKs. AHKs are autophosphorylated, and phosphate was transferred to the Asp residue in the receiver domain immediately. AHP1 to AHP5 subsequently relay the phosphate to type B ARRs to activate them. The activated type B ARRs promote the transcription of cytokinin response genes, including TIE1, TIE2, and type A ARRs. TIE1 and TIE2, in turn, act as bridges connecting type B ARRs with TPL/TPR co-repressors and HDAs to suppress the cytokinin response and prevent continuation of the response in the absence of cytokinin. H and D represent the conserved phosphor-accepting histidine and aspartate residues, respectively. P indicates phosphate.

MATERIALS AND METHODS

Plant materials and growth conditions

The Arabidopsis Columbia-0 (Col-0) ecotype type was used. Wild-type, tie1-1 (GABI_372F04), tie2-1, and CyclinB1;1:GUS (), TCSn::NLS-3×GFP seeds (), and other plant materials were first sterilized using 75% ethanol for 10 min and then dried by air flow using a clean bench. The dried seeds were sown on half-strength Murashige and Skoog (1/2 MS) medium and were cold-treated for 2 days in a 4°C refrigerator. The plates were then placed in an incubator at 22°C (16-hour white light and 8-hour dark cycle). For the analysis of root length and morphology, the plates were placed vertically in the incubator. Seedlings of Arabidopsis or Nicotiana benthamiana were grown in soil in a greenhouse at 22°C (16-hour white light and 8-hour dark cycle).

Root observation and phenotype characterization

For differential interference contrast (DIC) microscopy, the roots from seedlings were mounted in chloral hydrate solution (chloral hydrate:water:glycerol = 8:3:1, w/v/v) before observation. The size of the PZ is determined by the cell number from the initial cell to the first elongated cell characterized by the cell length exceeding its width at the boundary between the PZ and the TZ (). The boundary of the TZ and the EZ were defined by the cells’ increase in length by more than twofold (). Root elongation is investigated as previously described (). Briefly, cell length is determined on the shootward side of the EZ and DZ around the region of first root hair initiation cell, which is the marker of the boundary between EZ and DZ. ImageJ software is used to analyze the cell length. At least 20 roots were analyzed, and at least three independent experiments were performed. Student’s t test was used to evaluate the significance.

To test plant response to exogenous cytokinin, wild-type, tie1-1 tie2-1, and tie1-D seedlings were grown vertically on plates containing the different concentration of 6-benzylaminopurine (6-BA) for 7 days. The seedlings were photographed, and the length of primary roots was measured using ImageJ software. The inhibition rate was calculated by inhibition rate = (L0 − L)/L0 × 100%. L0 means the root length grown under the normal condition. L means the root length grown under the respective concentration of 6-BA.

To determine the phenotype of lateral roots, wild-type and tie1-1 tie2-1 seedlings were grown on vertical plates for different days and then photographed. The number of primary lateral roots was counted, and the length of main roots was measured. The density of lateral roots was calculated by the number of primary lateral roots/the length of the primary roots.

Generation of the tie2-1, tie3-1, and tie1-2 tie2-2 tie3-2 tie4-2 mutants using CRISPR-Cas9 technology

To generate tie2-1 and tie3-1, the sgRNA sequence targeting TIE2 or TIE3 was synthesized and inserted into the AtU6-26SK vector using Bbs I to generate AtU6-26SK-TIE2 (). The chimeric RNA expression cassette between the Kpn I and Sal I sites in AtU6-26SK-TIE2 was isolated and then cloned into the 35S-Cas9-pCAMBIA1300 vector ().

