Genetic and transformation studies reveal negative regulation of ERS1 ethylene receptor signaling in Arabidopsis

Abstract

Background: Ethylene receptor single mutants of Arabidopsis do not display a visibly prominent phenotype, but mutants defective in multiple ethylene receptors exhibit a constitutive ethylene response phenotype. It is inferred that ethylene responses in Arabidopsis are negatively regulated by five functionally redundant ethylene receptors. However, genetic redundancy limits further study of individual receptors and possible receptor interactions. Here, we examined the ethylene response phenotype in two quadruple receptor knockout mutants, (ETR1) ers1 etr2 ein4 ers2 and (ERS1) etr1 etr2 ein4 ers2, to unravel the functions of ETR1 and ERS1. Their functions were also reciprocally inferred from phenotypes of mutants lacking ETR1 or ERS1. Receptor protein levels are correlated with receptor gene expression. Expression levels of the remaining wild-type receptor genes were examined to estimate the receptor amount in each receptor mutant, and to evaluate if effects of ers1 mutations on the ethylene response phenotype were due to receptor functional compensation. As ers1 and ers2 are in the Wassilewskija (Ws) ecotype and etr1, etr2, and ein4 are in the Columbia (Col-0) ecotype, possible effects of ecotype mixture on ethylene responses were also investigated.

Results: Ethylene responses were scored based on seedling hypocotyl measurement, seedling and rosette growth, and relative Chitinase B (CHIB) expression. Addition of ers1 loss-of-function mutations to any ETR1-containing receptor mutants alleviated ethylene growth inhibition. Growth recovery by ers1 mutation was reversed when the ers1 mutation was complemented by ERS1p:ERS1. The addition of the ers2-3 mutation to receptor mutants did not reverse the growth inhibition. Overexpressing ERS1 receptor protein in (ETR1 ERS1)etr2 ein4 ers2 substantially elevated growth inhibition and CHIB expression. Receptor gene expression analyses did not favor receptor functional compensation upon the loss of ERS1.

Conclusions: Our results suggest that ERS1 has dual functions in the regulation of ethylene responses. In addition to repressing ethylene responses, ERS1 also promotes ethylene responses in an ETR1-dependent manner. Several lines of evidence support the argument that ecotype mixture does not reverse ethylene responses. Loss of ERS1 did not lead to an increase in total receptor gene expression, and functional compensation was not observed. The inhibitory effects of ERS1 on the ethylene signaling pathway imply negative receptor collaboration.

Figure 1

Seedling hypocotyl measurement and phenotypes of ethylene receptor mutants. (A) seedling hypocotyl length of air-grown seedlings in the presence and absence of the ethylene biosynthesis inhibitor AVG. (B) etiolated and (C) light-grown seedlings, and (D) rosettes of ethylene receptor mutants. Common receptor mutations are highlighted in the yellow box for each set of mutants. Error bars indicate standard deviation. Quintuple: the quintuple receptor mutant.

Figure 2

Relative receptor gene expression in wild type and ethylene receptor mutants. (A) expression of remaining wild-type receptor genes in receptor mutants relative to total receptor gene expression in wild type (Col-0). P value indicates the probability of a numerically larger value of t for the comparison (Student's t test) between two isogenic mutants highlighted with a box. (B), (C), (D), and (E) expression of individual receptor genes in isogenic receptor mutants relative to that in wild type. Box highlights the common receptor gene mutations of a set of isogenic mutants. Error bars indicate standard deviation. n = 3 × 3: each measurement was repeated three times from three independent biological materials. NA: the ERS1 expression is not measured in ers1 mutants. P: probability of a numerically larger value of t in a LSD-t test.

Figure 3

Effects of ers1 alleles on ethylene receptor mutant growth. (A) phenotype of dark-grown seedlings in the presence of AVG. (B), (C) adult phenotype of ers1-2 and ers1-3 mutants. Box highlights common mutations.

Figure 4

Effects of ecotype mixture on ers1-mediated growth recovery. (A) ethylene dose-response assay for ers1-2, ers1-3, Col-0, Ws, and the F1 of Col-0 and Ws. (B) seedling hypocotyl measurement of mutants respectively carrying and lacking ERS2. (C) phenotype and (D) hypocotyl measurement of etiolated seedlings of ers1-2 etr2 ein4, ERS1p:ERS1 ers1-2 etr2 ein4, and etr2 ein4. Error bars indicate standard deviation. L1 and L3: two independent ERS1 transformation lines. Box indicates common mutations.

Figure 5

Effect of ERS1 overexpression on etr2 ein4 ers2. (A) adult phenotype of etr2 ein4 ers2, class A (L1, L2, and L3) and class B (S1, S2, and S3) lines carrying the 35S:ERS1 transgene. (B) immunoassay of ERS1 level in individual transformed lines. The ERS1 protein is not detectable in the ers1 mutant ers1-2 etr2 ein4. (C) relative CHIB gene expression in etr2 ein4 ers2 and transformed lines. (D) relative RTE1 gene expression in receptor mutants. Chemiluminance for the immunoassay was captured by a cold CCD (at -110°C and displayed in pseudo-color; the pseudo-color bar (CHEM) indicates relative signal strength from weak (dark) to strong (bright). ERS1: relative ERS1 accumulation in immunoassay probed with ERS1 antibody. Stained blot: membrane was stained with Coomassie Blue after the immunoassay to indicate relative protein amount. NA: data not available. Arrows indicate senesced leaves. Error bars indicate standard deviation. P: probability of a larger F value. n = 3 × 3: each measurement was repeated three times from three independent biological materials. Common mutations are highlighted in a box.

Figure 6

Inhibitory effect of ERS1 on the repression of the ethylene response is not blocked by ethylene. (A), (B) ethylene dose-response curve for two sets of ERS1-lacking mutants. (C) under a high ethylene concentration, ERS1 still exerts an inhibitory effect on seedling hypocotyl elongation. (D) ERS1 accumulation is elevated upon ethylene treatment; a and b represent ERS1 level from two independent wild-type plants. Error bar indicates standard deviation. ERS1: the ERS1 protein. NA: no ethylene treatment. Stained blot: membrane was stained with Coomassie Blue after the immunoassay to indicate relative protein amount. L1, L2, and L3 represent three independently identified isogenic mutants. A molecular weight marker is indicated at the position of 72 kD. Common mutations are highlighted in a box. P: probability of a numerically larger t in Student's t test or LSD-t test.