A sweetpotato SRD1 promoter confers strong root-, taproot-, and tuber-

specific expression in Arabidopsis, carrot, and potato

Seol Ah Noh1, Haeng-Soon Lee2, Gyung Hye Huh3, Mi-Joung Oh1, Kyung-Hee Paek1, Jeong

Sheop Shin1, Jung Myung Bae1,*

1School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea

2Environmental Biotechnology Research Center, Korea Research Institute of Bioscience and

Biotechnology (KRIBB), Daejeon 305-806, Korea

3College of General Education, UHRC, Inje University, Gimhae, 621-749, Korea

*To whom correspondence should be addressed.

Dr. Jung Myung Bae

Address: School of Life Sciences and Biotechnology,

Korea University, Seoul 136-701,

Republic of Korea

Telephone: +82-2-3290-3952

E-mail: jmbae@korea.ac.kr

Fax: +82-2-927-9028

-2801 GGCTGGTTTCTAAGACATTTTTTGGTTTAATCCAAACCTAATTACAAATATTCCCAACAA ROOTMOTIFTAPOX1-2741 GATCGAATGATCTATGGCTACAAACCCTATCCCAACAAAAAACTACATTTAGTACATCAA

-2681 ATTAAGTGGCATGATTATTTTATTTTGTTCGACAAAGTAGCATCAAATAAACTACAAAAA

-2621 AAACTACATCATTACAAAAAGACTAATTATCAGGCATCAATGTTAGTATATGGGAGGTGG

-2561 TGGGTTCGAGCCTCAGTGGAGGCGTTGCTGTCTCTTTGTTCTTCAGTAGGTTGAGAGAGT ARFAT OSE2ROOTNODULE-2501 AATTTATGAACAGATACTACACTGTAATAGAGTCAGTAGCAATCAAAAAAAAATTTGTTT

-2441 TAATAATATCCTAATATTATATTTTTCTAACCAGTACTATGCTTTCGGCTTTCCAGAAGG ROOTMOTIFTAPOX1 ROOTMOTIFTAPOX1-2381 CAGAAGCCTAAAAAATTCAATTAAGTTTATAAACTTTAATCCACTTGTTTGAGTAATTGA NTBBF1ARROLB-2321 GTATCTTTCAGAACGGTTGTAGATTTAGGTGGGATGACAAATGGTATTCCAAAGTTCAAG

-2261 ATATTTCTTTTTAGATTTAGGAATTTGTAGTCTTTTAAGGTTAGAGGTTACTTAAAAGGA ROOTMOTIFTAPOX1-2201 TGAACAAATTTTTTATCCCATTCTATTTCTAGGAAGAATTTATAATCCGTACGTGTGACG

-2141 GCTGCCATTAATTATAGTGCCCATTCATTTTTATTGGGAAAAAGTACTCATCCATTATTT

-2081 CATTGGCACGGCAACCCAGTTTTAAATATTTTATAACAATAATAACATATGGAACCAAAT ROOTMOTIFTAPOX1 CATATGGMSAUR-2021 TGTAACCTTTATATCCCACAGACCCACACATTACACATCCAATAAAACTTGAGCCAAATT

-1961 ATATATTAGCGTTACTGAGTACTGACTAAAATATATTTTTAAAATATACTAAAATATTAT ROOTMOTIFTAPOX1 ROOTMOTIFTAPOX1 ROOTMOTIFTAPOX1-1901 TAAAAAAATATTAAAATAGTAAAATTATATTAAATAGAAAATTTAATTTAATCAAAGAAT ROOTMOTIFTAPOX1 ROOTMOTIFTAPOX1-1841 ACCAACTAAAACGTATAAAATGAGAAAATATAGGTATATAATATTGATGATTCCTTTTGA ROOTMOTIFTAPOX1-1781 TTTTTTTTTTATGATCCGAAAATTCTTTGGCCATAAGAAGAGTAAATGAACAATTTAAAC

-1721 TAAGAAAATAAGTAAGTTGCTCCTATGTGATTAATATATAAAGTGAGATTTGAGCTGTTG

-1661 ATCTATATTATTGAATTAGATCAACGACTCAAAATGAAGGATAATTTTTTTAAAAAATCG ROOTMOTIFTAPOX1-1601 CTTCCTGTTAATATTAATGCTTTAAAATTAAGCACATTAAACTTTAAAATAATGCACCTT ROOTMOTIFTAPOX1 NTBBF1ARROLB-1541 TTTTTTTAATACTATTGACCTTGTTACATGTAGTATCTGAAGTCCAACAAAGTCAACATT

