Fig 1.
YUC-dependent auxin production is required for ethylene-inhibited root growth.
(A) Primary root phenotypes of Nipponbare (Nip), overexpressing OsEIN2 (EIN2-OX) and overexpressing OsEIL1 (EIL1-OX) transgenic lines treated with or without 10 ppm ethylene, in the absence or presence of 10 μM yucasin. Rice seedlings were grown in the dark for 3 d. Bar = 10 mm. (B) Root length of the plants shown in (A). Values are shown as the mean ± SD of 20–30 seedlings. The experiment was repeated at least three times with similar results. ** indicates significant difference compared to air at P < 0.01. (C) Expression of YUC genes in 3-d-old etiolated seedling roots. 3-d-old etiolated seedlings were treated with or without 10 ppm ethylene for 3 h. The RNAs from roots were isolated for qPCR. The experiment was repeated at least five times with similar results. Bars indicate ± SD.
Fig 2.
The rein7-1, a truncation of YUC8 at the C-terminus, displays rolled leaves and salt tolerance.
(A) Plant morphology of Kitaake (KT) and rein7-1 at the heading stage. Bar = 10 cm. (B) Typical image of rolled leaf. (C) The 1000-grain weight. Each value is the average of 30–50 plants. (D) Phenotypes of KT and rein7-1 under salt stress. Control indicates that rice seedlings were grown under normal conditions, and NaCl indicates that seedlings were treated with 150 mM NaCl aqueous solution. Bar = 10 cm. (E) Survival rate after salt treatment in (D). Approximately 50–60 seedlings were used in each experiment. Bars indicate ± SD of three independent assays. ** indicates a significant difference compared to KT at P < 0.01. (F) Map-based cloning of the YUC8/REIN7 gene. The locus was mapped to chromosome 3 within a 63 kb region between M0721 and M0728. ‘n’ indicates the number of samples used for map-based cloning, ‘M’ represents marker. (G) Mutation sites of two allelic mutants are indicated in the schematic diagram of the YUC8/REIN7 protein. (H) Functional complementation of the rein7 mutant. The KT, rein7-1 and complementary lines were treated with 10 ppm ethylene for 3 d under dark conditions. Bar = 10 mm.
Fig 3.
Mutation of YUC8/REIN7 confers an insensitive phenotype of primary root growth in response to ethylene.
(A) and (C) Ethylene-response phenotypes of rein7 in dark or in normal growth conditions. KT and Hwayoung (HY) are the wild types. Seedlings were grown in the dark or in normal growth conditions for 3 d in the absence (air) or presence of 10 ppm of ethylene. Bar = 10 mm. (B) and (D) Root length in (A) and (C), correspondingly. Each column is the average of 20–30 seedlings, and bars indicate ± SD. The experiment was repeated at least three times with similar results. ** indicates significant difference compared to air at P < 0.01.
Fig 4.
YUC8/REIN7 overexpression enhances ethylene response in roots.
(A) Ethylene response phenotypes of KT and inducible transgenic line (PER8-REIN7) seedlings grown in the dark for 3 d in the presence or absence of 2.5 μM estradiol. Bar = 10 mm. (B) Root length and (C) relative root length (ethylene-treated versus untreated in each genotype, respectively) in (A). Each column is the average of 20–30 seedlings, and bars indicate ± SD. The experiment was repeated at least three times with similar results. * and ** indicate significant difference compared to KT at P < 0.05 and P < 0.01.
Fig 5.
The transcripts of YUC8/REIN7 are expressed in developmental tissues.
(A) YUC8/REIN7 expression in different rice tissues detected by qPCR. Bars indicate ± SD from five independent experiments. (B) Tissue-specific expression of YUC8/REIN7 revealed by transgenic line (YUC8 promoter-GUS) analysis. (a) 3-d-old etiolated seedlings. (b) Root tip. Bar = 100 μm. (c) Root. (d) Young stem nodes. (e) Leaf. (f) Stem. (g) Developing grains. Bar = 10 mm.
Fig 6.
YUC8/REIN7 is mainly required for IPA-dependent auxin biosynthesis.
(A) The simplified liner pathway of IAA biosynthesis. The red arrows indicate the function of YUC. Enzymatic assays with GST-AtYUC2, GST- REIN7, GST-REIN7m and the product IAA (B), the substrate TAM (C) were analyzed by LC-ESI-MS/MS. The bars represent ± SD from three independent experiments. (D) The content of remained NADPH in (C). The bars represent ± SD from three independent experiments. (E) Expression of auxin-inducible genes in 7-d-old normal grown seedlings. The bars represent ± SD from five independent experiments. (F) IAA content of 7-d-old normal grown seedlings. The bars represent ± SD from three independent experiments. ** indicates a significant difference compared to KT or HY at P < 0.01.
Fig 7.
