Supplemental Fig. 1

Alkaloid production of mutant strains JC and 1C2

The alkaloid content of non-elicited cultures (lower panels) and the increase caused by elicitor contact (upper panels) is shown for the wild type, the cell strain JC (class I) and 1C2 (class II). The data are obtained by the HPLC/ESI-MS assay described in Methods and are processed exactly as in Fig. 4 of the main manuscript. Paired columns in the upper graph indicate the response to low elicitor treatment (l, 1 µg/ml) or high elicitor treatment (h, 50 µg/ml). Class I mutants (middle panel) showed a measurable response only to high elicitor concentrations. Ordinate figures are ng alkaloid / per mg fresh weight.

Supplemental Figure 2

Alkaloid production of mutant strains 5b and 3C1

The increase of dihydroalkaloid content triggered by low and high elicitor-treatment of wild type and the mutant strains 5b (class I) and 3C1 (class II) was measured by the fluorescence assay described in Methods. Elicitor concentrations are 1 μg/ml (E1) and 50 μg/ml (E50), respectively. Data are differences between t0 and 24 h (mean ± SD, n = 3). The increase of alkaloid content in non-elicited cultures was negligable during this time.


Supplemental Figure 3

Southern blot used for the estimation of copy numbers of the incorporated sanguinarine reductase RNAi-vector

Genuine DNA was isolated from the wild type and each recombinant cell strain indicated on top. After digestion with Hind III each sample was hybridized to a DNA sequence specific of the RNAi vector and the bound, alkaline phosphatase-labelled probe detected by chemiluminescence. The used standard protocol is referred to in Methods. Left lane: DNA size standards, numbered in kbp.

Supplemental Table 1:

MS-based quantitation of UPLC-separated alkaloids

RT (min) / [M+H]+
(m/z) / compound / non-
elicited / elicitor / elicitor,
DHS
4.32 / 300 / N-Methylcoclaurine / 17 / 56 / 15
4.70 / 314 / 4’-O-Methyl-N-methylcoclaurine* / 67 / 436 / 120
5.33 / 324 / Stylopine / 19 / 3061 / 131
4.33 / 326 / Cheilanthifoline / 13 / 542 / 95
5.30 / 326 / Nandinine* / 3 / 140 / 19
4.08 / 328 / Scoulerine / 55 / 961 / 381
3.75 / 330 / Reticuline / 501 / 408 / 220
5.76 / 332 / Sanguinarine / n.d. / 11 / 1
11.49 / 334 / Dihydrosanguinarine / n.d. / n.d. / n.d.
5.26 / 338 / N-Methylstylopine / 843 / 1280 / 436
5.77 / 348 / 10-Hydroxysanguinarine / n.d. / 689 / 257
13.22 / 350 / Dihydrochelerythrine / 89 / 58 / 54
4.93 / 354 / Protopine / 1626 / 6901 / 1122
5.63 / 354 / N-Methylcanadine / 39 / 42 / 16
6.55 / 362 / Chelirubine / n.d. / 166 / 31
6.20 / 364 / 10-Hydroxychelerythrine / 18 / 343 / 178
11.48 / 364 / Dihydrochelirubine / 14 / 18 / 15
5.15 / 370 / Allocryptopine / 27 / 34 / 21
6.04 / 378 / 12-Hydroxychelirubine / 52 / n.d. / 79
6.53 / 392 / Macarpine / 36 / 226 / 51
13.21 / 394 / Dihydromacarpine / 162 / 95 / 87

Compounds are sorted by m/z.

columns 1,2: UPLC-retention times and [M+H]+ ions of alkaloids assayed in extracts of Eschscholzia

cultures by UPLC-ESI-MS/MS, and confirmed by ESI-FTICR-MS (cf. Methods);

columns 4,5, 6: peak areas per sample (arbitrary units), used for quantification in Fig. 7.

*Compounds represent side reactions of benzophenanthridine biosynthesis and are not considered in Fig. 7.

Supplemental Table 2:

Activities of dihydrobenzophenanthridine oxidase (DhbOx) and NAD(P)H-cytochrome c reductase in cell fractions

enzyme / 100 000 g
supernatant / plasma
membrane / cell
homogenate
DhbOx / 613 / 59 / 204
cyt c - reductase / 51 / 63 / 10

Data are enzyme activities in nkat/mg protein and were obtained after cell fractionation and activity assays described in Methods. Cyt c- reductase was assayed as described in Poenitz & Roos (1994). One typical experiment is shown which was repeated with another batch of cells and yielded the same relation of activities.

Note that DhbOx is enriched three times and the ER marker cyt c reductase five times from homogenate to supernatant. DhbOx is not enriched in the purified plasma membrane, in contrast to cyt c reductase which represents the plasma membrane-associated part of ER, as indicated in Schwartze & Roos (2008).

Supplemental Table 3:

Transcript levels of two biosynthetic enzymes in wild type and overproducing sanguinarine reductase RNAi-strain

Gene (mRNA) / Wild type / Strain 1C2 (class II)
BBE / 158 % / 154 %
4’OMT / 217 % / 127 %

RT-PCR with gene specific primers was performed with RNA from 6d old cultures of wild type or transgenic line 1C2, a member of class II strains. The experiment followed the protocol described in Fig.5 and in Methods, except that 20 PCR-cycles were used. Samples were taken shortly before or 6 hrs after a 30 min contact with low concentrated yeast elicitor (1 µg/ml). Data are mRNA/cDNA contents, normalized to the levels of elicitor-free culture that was set to 100 %. Figures are averaged from three cell suspensions taken from the same culture batch. SD of the cDNA assays ranged from 3 – 8 %.