Methods
Seed and Plant Material
The Papaver somniferum mutant was produced using EMS techniques (based on
ref. 3). 8000 M2 of
the EMS treated seed were analysed.
TLC of Poppy Alkaloids
About 50 L of latex from the mid vein of a torn leaf or lanced capsules was added to 200 L of Latex
extraction buffer (20 % EtOH, 0.25 % SDS and 0.2M NH 4H2PO4). The latex-buffer solution was
vortexed, centrifuged and 5 L loaded onto TLC plates (Merck Silca gel 60). The plates were
developed using toluene: acetone: ethanol: concentrated ammonia (40:40:6:2) and visualized with
Dragendorffs reagent. Standards used for the TLC were dissolved in 50 % EtOH/ 50 % NH 4OH in the
following concentrations: oripavine, 2.5 mg/mL; thebaine, 1.25 mg/mL; codeine, 5 mg/mL; and
morphine, 5 mg/mL. See Main Text Figure 1c.
HPLC of Poppy Alkaloids
Latex extracts (as above) were diluted 25 fold in extraction buffer before loading onto a Waters HPLC
with Partisphere SCX column (4.6 x 125 mm) at 40 °C. The HPLC phase was 30% aqueous methanol
containing ammonium acetate buffer (0.08-0.12 M), pH 4.5. The flow rate of the mobile phase was 0.81.5 mL/minute. Peaks were detected with a Waters 440 UV detector at 254 nm. The data was
interpreted and collated on a Waters Millennium Data Station. See Main Text Figure 1b.
Feeding Studies
Plants were grown from seed for 5-6 weeks (10-12 cm total length) before cutting the shoot just above
the root and placing in a 1.5 mL microcentrifuge tube filled with 300 µL of feeding solution. Shoots
were then placed in a transparent plastic lidded box containing enough Millipore water to obtain a
relative humidity of 80-90%.
Feeding solutions consisted of: 200 000 cpm 14C-oripavine and 9 nmol of oripavine; 215 000 cpm 14Cthebaine and 15 nmol of thebaine; 500 000 cpm 3H-codeine and 10 nmol codeine; 500 000 cpm 3Hsalutaridinol and 16 nmol salutaridinol; 260 000 cpm 3H-(R)-reticuline and 6 nmol (R)-reticuline; each
made up to a total volume of 300 µL with Millipore water. The duration of each of the feeding studies
was 24 hours for oripavine, thebaine, salutaridinol and R-reticuline. The codeine solution was fed for a
period of 72 hours. To prevent plants from running dry, Millipore water was used to top up the
microcentrifuge tube.
After the feeding assay, the plants were cut into small pieces and extracted in 80% ethanol for 15-20
minutes. The crude extract was lyophilised and dissolved in a small quantity of methanol and run on a
two dimensional TLC: 1st dimension solvent system was chloroform: acetone: diethylamine (5:4:1);
2nd dimension was toluene: acetone: ethanol: ammonia (45:45:7:3). See Supplementary Table 1.
RNA Isolation
RNA for the production of the cDNA library, microarray experiments and Real Time PCR was isolated
from CO52 total shoot when the flower bud had emerged and stalk was straight (prehook stage). The
NTES method1 was used. For quantitative Real Time PCR (qRTPCR), the Qiagen RNeasy Plant kit
was used. Poly A+ RNA (100 g at a concentration of 20 g/mL with an A260/280 of 1.9) was isolated
using the Promega PolyATract mRNA isolation System. Quality of the RNA and Poly A+ mRNA
was assured by running on MOPS agarose4. RNA for microarray analysis was reverse transcribed and
the cDNA labelled with Cy3 and Cy5 dyes using the methods as described in5.
cDNA Library and Microarrays
The directionally cloned P. somniferum plasmid cDNA library was provided in the pCMVSport 6
vector transformed into EMDH10B cells (Life Technologies). The library yielded 2.6 x 10 8 cfu with an
average insert size of 1.8 Kb and 96% of the colonies having an insert size  200 bp. 1000 random
clones from the library indicated a very low level of redundancy (0.4%).
DNAs for spotting on the microarray were generated by PCR using universal primers SP6 and T7.
Purified PCR products were printed on Corning CMT-GAP2 coated slides (# 40004) using a Bio-Rad
VersArray Chip Writer Pro. Slides were prehybridised and hybridised as per the Corning CMT
recommendations. RNA was extracted from tissue and pooled from six plants for each of the biological
replicates to ensure consistency of the biological material.
