E3 ubiquitin ligase SOR1 regulates ethylene response in rice root by modulating stability of Aux/IAA protein

Abstract

Plant hormones ethylene and auxin synergistically regulate plant root growth and development. Ubiquitin-mediated proteolysis of Aux/IAA transcriptional repressors by the E3 ubiquitin ligase SCF^TIR1/AFB triggers a transcription-based auxin signaling. Here we show that rice (Oryza sativa L.) soil-surface rooting 1 (SOR1), which is a RING finger E3 ubiquitin ligase identified from analysis of a rice ethylene-insensitive mutant mhz2/sor1-2, controls root-specific ethylene responses by modulating Aux/IAA protein stability. SOR1 physically interacts with OsIAA26 and OsIAA9, which are atypical and canonical Aux/IAA proteins, respectively. SOR1 targets OsIAA26 for ubiquitin/26S proteasome-mediated degradation, whereas OsIAA9 protects the OsIAA26 protein from degradation by inhibiting the E3 activity of SOR1. Auxin promotes SOR1-dependent degradation of OsIAA26 by facilitating SCF^OsTIR1/AFB2-mediated and SOR1-assisted destabilization of OsIAA9 protein. Our study provides a candidate mechanism by which the SOR1-OsIAA26 module acts downstream of the OsTIR1/AFB2-auxin-OsIAA9 signaling to modulate ethylene inhibition of root growth in rice seedlings.

Keywords: Aux/IAA protein; ethylene response; rice; root elongation; ubiquitination.

Fig. 1.

Characterization of mhz2/sor1 mutant and SOR1 protein. (A) Response of wild-type (WT) and sor1-2 etiolated seedlings to 10 ppm ethylene (ET). (Scale bar, 10 mm.) (B) Quantification of the ethylene response of WT and sor1-2 roots. The root lengths relative to the controls (0 ppm) are shown. Bars indicates SD (n ≥ 30). (C) Diagrams of SOR1 and its truncated version SOR1^T used for E3 ligase activity studies. Conserved amino acids of RING domain and their positions in full-length protein are shown. (D) GST-SOR1^T protein (∼45 kDa) displays E3 ubiquitin ligase activity in vitro. GST itself was used as a negative control. Coomassie Brilliant Blue (CBB) staining served as a loading control. (E) C127S mutation disrupts the E3 ligase activity of GST-SOR1^T. (F) Phenotypic analysis of sor1-2 transformed with SOR1-GFP with or without mutations in the RING domain. SOR1^SY harbors C127S and H148Y double mutations. (Scale bar, 10 mm.) (G) Quantification of the root ethylene response in WT, mutant, and rescued lines shown in F. The data show mean ± SD (n ≥ 30).

Fig. 2.

SOR1 mutation alters auxin response in rice root. (A) Quantification of the auxin response of WT and sor1-2 roots. The rates of root growth inhibition relative to the controls (0 µM) are shown. Bars indicate SD (n ≥ 30). (B) Ethylene inductions of OsIAA26, OsIAA9, and OsIAA20 fully require SOR1 function. Relative expression of Aux/IAA genes in WT and sor1-2 root tips was evaluated in response to 100 ppm ethylene. (C) Auxin inductions of these genes partially require SOR1 function. NAA (10 µM) was used for treatment. (D) Ethylene inductions of OsIAA26, OsIAA9, and OsIAA20 require auxin function. The auxin biosynthesis inhibitor l-Kyn (10 µM) was used for the treatment. The data show mean ± SE. Four independent biological repeats were performed and analyzed. All of the mRNA levels were normalized to OsACTIN2. (E–G) Venn diagram shows overlaps between genes induced by NAA or by ethylene (ET) treatment and the genes related by SOR1 or OsEIN2 in rice root tips. The genes regulated more than twofold were used for analysis. ET EIGs, ET ethylene-induced genes; NAA NIGs, NAA-induced genes; SOR1 Reg EIGs, SOR1-regulated ethylene-induced genes; SOR1 Reg NIGs, SOR1-regulated NAA-induced genes; and OsEIN2 Reg EIGs, OsEIN2-regulated ethylene-induced genes.

Fig. 3.