To generate tie1-2 tie2-2 tie3-2 tie4-2 mutant, the egg cell–specific promoter-controlled CRISPR-Cas9 system was used (). Briefly, the sgRNA sequence targeting TIE1, TIE2, TIE3, or TIE4 was cloned into pENTR-MSR vector to generate the construct pENTR-MSR-TIE1, pENTR-MSR-TIE2, pENTR-MSR-TIE3, or pENTR-MSR-TIE4. Next, the pENTR-MSR-TIE1-TIE4 was generated by isocaudamer enzyme ligation after digesting pENTR-MSR-TIE1 with Kpn I/Spe I and pENTR-MSR-TIE4 with Xba I/Hind III. The construct pENTR-MSR-TIE1-TIE4-TIE3 was generated from pENTR-MSR-TIE1-TIE4 and pENTR-MSR-TIE3 using the same method. Then pENTR-MSR-TIE1-TIE4-TIE3-TIE2 was generated from pENTR-MSR-TIE1-TIE4TIE3 and pENTR-MSR-TIE2. Last, pHEE401E-TIE1-TIE4-TIE3-TIE2 was generated from pHEE401E and pENTR-MSR-TIE1-TIE4-TIE3-TIE2 by Golden Gate Cloning. The constructs were introduced into wild-type Arabidopsis by Agrobacteria-mediated transformation to generate tie2-1, tie3-1 or tie1-2 tie2-2 tie3-2 tie4-2 mutants. The primers used in this study are listed in table S1.

Genotyping and gene expression analysis

To genotype the T-DNA insertion mutant tie1-1, the tie1-1-F and tie1-1-R primers were designed to amplify the wild-type fragment, while the tie1-1-R and O8760 primers were used to amplify the T-DNA insertion fragment. To genotype tie2-1, the tie2-1-F and tie2-1-R primers were used to amplify the genomic DNA of tie2-1, and the PCR product was sequenced to identify the mutation in tie2-1.

For gene expression analysis, 3- or 7-day-old wild-type and tie1-1 tie2-1 seedlings treated with or without 6-BA were cut and quickly frozen in liquid nitrogen. Total RNA was extracted using the Plant Total RNA Purification Kit (GeneMark, TR02-150), and M-MLV reverse transcriptase (Promega, M170A) was used to synthesize cDNA. RT-qPCR was carried out using SYBR reagent (CWBIO, CW2601M). The RT-qPCR cycle was as follows: 94°C for 20 s, 58°C for 30 s, and 72°C for 30 s.

Generation of binary constructs and plant transformation

For genetic complementation, TIE1pro-F/TIE1pro-R and TIE1geno-F/TIE1geno-R primer pairs were used to amplify the promoter and coding region of TIE1 from genomic DNA. The PCR fragments were cloned into pENTR/D-TOPO to generate pENTR-TIE1pro-TIE1nsc. TIE1pro-TIE1-GFP was generated by LR reaction between pENTR-TIE1pro-TIE1 and pKGWFS7. The promoter and coding region of TIE2 were amplified from genomic DNA using TIE2pro-F/TIE2pro-R or TIE2geno-F/TIE2geno-R. The TIE2 promoter fragment was cloned into the pDONR P4-P1R plasmid by BP reaction to generate TIE2pro-P4-P1R. The coding region of TIE2 was cloned into pENTR/D-TOPO to generate pENTR-TIE2geno. The GFP sequence was amplified from the pK7FWG0 plasmid by GFP-F/GFP-R primers and cloned into the pDONR-P2R-P3 plasmid to generate GFP-P2R-P3. TIE2pro-TIE2-GFP was generated by LR reaction with TIE2pro-P4-P1R, pENTR-TIE2geno, GFP-P2R-P3, and pB7m34GW.

To determine the subcellular localization of TIE2, 35S-GFP-TIE2 was generated by LR reaction between pK7FWG2 and pENTR-TIE2geno. The construct 35S-NLS-RFP as a nuclear marker was cotransformed into tobacco leaves with 35S-GFP-TIE2.

To decrease the cytokinin input in the double mutant tie1-1 tie2-1, the TIE1 promoter was amplified using TIE1pro-F/TIE1pro-R primers, and the PCR product was cloned into pDONR P4-P1R by BP reaction to generate TIE1pro-P4-P1R. The CKX1 gene was amplified from Arabidopsis cDNA and cloned into pENTR/D-TOPO to generate pENTR-CKX1. TIE1pro-CKX1-GFP and TIE2pro-CKX1-GFP were generated by LR reaction with TIE1pro-P4-P1R or TIE2pro-P4P1R, respectively, as well as pENTR-CKX1 and GFP-P2R-P3.

To generate ARR1pro-ARR1-TIE2C and ARR2pro-ARR2-TIE2C, the promoter of ARR1 or ARR2 was amplified using ARR1pro-F/ARR1pro-R or ARR2pro-F/ARR2pro-R, respectively, and cloned into P4-P1R by BP reaction to generate ARR1pro-P4-P1R or ARR2pro-P4-P1R, respectively. The ARR1 and ARR2 coding regions were amplified from genomic DNA using primer pairs ARR1-F/ARR1-R and ARR2-F/ARR2-R, respectively, and then cloned into pENTR/D-TOPO to generate pENTR-ARR1geno and pENTR-ARR2geno, respectively. The TIE2C sequence, encoding the C terminus of TIE2, was amplified using TIE2-C-F/TIE2-C-R primers and cloned into P2R-P3 to generate P2R-P3-TIE2C. ARR1pro-ARR1-TIE2C was generated by LR reaction with ARR1pro-P4-P1R, pENTR-ARR1geno, P2R-P3-TIE2C, and pB7m34GW. ARR2pro-ARR2-TIE2C was generated by LR reaction with ARR2pro-P4-P1R, pENTR-ARR2geno, P2R-P3-TIE2C, and pH7m34GW. TIE2pro-ARR1-TIE2C was generated by LR reaction with TIE2pro-P4P1R, pENTR-ARR1geno, P2R-P3-TIE2C, and pB7m34GW.

The TIE1pro-GUS construct was generated as described previously (). To generate TIE2pro-GUS, a 5104-bp-long TIE2 promoter was amplified from genomic DNA and cloned into pENTR/D-TOPO to generate pENTR-TIE2pro. TIE2pro-GUS was generated by LR reaction between pKGWFS7 and pENTR-TIE2.

Constructs were transformed into Agrobacterium tumefaciens GV3101/pMP90. Plant transformation was performed by an Agrobacterium-mediated floral dip method.

Co-IP and Western blotting

TIE2, ARR1, and ARR2 were amplified from wild-type cDNA and cloned into pENTR/D-TOPO to generate pENTR-TIE1, pENTR-TIE2, pENTR-ARR1, and pENTR-ARR2. To test the interaction between TIE2 and ARR1 or ARR2, TIE2-Myc was generated by LR reaction between pENTR-TIE2 and pK7MYCWG2. ARR1-Flag and ARR2-Flag were produced by LR reaction between pB7FLAGWG2 and pENTR-ARR1 or pENTR-ARR2.

Different combinations were transformed into tobacco leaves or Arabidopsis protoplasts. To perform co-IP assays, total protein was extracted with IP buffer [50 mM tris (pH 6.8), 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride, 1× cocktail, 1‰ MG132, and 1‰ NP-40] and immunoprecipitated using agarose beads conjugated with anti-Myc antibodies (Sigma-Aldrich, E6654) by incubation for 3 hours. Subsequently, the beads were washed five times and boiled with 2× SDS at 100°C for 10 min. The proteins were separated on a 12% polyacrylamide gel electrophoresis (PAGE) gel and transferred onto a polyvinylidene difluoride membrane (Millipore, ISEQ00010). A chemiluminescence detector (Tanon, 5200Multi) was used for detection. Anti-MYC (CWbio, CW0259), horseradish peroxidase (HRP)–conjugated anti-FLAG antibodies (Sigma-Aldrich, A8592), and goat-anti-mouse HRP (CWbio, CW0102S) were used for Western blot assays.

