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Supplemental Figures
Figure S1.
Nicotine biosynthetic pathway in tobacco.
Each solid arrow indicates single biochemically defined enzyme reaction, whereas a
dashed arrow represents a series of enzymatic reactions catalyzed more than one
enzyme or ill defined reactions.
Conversion of nicotinic acid mononucleotide to
nicotinic acid has been reported in protein extracts from tobacco roots (Wagner et al.,
1986), but is not characterized.
A622, a PIP-family oxidoredutase (Deboer et al.,
2009; Kajikawa et al., 2009), and BBL, a berberine bridge enzyme-like protein
(Kajikawa et al., 2011), are required for nicotine biosynthesis, and are presumably
involved in activation or subsequent condensation reactions of a nicotinic acid-derived
precursor.
the
boxed
The NIC2 regulatory locus and ERF189 regulate structural genes encoding
enzymes.
ODC,
ornithine
decarboxylase;
PMT,
putrescine
N-methyltransferase; MPO, N-methylputrescine oxidase; AO, aspartate oxidase; QS,
quinolinic acid synthase; QPT, qunolinate phosphoribosyltransferase.
REFERENCES
Deboer, K.D., Lye, J.C., Aitken, C.D., Su, A.K. and Hamill, J.D. (2009) The A622
gene in Nicotiana glauca (tree tobacco): Evidence for a functional role in pyridine
alkaloid biosynthesis. Plant Mol. Biol. 69, 299-312.
Kajikawa, M., Hirai, N. and Hashimoto, T. (2009) A PIP-family protein is required
for biosynthesis of tobacco alkaloids. Plant Mol. Biol. 69, 287-298.
Wagner, R., Feth, F. and Wagner, K.G. (1986) The pyridine-nucleotide cycle in
tobacco: Enzyme activities for the recycling of NAD. Planta 167, 226-232.
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Figure S2.
Relative transcript levels of QPT1 and QPT2 in tobacco.
A pair of PCR primers (arrowheads) which annealed to the sequences common to QPT1
and QPT2 were used to amplify by RT-PCR the intervening sequences (dotted lines)
from cDNAs derived from flower, leaf, or root samples.
After a part of the PCR
products were digested with ApoI, undigested (control) and digested DNA samples were
separated on a polyacrylamide gel.
The QPT1 cDNA fragment (257 bp) was intact,
while the QPT2 cDNA fragment was digested to 189-bp and 68-bp fragments.
Comparison of the fragment intensities indicates that the QPT2 cDNA is more abundant
than the QPT1 cDNA in the flower and the leaf.
In the root, the QPT2 cDNA vastly
predominates the QPT1 cDNA.
Figure S3. Histochemical GUS staining of tobacco hairy roots.
The GUS reporter was driven by the QPT2 promoter. This picture corresponds to an
enlarged part of Figure 7e. Scale bar, 0.05 mm. co, cortex; ep, epidermis.
Figure S4. Multiple alignment of amino acid sequences of AP2/ERF DNA binding
domains.
The sequences of tobacco clade 2 ERFs in the IXa family are aligned with Clustal W
(Chenna et al., 2003), together with the sequences of tobacco ERF32, Arabidopsis
thaliana AtERF1 (At4g17500), Catharansus roseus ORCA3 (GenBank accession
number EU072424), and five tomato (Solanum lycopersicum) ERFs (1g090300,
1g090310, 1g090320, 1g090340, and 1g090370) that are clustered on chromosome I.
Tobacco sequences can be found in the database of tobacco transcription factors
(TOBFAC) (Rushton et al., 2008) under the same names. Residues identical in at least
eleven sequences are shaded, and dashes indicate gaps introduced to maximize the
alignment. The secondary structures at the bottom are characterized for the DNA
2
binding domain of AtERF1 (Allen et al., 1998). An arrowhead indicates the critical
Arg/Lys residue that may contribute to the DNA binding preference (see the text for
details).
The first residues of the domain are numbered from N-termini, when
full-length protein sequences are available.
REFERENCES
Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T.J., Higgins, D.G. and
Thompson, J.D. (2003) Multiple sequence alignment with the Clustal series of
programs. Nucleic Acids Res. 31, 3497-3500.
Rushton, P.J., Bokowiec, M.T., Laudeman, T.W., Brannock, J.F., Chen, X. and
Timko, M.P. (2008) TOBFAC: the database of tobacco transcription factors. BMC
Bioinformatics 9, 53.
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