Transcriptional corepressor OsTPR1 regulates tillering and lateral root development in rice

OsTPR1 regulates plant architecture and flowering time in rice

Transcriptional corepressors regulate a variety of physiological processes by interacting with repressor proteins. We previously identified the rice corepressor OsTPR1 (NM_001056122) from a cDNA library generated from sucrose-starved rice suspension cells²⁹. In rice, three OsTPR homologs have been reported¹¹. Amino acid sequence alignment revealed that OsTPR1 shares 68% and 63% identity with OsTPR2 (NM_001067590) and OsTPR3 (NM_001049151), respectively. To investigate the functional role of OsTPR1 in rice development, we generated transgenic rice lines either overexpressing (OsTPR1-Ox) or silencing (OsTPR1-Ri) the gene under the control of the maize ubiquitin promoter³⁰ (Fig. 1A).

Comparison of plant height, tiller angle, and heading date among WT, OsTPR1-Ox, and OsTPR1-Ri lines. (A) Schematic representation of the overexpression construct (OsTPR1-Ox) and the RNA interference (RNAi) silencing construct (OsTPR1-Ri). (B) Growth phenotypes of WT and T₃ transgenic rice plants at 21 and 85 days of age. The 21-day-old seedlings were transplanted from culture pots into soil-filled buckets, and phenotypes were analyzed 64 days after transplantation (at 85-day-old). (C) Diagram illustrating how total tiller angle was calculated as the angle between the outermost tillers of each plant. (D) Quantification of total tiller angle among WT, OsTPR1-Ox, and OsTPR1-Ri lines. (E) Quantification of mean inter-plant tiller angles. (F) Comparison of heading date among genotypes. Data represent means ± SD from three independent experiments (n = 3). Statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc test. Different letters above the bars indicate statistically significant differences (P < 0.01).

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The T₂ transgenic plants were used for phenotypic analysis. To evaluate whether OsTPR1 affects rice development, 21-day-old WT, OsTPR1-Ox (OsTPR1-Ox-1, OsTPR1-Ox-2), and OsTPR1-Ri (OsTPR1-Ri-1, OsTPR1-Ri-2) seedlings were transplanted into soil (Fig. 1B). By 85 days, OsTPR1-Ox plants exhibited significantly reduced plant height (OsTPR1-Ox-1: 72 ± 2.8 cm; OsTPR1-Ox-2: 70 ± 2.6 cm) compared to WT (90 ± 3.1 cm), whereas OsTPR1-Ri lines (OsTPR1-Ri-1: 92 ± 3.4 cm; OsTPR1-Ri-2: 93 ± 2.7 cm) were slightly taller, with no significant difference from WT.

Heading date analysis revealed that OsTPR1-Ri-1 and OsTPR1-Ri-2 flowered earlier (84 ± 2.5 and 85 ± 2.2 days, respectively) compared to WT (98 ± 2.1 days), while OsTPR1-Ox-1 and OsTPR1-Ox-2 showed delayed flowering (105 ± 3.3 and 103 ± 2.6 days, respectively) (Fig. 1F). Notably, tiller angle was also influenced by OsTPR1. OsTPR1-Ox plants exhibited a significantly wider total tiller angle (~ 68.8°) and inter-plant tiller angle (~ 12.8°), compared to WT and OsTPR1-Ri lines, which had total angles of ~ 39.4° and 41.3° and inter-plant angles of − 8.6° and 7.8°, respectively (Fig. 1C-E). These findings indicate that OsTPR1 promotes increased tiller angle and delays heading date. Given the consistency of these phenotypes, OsTPR1-Ox-1 and OsTPR1-Ri-1 lines were selected for further experiments.

OsTPR1 negatively regulates lateral root development

Given the influence of OsTPR1 on shoot architecture, we next examined its role in root development. We constructed an OsTPR1::GUS reporter by fusing the OsTPR1 promoter (including its first intron within the 5′ UTR) to the GUS reporter gene (Fig. 2A). GUS staining was strong in 3-day-old germinating embryos but nearly absent in the endosperm and aleurone layers (Fig. 2B). In 7-day-old seedlings, intense GUS staining was observed in the elongation zones of seminal and crown roots, and in emerging lateral roots, but was weak in the differentiation zone and absent from the root cap (Fig. 2C). These patterns suggest a role for OsTPR1 in root development.