YUC8/REIN7-mediated auxin biosynthesis is required for ethylene-inhibited root growth in etiolated seedlings.
(A) DR5-GUS expression in root. Seedlings of 3-d-old etiolated transgenic lines containing DR5-GUS in the wild type or the rein7-1 background were treated with or without 10 ppm ethylene for 8 h before GUS activity was assayed. Bar = 10mm. (B) Root tip and elongation zone in (A). ‘RT’ represents root tip, ‘EZ’ represents elongation zone. Bar = 100 μm. (C) qPCR analysis of auxin-response gene expression in response to ethylene. Dark-grown 3-d-old wild-type seedlings were treated with 10 ppm ethylene for 3 h. The RNAs from roots were isolated for qPCR. (D) IAA levels in 3-d-old wild-type and rein7 etiolated seedlings in the absence or presence of 10 ppm ethylene. (E) Rescue of the reduced ethylene sensitivity of rein7-1 root by IAA. The wild-type and rein7-1 seedlings were grown in the dark for 3 d in the absence or presence of 10 ppm ethylene, with or without supplementation of 10 nM IAA. Bar = 10 mm. (F) Quantification of root inhibition in (D). Each column is the average of 20–30 seedlings. The data are shown as the mean ± SD of three biological replicates. * and ** indicate significant differences between the compared two samples at P < 0.05 and P < 0.01, respectively.
Fig 8.
Mutation of OsEIL1 abolishes ethylene-induced YUC8/REIN7 transcription.
(A) Expression of YUC8/REIN7 in response to ethylene. The wild-type seedlings were grown in the dark for 3 d and then treated with 10 ppm ethylene. The RNAs from roots and shoots were isolated and used for qPCR. (B) Ethylene-induced GUS activity in roots of transgenic plants harboring REIN7p-GUS. Etiolated seedlings of 3-d-old plants were treated with or without 10 ppm ethylene for 8 h before GUS activity was assayed. Bar = 10mm. (C) Root tip and elongation zone in (B). ‘RT’ represents root tip, ‘EZ’ represents elongation zone. Bar = 100 μm. (D) qPCR analysis of YUC8/REIN7 expression in primary roots and shoots of Nip, oseil1 and EIL1-OX seedlings grown in the dark for 3 d. (E) Expression of YUC8/REIN7 in primary roots of Nip, oseil1 and EIL1-OX seedlings grown in the dark for 3 d and then treated with 10 ppm ethylene. The data are shown as the mean ± SD of five biological replicates. * and ** indicate significant differences compared to 0 h at P < 0.05 and P < 0.01, respectively.
Fig 9.
OsEIL1 directly binds to YUC8/REIN7 promoter region.
(A) Schematic diagrams of putative EIN3 binding site (EBS) in the promoter of YCU8/REIN7. Black boxes indicate the positions of the EBS. P1-P4 are fragments of the YUC8/REIN7 promoter. (B) Anti-myc ChIP assays with DNA from 3-d-old etiolated seedling roots of Nip and overexpressing OsEIL1 with myc-tag (EIL1-myc) transgenic plants. (C) EMSA assay for binding to EBS sequence in the promoter of YUC8/REIN7 by OsEIL1 protein in vitro. Glutathione S-transferase (GST)-tagged OsEIL1 N-terminal fusion protein was incubated with biotin-labeled DNA fragments (Probe). Competition for the biotin-labeled promoter region was done by adding an excess of unlabeled probe (Competitor). Three biological replicates were performed with similar results. (D) The activation of OsEIL1 on the promoter activity of YUC8/REIN7 by transient expression assay in tobacco leaves. ‘EV’ represents empty vector. Three biological replicates were performed with similar results. (E) Quantitative analysis of luminescence intensity for each comparison in (D).
Fig 10.
YUC8/REIN7 genetically functions downstream of OsEIL1.
(A) Phenotypes of Nip, oseil1, oseil1 REIN7-OX and REIN7-OX dark-grown seedlings in the presence or absence of 10 ppm ethylene for 3 d. Bars = 10 mm. (B) Root length and relative root length (ethylene-treated versus untreated in each genotype, respectively) in (A). (C) Phenotypes of KT, rein7-1, rein7-1 EIL1-OX, EIL1-OX and Nip dark-grown seedlings in the presence or absence of 10 ppm ethylene for 3 d. Bars = 10 mm. (D) Root length and relative root length (ethylene-treated versus untreated in each genotype, respectively) in (C). In (B) and (D), each column is the average of 20–30 seedlings, and bars indicate ± SD. * and ** indicates a significant difference compared to air at P < 0.05 and P < 0.01. (E) A proposed model of ethylene-inhibited primary root elongation in rice. Ethylene signaling acts upstream of the auxin biosynthesis to regulate primary root elongation. YUC8/REIN7 is a key regulator required for ethylene-inhibited primary root elongation.