1kb

PpABA1 gene model

AtABA1 gene model

(a)

(b)

ATG TAA

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

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.

KO-1

KO-2

WT

23.1

9.4

6.5

4.3

2.3

(kbp)

2.0

KO-1

KO-2

WT

23.1

9.4

6.5

4.3

2.3

(kbp)

2.0

KO-1

λ/HindIII

λ/HindIII

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 NdeI8100 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).

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.

0h 2h 4h 6h

WT ppaba1

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).

0

20

40

60

80

100

*

0

20

40

60

80

100

Cold acclimation (d)

(a) (b)

WTppaba1

( -2˚ C ) ( -3˚C )

Ele

ctro

lyte

leak

age

(%)

Ele

ctro

lyte

leak

age

(%)

0 5 7

Cold acclimation (d)

0 5 7

AR7

WTppaba1AR7

*

*

**

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).