Histochemical staining of β-glucuronidase (GUS) activity in transgenic OsTPR1::GUS lines. (A) Diagram of the OsTPR1::GUS construct, including the OsTPR1 promoter and first intron located within the 5′ untranslated region. (B) GUS staining of 3-day-old germinating seeds from three independent lines. (C) GUS staining of 7-day-old seedlings. Dashed boxes indicate local differentiation zones in seminal roots (a), and developing crown roots (b). Corresponding enlarged views are shown in a1 and b1, respectively.

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To evaluate its functional role, we quantified lateral root traits in 20-day-old seedlings. OsTPR1-Ri-1 plants exhibited significantly more lateral roots in the crown root differentiation zone (18.3 ± 2.3 cm⁻¹) than WT (12.3 ± 1.8 cm⁻¹), while OsTPR1-Ox-1 plants had fewer lateral roots (6.3 ± 0.9 cm⁻¹) (Fig. 3A, B). Lateral root length also differed significantly: OsTPR1-Ri-1 roots were longest (0.95 ± 0.12 cm), followed by WT (0.45 ± 0.09 cm), with OsTPR1-Ox-1 showing the shortest roots (0.21 ± 0.04 cm) (Fig. 3C). These findings indicate that OsTPR1 negatively regulates lateral root formation and elongation in rice.

Comparison of lateral root development between WT and transgenic lines. (A) Representative images of crown root differentiation zones from 20-day-old seedlings. (B) Quantification of lateral root density. (C) Quantification of average lateral root length. Data are presented as means ± SD (n = 3). Statistical significance was evaluated using one-way ANOVA followed by Tukey’s post-hoc test. Different letters above the bars indicate statistically significant differences at P < 0.05 in (B) and P < 0.01 in (C).

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OsTPR1 modulates lateral root development through auxin signaling

Given the known role of auxin in root development, and the phenotypic effects observed in OsTPR1 transgenic lines, we hypothesized that OsTPR1 functions through auxin signaling pathways. To test this, OsTPR1-Ox-1 and OsTPR1-Ri-1 lines were crossed with the auxin-responsive DR5::GUS reporter line^31,32producing DR5::GUS/OsTPR1-Ox-1 and DR5::GUS/OsTPR1-Ri-1 hybrids. RT-PCR confirmed overexpression and silencing of OsTPR1 in the respective F₂ hybrid lines (Fig. 4A, B).

Expression of OsTPR1 and GUS staining of roots in hybrid rice lines. (A) RT-PCR analysis of OsTPR1 expression in hybrid rice lines DR5::GUS/OsTPR1-Ox-1 and DR5::GUS/OsTPR1-Ri-1. Total RNA was extracted from 7-day-old embryos and amplified using OsTPR1-specific primers targeting the 3′ untranslated region. (B) Quantification of OsTPR1 transcript levels from panel A. Relative gene expression was normalized to the internal control (OsActin1), with the DR5::GUS value set to 1.0. (C) GUS staining of 7-day-old germinated seedlings. Dashed boxes indicate the differentiation zones of seminal roots in DR5::GUS (a), DR5::GUS/OsTPR1-Ri-1 (b), and DR5::GUS/OsTPR1-Ox-1 (c). Corresponding enlarged views are shown in a1, b1, and c1, respectively. (D) The expression levels of the β-glucuronidase (GUS) gene in the roots of 14-day-old seedlings were analyzed by qRT-PCR. Total RNA was isolated from the differentiation zones (including lateral roots) of the seminal and crown roots of 14-day-old seedlings from the DR5::GUS, DR5::GUS/OsTPR1-Ox, and DR5::GUS/OsTPR1-Ri lines and subjected to qRT-PCR analysis. The relative expression level of the GUS gene was normalized to that of the internal control OsActin. The expression level of the GUS gene in the NAA untreated DR5::GUS line was set as 1.0. (E) GUS staining of 55-day-old roots. Dashed boxes indicate local differentiation zones in seminal roots of DR5::GUS (a), DR5::GUS/OsTPR1-Ri-1 (b), and DR5::GUS/OsTPR1-Ox-1 (c). Corresponding enlarged views are shown in a1, b1, and c1, respectively. (F) Quantification of S-type lateral root density in DR5::GUS, DR5::GUS/OsTPR1-Ri-1, and DR5::GUS/OsTPR1-Ox-1 lines. Data represent means ± SD from three independent experiments (n = 3). Statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc test. Different letters above the bars indicate statistically significant differences (P < 0.05).

