Overexpression of OsRAA1 causes pleiotropic phenotypes in transgenic rice plants, including altered leaf, flower, and root development and root response to gravity

Abstract

There are very few root genes that have been described in rice as a monocotyledonous model plant so far. Here, the OsRAA1 (Oryza sativa Root Architecture Associated 1) gene has been characterized molecularly. OsRAA1 encodes a 12.0-kD protein that has 58% homology to the AtFPF1 (Flowering Promoting Factor 1) in Arabidopsis, which has not been reported as modulating root development yet. Data of in situ hybridization and OsRAA1::GUS transgenic plant showed that OsRAA1 expressed specifically in the apical meristem, the elongation zone of root tip, steles of the branch zone, and the young lateral root. Constitutive expression of OsRAA1 under the control of maize (Zea mays) ubiquitin promoter resulted in phenotypes of reduced growth of primary root, increased number of adventitious roots and helix primary root, and delayed gravitropic response of roots in seedlings of rice (Oryza sativa), which are similar to the phenotypes of the wild-type plant treated with auxin. With overexpression of OsRAA1, initiation and growth of adventitious root were more sensitive to treatment of auxin than those of the control plants, while their responses to 9-hydroxyfluorene-9-carboxylic acid in both transgenic line and wild type showed similar results. OsRAA1 constitutive expression also caused longer leaves and sterile florets at the last stage of plant development. Analysis of northern blot and GUS activity staining of OsRAA1::GUS transgenic plants demonstrated that the OsRAA1 expression was induced by auxin. At the same time, overexpression of OsRAA1 also caused endogenous indole-3-acetic acid to increase. These data suggested that OsRAA1 as a new gene functions in the development of rice root systems, which are mediated by auxin. A positive feedback regulation mechanism of OsRAA1 to indole-3-acetic acid metabolism may be involved in rice root development in nature.

Figure 1.

The analysis of the OsRAA1 gene and its protein sequence. A, Nucleotide and putative protein sequence of OsRAA1 cDNA. B, Protein sequence multiple alignment of OsRAA1. BaLeP1 (BE421936), BaSpP1 (BG342901), and BaSpP2 (BE196402) are from barley leaf and spike; MuFPF1 (Y11987) is from mustard; WhRoP1 (BE428690) and WhRoP2 (BE428819) are from wheat root; ChrFPF1 (BE034410) and ChrFPF2 (BE033961) are from chrysanthemum; MossFPF1 (AW699964) is from moss (Physcomitrella patens); AtFPF1 (Y11988) is from Arabidopsis; ZeaTaP2 (BE552830) is from maize. C, Protein multialignment of rice putative OsRAA1/FPF1 family genes. OsRAAL1 (OsRAA like) is from rice contig AAAA01001788.1, which contains EST AU070455; OsRAAL2 is from rice chromosome 7 (AP003982). D, The phylogenetic tree of the FPF1/OsRAA1 gene family processed by software DNAMAN. In the phylogenetic tree at bottom, the numbers of amino acid substitutions per alignment site are indicated on the branches. E, OsRAA1 promoter structure. TAACAAA/TTTGTTA is a gibberellin response element; TGTCTC is an AuRE. Bar = 100 bp.

Figure 2.

OsRAA1 expression patterns. A, OsRAA1 northern blot of RNA from tissues of young shoots, roots, young spikes, and leaves in wild-type plants. Young spikes were harvested from 70-d-old plants grown in a growth chamber. The remaining materials were from plants grown for 14 d. Ethidium bromide (EtBr) staining shows equal RNA loading. B to G, In situ localization of OsRAA1 transcript in wild-type rice with an antisense probe. B, Longitudinal section of young root with lateral root. C, Longitudinal section of young root with lateral root primordium. D, Longitudinal section of young shoot, including the junction part between shoot and roots. E, Longitudinal section of young spike. F, Longitudinal sections of root tip. G, Details of young root meristem zone. b, e, and g are the control sections with a sense probe of OsRAA1 corresponding to B, E, and G sections, respectively. C and D photos were taken after mounting of slides using resin, so signals are blue. The remaining photos were taken before mounting of the slide, therefore, signals are brown.

Figure 3.

The localization of OsRAA1∷GUS gene expression in the transgenic rice. A, A primary root with lateral root. B, Apical region of a primary root. C, Mature spikelet. D, Young shoot with some young leaves. E, Floral organs of a spikelet. Arrow is a morphogenesis site of lateral root with GUS activity.

Figure 4.

The expression pattern of OsRAA1 and the phenotype of root in the Ubi∷OsRAA1 overexpression transgenic rice. A, Northern-blot analysis for a representative Ubi∷OsRAA1 transgenic plant to show an overexpression. Ethidium bromide (EtBr) staining shows equal RNA loading. B, Statistical analysis of primary root growth in Ubi∷OsRAA1 transgenic plant. More than 10 seedlings were examined for each point. C, Roots of 12-d-old seedlings grown in the half-strength MS medium. CK, control. OsRAA1, Representative of Ubi∷OsRAA1 transgenic line. D, Mature roots at flowering stage. E, The wild-type plant roots (CK), transgenic line 8 (OsRAA1-8), and transgenic line 11 (OsRAA1-11) at 9 d after germination in the half-strength MS medium. Arrow is a primary root.

Figure 5.

The phenotypes of Ubi∷OsRAA1 constitutive expression transgenic rice plant. A, Flag leaf of wild-type control plant (left) and the Ubi∷OsRAA1 transgenic plant (right). B, Silica cells of flag leaf of wild-type control plant. C, Silica cells of flag leaf of the transgenic plant. D, Statistical analysis of the length of silica cells in the flag leaves (P < 0.05; two samples t test proceeded by Origin 6.0). More than 50 cells were examined for each bar. E, Flowers of wild-type control plant. F and G, The transgenic plant with different lines (D–F) to show longer filaments.

Figure 6.

OsRAA1 expression is regulated by auxin. A, Northern-blot analysis for OsRAA1 expression treated with IAA of different concentration. Ethidium bromide staining (EtBr) shows equal RNA loading in the blot. The bar shows a relative amount of the gene expression at the bottom. B, Effect of GUS staining on treatment of 1 μm NAA (no. 2) and water as a control (no. 1) and on treatment of the auxin polar transport inhibitor (HFCA, 0.5 μm) for 0, 1, 2, and 4 h (corresponding to nos. 3, 4, 5, and 6, respectively). Data of auxin (nos.1 and 2) and HFCA (nos. 3–6) were from different experiments. C, Roots of wild-type plant (14 d). D, Effect of NAA (1 μm) on root growth of the wild-type plant. The seedling was incubated for 14 d after germination. Arrows show primary roots.

Figure 7.

Effects of auxin and HFCA on adventitious root in the OsRAA1 transgenic rice plants. A, Treatment of IAA. B, Treatment of HFCA, auxin polar transport inhibitor.

Figure 8.

Auxin levels of the OsRAA1 overexpression transgenic plant and wild type. A, IAA level in shoot. B, IAA level in root. The roots and shoots used were selected 9 d after germination.

Figure 9.

Overexpression of OsRAA1 inhibited gravitropism. Root gravitropic response treated with horizontal reorientation for 0 h, left, and 3 h, right (A–C). A, Wild-type control. B, Overexpression Ubi∷OsRAA1 transgenic seedling. C, Wild-type seedling treated with IAA (0.4 μm). D, Time course of gravitropic response (more than 20 plants were examined for each point).