-1481 GTCCCCACTGAGGCTCAAACCCGTGACCTCCCACTAGGGAGAATCGCTTCATGCCGCTTG

-1421 ACCACAAGTCCTTTGGTAAAAATAATGCACCTTAAAGATGTAAACTTACGCATCTTCGAT OSE1ROOTNODULE-1361 GAACTGACCACTTTGAGCTTGCAACTTATACTTTTTTGAAGATAAGCTTGTAACTTATTA

-1301 TAATGGTCTATTAACATTAAAAAAAAAAAAAGTTTCACAATCAAATTATAATATTTGTAG ROOTMOTIFTAPOX1-1241 CCAAATGAATTTACCGCGGGTGTGACTATTCAGGAATTTAAATACACTAAAGTTGGAGGG

-1181 GTAGTACACTCAATACACTATTGCTCATGACTTTTTTCTTCTTTTTTTTTTAGATTAGCT

-1121 AATATATTAATCCCAAATAGAAACGTTTACACCAAAGTTCGAAAAAATGTTGTGTCATTT ROOTMOTIFTAPOX1-1061 CTTACAGTTAGACACAAAAATAACATTTTTAGCTAAGTTACAGTAAACTTGATTGGCAGA

-1001 CTGTTTCACAAATTGGGAGCTTGGATCCTTGAAGGAACTTACTGCTTTCTTAGAGTCATT

-941 AATGGTTTGGCCAAACATAGAAAAGATTAGTTGAGCAGTCTTGCACACTACTTGAGTAAT OSE1ROOTNODULE-881 CATCTCCATTCTTCTACTTATTGACAATATTCTCTTATGAAAAAACACACTTGATCTTAT ROOTMOTIFTAPOX1 OSE2ROOTNODULE-821 ATCAGTTAGGGATTTGACCGGTTTATTAAAGGATAGCCTACCAACTTTGTTGAACGACAT

-761 ATCATCATATCATGATTCAAAAGATGCTCTTTTTTATTGTCATATTTGTGGCACAGGATG OSE1ROOTNODULE OSE2ROOTNODULE ROOTMOTIFTAPOX1-701 AGTACAGTTTCGCATACACCATGATCATTTTTATCAAATCATACTCTATAAAACCCTGTC

-641 AAAGAAAAGAGAGGAAGAAACGAGAAGAAGAAACTCATCCAAGAAACAAGAGGAACATTA

-581 TTGCTCATGATTAGATCGACTTGAACATGTACTAATGCCAATCTCAAATTACCTACATAG

-521 GTGTGTTAGACAAATATTTGTTAATTAGCTGATTGACTTAATGGATTTGACTAGTTGTTA ROOTMOTIFTAPOX1-461 ACATTAATTGATTGTAGGAAATTGTTTGGTAAATTAGTTGTTAGTTGATAGTTGATTACA

-401 TGAAAATTACTTTCTCAAAAAGCTTATCGAAAAAACTATTTTGAACAGCTTTTTGAATTT

-341 TAACATTTTATAACAATAAGTTGTTACAAAAAGCTAATTAATCAAATACTCATATCCATT CAAT box-281 GTTTAACCATGTCAAACAACTAATAATAATTAAATAATTTGTTTTTAAAATATAAGTTAA

-221 ATTTAATTGATAAGCTAACTATATTACCAAACATACCGTAATATTTTCTTAACCGCGGTA ROOTMOTIFTAPOX1 ROOTMOTIFTAPOX1-161 TGGGCTAAGATATGATTGTATACTATTTTTGTTGCGAGCATGATTAATACAGTAATACCA -101 TCATTTAAAAGTGGAAACCACATTCGCAGCTGTTTCCGAAAGCAAACAGCTAACATTTGC -41 TAGGTTCTTACTTATGCATTAATCTGGGTTATAAAATCCCCATTTCCATGTTGGTGTGAA TATA box 20 CAACCACCTAAACCTAGCGTCTTCAACAATTCTACCCTACTATCATCCCCCAAGACTTCC

80 CCGACCAgtaaataacccgctttcctctttcagtgatttcttcatttgactttgctatat 140 atatatataatctgatctgctttcatctttcagtgatttcttcatttggatttcttcagAG

200 GAGGAGAAGGATGGGGAGGGGCAAGGTTGAAATTAGGAGGATA

Fig. S1. Nucleotide sequence and structure of the SRD1 promoter. Numbers represent nucleotide positions relative to the transcriptional start site (+1). The translational start codon is in bold and boxed. The putative TATA box and CAAT box are underlined. Putative cis-elements are underlined and indicated according to the PLACE motif search software (http://www.dna.affrc.go.jp) . Lower case letters indicate putative intron sequence in 5’UTR.

