## 4 Discussion
Further evidence from the cryo-SEM micrographs revealed that storage circular cells at the adaxial surface experienced a considerable loss of cell integrity and turgidity (Figure 4E). Intact cell membranes were no longer distinguishable from the onion specimen and the effect of PEF under the investigated conditions on onion microstructure was even more severe than onion tissues processed with high pressure technology [23; 24]. Therefore, it can be suggested that the cell membrane and the tonoplast of onion cells within the bulb showed lesser resistance to PEF treatments, and hence, exhibited a rather significant PEF-induced cell disruption. Onion cells, depending on their cell size, cell type, and distribution of cells, have been found to exhibit different abilities to resist cell damage due to PEF [9; 15]. Therefore, cell viability staining tests have been successfully applied in other studies to visualize the uniform distribution of the PEF effect across all the onion scales within the bulb [9; 15]. An important finding from the cell viability test in this study was that the onion orientation with respect to the high voltage electrode at the time of PEF had an important role in causing uniform cell death (Figure 5). This was most possibly due to the fact that cells that were parallel to the applied electric field (onion bulb at \"FE\" position) were more prone to cell membrane breakdown [25]. Moreover, during the PEF treatment, onion bulbs with the root end facing the high voltage electrode may create less electrical resistance toward the incoming high voltage pulses and this onion orientation may confine the flow of electric voltage in one direction: i.e., entering from the root end, through the individual fleshy scales, and exiting at the foliar stem. As a result, onion cells within the bulb in the \"FE\" orientation had all areas across the scales that were found to be uniformly non-viable (Figure 5). In contrast, during the initiation of electric pulses, onion bulbs with the root end not facing the high voltage electrode (\"NFE\" orientation) may experience electrical resistance from the cuticle layer at each onion scale and the flow of electric charge can be easily dispersed in different directions within the onion bulb. It was not unexpected that terminal budswere unaffected by PEF, under any of the PEF conditions investigated, because the terminal bud has different cell types and arrangements compared to onion cells in the fleshy scale [26]. From the results of the present study, it is clear that PEF can causes major uniform cell damage and major structural changes across all onion cells within the bulb, provided the correct PEF conditions and sample orientation are chosen. Therefore, structural damage due to PEF may initiate enzymatic reactions, including those catalyzed by alliinase, within the whole and intact onion bulb. The breakdown of cell membranes and tonoplasts due to PEF, leading to the loss of cell integrity and absence of cell compartmentalization [10], most probably promoted the release of sulfur-containing precursors (ACSOs) and the alliinase enzyme from their localization in onion cells. As a result, this would facilitate the mixing of these compounds, resulting in the formation of the characteristic onion odor and flavor, and hence higher concentrations of sulfur-containing volatile compounds are expected to be detected. Onions have a complex volatile profile in which different classes of compounds can be detected either in the headspace or in the onions. In this study, PEF-treated onion bulbs were steeped in a phosphate buffer for a predefined duration following treatment in order to measure compounds that were generated within the onion bulb after PEF and released from the onion cells to the extracellular space. This method was selected to evaluate the changes occurring as a result of cellular disruption in the whole, intact onion bulb without the need for further destructive sample preparation, which could have generated more compounds as an artefact.