Rice CONSTITUTIVE TRIPLE-RESPONSE2 is involved in the ethylene-receptor signalling and regulation of various aspects of rice growth and development

Abstract

In Arabidopsis, the ethylene-receptor signal output occurs at the endoplasmic reticulum and is mediated by the Raf-like protein CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) but is prevented by overexpression of the CTR1 N terminus. A phylogenic analysis suggested that rice OsCTR2 is closely related to CTR1, and ectopic expression of CTR1p:OsCTR2 complemented Arabidopsis ctr1-1. Arabidopsis ethylene receptors ETHYLENE RESPONSE1 and ETHYLENE RESPONSE SENSOR1 physically interacted with OsCTR2 on yeast two-hybrid assay, and green fluorescence protein-tagged OsCTR2 was localized at the endoplasmic reticulum. The osctr2 loss-of-function mutation and expression of the 35S:OsCTR2 (1-513) transgene that encodes the OsCTR2 N terminus (residues 1-513) revealed several and many aspects, respectively, of ethylene-induced growth alteration in rice. Because the osctr2 allele did not produce all aspects of ethylene-induced growth alteration, the ethylene-receptor signal output might be mediated in part by OsCTR2 and by other components in rice. Yield-related agronomic traits, including flowering time and effective tiller number, were altered in osctr2 and 35S:OsCTR2 (1-513) transgenic lines. Applying prolonged ethylene treatment to evaluate ethylene effects on rice without compromising rice growth is technically challenging. Our understanding of roles of ethylene in various aspects of growth and development in japonica rice varieties could be advanced with the use of the osctr2 and 35S:OsCTR2 (1-513) transgenic lines.

Fig. 1.

Sequence and phylogenetic analyses of CTR1-related proteins. (A) Phylogenetic tree of plant CTR1-related proteins with accession number and genus names. Arabidopsis CTR1 (AtCTR1) and rice OsCTRs (OsCTR1, OsCTR2, and OsCTR3) are boxed. (B, C) Protein sequence similarity and identity at the N terminus (B) and C terminus (C) of rice OsCTRs compared with that of Arabidopsis CTR1.

Fig. 2.

Expression of CTR1p:OsCTR2 complements ctr1-1. (A, B) Phenotype (A) and hypocotyl measurements (B) of etiolated seedlings. L, transformation line. (C, D) Expression of the CTR1p:OSCTR2 transgene in ctr1-1 confirmed by RT-PCR (C) and quantified (D). (E, F) Phenotype of light-grown seedlings (E) and rosettes (F) of ctr1-1 and ctr1-1 expressing CTR1p:OsCTR2. (G) qRT-PCR analysis of mRNA level of ERF1. (H, I) Expression of CTR1 in Arabidopsis (H) and of OsCTR2 but not OsCTR3 in rice (I) is ethylene inducible. (J, K) Yeast two-hybrid assay (J) and kinetics of β-galactosidase activity (K) for the interaction between ETR1/ERS1 and the OsCTR2 N terminus. Data are means ±SD of three independent experiments performed in triplicate. pBTM and pGADT7 are the vectors for the yeast two-hybrid assay.

Fig. 3.

Phenotype analysis of osctr2. (A) Diagram of the OsCTR2 gene structure; the T-DNA insertion site in osctr2 is indicated. The positions of the PCR genotyping primers (L1, L2, and R1) are indicated, and the legend shows the PCR genotyping for the wild-type (DJ) and osctr2 mutant. (B) qRT-PCR analysis of relative mRNA levels of OsCTR2 in DJ and osctr2 seedlings. (C, D) Seedling phenotype (C) and leaf length measurement (D) of the DJ and osctr2 plants. Numbers on the x-axis in (D) indicate the order of the seedling leaves (1, first; 2, second; 3, third). (E) Primary root length in DJ and osctr2 seedlings. (F) Number of adventitious roots in the DJ and osctr2 seedlings. **P<0.01 for osctr2 compared with DJ. (G, H) Leaf senescence (G) and chlorophyll a content (H) of DJ and osctr2 seedlings. (I, J) Phenotype (I) and length (J) of etiolated seedling coleoptiles of DJ and osctr2 plants. (K) Coleoptile phenotype of light-grown seedlings. (L) Ethylene evolution of DJ and osctr2 seedlings. Data in are means ±SD of three independent experiments performed in triplicate. **P<0.01. (M, N) qRT-PCR analysis of mRNA expression of genes in rice seedlings with prolonged (M; 7 d) or short (N; 4h) ethylene treatment (100 µl l^–1). Data are means ±SEM of three independent experiments performed in triplicate. (This figure is available in colour at JXB online.)

Fig. 4.

Expression of 35S:OsCTR2 ^1–513 confers a constitutive ethylene response in rice. (A–E) qRT-PCR analysis of OsCTR2 mRNA level (A), seedling phenotype (B), primary root length (C), adventitious root number (D), and seedling leaf length (E) of the wild type (ZH11) and 35S:OsCTR2 ^1–513 transgenic lines. (F–I) Leaf senescence phenotype (F), relative chlorophyll a content (G), coleoptile phenotype (H), and coleoptile length (I) of the wild type (ZH11) and transgenic lines. (J) Coleoptile phenotype of light-grown seedlings. (K) Ethylene evolution in the wild-type (ZH11) and transgenic lines. Data are means ±SD of three independent experiments performed in triplicate. L, transformation line. (This figure is available in colour at JXB online.)

Fig. 5.

Subcellular localization of GFP–OsCTR2. Fluorescence of GFP–OsCTR2 (green) and ER-rk (red) in tobacco (A, B) and onion epidermal (C, D) cells. The cell for the co-localization study is indicated with an arrowhead in (A) and (B).

Fig. 6.

Quantification of yield-related agronomic traits. (A) Panicle length, (B) plant height, (C) effective tiller number, (D) panicle neck length, (E) 300-grain weight (gram), and (F) and (G) heading status of wild-type varieties (DJ and ZH11), osctr2, and 35S:OsCTR2 ^1–513 transgenic lines. Data are means ±SD of three independent experiments performed in triplicate. **P<0.01. Sample sizes are as indicated or are shown in parentheses.