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Fruit yield and quality of strawberry plants transformed with a fruit specific strawberry pectate lyase gene
Scientia Horticulturae 119 (2009) 120–125
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Fruit yield and quality of strawberry plants transformed with a fruit specific strawberry pectate lyase gene Sabry M. Youssef a,1, Silvia Jime´nez-Bermu´dez a, M. Luz Bellido b, Carmen Martı´n-Pizarro a, Marta Barcelo´ c, Samia Abdallah Abdal-Aziz a,2, Jose´ L. Caballero b, Jose´ M. Lo´pez-Aranda c, ˜ oz b, Miguel A. Quesada a, Jose´ A. Mercado a,* Fernando Pliego-Alfaro a, Juan Mun a
Dept. Biologı´a Vegetal, Universidad de Ma´laga, Campus Teatinos s/n, 29071 Ma´laga, Spain Dept. Bioquı´mica y Biologı´a Molecular, Universidad de Co´rdoba, 14071 Co´rdoba, Spain c CIFA Churriana, Junta de Andalucı´a, Churriana, Ma´laga, Spain b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 May 2007 Received in revised form 9 July 2008 Accepted 11 July 2008
Two transgenic strawberry lines (Pel 1 and Pel 3) containing the open reading frame of a fruit specific strawberry pectate lyase gene (FaplC) under the control of the CaMV35S promoter have been obtained to evaluate the role of this gene on fruit softening. Ripen fruits from both lines showed a significant downregulation of FaplC, being the percentage of silencing of 84 and 71% on Pel 1 and Pel 3, respectively. The agronomic behaviour of transgenic plants was evaluated during two consecutive years. Fruit set increased in the two transgenic lines when compared with control plants, although Pel 1 showed a significant reduction on fruit weight. Firmness of full ripen fruits from Pel lines was significantly higher than control fruits, while color and soluble solids were not affected. The increase of firmness in Pel lines was maintained when ripe fruits were stored for 3 days at 25 8C. Histological analysis of ripe fruits showed lower intercellular spaces and a higher degree of cell to cell contact area in transgenic fruits when compared with controls. Altogether, these results suggest a direct relationship between pectate lyase gene expression and strawberry fruit softening. ß 2008 Elsevier B.V. All rights reserved.
Keywords: Fragaria ananassa Co-suppression Fruit texture Fruit firmness Cell wall Pectin metabolism
1. Introduction Strawberry (Fragaria ananassa Duch.) is one of the most valuable and worldwide cultivated small fruits. This fruit is considered as non-climacteric (Perkins-Veazie, 1995) and its ripening is characterized by a fast softening, acquiring a melting texture in few days. The rapid loss of firm texture constrains the postharvest shelf life of strawberry and also the harvest practices, and frequently, fruits are harvested before being fully mature to accommodate shipping practices, with the consequent reduction in quality (Pritts, 2002). Improvement of textural properties is therefore one of the main objectives of strawberry breeders (Faedi et al., 2002; Graham, 2005; Mercado et al., 2007). The underlying
* Corresponding author. Tel.: +34 952 137522; fax: +34 952 131944. E-mail address: [email protected] (J.A. Mercado). 1 Present address: Department of Horticulture, Faculty of Agriculture, Ain Shams University, Egypt. 2 Present address: Genetic Engineering and Biotechnology Research Institute (GEBRI),Mubarak City for Scientific Research and Technology Applications, BorgalArab City, Alexandria, Egypt. 0304-4238/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2008.07.011
mechanism of strawberry softening still remains unclear. As observed in other fruits, cell wall degradation seems to be the main factor responsible for strawberry softening (Huber, 1984; PerkinsVeazie, 1995). The largest changes that occur in the fruit cell wall are the degradation of the middle lamella of cortical parenchyma cells as well as a dramatic increase in pectin solubilization, although the total quantity and length of pectins is little affected (Woodward, 1972; Huber, 1984; Redgwell et al., 1997). Polygalacturonase (PG) and pectate lyase are among the different enzymes that could be involved in pectin degradation during fruit ripening. These enzymes catalyze the cleavage of unesterified galacturonosyl linkages by hydrolysis (PG) or b-elimination (pectate lyase) mechanisms. PG seems to play a minor role on strawberry ripening, because this activity is low and decreased continuously during fruit development (Nogata et al., 1993). However, a recent study on cultivars differing in fruit firmness showed a positive correlation between PG activity and fruit softening (Lefever et al., 2004). Contrary to the role of PG on strawberry softening, studies performed with transgenic strawberry plants indicate a crucial role of pectate lyase on pectins metabolism and therefore fruit softening (Jime´nez-Bermu´dez
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et al., 2002; Santiago-Dome´nech et al., 2008). We have demonstrated that the inhibition of this enzyme by insertion of an antisense sequence of a fruit specific pectate lyase gene (FaplC) reduces strawberry softening, extends postharvest shelf life and improves quality of processed fruits (Jime´nez-Bermu´dez et al., 2002; Sesmero et al., 2007; Santiago-Dome´nech et al., 2008). However, most of transgenic lines containing the antisense sequence showed a significant reduction in fruit yield, due to a reduction of fruit set and fruit weight. Pectate lyase genes in plants were first reported in pollen (Rogers et al., 1992; Turcich et al., 1993; Dircks et al., 1996), suggesting a function for pectate lyase on pollen tube emergence and/or breakdown of the cell wall of transmitting tissue in the style to facilitate its penetration (Marı´nRodrı´guez et al., 2002). Therefore, the deficient fruit yield of antipel plants could be related to an inhibition of pectate lyase activity in pollen and/or style. In the present work, we have evaluated the effect of transformation with a sense sequence of a fruit specific strawberry pectate lyase gene on strawberry fruit yield and softening. We have found that sense transformation do not affect fruit yield although ripen fruits showed a significant inhibition of pectate lyase gene expression and, additionally, a reduction of fruit softening. 2. Materials and methods 2.1. Plant material and Agrobacterium-mediated transformation Leaves of strawberry (Fragaria ananassa Duch.) plants, cv. Chandler, micropropagated in vitro as described in Barcelo´ et al. (1998) were used as explants for transformation experiments. The Agrobacterium tumefaciens LBA4404 strain carrying a binary plasmid with the strawberry pectate lyase gene (FaplC) in the sense orientation under the control of the 35S promoter was used for transformation. To obtain this construct, a 1.2-kb DNA fragment containing the FaplC gene coding sequence was isolated from the clone pNJJS25C (Medina-Escobar et al., 1997) after digestion with EcoRV and purification in a 1% agarose gel. This fragment was subcloned into a pGUSINT-derived plasmid (Vancanneyt et al., 1990) previously digested with SmaI and KpnI to remove the GUS gene. The resulting plasmid, pJPJ4, was checked for the presence of FaplC gene in sense orientation by restriction analysis. Leaf disks were inoculated with A. tumefaciens containing the pJPJ4 and selected in 25 mg l1 kanamycin, as previously described by Barcelo´ et al. (1998). After 7–8 months of selection, kanamycin resistant plants were acclimated, transferred to the screenhouse and propagated vegetatively by runners to evaluate its agronomical behaviour. Transgenic plants were evaluated during two consecutive growing seasons, using non-transformed plants as control. To study fruit quality during the first year of analysis, a transgenic line (Apel 39) containing an antisense sequence of the FaplC gene which showed a 100% FaplC silencing was also used (Jime´nez-Bermu´dez et al., 2002; Benı´tez-Burraco et al., 2003). Runners were potted individually in 1 l pots, and after 3 months of culture in the screenhouse, plants were transplanted to 25 l pots, three plants per pot, containing a mixture of peat moss and perlite (3:1). The experiments were carried out in a screenhouse from January to June, under natural temperature and photoperiod. Thirty plants per line and experiment were used. 2.2. Molecular analysis of transgenic plants Genomic DNA was extracted from young strawberry leaves from plants growing in the screenhouse as described by Mercado et al. (1999). The transgenic nature of plants was confirmed by PCR amplification of both a 220-bp fragment corresponding to the nptII
