1. 1kb PpABA1 gene model AtABA1 gene model (a) (b) ATGTAA ATG TAA Fig. S1. Characterization of the PpABA1 gene. (a) AtABA1 and PpABA1 gene models. The PpABA1 gene has two exons, while AtABA1 has sixteen exons. (b) Amino acid sequence alignment of the polypeptides of AtABA1 and PpABA1. The red underline indicates the monooxygenase domain. Supporting Information Figs S1–S6

2. Fig. S2. Nucleotide sequence alignment of PpABA1 (Pp1s91_16V6.1) and Pp1s219 (Pp1s219_79V6.1). Nucleotide sequences of primers used for PCR analysis shown in Figs. 1 and 2 are indicated by red arrows. Nucleotides missing in Pp1s219 are indicated by a blue underline.

3. KO-1 KO-2WT 23.1 9.4 6.5 4.3 2.3 (kbp) 2.0 KO-1 KO-2WT 23.1 9.4 6.5 4.3 2.3 (kbp) 2.0 KO-1 λ/HindIII PvuII SpeI Nde I Bgl II (a)(b)(c) 23.1 9.4 6.5 4.3 2.3 (kbp) 2.0 NdeI 8100 bp (d) npt Fig. S3. DNA gel blot analysis of WT and ppaba1. Genomic DNA (2 µg) isolated from protonemata was digested with Nde I (a), Bgl II (b), Pvu II or Spe I (c) and hybridized with a labeled probe of npt. Restriction maps for the expected ppaba1 locus are shown in (d).

4. WT 17B9 Cold 0 3 0 3 0 3 (d) Ppblt101a Pptubulin AR7ppaba1 LEAIII LEA172 Fig. S4. RT-PCR analysis of cold-induced gene expression in WT, ppaba1 and AR7 (Bhyan et al., 2012). Oligonucleotide primers were used for amplification of transcripts of Pp1s294_52V6.1 (17B9), Pp1s118_232V6.1 (LEAIII), Pp1s370_29V6.1 (LEA172), Pp1s2_779V6.1 (Ppblt101a) and Pp1s252_90V6.1 (Pptubulin) from the cold-treated and non-treated protonemata.

5. 0h 2h4h6h WTppaba1 0h 2h 4h 6h LEA III PpNCED1 rRNA 0.5 M Man Fig. S5. Effects of hyperosmotic treatment on PpNCED1 transcript accumulation. P. patens protonemata were treated with 0.5 M mannitol for the indicated time and used for RNA gel blot analysis using radiolabeled probes of PpNCED1 (Pp1s412_7V6.1) and LEA III (Pp1s118_232V6.1).

6. * Cold acclimation (d) (a)(b) WT ppaba1 ( -2˚ C ) ( -3˚C ) Electrolyte leakage (%) 0 5 7 Cold acclimation (d) 0 5 7 AR7 WT ppaba1 AR7 * * ** Fig. S6. Cold acclimation of WT, ppaba1 and AR7. Protonemata acclimated to cold at 0˚C for the indicated times were subjected to freezing at -2˚C (a) and -3˚C (b). Survival after thawing was determined by measurement of electrolyte leakage. Error bars indicate ±SE (n = 3). Student’s t-test was used to determine the significance (*P<0.05, **P<0.01).