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First published online June 7, 2007; 10.1104/pp.107.096347 Plant Physiology 144:1960-1967 (2007) © 2007 American Society of Plant Biologists Silencing of the Major Salt-Dependent Isoform of Pectinesterase in Tomato Alters Fruit Softening1School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, United Kingdom
Pectinesterase (PE; E.C. 3.1.1.11) is an enzyme responsible for the demethylation of galacturonyl residues in high-molecular-weight pectin and is believed to play an important role in cell wall metabolism. In this study, Pmeu1, a ubiquitously expressed PE gene, has been characterized by antisense suppression in tomato (Solanum lycopersicum). Transgenic tomato plants showed reduced PE activity levels in both green fruit and leaf tissue to around 65% and 25% of that found in wild-type plants, respectively. Pmeu1 was observed to encode a salt-dependent PE isoform that correlated with PE1 as previously described in fruit tissue. Silencing of Pmeu1 did not result in any detectable phenotype within the leaf tissue despite the gene product representing the major isoform in this tissue. In comparison, silencing in fruit resulted in an enhancement to the rate of softening during ripening. The role of PMEU1 in fruit ripening is discussed.
Pectinesterase (PE) is a ubiquitous cell wall-associated enzyme thought to be responsible for the demethylation of galacturonyl residues in high-Mr pectin. This enzyme has been reported in many plant tissues and has been implicated in many developmental processes, including cellular adhesion, stem elongation (Micheli, 2001  ), pollen tube development (Bosch and Hepler, 2005), abscission (Wang et al., 2005), and fruit ripening (Brummell and Harpster, 2001; Tucker, 2004). The action of this enzyme and the consequences in terms of tissue integrity during many of these processes, however, remains to be fully elucidated. It is believed that many plant PE isoforms remove methyl esters in a block-wise fashion (Limberg et al., 2000). Such demethylation could allow the aggregation of pectin, via calcium interchelation, into the so-called "egg-box" structures as described by Grant et al. (1973) and, as such, could act to strengthen the wall. Alternatively, this could also make the pectin more susceptible to degradation by cell wall polygalacturonase (Pressey and Avants, 1982; Jarvis, 1984; Seymour et al., 1987; Koch and Nevins, 1989; Carpita and Gibeaut, 1993), thus acting to weaken the wall. However, not all pectin isolated from plant tissues demonstrates this block-wise deesterification pattern; some has clearly been subject to random deesterification (Willats et al., 2001), and this suggests the presence of plant PE isoforms capable of acting in this manner and, as such, mimicking the fungal isoforms of this enzyme. The extent and pattern of demethylation of the pectin is thought to be at least partially responsible for determining the mechanical properties and porosity of the cell wall (Goldberg et al., 2001). Thus, charges generated on the pectin would be expected to increase the osmotic stress and, hence, tend to drive swelling of the wall, while cross-linking of the pectin would be expected to strengthen the wall and resist expansion. In addition, the generation of localized pH changes due to the action of PE could act to activate wall hydrolases such as polygalacturonase or pectin lyase (Nari et al., 1986). It is thus apparent that the action of PE could serve to either strengthen or weaken the cell wall dependent on its mode of action and on the environment in which it is acting. It is therefore likely that the many PE isoforms, often found associated with plant tissues, could serve very diverse functions during development.
In tomato (Solanum lycopersicum), PE protein and activity is present throughout fruit development and ripening and is also present in leaf and root tissues (Harriman et al., 1991
It is clear that there is a temporal separation in the expression of the fruit isoforms. The PE1 activity represents the predominant isoform in developing fruit, while PE2 activity appears in fruit at around 20 to 25 DPA, increases rapidly in activity, and represents the major isoform in ripe fruit (G.A. Tucker, unpublished data). It is also apparent that these isoforms have different kinetic properties (Warrilow et al., 1994
A separate PE gene, Pmeu1 (GenBank accession no. U49330), has been identified (Gaffe et al., 1997
Generation and Screening of Primary Pmeu1 Antisense Transformants Out of the 25 primary Pmeu1 antisense transformants generated, 16 showed the PCR amplicon characteristic for the presence of the transgene (data not shown), and eight of these were selected at random for further analysis. These primary transformants were designated as p1 to p8. Mature leaf tissue was collected from primary transformants p1 to p8, total RNA was extracted, and a northern analysis was carried out to monitor the expression level of the endogenous Pmeu1 mRNA (Fig. 1 ). Plants 1, 2, 6, 7, and 8 all showed very low or no expression of Pmeu1 mRNA compared to the control, while plants 3, 4, and 5 showed expression.