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gene and a 540-bp fragment corresponding to the chimaeric gene 35S-FaplC. The primers sequences used for these analyses were: 50 CGCAGGTTCTCCGGCCGCTTGGGTG-30 and 50 -AGCAGCCAGTCCTTCCGCTTCAG-30 for nptII; 50 -CTCCGGATTATCTACTGCGTGCTCA-30 and 50 -GTTCAAGATGCCTCTGCCGACA-30 for 35S-FaplC. These last primers amplify 233 bp from the 35S promoter and 307 bp from the FaplC gene in sense orientation. For Southern blotting, 5–10 mg of DNA were digested overnight with EcoRI, fractionated in a 0.8% agarose gel and transferred to a Hybond N+ membrane. The filter was hybridized at 64 8C with a digoxigenin labelled probe obtained by PCR amplification of the 35S-FaplC gene from pJPJ4 plasmid, using the primers above described. The EcoRI digestion of our plasmid yields a DNA fragment of about 2 kb which hybridizes with the probe. RNA was isolated from a pool of 5–6 red fruits according to Manning (1991). FaplC gene expression was quantified by QRT-PCR analysis, using the iCycler system from Bio-Rad, as previously described by Benı´tez-Burraco et al. (2003). The reverse transcription reaction contained 1 RT buffer, 10 mM DTT, 1 mM each dNTP, 0.2 mM specific FaplC primers, 1 mg of DNase-treated total RNA and 40 U of SuperscriptTM RT II. The sequences of FaplC primers were 50 -GCGAAAGAGGTGACACATAGA-30 and 50 TTCTGGAACTTGTATATTATG-30 . The reaction was heated at 70 8C for 5 min and cooled to room temperature. Afterwards, the reaction was incubated at 42 8C for 5 min, followed by 50 min at 50 8C and 15 min at 70 8C. Transcribed cDNA was subjected to PCR amplification in a reaction mixture containing 30 ml of: 1 PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.2 mM of each specific primers, 3 ml of SYBR Green I (1:15000 diluted), 3 ml of transcribed cDNA, and 2 U of Taq polymerase. The PCR program was: 94 8C for 2 min, followed by 35 cycles of 94 8C 1 min, 55 8C for 30 s and 72 8C for 30 s, and a final step of 72 8C for 5 min. In QRT-PCR analysis, quantification is based on threshold cycle (Ct) values. The Ct is a measurement taken during the exponential phase of amplification when limiting reagents and small differences in starting material have not yet influenced the PCR efficiency. Ct is defined as the cycle at which fluorescence is first detectable above background and it is inversely proportional to the log of the initial copy number. Each reaction was performed in triplicate and the Ct values of each QRTPCR reactions were normalized in relation to the Ct value corresponding to an interspacer 26S–18S strawberry RNA gene (housekeeping gene). The change of gene expression between the different lines was calculated as previously described (Benı´tezBurraco et al., 2003). 2.3. Phenotypic analysis of transgenic plants Ripe fruits were harvested from March to June and yield estimated as total number of fruits per plant and g of fruits per plant. Fruit quality was evaluated using only normal shape fruits of uniform size and coloration, and weight larger than 5 g. Color was estimated using the CTIFL (Centre Technique Interprofessionel des Fruits et Legumes, France) code. This code comprise eight categories, increasing the red color from 1 (light orange-red) to 8 (dark wine-red). Soluble solids were measured by using a refractometer Atago N1, and firmness by using a hand-penetrometer (Effegi) with 9.62 mm2 surface needle. Eighty to 250 fruits per line were used each year to evaluate fruit quality parameters, including fruit firmness. To analyze fruit firmness at different developmental stages, fruits were collected at the green, white 1 (white receptacle with green achenes), white 2 (white receptacle with brown achenes), pink (lower than 25% surface red), ripe (full surface red) and overripe (harvested ripe fruits maintained 3 days at 25 8C in a growth chamber). In this experiment, different needles were used
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to estimate firmness (0.78 mm2 for green fruits, 3.16 mm2 for white and pink fruits and 9.62 mm2 for mature fruits) in a sample of 25–50 fruits per stage and line. This experiment was conducted the second year of study. For histological study, blocks of tissue were fixed in Bouin reagent for 24 h, dehydrated in an ethanol series to 95% ethanol and polymerized with the Historesin Embedding kit (Leica) following the manufacturer’s instruction. Sections (4 mm thick) were cut from the polymerized samples and stained with 0.2 g/l ruthenium red for 2 h. 2.4. Statistical analysis Data were subjected to analysis of variance using the SPSS program (version 14). Levene’s test for homogeneity of variances was performed prior to ANOVA. When variances were homogeneous, the Tukey test was used to determine significant differences among means. In the case of non-homogeneous variances, the Tamhane T2 test was employed, according with the SPSS software manual. In both cases, mean separation was performed at P = 0.05. 3. Results Two independent kanamycin resistant lines, Pel 1 and Pel 3, originated from different leaf explants, were obtained after 30 weeks of culture, and were acclimated to ex vitro conditions. The presence of the T-DNA in both lines was confirmed by PCR amplification of a DNA fragment belonging to the nptII gene (Fig. 1A). Additionally, DNA from both lines were subjected to PCR amplification of the chimaeric gene 35S-FaplC to confirm the integration of the pectate lyase gene in sense orientation. Both lines showed a positive signal whereas no amplification was observed in control DNA (Fig. 1A). The digestion of pJPJ4 plasmid with EcoRI yielded a DNA fragment of about 2 kb which contain the FaplC gene. Genomic DNA from control and transgenic lines was digested with EcoRI, and analyzed by Southern blot using a FaplC sequence as probe (Fig. 1B). Several hybridization fragments could be detected in the three genotypes, corresponding to the endogenous pectate lyase genes, as previously observed by Medina-Escobar et al. (1997) in strawberry DNA digested with EcoRI. Both transgenics showed one additional signal of the expected 2 kb, which corresponds to the foreign FaplC gene inserted. This result indicates the stable integration of the transgene in Pel lines. The same filter hybridized with a in cursive probe showed a different band pattern for both transgenic lines and also none hybridization signal in control (result not shown). The effect of transformation on FaplC gene expression was studied in ripen fruits by QRT-PCR following the experimental conditions and specific primers described by Benı´tez-Burraco et al. (2003). These authors have previously shown that pectate lyase expression measured by QRTPCR correlated with conventional Northern blot results in both control and transgenic antisense pectate lyase fruits (Benı´tezBurraco et al., 2003). Contrary to the expected result, both Pel lines showed a significant reduction in FaplC mRNA levels when compared with control untransformed fruits, with a gene silencing of 84% in Pel 1 and 71% in Pel 3 (Fig. 1C). Results of fruit yield on control and transgenic plants obtained during the 2 years evaluation are summarized in Table 1. Both Pel lines showed a significant increment in the number of fruits per plant when compared with control. Fruit weight in Pel 3 line was similar to the control, and therefore, its fruit yield was significantly higher. Contrary to this result, fruit weight from Pel 1 was significantly reduced in comparison to the rest of the lines. The number of achenes per weight of fruit was slightly reduced in the
Fig. 1. (A) DNA amplification by PCR of a 220 bp fragment from the nptII gene and a 540 bp fragment from the chimaeric 35S-FaplC gene. P: plasmid; C: control nontransformed DNA; 1: DNA from Pel 1 line; 3: DNA from Pel 3 line. (B) Southern blot analysis of DNA from control and transgenic Pel lines. Arrowhead on the right indicates the inserted copy of FaplC gene. (C) Relative FaplC gene expression estimated by QRT-PCR in ripen fruits from control and transgenic Pel lines.
Pel 1 line, 11.7 3.3 vs 13.0 3.7 achenes/g fw, (mean S.D.), in Pel 1 and control fruits, respectively. In the second year evaluation, the fruit yield and the number of fruits per plant were slightly lower in all genotypes when compared with the first evaluation (Table 1). Fig. 2 shows the fruit weight distribution in the different lines analyzed. In control plants, more than 40% of fruits had a fresh weight lower than 5 g and only a small percentage of fruits showed a weight higher than 15 g. Pel lines showed a contrasting behaviour. In the case of Pel 1 more than 60% of fruits were included in the lowest class, fruit weight lower than 5 g, meanwhile in the Pel 3 line the most abundant class was that corresponding to fruits with a small-medium size, 5–10 g. In the first year analysis, control and transgenic fruits of normal shape, uniform coloration, and weight higher than 5 g were harvested at full ripeness to record several quality parameters (Table 2). A transgenic line, Apel 39, transformed with an antisense sequence of the FaplC gene that showed a 100% FaplC silencing was included in this analysis. Fruits from Pel 1 and Apel 39 showed a significant reduction in fresh weight. Color and soluble solids in Pel lines were similar to control, although fruits from Pel 3 showed a slightly lower value of fruit color. On the other hand, as previously observed by Jime´nez-Bermu´dez et al. (2002), Apel 39 fruits displayed a significant increment in soluble solids. Fruit firmness was measured with a hand penetrometer in these selected fruits.
