THE ONSET OF GRAPEVINE BERRY RIPENING IS CHARACTERIZED BY
ROS ACCUMULATION AND LIPOXYGENASE-DERIVED GALACTOLIPID PEROXIDES
Pilati Stefaniaa, Brazzale Danielea, Guella Grazianob,c, Biasioli Franco a, Zottini Michelad, Moser Claudioa
a) Research and Innovation Centre, Fondazione Edmund Mach, via E. Mach 1, 38010 San Michele a/Adige (TN), Italy
b) Department of Physics, Bioorganic Chemistry Lab, University of Trento, Via Sommarive 14, 38123 Povo, Trento, Italy
c) CNR, Istituto di Biofisica Trento, Via alla Cascata 56/C, 38123 Povo, Trento, Italy
d) Department of Biology, University of Padova, Via U. Bassi 58/b - 35131 Padova – Italy
Green hard berries
Green soft berries
H2O2 (DCFDA) 1O2 (SOSG)
Chl Merge Green hard berries Green soft berries ROS sensor
Weeks post flowering
7 8 8.5 9 10 11 12 6 catalase 0 1 2 3 4 5 6 mmol H2O2/ min/ mg total protein H2O2 consumption O2 production 7 8 9 10 11 12 13
Weeks post flowering
0 2 4 6 8 10 12 nmol O2/min/mg total protein
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*
6 7 8 8.5 9 10 11 12Weeks post flowering
Galactolipid per oxidation (%) 0 2 4 6 8 MGDG mono−ox DGDG mono−ox MGDG di−ox
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**
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90 70 Weeks post flowering7 8 8.5 9 10 11 12 100 MW markers 50
Chl
YFP
Merge
leader
LOX-YFP leaderPLAT
LOX-YFP
*
*
*
*
*
*
*
*
MGDG 34:6 diOx MGDG 34:6 Ox DGDG 36:6 diOx DGDG 36:6 Ox MGDG 36:6 diOx MGDG 36:6 Ox MGDG 34:3 Ox DGDG 34:3 Ox Galactolipid per oxidation (%) 0 1 2 3 4 5 6 7 Control (pGreen) 35S−PnLOXA Leaf .Y Leaf .FS P etal Inflorescence .W D Inflorescence .Y Car pel Flo w er .F Flo w er .FB Bud.L Bud.B Bud.AB Seedling P ollen Stamen Stem.W Root Leaf .S Bud.W Seed.V Seed.MR Berr ySkin.PFS Tendr il.WD Berr yP er icar p.F S Tendr il.Y Rachis .FS Rachis .PFS Rachis .V Stem.G Tendr il.FS Berr yFlesh.PFSSeed.FS Bud.S Rachis
.R Rachis .MR Berr ySkin.MR Berr ySkin.V Berr yFlesh.MR Berr yFlesh.V Seed.PFS Berr yFlesh.R Berr ySkin.R Pn LOXA Vv01s0010g02750 Vv05s0020g03170 Vv09s0002g01080 Vv14s0128g00790 Vv06s0004g01500 Vv13s0064g01480 Vv06s0004g01470 Vv06s0004g01480 Vv06s0004g01450 Vv06s0004g01460 6 8 10 12 14 Value Color Key
Group:
A
B
C
D
9-Lox
13
-Lo
x
Figure 1A: Confocal images of Pinot Noir berries
(100-um sections) sampled at the green hard and green soft stages, stained for H2O2 and 1O
2. The sections were incubated with either 30 uM
DCFDA or 30 uM SOSG (ROS sensors). Chlorophyll fluorescence has been recorded (Chl) to localize chloroplasts inside the cells. Merge is the computed overlay of the two fluorescence images and the bright field. Reference bars are 75 um for all images.
Figure 1B: Zymogram of catalase activity during Pinot
Noir berry development.
Figure 1C: Catalase specific activity measured in vitro
by determining either H2O2 consumption (absorbance at 240 nm) or O2 production (in-line O2 recording using direct injection MS). n=2 ± se.
Hydrogen peroxide (H
2O
2) and singlet oxygen (
1O
2) accumulate
inside Pinot Noir skin cells at softening, in cytosol and chloroplasts.