Protein was also extracted from the mature leaves of wild type and primary transformants p1 to p8 and assayed for total PE activity (Fig. 2 ). It was observed that all eight primary transformants showed reduced levels of PE activity compared to the wild-type control but that this was most pronounced in plants 1, 2, 7, and 8.
Three primary transformants, p1, p7, and p8, which exhibited reductions in both Pmeu1 mRNA expression and PE activity in their leaf tissue, were selected for the production of tomato lines homozygous for the Pmeu1 antisense transgene.
Fruit from the T2 population were harvested at defined stages during development (15, 30, and 45 DPA) corresponding to expanding, fully expanded, and mature green fruit, respectively. Fruit were also harvested at defined stages during ripening: breaker, 5 d post breaker (B + 5), and 10 d post breaker (B + 10). Total RNA was extracted, and the expression of Pmeu1 in wild-type and transgenic fruit was determined by northern blotting. Figure 3 shows the results obtained from line 8; similar results were obtained for the other two lines. In wild-type fruit, the Pmeu1 transcript expression was highest in immature fruit 15 and 30 DPA. At this stage of development, expression in the fruit appears similar to that in mature leaf tissue. Expression in wild-type fruit decreased as the fruit matured and ripened, being practically undetectable from B + 5 onwards. In contrast, there was no detectable Pmeu1 transcript detectable at any stage of development or ripening in the transgenic fruit lines. Expression of Pmeu1 was also undetectable in mature leaf tissue from the transgenic lines, confirming inheritance of this trait through the generations.
Effect on PE Activity and Isoform Profiles in Fruit and Leaf Tissue Protein was extracted from the pericarp tissue of mature green fruit (45 DPA) harvested from the wild type and transgenic T2 population, and total PE activity was assayed (Fig. 4 ). The PE activity was found to be reduced compared to the wild-type control in all three transgenic lines tested, the level in each case being in the order of 60% to 70% of that extractable from wild-type mature green fruit. Total PE was also extracted and assayed from mature leaves of these three transgenic lines. The PE activity extractable in each case was equivalent to that detected in the corresponding primary transformant (Fig. 2). The effect of the antisense transformation on total PE activity was thus much more pronounced in leaf than in mature green fruit tissue. Leaf tissue from the T2 populations exhibited a 70% to 80% reduction in PE activity compared to wild type, whereas in mature green fruit, the silencing of Pmeu1 resulted in only a 30% to 40% reduction in PE activity.
The antisense Pmeu1 line 8 was chosen for analysis of the effect of the transformation on PE isoform profiles. Total PE was extracted from the leaf and mature green fruit of wild type and the T2 population of p8 transgenic line plants. The PE isoform profiles were then determined by heparin column chromatography (Fig. 5 ). It can be seen that the PE activity from wild-type fruit (Fig. 5A) resolved into three isoforms eluting between fractions 29 and 41, 44 and 53, and 60 and 75, respectively. Using the nomenclature of Tucker et al. (1982) , these correspond to PE2, PE3, and PE1, respectively. In contrast, the profile obtained for the mature green fruit from the transgenic line showed only two peaks corresponding to PE2 and PE3. The isoform peak corresponding to PE1 was completely missing. The PE isoform profile obtained from wild-type leaf tissue (Fig. 5B) shows two peaks of activity eluting between fractions 26 and 45 and 50 and 73, respectively. The profile from the leaves of transgenic plants also showed two peaks of activity, but it could be seen that in this instance their ratio had changed considerably, with the second peak, i.e. that eluting between fractions 53 and 73, being much reduced in activity.
Salt Dependency of the PMEU1-Associated Enzyme Activity
Given that the PE1 in tomato fruit has been shown to be a salt-dependent isoform (Warrilow et al., 1994
Effect on Fruit Morphology and Softening The effect of silencing the Pmeu1 gene on fruit morphology was examined. Fruit size in the transgenic plants was not significantly different to that of wild-type fruit, having equatorial diameters of 39.2 ± 5.4 mm compared to 42.5 ± 6.3 mm for the control wild-type fruit and a height of 32.3 ± 4.1 mm compared to 33.3 ± 4.5 mm for the wild type. Fruit from the wild type and the transgenic line were also analyzed for softening during ripening (Fig. 7 ). The compressibility of mature green fruit was identical in both the wild type and transgenic line. In both cases, the fruit softened during ripening; however, it can be seen that the rate of softening of the transgenic fruit appeared to be faster than that of the wild-type controls. This was most evident at the B + 5 stage of ripening, and there was a significant difference in the rate of softening (P = 0.019) between wild-type and transgenic fruit between B + 3 and B + 8. Compressibility at the later stages of ripening was again identical in both the wild-type and transgenic fruit. This observation has been repeated over two growing seasons.
Expression of the Pmeu1 gene has been demonstrated to occur in both the leaf and developing fruit of wild-type tomato plants. This confirms the results of Gaffe et al. (1997) . We successfully down-regulated the expression of pmeu1, resulting in transgenic plants that possessed reduced PE activity in both the leaf and fruit tissue. The total PE activity in mature transgenic leaves was reduced to around 25% of that in wild-type plants. The PE activity in fruit was monitored at the mature green stage of development because this represents the stage of ripening where pmeu1 expression starts to decline in the wild-type fruit. The PE activity in mature green fruit was thus shown to be reduced to around 65% of that found in wild-type plants. This observation would suggest that, in these two tissues at least, the PE isoform representing the Pmeu1 gene product is a major contributor to the total extractable PE activity.
The PE activity in tomato fruit tissue has been separated into at least three isoforms (Tucker et al., 1982
Much less is known concerning the PE isoform profile of wild-type tomato leaves. It has been shown, by isoelectric focusing, that there may be as many as five PE isoforms in tomato leaf tissue (Gaffe et al., 1994
Isoforms of PE can be classified into two groups: type 1, which are salt dependent, or type 2, which are salt independent. The two main isoforms in tomato fruit, PE1 and PE2, are representatives of group 1 and group 2, respectively (Warrilow et al., 1994
It is apparent that Pmeu1 expression provides a significant contribution to total PE activity in leaf and, indeed, seems to represent the major isoform in this tissue; yet suppression appears to have had no obvious phenotypic effect. It is possible that there is some genetic and enzymatic redundancy for PE in plant tissues. This may account for the apparently large number of genes, at least within the Arabidopsis (Arabidopsis thaliana) genome, that appear to encode PE isoforms (Henrissat et al., 2001
While it would appear that PMEU1 represents the major isoform in mature leaf tissue in ripe tomato fruit, it is PE2 that predominates (Tucker et al., 1982
One function of PE activity within the plant cell wall may be to generate blocks of deesterified GalUA residues within the pectin polymer (Limberg et al., 2000 It is possible that this pattern of pectin deesterification is related to the presence of multiple PE isoforms, each with very specific modes of action, within the fruit tissue. The demonstration in this report that suppression of PMEU1 activity has resulted in transgenic plants in which fruit soften faster would suggest that the action of PMEU1 would be to strengthen the wall. Given that this PE isoform is synthesized during fruit development, it can be postulated that the product of PMEU1 action (i.e. the deesterified pectin) is involved in egg box-like structures, formed during fruit development, which resist softening during the subsequent ripening process.
Tomato Plants
The tomato (Solanum lycopersicum) plants used in this work were var. Craigella-Tm-2a (S.A. Bowes, Glasshouse Crops Research Institute). This is an introgression line showing increased resistance to tobacco mosaic virus infection. Details of this introgression have been described by Young et al. (1988)
A single antisense construct, pK2Gwpmeu1, was made for the generation of transgenic tomato plants. The cDNA for Pmeu1 was obtained by reverse transcription-PCR from tomato leaf RNA using the published coding sequence (Gaffe et al., 1997
The 1,268-bp fragment was cloned in an antisense orientation driven by the cauliflower mosaic virus 35S promoter. The final construct, pK2Gwpmeu1, was used for transformation of tomato tissue using Agrobacterium tumefaciens as described by Seymour et al. (1993) Primary Pmeu1 antisense transformants were analyzed for the presence of the transgene by PCR, utilizing a forward primer within the cauliflower mosaic virus 35S promoter (5'-GATATCTCCACTGACGTAAGG-3') and an internal, reverse primer within the Pmeu1 transgene (5'-ACGAGACACTCGACGAGCTCC-3'), to give a characteristic 800-bp amplicon. Seeds were collected from fruit of the primary transformants (T0) and surface sterilized by immersion in 10% bleach for 10 min and then 70% ethanol for another 10 min. They were then rinsed with sterile distilled water and sown in sterile pots containing MSR3 medium in the presence of 50 µg/mL kanamycin. The seeds were allowed to germinate in a controlled environment with 16 h light at 25°C for approximately 2 weeks. After this period, they were scored for their ability to germinate with the establishment of roots. Selected seedlings were then grown to produce a T1 population. Seeds were collected from fruit of the T1 population and germinated in the presence of kanamycin as described above. T1 plants whose fruit gave seed showing 100% germination were considered as being homozygous for the transgene, and their seed was used to generate a T2 population.
