Manipulation of strawberry fruit softening by antisense expression of a pectate lyase gene

Abstract

Strawberry (Fragaria x ananassa, Duch., cv Chandler) is a soft fruit with a short postharvest life, mainly due to a rapid lost of firm texture. To control the strawberry fruit softening, we obtained transgenic plants that incorporate an antisense sequence of a strawberry pectate lyase gene under the control of the 35S promoter. Forty-one independent transgenic lines (Apel lines) were obtained, propagated in the greenhouse for agronomical analysis, and compared with control plants, non-transformed plants, and transgenic lines transformed with the pGUSINT plasmid. Total yield was significantly reduced in 33 of the 41 Apel lines. At the stage of full ripen, no differences in color, size, shape, and weight were observed between Apel and control fruit. However, in most of the Apel lines, ripened fruits were significantly firmer than controls. Six Apel lines were selected for further analysis. In all these lines, the pectate lyase gene expression in ripened fruit was 30% lower than in control, being totally suppressed in three of them. Cell wall material isolated from ripened Apel fruit showed a lower degree of in vitro swelling and a lower amount of ionically bound pectins than control fruit. An analysis of firmness at three different stages of fruit development (green, white, and red) showed that the highest reduction of softening in Apel fruit occurred during the transition from the white to the red stage. The postharvest softening of Apel fruit was also diminished. Our results indicate that pectate lyase gene is an excellent candidate for biotechnological improvement of fruit softening in strawberry.

Figure 1

Plots of fruit weight with external and internal firmness in controls (non-transformed and GUS-transformed lines) and Apel transgenic lines.

Figure 2

Southern-blot analysis of DNA extracted from selected Apel clones. Membrane was hybridized with the ³²P-labeled 35S-antipel-nos chimeric gene (for details, see “Materials and Methods”).

Figure 3

The effect of antisense transformation in pectate lyase gene expression in fruit. A, Northern-blot analysis of RNA isolated from full ripe fruit of selected Apel clones. Membrane was hybridized with the ³²P-labeled pectate lyase gene (for details, see “Materials and Methods”). The same blot was hybridized with a ³²P-labeled 18S RNA probe as a control. B, Percentage of pectate lyase gene expression in full ripe fruit of selected Apel clones. C, Western blot of ripened fruit-soluble protein extracts prepared from Control and Apel lines 23 and 3, probed with a 1:100 dilution of antiserum raised to a strawberry pectate lyase protein. Each lane contained 100 μg of total fruit protein. In all cases, C represents a control, non-transformed fruit.

Figure 4

The effect of antisense down-regulation of a pectate lyase gene in the external and internal firmness of fruit at different stages of maturation (green, white, and full red). The values of firmness are expressed as percentage of control, non-transformed fruit. Values are means ± sd of a minimum of 20 fruit per clone.

Figure 5

Postharvest softening of controls (C and G1) and Apel transgenic fruit (A22, A23, and A39). Fruit were harvested at ripening and maintained for 4 d at 25°C. The percentage of soft fruit was calculated as the percentage of fruit with internal firmness lower than 15 g mm⁻². C corresponds to non-transformed control and G1 corresponds to the GUS1 line. A minimum of 20 fruit per line were analyzed. Bars represent the se.