Molecular association of Arabidopsis RTH with its homolog RTE1 in regulating ethylene signaling

Abstract

The plant hormone ethylene affects many biological processes during plant growth and development. Ethylene is perceived by ethylene receptors at the endoplasmic reticulum (ER) membrane. The ETR1 ethylene receptor is positively regulated by the transmembrane protein RTE1, which localizes to the ER and Golgi apparatus. The RTE1 gene family is conserved in animals, plants, and lower eukaryotes. In Arabidopsis, RTE1-HOMOLOG (RTH) is the only homolog of the Arabidopsis RTE1 gene family. The regulatory function of the Arabidopsis RTH in ethylene signaling and plant growth is largely unknown. The present study shows Arabidopsis RTH gene expression patterns, protein co-localization with the ER and Golgi apparatus, and the altered ethylene response phenotype when RTH is knocked out or overexpressed in Arabidopsis. Compared with rte1 mutants, rth mutants exhibit less sensitivity to exogenous ethylene, while RTH overexpression confers ethylene hypersensitivity. Genetic analyses indicate that Arabidopsis RTH might not directly regulate the ethylene receptors. RTH can physically interact with RTE1, and evidence supports that RTH might act via RTE1 in regulating ethylene responses and signaling. The present study advances our understanding of the regulatory function of the Arabidopsis RTE1 gene family members in ethylene signaling.

Keywords: Arabidopsis; RTE1 homolog; ethylene; receptor; signaling.

Fig. 1.

RTHpromoter–GUS gene expression patterns. Representative images of RTHpromoter–GUS expression are shown in Arabidopsis plant samples: (A) 1-day-old light-grown seedlings; (B) 3-day-old light-grown seedling; (C) 9-day-old light-grown seedling; (D) 3-day-old dark-grown seedling; (E) root tip of 3-day-old dark-grown seedling; (F) shoot of 7-day-old light-grown seedling; (G) mature flowers show no GUS activity. Scale bars=1 mm (A–D, F, G), and 100 µm (E).

Fig. 2.

Co-localization of RTH with Golgi and ER markers in plant cells. (A) Representative images showing the fluorescent Golgi marker (ST–GFP, left panel), RFP–RTH (middle panel), and merged images (right panel) in the root epidermal cells of an 8-day-old seedling co-expressing both ST–GFP and RFP–RTH. (B) Representative images showing the fluorescent ER marker (GFP–HDEL, left panel), RFP–RTH (middle panel), and merged images (right panel) in the root epidermal cells of an 8-day-old seedling co-expressing both GFP–HDEL and RFP–RTH. Scale bars=10 µm.

Fig. 3.

Molecular association of Arabidopsis RTH with RTE1. (A) Molecular interaction between RTH and RTE1 in the yeast split-ubiquitin assay. Positive interaction is indicated by growth on medium lacking leucine, tryptophan, histidine, and alanine. Undiluted and 1:10, 1:100, and 1:1000 diluted liquid cultures were spotted on the indicated plates and incubated for 5 d at 30 °C. As a control, the ETR1 fusion paired with RTE1 or RTH was included. (B, C) Molecular interaction between RTH and RTE1 in plant cells by BiFC assay. Constructs expressing the N- and C-terminal halves of YFP fused to the N-terminus of RTH and RTE1, respectively, were co-infiltrated into onion peel cells (B) or tobacco leaf epidermal cells (C). Fluorescent YFP signals were detected by laser scanning confocal microscopy at 505–530 nm.

Fig. 4.

