Journal of Experimental Botany, Vol. 52, No. 357, pp. 663-668,
April 15, 2001
© 2001 Oxford University Press
Original Papers |
Down-regulation of a ripening-related ß-galactosidase gene (TBG1) in transgenic tomato fruits
1 Department of Plant Genetics and Biotechnology, Horticulture Research International, Wellesbourne, Warwick CV35 9EF, UK
2 Horticultural Crops Quality Laboratory, Plant Sciences Institute, Agricultural Research Service, United States Department of Agriculture, 10300 Baltimore Avenue, Beltsville, Maryland 20705-2350, USA
3 Zeneca Plant Science, Jealott's Hill Research Station, Bracknell, Berkshire RG 12 6EY, UK
4 School of Biological Sciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leics LE12 5RD, UK
Received 28 June 2000; Accepted 28 September 2000
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Abstract |
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Exo-galactanase/ß-galactosidase (EC 3.2.1.23) activity is thought to be responsible for the loss of galactosyl residues from the cell walls of ripening tomatoes. Transgenic tomato plants (Lycopersicon esculentum Mill cv. Ailsa Craig) with reduced exo-galactanase/ß-galactosidase mRNA were generated to test this hypothesis and to investigate the role of the enzyme in fruit softening. A previously identified tomato ß-galactosidase cDNA clone, TBG1, was used in the experiments. Heterologous expression of the clone in yeast demonstrated that TBG1 could release galactosyl residues from tomato cell wall galactans. Transgenic plants showed a reduction in TBG1 mRNA to 10% of normal levels in the ripening fruits. However, despite the reduction in message, total ß-galactosidase and exo-galactanase activities were unaffected. Furthermore, there was no apparent effect on levels of cell wall galactosyl residues when compared with the control. It was concluded that during the ripening of tomato fruits a family of ß-galactosidases capable of degrading cell wall galactans are active and down-regulation of TBG1 message to 10% was insufficient to alter the degree of galactan degradation.
Key words: ß-galactosidase, TBG1, tomato, fruit ripening, transgenic plants, cell walls.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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The texture of fruit and vegetables determines not only their palatability, but also their shelf-life, transportability and disease resistance. Currently, traditional breeding strategies and the optimization of storage conditions have provided some control over the texture of fresh produce. However, further progress in this ability to manipulate texture relies on gaining an insight into the biochemical and molecular events which regulate this process.
Texture changes in plant tissues are likely to result from alterations in cell wall structure, although other factors such as turgor pressure also play a role. Breeding studies show that in tomato, firmness is a multigene trait (Stevens and Rick, 1986
). A wide range of cell wall-degrading enzymes have been reported to be active during tomato fruit ripening with the major activities in tomato including polygalacturonase (PG), pectinesterase (PE) and ß-galactosidase (Seymour and Gross, 1996). Expansins are also expressed in ripening tomatoes (Rose et al., 1997). In the case of PG and PE, individual enzymes have been the subject of transgenic experiments designed to investigate their role in fruit texture (Smith et al., 1988, 1990; Tieman et al., 1992; Hall et al., 1993). These experiments have indicated that while softening in tomato is not greatly affected by altering the levels of these enzymes, important quality traits are enhanced, for example, low PG fruits show reduced cracking and improved paste quality (Schuch et al., 1991). Down-regulation of the expression of the ripening-specific expansin Exp1 had a significant effect on tomato fruit texture, suggesting that Exp1 is a component of the wall modifications involved in fruit softening (Brummell et al., 1999). The role of ß-galactosidase is less well understood and no transgenic experiments have been reported.
