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. 2012 Jun;63(11):4151-64.
doi: 10.1093/jxb/ers098. Epub 2012 Mar 26.

Modulation of ethylene responses by OsRTH1 overexpression reveals the biological significance of ethylene in rice seedling growth and development

Wei Zhang  1 , Xin Zhou, Chi-Kuang Wen
Affiliations

Modulation of ethylene responses by OsRTH1 overexpression reveals the biological significance of ethylene in rice seedling growth and development

Wei Zhang et al. J Exp Bot. 2012 Jun.

Abstract

Overexpression of Arabidopsis Reversion-To-ethylene Sensitivity1 (RTE1) results in whole-plant ethylene insensitivity dependent on the ethylene receptor gene Ethylene Response1 (ETR1). However, overexpression of the tomato RTE1 homologue Green Ripe (GR) delays fruit ripening but does not confer whole-plant ethylene insensitivity. It was decided to investigate whether aspects of ethylene-induced growth and development of the monocotyledonous model plant rice could be modulated by rice RTE1 homologues (OsRTH genes). Results from a cross-species complementation test in Arabidopsis showed that OsRTH1 overexpression complemented the rte1-2 loss-of-function mutation and conferred whole-plant ethylene insensitivity in an ETR1-dependent manner. In contrast, OsRTH2 and OsRTH3 overexpression did not complement rte1-2 or confer ethylene insensitivity. In rice, OsRTH1 overexpression substantially prevented ethylene-induced alterations in growth and development, including leaf senescence, seedling leaf elongation and development, coleoptile elongation or curvature, and adventitious root development. Results of subcellular localizations of OsRTHs, each fused with the green fluorescent protein, in onion epidermal cells suggested that the three OsRTHs were predominantly localized to the Golgi. OsRTH1 may be an RTE1 orthologue of rice and modulate rice ethylene responses. The possible roles of auxins and gibberellins in the ethylene-induced alterations in growth were evaluated and the biological significance of ethylene in the early stage of rice seedling growth is discussed.

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Figures

Fig. 1.
Fig. 1.
RTH sequence and gene structure. (A) Sequence alignment of evolutionarily representative plant RTHs. NCR, non-conserved region; CR, conserved region; TM, transmembrane domain; NΔ49rte1, the rte1 isoform lacking the N-terminus; the arrow indicates the start of NΔ49 rte1. (B) RTH gene structure. Rectangle indicate the exons and lines indicate the introns. (C) Sequence identity and similarity of plant RTHs compared with RTE1. Os, rice (Oryza sativa); At, Arabidopsis; Sm, Selaginella; Pp, Physcomitrella.
Fig. 2.
Fig. 2.
Functional analyses of plant RTHs in Arabidopsis. The seedling triple-response phenotype of ethylene-grown wild type (Col-0) and 35S:OsRTH1 transformation lines (A), (ETR1)4LOF and a mutant expressing 35S:OsRTH1 (B), etr1-7 and a mutant expressing 35S:OsRTH1 (C), and etr1-2 rte1-2 and a mutant expressing 35S:OsRTH1 (D). (E) The seedling triple-response phenotype of the wild type (Col-0) expressing 35S:OsRTH2 and 35S:OsRTH3. (F) Seedling triple-response phenotype of etr1-2 rte1-2 expressing 35S:OsRTH2 and OsRTH3. Leaf senescence phenotype of the wild type (Col-0) (G) and ethylene-insensitive etr1-2 (H); phenotype of wild type (Col-0), (ETR1)4LOF (J), etr1-2 rte1-2 (K), and etr1-7 (L) expressing 35S:OsRTH1. Air and ethylene indicate the phenotype of the same plants before and after the treatment, respectively. Chlorophyll a measurement (M) and SAG12 expression (N) of the wild type (Col-0), etr1-2, and 35S:OsRTH1 transformants in the corresponding mutation background as indicated. Error bars indicate the standard error (SE) for the means of five measurements. RT-PCR, analysis of the mRNA level of corresponding transgenes at the translational level. a (air) and b (ethylene) indicate a statistically significant difference (α=0.01) between the wild type and mutant or transformation lines.
Fig. 3.
Fig. 3.
Subcellular localizations of GFP–RTHs in onion epidermal cells. Subcellular localizations of GFP–OsRTH1 (A), GFP–RTH2 (B), GFP–RTH3 (C), and GFP–AtRTH (E) determined by laser scanning confocol miscroscopy in onion epidermal cells co-expressing G-kb, the Golgi marker, and ER-rb, the ER marker.
Fig. 4.
Fig. 4.
Rice leaf senescence test. (A) Senescence phenotypes of rice leaf in air, ethylene (ET), and 1-MCP. (B) Chlorophyll a content (%) relative to that before treatment (0 h). Data are the mean ±SD of five biological repeats. (C) Ethylene evolution of ZH11 and transformation rice lines. (D) Relative OsRTH1 expression in ZH11 and transformation rice lines (L). Data are the mean ±SD of each measurement. Ethylene, 100 μl l−1; 1-MCP, 5 μl l−1. a, b, and c, statistically significant difference (Fisher’s LSD, α=0.01) between the wild type (ZH11) and transformation rice lines for air (a), ethylene (ET, b), and 1-MCP (c) treatments. **Significant difference (Fisher’s LSD, α=0.01) among ZH11 and transformation rice lines.
Fig. 5.
Fig. 5.
Gene expression analyses. (A) Kinetics of Sub1C induction by ethylene treatment. Expression of Sub1C (B), ADH2 (C), and SC129 (D) of ZH11 and 35S:OsRTH1 transformation lines in air (white bars) and ethylene (grey bars). Data are the mean ±SE of three independent measurements with three repeats (n=3×3). a (air) and b (ethylene): significant difference (Fisher’s LSD, α=0.01) between ZH11 and transformation rice lines.
Fig. 6.
Fig. 6.
Seedling coleoptile growth. Measurement of the coleoptile length of etiolated rice seedlings grown in air (A) and ethylene (B). The coleoptile phenotype of light-grown ZH11 and 35S:OsRTH1 (line L6) seedlings in air and ethylene, viewed from the side (C) and back (D). (E) Chlorophyll content of coleoptiles of light-grown ZH11 and 35S:OsRTH1 line L6. Numbers in (C), (D), and on the x-axis (E) indicate the ethylene concentration (μl l−1). Data are the mean ±SD for (A) and (B), n ≥ 15, and mean ± SE for (E) of 3–5 measurements. **Significant difference (Fisher’s LSD, α=0.01) between 1-MCP-treated ZH11 and air-grown ZH11 and transformation rice lines (A), or ethylene-treated ZH11 and transformation rice lines (B). a (chlorophyll a) and b (chlorophyll b) indicate identical chlorophyll contents between ZH11 treated with 10 μl l−1 and 100 μl l−1 ethylene (Student’s t-test, P > 0.05).
Fig. 7.
Fig. 7.
Ethylene promotes the leaf growth of ZH11. (A) Seedling phenotype of ZH11 and 35S:OsRTH1 transformation lines in air and ethylene. (B) The internode is not visible in rice seedlings with the outer leaves removed. Length of individual leaves of rice seedlings grown in air (C), 1-MCP (D), and ethylene (E); the x-axis indicates the leaf order. (F) Shoot phenotype of ZH11 rice seedlings grown in air, 1-MCP, and ethylene (ET). The second blade is indicated by a white arrow; the third sheath by a grey arrow; the third blade by a yellow arrow; and the fourth leaf by a white box. **Significant difference (Fisher’s LSD, α=0.01) comparing the third-leaf sheath length between ZH11 and transformation rice lines.
Fig. 8.
Fig. 8.
Effect of gibberellins on rice seedling growth. (A) Leaf length of ZH11 seedlings grown with GA3. (B) Leaf length of ZH11 and 35S:OsRTH1 transformation lines with 1 μM GA3. The x-axis indicates leaf order. (C) Leaf length of ZH11 seedlings with GA3 and 1-MCP. The x-axis indicates leaf order. ZH11, wild type; L2, L10, and L17 are three independent transformation lines. At least 15 seedlings were scored for each measurement (n ≥ 15). Data are the mean ±SD for each measurement. Ethylene: 100 μl l−1. **Significant difference (Fisher’s LSD, α=0.01) comparing the third-leaf sheath length between ZH11 and transformation lines in (B).
Fig. 9.
Fig. 9.
Adventitious root growth of ZH11 and 35S:OsRTH1 lines. Adventitious root number of hydroponically grown (A) and wet tissue-grown (B) ZH11 and 35S:OsRTH1 lines. ZH11, wild type; L2, L10, and L17 are three independent transformation lines. At least 15 seedlings were scored for each measurement (n ≥ 15). NAA, 0.1 μM; NPA, 1 μM; ethylene, 100 μl l−1; 1-MCP, 5 μl l−1.
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