Analysis of Arabidopsis rth-1 mutants. (A) The relative expression of RTH in Col-0, rth-1, Ler, and rth-2 by qRT-PCR. Values are means ± SD. Significant differences between measurements (P<0.05) are indicated by different letters above the bars. (B) Hypocotyl length measurements for the 4-day-old dark-grown seedlings of Col-0, rth-1, Ler, and rth-2 germinated in the presence of the ethylene precursor ACC (100 µM). For each genotype, values are means ± SD (n=25). *P<0.05. (C) Comparison of 11-day-old light-grown seedlings of Col-0, rth-1, Ler, and rth-2. Three representative seedlings are shown. Scale bar=5 mm. (D, E) The relative expression of AtERF8 and AtERF9 in Col-0 and rth-1 by qPCR. The seedlings were germinated on the medium with different concentrations of ACC (0, 0.5, 5, 20, and 100 µM). Values are means ± SD. Significant differences between measurements (P<0.05) are indicated by different letters above the bars. (F) Comparison of 4-day-old dark-grown seedlings germinated in the presence of the ethylene precursor ACC (0.5 µM). The representative seedlings of the wild type (WT) and three RTH-overexpressing lines (RTH-ov, lines #1, #2, and #4) are shown. Scale bar=1 mm. (G) Hypocotyl length measurements for the 4-day-old dark-grown seedlings in the wild type (WT) and three RTH-overexpressing lines (RTH-ov, lines #1, #2, and #4) germinated in the presence of the ethylene precursor ACC (0, 0.5 µM). For each genotype, the mean value ± SD is shown for >30 seedlings. Significant differences between measurements (P<0.05) are indicated by different letters above the bars.

Fig. 5.

Ethylene ‘triple response’ assays in double mutants. (A) Comparison of 4-day-old dark-grown seedlings germinated in the presence of the ethylene precursor ACC (100 µM). Three representative seedlings of the wild type (WT), etr1-2, etr1-2 rte1-3, and etr1-2 rth-1 are shown. Scale bar=2 mm. (B) Comparison of 4-day-old dark-grown seedlings of different double mutants germinated in the presence of the ethylene precursor ACC (100 µM). Three representative seedlings of the mutants are shown. Scale bar=2 mm. (C) Comparison of 4-day-old dark-grown seedlings germinated in the presence of the ethylene precursor ACC (100 µM). Scale bar=2 mm. (D) Hypocotyl length measurements for the 4-day-old dark-grown seedlings in (C). For each genotype, the mean value ± SD is shown for >30 seedlings. *P<0.05.

Fig. 6.

RTH probably regulates ethylene signaling via RTE1. (A) Semi-quantitative RT-PCR analysis for RTH transcripts in rte1-3 and rte1-3-RTH-OV lines. Actin2 was used as an internal control. (B) The relative expression of RTH in rte1-3 and rte1-3-RTH-OV lines by qRT-PCR. Values are means ± SD; *P<0.05. (C) Representative seedlings of the wild type (WT), rte1-3, and rte1-3-RTH1-OV transgenic lines germinated in the presence of the ethylene precursor ACC (100 µM) in darkness. Scale bar=2 mm. (D) The RTH overexpression in rte1-3 does not change the ethylene sensitivity in rte1-3. The 4-day-old etiolated seedlings of the wild type, rte1-3, and three transgenic lines of RTH-OV (#1, #3, and #6) in the rte1-3 background were germinated on medium with different concentrations of ACC (0, 0.5, 5, 20, and 100 µM). Quantitative analysis of hypocotyl lengths is shown. Significant differences between measurements (P<0.05) are indicated by different letters above the bars (n=25). (E) The relative expression of RTE1 in rth-1 and rth-1-RTE1-OV lines by qPCR. Values are means ± SD; *P<0.05. (F) Representative 4-day-old etiolated seedlings of the wild type (WT), rth-1, and rth-1-RTE1-OV transgenic lines germinated in the presence of the ethylene precursor ACC (100 µM). Scale bar=2 mm. (G) Ethylene sensitivity in RTE1 overexpression lines. The 4-day-old etiolated seedlings of the WT, rth-1, and three transgenic lines of RTE1-OV (#4, #5, and #7) in the rth-1 background were germinated on the medium with different concentrations of ACC (0, 0.5, 5, 20, and 100 µM). Quantitative analysis of hypocotyl lengths is shown. Significant differences between measurements (P<0.05) are indicated by different letters above the bars (n=25).