Leaf tissue (100 mg) was ground with liquid nitrogen to a fine powder using a pestle and mortar. The genomic DNA was subsequently extracted using GenElute Plant Genomic DNA Miniprep kit (Sigma) according to the manufacturers' instructions.
Tissue, 4 g of either tomato fruit pericarp or leaf, was frozen in liquid nitrogen and ground to a fine powder using a pestle and mortar. The powder was then transferred to a 50-mL Falcon tube, and 15 mL of extraction buffer (6% [w/v] 4-aminosalicyclic acid, 1% [w/v] TNS, 5% [v/v] phenol mixture [100 g of phenol, 14 mL of m-cresol, 0.1 g of 8-hydroxyquinoline, and 30 mL of distilled water] in 50 mM Tris-HCl, pH 8.3) was added. After vigorously vortexing the mixture, 1 volume phenol:chloroform (1:1 [v/v]) was added, vortexed, and then centrifuged at 3,000g for 15 min. The aqueous fraction was collected, and a second phenol:chloroform extraction was performed as above. The supernatant was carefully removed and placed in a Sorvall tube where 1 volume of isopropanol and 1/10 volume of 3 M NaOCOCH3 were added. The mixture was then vortexed and placed at –20°C for at least 1 h. The nucleic acid was then sedimented at 12,000g for 20 min and subsequently washed with 70% ethanol. Care was taken not to completely dry the pellet, otherwise great difficulties were encountered in attempting to redissolve it. The pellet was then resuspended in 0.7 mL of distilled water and transferred to a 1.5-mL Eppendorf tube. To precipitate the RNA, 1 volume of 8 M LiCl was added, the tube was vortexed, and then placed at –20°C for at least 1 h. The precipitated RNA was sedimented at 8,000g, washed with 70% ethanol, and allowed to dry on the bench. The total RNA was resuspended in distilled water.
Total RNA (10 µL containing 10 µg) was mixed with 10 µL of RNA loading buffer (10 mM EDTA, 40 mM NaH2PO4/Na2HPO4, pH 6.5, 200 µg/mL ethidium bromide, 16.5% [v/v] formaldehyde, and 50% [v/v] formamide). The samples were then incubated at 15°C for 15 min and quenched on ice for 5 min. Then 4 µL of bromphenol blue (0.02% [w/v]) was added and the samples loaded onto a 1.5% agarose gel containing running buffer (0.1 M NaH2PO4/Na2HPO4, pH 6.5, 8% [v/v] formaldehyde). The gel was run for 3 h at 100 V with recirculation of the buffer. The RNA was then transferred using 20 mM NaH2PO4/Na2HPO4, pH 6.5, onto a nylon membrane (Hybond N+; Amersham Bioscience) by capillary action overnight, fixed using a UV cross-linker (Stratalinker; Stratagene), and allowed to dry in air.
Complementary sequences for the probes were generated by PCR amplification; they were then run on a 0.7% TBE (10x TBE 0.89 M Tris-HCl, 0.028 M EDTA, 0.89 M boric acid) gel and gel purified. Radioactively labeled probes were generated using a Rediprime kit (Amersham Biosciences) according to the manufacturer's instructions, using 50 ng of denatured template DNA and 5 µL of [ Once the probe was prepared, 50 µL was added to the hybridization tube containing both the nylon membrane and the prehybridization buffer (5x sodium chloride/sodium phosphate/EDTA [SSPE; 20x SSPE, 3 M NaCl, 0.2 M NaH2PO4/Na2HPO4, 0.02 M EDTA, pH 7.4], 5x Denhardt's reagent [100x Denhardt's reagent, 2% ficoll 400, 2% polyvinylpyrrolidone, 2% bovine serum albumin], 1% SDS, 0.8 mL of 0.2 mg/mL salmon sperm DNA). The nylon membrane with the fixed RNA samples was rolled up neatly and placed in a hybridization tube. To this tube, 20 mL of prehybridization buffer was added (5x SSPE, 5x Denhardt's reagent, 1% SDS, 8 µg/mL salmon sperm DNA) and incubated at 65°C for at least 4 h. After this prehybridization period, the DNA probe was added directly to the prehybridization solution and left overnight under similar conditions. Following hybridization, the membrane was washed with increasing stringency to remove nonspecific binding of the probe. The membrane was washed with 2x SSC (0.3 M NaCl, 30 mM Na2 citrate), 0.1% (w/v) SDS at room temperature, 1x SSC, 0.1% (w/v) SDS at 42°C, and 0.1x SSC, 0.1% (w/v) SDS at 65°C, each for 5 min. Having removed nonspecific binding, the membrane was moistened with washing solution (1x SSC, 0.1% [w/v] SDS at room temperature) and placed in a plastic bag and heat-sealed, ready for autoradiography. The sealed and probed membrane was placed in an autoradiography cassette and overlaid with a sheet of x-OMAT film (Kodak) at –70°C.