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Table 1 Fruit yield of control and transgenic plants (Pel) transformed with a sense sequence of FaplC gene, during a 2 years evaluation First year evaluation
Control Pel 1 Pel 3
Second year evaluation
Yield (g/plant)
Fruit weight (g)
Fruits per plant
Yield (g/plant)
Fruit weight (g)
Fruits per plant
156.4 43.3 b 150.1 36.9 b 419.3 50.0 a
7.5 0.9 a 4.5 0.3 b 8.1 0.6 a
20.7 4.7 c 33.3 6.7 b 51.6 5.5 a
151.8 16.7 b 134.2 11.9 b 178.3 28.0 a
8.8 0.6 a 5.1 0.3 b 8.7 0.5 a
17.2 1.2 c 26.1 2.4 a 20.4 2.3 b
Thirty plants per genotype and year were employed. Mean separation was performed by Tukey test at P = 0.05.
Table 2 Fresh weight, color, soluble solids and firmness of full ripen selected fruits from control and transgenic pectate lyase plants
Control Apel 39 Pel 1 Pel 3
Fruit fresh weight (g)
Color
12.5 4.8 9.2 2.4 8.0 2.4 13.9 4.4
5.4 0.7 5.5 0.5 5.4 0.6 5.0 0.6
a b b a
a a a b
Soluble solids
Fruit firmness (g mm2)
8.5 1.6 9.4 1.4 8.6 1.3 8.2 1.5
61.14 11.3 81.8 12.6 75.7 11.3 72.0 16.0
b a b b
c a b b
Data correspond to the first year of analysis. Mean separation was performed by Tamhane T2 test at P = 0.05.
Fruits from the three transgenic pectate lyase lines were significantly firmer than those of control non-transformed plants, ranging the increments between 33% for Apel 39 and 18% for Pel 3. A positive correlation between pectate lyase gene inhibition and fruit firmness can be drawn from these results. Fruit firmness was also measured in the Pel lines during the second year evaluation and a similar behaviour could be observed. Fruits from both transgenic lines were significantly firmer than control, being fruits from Pel 1, which showed the highest pectate lyase suppression, the firmest followed by Pel 3 fruits (data not shown). Microscopy examinations of cortical cells from ripen nontransformed fruits showed most cells with little or no visible contact area between them (Fig. 3A). Furthermore, cell walls were poorly stained with ruthenium red, a pectin specific staining. By contrast, histological sections from ripen transgenic Pel fruits showed cells with an extensive area of cell to cell contact and a dense cell wall staining (Fig. 3B and C), indicating that these walls were enriched with pectin when compared with control fruits. Firmness was also measured in control and Pel fruits at different developmental stages, from small green to ripe, including a 3 days postharvest period (Fig. 4). No differences between control and transgenics were observed at the green and white stages. Firmness of transgenic fruits was slightly higher than control at the pink stage and significantly higher at ripe stage. In harvested fruits stored for 3 days at 25 8C, overripe fruits, the differences in firmness between control and transgenic diminished, but even at this stage, both Pel lines also showed higher firmness values than control. 4. Discussion
Fig. 2. Fruit weight distribution of control and transgenic pectate lyase plants (Pel), obtained during the first year assay. Bars with different letters within each fruit weight category indicate statistically significant differences by Tamhane T2 test at P = 0.05.
It has previously been demonstrated that pectate lyase genes play a crucial role in strawberry fruit ripening. Following transformation with an antisense sequence of FaplC, the same gene used in this research, Jime´nez-Bermu´dez et al. (2002) obtained transgenic fruits which showed a reduced softening and an extended postharvest shelf life. We have transformed the same cultivar with a sense sequence of FaplC, and interestingly, we have obtained fruits with a significant down-regulation of pectate lyase gene. This phenomenon, called co-suppression, is relatively frequent even in single transgene copy plants, provided that the sense transgene expression is driven by a strong promoter (Fagard and Vaucheret, 2000), as it is the case of CaMV35S promoter used in this work. A similar co-suppression effect on strawberry fruit
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Fig. 4. Firmness of transgenic pectate lyase fruits at different developmental stages. Firmness is expressed as percentage of control, non-transformed fruits. Data correspond to mean S.D. of a minimum of 25–50 fruits per line and stage.
Fig. 3. Cross-sections of cortical cells from full ripen fruits. (A) Control nontransformed fruits; (B) Pel 1 fruits; (C) Pel 3 fruits. Bars correspond to 50 mm.
was observed by Woolley et al. (2001) following transformation with a sense sequence of a endo-b-1,4-glucanase gene. Among the three reported strawberry pectate lyase genes (Benı´tez-Burraco et al., 2003), FaplC and FaplB shares a 98% of DNA sequence homology. Thus, the gene silencing obtained in Pel lines corresponds to the global expression of both genes. The level of FaplA gene expression, which has a 78% homology with the other two genes, has not been measured. Interestingly, the percentage of pectate lyase silencing in Pel fruits was lower than that obtained in most lines containing the antisense sequence (Jime´nez-Bermu´dez et al., 2002). Most transgenic lines containing FaplC gene in antisense orientation showed a significant reduction in fruit set, and even a 32% of the lines did not produce any fruit (Jime´nez-Bermu´dez et al., 2002). By contrast, both transgenic Pel lines obtained in this work did not show a reduction in the number of fruits per plant. Although the expression of FaplC gene in pollen has not been
reported (Medina-Escobar et al., 1997), it was hypothesized that the low fruit set in antipectate lyase plants could be related to a poor pollination due to a strong inhibition of pectate lyase genes expression in pollen (Jime´nez-Bermu´dez et al., 2002). As inhibition of FaplC expression by sense transgene was lower than that obtained through antisense transformation, it is likely that the pollination process might have not been altered in Pel lines. Mean fruit weight differed notably in the two pectate lyase transgenic lines. Fruits from Pel 3 line were similar in weight than those of control non-transformed plants, although the production of fruits with medium weight, 5–15 g, was increased in this transgenic line. Consequently, fruit yield increased significantly in this line when compared with controls plants. Contrary to Pel 3, fruits from Pel 1 line were smaller than controls and similar to those obtained in the Apel 39 line. Average fruit size and weight generally decreased when micropropagated strawberry plants are used directly for fruit production, but no reduction in fruit quality is detected when the progeny of micropropagated plants is evaluated (Cameron et al., 1989; Lo´pez-Aranda et al., 1994). Thus, fruit weight reduction in Pel 1 transgenic line should not be ascribed to an effect of in vitro micropropagation, since this reduction was observed in the two growing seasons, using plants obtained through runner propagation. In general, fruit size increase is due to a combination of cell division and cell expansion, and, in strawberry depends mainly on the number of achenes per fruit, flower position and receptacle sensitivity to auxin (PerkinsVeazie, 1995, and references herein). Fruits from Pel 1 line showed a slight reduction in the number of achenes per weight of fruit when compared with the rest of lines. However, histological analysis of cortical cells from transgenic pectate lyase ripe fruits showed no differences in cell area among control and transgenic lines, as observed in Fig. 3. Cheng and Breen (1992) demonstrated that the genotypic variation in the size of mature strawberry fruit was due to the number of receptacle cells established by anthesis. Thus, it can be possible that differences in size between lines could be due to a lower number of cells and/or slower cell division rate in those transgenic lines with higher FaplC gene down-regulation. Firmness of transgenic Pel ripe fruits was significantly higher than control, while no differences in other quality attributes such as color and soluble solids could be observed. The increase of firmness in Pel lines was lower than the one obtained in the Apel 39 line which showed almost a 100% of FaplC gene inhibition. The results of fruit firmness in Pel lines at different developmental stages obtained in this study are consistent with the expression
