Figure 1. Mutations in two different genes are responsible for the ET-insensitivity in ein6-1 plants.

(A) Triple response phenotype of 3-day-old etiolated seedlings of Ler, ein6-1, een-1, ein6-1 een-1, ein2-45 (upper row) and of the indicated complementation lines, either driven by the respective native promoter or by the Cauliflower mosaic virus 35S promoter (35S) (lower row). Seedlings were grown on control LS media or LS media supplemented with 10 µM ACC. (B) Schematic illustration of a Bionano Genomics Irys optical map of the een-1 inversion region at the end of the fourth chromosome in ein6-1 een-1 (blue) aligned to an optical map of Ler (green). Original output is shown in Figure 1—figure supplement 1D. Nick sites are indicated as black lines within the respective optical map. Matching nick sites between the maps are indicated as gray lines. Numbered nick sites are used to better visualize the inversion event. The approximate position of EEN and AQI is indicated as well. (C) Heatmap visualizes the log₂ fold change of expression in Ler, ein6-1, een-1, ein6-1 een-1 and ein2-45 seedlings in response to 4 hr of ethylene (ET) treatment detected by RNA-seq. Differentially expressed genes (DE genes) that are significantly induced (1803 genes) or repressed (2598 genes) after 4 hr of ET treatment in Ler seedlings are shown. Cluster dendogram below the heatmap indicate similarities between the tested genotypes. (D), (E), Western blot analysis of nuclear extracts of 3-day-old etiolated Ler, ein6-1, een-1, ein6-1 een-1 and ein2-45 seedlings that were either treated for 4 hr with hydrocarbon-free air (control) or ET gas. Antibodies against EIN3 and EIN2 were used to detect nuclear-localized EIN3 (D) and EIN2C (E), respectively. Amounts of histone H3 were detected with an anti-H3 antibody and served as a loading control. (F) EIN2 expression in response to 4 hr of ET treatment in the indicated genotypes using RNA-seq is shown. EIN2 transcripts can still be detected in ein2-45 mutants because they only harbor a point mutation in the C-terminus of EIN2 (Beaudoin et al., 2000). Expression is measured in TPM (Transcripts Per Kilobase Million) and results from two biological replicates are shown.

Figure 1—figure supplement 1. Characterization of the two causal mutations in ein6-1 een-1 double mutants.

(A) SHOREmap analysis of the ET-insensitivity quantitative trait locus (QTL) revealed the genomic location of the ein6-1 and een-1 mutation. At even allele frequency of both parents (Col-0, Ler) the homozygosity estimator is 0. At +1 it’s homozygous for Col-0; at −1 is homozygous for Ler. The five chromosomes of the Arabidopsis genome are shown in 5 Mb increments. (B) Graphic illustration shows the known T-DNA insertions (ref6-1, ref6-2, ref6-3) and the 7bp-deletion (ein6-1) in the EIN6 (REF6) gene. The premature stop codon in the fifth exon of EIN6 (REF6) that results from the 7bp-deletion is indicated. (C) Agarose gel image shows the TAIL-PCR amplicons of the secondary PCR (1) and tertiary PCR (2) amplified with the AD2 primer. 2 kb DNA ladder indicates the approximate size of the amplicons. (D) Optical map of ein6-1 een-1 aligned to Ler indicates the 83 kb inversion event on the fourth chromosome. (E) Agarose gel image shows the chimeric AQI-EEN fusion that is only present in een-1 mutants, on the gene (upper row) and cDNA (middle row) level detected by PCR and RT-PCR, respectively. Level of Actin2 cDNA (lower row) indicates equal loading of Ler and een-1 cDNA. (F) Scheme of the inversion event that led to the AQI-EEN fusion is illustrated. The fast neutron mutagenesis induced a double strand break (DSB) in the start of fourth intron of AQI and another DSB in the 5’UTR of EEN. Consequently, incorrect repair of the DSBs resulted in a 83 kb inversion event leading to the fusion of AQI with EEN. (G) Detailed illustration of the AQI-EEN fusion at the gene, cDNA and protein level in nucleotide and amino acid resolution, respectively. Sequence comparison between the AQI-EEN fusion at gene and cDNA level revealed that the 5’UTR of EEN was incompletely spliced after the fusion with the residual fourth intron of AQI. The remaining 14 bp of the 5’UTR became part of the coding sequence causing an open reading frame shift in the first exon of EEN.