Firefly luciferase complementation imaging assay

To confirm the protein interaction in vivo, nLUC-TIE2 was generated by LR reaction between pENTR-TIE2 and pCAMBIA-nLUC. cLUC-ARRx was generated by LR reaction between pCAMBIA-cLUC and pENTR-ARRx, respectively. cLUC-ARR3 was generated as a negative control. The constructs were transformed into A. tumefaciens GV3101. Different combinations were coinfiltrated into tobacco leaves with pCAM-P19. The transformed plants were grown in the dark for 12 hours and then put into a greenhouse with long-day conditions for 48 hours. The isolated tobacco leaves were treated with 1 mM luciferin at the abaxial surface in the dark for 10 min. Luciferase luminescence was detected by a low-light cooled CCD imaging apparatus (NightOWL II LB 983) with indiGO software.

Yeast two-hybrid assays

ARR10, ARR11, ARR12, ARR14, and ARR18 were amplified from wild-type cDNA and cloned into pENTR/D-TOPO to generate pENTR-ARRs. Truncated TIE2 (TIE2-N and TIE2-C) and ARR2 (ARR2-DDK, ARR2-Ac-M, and ARR2-Q) were cloned into pENTR/D-TOPO to generate pENTR-TIE2-N, pENTR-TIE2-C, pENTR-ARR2-DDK, pENTR-ARR2-Ac-M, and pENTR-ARR2-Q.

To test the interaction between TIE2 and ARRs, TIE2 was cloned into pDEST32 (Invitrogen) as bait. AD-ARRs were generated by LR reaction between pENTR-ARRs and pDEST22 as prey. To further confirm the interaction between TIE2 and type B ARRs, TIE2 was cloned into pDEST22 as prey, while type B ARRs into pDEST32 as bait. To test the interaction between TIE2 and ARR2, pENTR-TIE2-N, and pENTR-TIE2-C were cloned into pEDST32 as bait by LR reaction. pENTR-ARR2-DDK, pENTR-ARR2-Ac-M, and pENTR-ARR2-Q were cloned into pDEST22 as prey by LR reaction. Different combinations of bait and prey constructs were cotransformed into yeast strain AH109. The blank construct pDEST22 was cotransformed with the bait constructs as a negative control. The transformed yeast cells were grown on selective medium supplemented with SD-Leu-Trp-His with or without 2.5 mM 3-AT at 30°C for 3 days.

Transient dual-luciferase reporter assay

To assess the transcriptional repression activity of TIE2 in tobacco leaves, the sequence encoding G4BD-TIE2 (G4BD, GAL4 DNA binding domain) was amplified from the TIE2-pDEST32 construct and cloned into pENTR/D-TOPO to generate pENTR-G4BD-TIE2. 35S-G4BD-TIE2 was generated by LR reaction between pENTR-G4BD-TIE2 and pK2GW7. The sequence encoding G4BD was cloned into pENTR/D-TOPO to generate pENTR-G4BD, and 35S-G4BD was generated by LR reaction between pK2GW7 and pENTR-G4BD. The sequence encoding TIE2mEAR, in which the conserved Leu residues were mutated, was amplified using TIE2-F and TIE2mEAR-R primers, and subsequently cloned into pENTR-D/TOPO to generate pENTR-TIE2mEAR. G4BD-TIE2mEAR was generated by LR reaction between pDEST32 and pENTR-TIE2mEAR. The sequence encoding G4BD-TIE2mEAR was amplified to generate pENTR-BD-TIE2mEAR. 35S-BD-TIE2mEAR was obtained by LR reaction between pK2GW7 and pENTR-BD-TIE2mEAR. Different combinations were coinfiltrated into tobacco leaves with pCAM-P19. The treatment and the detection of luminescence were as described above.

