Cell wall structures leading to cultivar differences in softening rates develop early during apple (Malus x domestica) fruit growth

Abstract

Background: There is a paucity of information regarding development of fruit tissue microstructure and changes in the cell walls during fruit growth, and how these developmental processes differ between cultivars with contrasting softening behaviour. In this study we compare two apple cultivars that show different softening rates during fruit development and ripening. We investigate whether these different softening behaviours manifest themselves late during ethylene-induced softening in the ripening phase, or early during fruit expansion and maturation.

Results: 'Scifresh' (slow softening) and 'Royal Gala' (rapid softening) apples show differences in cortical microstructure and cell adhesion as early as the cell expansion phase. 'Scifresh' apples showed reduced loss of firmness and greater dry matter accumulation compared with 'Royal Gala' during early fruit development, suggesting differences in resource allocation that influence tissue structural properties. Tricellular junctions in 'Scifresh' were rich in highly-esterified pectin, contributing to stronger cell adhesion and an increased resistance to the development of large airspaces during cell expansion. Consequently, mature fruit of 'Scifresh' showed larger, more angular shaped cells than 'Royal Gala', with less airspaces and denser tissue. Stronger cell adhesion in ripe 'Scifresh' resulted in tissue fracture by cell rupture rather than by cell-to-cell-separation as seen in 'Royal Gala'. CDTA-soluble pectin differed in both cultivars during development, implicating its involvement in cell adhesion. Low pectin methylesterase activity during early stages of fruit development coupled with the lack of immuno-detectable PG was associated with increased cell adhesion in 'Scifresh'.

Conclusions: Our results indicate that cell wall structures leading to differences in softening rates of apple fruit develop early during fruit growth and well before the induction of the ripening process.

Figure 1

Physiological parameters during growth and maturation of ‘Royal Gala’ and ‘Scifresh’. Flesh firmness (A), fruit weight (B), dry matter concentration (C), internal ethylene production (D), and percent increase in fruit weight (E) during development of ‘Royal Gala’ and ‘Scifresh’ apples (n = 20 ± SE). Probe size for measuring firmness was 5 mm. The percentage increase in fruit weight (E) was calculated as the change in mean fruit weight relative to the weight at the start of each period per day.

Figure 2

Firmness loss and ethylene production of ‘Royal Gala’ and ‘Scifresh’ fruit during ripening. Flesh firmness (A) and internal ethylene production (B) during ripening at 0.5°C (n = 20 ± SE). Note: probe size for measuring firmness was 11 mm.

Figure 3

Size and density of cortical cells of ‘Royal Gala’ and ‘Scifresh’ at the fruitlet and mature stage. Cryo-scanning electron micrographs of fruitlet (A, C) and mature fruit (B, D) of ‘Royal Gala’ (A, B) and ‘Scifresh’ (C, D), and cortical density of cells at the mature fruit stage (E). Bars = 200 μm for all micrographs. Apples were matched for size at each stage of development. Values in (E) are the mean of 15 measurements ± SE.

Figure 4

Fracture pattern and tensile strength of cortical tissue of ‘Royal Gala’ and ‘Scifresh’ fruit during ripening. Scanning electron micrographs of ripe ‘Royal Gala’ (A, C) and ‘Scifresh’ fruit (B, D) (20 weeks at 0.5°C), showing a different fracture pattern between cells with the appearance of more intact cells in ‘Royal Gala’ and more broken open cells in ‘Scifresh’. (E) Tensile tests to quantify the force required to pull cortex tissue apart (expanding fruit 100 DAFB, mature and ripe fruit). Bars A, B = 500 μm; bars C, D = 100 μm. Values in (E) are the mean of 15 measurements ± SE.

Figure 5

Immunofluorescence labelling for lowly-esterified homogalacturonan during apple fruit development. LM19 antibody labelling (pink) and anti-cellulose stain calcofluor (blue) in ‘Royal Gala’ (A-D) and ‘Scifresh’ (E-H) apple cortex tissue. Fruitlet: 40 DAFB; Expanding fruit: 70 DAFB; Mature fruit: 120 DAFB (RG) 140 DAFB (SF); Ripe fruit: 20 weeks at 0.5°C. Bar in (A) = 10 μm for all micrographs. cr, corner of tricellular junction; ml, middle lamella; is intercellular space.

Figure 6

Immunofluorescence labelling for highly-esterified homogalacturonan during apple fruit development. LM20 antibody labelling (pink) and anti-cellulose stain calcofluor (blue) in ‘Royal Gala’ (A-D) and ‘Scifresh’ (E-H) apple cortex tissue. Fruitlet: 40 DAFB; Expanding fruit: 70 DAFB; Mature fruit: 120 DAFB (RG) 140 DAFB (SF); Ripe fruit: 20 weeks at 0.5°C. Bar in (A) = 10 μm for all micrographs. cr, corner of tricellular junction; tj, tricellular junction; is, intercellular space.

Figure 7

Immunofluorescence labelling for calcium-associated HG regions with antibody 2F4 in mature and ripe apple fruit. ‘Royal Gala’ (A, B) and ‘Scifresh’ (C, D) apple cortex tissue of mature fruit and ripe fruit. 2F4-labelling (green) was concentrated at tricellular junctions as indicated by the arrows. No labelling was detected in ripe ‘Royal Gala’ (B). Bar in (A) = 10 μm for all micrographs.

Figure 8

Size, yield and composition of CDTA-soluble pectin in ‘Royal Gala’ and ‘Scifresh’ fruit during development. Molecular weight distribution (A-D), yield and uronic acid (UA) content (E) of CDTA-soluble pectin in ‘Royal Gala’ (RG) and ‘Scifresh’ (SF). Fruitlet: 40 DAFB; Expanding fruit: 70 DAFB; Mature fruit: 120 DAFB (RG) 140 DAFB (SF); Ripe fruit: 20 weeks at 0.5°C. Yields in (E) are means of 3 extractions ± standard deviation; UA values are the mean of 3 CDTA extracts with duplicate assays ± standard deviation.

Figure 9

Pectin methylesterase (PME) activity and degree of methyl esterification of ‘Royal Gala’ and ‘Scifresh’ fruit cell walls during development. PME activity (A), degree of methyl esterification of cell wall material (B) and CDTA-soluble pectin (C). Fruitlet: 40 DAFB; Expanding fruit: 70 DAFB; Mature fruit: 120 DAFB (RG) 140 DAFB (SF); Ripe fruit: 20 weeks at 0.5°C. Values in (A) are the means of 3 assays ± standard deviation; values in (B) and (C) are the means of 6 assays ± standard deviation. Asterisks indicate significantly different values at a level of P ≤ 0.05 using a protected Fisher’s least significant difference test.

Figure 10

Western blot stained with a rabbit polyclonal antibody for polygalacturonase. Lane 1, Precision Plus Dual Protein Standard; lanes 2, 3, 4, ‘Royal Gala’ in the order of fruitlet, mature and ripe fruit; lanes 5, 6, 7, ‘Scifresh’ in the order of fruitlet, mature and ripe fruit. An arrow indicates the presence of PG protein at 45 kDa only detected in ripe ‘Royal Gala’ fruit.