The onset of grapevine berry ripening is characterized by ROS accumulation and lipoxygenase-mediated membrane peroxidation in the skin.
BACKGROUND
The ripening of fleshy fruits is a complex developmental program characterized by extensive transcriptomic and metabolic remodeling in the pericarp tissues (pulp and skin) making unripe green fruits soft, tasteful and colored. The onset of ripening is regulated by a plethora of endogenous signals tuned to external stimuli. In grapevine and tomato, which are classified as non-climacteric and climacteric species respectively, the accumulation of hydrogen peroxide (H2O2) and extensive modulation of reactive oxygen species (ROS) scavenging enzymes at the onset of ripening has been reported, suggesting that ROS could participate to the regulatory network of fruit development. In order to investigate this hypothesis, a comprehensive biochemical study of the oxidative events occurring at the beginning of ripening in Vitis vinifera cv. Pinot Noir has been undertaken.


RESULTS
ROS-specific staining allowed to visualize not only H2O2 but also singlet oxygen (1O2) in berry skin cells just before color change in distinct subcellular locations, i.e. cytosol and plastids. H2O2 peak in sample skins at véraison was confirmed by in vitro quantification and was supported by the concomitant increase of catalase activity. Membrane peroxidation was also observed by HPLC-MS on galactolipid species at véraison. Mono- and digalactosyl diacylglycerols were found peroxidized on one or both α-linolenic fatty acid chains, with a 13(S) absolute configuration implying the action of a specific enzyme. A lipoxygenase (PnLOXA), expressed at véraison and localizing inside the chloroplasts, was indeed able to catalyze membrane galactolipid peroxidation when overexpressed in tobacco leaves.


CONCLUSIONS
The present work demonstrates the controlled, harmless accumulation of specific ROS in distinct cellular compartments, i.e. cytosol and chloroplasts, at a definite developmental stage, the onset of grape berry ripening. These features strongly candidate ROS as cellular signals in fruit ripening and encourage further studies to identify downstream elements of this cascade. This paper also reports the transient galactolipid peroxidation carried out by a véraison-specific chloroplastic lipoxygenase. The function of peroxidized membranes, likely distinct from that of free fatty acids due to their structural role and tight interaction with photosynthesis protein complexes, has to be ascertained.
Research and Innovation Centre, Fondazione Edmund Mach, via E, Mach 1, 38010 San Michele a/Adige, TN, Italy. stefania.pilati@fmach.it.
Stefania
S
Pilati
Daniele
D
Brazzale
Graziano
G
Guella
Alberto
A
Milli
Cristina
C
Ruberti
Franco
F
Biasioli
Michela
M
Zottini
Claudio
C
Moser
D055162:Biocatalysis; D015153:Blotting, Western; D002374:Catalase / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism; D005638:Fruit / Q000201:enzymology / Q000235:genetics / Q000254:growth & development*; D038983:Galactolipids / Q000737:chemistry / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D006861:Hydrogen Peroxide / Q000378:metabolism; D006868:Hydrolysis; D015227:Lipid Peroxidation; D008084:Lipoxygenase / Q000378:metabolism; D013058:Mass Spectrometry; D018613:Microscopy, Confocal; D019441:Plant Epidermis / Q000201:enzymology / Q000378:metabolism*; D018515:Plant Leaves / Q000378:metabolism; D018087:Plastids / Q000201:enzymology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D026082:Singlet Oxygen / Q000378:metabolism; D014026:Nicotiana / Q000378:metabolism; D027843:Vitis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development*
D005227:Fatty Acids; D038983:Galactolipids; D017382:Reactive Oxygen Species; D011993:Recombinant Fusion Proteins; D026082:Singlet Oxygen; D006861:Hydrogen Peroxide; D002374:Catalase; D008084:Lipoxygenase