Exo-galactanases/ß-galactosidases are thought to be responsible for the conspicuous reduction in the level of galactosyl residues which occurs in many ripening fruits including tomato (Gross and Sams, 1984). These enzymes have also been detected in a wide range of plant organs and tissues undergoing developmental changes such as seeds (Sekimata et al., 1989; Simos et al., 1989), cotyledons (Buckeridge and Reid, 1994), elongating epicotyls (Dopico et al., 1989), and in freshly harvested asparagus spears (King and Davies, 1995). So far, only exo-acting activities have been found in plants and these enzymes are characterized by their ability to hydrolyse terminal non-reducing ß-D-galactosyl residues from ß-D-galactosides. They are usually assayed by their ability to release galactosyl residues from artificial substrates such as p-nitrophenyl-ß-D-galactopyranoside, 4-methyl umbelliferyl-ß-D-galactopyranoside and X-gal. In many cases, however, their natural substrates are unknown, but probably include chloroplast galactolipids (Bhalla and Dalling, 1984) as well as cell wall galactans. Three isoforms of ß-galactosidase have been identified from ripening tomato fruits (Pressey, 1983). One of these isoforms, ß-galactosidase II is active against tomato cell wall (1-4)-ß-D-galactan (Pressey, 1983; Pressey and Himmelsbach, 1984). Isoform II has been purified and a related cDNA, pTomßgal1 (now renamed TBG1) has been cloned (Carey et al., 1995). This clone did not, however, encode exactly the N-terminal amino acid sequence of the purified ß-galactosidase II protein. More recently, the cloning of pTomßgal4 (TBG4) has been reported (Smith et al., 1998), which unlike TBG1, matches the N-terminal sequence for the ß-galactosidase II isoform and is likely to encode this protein. Also, several other closely related ß-galactosidase-like cDNA clones have been isolated (Smith et al., 1998; Smith and Gross, 2000). It is unclear whether all of these messages encode proteins with galactan degrading activity and the role of ß-galactosidases in tomato fruit ripening is not known.
This paper describes the heterologous expression of TBG1 in yeast and demonstrates that the expressed protein has exo-galactanase/ß-galactosidase activity, including the ability to release galactosyl residues from a variety of cell wall substrates. The pattern of expression of this transcript in ripening tomato fruit is described along with the effects of down-regulating the message in transgenic tomato plants.
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Expression in yeast, Western analysis and enzyme activity
TBG1 protein was expressed using the FLAG N-terminal Expression System (Sigma-Aldrich, St Louis, MO, USA). The open reading frame of TBG1 (cDNA clone pTomßgal-10; accession no. AF023847) was PCR-amplified using oligonucleotides (primer sequences: 5' forward TGT GGA GAA TTC TCT GTT TCA TAT GAC CAT AAA G, 3' reverse TAC TCA GTC GAC TCA ACT ACA AAT GGC TTC CA) so that the predicted signal peptide (aa 1–22) was removed and a EcoRI and SalI restriction site was created at the 5' and 3' end of the open reading frame, respectively. The digested PCR fragment was cloned into an EcoRI plus SalI digested YEpFLAG1 vector to produce an extracellular N-terminal FLAG fusion protein when expressed. The vector was transformed into the Saccharomyces cerevisiae host strain BJ3505. Yeast cells transformed with the YEpFLAG1 vector were used as a negative control. Cultures were grown and protein harvesting was done as described by the manufacturer except that the cells were grown for 5–6 d at 20 °C for maximal expression.
TBG1-FLAG fusion protein was affinity purified using an ANTI-FLAG M1 affinity gel according to the manufacturer's instructions. Briefly, 12 ml of TBG1-FLAG protein-containing yeast growth medium was equilibrated by the addition of 1/10 volume 10xTBS/Ca (0.5 M TRIS, pH 7.4, 1.5 M NaCl, 100 mM CaCl2) and was passed through a 1 ml bed volume of ANTI-FLAG M1 affinity gel three times. The gel was washed three times with 12 ml 1xTBS/Ca and TBG1-FLAG eluted by competition with five one-column aliquots of a solution containing 100 µg ml-1 FLAG peptide. Yeast culture medium samples or affinity-purified TBG1 protein, was subjected to SDS-PAGE and transferred to nitrocellulose. Blots were subjected to protein blot analysis using M1 anti-FLAG primary antibody, rabbit anti-mouse secondary antibody conjugated to alkaline phosphatase and colorimetric detection using Sigma-Fast substrate following the manufacturers recommendations.
