Glucose and ethylene signal transduction crosstalk revealed by an Arabidopsis glucose-insensitive mutant

Abstract

Glucose is an essential signaling molecule that controls plant development and gene expression through largely unknown mechanisms. To initiate the dissection of the glucose signal transduction pathway in plants by using a genetic approach, we have identified an Arabidopsis mutant, gin1 (glucose-insensitive), in which glucose repression of cotyledon greening and expansion, shoot development, floral transition, and gene expression is impaired. Genetic analysis indicates that GIN1 acts downstream of the sensor hexokinase in the glucose signaling pathway. Surprisingly, gin1 insensitivity to glucose repression of cotyledon and shoot development is phenocopied by ethylene precursor treatment of wild-type plants or by constitutive ethylene biosynthesis and constitutive ethylene signaling mutants. In contrast, the ethylene insensitive mutant etr1-1 exhibits glucose hypersensitivity. Epistasis analysis places GIN1 downstream of the ethylene receptor, ETR1, and defines a new branch of ethylene signaling pathway that is uncoupled from the triple response induced by ethylene. The isolation and characterization of gin1 reveal an unexpected convergence between the glucose and the ethylene signal transduction pathways. GIN1 may function to balance the control of plant development in response to metabolic and hormonal stimuli that act antagonistically.

Figure 1

Phenotypes of gin1-1 plants. (A) Cotyledon development. gin1-1 and wild-type (Ws-0) seeds were germinated and grown on MS medium containing 6% glucose or mannitol for 4 days in the light (60 μE m⁻² s⁻¹). (Bar = 1.3 mm.) (B) The gin1-1 mutant population is uniformly insensitive to glucose. Approximately 150 seeds of wild type (Left) and gin1-1 (Right) were germinated and grown on MS medium containing 6% glucose for 4 days in the light. (C) True leaf development. Wild-type (Left) and gin1-1 (Right) seedlings were grown as in B for 10 days. (Bar = 1.9 mm.) (D) Greenhouse phenotype. Both wild-type (left) and gin1-1 (right) plants were grown in Metro-Mix 200 under continuous light (60 μE m⁻² s⁻¹) for 3 weeks.

Figure 2

Reduced glucose sensitivity in gin1-1. (A) Glucose repression of photosynthetic genes. Total RNA was isolated from gin1-1 and wild-type (Ws-0) seedlings grown on MS medium containing 0 (−) or 4% glucose (+) in the dark for 3 days. RNA blot analysis was performed by using 5 μg of RNA per lane. Ribosomal RNA stained with ethidium bromide was used as a loading control. (B) Glucose effect on flowering. Lighting was continuous (60 μE m⁻² s⁻¹) for LD, or day/night cycles (8 hr/16 hr) for SD treatment. Bolting time was determined by the appearance of an inflorescence stem. Data were obtained from 10 replicates. Standard deviation was zero as all plants bolted within the same day under each condition. (C) Flowering is not delayed by glucose in gin1-1. gin1-1 and wild-type (Ws-0) plants were grown on MS medium with 6% glucose for 4 weeks under continuous light.

Figure 3

GIN1 as a positive regulator in the glucose signaling pathway mediated by HXK. (A) Glucose uptake. The rates of glucose uptake in the seedlings and protoplasts of gin1-1 (filled symbols) and wild-type (Ws-0) (opened symbols) plants was determined (10). Values are the means of three measurements (each with duplicated samples). Error bars indicate the standard deviation. (B) Immunoblot analysis of HXK. HXK levels were determined in wild-type Ws-0 (lane 1), gin1-1 (lane 2), wild-type Bensheim (lane 3), 35S-AtHXK1 transgenic plant (Bensheim ecotype) (lane 4), and two independent lines of 35S-AtHXK1 gin1-1 (lanes 5 and 6). Proteins were extracted from shoots of 14-day-old seedlings grown in liquid culture (11). (C) 35S-AtHXK1 gin1-1 can overcome the glucose induced developmental arrest. 35S-AtHXK1, gin1-1, and 35S-AtHXK1 gin1-1 seedlings were grown on MS medium with 6% glucose. (Bar = 1.9 mm.)

Figure 4

Crosstalk between ethylene and glucose signaling. (A) ACC treatment can phenocopy gin1 phenotype. Glucose-induced developmental arrest was examined in wild-type seedlings, gin1-1, and ethylene mutants on MS medium containing 6% glucose in the absence (Upper) or presence (Lower) of 50 μM of ACC. Wild-type (Ws-0) and gin1-1 seedlings were 4 days old and all others were 5 days old. (Bar = 1.3 mm.) (B) etr1-1 is hypersensitive to glucose. Wild-type, gin1-1, and etr1-1 seeds were germinated and grown on MS medium containing 0%, 4% or 6% glucose for 4 days in the light (60 μE m⁻² s⁻¹). (Bar = 1.3 mm.)

Figure 5

Phenotypes of the etr1-1 gin1-1 double mutant. (A) gin1-1 is epistatic to etr1-1. etr1-1, gin1-1, and etr1-1 gin1-1 seedlings were grown on MS medium containing 6% glucose for 5 days under constant light. (Bar = 1.3 mm.) (B) Greenhouse phenotype of etr1-1 gin1-1 Four-week-old etr1-1, gin1-1, and etr1-1 gin1-1 plants grown in the greenhouse are shown. (C) The GIN1 pathway is uncoupled from the ethylene triple response. etr1-1 and gin1-1 seedlings were grown on MS medium containing 1% sucrose in the absence or presence of 50 μM ACC for 3 days in the dark. (Bar = 1.6 mm.) (D) The GIN1 pathway is genetically separable from the ethylene root hair response. gin1-1 and etr1-1 gin1-1 seedlings from A were transferred to MS medium containing 1% sucrose and 50 μM ACC for an additional 2 days. (Bar = 1.9 mm.)

Figure 6

Model for glucose and ethylene signaling.