JXB Advance Access originally published online on July 16, 2003
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Journal of Experimental Botany, Vol. 54, No. 390, pp. 2025-2033,
September 1, 2003
© 2003 Oxford University Press
Antisense suppression of a ß-galactosidase gene (TB G6) in tomato increases fruit cracking
Received 30 December 2002; Accepted 15 May 2003
Produce Quality and Safety Laboratory, USDA-ARS, Building 002, Henry A. Wallace Beltsville Area Research Center, 10300 Baltimore Avenue, Beltsville, MD 20705-2350, USA
* To whom correspondence should be addressed. Fax: +1 301 504 5107. E-mail: grossk{at}ba.ars.usda.gov
Abbreviations: Br+3, Breaker stage plus 3 d; dap, days after pollination; MG, mature green stage; TBG, tomato ß-galactosidase.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Antisense suppression of a tomato ß-galactosidase gene (TBG6) was used to study its role in fruit development, cell wall-modification, and fruit firmness. TBG6 mRNA is highly abundant during the early stages of fruit development, but the levels decline sharply after the breaker stage with the start of the respiratory climacteric and a concomitant increase in ethylene production. Two antisense lines were obtained with significantly reduced levels of TBG6 mRNA at all stages of fruit development. At 30 d after pollination (dap), TBG6 mRNA levels were reduced by up to 98% and 88% in lines 6-2 and 6-10, respectively. Morphological phenotypes observed in the antisense lines included increased fruit cracking, reduced locular space, and a doubling in the thickness of the fruit cuticle. Two biochemical changes in antisense lines, compared with wild-type lines, were a reduction of exo-galactanase activity at the breaker +3 d stage and a reduction in the cell wall galactosyl content at the 20 dap stage. In addition, transgenic lines exhibited a 35–39% reduction in fruit firmness at the 20 dap stage, but their texture was equivalent to the wild type at 30 dap and beyond. Although the exact function of the TBG6 product is still unknown, these results implicate an important role for this enzyme in early fruit growth and development in tomato.
Key words: Antisense suppression, fruit cracking, tomato.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Fruit growth and development occur as a result of a myriad of biochemical and physiological events. The texture of the tomato (Lycopersicon esculentum Mill.) fruit, for example, is controlled by cell turgor pressure and changes in cell wall structure and composition. Several cell wall-modifying enzymes active during ripening have been identified. Some cell wall-degrading enzymes include polygalacturonase (PG), pectinesterase (PE), expansin, and ß-galactosidase (Brummell and Harpster, 2001). Decreasing levels of PG and PE by transgenic antisense technology significantly altered traits such as cracking and viscosity, but had little effect on fruit firmness (Schuch et al., 1991). On the other hand, suppression of the ripening-related expansin Exp1 and tomato ß-galactosidase 4 (TBG4) resulted in significantly firmer fruit (Brummell et al., 1999; Smith et al., 2002).
ß-Galactosidases are a large family of glycosyl hydrolases that reduce the levels of cell wall galactosyl residues in ripening tomato fruit (Gross and Sams, 1984; Smith et al., 1998; Carey et al., 2001). Genes from a family of at least seven tomato ß-galactosidases (TBG) show unique patterns of expression during fruit development (Smith and Gross, 2000). Antisense technology has been used to study these genes and promises to be a useful tool in attempts to produce fruit with attenuated softening. Down-regulation of TBG1 and TBG3 did not result in significant modifications in fruit firmness: however, reduced levels of TBG3 did result in lower levels of enzyme activity, altered cell wall composition, and increased serum viscosity of processed paste (Carey et al., 2001; de Silva and Verhoeyen, 1998). Antisense down-regulation of TBG4 resulted in fruit that were up to 40% firmer than controls, thus confirming a role for this gene’s product in determining fruit firmness (Smith et al., 2002). Further antisense studies of this nature are needed to elucidate the function(s) of the remaining members of the TBG family.
