Transcriptional corepressor OsTPR1 regulates tillering and lateral root development in rice

Abstract

TOPLESS (TPL) and TOPLESS-Related (TPR) corepressors are key regulatory proteins that interact with a variety of transcription factors to form specific complexes, thereby modulating a wide range of signaling pathways and metabolic processes. This study explored the function of the rice TPR gene OsTPR1. Transgenic rice lines overexpressing OsTPR1 (OsTPR1-Ox) exhibited reduced lateral root density, whereas OsTPR1 RNA interference lines (OsTPR1-Ri) showed increased lateral root density. To gain further insight, these transgenic lines were crossed with the DR5::GUS auxin reporter line. In 7-day-old seedlings, lateral root formation occurred in the differentiation zone of seminal roots, with GUS staining prominently localized in the lateral root primordia of the DR5::GUS/OsTPR1-Ri line. Similar results were observed in 45-day-old seedlings, where the DR5::GUS/OsTPR1-Ri line exhibited stronger GUS staining and a higher number of lateral roots in the crown root differentiation zones. In contrast, the DR5::GUS/OsTPR1-Ox line showed weaker GUS signals and fewer lateral roots. Additionally, the expression levels of several auxin efflux transporter genes encoding PIN-FORMED (PIN) proteins, including OsPIN1a, OsPIN1b, OsPIN1c, OsPIN2, and OsPIN5a, were increased in the OsTPR1-Ri line but decreased in the OsTPR1-Ox line. These results suggest that OsTPR1 also modulates the expression of multiple OsPIN genes, thereby potentially influencing auxin responses and lateral root development. Beyond root development, OsTPR1 overexpression led to a significant increase in tiller angle and a delay in flowering time, whereas OsTPR1-Ri plants exhibited earlier flowering. These findings indicate that OsTPR1 acts as a negative regulator of the auxin response, with its overexpression leading to reduced auxin sensitivity and altered plant architecture. Our results show that OsTPR1 modulates lateral root development, tiller angle, and flowering time, contributing to the coordinated growth and development in rice.

Keywords: Auxin; Heading date; Lateral root; Rice; TOPLESS-related protein (OsTPR1); Tiller angle.

Fig. 1

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).

Fig. 2

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.

Fig. 3

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).

Fig. 4

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).

Fig. 5

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.