1A
1B
1C
13-S Peroxidized membrane galactolipids accumulate
inside Pinot Noir skin cells at the onset of ripening.
Figure 2A: Overview of the characterization study
of galactolipids extracted from Pinot Noir berry skins. The chromatogram shows eluted peaks recorded at 210 nm, with retention time shown
on the x-axis. The mass spectra of the indicated peaks revealed that peaks 1 and 2 are attributable to
MGDG 36:6 and DGDG 36:6. Peaks 3 and 4 are
attributable to the corresponding mono-oxidized forms and peak 5 to the di-oxidized MGDG 36:6. Di-oxidized DGDG 36:6 has been identified but was nearly
detectable in the chromatogram.
Figure 2B: Galactolipid peroxidation profiles during
Pinot Noir berry development. The mono-oxidized and di-oxidized forms of MGDG 36:6 and
DGDG 36:6 are shown as percentage of total MGDG and DGDG, respectively. n=3 ± sd. Lipid peroxidation at pre-véraison (6 and 7 wpf),
véraison (8.5 and 9 wpf) and ripening (11 and
12 wpf) were analyzed by ANOVA and Tukey's HSD test. Asterisks indicate that the amount of peroxidized species accumulated at véraison is significantly
different from that of the other two moments (p<0.01).
2A
2B
A 13- lipoxygenase is expressed at the onset of ripening
in the skin of Pinot Noir berries.
PnLoxA is a chloroplastic lipoxygenase able to peroxidize
membrane galactolipids.
Is PnLoxA the homologue of Tomato Lipoxygenase C? Are they involved in fleshy fruits aroma production or in ripening regulation, or both?
* *
B. Skin 6 wpf B. Skin 7 wpf B. Skin 8 wpf B. Skin 8.5 wpf B. Skin 9 wpf B. Skin 10 wpf B. Skin 11 wpf B. Skin 12 wpf Pn LO XA gene expression (NRQ) 0 2 4 6 83A
3B
Figure 3A: Western blot analysis of lipoxygenase expression in Pinot Noir berry skin extracts using a
commercial antibody against Arabidopsis LOX2 and 10 ug of total protein extracts per lane. The band has been sequenced (nanoLC-MC) and identified as Vv06s0004g01510, named PnLoxA.
Figure 3B: RT-PCR analysis of PnLOXA gene expression in the berry skin along development.
Normalized relative quantities ± se were calculated using three reference genes; n=3. PnLOXA expression at véraison (marked by asterisks) was significantly different from pre-véraison (6-7 wpf) and ripening (11-12 wpf) as assessed by ANOVA and Tukey HSD test (p<0.01).
Figure 4A: PnLOXA localization demonstrated by the
expression of YFP fusion constructs in grapevine leaves. Leaves were infiltrated with Agrobacterium tumefaciens carrying the pGreen[PnLOXAtransitpeptide1-47-YFP] and pGreen[PnLOXAtransitpeptidePLAT1-220-YFP] constructs. Chlorophyll (Chl) and YFP fluorescence were recorded
using Leica SP II confocal microscope. Merge is the computed overlay of the two fluorescence images. Reference bar is 10 um.
Figure 4B: Galactolipid analysis of tobacco leaves
transiently expressing PnLOXA. Leaves transformed either with the PnLOXA or the empty vector (pGreen) as control were collected 7 days after Agrobacterium inoculation. Galactolipid peroxidation is reported as a percentage of mono- and di-oxidized species within each class, normalized on the amount of PnLOXA protein.
n=3 ± sd. Asterisks indicate significant differences from
control at p<-0.05 (ANOVA and Tukey HSD's test).
4A
4B
Figure 5A: Heatmap of the grapevine LOX gene family expression
in the V. vinifera cv Corvina atlas (Fasoli et al., 2012).
Figure 5B: Phylogenetic analysis of Vitis vinifera, Solanum
lycopersicum and Arabidopsis thaliana Lox families. Bayesian tree
using PhyloBayes3 and the LG+Gamma model. Support at nodes are posterior probabilities from the consensus of two independent, satisfactorily converged Bayesian analyses (maxdiff statistics lower than 0.3). The alignment was done using Muscle
(http://www.ebi.ac.uk/Tools/msa/muscle/help/).