Slides were scanned with a GenePix 4000A microarray scanner (Axon Instruments, Union, CA), and
spots were analysed using GenePix Pro 3 software. Manual adjustment of any spots that were poorly
read by the software was undertaken to ensure quality. Cy3 and Cy5 labels were interchanged between
sample and control cDNA in complementary experiments to reduce any of the effects from
photobleaching of the fluorescent dyes and cDNA incorporation inequalities. Labelled cDNA probes
did not hybridize to several randomly spotted negative controls.
Data Normalisation and Median Value Calculation
The data was normalised using the spatial-normalisation protocol from the tRMA package 6. Using the
relevant function in tRMA the spatially normalised back to ratio differentially-expressed genes were
extracted from the complete list of 17,000 clones’ median values. Gaussian distribution outliers were
selected. Log Red dye/log Green dye [log(R/G)] data were compared using GenePix Pro 3 software
(after spatial normalisation). We used a ratio threshold cut-off of 2. See Supplementary Table 2. Data
has been submitted to the GEO website http://www.ncbi.nlm.nih.gov/geo/query/. See
Supplementary Table 3.
Sequencing of Differentially-Expressed Genes
Primers to the vector at the 5’ and 3’ regions of the inserted cDNA were used to get sequence data of
the differentially-expressed genes. Sequences were analysed on BioManager by ANGIS
(http://www.angis.org.au). against the NCBI database (GenBank main, SwissProt and SpTr EMBL
databases). See Supplementary Table 2.
2D protein Analysis.
Latex was collected into water from young green capsules of top1 and CO52, pooled from 8 plants and
matched for developmental stage. Samples were separated into serum and 1000g pellet fractions. The
serum proteins were acetone precipitated and dried. The 1000g pellet was washed, solubilised with 1%
Triton X100 and sonicated, acetone precipitated and dried. At the Australian Proteome Analysis
Facility, 100µg samples were separated first with isoelectric focusing pH 3-10 (87 kV hours), and then
in the second dimension on 8-18% polyacrylamide gradients (3 h at 3mA, 14 h at 15 mA). Proteins
were visualized with SYPRO Ruby fluorescent stain. Data not shown.
Real-Time Quantitative Reverse Transcriptase-Mediated PCR and Qualitative RT-PCR
Gene-specific primer sets were designed using the Primer3 software package (http://wwwgenome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) and made by Sigma (Sigma-Aldrich, Sydney,
Australia). These primers were designed to have a T m of 59-63 C (with the optimal being =60 C),
yield a PCR product around 225-275 bp, GC content from 40-60%, self-complementarity <4, and 3’
self-complementarity <3. Quantitative Real Time PCR (qRT-PCR) reactions were carried out as per 2.
Ratio values were determined by comparing gene expression in C052 to top1. RNA was extracted from
de-veined leaf two days after petal drop. Three technical replicates of each sample were tested.
For Reverse transcriptase PCR analysis, RNA was isolated at two days post petal drop from the leaf,
stem and roots. 20 ng of cDNA was used per PCR reaction using the primers (each at 6 pmoles)
designed for qRT-PCR. Qiagen Hotstar Taq Polymerase mix was used in each 20 µL reaction. An
annealing temperature of 50º C was used in the PCR reaction. The number of cycles was adjusted to
discern differences in expression levels See Supplementary Figure 1. Two biological and two
technical replicates were run for each sample and set of primers. RNA was quantified by comparison of
the ribosomal loading on an RNA gel. See Supplementary Table 2.
Reference List
1. Higgins TJV, Zwar JA, and Jacobsen JV. Nature 260, 166-169 (1976).
2. Klok, E.J. et al. Plant Cell 14, 2481-2494 (2002).
3. Mikaelson, K. Manual on Mutation Breeding, 2nd ed. I.A.E.A., Vienna (1977).
4. Sambrook J, Fritsch EF, and Maniatis T. Molecular cloning: a laboratory manual (2 nd Edition).
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. (1989).
5. Schenk, P.M. et al. Proceedings of the National Academy of Sciences of the United States of
America 97, 11655-11660 (2000).
6. Wilson, D.L., Buckley, M.J., Helliwell, C.A., and Wilson, I.W. Bioinformatics 19, 1325-1332
(2003).