Crown rootless1, which is essential for crown root formation in rice, is a target of an AUXIN RESPONSE FACTOR in auxin signaling

Abstract

Although the importance of auxin in root development is well known, the molecular mechanisms involved are still unknown. We characterized a rice (Oryza sativa) mutant defective in crown root formation, crown rootless1 (crl1). The crl1 mutant showed additional auxin-related abnormal phenotypic traits in the roots, such as decreased lateral root number, auxin insensitivity in lateral root formation, and impaired root gravitropism, whereas no abnormal phenotypic traits were observed in aboveground organs. Expression of Crl1, which encodes a member of the plant-specific ASYMMETRIC LEAVES2/LATERAL ORGAN BOUNDARIES protein family, was localized in tissues where crown and lateral roots are initiated and overlapped with beta-glucuronidase staining controlled by the DR5 promoter. Exogenous auxin treatment induced Crl1 expression without de novo protein biosynthesis, and this induction required the degradation of AUXIN/INDOLE-3-ACETIC ACID proteins. Crl1 contains two putative auxin response elements (AuxREs) in its promoter region. The proximal AuxRE specifically interacted with a rice AUXIN RESPONSE FACTOR (ARF) and acted as a cis-motif for Crl1 expression. We conclude that Crl1 encodes a positive regulator for crown and lateral root formation and that its expression is directly regulated by an ARF in the auxin signaling pathway.

Figure 1.

Phenotypes of crl1. (A) and (B) Two-week-old wild-type (A) and crl1 (B) seedlings. SR, seminal root; CR, crown root; LR, lateral root. Bars = 2 cm. (C) and (D) Cross sections through the second nodes in 2-week-old wild-type (C) and crl1 (D) seedlings. The arrow and arrowheads indicate the crown root primordium and peripheral cylinder of vascular bundles, respectively. Bars = 150 μm. (E) and (F) Two-month-old wild-type (E) and crl1 (F) plants. Bar = 5 cm. (G) and (H) Wild-type (G) and crl1 (H) plants at the grain filling stage. Bars = 30 cm.

Figure 2.

Gravitropic Phenotype in Seminal Roots of crl1. (A) Wild-type and crl1 seedlings were grown vertically for 3 d and then rotated 90°. (B) and (C) The root tip angle (θ) in (B) was measured 24 h after reorientation (C). Each data point is the average of 20 plants. The average values of the wild type and crl1 were 33.6 and 66.4, respectively, and there was a significant difference at P < 0.01 (t test).

Figure 3.

Map-Based Cloning and Phenotypic Complementation by the Introduction of Crl1. (A) High-resolution linkage and physical map of the Crl1 locus. The vertical bars represent molecular markers, and the numbers of recombinant plants are indicated under the linkage map. The Crl1 locus was mapped between two markers, SI56 and SI52, on BAC clone b0050N02. cM, centimorgan. (B) crl1 mutant plants containing the empty vector (left) and the genomic DNA fragment encompassing the entire Crl1 gene (right) are shown. Bar = 5 cm.

Figure 4.

Structure of Crl1. (A) Predicted amino acid sequence of Crl1. The AS2/LOB domain is boxed. Asterisks show the positions of Cys residues in the C-motif. The amino acid residue mutated in crl1 is indicated in italics above the wild-type sequence. Conserved C- and GAS-motifs are underlined with solid and dashed lines, respectively. (B) Comparison of the amino acid sequences of the AS2/LOB domains for AS2/LOB proteins in Arabidopsis (ASL18/LBD16 and ASL16/LBD29), maize (ZmCrll1), and rice (Crl1 and OsCrll1). Amino acid residues conserved in the AS2/LOB proteins are indicated by white characters on a black background. Conserved C- and GAS-motifs are bracketed. Asterisks show the conserved Cys residues, and the arrow indicates the mutated Ala with Thr in crl1. (C) An unrooted dendogram of the AS2/LOB family proteins in Arabidopsis (ASL4/LOB, AS2/LBD6, ASL18/LBD16, ASL20/LBD18, ASL23/LBD19, ASL3/LBD25, and ASL16/LBD29), maize (ZmCrll1 and ZmCrll2), and rice (Crl1, OsCrll1, OsCrll2, OsCrll3, and OsCrll4).

Figure 5.

Expression Pattern of Crl1. (A) Crl1 expression in various organs of wild-type rice. Semiquantitative RT-PCR was conducted, and Actin1 was used as a control. (B) to (L) Localization of GUS activity under the control of the Crl1 promoter. Leaves (B), stem (C), transverse sections through nodes of the stem ([D] and [E]), young seminal root (F), and transverse sections through a seminal root ([G], [I], and [K]) are shown. (H), (J), and (L) are schematic diagrams of (G), (I), and (K), respectively. Arrows and arrowheads in (D) and (E) indicate parenchyma cells and peripheral vascular cylinders, respectively. Gray, yellow, and blue regions in (H), (J), and (L) indicate lateral root primordia, endodermis, and pericycle, respectively.

Figure 6.

Auxin Induces Crl1 Expression. (A) Induction of Crl1 and OsIAA4 by auxin. Seedlings were treated with 10⁻⁵ M IAA, and whole plants were sampled after the indicated time. (B) Effects of auxin and CHX on Crl1 transcript levels. Seedlings were treated with or without 10⁻⁵ M IAA and 10⁻⁶ M CHX. (C) and (D) Localized GUS staining controlled by Crl1 (C) and the DR5 (D) promoter in the seminal root developing lateral roots of transgenic rice. Arrows and arrowheads indicate outer parenchyma cells and peripheral vascular cylinders, respectively. (E) and (F) Auxin-induced Crl1 expression requires the degradation of AUX/IAA proteins. (E) The chimeric gene consists of an in-frame fusion of the mutated OsIAA3 cDNA and the steroid binding domain of the human GR. The conserved Pro at position 58 of OsIAA3, which is located in the degradation-related domain (domain II), was exchanged with Leu. (F) Transgenic seedlings were incubated without (−) or with (+) IAA and CHX, respectively. Semiquantitative RT-PCR was conducted, and Actin1 was used as a control.

Figure 7.

AuxRE in the CrlI Promoter Is Essential for Interaction of OsARF1 and Expression of Crl1. (A) The fragments used as probes for electrophoresis mobility shift assays are numbered from 1 to 4. The M3 fragment contains a single nucleotide substitution from A to T in the AuxRE2 sequence. (B) Fragment 3 was shifted by incubation with OsARF1. (C) and (D) GUS staining controlled by the wild type (C) and the mutated (D) Crl1 promoter at the AuxRE2 sequence in the transgenic rice seedlings.

Figure 8.

Auxin Signaling Pathway Leading to Crl1 Expression in Root Formation. AUX/IAAs function as negative regulators in auxin signaling to prevent the function of ARFs by direct interaction. Auxin treatment promotes the degradation of AUX/IAAs to release ARF. The released ARFs interact with the AuxRE in the Crl1 promoter to trigger its transcription, resulting in root initiation.