The yeast culture medium from YEpFLAG1 and YEpFLAGTBG1 transformed cells was concentrated and desalted using Centriprep30 columns (Amicon) and affinity-purified TBG1-FLAG protein was concentrated and desalted using Ultrafree-4 filter units (Millipore). Desalting was performed by three washes with 0.1xTBS. Enzyme assays were performed using the desalted medium and were repeated using column-purified TBG1 enzyme to confirm the specificity of TBG1 enzyme's activity. ß-Galactosidase activity was assayed using p-nitrophenol-ß-D-galactopyranoside (PNP-gal) as substrate. Assays consisted of 1 mg ml-1 substrate, 50 mM sodium acetate pH 4.5, 0.02% BSA, and 5 µl of column pure enzyme (approximately 1 µg protein determined by Bradford assay). One unit of activity was defined as the amount of enzyme that liberated 1 mol of p-nitrophenol min-1 at 37 °C. Exo-galactanase activity was determined by incubating cell wall material with the enzyme samples essentially as described previously (Carey et al., 1995). One ml assays consisted of 2 mg substrate, 0.005 units enzyme (approximately 17 µg affinity-purified protein), 0.1% (w/v) BSA, and 50 mM sodium acetate buffer at pH 4.75. Cell wall material was purified from mature green tomato fruit using the methods described earlier (Gross, 1984). A lupin galactan, pretreated with 
-L-arabinofuranosidase, was obtained from Megazyme (Wicklow, Ireland). Assays were carried out at 37 °C for 4 h. Free galactose was identified and quantified by GC/MS-SIM of the galactitol acetate derivative prepared from the released galactose product in reaction mixtures (Gross and Acosta, 1991).
Plant material
Tomato plants (Lycopersicon esculentum Mill. cv. Ailsa Craig) were grown in a heated glasshouse using standard cultural practices. The fruits were harvested at different stages of maturity, as determined by time from anthesis and at various stages of ripeness as determined from days after the breaker stage.
Construction of the transformation vector and selection of transgenic plants
A PCR product corresponding to the 376 bp at the 5' end of TBG1 (Carey et al., 1995) was cloned, in the sense orientation, into the binary vector Bin 19 carrying an expression cassette containing the CaMV 35S promotor and Nos terminator in the sense orientation. This vector was then transformed into Agrobacterium tumefaciens (LBA4404). Tomato stem explants were then transformed using Agrobacterium following standard protocols for tomato transformation and regeneration as described previously (Bird et al., 1988). Genomic DNA was prepared as described earlier (Clarke et al., 1989). PCR was used to identify transformed plants and azygous segregants. These results were confirmed by identification of the expression of the 376 bp sense transgene in leaves by Northern analysis. The endogenous transcript was c. 3 kb. Seeds from primary transformants with low levels of TBG1 message were collected and shown on kanamycin selective media. Seedlings that grew vigorously on the selective media were grown to fruiting and represented the T1 generation.
RNA extraction
Total RNA was extracted from tomato fruit tissues as described earlier (Smith et al., 1986) and contaminating carbohydrate and phenolic compounds being removed by differential cetyltrimethylammonium bromide precipitation. The RNA samples (20 g) were separated on formaldehyde–agarose (1.5%) gels and transferred to Hybond N+ membrane. Prehybridization and hybridization steps were conducted in 5xDenhardt's solution, 5xSSC at 65 °C and the membrane was probed with radiolabelled TBG1. Following hybridization blots were washed twice in 3xSSC, 0.1% SDS for 20 min and twice in 0.3xSSC, 0.1% SDS at 65 °C for 10 min.
Enzyme assays and cell wall preparation
Cell wall bound proteins were extracted from tomato pericarp with 1.0 M NaCl and precipitated with ammonium sulphate as described earlier (Pressey, 1993). PNP-gal, spruce galactan (Dextra Laboratories, Reading, UK) and lupin galactan were used as substrates for determination of enzyme activity following the methods described earlier (Carey et al., 1995). There was no apparent difference in the activity of the enzyme preparations from tomato pericarp against either spruce or lupin galactan. Only lupin galactan was used in the yeast experiments. Acetone-insoluble cell wall preparations were prepared from tomato pericarp with precautions taken to remove endogenous wall hydrolase activity as described previously (Seymour et al., 1987). The sugar composition of this acetone-insoluble material was then determined after hydrolysis in H2SO4 (Selvendran et al., 1979) and the monosaccharide components were separated and quantified by GC after conversion to alditol acetates as described earlier (Englyst and Cummings, 1984).
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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A loss of cell wall galactosyl residues is a characteristic of ripening in many types of fruit (Seymour and Gross, 1996
). The cloning of several ripening-related ß-galactosidase genes, including seven from tomato (Smith and Gross, 2000) provides an opportunity to determine whether galactosyl loss is linked to the action of ß-galactosidases and to investigate the role of these enzymes in fruit softening. This study has focused on the role of TBG1, which was the first tomato ß-galactosidase to be cloned (Carey et al., 1995).
Activity of TBG1 expressed in yeast
In order to determine the role of the TBG1 encoded product, experiments to express the cDNA encoded enzyme using a heterologous expression system were undertaken. A yeast expression system that secretes a mature amino-terminal-FLAG fusion protein into the culture medium was successfully tested and a FLAG-TBG1-encoded fusion protein construct resulted in the production of approximately 0.25 mg of active enzyme per 50 ml culture. The activity of the expressed protein could not be compared with that of native TBG1 enzyme since this has not been purified. The expressed TBG1 protein was affinity-purified using an anti-FLAG affinity resin (Fig. 1). Both the crude protein mix from the FLAG-TBG1 transformed yeast culture medium and the affinity-purified TBG1 enzyme had ß-(1-4)-D-galactosidase activity by virtue of its ability to hydrolyse the synthetic substrates PNP-gal (Table 1) and X-gal (not shown). The enzyme was not able to hydrolyse lactose (Table 1). However, the enzyme was able to release free galactose from a variety of galactosyl-containing cell wall substrates and therefore had exo-galactanase activity (Table 1). The concentrated and desalted yeast culture medium from YEpFLAG1 transformed cells had no detectable ß-(1-4)-D-galactosidase or exo-galactanase activity under the conditions tested. These data demonstrate that the TBG1 clone expressed in yeast yields a protein that has ß-galactosidase activity against artificial substrates and the ability to degrade pectic galactans.
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Expression of TBG1 in fruit and analysis of transgenic plants
Earlier findings indicated that the TBG1 gene expression was ripening-related (Carey et al., 1995) and this has been confirmed previously (Smith and Gross, 2000). Transgenic plants were generated to determine if the accumulation of TBG1 message could be suppressed and to examine the effects of this altered gene expression on galactan degradation and fruit softening.
Three fruits at the red ripe stage of development (breaker+7 d) were harvested from each of 27 primary transformants containing a TBG1 partial sense construct. There was no transgene transcript detectable in plants 1, 3, 5, 18, 22, and 26, as confirmed by Southern hybridization and expression of the transgene in leaf tissue (data not shown). All these plants had fruit with similar levels of activity against p-nitrophenyl-ß-D-galactopyranoside and spruce galactan and plant 26 was chosen as a representative control for the remaining experiments. Northern analysis indicated a reduction in endogenous TBG1 gene expression in all transgenic lines, to as little as 10% of the control in lines 10 and 27 (Fig. 2). The sense transgene mRNA was visible in all the transformed lines, but the intensity of expression varied, for example, lines 10 and 27 (Fig. 2).