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GUS staining of 7-day-old roots revealed strong GUS activity in the elongation zones of all genotypes (DR5::GUS as control, DR5::GUS/OsTPR1-Ox-1, and DR5::GUS/OsTPR1-Ri-1) (Fig. 4C, panels a-c). However, GUS activity in the differentiation zone, particularly in the lateral root primordia, was observed only in DR5::GUS/OsTPR1-Ri-1 roots (Fig. 4C, panel b, b1). No such activity was detected in DR5::GUS or DR5::GUS/OsTPR1-Ox-1 lines (Fig. 4C, panels a and a1, c and c1). These results suggest that OsTPR1 plays a negative role in auxin signaling. To further confirm this finding, we analyzed the expression levels of the GUS gene in these three lines treated with or without 1-naphthaleneacetic acid (NAA). Total RNA was isolated from the differentiation zones (including lateral roots) of the seminal and crown roots of 14-day-old seedlings from the DR5::GUS, DR5::GUS/OsTPR1-Ox, and DR5::GUS/OsTPR1-Ri lines and subjected to qRT-PCR analysis. As shown in Fig. 4D, the GUS gene expression was significantly induced in all three lines following NAA treatment compared to the untreated conditions. Although the differences in fold induction of GUS expression among the three lines were not statistically significant. However, when compared to the DR5::GUS control line, GUS expression was increased in the DR5::GUS/OsTPR1-Ri line and decreased in the DR5::GUS/OsTPR1-Ox line, under both treated and untreated conditions. These results suggest that the negative effect of OsTPR1 on auxin signaling in the roots of rice seedlings.

To assess long-term effects on lateral root development, 10-day-old seedlings were transferred to soil and grown for an additional 45 days. At 55 days, roots were carefully excavated and subjected to GUS staining. DR5::GUS/OsTPR1-Ri-1 roots exhibited stronger GUS activity and more S-type lateral roots in crown root differentiation zones compared to DR5::GUS controls, whereas DR5::GUS/OsTPR1-Ox-1 roots showed weaker GUS staining and fewer lateral roots (Fig. 4E, F). These results support the hypothesis that OsTPR1 negatively regulates lateral root development through modulation of auxin responses.

PIN-FORMED proteins (PINs) are essential for auxin transport, functioning as efflux carriers that mediate the polar movement of auxin from shoot to root tissues³³. This directional transport establishes auxin gradients that regulate both shoot growth and root development³⁴. In Fig. 3, analysis of 20-day-old seedlings revealed a significant difference in lateral root number among WT, OsTPR1-Ox, and OsTPR1-Ri lines. To investigate whether OsTPR1 influences the expression of rice OsPIN genes, potentially explaining the observed differences in lateral root development among the three lines. We analyzed the expression of OsPIN genes in the roots of 20-day-old seedlings using qRT-PCR. The results (Fig. 5) show that the expression levels of OsPIN1a, OsPIN1b, OsPIN1c, OsPIN2, and OsPIN5a were upregulated in OsTPR1-Ri lines and downregulated in OsTPR1-Ox lines (Fig. 5B, C, D, F, and G). In contrast, OsPIN10a expression was reduced in OsTPR1-Ri and increased in OsTPR1-Ox plants (Fig. 5H). No significant change was observed in OsPIN1d expression (Fig. 5E). These findings suggest that OsTPR1 affects the expression of multiple OsPIN family members, thereby potentially influencing lateral root development.

Expression levels of OsPIN genes in roots of 20-day-old seedlings. Root samples were collected by pooling ten seedlings per line for each biological replicate. Total RNA was isolated from roots of 20-day-old seedling of WT, OsTPR1-Ox-1, and OsTPR1-Ri-1 plants and subjected to qRT-PCR analysis. Relative gene expression was normalized to the internal control (OsActin1), with the WT value set to 1.0. Data represent means ± SD from single biological replicate with three technical repeats (n = 3). Statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc test. Different letters above the bars indicate statistically significant differences (P < 0.01). Primer sequences and gene accession numbers are provided in Supplementary Table S1.

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The SMALL AUXIN UP RNA (SAUR) gene family comprises early auxin-responsive genes that play diverse roles in plant development^35,36. To further evaluate whether OsTPR1 influences auxin response and, consequently, affects the expression of OsSAUR genes in roots of rice seedling. We examined the differential expression of OsSAUR genes in WT, OsTPR1-Ri, and OsTPR1-Ox lines. There are 58 OsSAURs in rice³⁵based on expression profiles from the RiceXPro database³⁷ and heatmap analysis³⁶five OsSAUR genes were selected for expression analysis during rice root development, three predicted auxin-induced genes (OsSAUR10, OsSAUR11, and OsSAUR30), and two predicted auxin-repressed genes (OsSAUR9 and OsSAUR55). The results showed that NAA treatment increased OsSAUR10 expression in the roots by 0.28 ± 0.08-fold in WT plants and 0.67 ± 0.06-fold in OsTPR1-Ri plants, but had no effect in OsTPR1-Ox roots (Fig. S1). In contrast, the other four OsSAUR genes examined did not show significant changes in expression in response to NAA treatment. If OsTPR1 influences the auxin response to regulate lateral root development through an OsSAUR-dependent pathway, OsSAUR10 is likely the candidate gene involved.

To date, many studies have shown that the TPR family can act as universal corepressors to regulate various biotic and abiotic stresses, plant growth and development, and hormone signaling responses. This regulation is mediated through the TPD domain’s interaction with the EAR motif located in different transcriptional repressors³⁸. Therefore, it is important to understand how TPR corepressors respond to various signals to balance plant growth, development, and environmental stress responses. In this study, we isolated a TPR orthologous gene, OsTPR1, from rice. Transgene overexpression and gene silencing experiments revealed that OsTPR1 increases the tiller angle during the tillering stage and delays the heading date during the flowering stage (Fig. 1). Our results also indicate that OsTPR1 negatively regulates rice lateral root development (Figs. 2, 3 and 4). These findings suggest that OsTPR1 plays multifaceted roles in rice lateral root development, tillering angle, and heading date by influences auxin signaling responses.

Root system is a key agronomic trait that plays an important role in soil conservation, water and nutrient uptake, plant-microbe interactions, and supporting overall plant growth³⁹. There are two main types of root systems in plants, taproot systems in dicot and fibrous root systems in monocot. The fibrous root system consists of adventitious roots (crown roots) growing from the base of the stem, lateral roots, and root hairs⁴⁰. Among which the lateral roots are a very important part of the plant root system. They can increase the biomass of root hairs, allowing the plant to absorb more water and nutrients, and provide better fixation in the soil^41,42. Auxin is required for the initial of lateral root primordium^43,44. Auxin transport inhibitors suppress lateral root formation, suggesting that polar auxin transport is required for lateral root initiation^45,46. Mutations in the rice cyclophilin gene (OsCYP2) exhibit defective auxin responses and block lateral root initiation⁴⁷. In this study, we found that OsTPR1 may negatively regulate the sensitivity of auxin signaling, thereby influencing root development in rice seedlings. Results (Fig. 2) show that OsTPR1::GUS is specifically expressed in developing rice roots, indicating that OsTPR1 is functionally related to root development. A synthetic promoter (DR5), composed of nine copies of the auxin response element (TGTCGG), has been shown to exhibit higher affinity for auxin response factor (ARF) binding, resulting in increased sensitivity to the auxin response³². The DR5::GUS construct contains a reporter gene that expresses β-glucuronidase (GUS) in response to auxin, enabling the monitoring of auxin distribution and signaling in different tissues and developmental stages through GUS activity staining^48,49,50. The intensity and location of the GUS staining typically reflect the distribution and concentration of auxin in that region. To investigate the functional correlation between OsTPR1 and auxin, we conducted the DR5::GUS reporter line to hybrid with the OsTPR1-Ox-1 and OsTPR1-Ri-1 lines, respectively. The hybrid line DR5::GUS/OsTPR1-Ri-1 shows stronger GUS activity staining, increased GUS gene expression, and more lateral roots in most differentiation zones of crown roots, while the DR5::GUS/OsTPR1-Ox-1 line exhibits weaker GUS staining, decreased GUS gene expression, and fewer lateral roots (Fig. 4). The results indicate that OsTPR1 plays a functional role in rice lateral root development by negatively regulating auxin signaling responses.

