Suppl. Fig. S1

Suppl. Fig. S1 The nucleotide sequence and its deduced amino acid se-quences of CaSAMDC. The full-length of CaSAMDC (GenBank Accession No. JX875948) was 1,681 bp in length and consisted of a 523-bp 5’-UTR, a 1,095-bp coding region of 364 amino acids, and a 63-bp 3’-UTR. CaSAMDC contained overlapping two uORFs consisting of two and 50 amino acids that overlap by one base in the 5’-UTR

Suppl. Fig. S2a

b

Suppl. Fig. S2 Sequence analysis of hot pepper CaSAMDC. a Multiple alignments of 10 plant SAMDC genes. The predicted amino acid sequence of CaSAMDC (GenBank Accession No. JX875948) was compared with those of proteins from carnation (CSAMDC1, U38526; CSAMDC2, U38527), Arabidopsis (ASAMDC1, U63633; ASAMDC2, AJ251915), morning glory (InSAMDC, AAC04611), tobacco (NtSAMDC, U91924), soybean (GmSAMDC, AF488307), cot-tonwood (PtSAMDC, EEF07896) and tomato (SiSAMDC, ABY55855). Amino acid sequences conserved in all ten proteins are indicated in black. b Phylogenetic relationship of the ten SAMDCs from various plant species

NPTII 35S CaSAMDCRB LB

NOS-pro NOS-ter NOS-ter

BamHIHindIII

pBI121

1 437 950 1681524 1618

ATG TAA

1544

227 379

150

HindIII

Suppl. Fig. S3

Suppl. Fig. S3 A Schematic representation of the CaSAMDC-overexpressing gene construct in the pBI121 vector. LB, left border; NOS-Ter, terminator se-quence from nopaline synthase gene; 35S, CaMV 35S promoter; NPT II, neomycin phosphotransferase; NOS-Pro, promoter sequence from nopaline syn-thase gene; RB, right border

10.0

3.0

8.0

5.0

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2.5

KbCol-0 #2 #4Col-0 #2 #4

SacIScaI

Suppl. Fig. S4

Suppl. Fig. S4 Genomic Southern blot analysis of WT and 35S::CaSAMDC trans-genic Arabidopsis lines #2 and #4. Genomic DNA (10 μg per lane) from Arabidopsis seedlings was digested with ScaI and SacI enzymes, blotted onto ny-lon membranes, and hybridized with the [32P]-labeled cDNA probe under normal stringent conditions. The blot was visualized by autoradiography

35S:CaSAMDCCol-0

AtUBC10

CaSAMDC

#2 #3 #4 #5 #6 #10

b

c

d

0 1 2 h

Drought

CaLEA-1

CaActin

CaSAMDC

CaDhn

CaActin

0 12 24 36 h

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CaSAMDC

CaDhn

CaActin

0 1 4 1 4 h

ABAMock

a

Put Spd Spm Total0

30

60

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150 Col-0 #2 #4

Free Polyamines

Poly

amin

e co

nten

ts (n

mol

g-1

FW

)

****

****

****

Col-0 #2 #40

500 1,000 1,500 2,000 2,500 3,000 3,500 4,000 4,500 5,000

SAM

DC a

ctiv

ity (n

mol

mg-

1h-1

)

***

Figure S5.

Suppl. Fig. S5 Patterns of transcription and PA biosynthesis in response to abiotic stresses in aerial parts of hot pepper (Capsicum annum) and transgenic Arabidopsis plants overexpressing CaSAMDC. a Semi-quantitative RT-PCR analysis of CaSAMDC, CaLEA-1, and CaDhn transcription after drought stress (upper panel), cold stress (middle panel), and ABA treatment (lower panel) using total RNAs from aerial parts of hot pepper. CaActin was used as an equal loading control. b Semi-quantitative RT-PCR analysis of CaSAMDC tran-scription in WT (Col-0) and CaSAMDC-overexpressing Arabidopsis plants without stress treatment. Total RNA from each Arabidopsis line was used, and AtUBC10 was used as a loading control. Primers used in RT-PCR analyses are listed in Suppl. Table S1. c Changes in SAMDC activity in WT and transgenic Arabidopsis plants (#2 and #4). SAMDC activity was measured in the aerial parts of 2-week-old Arabidopsis seedlings without any abiotic stress. SAMDC activity is shown as the mean ± SD. Asterisk indicates significant difference at P < 0.06 when comparing data by Student’s t test for each measurement between WT and transgenic plants (#2 and #4). d Changes in PA levels in WT and transgenic Arabidop-sis plants (#2 and #4). PAs were measured in the aerial parts of 2-week-old Arabidopsis plants without any abiotic stress. Total PA level is the sum of three PA levels. PAs levels are shown as the meas ± SD. Put, putrescine; Spd, spermidine; Spm, spermine. Asterisk indicates significant difference at one (P < 0.05) or two (P < 0.01) when comparing data by Student’s t test for each measurement between WT and transgenic plants (#2 and #4)

11 d

ays

with

out w

ater

ing

3-w

eek-

old

plan

ts 3

day

s af

ter r

e-w

ater

ing

35S::CaSAMDC

20 %(8/40)

90 %(33/40)

65 %(26/40)

6 0%(24/40)

50 %(20/40)

Col-0 #2 #4 #5 #10

0 60 120 180 240 300 40

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Time (min)

Wat

er c

onte

nt (%

initi

al fr

esh

wei

ght)

a

b

*

***

******

**

Suppl. Fig. S6

Suppl. Fig. S6 Phenotypic analyses of WT and CaSMADC-overexpressing transgenic plants in response to drought stress. a Drought tolerance of WT and transgenic plant lines #2, #4, #5, and #10. WT and transgenic plants were grown under normal growth conditions for 3 weeks and then exposed to drought stress by withholding water for 11 days. Surviving plants were counted 3 days after rewatering. b Water contents of detached leaves from transgenic Ara-bidopsis plants that were incubated at room temperature under dim light. Reduction of fresh weights was measured at the indicated time points. Water loss rates were estimated as the percentage of initial to final fresh weights. Data are shown as the mean ± SD. Asterisk indi-cates significant difference at one (P < 0.05) or two (P < 0.01) when comparing data by Stu-dent’s t test for each measurement between WT and transgenic plants (#2 and #4)