Among the TBG genes, TBG6 exhibits the highest expression levels, but only during the green stages of tomato fruit development, prior to visible signs of ripening (Smith and Gross, 2000). At the start of the breaker stage, the levels of TBG6 mRNA decline dramatically and little or no mRNA (depending on the cultivar) is detected during ripening (from breaker to the red ripe stage). The climacteric rise of ethylene in ripening tomato fruit may play a role in the developmental down-regulation of TBG6; wild-type fruit treated with ethylene at 25 and 33 dap show a dramatic decrease in TBG6 mRNA accumulation (Moctezuma et al., 2003). cDNA nucleotide and deduced amino acid sequence data predict the TBG6 product to be 91 kDa and secreted from the cell (Smith and Gross, 2000). Because of its unique temporal expression pattern and likely presence in the wall during fruit development, it is suspected that TBG6 is involved in early fruit growth (possibly wall expansion in immature fruit) and/or modifying fruit texture. To determine TBG6’s function in fruit growth and development, morphological and anatomical changes, cell wall modification, and fruit firmness were studied in antisense transgenic plants with suppressed TBG6 mRNA level.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant material
Tomato plants (Lycopersicon esculentum Mill. cv. ‘UC82B’) were grown in a greenhouse using standard cultural practices. Flowers were pollinated at anthesis using a vibrating device, and fruit were harvested at 10, 20 and 30 d after pollination (dap), as well as 3 d after the breaker stage (Br+3). The breaker stage was determined by incipient red coloration at the blossom end of the fruit. For molecular and biochemical analysis, five fruit from at least three plants of each line were collected and processed together by mixing peel and pericarp tissues and removing samples for different analysis.
Transformation vector and transgenic plant selection
The transformation vector was constructed by modifying the binary vector PBI121 (Clontech, Palo Alto, CA). The ß-glucuronidase coding region was first removed by digesting with BamHI and SacI, then a 749 bp fragment (nt 254–1003) from the 5' end of the TBG6 cDNA (accession number AF154424
[GenBank]
) was inserted in the antisense orientation with respect to the 35S CaMV promoter. Seven-day-old cotyledons were transformed using Agrobacterium tumefaciens (strain LBA4404) and plantlets were regenerated as in Deikman and Fischer (1988). Transformation was confirmed by DNA gel-blot analysis, using genomic DNA isolated from T0 plants. Seeds from six T1 generation plants were grown on kanamycin-containing media to select for antibiotic-resistant plants with the T-DNA insertion. After preliminary RNA gel-blot analysis of TBG6 mRNA suppression, two antisense lines (6-2 and 6-10) were selected for further study. Control plants were the parental wild type and azygous, kanamycin-sensitive T1 plants of lines 6-2 and 6-10, which were rescued from kanamycin-containing media.
RNA gel-blot analysis
Control and transgenic fruit were harvested at 10, 20, and 30 dap and Br+3, and 4 g of pericarp and peel tissue (combined) were chopped and immediately frozen in liquid nitrogen. Total RNA extraction and gel-blot analysis were as described in Smith and Gross (2000). Briefly, 20 µg of total RNA per sample were separated in a formaldehyde-agarose (1.5%) gel, transferred to a Hybond-N+ nylon membrane (Amersham, UK), and fixed by incubating for 2 h at 80 °C. Membranes were washed in 0.3 M Na-acetate (pH 5.2), methylene blue and 0.2x SSC (0.5% SDS) as described in Smith et al. (2002). TBG6-specific probes were synthesized using DNA fragments derived from PCR-amplification of the cDNA nucleotides 254–1003 (to hybridize to antisense and sense mRNA) and nucleotides 2551–3023 (to hybridize to sense mRNA 3'UTR only) using a Random Primed DNA Labeling Kit (Roche, Indianapolis, IN) with [32P]dATP (3000 Ci mmol–1) as the label. Probes for TBG4 and TBG5 were synthesized as described in Smith et al. (2002). Biomax MS X-ray film (Kodak, Rochester, NY) was used to expose the blots in an intensifying screen for 36 h at –80 °C. Finally, blots were stripped and hybridized using an apple 18S rDNA fragment to allow normalization for differences in loading of total RNA in each lane. Autoradiographs were quantified using a Fluor-S MultiImager scanner and analysed with Quantity One software (Bio-Rad, Hercules, CA). To corroborate the results, RNA gel blot experiments were repeated twice.
Anatomical sections, cracking and cuticle measurements
Peel/pericarp tissue samples were collected from five fruit of wild type and line 6-2 at the mature green and red stages of development. The tissue samples were fixed in 50% formalin-acetic acid-alcohol (FAA) under a vacuum for 16 h. Next, tissue samples were dehydrated in an increasing concentration series of ethanol:water, infiltrated with CitriSolv (Fisher, Pittsburgh, PA), embedded in paraffin, and sectioned at 12 µm on a rotary microtome. Sections were mounted on glass slides and stained with aqueous Safranin O (1%) and Fast Green (0.1% in 95% ethanol), as described by Ruzin (1999). Safranin O stains cutin, a major component of the cuticle. The thickness of the cuticle of each sample was measured using a calibrated light microscope at 100x magnification. Three tissue samples (blocks) from each fruit were processed. Five to ten measurements were made from each section for a total of 64 and 80 measurements for wild type and line 6-2, respectively.
Per cent fruit cracking was measured on 6-10 plants per line. An average of 15 fruit per plant (more than 100 total fruit per line), at all stages of development, were visually examined for cracks or scars on the surface of the pericarp. Typically one to three cracks and/or scars were found per fruit.
Enzyme assays and cell wall analysis
Cell wall-associated enzymes were extracted from tomato fruit peel and outer pericarp tissue according to the technique described in Carey et al. (1995) and Kim et al. (1991). Briefly, 20 g of fruit pericarp tissue were homogenized in a blender with 30 ml of water, NaCl was added to 1 M, and the pH adjusted to 6.0. After filtration and centrifugation, (NH4)2SO4 was added to 80% saturation. A suspension of precipitated proteins was dialysed against water for 4, 16 and 2 h at 4 °C. For estimating ß-galactosidase activity, p-nitrophenol-ß-D-galactopyranoside (PNP-gal) was used as a substrate (Pressey, 1983), and one unit of activity was defined as the amount of enzyme that liberated 1 µmol PNP min–1 at 37 °C. For exo-galactanase activity, lupin galactan (Megazyme, Wicklow, Ireland) was used as a substrate, following the method described by Carey et al. (1995) and Smith et al. (2002). Assays were run under conditions in which product formation was linear with time and proportional to the amount of enzyme in the assay.
Cell wall preparations were obtained from peel and outer fruit pericarp tissue using the methods described by Gross (1983). Ten grams of fruit pericarp tissue from wild-type and transgenic lines at different stages of development were chopped, frozen immediately in liquid nitrogen, and pulverized. The glycosyl composition of the acetone-insoluble cell wall material was established by hydrolysis with 2 N TFA followed by gas chromatography–mass spectrometry–selected ion monitoring of the alditol acetate derivatives of the monosaccharides released by hydrolysis (Gross and Acosta, 1991). Each enzyme assay and cell wall analysis was performed twice.
Texture measurements
Firmness measurements were taken from 12–20 whole fruit from each line, harvested at 20 and 30 dap. Each fruit was tested one to three times by indentation with a 4 mm diameter steel cylinder probe centred on the middle of a locule, as described in Smith et al. (2002). Texture measurements were determined using an instrument (TA-XT2i, Stable Microsystems Texture Analyser, Surrey, UK) loading at 1 mm s–1 to an indentation of 3 mm in depth. Firmness was defined as the maximum force required to deform the pericarp tissue 3 mm. Statistical analysis of maximum force measurements was performed using SAS software (SAS Institute, Cary, NC), and the statistical significance of the results was determined using the Tukey–Kramer multiple comparison test.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Antisense suppression of TBG6
Tomato plants were transformed with an antisense-oriented TBG6 cDNA fragment and a gene for kanamycin resistance. After regeneration, several T0 lines were chosen and grown to maturity. From these lines, T1 seed were collected and plated onto kanamycin-containing growth medium. Lines having a 3:1 resistant:sensitive ratio, meaning they had a single locus insertion of the T-DNA, were chosen for further study. Two lines (6-2 and 6-10) were selected and DNA gel-blot analysis was performed in these lines to confirm the T-DNA copy number and integrity (data not shown). Both the wild-type line used for transformation and an azygous line derived by rescuing T1 kanamycin-sensitive plants from lines 6-2 and 6-10 served as controls.
RNA gel-blot analysis was carried out using the 3'UTR as a probe to detect TBG6-specific sense mRNA only. In wild-type and azygous fruit, TBG6 mRNA levels were high in the early stages of development (10, 20 and 30 dap), but then dropped dramatically after the breaker stage (Fig. 1). Fruit from transgenic line 6-3 had levels of TBG6 mRNA similar to wild type, whereas lines 6-2 and 6-10 had significantly reduced levels of TBG6 mRNA. In antisense line 6-2, mRNA levels of TBG6 are greatly down-regulated at all stages of development; up to 98% suppression at 30 dap. Line 6-10 showed moderate suppression of TBG6 mRNA at the early stages of development: approximately 93% and 88% suppression at 20 and 30 dap, respectively (Fig. 1). An additional RNA gel-blot was hybridized using a probe that hybridizes to both sense and antisense TGB6 mRNAs. All antisense lines had abundant levels of antisense transcript at all stages of fruit development (data not shown).
|
Although unlikely, since a segment from the ORF of the TBG6 cDNA was used to construct the antisense cassette, it was possible that other ß-galactosidase gene family mRNAs might be suppressed as well, especially TBG4 and TBG5 (which share significant sequence identity) are expressed before ripening, and have exo-galactanase activity. The line with the greatest suppression of TBG6, line 6-2, was therefore tested for TBG4 and TBG5 mRNA abundance. It was found that TBG4 mRNA levels were similar to wild-type levels before ripening (Fig. 2). However, TBG4 mRNA levels in line 6-2 were 43% lower than wild type at the Br+3 stage. Levels of TBG5 mRNA in line 6-2 were similar to wild-type levels except for a slight increase at 20 dap.
|
Morphological and anatomical changes
Fruit from transgenic line 6-3 (with normal levels of TBG6 mRNA) had no altered physiological, biochemical or morphological phenotypes. However, several morphological and anatomical changes were observed in fruit of the transgenic lines with suppressed TBG6 mRNA. Transgenic lines 6-2 and 6-10 exhibited cracks in the epidermis of the fruit at early stages of development. These cracks eventually healed and formed ‘zipper-like’ scars (Fig. 3A). Most scars were oriented vertically, however, they also appeared with a horizontal or diagonal orientation. Anatomical sections revealed that the cracks and scars were several cell layers deep, of possible internal origin, and that they may involve changes in the cell wall (lignification, cutinization, etc.) (Fig. 3B). The number of fruit with one or several scars, as a percentage of the entire fruit per plant, increased with increased TBG6 suppression: 40% of the fruit of line 6-2 exhibited cracking, whereas approximately 14.5% of fruit in line 6-10 did so (Fig. 3C). In the wild-type and azygous lines only 1.8% or 1% of fruit, respectively, exhibited surface cracks. In addition, in transgenic line 6-3 (which has normal levels of TBG6 mRNA) only 3.3% of fruit had cracks.
|
The internal fruit anatomy of transgenic lines 6-2 and 6-10 was characterized by a lack of open locular space (Fig. 4). In cross-section, fruit of lines 6-2 and 6-10 consistently had a smaller open locular space than the wild type or azygous lines, possibly due to a thicker pericarp and larger columella and locules. Despite the increased volume of internal tissues in line 6-2, the average number of seeds per fruit was approximately 80% lower than the wild type (data not shown).
|
One of the most dramatic phenotypes observed in the transgenic lines was the glossiness of the fruit surface. This phenotype was first observed by the inability to write on green fruit from antisense lines 6-2 and 6-10 with a felt-tip permanent marker. Whereas ink flowed smoothly on the surface of control and line 6-3 fruit, the ink formed small droplets on the surface of fruit from lines 6-2 and 6-10. Upon further anatomical analysis, a doubling in the thickness of the cuticle at the mature green stage was found (Fig. 5). The cuticle of the parental wild type and azygous lines measured approximately 4.96 µm, whereas the cuticle of line 6-2 measured 10.06 µm. As a probable consequence of the thicker cuticle, transgenic fruit at the mature green stage appeared glossier than the wild type. It is interesting to note that this difference in cuticle thickness was seen only during the green stages of development: the cuticle at the red stage of development was not statistically different in control and transgenic fruit. The cuticle measured 7.7 µm (SE=1.07) in the wild type, and 8.3 µm (SE=1.29) in line 6-2, during the red ripe stage of development.
|
Physiological and biochemical changes
At 20 dap, ß-galactosidase activity was twice as high in transgenic line 6-2, and 3-fold higher in line 6-10, compared with the wild type (Fig. 6A). However, ß-galactosidase activity was virtually the same in transgenics and wild type at later developmental stages (30 dap and Br+3). Exo-galactanase activity was the same in the wild-type and transgenic lines at 20 dap and 30 dap, but at the Br+3 stage, it was reduced in lines 6-2 and 6-10 to 19% and 57% of wild-type levels, respectively (Fig. 6B).
|
The galactosyl content of the cell wall decreased continually in wild type and transgenic lines throughout development (Fig. 7). However, at 20 dap, transgenic lines 6-2 and 6-10 showed a more marked reduction in cell wall galactosyl content compared with wild type (Fig. 7). Line 6-2 exhibited a 22% decrease, whereas line 6-10 showed a 40% decrease. In all other developmental stages, the galactosyl residue content was the same as in the wild type or azygous fruit.
|
Lastly, fruit from transgenic lines with suppressed TBG6 mRNA were significantly softer than control fruit at 20 dap, as shown by the lower maximum force required by a 4 mm cylinder to penetrate 3 mm into the pericarp of the fruit (Fig. 8). Line 6-2 exhibited a 35% reduction in the maximum force, whereas 6-10 showed a 39% reduction. Interestingly, the difference in texture was only found at this stage of development, but not at 30 dap (Fig. 8) or Br+3 (data not shown).
|
=0.05. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The role of the TBG6 gene product in fruit development was explored by down-regulating its expression. Two antisense lines were found with significantly reduced levels of TBG6 mRNA. When compared with control fruit at 30 dap, TBG6 mRNA levels were reduced to 2% and 12% of normal in lines 6-2 and 6-10, respectively. The number of fruit that developed cracks was correlated with decreasing levels of TBG6 mRNA in the transformed lines; the stronger the suppression, the higher the percentage of fruit exhibiting cracking. Both suppressed lines also exhibited changes in internal fruit morphology (reduced air space in locules) and a doubling of the cuticle thickness in green fruit. A third transgenic line, line 6-3, served as an additional control by expressing TBG6 antisense mRNA, but not suppressing TBG6 mRNA or having altered fruit phenotypes.
Several members of the TBG gene family (TBG1, TBG3 and TBG4) have been studied by sense co-suppression and antisense suppression. Carey et al. (2001) analysed transgenic lines with TBG1 mRNA levels that were reduced by up to 90%. Even though a yeast-expressed TBG1 gene product was shown to have both ß-galactosidase and exo-galactanase activity in vitro, total ß-galactosidase and exo-galactanase activities, as well as levels of cell wall galactosyl residues and softening, were not affected in the TBG1-suppressed lines (Carey et al., 2001). In another study, down-regulation of TBG3 mRNA levels resulted in transgenic lines with reduced levels of exo-galactanase activity and increased levels of cell wall galactosyl content (de Silva and Verhoeyen, 1998). Even though the firmness of antisense TBG3 fruit was not significantly affected, the rate of deterioration during long-term storage decreased. Antisense down-regulation of TBG4 mRNA levels resulted in transgenic plants with fruit up to 40% firmer than controls (Smith et al., 2002). In addition, TBG4 mRNA suppression was correlated with reductions in exo-galactanase activity and levels of free galactose at the mature green stage. These results implied an involvement of TBG4 in cell wall modification and eventual fruit softening (Smith et al., 2002).
Unfortunately, it was not possible to express TBG6 using several yeast and E. coli expression systems and, therefore, have not confirmed or characterized its activity (data not presented). Nevertheless, of the seven TBGs studied so far, TBG6 mRNA abundance is highest at the early stages of green fruit development and is, by far, the most abundantly expressed TBG before ripening. The deduced amino acid sequence data predicts the TBG6 product to be 91 kDa and secreted from the cell (Smith and Gross, 2000). More importantly, the TBG6 product is predicted to have ß-galactosidase activity because it shares more than 64% sequence identity with other TBGs known to have ß-galactosidase and exo-galactanase activity against both native and lupin galactans, such as TBG1, TBG4 and TBG5 (Carey et al., 2001; Smith et al., 1998; Moctezuma et al., 2003) and contains the putative active site-containing consensus sequence pattern G-G-P-[LIVM]- x-Q-x-E-N-E-[FY] belonging to glycosyl hydrolase family 35; where amino acids separated by dashes are conserved, amino acids [LIVM] and [FY] are conserved substitutions, and ‘x’ is any amino acid (Henrissat, 1998).
For most of the developmental stages tested, ß-galactosidase/exo-galactanase activity, cell wall composition, and fruit firmness of control and TBG6-suppressed lines were similar. The most intriguing results obtained in this study were the changes in ß-galactosidase activity, cell wall galactosyl content, and fruit texture observed in lines 6-2 and 6-10 at 20 dap. It is interesting that at 20 dap, ß-galactosidase activity was higher in lines 6-2 and 6-10 than in the control lines, even though both transgenic lines had very low levels of TBG6 mRNA, which is the most abundant TBG transcript in the wild type at that particular stage of fruit development. When the mRNA abundance of a target gene is down-regulated by antisense, overall enzyme activity levels may remain normal due to redundancy and/or compensation provided via other members of the gene family. Transcript levels of TBG4 and TBG5, which are expressed in 20 dap fruit and are known to have ß-galactosidase activity in vitro, were, therefore, examined. However, increased levels of TBG4 and TBG5 mRNA were not found in the transgenic lines (Fig. 2). Thus, increased expression of TBG4 and TBG5 can not explain why ß-galactosidase activity in the transgenic lines was higher than in the wild type in 20 dap fruit. It is possible that the TBG6 gene product activity is not detectable because it does not have activity against the substrates that were tested or activity is lost during the extraction process. It is also not clear why cell wall galactosyl content and fruit firmness are reduced in transgenic fruit only at 20 dap. While the galactosyl levels of the wall were reduced at 20 dap in the antisense lines, free galactose levels remained constant throughout the immature stages of fruit development in all lines (data not shown). However, these observations may have a significant connection with the increased incidence of cracked fruit and thickening of the cuticle in the transgenic lines.
Based on TBG6 mRNA expression patterns at the early stages of fruit development and the incidence of cracks correlating to decreased levels of TBG6 mRNA, it is possible that the TBG6 gene product may help to facilitate cell expansion. Previous studies indicate that changes in ß-galactosidase activities occur in elongating and rapidly- growing tissues such as epicotyls (Dopico et al., 1989). Absence of the TBG6 product may create disturbances in cell wall metabolism during the cell expansion phase of fruit development that are responsible for the morphological changes observed in the antisense lines, such as fruit cracking and the more compact internal fruit anatomy (reduction in open locular space). In addition, it is possible that the TBG6-encoded product is especially critical during the most rapid period of cell elongation and fruit expansion, and its absence may modify fruit texture and/or elasticity, resulting in softer fruit at 20 dap. As fruit continue to develop, the TBG6 product may no longer play a major role in cell expansion, or alternatively, the reduced but not eliminated level of TBG6 product in antisense lines is enough for complete function, and the fruit have normal texture characteristics.
One of the most interesting phenotypes observed was the doubling in cuticle thickness of fruit from TBG6-suppressed lines. The cuticle is a film of soluble and polymerized lipids that prevents water loss and provides protection against pathogens and insects (Somerville et al., 2000). The outside-facing surface of a typical epidermal cell has a cap of secondary cell wall which is suspected to play a mechanical role in preventing deformation of the epidermis (Kunst and Samuels, 2003). Above the secondary wall, in direct contact with the cuticular lipids and continuous with the pectin-rich middle lamella of the anticlinal walls between adjacent cells, is a pectin-rich layer composed of homogalacturonans and rhamnogalacturonans with cellulose microfibrils branching through it (Kunst and Samuels, 2003). Any modifications to galactan metabolism in these pectin-rich layers could result, for example, in enhanced cuticle deposition to repair the cracks and/or strengthen a weaker structure. Perturbations in fruit growth are known to lead to drastic changes in the composition and depth of the cuticle (E Curry, personal communication). It has also been observed that severe changes in environmental conditions during early apple fruit growth can lead to surface cracking and excess cuticle deposition (E Curry, personal communication). These observations support the idea that the phenotypes observed in TBG6-suppressed lines may be due to alterations in cell expansion or orientation during the early stages of fruit development.
The results presented in this study indicate that the presence of a TBG6 antisense construct may have resulted in decreased levels of TBG4 and TBG5 mRNA in fruit, but only at the Br+3 stage. The observed decrease in exo-galactanase activity measured in the transgenic lines at the Br+3 stage of development (Fig. 6), is most likely related to the decrease in TBG4 and possibly TBG5 mRNA abundance seen in transgenic line 6-2 at this time point. Smith et al. (2002) found that reduced exo-galactanase activity was correlated with reduced TBG4 mRNA levels in TBG4 antisense lines, and that the TBG4 product is mostly responsible for the observed exo-galactanase activity in fruit.
