Title:The apoplastic oxidative burst as a key factor of hyperhydricity in garlic plantletin vitro

Supplementary Data

SupplementaryTable S1.Cell fraction purities according to the enrichment of the marker enzyme activities before treatment (day 0).After cell fractioning, the measurement of the marker enzyme activity has been performed in all isolated compartments. The chlorophyll content (chloroplast marker) and activity of cytochrome c oxidase (mitochondrion marker enzyme) andalcohol dehydrogenase (cytosolmarker enzyme) has been measured as reported in Supplementary Materials and methods. Each value is the mean of three samples of thirty randomly selected shoots ± standard error (SE)

Subcellular compartment / Chlorophyll
Content distribution（%） / Cytochrome c oxidase
Activity distribution（%） / Alcohol dehydrogenase
Activity distribution（%）
Chloroplasts / 87.9±0.6 / 4.2±0.6 / 3.4±0.2
Mitochondria / 5.3±0.6 / 86.7±0.3 / 4.1±0.4
Apoplasts / 2.1±0.1 / 2.3±0.3 / 4.6±0.5
Cytosol / 4.7±0.2 / 6.8±0.6 / 87.9±1.0

SupplementaryTable S2.Cell fraction purities according to the enrichment of the marker enzyme activities on day 4of H2O2 treatment.After cell fractioning, the measurement of the marker enzyme activity has been performed in all isolated compartments. The chlorophyll content (chloroplast marker) and activity of cytochrome c oxidase (mitochondrion marker enzyme) and alcohol dehydrogenase (cytosolmarker enzyme) has been measured as reported in Supplementary Materials and methods.Each value is the mean of three samples of thirty randomly selected shoots ± standard error (SE)

Subcellular compartment / Chlorophyll
Content distribution（%） / Cytochrome c oxidase
Activity distribution（%） / Alcohol dehydrogenase
Activity distribution（%）
Control / H2O2 / Control / H2O2 / Control / H2O2
Chloroplasts / 88.6±0.3 / 86.9±02. / 3.6±0.4 / 4.1±0.6 / 3.0±0.5 / 3.2±0.8
Mitochondria / 4.6±0.2 / 5.5±0.1 / 87.7±1.0 / 88.3±0.6 / 3.3±0.5 / 3.7±0.6
Apoplasts / 2.5±0.4 / 3.5±0.3 / 2.3±0.4 / 1.7±0.1 / 3.2±0.4 / 3.9±0.6
Cytosol / 4.3±0.5 / 4.1±0.1 / 6.4±0.6 / 5.9±0.5 / 90.5±0.6 / 89.2±1.7

SupplementaryTable S3.Cell fraction purities according to the enrichment of the marker enzyme activities on day 8of H2O2 treatment.After cell fractioning, the measurement of the marker enzyme activity has been performed in all isolated compartments. The chlorophyll content (chloroplast marker) and activity of cytochrome c oxidase (mitochondrion marker enzyme) and alcohol dehydrogenase (cytosolmarker enzyme) has been measured as reported in Supplementary Materials and methods.Each value is the mean of three samples of thirty randomly selected shoots ± standard error (SE)

Subcellular compartment / Chlorophyll
Content distribution（%） / Cytochrome c oxidase
Activity distribution（%） / Alcohol dehydrogenase
Activity distribution（%）
Control / H2O2 / Control / H2O2 / Control / H2O2
Chloroplasts / 91.1±0.6 / 89.2±0.5 / 4.2±0.5 / 4.6±0.4 / 2.5±0.3 / 3.9±0.4
Mitochondria / 2.8±0.3 / 3.8±0.2 / 89.1±0.8 / 86.5±0.1 / 3.4±0.5 / 4.1±0.4
Apoplasts / 2.3±0.3 / 3.0±0.3 / 2.0±0.3 / 2.0±0.2 / 5.4±0.6 / 4.4±0.5
Cytosol / 3.8±0.2 / 4.0±0.5 / 4.7±0.2 / 6.9±0.4 / 88.7±0.7 / 87.6±0.7