The PIN protein family mediates polar auxin transport and the asymmetric distribution of auxin within plant tissues. The expression of most PIN genes is tissue-specific and induced by auxin³³. In rice, there are 12 putative OsPIN genes encoding auxin efflux transporters^34,51which perform diverse functions. These include mediating asymmetric auxin distribution (e.g., OsPIN2, OsPIN5a)^34,51,52,53 and regulating seminal, crown, and lateral root development, tillering, and shoot growth (OsPIN1a, OsPIN1b, OsPIN2)^{34,51,52,54,55}. OsPIN2 also plays a role in gravitropic responses^51,56. Additionally, OsPIN1c and OsPIN1d are associated with inflorescence development⁵⁷while OsPIN10a is involved in regulating auxin flux to maintain growth under abiotic stress conditions⁵⁸. Our results (Fig. 5) show that the expression of OsPIN1a, OsPIN1b, OsPIN1c, OsPIN2, and OsPIN5a is significantly upregulated in the OsTPR1-Ri line and downregulated in the OsTPR1-Ox line. This pattern suggests that in the OsTPR1-Ri line, which exhibits enhanced auxin sensitivity, the increased expression of these five genes may promote lateral root formation (Figs. 3 and 4), as well as increased plant height and an earlier heading date (Fig. 1). In contrast, the expression level of OsPIN10a is increased in the OsTPR1-Ox line but decreased in the OsTPR1-Ri line, while OsPIN1d expression remains unchanged among the lines (Fig. 5E and H). These results suggest that different members of the rice OsPIN gene family exhibit distinct auxin-responsive expression patterns, contributing to the regulation of plant growth in rice.

SAUR proteins act downstream of auxin signaling by promoting cell expansion through the activation of H⁺-ATPases, which acidify the cell wall to facilitate expansion and enhance plant growth^59,60. As shown in Fig. S1, among the five OsSAUR genes examined, only OsSAUR10 showed enhanced expression in OsTPR1-Ri roots under NAA treatment. This finding is consistent with previous reports indicating that OsSAUR10 is upregulated by auxin in roots^36,61. Given that there are 58 OsSAUR paralogs in rice, it is possible that the other four genes are simply not involved in auxin-responsive expression in roots. However, if OsTPR1 influences root development through a SAUR-dependent pathway, it is likely mediated by OsSAUR10.

The optimal tiller angle constitutes an ideal plant architecture, is crucial for increasing grain yield in rice⁶². Rice tiller angle is determined as the angle between the middle vertical line and the plants on both sides⁶³. Auxin also regulates tillering and tiller angle in rice. The study found that the lazy1 mutants display defects in the lateral distribution of auxin in the stem in response to gravity, resulting in larger tiller angles in rice^64,65. Mutations in the nuclear protein Tiller Angle Control 4 (TAC4) reduce endogenous auxin content, ultimately leading to a larger tiller angle phenotype⁶². These previous studies confirmed that auxin negatively regulates tiller angle in rice. Consistent with the findings in our study (Fig. 1), the tiller angle in OsTPR1-Ox line was significantly larger than that of the WT and OsTPR1-Ri lines. Combining the results in Figs. 1, 4 and 5, suggests that OsTPR1 reduces the responses of auxin in tiller buds by changing the expression of OsPINs, thereby causing tillering plants to expand outward and produce larger tiller angles.

