Volume 3, Issue 5, Pages 927-939 (September 2010) Overproduction of Alkaloid Phytoalexins in California Poppy Cells Is Associated with the Co-Expression of Biosynthetic and Stress-Protective Enzymes  Angelova Sornitza , Buchheim Marcus , Frowitter Doreen , Schierhorn Angelika , Roos Werner   Molecular Plant  Volume 3, Issue 5, Pages 927-939 (September 2010) DOI: 10.1093/mp/ssq043 Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 1 Overview of the Biosynthesis of Benzophenanthridine Alkaloids. A selection of biosynthetic enzymes is notified, according to Zenk (1994) and Facchini and St-Pierre (2005): TYDC, tyrosine decarboxylase; NCS, norcoclaurine synthase; 6OMT, norcoclaurine 6-O-methyltransferase; CNMT, coclaurine N-methyltransferase; NMCH, N-methylcoclaurine 3'-hydroxylase; 4'OMT, 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase; BBE, berberine bridge enzyme; CFS, cheilanthifoline synthase; STS, stylopine synthase; TNMT, tetrahydroprotoberberine cis-N-methyltransferase; MSH, N-methylstylopine 14-hydroxylase; P6H, protopine 6-hydroxylase; DBOX, dihydrobenzophenanthridine oxidase. Sanguinarine is the first benzophenanthridine formed; macarpine is the most oxidized of these alkaloids. Between dihydrosanguinarine and dihydromacarpine, ornamental reactions (indicated by arrows) form intermediates as 10-hydroxy-dihydrosanguinarine, 10-hydroxy-dihydrochelerythrine, or dihydrochelirubine; all of them are finally oxidized by dihydrobenzophenanthridine oxidase. Molecular Plant 2010 3, 927-939DOI: (10.1093/mp/ssq043) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 2 Longitudinal Section of a Root from a 63-Day-Old California Poppy Plant. The red/orange color in the cortex region indicates accumulated benzophenanthridines. Cutting lines indicate the root collar region. The present cell suspension culture was established via a callus obtained from a disk of this area. Molecular Plant 2010 3, 927-939DOI: (10.1093/mp/ssq043) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 3 Response to Yeast Elicitor of Roots from Different Growth Stages. Roots from greenhouse plants of the indicated age were washed with sterile tap water, cut into disk segments of 10–50 mg fresh weight, and incubated with and without yeast elicitor (50–100 mg fresh weight in 3 ml of 75% phosphate-free nutrient solution). Seedlings were grown from sterilized seeds on agar plates containing the same nutrient solution with and without elicitor. Cell suspensions were harvested after 7-d growth by suction, re-suspended in the same nutrient solution (50 mg fwt ml−1) with and without yeast elicitor and incubated on a rotary shaker. After 30 min of elicitor contact, the cells were filtered and re-suspended in elicitor-free medium. Alkaloids were assayed in all samples 24 h after elicitor contact. Primary roots were cut off the seedlings and used for analysis. Columns represent the alkaloid content, namely the sum of dihydrobenzophenanthridines (the penultimate biosynthetic product, dominating species in young roots and cell cultures) plus benzophenanthridines (the final biosynthetic product, dominating species in adult roots). The experiment was performed with a series of elicitor concentrations (1, 10, 50, 100, 150 μg ml−1). Hatched columns show the highest and lowest response to elicitor contact, with the respective elicitor concentration (μg ml−1) given on top (* = 30 min of elicitor contact). Open columns represent non-elicited samples. Data are means ± SD, n = 4 (roots), n = 8 (cell culture). Molecular Plant 2010 3, 927-939DOI: (10.1093/mp/ssq043) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 4 Shifts of Cytoplasmic pH Triggered by Changing External pH and K+ Concentration in the Presence of Nigericin. Local pH values were derived from pH maps obtained by confocal fluorescence imaging (cf. Experimental Procedures, and Viehweger et al., 2006). Each trace represents the cytoplasmic pH (top) or vacuolar pH (bottom) of one single cell. Two typical cells are shown that responded similarly to most cells in a 2-ml suspension as well as in another batch. Cells preloaded with the pH probe SNARF-4F were perfused in a flow-through chamber with 75% phosphate-free nutrient solution. As indicated by the arrows, the medium was replaced by Na-MES buffer, 20 mM, pH 5.5, containing 4 μM nigericin (allowing an outward-directed K+-gradient), and 3 min later by 20 mM K-HEPES, pH 7.5, containing 40 mM KCl (which is considered to stop the K+-efflux and allow recovery of the intracellular pH). Neither pH shifts nor alkaloid production was measurable if 100 mM external K+ or an external pH of 7.5 was present during the incubation with nigericin or no K+ was present in the following nigericin-free medium. The filtrate obtained after a 5-min incubation of the cell suspension with nigericin caused a similar pH shift if used with another batch of cells, whereas filtrates obtained after a 6-h incubation had lost this activity (data not shown). We therefore assume that the nigericin remaining in the cells after treatment was deactivated and thus caused no irreversible damage. Molecular Plant 2010 3, 927-939DOI: (10.1093/mp/ssq043) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 5 Overproduction of Benzophenanthridine Alkaloids Caused by Elicitor or Nigericin Treatment. Cell suspensions were treated with the indicated stressors as described in Methods. eli 1: low-elicitor treatment, 1 μg ml−1; eli 50: high-elicitor treatment, 50 μg ml−1, nig: nigericin treatment as described in Methods; nig pH 7.5: nigericin treatment at an external pH of 7.5. Data represent the total alkaloid content, namely the sum of dihydrobenzophenanthridines and benzophenanthridines, assayed by their specific fluorescence. They are normalized to the response to 1 μg ml−1 yeast elicitor (low-elicitor treatment) within 24 h, which is set to 100%. The dominating species are dihydroalkaloids (the penultimate products) whereas benzophenanthridines (the final products) constitute between 10 and 30% of the total alkaloids. Non-viable cells are unable to accumulate 5'carboxyfluorescein from a 2-μM solution of its diacetate during a 20-min incubation, as assayed by classical fluorescence microscopy. Molecular Plant 2010 3, 927-939DOI: (10.1093/mp/ssq043) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 6 Typical Two-Dimensional Electrophoresis of Genuine Proteins and In vitro Translation Products of mRNAs, Collected 6 h after Low-Elicitor Treatment. Left: SYPRO Ruby fluorescence of genuine proteins; right: autoradiogram of the 35S proteins that were in vitro translated from the mRNAs in the same sample. The range of IEF (first dimension) reached from pH 3 to pH 10. Numbers refer to some identified proteins. 1: HSP 70; 6: 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase (4'OMT); 9:14–3–3 protein; 11: glyceraldehyde 3-phosphate dehydrogenase (GAPDH); 12: peptidylprolyl-cis/trans-isomerase (cyclophilin). Molecular Plant 2010 3, 927-939DOI: (10.1093/mp/ssq043) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 7 RT–PCR Assay of the GAPDH mRNA in Elicitor-Treated Cultures. mRNA was isolated from cell suspensions immediately after a 30-min contact with 1 μg ml−1 yeast elicitor (t = 0) and at the times indicated. cDNA was generated by reverse transcriptase, followed by PCR with primers that allow amplification of the entire cDNA of the gene encoding GAPDH (cf. Methods). The figure shows the PCR product of the expected size (980 bp). No other bands were detectable. One typical experiment is shown, which was repeated twice and yielded a similar result, namely a transient increase in GAPDH mRNA followed by a strong decrease. In non-elicited cell suspensions, no increase in this mRNA was detectable. Molecular Plant 2010 3, 927-939DOI: (10.1093/mp/ssq043) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions