The ERF (ethylene responsive factor) family is composed of transcription factors (TFs) that are critical for appropriate Arabidopsis thaliana responses to biotic and abiotic stresses. Here we identified and characterized a member of the ERF TF group IX, namely ERF96, that when overexpressed enhances Arabidopsis resistance to necrotrophic pathogens such as the fungus Botrytis cinerea and the bacterium Pectobacterium carotovorum. ERF96 is jasmonate (JA) and ethylene (ET) responsive and ERF96 transcripts accumulation was abolished in JA-insensitive coi1-16 and in ET-insensitive ein2-1 mutants. Protoplast transactivation and electrophoresis mobility shift analyses revealed that ERF96 is an activator of transcription that binds to GCC elements. In addition, ERF96 mainly localized to the nucleus. Microarray analysis coupled to chromatin immunoprecipitation-PCR of Arabidopsis overexpressing ERF96 revealed that ERF96 enhances the expression of the JA/ET defence genes PDF1.2a, PR-3 and PR-4 as well as the TF ORA59 by direct binding to GCC elements present in their promoters. While ERF96-RNAi plants demonstrated wild-type resistance to necrotrophic pathogens, basal PDF1.2 expression levels were reduced in ERF96-silenced plants. This work revealed ERF96 as a key player of the ERF network that positively regulates the Arabidopsis resistance response to necrotrophic pathogens.

The plant hormone ethylene plays a regulatory role in development in light- and dark-grown seedlings. We previously isolated a group of small-molecule compounds with a quinazolinone backbone, which were named acsinones (for AC C s ynthase in hibitor quinazoli nones ), that act as uncompetitive inhibitors of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS). Thus, the triple response phenotype, which consists of shortened hypocotyls and roots, radial swelling of hypocotyls and exaggerated curvature of apical hooks, was suppressed by acsinones in dark-grown (etiolated) ethylene overproducer ( eto ) seedlings. Here, we describe our isolation and characterization of an Arabidopsis r evert to et o1 9 ( ret9 ) mutant, which showed reduced sensitivity to acsinones in etiolated eto1 seedlings. Map-based cloning of RET9 revealed an amino acid substitution in CHITINASE LIKE1 ( CTL1 ), which is required for cell wall biogenesis and stress resistance in Arabidopsis. Etiolated seedlings of ctl1 ret 9 showed short hypocotyls and roots, which were augmented in combination with eto1-4 . Consistently, ctl1 ret 9 seedlings showed enhanced sensitivity to exogenous ACC to suppress primary root elongation as compared with the wild type. After introducing ctl1 ret 9 to mutants completely insensitive to ethylene, genetic analysis indicated that an intact ethylene response pathway is essential for the alterations in root and apical hook but not hypocotyl in etiolated ctl1 ret 9 seedlings. Furthermore, a mild yet significantly increased ethylene level in ctl1 mutants was related to elevated mRNA level and activity of ACC oxidase (ACO). Moreover, genes associated with ethylene biosynthesis ( ACO1 and ACO2 ) and response ( ERF1 and EDF1 ) were upregulated in etiolated ctl1 ret 9 seedlings. By characterizing a new recessive allele of CTL1 , we reveal that CTL1 negatively regulates ACO activity and the ethylene response, which thus contributes to understanding a role for ethylene in root elongation in response to perturbed cell wall integrity.

ETHYLENE OVERPRODUCER1 (ETO1), ETO1-LIKE1 (EOL1), and EOL2 are members of the Broad complex, Tramtrack, Bric-a-brac (BTB) protein family that collectively regulate type-2 1-aminocyclopropane-1-carboxylic acid synthase (ACS) activity in Arabidopsis thaliana. Although ETO1 and EOL1/EOL2 encode structurally related proteins, genetic studies suggest that they do not play an equivalent role in regulating ethylene biosynthesis. The mechanistic details underlying the genetic analysis remain elusive. In this study, we reveal that ETO1 collaborates with EOL1/2 to play a key role in the regulation of type-2 ACS activity via protein-protein interactions. ETO1, EOL1, and EOL2 exhibit overlapping but distinct tissue-specific expression patterns. Nevertheless, neither EOL1 nor EOL2 can fully complement the eto1 phenotype under control of the ETO1 promoter, which suggests differential functions of ETO1 and EOL1/EOL2. ETO1 forms homodimers with itself and heterodimers with EOLs. Furthermore, CULLIN3 (CUL3) interacts preferentially with ETO1. The BTB domain of ETO1 is sufficient for interaction with CUL3 and is required for homodimerization. However, domain-swapping analysis in transgenic Arabidopsis suggests that the BTB domain of ETO1 is essential but not sufficient for a full spectrum of ETO1 function. The missense mutation in eto1-5 generates a substitution of phenylalanine with an isoleucine in ETO1 F466I that impairs its dimerization and interaction with EOLs but does not affect binding to CUL3 or ACS5. Overexpression of ETO1 F466I in Arabidopsis results in a constitutive triple response phenotype in dark-grown seedlings. Our findings reveal the mechanistic role of protein-protein interactions of ETO1 and EOL1/EOL2 that is crucial for their biological function in ethylene biosynthesis.