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pattern of the FaplC gene from the previous studies. The expression of this gene is low in green fruits, increases at the white stage and shows the maximum level at red stage (Medina-Escobar et al., 1997; Benı´tez-Burraco et al., 2003). Altogether, these results confirm that pectate lyase activity is the key factor in controlling strawberry fruit texture during ripening, and even more, firmness can be negatively correlated with FaplC gene expression. Regarding the molecular basis of this finding, cell wall analysis of antipectate lyase fruits showed a reduction of pectin solubilization, an increase of the pectin fraction covalently bound to cell wall, and a slight decrease of pectin depolymerization (Santiago-Dome´nech et al., 2008). In the case of Pel fruits, the study of cell wall is still lacking, but histological analysis of parenquima cells from red fruits showed a reduction of intercellular spaces, a higher degree of cell to cell adhesion, and a pectin enrichment of cell wall. Pectin solubilization, increase of intercellular spaces as results of middle lamella disintegration, and a reduction of cell to cell contact area, are main characteristics of strawberry softening (Redgwell et al., 1997). In conclusion, a significant inhibition of FaplC gene expression has been achieved by co-suppression. In spite of the fact that the inhibition is lower than that obtained with antisense transformation, ripe fruits also showed an increment in fruit firmness that was maintained after 3 days of postharvest. The use of this approach to manipulate pectate lyase gene expression does not have negative effects on yield, and lines with higher fruit set, similar fruit size, and increased fruit firmness can be obtained. Acknowledgements This investigation was funded by RTA01-122 and AGL 200508128 projects (INIA and Ministerio de Educacio´n y Ciencia of Spain, respectively, and FEDER EU Funds), and AGR-226 Research Group (Junta de Andalucı´a, Spain). Sabry M. Youssef was a graduate student from IAMZ, Zaragoza. S.M.Y. and Samia A. Abdal-Aziz also ˜ ola de Cooperacio´n Internacional (AECI) for thank Agencia Espan their fellowship. References Barcelo´, M., El-Mansouri, I., Mercado, J.A., Quesada, M.A., Pliego-Alfaro, F., 1998. Regeneration and transformation via Agrobacterium tumefaciens of the strawberry cultivar Chandler. Plant Cell Tiss. Org. Culture 54, 29–36. Benı´tez-Burraco, A., Blanco-Portales, R., Redondo-Nevado, J., Bellido, M.L., Moyano, ˜ oz-Blanco, J., 2003. Cloning and characterization of two E., Caballero, J.L., Mun ripening-related strawberry (Fragaria ananassa cv. Chandler) pectate lyase genes. J. Exp. Bot. 54, 633–645. Cameron, J.S., Hancock, J.F., Flore, J.A., 1989. The influence of micropropagation on yield components, dry matter partitioning and gas exchange characteristics of strawberry. Sci. Hort. 38, 61–67. Cheng, G.W., Breen, P.J., 1992. Cell count and size in relation to fruit size among strawberry cultivars. J. Am. Soc. Hort. Sci. 117, 946–950. Dircks, L.K., Vancanneyt, G., McCormick, S., 1996. Biochemical characterization and baculovirus expression of the pectate lyase-like LAT56 and LAT59 pollen proteins of tomato. Plant Physiol. Biochem. 34, 509–520.
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Fagard, M., Vaucheret, H., 2000. (Trans)Gene silencing in plants: how many mechanisms? Ann. Rev. Plant Biol. 51, 167–194. Faedi, W., Mourgues, F., Rosati, C., 2002. Strawberry breeding and varieties: situation and perspectives. Acta Hort. 567, 51–59. Graham, J., 2005. Fragaria strawberry. In: Litz (Eds.), Biotechnology in Agriculture Series, no. 29, Biotechnology of Fruit and Nut Crops. CABI Publishing, pp. 456– 474. Huber, D.J., 1984. Strawberry fruit softening: the potential roles of polyuronides and hemicelluloses. J. Food. Sci. 49, 1310–1315. ˜ oz-Blanco, J., Caballero, J.L., Lo´pezJime´nez-Bermu´dez, S., Redondo-Nevado, J., Mun Aranda, J.M., Valpuesta, V., Pliego-Alfaro, F., Quesada, M.A., Mercado, J.A., 2002. Manipulation of strawberry fruit softening by antisense expression of a pectate lyase gene. Plant Physiol. 128, 751–759. Lefever, G., Vieuille, M., Delage, N., D’Harlingue, A., de Monteclerc, J., Bompeix, G., 2004. Characterization of cell wall enzyme activities, pectin composition, and technological criteria of strawberry cultivars (Fragaria ananassa Duch). J. Food Sci. 69, 221–226. ˜ oz, M., 1994. Lo´pez-Aranda, J.M., Pliego-Alfaro, F., Lo´pez-Navidad, I., Barcelo´-Mun Micropropagation of strawberry (Fragaria ananassa Duch.). Effect of mineral salts, benzyladenine levels and number of subcultures on the in vitro and field behaviour of the obtained microplants and the fruiting capacity of their progeny. J. Hort. Sci. 69, 625–637. Manning, K., 1991. Isolation of nucleic acids from plants by differential solvent precipitation. Anal. Biochem. 195, 45–50. Marı´n-Rodrı´guez, M.C., Orchard, J., Seymour, G.B., 2002. Pectate lyases, cell wall degradation and fruit softening. J. Exp. Bot. 53, 2115–2119. ˜ oz-Blanco, J., 1997. Medina-Escobar, N., Ca´rdenas, J., Moyano, E., Caballero, J.L., Mun Cloning, molecular characterization and expression pattern of a strawberry ripening-specific cDNA with sequence homology to pectate lyase from higher plants. Plant Mol. Biol. 34, 867–877. Mercado, J.A., El Mansouri, I., Jime´nez-Bermu´dez, S., Pliego-Alfaro, F., Quesada, M.A., 1999. A convenient protocol for extraction and purification of DNA from Fragaria. In Vitro Cell Dev. Biol.—Plant 35, 152–153. Mercado, J.A., Pliego-Alfaro, F., Quesada, M.A., 2007. Strawberry. In: Pua, E.C., Davey, M.R. (Eds.), Biotechnology in Agriculture and Forestry, vol. 60, Transgenic Crops V. Springer-Verlag, Berlin, Heidelberg, pp. 309–328. Nogata, Y., Ohta, H., Voragen, A.G.J., 1993. Polygalacturonase in strawberry fruit. Phytochemistry 34, 617–620. Perkins-Veazie, P., 1995. Growth and ripening of strawberry fruit. Hort. Rev. 17, 267–297. Pritts, M., 2002. Growing strawberries, healthy communities, strong economies and clean environments: what is the role of the researcher? Acta Hort. 567, 411– 417. Redgwell, R.J., MacRae, E., Hallett, I., Fisher, M., Perry, J., Harker, R., 1997. In vivo and in vitro swelling of cell walls during fruit ripening. Planta 203, 162–173. Rogers, H.J., Harvey, A., Lonsdale, D.M., 1992. Isolation and characterization of a tobacco gene with homology to pectate lyase which is specifically expressed during microsporogenesis. Plant Mol. Biol. 20, 493–502. ˜ ozSantiago-Dome´nech, N., Jime´nez-Bermu´dez, S., Matas, A.J., Rose, J.K.C., Mun Blanco, J., Mercado, J.A., Quesada, M.A., 2008. Antisense inhibition of a pectate lyase gene supports a role for pectin depolymerization in strawberry fruit softening. J. Exp. Bot. 59, 2769–2779. Sesmero, R., Quesada, M.A., Mercado, J.A., 2007. Antisense inhibition of pectate lyase gene expression in strawberry fruit: characteristics of fruits processed into jam. J. Food Eng. 79, 194–199. Turcich, M.P., Hamilton, D.A., Mascarenhas, J.P., 1993. Isolation and characterization of pollen-specific maize genes with sequence homology to ragweed allergens and pectate lyases. Plant Mol. Biol. 23, 1061–1065. Vancanneyt, G., Schmidt, R., O’Connor-Sanchez, A., Willmitzer, L., Rocha-Sosa, M., 1990. Construction of an intron-containing marker gene: splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacteriummediated plant transformation. Mol. Gen. Genet. 220, 245–250. Woodward, J.R., 1972. Physical and chemical changes in developing strawberry fruits. J. Sci. Food Agric. 23, 465–473. Woolley, L.C., James, D.J., Manning, K., 2001. Purification and properties of an endob-1,4-glucanase from strawberry and down-regulation of the corresponding gene, cel1. Planta 214, 11–21.
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Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Fruit yield and quality of strawberry plants transformed with a fruit specific strawberry pectate lyase gene Sabry M. Youssef a,1, Silvia Jime´nez-Bermu´dez a, M. Luz Bellido b, Carmen Martı´n-Pizarro a, Marta Barcelo´ c, Samia Abdallah Abdal-Aziz a,2, Jose´ L. Caballero b, Jose´ M. Lo´pez-Aranda c, ˜ oz b, Miguel A. Quesada a, Jose´ A. Mercado a,* Fernando Pliego-Alfaro a, Juan Mun a
Dept. Biologı´a Vegetal, Universidad de Ma´laga, Campus Teatinos s/n, 29071 Ma´laga, Spain Dept. Bioquı´mica y Biologı´a Molecular, Universidad de Co´rdoba, 14071 Co´rdoba, Spain c CIFA Churriana, Junta de Andalucı´a, Churriana, Ma´laga, Spain b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 May 2007 Received in revised form 9 July 2008 Accepted 11 July 2008
Two transgenic strawberry lines (Pel 1 and Pel 3) containing the open reading frame of a fruit specific strawberry pectate lyase gene (FaplC) under the control of the CaMV35S promoter have been obtained to evaluate the role of this gene on fruit softening. Ripen fruits from both lines showed a significant downregulation of FaplC, being the percentage of silencing of 84 and 71% on Pel 1 and Pel 3, respectively. The agronomic behaviour of transgenic plants was evaluated during two consecutive years. Fruit set increased in the two transgenic lines when compared with control plants, although Pel 1 showed a significant reduction on fruit weight. Firmness of full ripen fruits from Pel lines was significantly higher than control fruits, while color and soluble solids were not affected. The increase of firmness in Pel lines was maintained when ripe fruits were stored for 3 days at 25 8C. Histological analysis of ripe fruits showed lower intercellular spaces and a higher degree of cell to cell contact area in transgenic fruits when compared with controls. Altogether, these results suggest a direct relationship between pectate lyase gene expression and strawberry fruit softening. ß 2008 Elsevier B.V. All rights reserved.
Keywords: Fragaria ananassa Co-suppression Fruit texture Fruit firmness Cell wall Pectin metabolism
1. Introduction Strawberry (Fragaria ananassa Duch.) is one of the most valuable and worldwide cultivated small fruits. This fruit is considered as non-climacteric (Perkins-Veazie, 1995) and its ripening is characterized by a fast softening, acquiring a melting texture in few days. The rapid loss of firm texture constrains the postharvest shelf life of strawberry and also the harvest practices, and frequently, fruits are harvested before being fully mature to accommodate shipping practices, with the consequent reduction in quality (Pritts, 2002). Improvement of textural properties is therefore one of the main objectives of strawberry breeders (Faedi et al., 2002; Graham, 2005; Mercado et al., 2007). The underlying
* Corresponding author. Tel.: +34 952 137522; fax: +34 952 131944. E-mail address: [email protected] (J.A. Mercado). 1 Present address: Department of Horticulture, Faculty of Agriculture, Ain Shams University, Egypt. 2 Present address: Genetic Engineering and Biotechnology Research Institute (GEBRI),Mubarak City for Scientific Research and Technology Applications, BorgalArab City, Alexandria, Egypt. 0304-4238/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2008.07.011
mechanism of strawberry softening still remains unclear. As observed in other fruits, cell wall degradation seems to be the main factor responsible for strawberry softening (Huber, 1984; PerkinsVeazie, 1995). The largest changes that occur in the fruit cell wall are the degradation of the middle lamella of cortical parenchyma cells as well as a dramatic increase in pectin solubilization, although the total quantity and length of pectins is little affected (Woodward, 1972; Huber, 1984; Redgwell et al., 1997). Polygalacturonase (PG) and pectate lyase are among the different enzymes that could be involved in pectin degradation during fruit ripening. These enzymes catalyze the cleavage of unesterified galacturonosyl linkages by hydrolysis (PG) or b-elimination (pectate lyase) mechanisms. PG seems to play a minor role on strawberry ripening, because this activity is low and decreased continuously during fruit development (Nogata et al., 1993). However, a recent study on cultivars differing in fruit firmness showed a positive correlation between PG activity and fruit softening (Lefever et al., 2004). Contrary to the role of PG on strawberry softening, studies performed with transgenic strawberry plants indicate a crucial role of pectate lyase on pectins metabolism and therefore fruit softening (Jime´nez-Bermu´dez
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et al., 2002; Santiago-Dome´nech et al., 2008). We have demonstrated that the inhibition of this enzyme by insertion of an antisense sequence of a fruit specific pectate lyase gene (FaplC) reduces strawberry softening, extends postharvest shelf life and improves quality of processed fruits (Jime´nez-Bermu´dez et al., 2002; Sesmero et al., 2007; Santiago-Dome´nech et al., 2008). However, most of transgenic lines containing the antisense sequence showed a significant reduction in fruit yield, due to a reduction of fruit set and fruit weight. Pectate lyase genes in plants were first reported in pollen (Rogers et al., 1992; Turcich et al., 1993; Dircks et al., 1996), suggesting a function for pectate lyase on pollen tube emergence and/or breakdown of the cell wall of transmitting tissue in the style to facilitate its penetration (Marı´nRodrı´guez et al., 2002). Therefore, the deficient fruit yield of antipel plants could be related to an inhibition of pectate lyase activity in pollen and/or style. In the present work, we have evaluated the effect of transformation with a sense sequence of a fruit specific strawberry pectate lyase gene on strawberry fruit yield and softening. We have found that sense transformation do not affect fruit yield although ripen fruits showed a significant inhibition of pectate lyase gene expression and, additionally, a reduction of fruit softening. 2. Materials and methods 2.1. Plant material and Agrobacterium-mediated transformation Leaves of strawberry (Fragaria ananassa Duch.) plants, cv. Chandler, micropropagated in vitro as described in Barcelo´ et al. (1998) were used as explants for transformation experiments. The Agrobacterium tumefaciens LBA4404 strain carrying a binary plasmid with the strawberry pectate lyase gene (FaplC) in the sense orientation under the control of the 35S promoter was used for transformation. To obtain this construct, a 1.2-kb DNA fragment containing the FaplC gene coding sequence was isolated from the clone pNJJS25C (Medina-Escobar et al., 1997) after digestion with EcoRV and purification in a 1% agarose gel. This fragment was subcloned into a pGUSINT-derived plasmid (Vancanneyt et al., 1990) previously digested with SmaI and KpnI to remove the GUS gene. The resulting plasmid, pJPJ4, was checked for the presence of FaplC gene in sense orientation by restriction analysis. Leaf disks were inoculated with A. tumefaciens containing the pJPJ4 and selected in 25 mg l1 kanamycin, as previously described by Barcelo´ et al. (1998). After 7–8 months of selection, kanamycin resistant plants were acclimated, transferred to the screenhouse and propagated vegetatively by runners to evaluate its agronomical behaviour. Transgenic plants were evaluated during two consecutive growing seasons, using non-transformed plants as control. To study fruit quality during the first year of analysis, a transgenic line (Apel 39) containing an antisense sequence of the FaplC gene which showed a 100% FaplC silencing was also used (Jime´nez-Bermu´dez et al., 2002; Benı´tez-Burraco et al., 2003). Runners were potted individually in 1 l pots, and after 3 months of culture in the screenhouse, plants were transplanted to 25 l pots, three plants per pot, containing a mixture of peat moss and perlite (3:1). The experiments were carried out in a screenhouse from January to June, under natural temperature and photoperiod. Thirty plants per line and experiment were used. 2.2. Molecular analysis of transgenic plants Genomic DNA was extracted from young strawberry leaves from plants growing in the screenhouse as described by Mercado et al. (1999). The transgenic nature of plants was confirmed by PCR amplification of both a 220-bp fragment corresponding to the nptII
