Insights into the effects of polygalacturonase FaPG1 gene silencing on pectin matrix disassembly, enhanced tissue integrity, and firmness in ripe strawberry fruits

Abstract

Antisense-mediated down-regulation of the fruit-specific polygalacturonase (PG) gene FaPG1 in strawberries (Fragaria×ananassa Duch.) has been previously demonstrated to reduce fruit softening and to extend post-harvest shelf life, despite the low PG activity detected in this fruit. The improved fruit traits were suggested to be attributable to a reduced cell wall disassembly due to FaPG1 silencing. This research provides empirical evidence that supports this assumption at the biochemical, cellular, and tissue levels. Cell wall modifications of two independent transgenic antisense lines that demonstrated a >90% reduction in FaPG1 transcript levels were analysed. Sequential extraction of cell wall fractions from control and ripe fruits exhibited a 42% decrease in pectin solubilization in transgenic fruits. A detailed chromatographic analysis of the gel filtration pectin profiles of the different cell wall fractions revealed a diminished depolymerization of the more tightly bound pectins in transgenic fruits, which were solubilized with both a chelating agent and sodium carbonate. The cell wall extracts from antisense FaPG1 fruits also displayed less severe in vitro swelling. A histological analysis revealed more extended cell-cell adhesion areas and an enhanced tissue integrity in transgenic ripe fruits. An immunohistological analysis of fruit sections using the JIM5 antibody against low methyl-esterified pectins demonstrated a higher labelling in transgenic fruit sections, whereas minor differences were observed with JIM7, an antibody that recognizes highly methyl-esterified pectins. These results support that the increased firmness of transgenic antisense FaPG1 strawberry fruits is predominantly due to a decrease in pectin solubilization and depolymerization that correlates with more tightly attached cell wall-bound pectins. This limited disassembly in the transgenic lines indicates that these pectin fractions could play a key role in tissue integrity maintenance that results in firmer ripe fruit.

Keywords: Cell wall; Fragaria×ananassa; fruit ripening; fruit softening; pectins; polygalacturonase..

Fig. 1.

Firmness of control and transgenic ripe fruits of the APG29 and APG62 lines was evaluated for three consecutive years. The data represent the means ±SD of 30 fruits per line. The bars with different letters within each year are significantly different by the Tukey-HSD test (P=0.05).

Fig. 2.

Amount of uronic acids (A) and neutral sugars (B) in the cell wall fractions extracted from ripe strawberry fruits. The data correspond to the mean ±SD from three independent extractions. The different letters within the same fraction indicate significant differences by the Tukey-HSD test (P=0.05).

Fig. 3.

Molecular mass profiles of soluble polyuronides extractable by PAW (A, B) and water (C, D) from fruit cell walls of control and two independent APG transgenic lines. The profiles were obtained by gel filtration chromatography on Sepharose CL6B. The fractions were assayed for uronic acid, and the pectin contents were estimated by the absorbance values at 515nm. The elution volumes for the different dextran standards and acetone used for column calibration are presented.

Fig. 4.

Molecular mass profiles of bound polyuronides extractable by CDTA (A, B) and carbonate (C, D) from fruit cell walls of control and two independent APG transgenic lines. The profiles were obtained by gel filtration chromatography on Sepharose CL2B. The fractions were assayed for uronic acid, and the pectin contents were estimated by the absorbance values at 515nm. The elution volumes for the different dextran standards and acetone used for column calibration are presented.

Fig. 5.

Molecular mass profiles of hemicellulosic polyuronides extractable by 1M KOH (A, B) and 4M KOH (C, D) from fruit cell walls of control and two independent APG transgenic lines. The profiles were obtained by gel filtration chromatography on Sepharose CL2B. The fractions were assayed for uronic acid, and the pectin contents were estimated by the absorbance values at 515nm. The elution volumes for the different dextran standards and acetone used for column calibration are presented.

Fig. 6.

PACE analysis of CDTA-soluble pectins that were isolated from control and transgenic ripe fruits. The pectin samples were digested with fungal endo-PG, derivatized with AMAC, and separated in polyacrylamide gels. Lane 1, undigested sample; lanes 2 and 3, samples digested with endo-PG for 30min and 60min, respectively. The same amount of pectin was loaded in each lane.

Fig. 7.

In vitro swelling of cell walls from control and transgenic APG lines, estimated as the height reached by the cell wall column after sequential extraction with water, CDTA, Na₂CO₃, and 1M KOH. The data represent the average of three replicates, and the bars represent the SD. Different letters indicate statistically significant differences by ANOVA and the Tukey-HSD test (P=0.05).

Fig. 8.

Light microscopy micrographs (A, B) and scanning electron micrographs (C, D) of control (A, C) and APG (B, D) strawberry fruit tissues. Tissue sections for light microscopy were stained with toluidine blue. Arrows indicate vascular bundles, and arrowheads indicate small, well-stained tri-cellular junctions. The large spaces without cells are indicated by asterisks. Scale bars correspond to 10 μm (A, B) or 200 μm (C, D). (This figure is available in colour at JXB online.)

Fig. 9.

Immunolabelling of pectic components in cell walls in ripe controls (A, D) and transgenic fruit lines APG29 (B, E) and APG62 (C, F). Tissue sections were incubated with JIM5 (A–C) or JIM7 (D–F). Scale bars correspond to 50 μm. (This figure is available in colour at JXB online.)