DOI: 10.21950/jxdltn
title: Pear Stone cells

description: Regulated deficit irrigation (RDI) strategies aim to improve water usage without reducing yield. Generally, irrigation strategy effectiveness is measured as fruit yield, with little consideration of fruit quality. As water deficit and increased plant cell sclerification are often associated, this study explored the effect of RDI on pear fruit stone cells, a crucial trait affecting flesh texture. The presence, distribution, and development of pear fruit stone cells under RDI and full irrigation were compared using Pyrus communis L. cv. Barlett trees, employing recently developed microscope image analysis technology. The control treatment was maintained under non-stress conditions, while the RDI treatment received an average of 15% of the control water during the latter part of Stage I fruit development. Observations at the end of Stage I and at harvest revealed no effect on stone cell presence under the RDI strategy tested. The relative area of stone cells within the flesh was greater at Stage I than at harvest, as stone cell expansion occurred early in development, while the (unsclerified) parenchyma cells, a dominant component of the fruit flesh, expanded until harvest. Stone cell cluster density was higher near the fruit core than in the cortex center and exterior. These initial results suggest that well-planned RDI strategies will generally not affect pear fruit stone cell content and, thus, textural quality. Microscope image analysis supported the results from previously used analytical techniques, mainly chemical, while providing a tool for better understanding the process and factors involved in the timing of stone cell differentiation.
description: CellSens Imaging Software, V.5.10
description: Image J software, V.2.1.0
description: Metolodogía (empleada para la recogida o generación de los datos)

Fruits were collected at the end of Stage I and Stage II (at harvest). The Stage I harvest was at 60 DAFB (days after full bloom), corresponding to the end of Stage I and the beginning of rehydration via irrigation. The stage II harvest was at 120 DAFB, corresponding to the end of Stage II and commercial harvest time. At Stage I, 9 fruits/treatment (3 fruits per tree in 3 trees per treatment) were taken, while at Stage II, 4 fruits/treatment (2 fruits per tree in 2 trees per treatment) were sampled. A 5 mm transverse slice at the widest portion of each fruit was taken, and the slices were fixed in FAE (formalin: acetic acid: 60% ethanol, 2:1:17, v/v/v) and preserved in 70% ethanol. Standardized portions of the fruit cortex were identified by first staining the complete slices for 2–3 h in 70% ethanol with weak toluidine blue to show the different vascular bundles (associated with sepals, petals, and carpels) present in pome fruit. One (in Stage I) and two (in Stage II) wedge-shaped radial sectors extending from the sepal vascular bundle (SB) to the fruit exterior were cut per fruit slice. The sectors were dehydrated in tertiary butyl alcohol and processed according to standard paraffin procedures, obtaining microtome sections of 10–12 µm, which were stained with tannic acid, iron chloride, safranin, and fast green, adapted from Johansen and Ruzin.


For histological evaluation, the fruit sectors were divided at equal distances along the radius into three concentrically oriented zones: the area closest to the fruit exterior (zone Ex), the intermediate, or middle zone (zone Md), and the area closest to the fruit interior (zone In), as depicted in. Two square images per zone, each 0.06 cm2, were observed in each sector for Stage I, and six images per zone were obtained for Stage II. Microscopic images were acquired with an Olympus BX51 light microscope and recorded using a digital camera (Olympus SC50) attached to the microscope using CellSens Imaging Software (V.5.10, Olympus Corporation, Tokyo, Japan). Finally, 54 images per treatment were analyzed in Stage I and 144 in Stage II. All parameters were first determined for the three different fruit flesh zones, and then overall (globally) for the total area studied. The global values were calculated by considering the proportional area contribution of each zone.


Individual stone cells and stone cell clusters were observed. Stone cell clusters, referred to as SCCs (following the nomenclature of Nii et al.), were considered as such when the cluster contained more than three adjoining stone cells. Individual stone cells and those found in groups of two or three, in all cases separated from other stone cells by at least one parenchyma cell, were designated as isolated. Given the relatively small number of isolated stone cells (Figure 1D), the majority of the stone cell analyses only utilized the SCCs. A macro was developed for use with Image J software (V.2.1.0, National Institutes of Health, Bethesda, USA) to automatically determine the size of the SCCs, the number of SCCs and isolated stone cells, the area occupied by the SCCs, and the number of SCCs per area. In addition, the average stone cell size was calculated from the data. Data automatically determined via software were compared with data obtained via manual determination, achieving a high correlation (R2 = 0.9). 


The parenchyma cells were divided into two types: those with and without direct contact with stone cells, and a second Image J software (V.2.1.0, National Institutes of Health, Bethesda, USA) macro was established for measuring parenchyma cell size and circularity index. The internal size of each cell was identified and measured, and the average cell size was subsequently calculated. A circularity shape index ranging from 0 to 1 was used to assess the roundness of the cells, in which values closer to 1 indicate a more circular shape and values closer to 0 imply a more elliptical shape. To obtain this value, the largest and smallest diameters of each parenchyma cell were automatically measured, and the following equation was used