1 1 Supplementary Figure S1. Potential SAD promoter elements conferring gene expression in seeds. SAD-A promoter (GenBank accession no. AF242183) SAD-C promoter (GenBank accession no. AF242182) a b

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1 Supplementary Figure S1. Potential SAD promoter elements conferring gene expression

in seeds.

a

SAD-A promoter (GenBank accession no. AF242183)

TGGTCCTTTTAGGCCTAGGATTCTSCTTGGAAAGGGTCATCGACCACTCAGTCAATGGTGGAGCATTGAATC CAAATTTACAAGGAAGATGTGAGTTAGTTAGTGACCTTGGCTTCTCTCTACCAGAAATTTCTTATCCCATGT TTCCAAGTGAGGCGATACCGTCCTTGAGGCGACCTATTATCGTCTCCTCTCCTAGGACGTCCTATAGTCATC GCGTTAGACAAATCCTTTTACAAATATAACACTACATATATATAATACATTGATTAACTACTACAACATGAC ATATTTTTCTACACCTACGCCAATCACCATGACACACACTCGYTTGATGAGAAATACTTATATTGTGGTTGG ATTGATTATGAATTATGTATGGTGAAACATTTATTCGGGTGATTCATGCATCAAATCGAATATGTACAAACT ATTCACAGGAATTTGTATGTCTTTGCAAAATCTATGATCACTTGATAACAGGTTGACCAAAGTTATGATGAG TGTTTTGATCATGTGTTAGGATCAAGACTACCATATTCTATACAGTTGAGAGATTGAGGTGAGATACATGAG GTAATATATTAAGATATCACATCCTTACATGTTTCTCACGCTTCAAGGAGAGTTCTTGAGGGCAAATGAGGA GGGAACAATTGCGGAGAAATCAACGGAAGAGGGAAGGAAGTCATGAAGGTAGTCTAGTCCATACAAACGCAT GTATCCAAGACATTACAATTACACTCTTGYTTGGAAGGGGTTTGAAGCCCGTCACAAGATATGTAGTTGTTG TGCATCAAATTGTAAAAGAGACTAAGAATGCCTCATATCACAAGAGATTGAGGATGATGAGAGTATGTCATT GAACCCAATCAAGAAACTAGTTTATTTTTTTCGTCATTTTGTTTGTGTTTMATTAATAATATTAATAAATTA TTATTGTTATTTATTTTAATTTAATAAAATATGTTAAATATTTATTCACTATGTTTACAATATGTTAGAATT ATATAATTTGAAAGAGATTAAAAATAATACATAAACAATTTAAAACAATATTAGAGGTACAATTTGGTTTTG AATGTCAATTTTTTTATAAAAAAAACCCTGAAAAAAAGTCTTGACATCTCTTAGTCTTCTCTATGACACCGC ATCTTTCAAGAAAGCAATGTTACAACAACAGTCTTGCATACTACGTACTAGTATTCATTTGGAAGGTTCTGG ACAAGCAATGAGAACGTCAGATCAAACTGTGGTTAAAAATATTCAACGAAAACATTCAACTAAAAACAACTT TGCAATAACTCATTCACATGGTCATGGATATGGATGTATATATTTTGTGGTGCCCACTTTTGTTGACAATTT GGAGTACTTTAATAGTAAATACTACCATTATTTTATCTAGAGAAAGCGATGGAAATTCGGACAAAAAAATAC ACTTTTCGTGTTGAATTGAAAGTAATGTTTGATTCATGAGAGCCGCGGTTGACGGTCCACCCAATCTGAAAA GATCAAGATGACTTAGTATCATATTCCATCACGTCTCGTAGACCCAATCAGTATAAAAGGATAGTAAGGATC CATTTTCTGTACATCAATAAACAAATCATTTTCCATACATACAAAAATGGCAGAATCTTCATCAACCAAAAC CGGTCTTAGGTTAGCCGGCAAAGTAGCCATCGTCACCGGAGGTGCCAGCGGTATCGGCAAAGAGACTGCGCA TCTCTTTGCCAATCAAGCTGCACGTATGG

b

SAD-C promoter (GenBank accession no. AF242182)

ATCTCGTTGTTGTGCATCAAATTATAAAGAAAACTAAGAATGCCTCACTTCACAAGAGGTTGAGGATGATGA AAGTATATCATTGAACCCAATATAAAGACTAATTTGTTTTTTTTCATATTTTTAATTGTGTTTAATTAATAC TATCGTCCACGTGCTCGTCTGTCCGTTTTTTTTAATGCGCATACGGATAAAAATATGAGCGGTATTTTTGTG TRTCTATTTTAATTTAAATTCAATTGATTTTGAATATCAAATTTAAGAATATAAATTTTCCAAAAAATTGTT TGACATCTCTTAGTCTTCTCCATGACACCGCGTCTATCAAGAAAGCARTGTTACTAGTAATACATTTTGGAA GGTTCTGAACCCGCAATGACAACATCAGATCAAACTGTCGTTAAAAATATTCAACGAAAACATTCAACTAAA AACAACTTTTCAATAACTCATGATCATGGATATGGATGTATTTATTTTGTGGCGCCCACTTTTGTTGACAAT TTAAATCAATTTGGCGTGCTACTATTTTTGGTGACATATTTTATAAATTAAATAACAAGCCAAATATGAAAC ATTGATTACACTATGCTAAAATACACTTTCCGTGTTCAAAGGAATGTTTGATTCATGAGAGCCGCGGTTGAC GGTCCACCCAATCTGAAAAGATCAACCTGACTTAGTATCATATTCCATCACGTCTCGTAGACCCAATCAGTA TAAAAGGATAGTAAGGATCCATTTTCTGTACATCAATAAACAAATCATTTTCCATTATACATACAAAAATGG CAGAATCTTCATCAACCAAAACCGGTCTTAGGTTAGCCGGCAAAGTAGCCATCGTCACCGGAGGTGCCAGTG GTATCGGCAAAGAGACGGCGCATCTCTTTGCCAATCAAGCTGCACGTAT

ATGTGTT – reverse complement of AACACAT. One base mismatch (permitted) of TACACAT activating expression of seed storage protein (Josefsson et al., 1987).