+1

Motif No. Sequence Description Reference

Root motif 20 ATATT Root-specific expression Elmayan and Tepfer, 1995

OSE1 3 AAAGAT Root nodule expression Vieweg et al., 2004

OSE2 3 CTCTT Root nodule expression Vieweg et al., 2004

ARFAT 1 TGTCTC Auxin responsive factor Goda et al., 2004

NTBBF1 2 ACTTTAAuxin regulated expression element

Baumann et al., 1999

Table S1. Motifs related to root expression and auxin responsive activity in the SRD1 promoter.

0

500

1000

1500

2000

2500

3000

3500

4000

4500

GU

S a

ctiv

ity (

nmo

l 4-M

U m

g-1m

in-1)

C JA IAA BAC JA IAA BA

#3 #5

Fig. S2. Effect of various hormones on the activity of the SRD1 promoter. Two-week-old T2 transgenic Arabidopsis seedlings (lines #3 and #5) harboring the 3.0-kb SRD1 promoter (D2801) were incubated in the solution containing JA (50 μM), IAA (25 μM), or BA (25 μM) for 24 h at 25oC in the dark. Total proteins were extracted from root tissues to determine GUS activity. Error bars indicate the standard deviation between three technical replicates measured on root tissues collected from at least three different Arabidopsis seedlings and subsequently pooled for analysis. C Control (Seedlings were incubated in distilled water).

3 h0 h 6 h 12 h 24 h

A

B

Fig. S3. Effect of IAA on the activity of the SRD1 promoter. Two-week-old T3 homozygous transgenic Arabidopsis seedlings harboring the 3.0-kb SRD1 promoter (D2801) were incubated in a solution containing IAA (25 or 50 μM) for various time periods (0–48 h) at 25oC in the dark. a Time-course of the activity of the SRD1 promoter in response to exogenously applied IAA. Total proteins were extracted from root tissues to determine GUS activity. Error bars indicate the standard deviation between three technical replicates measured on root tissues collected from at least three independent transgenic lines and subsequently pooled for analysis. b Histochemical analysis of the SRD1 promoter in response to exogenously applied IAA (50 μM) for various time periods (0–24 h).

GU

S a

ctiv

ity(n

mol

e 4-

MU

mg-1

min

-1)

Fig. S4. Histochemical analysis of the two-week-old T3 homozygous transgenic Arabidopsis seedlings harboring the 3.0-kb SRD1 promoter (D2801) and 5’ truncated SRD1 promoter derivatives (D2303, D1844, D1385, D862, D321 and D83).

D2801 D2303 D1844 D1385

D862 D321 D83

C T L

Carrot

Radish

Fig. S5. Transient expression of the SRD1 promoter in carrot and radish. Plasmid DNAs of the 3-kb SRD1 promoter (D2801-221) were transiently expressed in taproot discs (T) and leaves (L) from carrot and radish. Promoter activity was visualized by histochemical analysis of GUS protein. C, Control (non-bombarded taproot discs).

0

1000

2000

3000

4000

5000

6000

0.5 1.0 1.5 2.0

GU

S a

ctiv

ity

(nm

ol 4

-MU

mg-1

min

-1)

Root Diameter (cm)

Fig. S6. Size-dependent activity of the SRD1 promoter in transgenic carrot. GUS activity determined in the taproots from the T1 transgenic carrot plants harboring the 3.0-kb SRD1 promoter (D2801). Taproots from three independent transgenic lines for each designated diameter were used for the assay. Error bars indicate the standard deviation between three technical replicates measured on taproots collected from at least three independent transgenic lines and subsequently pooled for analysis.

5 mm

Pi Vb

Ct

Wild-type D2801

Leaf

Stem

Tuber

Fig. S7. Histochemical analysis of the SRD1 promoter in the transgenic potato plants. GUS staining of the T1 transgenic potato plants harboring the 3.0-kb SRD1 promoter (D2801). Tubers were cut perpendicularly prior to staining. Ct, Cortex; Pi, Pith; Vb, Vascular bundle.