It has been suggested that the pectin matrix establishes the ‘pore size’ in the cell wall, i.e. the relative size of the channels formed by the wall matrix that permit polymeric molecules to diffuse freely through the matrix. This pore size can be established by a combination of the frequency and length of the junction zones, the degree of methyl esterification, and the length of arabinans, galactans, and arabinogalactans attached to rhamnogalacturonan I (RGI) that extend into the pores (Carpita and McCann, 2000). An increased content of pectic galactan side chains has also been linked to increased mechanical strength of the wall in pea cotyledons (McCartney et al., 2000). Despite repeated tries using yeast and E. coli systems, it has not been possible to express the TBG6 protein product, in order to characterize enzyme activity, substrate specificity, etc. Even so, the data presented here suggest that the TBG6 product may have an important function in cell wall galactosyl residue metabolism during cell elongation. It is interesting to note that many ß-galactosidases have additional specific biosynthetic activities, such as transglycosylation and reverse hydrolysis (Yoon and Ajisaka, 1996). These functions, although not yet identified in plant cell wall metabolism, may have important roles in modifying cell wall structure to accommodate periods of rapid wall modifications which occur, for example, during cell expansion.
In addition, it would be useful to generate transgenic plants that over-express TBG6 during fruit ripening in order to investigate if this leads to reduced cracking during the handling and shipping of post-breaker fruit. It would also be interesting to show how the observed morphological phenotypes can be used for improving commercial tomato varieties. For example, decreased locular space can lead to developing ‘meatier’ tomatoes, which could have significant impact in the fresh-cut slice industry. Further study in the genetic and molecular origins of increased cuticle thickness, without the deleterious effects of increased cracking, may be helpful in developing fruit with improved resistance to water loss and infection by pathogens. Summing up, the various altered phenotypes observed as a result of TBG6 down-regulation in tomato fruit are further evidence that ß-galactosidases have important functions in the overall growth and development of tomato fruit.
|
Acknowledgements |
|---|
We wish to thank Virginia Boulais, Norman Livsey, Heather Torlina, and Eunhee Park for their valuable technical help. We are grateful to Zeneca (now Syngenta) for producing the transgenic plants and Dr Graham Seymour for growing and collecting seed from the T0 plants. We are also thankful to Dr Judy Abbott for statistical advice, Dr Eric Curry for valuable discussions, Drs James McEvoy and Rob Griesbach for help with the micrographs.
Note: Use of a product name by the US Department of Agriculture does not imply approval or recommendation of such product to the exclusion of others, which may also be suitable.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Brummell DA, Harpster MH. 2001. Cell wall metabolism in fruit softening and quality and its manipulation in transgenic plants. Plant Molecular Biology 47, 311–340.[CrossRef][Web of Science][Medline]
Brummell DA, Harpster MH, Civello PM, Palys JM, Bennett AB Dunsmuir P. 1999. Modification of expansin protein abundance in tomato fruit alters softening and cell wall polymer metabolism during ripening. The Plant Cell 11, 2203–2216.
Carey AT, Holt K, Picard S, Wilde R, Tucker GA, Bird CR, Schuch W, Seymour GB. 1995. Tomato exo- (1
4)-D-galactanase: isolation, changes during ripening in normal and mutant tomato fruit, and characterization of a related cDNA clone. Plant Physiology 108, 1099–1107.[Abstract]
Carey AT, Smith DL, Harrison E, Bird CR, Gross KC, Seymour GB, Tucker GA. 2001. Down-regulation of a ripening-related ß-galactosidase gene (TBG1) in transgenic tomato fruits. Journal of Experimental Botany 52, 663–669.
Carpita N, McCann M. 2000. The cell wall. In: BB Buchanan, W Gruissem, RL Jones, eds. Biochemistry and molecular biology of plants. Rockville: American Society of Plant Physiologists, 52–108
de Silva J, Verhoeyen ME. 1998. Production and characterization of antisense-exogalactanase tomatoes. In: Kuiper HA, ed. Report of the demonstration programme on food safety evaluation of genetically modified foods as a basis for market introduction. The Hague, The Netherlands: Ministry of Economic Affairs, 99–106.