To test the transcriptional repression activity of TIE2 in Arabidopsis protoplasts, 35S-G4BD, 35S-G4BD-TIE2, and 35S-G4BD-TIE2mEAR were cloned into pGreen II 62-SK using the FastClone method. Arabidopsis protoplasts were prepared as described previously (). Next, the protoplasts were cotransformed with 10 μg of different combinations of plasmids. The transformed protoplasts were collected and resuspended with 1 ml of W5 solution. The LUC/REN ratio was detected by a dual-luciferase reporter assay system (Promega, GLO-MAX 20/20 luminometer) after incubation at room temperature for 16 hours under mild light conditions.

To test whether TIE2 represses ARR1 or ARR2 transactivation activity, the sequences encoding ARR1, ARR2, TIE2, and TIE2mEAR were cloned into pGreen II 62-SK to generate 35S-ARR1, 35S-ARR2, 35S-TIE2, and 35S-TIE2mEAR using the FastClone method. The ARR15 promoter was amplified by ARR15pro-F and ARR15pro-R and then cloned into pENTR/D-TOPO to generate pENTR-ARR15pro. 35S-REN-ARR15pro-LUC was generated by LR reaction between pENTR-ARR15pro and pGreen II 0800 LUC-GW. The transient expression of different combinations of plasmids and detection of luminescence were performed as described above.

To detect whether TIE2 repressed the transactivation activity of ARR2, the protoplasts coexpressing different combinations of plasmids were collected and resuspended with 100 μl of W5 solution. Ten microliters of 10 mM d-luciferin was mixed with 90 μl of protoplast suspension in 96-well plates. The LUC activity was then detected with Centro XS3 LB 960 microplate Luminometer continuously in time course.

GUS staining, propidium iodide staining, EdU staining, and GFP observation

TIE1pro-TIE1-GFP and TIE2pro-TIE2-GFP seedlings were fixed in 90% (v/v) acetone for 20 min on ice, washed twice with phosphate buffer, and incubated in GUS staining solution containing 5-bromo-4-chloro-3-indolyl glucuronide at 37°C for 2 hours. The staining solution was then changed to 75% ethanol for decolorizing. The root staining was observed using a Leica M205 FCA stereoscope.

For propidium iodide (PI) staining and GFP observation, Arabidopsis seedlings of TIE1pro-TIE1-GFP or TIE2pro-TIE2-GFP were grown vertically on 1/2 MS medium. Roots were stained with PI solution (1 mg/ml; Sigma-Aldrich, P4170) for 30 s. PI staining and GFP were then observed using a confocal laser scanning microscope (Zeiss LSM 710 NLO).

Click-iT EdU Alexa Flour 488 Imaging Kit (Invitrogen, C10337) was used to perform EdU staining. In brief, 5-day-old wild-type and tie1-1 tie2-1 seedlings were submerged in liquid MS medium containing 20 μM EdU for 30 min. The samples were fixed in phosphate-buffered saline (PBS; pH 7.2 to 7.6) containing 3.7% formaldehyde and 0.1% Triton X-100 for 30 min. The fixative was then removed, and the samples were washed twice with PBS containing 3% bovine serum albumin (BSA). The seedlings were incubated in the Click-iT reaction cocktail for 30 min at room temperature without light. The reaction cocktail was removed, and the samples were washed three times with PBS.

RNA-seq and ChIP-seq analysis

The roots of 7-day-old tie1-1 tie2-1 and wild-type seedlings were collected for RNA extraction. RNA-seq was carried out by Novogene Corporation with three replicates. RNA-seq libraries consisting of 150-bp paired-end reads were generated on an Illumina NovaSeq 6000 platform. RNA-seq data analysis was described previously (). Briefly, quality control of the raw reads was performed by fastp (0.20.1). The clean reads were mapped to the Arabidopsis reference genome (TAIR10) by STAR (2.7.3a). Expression counts were generated by featureCounts in Subread 2.0.1 with default parameters. The R package DEseq2 (1.32.0) was used for differential gene expression analysis. Genes with a Bonferroni-adjusted P value (padj) < 0.05 and a log2 fold change <−1 or > 1 were considered as significantly DEGs. Volcano plots were generated by R package ggpubr (0.4.0). To perform promoter analysis, motif enrichment was analyzed in the 500-bp promoter regions of up-regulated genes in tie1-1 tie2-1 using MEME software online (https://meme-suite.org/meme/tools/sea) ().