Heading date is a critical agronomic trait regulated by environmental factors such as photoperiod, temperature, and nutrient availability, as well as genetic factors including hormones and key photoperiod-related genes such as Hd3a (Heading date 3a), RFT1 (RICE FLOWERING LOCUS T1), Ehd1 (Early heading date 1), OsGI (GIGANTEA), Hd1 (Heading date 1), and Ghd7 (Grain number, plant height, and heading date 7)^66,67,68. Among these, Ghd7 can interact with Hd1 to form a strong transcriptional repressor complex that inhibits the expression of Ehd1, a key floral inducer under non-inductive long-day conditions, thereby delaying the heading date⁶⁹. Further studies have shown that the auxin signaling repressor OsIAA23 downregulates Ghd7 expression, resulting in a delayed heading date under long-day conditions⁷⁰revealing that auxin also plays a regulatory role in the rice heading stage. Our results demonstrated that the OsTPR1-Ri-1 and OsTPR1-Ox-1 lines exhibited early and delayed heading dates, respectively. Based on the data (Figs. 4 and 5), the corepressor OsTPR1 appears to play a negative regulatory role in auxin signaling. This suggests that OsTPR1-Ri plants may have an enhanced auxin response, leading to earlier flowering, while OsTPR1-Ox plants show a delayed flowering phenotype.

Plant materials

The rice cultivar Oryza sativa L. cv. Tainung 67 was used throughout this study. Among the OsTPR1 overexpression (OsTPR1-Ox) and RNA interference (OsTPR1-Ri) transgenic lines, three independent T₂ lines carrying a single-copy transgene were selected to evaluate the functional effects of OsTPR1 on rice development.

Callus induction.

Immature rice seeds, collected 10 days after flowering, were air-dried for 7 days. Thirty seeds were dehulled, surface-sterilized with 2.4% NaOCl for 30 min, and washed thoroughly with sterile water. The sterilized seeds were cultured on callus induction medium composed of N6 salts⁷¹10 µM 2,4-D, and 3% sucrose. Cultures were maintained at 28 °C under a 16 h/8 h light/dark photoperiod with 3500 lx light intensity⁶⁴. After 35 days, calli were transferred to fresh medium and cultured for an additional 7 days before Agrobacterium-mediated transformation.

Primer sequences

The nucleotide sequences of all primers used for PCR, RT-PCR, and qRT-PCR are listed in Supplementary Table S1.

Vector construction

To construct the OsTPR1 overexpression vector (OsTPR1-Ox), a 3357-bp fragment containing the full coding region of OsTPR1 was amplified using primers OsTPR1-F1 and OsTPR1-R1. The PCR product was digested with BamHI and ligated into the BamHI-digested pAHC18 vector under the control of the maize ubiquitin promoter⁷². The recombinant plasmid was then linearized with HindIII and inserted into the HindIII site of the binary vector pSMY1H⁷³.

To generate the OsTPR1 RNAi construct (OsTPR1-Ri), a 405-bp fragment comprising 65 bp of the 3′ coding region and 340 bp of the 3′ untranslated region of OsTPR1 was amplified with primers OsTPR1-F2 and OsTPR1-R2. The fragment was inserted downstream of the maize ubiquitin promoter in both sense and antisense orientations, separated by a truncated GFP sequence as a spacer⁷⁴. The construct was linearized with HindIII and inserted into the HindIII site of the pSMY1H binary vector.

To construct the OsTPR1::GUS reporter vector, a 2407 bp promoter fragment, including the 5′ untranslated region (with an 87 bp intron), was amplified using primers OsTPR1-5P and OsTPR1-3B. The PCR product was digested with PstI and BamHI and ligated upstream of the GUS reporter gene in the pBX-2 vector⁷³. The final construct was linearized with PstI and inserted into the PstI site of the pSMY1H binary vector.

Plant transformation

Transgenic rice plants were generated by Agrobacterium-mediated transformation. The recombinant plasmids were introduced into Agrobacterium tumefaciens strain EHA101 via electroporation. Transformed Agrobacterium was co-cultivated with rice calli to deliver the T-DNA, following established protocols⁶⁴.

Phenotypic analysis

Dehulled rice seeds were surface-sterilized with 2.4% NaOCl for 30 min, rinsed thoroughly with sterile water, and sown on half-strength MS solid medium supplemented with 1.5% sucrose. Cultures were maintained at 28 °C under a 16 h photoperiod with a light intensity of 3500 lx. After 7 days, seedlings were transferred to soil-filled pots and grown for either 14 days (21-day-old plants; Figs. 1B and 3) or 78 days (85-day-old plants; Fig. 1B). Phenotypic traits including plant height, tiller number, tiller angle, root length, and lateral root number were measured. Statistical analyses were performed using one-way ANOVA.

Hydroponic cultivation of the rice seedlings

Ten-day-old seedlings grown in Kimura B hydroponic solution⁷⁵ were treated with or without 10 µM NAA (1-Naphthaleneacetic acid). After 3 h of treatment (the optimal auxin concentration and induction time for OsSAUR genes are based on previous studies^36,61), seminal and crown roots were collected from WT, OsTPR1-overexpressing (OsTPR1-Ox), and RNAi knockdown (OsTPR1-Ri) lines. Quantitative RT-PCR was performed to evaluate the expression levels of selected OsSAUR genes.

Histochemical GUS staining

Sterilized, dehulled rice seeds were germinated on half-strength MS solid medium. Roots from 3-day-old seedlings, 7-day-old roots, and 55-day-old soil-grown plants were collected for GUS staining. Tissues were incubated at 37 °C in the dark for 4 h in staining buffer containing 100 mM sodium phosphate (pH 7.0), 10 mM EDTA, 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, 0.1% Triton X-100, and 1 mM 5-bromo-4-chloro-3-indolyl β-D-glucuronide. After staining, samples were stored in 70% ethanol, rinsed with water, and imaged.

Quantitative RT-PCR

Thirty dehulled, sterilized rice seeds were sown on half-strength MS medium with 1.5% sucrose and cultured under a 16 h/8 h photoperiod at 28 °C with a light intensity of 3500 lx. Root samples were collected by pooling ten seedlings per line for each biological replicate. For Figs. 4 and 14-day-old seedlings of DR5::GUS, DR5::GUS/OsTPR1-Ri, and DR5::GUS/OsTPR1-Ox were used. For Figs. 5 and S1, roots from ten seedlings per line of 20-day-old (Fig. 5) or 10-day-old (Fig. S1) WT, OsTPR1-Ri, and OsTPR1-Ox plants were isolated and pooled. Total RNA was extracted using TRIzol reagent (Invitrogen, USA), and genomic DNA was removed with a TURBO DNA-free kit (Ambion, USA). First-strand cDNA synthesis was performed using 5 µg of RNA, M-MuLV reverse transcriptase (New England Biolabs, USA), and oligo(dT) primers. Quantitative RT-PCR was conducted using an Eco Real-Time PCR System (Illumina, USA) following the manufacturer’s protocol. Gene-specific primers (Table S1) targeting the 3′ untranslated region were used. Expression levels were normalized to OsActin1 as an internal control, and expression in WT was set as 1.0. All reactions were performed in three technical replicates.

Statistical analysis

Each experiment was performed with a minimum of three experimental (biological) replicates. Data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s test. Statistical analyses were conducted using SPSS version 13.0 (SPSS Inc., Chicago, IL, USA). Significance thresholds were set at P < 0.01 (Figs. 1 and 5) or P < 0.05 (Figs. 3 and 4).

TPR :

TOPLESS-related

Ox:

Overexpression

Ri:

RNA interference

GUS:

β-glucuronidase

PIN:

PIN-FORMED

SAUR:

SMALL AUXIN UP RNA

qRT-PCR:

Quantitative reverse transcription polymerase chain reaction

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Authors and Affiliations

1. Institute of Biotechnology, National Taiwan University, Taipei, Taiwan

   Yi Hsuan Hou & Tin Yau Edwin Lai

2. Agriculture Biotechnology Research Center, Academia Sinica, Taipei, Taiwan

   Yi Hsuan Hou

3. Department of Agronomy, National Chiayi University, Chiayi, 600, Taiwan

   Chia Ying Kuo, Tin Yau Edwin Lai, Ming Hong Xiao & Shin Lon Ho

Contributions

Conceived and designed the experiments: Shin Lon Ho. Performed the experiments: Yi Hsuan Hou, Chia Ying Kuo, Tin Yau Edwin Lai and Ming Hong Xiao. Analyzed the data and wrote the paper: Shin Lon Ho. All authors reviewed the manuscript.

Corresponding author

Correspondence to Shin Lon Ho.

Conflict of interest

The authors declare no competing interests.

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