Fig 1.
Sketches (AC) and micrographs of longitudinal sections (B) and cross-sections (D-F) through mature ‘Nicoter’ apples. (A) Sketch of longitudinal section of the whole fruit. Grey box indicates detailed view of calyx region shown in micrograph in (B). Abbreviations are: cc, calyx cavity; co, cortex; pi, pith; sb, sepal bundle; pb, petal bundle; ip, point of inflection of vascular bundles; ca, cavity with surrounding browning of cortex. (C) Sketch of cross-section of fruit in calyx region and corresponding micrograph (D). Five sepal bundles (sb, red) and five petal bundles (pb, yellow) are arranged in two circles of differing radii with the sepal bundles being the outermost. Pith (pi) with small cells is separated from elongated cells (dotted line) of the ray parenchyma (rp). ca, cavity in symptomatic fruit; cc, calyx cavity as indicated by green circle. The asterisk in the center indicates the fruit axis. (EF) Cross-sections overview (E) and detail (F) of symptomatic ‘Nicoter’ apple having numerous cavities (ca) surrounded by brown tissue. cc, calyx cavity; co, cortex; pi, pith; rp, ray parenchyma; vb, vascular bundle. Scale bars in (A-C) 5 mm, in (D, E) 2 mm and in (F) 0.5 mm. Images were obtained from a minimum of ten symptomatic apples.
Table 1.
Frequency of ‘Nicoter’ apples affected by vascular browning at three different sites without and with application of promalin.
Table 2.
Effect of harvest date on the frequency of vascular browning in ‘Nicoter’ apples.
Fruit were harvested either at the optimum harvest date (‘optimum’) or six weeks later (‘Delayed by 6 weeks’). Vascular browning was assessed both on a whole-fruit basis and on an individual-segment basis where one segment corresponds to a tissue segment bounded by two adjacent vascular bundles.
Fig 2.
Light micrographs of longitudinal sections of ‘Nicoter’ (AB), ‘Gala’ (CD), ‘Braeburn’ (EF) apples in the calyx region during early (14 to 17 DAFB, days after full bloom; ACE) and late fruit development (122 to 153 DAFB; BDF). Sections were stained with toluidine blue and subsequently viewed in incident light. cc, calyx cavity; st, remains of the style; lo, locule with ovules; pi, pith; rp, ray parenchyma; co, cortex; ip, point of inflection of vascular bundles. Scale bars in (A-F) 1.0 mm. A minimum of eight fruit per cultivar was inspected.
Fig 3.
Frequency distribution of the tissue volumes with browning (A), of the volume of cavities, (B) and of the number of cavities (C) in mature symptomatic ‘Nicoter’ apples. The number of fruit inspected with tissue browning and/or cavities was 103 in (A) and (B), and 20 in (C). Inset in (B): Relationship between the volume of the brown tissue cavity and the volume of surrounding the cavity, volumes were cube root transformed (n = 103).
Fig 4.
Schematic drawings (AC) and frequency distributions of the angles of orientation of cavities (BD) in mature ‘Nicoter’ apples. (AB) Orientation of cavities (ca) relative to the calyx/pedicel axis (green dashed line, asterisk); n = 83. (CD) Orientation of cavities (ca) in cross-section relative to the position of a bundle leading to the sepals (outer ring, see Fig 1C) or petals (inner ring, see Fig 1C); n = 40.
Fig 5.
Schematic drawing (A) illustrating the position of sepal bundles (sb) and petal bundles (pb) in a cross-section in the calyx region of ‘Nicoter’ apples. Rsb, radii of sepal bundles; Rpb, radii of petal bundles; γ, angle between neighboring sepal and petal bundles; asterisk, calyx/pedicel axis. (BC) Normal probability plots of the angle between sepal bundles and petal bundles (B), of the radii of sepal bundles (C) and that of petal bundles (D) and of the difference in radii of neighboring sepal and petal bundles (dR; inset in D). Data were obtained from fruit without (n = 72) and with cavities (n = 108).
Fig 6.
Light (AB) and fluorescence (C-K) micrographs of sections of symptomatic ‘Nicoter’ apple fruit. Longitudinal section (A) and cross-section in the calyx region (B) showing cavities (ca) that formed by rupture (arrows) of cell walls. Sections were stained with toluidine blue and subsequently viewed in incident light. pi, pith; rp, ray parenchyma.
Table 3.
Rating of fluorescence staining in cross-sections of ‘Nicoter’ apples, without and with cavities.
Fruit were harvested when the frequency of symptomatic fruit averaged 54%, six weeks after the optimum harvest date. The tissue investigated was in the ray/cortex transition zone in a region without browning and without cavities. The antibodies against pectins were: LM7 (anti-homogalacturonan) [13], LM8 (anti-xylogalacturonan) [14], LM19 (anti-homogalacturonan) [15], LM20 (anti-homogalacturonan) [15] and 2F4 for dimeric associations of Ca with homogalacturonan [16]. Those reacting with hemicelluloses were: LM11 (anti-xylan/arabinoxylan) [17], LM21 (anti-mannan) [18] and LM25 (anti-xyloglucan) [19]. The GFP fluorescence was rated on a three-point scale: 0 no fluorescence, + some cell walls fluorescing, ++ all cell walls fluorescing; n = 3.
