Antisense down-regulation of the FaPG1 gene reveals an unexpected central role for polygalacturonase in strawberry fruit softening

Abstract

The strawberry (Fragaria x ananassa 'Chandler') fruit undergoes a fast softening during ripening. Polygalacturonase (PG) activity is low during this process, but two ripening-related PG genes, FaPG1 and FaPG2, have been cloned. Both genes were up-regulated during fruit ripening and were also negatively regulated by auxin. To further assess the role of FaPG1 on strawberry softening, transgenic plants containing an antisense sequence of this gene under the control of the 35S promoter (APG lines) were obtained. Sixteen out of 30 independent transgenic lines showed fruit yields similar to those of the control. Several quality parameters were measured in ripe fruits from these 16 lines. Fruit weight was slightly reduced in four lines, and most of them showed an increase in soluble solid content. Half of these lines yielded fruits significantly firmer than did the control. Four APG lines were selected, their ripened fruits being on average 163% firmer than the control. The postharvest softening of APG fruits was also diminished. Ripened fruits from the four selected lines showed a 90% to 95% decrease in FaPG1 transcript abundance, whereas the level of FaPG2 was not significantly altered. Total PG activity was reduced in three of these lines when compared with control fruits. Cell wall extracts from APG fruits showed a reduction in pectin solubilization and an increase in pectins covalently bound to the cell wall. A comparative transcriptomic analysis of gene expression between the ripened receptacle of the control and those of the APG fruits (comprising 1,250 receptacle expressed sequence tags) did not show any statistically significant change. These results indicate that FaPG1 plays a central role in strawberry softening.

Figure 1.

Expression of FaPG1 and FaPG2 genes, in relative units, and evolution of fruit firmness during fruit development. Bars represent means ± sd of three independent RNA quantifications.

Figure 2.

Effect of auxin on FaPG gene expression, expressed as relative units. –Ach, Deachened fruits; –Ach + NAA, deachened fruits treated with lanolin containing 1% NAA. Bars represent means ± sd of three independent RNA quantifications.

Figure 3.

Fruit firmness at different stages of maturation in two transgenic APG lines. Firmness was evaluated during the second year of analysis and is expressed as the percentage of control fruit firmness at each developmental stage. G, Green fruit; W-1, white fruit with green achenes; W-2, white fruit with brown achenes; T, turning fruit; R, full red fruit. Bars represent means ± se of a minimum of 20 fruits per line and stage.

Figure 4.

Firmness of control and transgenic ripe APG fruits at harvest and after storage for 4 d at 5°C plus 2 d at 25°C. Firmness was measured with a TAXT-Plus texturometer using an Ottawa cell. Bars with different letters indicate statistically significant differences by T2-Tamhane in the case of fruits at harvest or by lsd in postharvest fruits, both at P = 0.05. Uppercase letters represent mean separation at harvest, and lowercase letters represent mean separation at postharvest.

Figure 5.

Southern-blot analysis of DNA isolated from selected APG lines. Filter was hybridized with a 2.3-kb probe, including part of the 35S promoter and the nptII gene. M, Molecular mass marker.

Figure 6.

Expression of FaPG genes in ripened fruits from transgenic APG lines showing the “firmer fruit” phenotype (APG2, -5, -29, and -62) and those not showing the phenotype (APG1, -14, -25, and -27). The level of gene expression was evaluated by QRT-PCR; it is expressed as the percentage of gene expression in control nontransformed fruits. Bars represent means ± sd of three independent quantifications.

Figure 7.

Total PG activity in ripened fruits from control and selected APG lines. Data correspond to three independent extractions per line. Bars with different letters indicate statistically significant differences by lsd at P = 0.05.

Figure 8.

Cell wall analysis of ripe fruits from control and transgenic APG lines APG29 and APG62. A, Yield of the different fractions obtained after sequential extraction of the CWM. B, Uronic acid content, expressed as mg of GalUA per 100 g of fruit, in the different cell wall fractions. Bars represent means ± sd of five independent fractionations. Within each fraction, bars with different letters indicate statistically significant differences by lsd at P = 0.05.