SupplementaryTable S4.Cell fraction purities according to the enrichment of the marker enzyme activities on day 12of H2O2 treatment.After cell fractioning, the measurement of the marker enzyme activity has been performed in all isolated compartments. The chlorophyll content (chloroplast marker) and activity of cytochrome c oxidase (mitochondrion marker enzyme) and alcohol dehydrogenase (cytosolmarker enzyme) has been measured as reported in Supplementary Materials and methods.Each value is the mean of three samples of thirty randomly selected shoots ± standard error (SE)

Subcellular compartment / Chlorophyll
Content distribution（%） / Cytochrome c oxidase
Activity distribution（%） / Alcohol dehydrogenase
Activity distribution（%）
Control / H2O2 / Control / H2O2 / Control / H2O2
Chloroplasts / 89.7±1.2 / 87.8±0.2 / 5.0±0.5 / 3.9±0.7 / 3.7±0.4 / 3.2±0.8
Mitochondria / 3.7±0.8 / 5.1±0.3 / 86.9±0.6 / 88.6±0.5 / 4.3±0.4 / 2.6±0.7
Apoplasts / 3.1±0.5 / 2.8±0.1 / 2.0±0.1 / 1.9±0.1 / 6.8±0.3 / 6.5±0.5
Cytosol / 3.5±0.4 / 4.3±0.2 / 6.1±0.3 / 5.6±0.4 / 85.2±0.4 / 87.7±1.2

SupplementaryTable S5.Cell fraction purities according to the enrichment of the marker enzyme activities on day 16of H2O2 treatment.After cell fractioning, the measurement of the marker enzyme activity has been performed in all isolated compartments. The chlorophyll content (chloroplast marker) and activity of cytochrome c oxidase (mitochondrion marker enzyme) and alcohol dehydrogenase (cytosolmarker enzyme) has been measured as reported in Supplementary Materials and methods.Each value is the mean of three samples of thirty randomly selected shoots ± standard error (SE)

Subcellular compartment / Chlorophyll
Content distribution（%） / Cytochrome c oxidase
Activity distribution（%） / Alcohol dehydrogenase
Activity distribution（%）
Control / H2O2 / Control / H2O2 / Control / H2O2
Chloroplasts / 86.3±0.7 / 88.0±0.6 / 4.8±0.2 / 4.4±0.5 / 3.0±0.4 / 2.9±0.6
Mitochondria / 5.2±0.2 / 5.4±0.2 / 88.5±0.2 / 87.2±0.7 / 1.6±0.3 / 2.9±0.5
Apoplasts / 3.7±0.4 / 2.5±0.1 / 1.9±0.2 / 1.9±0.1 / 5.8±0.9 / 3.1±0.5
Cytosol / 4.8±0.5 / 4.1±0.4 / 4.8±0.3 / 6.5±0.1 / 89.6±0.8 / 91.1±1.5

Supplementary Material and Methods

Chloroplastic marker Enzyme – Chlorophyll content assay

Chlorophyllcontentwas measured according toPorra and others (1989).

Mitochondrial marker Enzyme – Cytochrome C oxidase activity assay

Cytochrome c oxidase activity was performed following the decrease in absorbance at 550 nm because of the oxidation of reduced cytochrome c. The reaction mixture contained 0.1 M phosphate buffer pH 7 and the sample pretreated with 0.1% Triton-X-100 in ice for 1 min. The reaction started with the addition of 50 mM reduced cytochrome c. An extinction coefficient of21 mM-1 cm-1 was used (Locato et al. 2009).

Cytosolic marker enzyme –Alcohol dehydrogenase activity assay

Alcohol dehydrogenase activity was tested measuring the increase of absorbance at340 nm because of NAD+reduction. The reaction mixture contained 50 mM Tris-HCl pH 9, 0.867 mM NAD+. The reaction started adding 20% ethanol. An extinction coefficient of21 mM-1 cm-1 was used (Chung and Robert 1999).

References

Chung HJ, Robert JF (1999) Arabidopsis alcohol dehydrogenase expression in both shoots and roots is conditioned by root growth environment. Plant Physiol 121: 429-436

Locato V, de Pinto MC, de Gara L (2009) Different involvement of the mitochondrial, plastidial and cytosolic ascorbate–glutathione redox enzymes in heat shock responses. Physiol Plant 135:296-306

Porra RJ, ThompsonWA, Kriedemann PE (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectrometry. BiochimBiophysActa 975: 384-394