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gene and a 540-bp fragment corresponding to the chimaeric gene 35S-FaplC. The primers sequences used for these analyses were: 50 CGCAGGTTCTCCGGCCGCTTGGGTG-30 and 50 -AGCAGCCAGTCCTTCCGCTTCAG-30 for nptII; 50 -CTCCGGATTATCTACTGCGTGCTCA-30 and 50 -GTTCAAGATGCCTCTGCCGACA-30 for 35S-FaplC. These last primers amplify 233 bp from the 35S promoter and 307 bp from the FaplC gene in sense orientation. For Southern blotting, 5–10 mg of DNA were digested overnight with EcoRI, fractionated in a 0.8% agarose gel and transferred to a Hybond N+ membrane. The filter was hybridized at 64 8C with a digoxigenin labelled probe obtained by PCR amplification of the 35S-FaplC gene from pJPJ4 plasmid, using the primers above described. The EcoRI digestion of our plasmid yields a DNA fragment of about 2 kb which hybridizes with the probe. RNA was isolated from a pool of 5–6 red fruits according to Manning (1991). FaplC gene expression was quantified by QRT-PCR analysis, using the iCycler system from Bio-Rad, as previously described by Benı´tez-Burraco et al. (2003). The reverse transcription reaction contained 1 RT buffer, 10 mM DTT, 1 mM each dNTP, 0.2 mM specific FaplC primers, 1 mg of DNase-treated total RNA and 40 U of SuperscriptTM RT II. The sequences of FaplC primers were 50 -GCGAAAGAGGTGACACATAGA-30 and 50 TTCTGGAACTTGTATATTATG-30 . The reaction was heated at 70 8C for 5 min and cooled to room temperature. Afterwards, the reaction was incubated at 42 8C for 5 min, followed by 50 min at 50 8C and 15 min at 70 8C. Transcribed cDNA was subjected to PCR amplification in a reaction mixture containing 30 ml of: 1 PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.2 mM of each specific primers, 3 ml of SYBR Green I (1:15000 diluted), 3 ml of transcribed cDNA, and 2 U of Taq polymerase. The PCR program was: 94 8C for 2 min, followed by 35 cycles of 94 8C 1 min, 55 8C for 30 s and 72 8C for 30 s, and a final step of 72 8C for 5 min. In QRT-PCR analysis, quantification is based on threshold cycle (Ct) values. The Ct is a measurement taken during the exponential phase of amplification when limiting reagents and small differences in starting material have not yet influenced the PCR efficiency. Ct is defined as the cycle at which fluorescence is first detectable above background and it is inversely proportional to the log of the initial copy number. Each reaction was performed in triplicate and the Ct values of each QRTPCR reactions were normalized in relation to the Ct value corresponding to an interspacer 26S–18S strawberry RNA gene (housekeeping gene). The change of gene expression between the different lines was calculated as previously described (Benı´tezBurraco et al., 2003). 2.3. Phenotypic analysis of transgenic plants Ripe fruits were harvested from March to June and yield estimated as total number of fruits per plant and g of fruits per plant. Fruit quality was evaluated using only normal shape fruits of uniform size and coloration, and weight larger than 5 g. Color was estimated using the CTIFL (Centre Technique Interprofessionel des Fruits et Legumes, France) code. This code comprise eight categories, increasing the red color from 1 (light orange-red) to 8 (dark wine-red). Soluble solids were measured by using a refractometer Atago N1, and firmness by using a hand-penetrometer (Effegi) with 9.62 mm2 surface needle. Eighty to 250 fruits per line were used each year to evaluate fruit quality parameters, including fruit firmness. To analyze fruit firmness at different developmental stages, fruits were collected at the green, white 1 (white receptacle with green achenes), white 2 (white receptacle with brown achenes), pink (lower than 25% surface red), ripe (full surface red) and overripe (harvested ripe fruits maintained 3 days at 25 8C in a growth chamber). In this experiment, different needles were used
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to estimate firmness (0.78 mm2 for green fruits, 3.16 mm2 for white and pink fruits and 9.62 mm2 for mature fruits) in a sample of 25–50 fruits per stage and line. This experiment was conducted the second year of study. For histological study, blocks of tissue were fixed in Bouin reagent for 24 h, dehydrated in an ethanol series to 95% ethanol and polymerized with the Historesin Embedding kit (Leica) following the manufacturer’s instruction. Sections (4 mm thick) were cut from the polymerized samples and stained with 0.2 g/l ruthenium red for 2 h. 2.4. Statistical analysis Data were subjected to analysis of variance using the SPSS program (version 14). Levene’s test for homogeneity of variances was performed prior to ANOVA. When variances were homogeneous, the Tukey test was used to determine significant differences among means. In the case of non-homogeneous variances, the Tamhane T2 test was employed, according with the SPSS software manual. In both cases, mean separation was performed at P = 0.05. 3. Results Two independent kanamycin resistant lines, Pel 1 and Pel 3, originated from different leaf explants, were obtained after 30 weeks of culture, and were acclimated to ex vitro conditions. The presence of the T-DNA in both lines was confirmed by PCR amplification of a DNA fragment belonging to the nptII gene (Fig. 1A). Additionally, DNA from both lines were subjected to PCR amplification of the chimaeric gene 35S-FaplC to confirm the integration of the pectate lyase gene in sense orientation. Both lines showed a positive signal whereas no amplification was observed in control DNA (Fig. 1A). The digestion of pJPJ4 plasmid with EcoRI yielded a DNA fragment of about 2 kb which contain the FaplC gene. Genomic DNA from control and transgenic lines was digested with EcoRI, and analyzed by Southern blot using a FaplC sequence as probe (Fig. 1B). Several hybridization fragments could be detected in the three genotypes, corresponding to the endogenous pectate lyase genes, as previously observed by Medina-Escobar et al. (1997) in strawberry DNA digested with EcoRI. Both transgenics showed one additional signal of the expected 2 kb, which corresponds to the foreign FaplC gene inserted. This result indicates the stable integration of the transgene in Pel lines. The same filter hybridized with a in cursive probe showed a different band pattern for both transgenic lines and also none hybridization signal in control (result not shown). The effect of transformation on FaplC gene expression was studied in ripen fruits by QRT-PCR following the experimental conditions and specific primers described by Benı´tez-Burraco et al. (2003). These authors have previously shown that pectate lyase expression measured by QRTPCR correlated with conventional Northern blot results in both control and transgenic antisense pectate lyase fruits (Benı´tezBurraco et al., 2003). Contrary to the expected result, both Pel lines showed a significant reduction in FaplC mRNA levels when compared with control untransformed fruits, with a gene silencing of 84% in Pel 1 and 71% in Pel 3 (Fig. 1C). Results of fruit yield on control and transgenic plants obtained during the 2 years evaluation are summarized in Table 1. Both Pel lines showed a significant increment in the number of fruits per plant when compared with control. Fruit weight in Pel 3 line was similar to the control, and therefore, its fruit yield was significantly higher. Contrary to this result, fruit weight from Pel 1 was significantly reduced in comparison to the rest of the lines. The number of achenes per weight of fruit was slightly reduced in the
Fig. 1. (A) DNA amplification by PCR of a 220 bp fragment from the nptII gene and a 540 bp fragment from the chimaeric 35S-FaplC gene. P: plasmid; C: control nontransformed DNA; 1: DNA from Pel 1 line; 3: DNA from Pel 3 line. (B) Southern blot analysis of DNA from control and transgenic Pel lines. Arrowhead on the right indicates the inserted copy of FaplC gene. (C) Relative FaplC gene expression estimated by QRT-PCR in ripen fruits from control and transgenic Pel lines.
Pel 1 line, 11.7 3.3 vs 13.0 3.7 achenes/g fw, (mean S.D.), in Pel 1 and control fruits, respectively. In the second year evaluation, the fruit yield and the number of fruits per plant were slightly lower in all genotypes when compared with the first evaluation (Table 1). Fig. 2 shows the fruit weight distribution in the different lines analyzed. In control plants, more than 40% of fruits had a fresh weight lower than 5 g and only a small percentage of fruits showed a weight higher than 15 g. Pel lines showed a contrasting behaviour. In the case of Pel 1 more than 60% of fruits were included in the lowest class, fruit weight lower than 5 g, meanwhile in the Pel 3 line the most abundant class was that corresponding to fruits with a small-medium size, 5–10 g. In the first year analysis, control and transgenic fruits of normal shape, uniform coloration, and weight higher than 5 g were harvested at full ripeness to record several quality parameters (Table 2). A transgenic line, Apel 39, transformed with an antisense sequence of the FaplC gene that showed a 100% FaplC silencing was included in this analysis. Fruits from Pel 1 and Apel 39 showed a significant reduction in fresh weight. Color and soluble solids in Pel lines were similar to control, although fruits from Pel 3 showed a slightly lower value of fruit color. On the other hand, as previously observed by Jime´nez-Bermu´dez et al. (2002), Apel 39 fruits displayed a significant increment in soluble solids. Fruit firmness was measured with a hand penetrometer in these selected fruits.
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Table 1 Fruit yield of control and transgenic plants (Pel) transformed with a sense sequence of FaplC gene, during a 2 years evaluation First year evaluation
Control Pel 1 Pel 3
Second year evaluation
Yield (g/plant)
Fruit weight (g)
Fruits per plant
Yield (g/plant)
Fruit weight (g)
Fruits per plant
156.4 43.3 b 150.1 36.9 b 419.3 50.0 a
7.5 0.9 a 4.5 0.3 b 8.1 0.6 a
20.7 4.7 c 33.3 6.7 b 51.6 5.5 a
151.8 16.7 b 134.2 11.9 b 178.3 28.0 a
8.8 0.6 a 5.1 0.3 b 8.7 0.5 a
17.2 1.2 c 26.1 2.4 a 20.4 2.3 b
Thirty plants per genotype and year were employed. Mean separation was performed by Tukey test at P = 0.05.
Table 2 Fresh weight, color, soluble solids and firmness of full ripen selected fruits from control and transgenic pectate lyase plants
Control Apel 39 Pel 1 Pel 3
Fruit fresh weight (g)
Color
12.5 4.8 9.2 2.4 8.0 2.4 13.9 4.4
5.4 0.7 5.5 0.5 5.4 0.6 5.0 0.6
a b b a
a a a b
Soluble solids
Fruit firmness (g mm2)
8.5 1.6 9.4 1.4 8.6 1.3 8.2 1.5
61.14 11.3 81.8 12.6 75.7 11.3 72.0 16.0
b a b b
c a b b
Data correspond to the first year of analysis. Mean separation was performed by Tamhane T2 test at P = 0.05.
Fruits from the three transgenic pectate lyase lines were significantly firmer than those of control non-transformed plants, ranging the increments between 33% for Apel 39 and 18% for Pel 3. A positive correlation between pectate lyase gene inhibition and fruit firmness can be drawn from these results. Fruit firmness was also measured in the Pel lines during the second year evaluation and a similar behaviour could be observed. Fruits from both transgenic lines were significantly firmer than control, being fruits from Pel 1, which showed the highest pectate lyase suppression, the firmest followed by Pel 3 fruits (data not shown). Microscopy examinations of cortical cells from ripen nontransformed fruits showed most cells with little or no visible contact area between them (Fig. 3A). Furthermore, cell walls were poorly stained with ruthenium red, a pectin specific staining. By contrast, histological sections from ripen transgenic Pel fruits showed cells with an extensive area of cell to cell contact and a dense cell wall staining (Fig. 3B and C), indicating that these walls were enriched with pectin when compared with control fruits. Firmness was also measured in control and Pel fruits at different developmental stages, from small green to ripe, including a 3 days postharvest period (Fig. 4). No differences between control and transgenics were observed at the green and white stages. Firmness of transgenic fruits was slightly higher than control at the pink stage and significantly higher at ripe stage. In harvested fruits stored for 3 days at 25 8C, overripe fruits, the differences in firmness between control and transgenic diminished, but even at this stage, both Pel lines also showed higher firmness values than control. 4. Discussion
Fig. 2. Fruit weight distribution of control and transgenic pectate lyase plants (Pel), obtained during the first year assay. Bars with different letters within each fruit weight category indicate statistically significant differences by Tamhane T2 test at P = 0.05.
It has previously been demonstrated that pectate lyase genes play a crucial role in strawberry fruit ripening. Following transformation with an antisense sequence of FaplC, the same gene used in this research, Jime´nez-Bermu´dez et al. (2002) obtained transgenic fruits which showed a reduced softening and an extended postharvest shelf life. We have transformed the same cultivar with a sense sequence of FaplC, and interestingly, we have obtained fruits with a significant down-regulation of pectate lyase gene. This phenomenon, called co-suppression, is relatively frequent even in single transgene copy plants, provided that the sense transgene expression is driven by a strong promoter (Fagard and Vaucheret, 2000), as it is the case of CaMV35S promoter used in this work. A similar co-suppression effect on strawberry fruit
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Fig. 4. Firmness of transgenic pectate lyase fruits at different developmental stages. Firmness is expressed as percentage of control, non-transformed fruits. Data correspond to mean S.D. of a minimum of 25–50 fruits per line and stage.
Fig. 3. Cross-sections of cortical cells from full ripen fruits. (A) Control nontransformed fruits; (B) Pel 1 fruits; (C) Pel 3 fruits. Bars correspond to 50 mm.
was observed by Woolley et al. (2001) following transformation with a sense sequence of a endo-b-1,4-glucanase gene. Among the three reported strawberry pectate lyase genes (Benı´tez-Burraco et al., 2003), FaplC and FaplB shares a 98% of DNA sequence homology. Thus, the gene silencing obtained in Pel lines corresponds to the global expression of both genes. The level of FaplA gene expression, which has a 78% homology with the other two genes, has not been measured. Interestingly, the percentage of pectate lyase silencing in Pel fruits was lower than that obtained in most lines containing the antisense sequence (Jime´nez-Bermu´dez et al., 2002). Most transgenic lines containing FaplC gene in antisense orientation showed a significant reduction in fruit set, and even a 32% of the lines did not produce any fruit (Jime´nez-Bermu´dez et al., 2002). By contrast, both transgenic Pel lines obtained in this work did not show a reduction in the number of fruits per plant. Although the expression of FaplC gene in pollen has not been
reported (Medina-Escobar et al., 1997), it was hypothesized that the low fruit set in antipectate lyase plants could be related to a poor pollination due to a strong inhibition of pectate lyase genes expression in pollen (Jime´nez-Bermu´dez et al., 2002). As inhibition of FaplC expression by sense transgene was lower than that obtained through antisense transformation, it is likely that the pollination process might have not been altered in Pel lines. Mean fruit weight differed notably in the two pectate lyase transgenic lines. Fruits from Pel 3 line were similar in weight than those of control non-transformed plants, although the production of fruits with medium weight, 5–15 g, was increased in this transgenic line. Consequently, fruit yield increased significantly in this line when compared with controls plants. Contrary to Pel 3, fruits from Pel 1 line were smaller than controls and similar to those obtained in the Apel 39 line. Average fruit size and weight generally decreased when micropropagated strawberry plants are used directly for fruit production, but no reduction in fruit quality is detected when the progeny of micropropagated plants is evaluated (Cameron et al., 1989; Lo´pez-Aranda et al., 1994). Thus, fruit weight reduction in Pel 1 transgenic line should not be ascribed to an effect of in vitro micropropagation, since this reduction was observed in the two growing seasons, using plants obtained through runner propagation. In general, fruit size increase is due to a combination of cell division and cell expansion, and, in strawberry depends mainly on the number of achenes per fruit, flower position and receptacle sensitivity to auxin (PerkinsVeazie, 1995, and references herein). Fruits from Pel 1 line showed a slight reduction in the number of achenes per weight of fruit when compared with the rest of lines. However, histological analysis of cortical cells from transgenic pectate lyase ripe fruits showed no differences in cell area among control and transgenic lines, as observed in Fig. 3. Cheng and Breen (1992) demonstrated that the genotypic variation in the size of mature strawberry fruit was due to the number of receptacle cells established by anthesis. Thus, it can be possible that differences in size between lines could be due to a lower number of cells and/or slower cell division rate in those transgenic lines with higher FaplC gene down-regulation. Firmness of transgenic Pel ripe fruits was significantly higher than control, while no differences in other quality attributes such as color and soluble solids could be observed. The increase of firmness in Pel lines was lower than the one obtained in the Apel 39 line which showed almost a 100% of FaplC gene inhibition. The results of fruit firmness in Pel lines at different developmental stages obtained in this study are consistent with the expression