PCR was performed in a 50-µL volume containing 30 ng of template DNA, the appropriate forward and reverse primers (0.2 mM each), and ReadyMix Taq PCR Reaction mix with MgCl2 (Sigma), according to the manufacturer's instructions. The reaction conditions were 94°C for 1 min 30 s for 1 cycle, then 35 cycles of 94°C for 40 s, 55°C for 40 s, and 72°C for 40 s.
Tomato pericarp or leaf tissue (10–15 g) was homogenized in 4 volumes of acetone at –20°C, filtered through Miracloth (Calicoes), and washed with 10 volumes of 80% acetone at 4°C. A further wash with 10 volumes of 100% acetone at 4°C was performed before drying the acetone insoluble solids (AIS) under vacuum overnight in the presence of P2O5. The dried AIS was weighed and then resuspended, with homogenization, in 20 mL of extraction buffer (1 M NaCl, 0.05 M NaOCOCH3). The pH was adjusted to 6.0 (with 0.1 N NaOH) and left at 4°C for 3 h with stirring. The samples were then spun down at 13,000g for 20 min, and the resulting supernatant was made to 80% saturation with NH4SO4 (0.57 g/mL). The samples were then left overnight at 4°C. The precipitate was spun down at 20,000g for 20 min and resuspended in 5 mL of dialysis buffer (0.15 M NaCl; 0.05 M NaOCOCH3, pH 6), dialyzed overnight, and then stored at –20°C until required. Protein was determined using the Bio-Rad protein assay dye (catalog no. 500–0001) using bovine serum albumin as the standard.
PE enzyme activity was determined by titration as described by Tucker et al. (1982)
Total protein was extracted from AIS as described above. The protein extract was then dialyzed overnight against 50 mM NaOCOCH3, 10 mM NaCl, pH 6.0. PE isoform separation was carried out using Bio-Rad heparin affinity chromatography. The system consisted of an Econo system controller model ES-1, Econo pump model EP-1, Econo UV monitor model EM-1, Econo buffer selector model EV-1, six-port sample injection valve model MV-6, diverter valve model SV-3, and fraction collector model 2128. The column, with 5-mL bed volume, was equilibrated with buffer A (10 mM Tris-HCl, 10 mM NaCl, pH 7.5). Samples (2 mL) were applied in buffer A and isoforms eluted at a flow rate of 1 mL/min using a linear gradient of NaCl from 10 mM (buffer A) to 300 mM (10 mM Tris-HCl, 300 mM NaCl, pH 7.5). Fractions (75 x 2 mL) were collected and assayed for PE activity using a microtiter plate method. Then 20 µL of each fraction was placed into wells in a 96-well microtiter plate. A total of 200 µL of assay buffer (0.5% citrus pectin, 2 mM Tris-HCl, 150 mM NaCl, 0.002% phenol red, pH 8.0) was added into each well. The plate was read on a Dynatech MR 5000 microtiter plate reader at 405 nm every 20 min for 2 h with 2-s shaking before each reading.
Fruit were harvested at the mature green/breaker stage of ripening and allowed to ripen at 20°C. Texture was measured daily, in the equatorial plane, using a Stevens texture analyzer, fitted with a 6-mm-diameter flat probe, and results are expressed as the weight required to compress the fruit by 4 mm.
Fruit size and texture data were analyzed using a two-way ANOVA using the Genstat 5.1 statistical program. Received January 24, 2007; accepted May 19, 2007; published June 7, 2007.
1 This work was supported by the Agri-Food Committee of the Biotechnology and Biological Sciences Research Council (studentship to T.D.P.) and by Overseas Research Studentship (to W.B.).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Gregory Tucker (gregory.tucker{at}nottingham.ac.uk). www.plantphysiol.org/cgi/doi/10.1104/pp.107.096347 * Corresponding author; e-mail gregory.tucker{at}nottingham.ac.uk; fax 44(0)1159–516122.
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) or Pmeu1 antisense line 8 (
) plants.
-32P]dCTP. 