SOR1 interacts with and ubiquitinates OsIAA26 protein. (A) SOR1 interacts with OsIAA26 and OsIAA9 in a yeast two-hybrid assay. Yeast transformants were grown on the DDO media [synthetic defined (SD) –Trp −Leu] and on the DQO/X/A media (SD −Trp −Leu −His −Ade plus X-α-gal and aureobasidin A) with or without 100 µM IAA. Greenish blue indicates positive interactions. OsIAA9 and OsIAA20 are canonical Aux/IAA proteins with a conserved domain II. OsIAA26 is a noncanonical Aux/IAA protein. Interactions of the three Aux/IAA proteins with auxin receptors OsTIR1 and OsAFB2 were also tested. (B) The truncated SOR1 version containing the RING domain (GST-SOR1^T) ubiquitinates OsIAA26, but not OsIAA9. Asterisks indicate ubiquitinated MBP-OsIAA26 proteins. (C) Ubiquitination of OsIAA26 depends on the intact RING finger of SOR1. (D) SOR1 associates with OsIAA26 in a pull-down assay. The assay was performed using MBP-OsIAA26 recombinant protein and extracts were prepared from tobacco leaves expressing Myc-SOR1 protein. (E) SOR1^C127S, but not SOR1, can be detected to interact with OsIAA26 in the BiFC assay. Yellow fluorescence indicates positive interactions. (Scale bar, 50 µm.)

Fig. 4.

OsIAA26 is a substrate of SOR1. (A) OsIAA26-GFP protein levels in the absence or presence of SOR1. Crude plant protein extracts from tobacco leaves coexpressing SOR1 and OsIAA26-GFP or GFP were incubated at 30 °C for degradation assays. Coomassie Brilliant Blue (CBB) staining served as a loading control. Three independent biological repeats were performed and analyzed, and the relative protein levels were measured using ImageJ software. (B) OsIAA26-GFP protein levels in response to IAA at different time points. Ethanol solvent (EtOH) was used as a control. (C) OsIAA26-GFP protein levels in response to different concentrations of IAA. Crude plant protein extracts from tobacco leaves expressing OsIAA26-GFP were incubated with IAA for 4 h. (D) Stability of MBP-OsIAA26 in protein extracts from various indicated rice seedling roots. E. coli-expressed MBP-OsIAA26 proteins were incubated with different plant protein extracts prepared from indicated materials. MBP itself was used as a control. (E) Analysis of MBP-OsIAA26 ubiquitination in protein extracts prepared from WT and sor1-2 with or without MG132 (100 µM) treatment. DMSO was used as a control. MBP-OsIAA26-Ubn indicates ubiquitinated MBP-OsIAA26.

Fig. 5.

OsIAA9 protects OsIAA26 from SOR1-mediated degradation. (A) SOR1 interacts with OsIAA9 in a BiFC assay. Yellow fluorescence indicates positive interactions. (Scale bar, 50 µm.) (B) The truncated SOR1 version containing the RING domain (GST-SOR1^T) interacts with MBP-OsIAA9 in a pull-down assay. (C) OsIAA9 inhibits the E3 ligase activity of GST-SOR1^T in vitro. MBP was used as a control. (D) OsIAA9 reduces SOR1-mediated degradation of OsIAA26. Assays were performed using crude plant protein extracts from tobacco leaves coexpressing SOR1 and OsIAA26-GFP, with or without OsIAA9. Coomassie Brilliant Blue (CBB) staining served as a loading control. Three independent biological repeats were performed and analyzed, and the relative protein levels were measured using ImageJ software. (E) Degradation of OsIAA26 in the presence of both SOR1 and OsIAA9 in response to auxin. Assays were performed using protein extracts from tobacco leaves coexpressing SOR1, OsIAA26-GFP, and OsIAA9. Ethanol solvent (EtOH) was used as a control. (F–G) Real-time qPCR analysis for expression of OsIAA20 and OsIAA9 in their corresponding overexpressed transgenic lines. The mRNA levels were normalized to OsACTIN2. (H) Stability of GST-OsIAA26 in protein extracts from indicated transgenic rice seedling roots. E. coli-expressed GST-OsIAA26 proteins were incubated with plant protein extracts prepared from indicated materials. GST itself was used as a control.

Fig. 6.

Regulation of OsIAA9 stability and a working model for SOR1. (A) OsIAA9 interacts with OsAFB2 in the presence of auxin. Crude protein extracts prepared from tobacco leaves expressing OsAFB2 were used to do MBP pull-down assays. (B) Stability of MBP-OsIAA9 in protein extracts from various rice materials. E. coli-expressed MBP-OsIAA9 proteins were incubated with different plant protein extracts prepared from indicated materials. MBP itself was used as a control. (C) A working model for SOR1 functions in auxin-mediated ethylene response. In the absence of ethylene and auxin (Left), OsIAA9 inhibits the E3 ligase activity of SOR1 and thus stabilizes OsIAA26, facilitating normal root elongation. In the presence of ethylene and auxin (Right), ethylene-triggered IAA accumulation enhanced OsAFB2-mediated degradation of OsIAA9, with the help of SOR1. OsIAA9 removal released the E3 ligase activity of SOR1 and then promoted SOR1-mediated degradation of OsIAA26. The normal root elongation is thus repressed.