Evidence for substantial maintenance of membrane integrity and cell viability in normally developing grape (Vitis vinifera L.) berries throughout development

Fluorescein diacetate (FDA) was used as a vital stain to assaymembrane integrity (cell viability) in mesocarp tissue of thedeveloping grape (Vitis vinifera L.) berry in order to testthe hypothesis that there is a substantial loss of compartmentationin these cells during ripening. This technique was also usedto determine whether loss of viability was associated with symptomsof a ripening disorder known as berry shrivel. FDA fluorescenceof berry cells was rapid, bright, and stable for over 1 h atroom temperature. Confocal microscopy detected FDA stainingthrough two to three intact surface cell layers (300–400µm) of bisected berries, and showed that the fluorescencewas confined to the cytoplasm, indicating the maintenance ofintegrity in both cytoplasmic as well as vacuolar membranes,and the presence of active cytoplasmic esterases. FDA clearlydiscriminated between living cells and freeze-killed cells,and exhibited little, if any, non-specific staining. Propidiumiodide and DAPI, both widely used to assess cell viability,were unable to discriminate between living and freeze-killedcells, and did not specifically stain the nuclei of dead cells.For normally developing berries under field conditions therewas no evidence of viability loss until about 40 d after veraison,and the majority (80%) of mesocarp cells remained viable pastcommercial harvest (26 °Brix). These results are inconsistentwith current models of grape berry development which hypothesizethat veraison is associated with a general loss of compartmentationin mesocarp cells. The observed viability loss was primarilyin the locule area around the seeds, suggesting that a localizedloss of viability and compartmentation may occur as part ofnormal fruit development. The cell viability of berry shrivel-affectedberries was similar to that of normally developing berries untilthe onset of visible symptoms (i.e. shrivelling), at which timeviability declined in visibly shrivelled berries. Berries withextensive shrivelling exhibited very low cell viability (15%). Key words: Apoplast, berry shrivel, compartmentation, DAPI, FDA, fluorescence, fruit ripening, locule, propidium iodide Received 19 September 2007; Revised 16 December 2007 Accepted 26 December 2007