Table 4.
Rating of fluorescence staining of fracture surfaces of skin strips excised from the calyx region of ‘Nicoter’, ‘Gala’ and ‘Braeburn’ apples.
Fruit were harvested at the optimum harvest date and 6 weeks thereafter. The regions were: A = pith distal, B = pith proximal, C = cortex distal, D = cortex proximal; distal or proximal regions were on the level or below the calyx cavity, comprising the area above or below the inflexion point (ip) of the vascular bundle, respectively; the cortex included the ray parenchyma embedding the vascular bundles. The monoclonal antibodies (mAB) against pectins were: LM7 (anti-homogalacturonan) [13], LM8 (anti-xylogalacturonan) [14], LM19 (anti-homogalacturonan) [15], LM20 (anti-homogalacturonan) [15] and 2F4 for dimeric associations of Ca with homogalacturonan [16]. Those reacting with hemicelluloses were: LM11 (anti-xylan/arabinoxylan) [17], LM21 (anti-mannan) [18]; Marcus et al., 2010) and LM25 (anti-xyloglucan) [19]. The GFP fluorescence was rated on a three-point scale: 0 –no fluorescence, + some cell walls fluorescing, ++ all cell walls fluorescing; n = 3.
Fig 7.
Time course of change in mass, n = 49–50 (A), diameter, n = 10 (B), length, n = 10 (C) of ‘Nicoter’, ‘Gala’ and ‘Braeburn’ apples. (A-C) X axis scale in days after full bloom (DAFB). (D) Plot of log length vs. log diameter, n = 70 (‘Nicoter’, ‘Braeburn’), n = 60 (‘Gala’).
Fig 8.
Allometric relationships between different longitudinal measures in the calyx region of ‘Nicoter’ (n = 70), ‘Gala’ (n = 60) and ‘Braeburn’ apples (n = 70).
(A) Sketch of longitudinal section. D, maximum fruit diameter; La, distance between calyx lob and base of sepal; Lb, distance between base of sepal and point of inflection of vascular bundle (ip); Lc, distance between point of inflection of vascular bundle and distal end of locule (lo). (B-D) Allometric relationships between the logarithms of La and of D (B), between the logarithms of Lb and of D (C) and the logarithm of Lc and of D (D). For regression equations see Table 5.
Table 5.
Parameters of linear regression equations describing the allometric relationships in ‘Nicoter’, ‘Gala’ and ‘Braeburn’ apples between the logarithms of the distance between the calyx lobes and the base of the sepals (La) and the maximum fruit diameter, between the logarithms of the distance between the base of the sepals and the point of inflection of the vascular bundle (Lb) and the maximum fruit diameter, and between the logarithms of the distance between the point of inflection of the vascular bundles and the distal end of the locule (Lc) and the maximum fruit diameter.
The data were obtained between 17 and 163 days after full bloom (DAFB) in ‘Nicoter’ (n = 70), 14 and 136 DAFB in ‘Gala’ (n = 60), and 16 and 166 DAFB in ‘Braeburn’ (n = 70). For details see Fig 8.
Fig 9.
Allometric relationships between different radial measures in the calyx region of ‘Nicoter’ (n = 70), ‘Gala’ (n = 60) and ‘Braeburn’ apples (n = 70).
(A) Sketch of longitudinal section. D, maximum fruit diameter; Ld, distance between calyx-pedicel axis and surface of calyx cavity; Le, distance between surface of calyx cavity and point of inflection of vascular bundle (ip); Lf, distance between point of inflection of vascular bundles and fruit surface. (B-D) Allometric relationships between the logarithms of Ld and of D (B), between the logarithms of Le and of D (C) and the logarithms of Lf and of D (D). For regression equations see Table 6.
Table 6.
Parameters of regression equations describing allometric relationships in ‘Nicoter’, ‘Gala’ and ‘Braeburn’ apples between the logarithms of the distance between the calyx-stem axis and the surface of the calyx cavity (Ld) and the maximum fruit diameter, between the logarithms of the distance between the surface of the calyx cavity and the point of inflection of the vascular bundles (Le) and the maximum fruit diameter, and the logarithms of the distance between the point of inflection of the vascular bundle and the fruit surface (Lf) and the maximum fruit diameter.
Linear regression models were used throughout. The only exception was for Le in ‘Nicoter’, where a segmented linear regression model was used. The data were obtained between 17 and 163 days after full bloom (DAFB) in ‘Nicoter’ (n = 70), 14 and 136 DAFB in ‘Gala’ (n = 60) and 16 and 166 DAFB in ‘Braeburn’ (n = 70). For details see Fig 9.
Fig 10.
Sketch of cross-section of ‘Nicoter’ apples in the calyx region illustrating development of the vascular browning disorder.
(A) Growth of the pith (pi) displaces the sepal bundles (sb) and in particular the petal bundles (pb). (B) The outward movement of the vascular bundles is particularly large for the petal bundles and the ray parenchyma (rp). (C) The diverging bundles subject the ray parenchyma between the vascular bundles to tangential growth stress normal to the calyx/stem axis. The stress-induced strain results in rupture of the cortex (co) as indexed by tissue browning and cavity formation (ca). Arrows in (A-C) indicate directions of stress and strain.