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pattern of the FaplC gene from the previous studies. The expression of this gene is low in green fruits, increases at the white stage and shows the maximum level at red stage (Medina-Escobar et al., 1997; Benı´tez-Burraco et al., 2003). Altogether, these results confirm that pectate lyase activity is the key factor in controlling strawberry fruit texture during ripening, and even more, firmness can be negatively correlated with FaplC gene expression. Regarding the molecular basis of this finding, cell wall analysis of antipectate lyase fruits showed a reduction of pectin solubilization, an increase of the pectin fraction covalently bound to cell wall, and a slight decrease of pectin depolymerization (Santiago-Dome´nech et al., 2008). In the case of Pel fruits, the study of cell wall is still lacking, but histological analysis of parenquima cells from red fruits showed a reduction of intercellular spaces, a higher degree of cell to cell adhesion, and a pectin enrichment of cell wall. Pectin solubilization, increase of intercellular spaces as results of middle lamella disintegration, and a reduction of cell to cell contact area, are main characteristics of strawberry softening (Redgwell et al., 1997). In conclusion, a significant inhibition of FaplC gene expression has been achieved by co-suppression. In spite of the fact that the inhibition is lower than that obtained with antisense transformation, ripe fruits also showed an increment in fruit firmness that was maintained after 3 days of postharvest. The use of this approach to manipulate pectate lyase gene expression does not have negative effects on yield, and lines with higher fruit set, similar fruit size, and increased fruit firmness can be obtained. Acknowledgements This investigation was funded by RTA01-122 and AGL 200508128 projects (INIA and Ministerio de Educacio´n y Ciencia of Spain, respectively, and FEDER EU Funds), and AGR-226 Research Group (Junta de Andalucı´a, Spain). Sabry M. Youssef was a graduate student from IAMZ, Zaragoza. S.M.Y. and Samia A. Abdal-Aziz also ˜ ola de Cooperacio´n Internacional (AECI) for thank Agencia Espan their fellowship. References Barcelo´, M., El-Mansouri, I., Mercado, J.A., Quesada, M.A., Pliego-Alfaro, F., 1998. Regeneration and transformation via Agrobacterium tumefaciens of the strawberry cultivar Chandler. Plant Cell Tiss. Org. Culture 54, 29–36. Benı´tez-Burraco, A., Blanco-Portales, R., Redondo-Nevado, J., Bellido, M.L., Moyano, ˜ oz-Blanco, J., 2003. Cloning and characterization of two E., Caballero, J.L., Mun ripening-related strawberry (Fragaria ananassa cv. Chandler) pectate lyase genes. J. Exp. Bot. 54, 633–645. Cameron, J.S., Hancock, J.F., Flore, J.A., 1989. The influence of micropropagation on yield components, dry matter partitioning and gas exchange characteristics of strawberry. Sci. Hort. 38, 61–67. Cheng, G.W., Breen, P.J., 1992. Cell count and size in relation to fruit size among strawberry cultivars. J. Am. Soc. Hort. Sci. 117, 946–950. Dircks, L.K., Vancanneyt, G., McCormick, S., 1996. Biochemical characterization and baculovirus expression of the pectate lyase-like LAT56 and LAT59 pollen proteins of tomato. Plant Physiol. Biochem. 34, 509–520.
125
Fagard, M., Vaucheret, H., 2000. (Trans)Gene silencing in plants: how many mechanisms? Ann. Rev. Plant Biol. 51, 167–194. Faedi, W., Mourgues, F., Rosati, C., 2002. Strawberry breeding and varieties: situation and perspectives. Acta Hort. 567, 51–59. Graham, J., 2005. Fragaria strawberry. In: Litz (Eds.), Biotechnology in Agriculture Series, no. 29, Biotechnology of Fruit and Nut Crops. CABI Publishing, pp. 456– 474. Huber, D.J., 1984. Strawberry fruit softening: the potential roles of polyuronides and hemicelluloses. J. Food. Sci. 49, 1310–1315. ˜ oz-Blanco, J., Caballero, J.L., Lo´pezJime´nez-Bermu´dez, S., Redondo-Nevado, J., Mun Aranda, J.M., Valpuesta, V., Pliego-Alfaro, F., Quesada, M.A., Mercado, J.A., 2002. Manipulation of strawberry fruit softening by antisense expression of a pectate lyase gene. Plant Physiol. 128, 751–759. Lefever, G., Vieuille, M., Delage, N., D’Harlingue, A., de Monteclerc, J., Bompeix, G., 2004. Characterization of cell wall enzyme activities, pectin composition, and technological criteria of strawberry cultivars (Fragaria ananassa Duch). J. Food Sci. 69, 221–226. ˜ oz, M., 1994. Lo´pez-Aranda, J.M., Pliego-Alfaro, F., Lo´pez-Navidad, I., Barcelo´-Mun Micropropagation of strawberry (Fragaria ananassa Duch.). Effect of mineral salts, benzyladenine levels and number of subcultures on the in vitro and field behaviour of the obtained microplants and the fruiting capacity of their progeny. J. Hort. Sci. 69, 625–637. Manning, K., 1991. Isolation of nucleic acids from plants by differential solvent precipitation. Anal. Biochem. 195, 45–50. Marı´n-Rodrı´guez, M.C., Orchard, J., Seymour, G.B., 2002. Pectate lyases, cell wall degradation and fruit softening. J. Exp. Bot. 53, 2115–2119. ˜ oz-Blanco, J., 1997. Medina-Escobar, N., Ca´rdenas, J., Moyano, E., Caballero, J.L., Mun Cloning, molecular characterization and expression pattern of a strawberry ripening-specific cDNA with sequence homology to pectate lyase from higher plants. Plant Mol. Biol. 34, 867–877. Mercado, J.A., El Mansouri, I., Jime´nez-Bermu´dez, S., Pliego-Alfaro, F., Quesada, M.A., 1999. A convenient protocol for extraction and purification of DNA from Fragaria. In Vitro Cell Dev. Biol.—Plant 35, 152–153. Mercado, J.A., Pliego-Alfaro, F., Quesada, M.A., 2007. Strawberry. In: Pua, E.C., Davey, M.R. (Eds.), Biotechnology in Agriculture and Forestry, vol. 60, Transgenic Crops V. Springer-Verlag, Berlin, Heidelberg, pp. 309–328. Nogata, Y., Ohta, H., Voragen, A.G.J., 1993. Polygalacturonase in strawberry fruit. Phytochemistry 34, 617–620. Perkins-Veazie, P., 1995. Growth and ripening of strawberry fruit. Hort. Rev. 17, 267–297. Pritts, M., 2002. Growing strawberries, healthy communities, strong economies and clean environments: what is the role of the researcher? Acta Hort. 567, 411– 417. Redgwell, R.J., MacRae, E., Hallett, I., Fisher, M., Perry, J., Harker, R., 1997. In vivo and in vitro swelling of cell walls during fruit ripening. Planta 203, 162–173. Rogers, H.J., Harvey, A., Lonsdale, D.M., 1992. Isolation and characterization of a tobacco gene with homology to pectate lyase which is specifically expressed during microsporogenesis. Plant Mol. Biol. 20, 493–502. ˜ ozSantiago-Dome´nech, N., Jime´nez-Bermu´dez, S., Matas, A.J., Rose, J.K.C., Mun Blanco, J., Mercado, J.A., Quesada, M.A., 2008. Antisense inhibition of a pectate lyase gene supports a role for pectin depolymerization in strawberry fruit softening. J. Exp. Bot. 59, 2769–2779. Sesmero, R., Quesada, M.A., Mercado, J.A., 2007. Antisense inhibition of pectate lyase gene expression in strawberry fruit: characteristics of fruits processed into jam. J. Food Eng. 79, 194–199. Turcich, M.P., Hamilton, D.A., Mascarenhas, J.P., 1993. Isolation and characterization of pollen-specific maize genes with sequence homology to ragweed allergens and pectate lyases. Plant Mol. Biol. 23, 1061–1065. Vancanneyt, G., Schmidt, R., O’Connor-Sanchez, A., Willmitzer, L., Rocha-Sosa, M., 1990. Construction of an intron-containing marker gene: splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacteriummediated plant transformation. Mol. Gen. Genet. 220, 245–250. Woodward, J.R., 1972. Physical and chemical changes in developing strawberry fruits. J. Sci. Food Agric. 23, 465–473. Woolley, L.C., James, D.J., Manning, K., 2001. Purification and properties of an endob-1,4-glucanase from strawberry and down-regulation of the corresponding gene, cel1. Planta 214, 11–21.