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Fig. 1. Optical microscopy images of protoplasts and vacuoles isolated from C. roseus leaves. Protoplast fraction (A), vacuole fraction (B), and Western.

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Presentation on theme: "Fig. 1. Optical microscopy images of protoplasts and vacuoles isolated from C. roseus leaves. Protoplast fraction (A), vacuole fraction (B), and Western."— Presentation transcript:

1 Fig. 1. Optical microscopy images of protoplasts and vacuoles isolated from C. roseus leaves. Protoplast fraction (A), vacuole fraction (B), and Western blot of protein extracts from protoplasts (Pro) and vacuoles (Vac), probed with an anti-calreticulin antibody (ER-resident protein) (C). The marker was only observed in protoplasts (arrow), demonstrating the absence of other cellular endomembranes in the vacuolar fraction. Bar, 50 μm. Identification of phenolic compounds in isolated vacuoles of the medicinal plant Catharanthus roseus and their interaction with vacuolar class III peroxidase: an H2O2 affair? J Exp Bot. 2011;62(8): doi: /jxb/erq458 J Exp Bot | © 2011 The Author(s).This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see for further details)

2 Fig. 2. Hydro-alcoholic extract of C. roseus vacuoles
Fig. 2. Hydro-alcoholic extract of C. roseus vacuoles. UV chromatogram, 340 nm (A), Vis chromatogram, 520 nm (B), and Extraction Ion Chromatogram (EIC) of [353.2]<sup>–</sup>, [755.5]<sup>–</sup>, [739.6]<sup>–</sup>, [309.0]<sup>+</sup>, [253.0]<sup>+</sup> (C–G). Identity of peaks corresponds to compounds in Table 1. Identification of phenolic compounds in isolated vacuoles of the medicinal plant Catharanthus roseus and their interaction with vacuolar class III peroxidase: an H2O2 affair? J Exp Bot. 2011;62(8): doi: /jxb/erq458 J Exp Bot | © 2011 The Author(s).This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see for further details)

3 Fig. 3. Chemical structures of the phenolic compounds identified in C
Fig. 3. Chemical structures of the phenolic compounds identified in C. roseus leaf vacuoles. 3-O-Caffeoylquinic acid (compound 1), 4-O-caffeoylquinic acid (compound 2), 5-O-caffeoylquinic acid (compound 3), quercetin-3-O-(2,6-di-O-rhamnosyl)galactoside (compound 4), and kaempferol-3-O-(2,6-di-O-rhamnosyl)galactoside (compound 5). Identification of phenolic compounds in isolated vacuoles of the medicinal plant Catharanthus roseus and their interaction with vacuolar class III peroxidase: an H2O2 affair? J Exp Bot. 2011;62(8): doi: /jxb/erq458 J Exp Bot | © 2011 The Author(s).This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see for further details)

4 Fig. 4. Time-courses of the CrPrx1-mediated oxidation of 0
Fig. 4. Time-courses of the CrPrx1-mediated oxidation of 0.1 mM 5-O-caffeoylquinic acid (A), of 0.1 mM ascorbic acid (B), and of 0.1 mM 5-O-caffeoylquinic acid+0.1 mM ascorbic acid (C). CrPrx1 concentration was 0.6 nM. Identification of phenolic compounds in isolated vacuoles of the medicinal plant Catharanthus roseus and their interaction with vacuolar class III peroxidase: an H2O2 affair? J Exp Bot. 2011;62(8): doi: /jxb/erq458 J Exp Bot | © 2011 The Author(s).This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see for further details)

5 Fig. 5. Peroxidase isoenzyme pattern of C
Fig. 5. Peroxidase isoenzyme pattern of C. roseus extracts after isoelectric focusing. Leaf protein extract (Leaf), extracellular protein extract (ExtCel), protoplast extract (Pro), and vacuole extract (Vac). The basic band corresponds to CrPrx1 and is the only isoenzyme present in protoplasts and vacuoles. Different lanes correspond to non contiguous parts of the same gel. Identification of phenolic compounds in isolated vacuoles of the medicinal plant Catharanthus roseus and their interaction with vacuolar class III peroxidase: an H2O2 affair? J Exp Bot. 2011;62(8): doi: /jxb/erq458 J Exp Bot | © 2011 The Author(s).This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see for further details)

6 Fig. 6. Localization of peroxidase activity representative of CrPrx1 in C. roseus mesophyll cells. All sections were incubated with DAB and H<sub>2</sub>O<sub>2</sub>, and they show electron-dense deposits representative of peroxidase activity mainly in spots of the inner surface of the tonoplast (B–D), and rare deposits in the external face of the plasma membrane (arrows in A and E). Identification of phenolic compounds in isolated vacuoles of the medicinal plant Catharanthus roseus and their interaction with vacuolar class III peroxidase: an H2O2 affair? J Exp Bot. 2011;62(8): doi: /jxb/erq458 J Exp Bot | © 2011 The Author(s).This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see for further details)

7 Fig. 7. Peroxidase activity in leaves of C
Fig. 7. Peroxidase activity in leaves of C. roseus plants adapted to fluorescent light with a maximum intensity of 70 μmol m<sup>−2</sup> s<sup>−1</sup> (Low light) and after exposure of the same plants to sunlight with a maximum intensity of 1400 μmol m<sup>−2</sup> s<sup>−1</sup> for 7 d (High light). Percentage values in the graph refer to the increase observed in peroxidase activity for each plant or the average after exposure to high light conditions. Identification of phenolic compounds in isolated vacuoles of the medicinal plant Catharanthus roseus and their interaction with vacuolar class III peroxidase: an H2O2 affair? J Exp Bot. 2011;62(8): doi: /jxb/erq458 J Exp Bot | © 2011 The Author(s).This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see for further details)

8 Fig. 8. Co-operative action of ascorbic acid with the couple Prx/phenols, generating a self-sustained Prx substrate regeneration cycle. A phenolic compound (Ph) is oxidized into a phenolic radical (Ph·) by Prx, which in turn oxidizes ascorbic acid (AsA) to monodehydroascorbate (MDHA) returning to the original form. MDHA will interact spontaneously to produce AsA+dehydroascorbic acid (DHA) which may be reduced back to AsA by a cytosolic dehydroascorbic acid reductase (cDHAR) (adapted from Yamasaki et al. 1997and reproduced by kind permission of The American Society of Plant Biologists © 1997). Identification of phenolic compounds in isolated vacuoles of the medicinal plant Catharanthus roseus and their interaction with vacuolar class III peroxidase: an H2O2 affair? J Exp Bot. 2011;62(8): doi: /jxb/erq458 J Exp Bot | © 2011 The Author(s).This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see for further details)

9 Fig. 9. Model proposed for the role of the vacuolar couple Prx/SMs as a sink/buffer of H<sub>2</sub>O<sub>2</sub> levels in plant leaf cells. H<sub>2</sub>O<sub>2</sub> produced in the chloroplasts during excess light will diffuse spontaneously to sink spots in the vacuole, where Prx will reduce H<sub>2</sub>O<sub>2</sub> to water through oxidation of available secondary metabolite substrates (SM) into secondary metabolite radicals (SM·). Identification of phenolic compounds in isolated vacuoles of the medicinal plant Catharanthus roseus and their interaction with vacuolar class III peroxidase: an H2O2 affair? J Exp Bot. 2011;62(8): doi: /jxb/erq458 J Exp Bot | © 2011 The Author(s).This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see for further details)

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