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Figure S1. Phylogenetic analysis of candidate ethylene receptors from land-plants and charophytic algae. A BLAST database search identified sequences encoding seven putative ethylene receptor proteins in the moss P.  patens (red) (Ishida et al. 2010), two in the liverwort M.  polymorpha (blue) and four in the lycophyte S.  moellendorffii (green). Two partial-length sequences from the charophytic algae Klebsormidium flaccidum and Spirogyra pratensis were also included in the analysis. An unrooted phylogenetic tree was constructed using Bayesian methods on aligned amino acid sequences. Numbers indicated are Bayesian posterior probability. Accession numbers and names of the species for the sequences used are listed in Table S6. Scale bar, 0.1 substitutions. (a) A rectangular phylogram showing the relationships of non-seed plant putative ethylene receptor sequences with angiosperm subfamily I and II ethylene receptor sequences. (b) A radial phylogram with branch length proportional to the amount of evolutionary change between successive nodes on the tree. MpETRa, PpETR4, 5 and SmETR4 were positioned away from the two clusters representing subfamilies I and II, although they appeared to be more closely related to subfamily I than to subfamily II. Some taxa names are omitted due to space constraints.

Figure S2. Phylogenetic analysis of CTR1 proteins and related kinases from land-plants and charophytic alagae. Sequences encoding three P.  patens proteins (red), three M.  polymorhpa proteins (blue) and four S.  moellendorffii proteins (green) were identified by BLAST search to share homology with sequences encoding angiosperm CTR1 proteins and related MAPKKK proteins. A phylogenetic tree was constructed using Bayesian methods on aligned amino acid sequences. The tree is rooted on a related algal kinase sequence (Corb6867 from Coleochaete orbicularis). Numbers indicated are Bayesian posterior probability. Accession numbers and names of the species for the sequences used are listed in Table S7. Scale bar, 0.1 substitutions.

Figure S3. Phylogenetic analysis of EIN3 and EIN3-like proteins from land-plants and charophytic algae. BLAST Database search identified sequences encoding two putative EIN3-like proteins in P.  patens (red), one in M.  polymorhpa (blue) and two in S.  moellendorffii. A sequence encoding a putative EIN3-like protein found in the Spirogyra pratensis EST database was also included. An unrooted phylogenetic was tree constructed using Bayesian methods on aligned amino acid sequences. Numbers indicated are Bayesian posterior probability. Accession numbers and names of the species for the sequences used are listed in Table S8. Scale bar, 0.1 substitutions.

Figure S4. Phylogenetic analysis of Arabidopsis group VII-IX ERF proteins and P.  patens ERF-like proteins. Forty-six protein sequences (red) were identified through BLAST search in the P.  patens genome database as encoding proteins related to group VII (blue), VIII (orange) and IX (pale green) ERF proteins of Arabidopsis. Although the resolution may have been compromised by the phylogenetic distance between Arabidopsis and P.  patens, overall groupings were largely maintained (except that At4g18450, a group IX ERF (Nakano et al., 2006) was misplaced). A phylogenetic tree was constructed using Bayesian methods on aligned amino acid sequences, and rooted on a related Arabidopsis protein (AtSHN1, a group V ERF). Numbers indicated are Bayesian posterior probability. Accession numbers and names of the species for the sequences used are listed in Table S9. Scale bar, 0.1 substitutions.

Figure S5. PUbi:PpETR7 and PUbi:Ppetr7-1 transgenes were integrated in the genome and expressed in PUbi:PpETR7 and PUbi:Ppetr7-1 lines. (a) Schematic diagram of PpETR7 and Ppetr7-1 expressing transgene constructs. Sequences were cloned into pBRACT211. The triangle shows where the mutation was introduced in Ppetr7-1 sequence. The 7 kb construct (PUbi:PpETR7/Ppetr7-1:Tnos:P35S:HygRint:T35S) was cleaved from the vector sequence prior to transformation with NotI and HaeII. NcoI cuts within the construct. Block arrows indicate NotI, HaeII and NcoI restriction sites as indicated. PUbi, maize ubiquitin promoter sequence; Tnos, Nos terminator; P35S, Ca35S promoter; HygRint, aphIII gene with an intron; T35s, Ca35S terminator; LB, left border sequence; RB, right border sequence. (b) DNA gel-blot analysis of genomic NcoI digests of WT, two independent PUbi:PpETR7 (5 and 8) and PUbi:Ppetr7-1 lines (7 and 17). The blot was hybridized with a radiolabelled PpETR7 fragment. Two bands labelled PpETR1 and PpETR7 indicate endogenous WT genes present in WT and all transgenic lines. Hybridised bands a–c indicate integrated transgenes hybridized with the radiolabelled PpETR7 fragment. Strong signals marked with a suggest the presence of tandem repeats of the construct (5 kb) in each transgenic line, and an extra band marked with c in PUbi:PpETR7 line 8 may be due to tandem repeats of rearranged construct. (c) RNA gel-blot analysis of transcripts in WT and transgenic lines. Total RNA was extracted from eight day-old protonema of WT, two independent PUbi:PpETR7 (5 and 8) and PUbi:Ppetr7-1 lines (7 and 17). Following electrophoretic separation, RNA was hybridized with a radiolabelled PpETR7 fragment. An ethidium bromide-stained gel showing ribosomal RNA (below) is shown as loading control. The radiolabelled probe does not distinguish the transgenic from endogenous PpETR7 expression. However, the strong signal detected in the four transgenic lines (versus no detectable signal in WT) suggests that the transgenes are expressed in the transgenic lines.

Figure S6. Filamentous cell size of WT, PUbi:PpETR7 and PUbi:Ppetr7-1 lines. Cell length of the third cell from the tip of each filament was measured for caulonemal and chloronemal filaments (n = 20).

Figure S7. Increased osmotic stress tolerance of PUbi:Ppetr7-1 cells. WT and PUbi:Ppetr7-1 plants were grown from small inoculums of filamentous tissue for two weeks on media with no added mannitol (control) or media containing 0.4, 0.6, 0.8 and 1 M mannitol. PUbi:Ppetr7-1 cells remained green at 0.6 M and higher concentrations of mannitol, at which concentrations WT P.  patens senesced. Scale bar, 1 mm.

Figure S8. Examples of P.  patens plant pictures with radial sectors. Plant structures detected for intensity measurements were defined by red outlines. The examples are 14 days-old P.  patens transferred on the 5th day following inoculation to an airflow chamber filled with air (a) or with air containing 3 ppm ethylene (b). Scale bar, 2 mm.

Figure S9. Full result of semi-quantitative RT-PCR analysis with amplification status at ± two cycles. For each transcript accumulation levels studied in the Figures 3b, 7a, 7b and 9, amplification status at three different stages of PCR at the intervals of two cycles is shown in (a,b,c,d) respectively. Gel photos with underlined cycle numbers were included in the main figures, and the increasing levels of amplification from −2 cycle stage to +2 cycle stage shows that the data were taken from the log phase of PCR amplification.

Table S1. Sequences of oligos used in this work.

Table S2. Unedited sequence alignment of ethylene receptor family proteins.

Table S3. Unedited sequence alignment of CTR1 proteins and related kinases.

Table S4. Unedited sequence alignment of EIN3 and EIN3-like proteins.

Table S5. Unedited sequence alignment of Arabidopsis group VII-IX ERF proteins and P.  patens ERF-like proteins.

Table S6. Accession numbers and descriptions of the sequences used in the phylogenetic analysis of ethylene receptor family shown in Figures 2 and S1.

Table S7. Accession numbers and descriptions of the sequences used in the phylogenetic analysis of CTR1 proteins and related kinases shown in Figure S2.

Table S8. Accession numbers and descriptions of the sequences used in the phylogenetic analysis of EIN3 and EIN3-like proteins shown in Figure S3.

Table S9. Accession numbers and descriptions of the sequences used in the phylogenetic analysis of Arabidopsis group VII-IX proteins and P.  Patens ERF-like proteins shown in Figure S4.

Table S10. Sequence alignment generated for the phylogenetic analysis of ethylene receptor family shown in Figures 2 and S1.

Table S11. Sequence alignment generated for the phylogenetic analysis of CTR1 proteins and related kinases shown in Figure S2.

Table S12. Sequence alignment generated for the phylogenetic analysis of EIN3 and EIN3-like proteins shown in Figure S3.

Table S13. Sequence alignment generated for the phylogenetic analysis of Arabidopsis group VII-IX ERF proteins and P.  patens ERF-like proteins shown in Figure S4.

Methods S1. Experimental procedures.

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