CANNTG – E-box cooperating with other cis-elements to activate seed-specific promoters for heterologous expression (Kawagoe and Murai, 1992).

CATGCAT – Almost complete RY repeat element (CATGCATG; Chamberland et al., 1992, Reidt et al., 2000).

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2

References

Chamberland S, Daile N, Bernier F (1992). The legumin boxes and the 3′ part of a soybean β-conglycinin

promoter are involved in seed gene expression in transgenic tobacco plants. Plant Molecular Biol 19: 937–949

Josefsson LG, Lenman M, Ericson ML, and Rask L (1987) Structure of a gene encoding the 1.7 S storage protein, napin, from Brassica napus. J Biol Chem 262: 12196-12201.

Kawagoe Y, Murai N (1992) Four distinct nuclear proteins recognize in vitro the proximal promoter of the bean seed storage protein beta-phaseolin gene conferring spatial and temporal control. Plant J 2: 927–936

Reidt W, Wohlfarth T, Ellerström M, Czihal A, Tewes A, Ezcurra I, Rask L, Bäumlein H (2000) Gene regulation during late embryogenesis: the RY motif of maturation-specific gene promoters is a direct target of the FUS3 gene product. Plant J 21: 401-408.

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3 Supplementary Figure S2. MALDI-TOF MS analysis chromatograms (a,b) and analysis

reports (c,d) for the SAD content of immunoprecipitates from pea seeds (a,c) and leaves

of pea plants irradiated for 5d with UV-B radiation (b,d). Peaks in the chromatograms

indicated with red numbers represent peptides derived from SAD proteins that were used

in their identification.

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7 Supplementary Table S1. Substrates tested in enzymatic activity assays with

recombinant SAD-A and SAD-C proteins. For alcohols and hydroxylic compounds,

NAD

+

was used as co-factor. For carbonyls and quinones, NADH was used.

Chemical compound

Activity with

SAD

Chemical compound

Activity with

SAD

Steroids

Plant hormones

β-sitosterol

No

Gibberellic acid 3

No

campesterol

No

Gibberellic acid 4

2

_No

17α-hydroxyprogesterone

No

Gibberellic acid 7

2

_No

Deoxycorticosterone

No

Gibberellic acid 19

2

No

5α-androstane-3β,17β-diol

No

Gibberellic acid 24

2

No

Hydrocortisone

No

(+-)-cis,trans-Abscisic acid

No

4-pregnen-20β-ol-3-one

No

Teasterone

3

No

Corticosterone

No

3-dehydroteasterone

3

_No

Testosterone

No

Typhasterol

3

_No

6-deoxoteasterone

3

_No

Monoterpenes

3-dehydro-6-deoxoteasterone

3

_No

Pulegone

No

6-deoxotyphasterol

3

_No

Quinones and aromatic

Sugars and sugar alcohols

carbonyls

Adonitol

No

1,4-Benzoquinone

-

a

_Arabinose

_No

2,5-Dimethylbenzoquinone

Yes

Glucose

4

_No

2,6-Dimethylbenzoquinone

Yes

Sucrose

5

_No

2,6-Dimethoxybenzoquinone

Yes

Sorbitol

No

Duroquinone

Yes

1,4-Naphthoquinone

Yes

Flavonoids

1,2-Naphthoquinone

-

a

_Kaempferol

_No

Anthraquinone

_No

_{4’,5,7-Trihydroxyflavanone}

_No

Thymoquinone

_Yes

Menadione, vit K

3

Yes

Aromatic hydroxyls

Menaquinone, vit K

2

No

Pyrogallol

No

Phylloquinone, vit K

1

No

Hydroquinone

No

Decylplastoquinone

No

Pyrocatechol

No

Ubiquinone (Q10)

_No

8-Methoxypsoralen

_No

Alcohols

Methoxatin

_-

a

_Ethanol

6

_No

4-nitrobenzaldehyde

Yes

Propanol

7

No

Metyrapone

No

Butanol

No

Glycerol

8

No

Lignans

Methanol

9

No

Matairesinol

1

No

a

_{reacts non-enzymatically with NADH (Scherbak, N., Strid, Å. and Eriksson, L.A. (2005)}

Non-enzymatic oxidation of NADH by quinones. Chem. Phys. Lett. 414, 243-247).

Suppliers of compounds: Unless otherwise stated, all chemicals were of the Sigma, Aldrich,

Fluka or Supelco brands of the Sigma-Aldrich Company, St Louis, MO. Other suppliers were:

1

_{ArbaNova, Turku, Finland;}

2

_{Prof. Lewis Mander, Australian National University, Canberra}

provided us with these compounds;

3

Prof. Shuzo Fujioka, RIKEN, Saitama, Japan provided us

with these compounds;

4

_{ICN, Aurora, OH;}

5

_{Serva, Heidelberg, Germany;}

6

_{Solveco, Stockholm,}

Sweden;

7

_{AppliChem, Darmstadt, Germany;}

8

_{Duchefa, Haarlem, The Netherlands;}

9