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Cell wall proteins were extracted from pericarp of the ripe (breaker+7 d) tomato fruit from selected transgenic plant lines and the control for measurement of activity against p-nitrophenyl-ß-D-galactopyranoside and spruce galactan. While variation was observed in the specific activities between lines, there was no general reduction in activity in lines having the transgene compared with the control (Fig. 3A
, B). Furthermore, there was no absolute correlation between reduction in TBG1 mRNA and enzyme activity. Reduced levels of TBG1 expression also appeared to have no significant effect on overall levels of cell wall galactosyl residues (Table 2) or fruit softening as determined by compression tests using an automated texture analyser (data not shown). Thus the transgene appeared to have no detectable effect on ß-galactosidase/exo-galactanase activity or cell wall galactosyl loss. The yeast expression data demonstrates, however, that TBG1 has the ability to degrade cell wall galactans and the northern analysis confirms that there are substantially reduced levels of this message in some of the transgenic lines. There are several possible explanations for these observations: (1) TBG1 enzyme is not secreted and therefore is not in contact with the cell wall. TBG1's cellular localization is not known and the targeting prediction programs PSORT and SignalP predicted conflicting targeting information. SignalP predicted that aa 1–22 forms a signal sequence and that a possible cleavage site exists between aa 22 and 23 (GIA-SV). However, PSORT predicted a non-cleavable signal sequence from aa 1–26 containing a transmembrane domain from aa 5–21 and an endoplasmic reticulum target. (2) TBG1 is secreted, but acts on a highly specific substrate in vivo resulting in limited galactosyl residue hydrolysis by this TBG isoform. This idea is not, however, supported by TBG1's in vitro activity since the enzyme was capable of releasing free galactose from a variety of tomato fruit cell wall substrates. (3) Based on the northern analysis of a family of ß-galactosidase genes, at least one is known to have exo-galactanase activity and was expressed in fruit during ripening (Smith et al., 1998; Smith and Gross, 2000), TBG1 mRNA levels were a minor constituent of the total mRNA of all the ß-galactosidase genes expressed. If it is assumed that mRNA levels of each of the ß-galactosidase/exo-galactanase genes corresponds directly to the amount of each ß-galactosidase/exo-galactanase enzyme then the TBG1 enzyme would be a minor constituent of the total activity (Smith and Gross, 2000). Therefore a lack of TBG1 enzyme activity might not be detectable by evaluation of galactosyl content in crude cell wall preparations. Alternatively, other isoforms of ß-galactosidase may be up-regulated in transgenic plants to compensate for the reduction in TBG1 message as observed with ß-1,3-glucanase in tobacco (Neuhaus et al., 1992; Beffa et al., 1993) or the effects of the transgene are more pronounced at either earlier stages of ripening or in a homozygous line. No evidence was found in the present study for changes in isoform profiles of the ß-galactosidases (data not shown). Analysis of T1 plants from those lines showing the greatest reduction in TBG1 mRNA abundance also failed to reveal any reduction in total ß-galactosidase/exo-galactanase activity in comparison with the control during ripening. Thus at breaker+3 d the mean galactanase activities measured as µg galactose released per 4 h mg-1 protein were 125±38 and 112±24 for T1 plants from T0 lines 26 and 27, respectively (±SE, n=3). In conclusion, TBG1 has a different amino acid sequence to the main tomato cell wall degrading ß-galactosidase, isoform II, which has recently been cloned and is represented by TBG4 (Smith et al., 1998). This demonstrates that there are at least two galactan degrading ß-galactosidases present in tomato, although the major protein product appears to be that encoded by TBG4 (Carey et al., 1995; Smith et al., 1998). Thus TBG1 may not contribute substantially to the total level of ß-galactosidase/exo-galactanase activity during ripening, but may act on a small, but specific set of galactan-rich polymers in the wall. Further transgenic experiments with other members of the tomato ß-galactosidase gene family are underway (DL Smith and KC Gross, unpublished results) and should help to answers these questions.
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Acknowledgments |
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We would like to thank the Biotechnology and Biological Science Research Council (UK) for financial support in the form of an ASD grant to GAT and GBS. We also wish to thank Norman Livsey and Heather Torlina for excellent technical assistance.
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Notes |
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5 To whom correspondence should be addressed. Fax: +44 1789 470552. E-mail: graham.seymour{at}hri.ac.uk
6 Present address: Horticulture Development Council, Bradbourne House, Stable Block, East Malling, Kent ME19 6DZ, UK.
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