Deikman J, Fischer RL. 1988. Interaction of a DNA binding factor with the 5'-flanking region of an ethylene-responsive fruit ripening gene from tomato. EMBO Journal 7, 3315–3320.[Web of Science][Medline]
Dopico B, Nicolas G, Labrador E. 1989. Partial purification of cell wall ß-galactosidase from Cicer arietinum epicotyls. Relationship with cell wall autolytic processes. Physiologia Plantarum 75, 458–464.
Gross KC. 1983. Changes in free galactose, myo-inositol and other monosaccharides in normal and non-ripening mutant tomatoes. Phytochemistry 22, 1137–1139.[CrossRef]
Gross K, Acosta PB. 1991. Fruits and vegetables are a source of galactose: implications in planning the diets of patients with galactosemia. Journal of Inherited Diseases 14, 253–258.[CrossRef]
Gross KC, Sams CE. 1984. Changes in cell wall neutral sugar composition during fruit ripening: a species survey. Phytochemistry 23, 2457–2461.[CrossRef]
Henrissat B. 1998. Glycosidase families. Biochemical Society Transactions 26, 153–156.[Web of Science][Medline]
Kim J, Gross KC, Solomos T. 1991. Galactose metabolism and ethylene production during development and ripening of tomato fruit. Postharvest Biology and Technology 1, 67–80.[Medline]
Kunst L, Samuels AL. 2003. Biosynthesis and secretion of plant cuticular wax. Progress in Lipid Research 42, 51–80.[CrossRef][Web of Science][Medline]
McCartney L, Ormerod AP, Gidley MJ, Knox JP. 2000. Temporal and spatial regulation of pectic (1-4)-ß -D-galactan in cell walls of developing pea cotyledons: implications for mechanical properties. The Plant Journal 22, 105–113.[CrossRef][Web of Science][Medline]
Moctezuma E, Smith DL, Gross KC. 2003. Effect of ethylene on mRNA abundance of three ß-galactosidase genes in wild-type and mutant tomato fruit. Postharvest Biology and Technology (in press).
Pressey R. 1983. ß-galactosidases in ripening tomatoes. Plant Physiology 71, 132–135.
Ruzin SE. 1999. Staining techniques. In: Ruzin SE, ed. Plant microtechnique and microscopy. New York: Oxford University Press, 87–116.
Schuch W, Kanczler J, Robertson D, Hobson G, Tucker G, Grierson D, Bright S, Bird C. 1991. Fruit quality characteristics of transgenic tomato fruit with altered polygalacturonase activity. HortScience 26, 1517–1520.
Smith DL, Starrett DA, Gross KC. 1998. A gene coding for tomato fruit -galactosidase II is expressed during fruit ripening. Plant Physiology 117, 417–423.
Smith DL, Gross KC. 2000. A family of at least seven ß-galactosidase genes is expressed during tomato fruit development. Plant Physiology 123, 1173–1183.
Smith DL, Abbott JA, Gross KC. 2002. Down-regulation of tomato ß-galactosidase 4 results in decreased fruit softening. Plant Physiology 129, 1755–1762.
Somerville C, Browse J, Jaworski JG, Ohlrogge JB. 2000. Lipids. In, Buchanan RB, Gruissem W, Jones RL, eds. Biochemistry and molecular biology of plants. Rockville, American Society of Plant Physiologists, 507–509.
Yoon JH, Ajisaka K. 1996. The synthesis of galactopyranosyl derivatives with ß-galactosidases of different origins. Carbohydrate Research 292, 153–163.[Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Mintz-Oron, T. Mandel, I. Rogachev, L. Feldberg, O. Lotan, M. Yativ, Z. Wang, R. Jetter, I. Venger, A. Adato, et al. Gene Expression and Metabolism in Tomato Fruit Surface Tissues Plant Physiology, June 1, 2008; 147(2): 823 - 851. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Carrari and A. R. Fernie Metabolic regulation underlying tomato fruit development J. Exp. Bot., June 1, 2006; 57(9): 1883 - 1897. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. GIBERT, F. LESCOURRET, M. GENARD, G. VERCAMBRE, and A. PEREZ PASTOR Modelling the Effect of Fruit Growth on Surface Conductance to Water Vapour Diffusion Ann. Bot., March 1, 2005; 95(4): 673 - 683. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||