To determine whether ARR1 could bind to the promoter of TIE1 and TIE2, raw ARR1 ChIP-seq data were downloaded from the Gene Expression Omnibus (accession number GSE94486) (). The analysis of ChIP-seq data was described previously (). Briefly, the quality control of the raw reads was performed by fastp (0.20.1). The clean reads were mapped to the Arabidopsis reference genome TAIR10 by bowtie2 (2.3.5). The unique mapped reads were used for downstream analysis in Samtools (1.9). BigWig files were generated with binSize 10 and the option normalizeUsingBPM using bamCoverage in deepTools (3.3.1). Typical screenshots were captured with Integrative Genomics Viewer (2.6.2). ChIP-seq raw data of EIN3 were downloaded from the Sequence Read Archive (SRA) with the accession number SRA063695 (). The FASTQ files were extracted from SRA files by fasterq-dump (2.9.6) with parameter split-3. The adaptors were removed with trim_galore (0.6.6). The clean reads were mapped the Arabidopsis reference genome TAIR10 by bowtie2 (2.3.5). Replicated reads were removed by sambamba (0.8.2). BW files were generated with binSize 10 and normalizeUsinfBPM by bam coverage from deepTools (3.3.1). Typical screenshots were captured with IGV (2.6.2).

ChIP-PCR assay

ChIP experiments were performed as described previously (). The seedlings of 5-day-old wild-type and 35S-GFP-ARR1 seedlings were collected to perform ChIP-PCR. The sonicated chromatin was incubated with 5 μl anti-GFP (Abcam, ab290) at 4°C overnight. Protein A-Sepharose (GE Healthcare 17-1279-01) was used as a negative control and the supernatant after incubation was taken as the input. TUBULIN ALPHA-4 CHAIN (TUA4) was used as the negative control and ARR7 as the positive control. The primer pairs used for ChIP-PCR were listed in table S1.

Electrophoretic mobility shift assay

The DNA sequence encoding the ARR1 DNA binding domain (DBD) was cloned into pGEX-4T with an N-terminal glutathione S-transferase (GST) tag. The vector was transformed into Escherichia coli strain BL21. The bacteria were cultured overnight at 18°C, and proteins were induced by adding isopropyl β-d-1-thiogalacto-pyranoside (IPTG) to a final concentration of 0.5 mM. The cells were collected and resuspended in binding buffer (PBS, pH 7.3, 1 mM DTT). The target protein was eluted with elution buffer [50 mM tris-HCl, 10 mM reduced glutathione (pH 8.0), and 1 mM DTT] using a GSTrap HP column (Cytiva, 17-5282-01).

The 18- or 30-bp DNA fragments containing ARR1 binding motifs 5′-(A/G)GAT(T/C)-3′ in the TIE2 promoter region were synthesized. The binding motifs were mutated to AAAAA in the control DNA fragments. Double-stranded DNA was obtained by annealing equimolar concentrations in binding buffer.

To carry out EMSA, the purified GST-ARR1-DBD proteins (about 550 ng) were mixed with double-stranded DNA (200 ng). The mixture was incubated at 4°C for 20 to 30 min and then loaded onto a 7% nondenaturing polyacrylamide gel and subjected to electrophoresis (PAGE) using 0.5× tris-borate EDTA buffer (Sangon, B548102). Electrophoresis was performed at 120 V for 40 hours. Ethidium bromide staining was used to detect DNA under Tanon2500.