beta-Galactosidases (EC 3.2.1.23) constitute a widespread family of enzymes characterized by their ability to hydrolyze terminal, nonreducing beta-D-galactosyl residues from beta-D-galactosides. Several beta-galactosidases, sometimes referred to as exo-galactanases, have been purified from plants and shown to possess in vitro activity against extracted cell wall material via the release of galactose from wall polymers containing beta(1-->4)-D-galactan. Although beta-galactosidase II, a protein present in tomato (Lycopersicon esculentum Mill.) fruit during ripening and capable of degrading tomato fruit galactan, has been purified, cloning of the corresponding gene has been elusive. We report here the cloning of a cDNA, pTombetagal 4 (accession no. AF020390), corresponding to beta-galactosidase II, and show that its corresponding gene is expressed during fruit ripening. Northern-blot analysis revealed that the beta-galactosidase II gene transcript was detectable at the breaker stage of ripeness, maximum at the turning stage, and present at decreasing levels during the later stages of normal tomato fruit ripening. At the turning stage of ripeness, the transcript was present in all fruit tissues and was highest in the outermost tissues (including the peel). Confirmation that pTombetagal 4 codes for beta-galactosidase II was derived from matching protein and deduced amino acid sequences. Furthermore, analysis of the deduced amino acid sequence of pTombetagal 4 suggested a high probability for secretion based on the presence of a hydrophobic leader sequence, a leader-sequence cleavage site, and three possible N-glycosylation sites. The predicted molecular mass and isoelectric point of the pTombetagal 4-encoded mature protein were similar to those reported for the purified beta-galactosidase II protein from tomato fruit.

During our search for a cDNA encoding beta-galactosidase II, a beta-galactosidase/exogalactanase (EC 3.2.1.23) present during tomato (Lycopersicon esculentum Mill.) fruit ripening, a family of seven tomato beta-galactosidase (TBG) cDNAs was identified. The shared amino acid sequence identity among the seven TBG clones ranged from 33% to 79%. All contained 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. Six of the seven single-copy genes were mapped using restriction fragment length polymorphisms of recombinant inbred lines. RNA gel-blot analysis was used to evaluate TBG mRNA levels throughout fruit development, in different fruit tissues, and in various plant tissues. RNA gel-blot analysis was also used to reveal TBG mRNA levels in fruit of the rin, nor, and Nr tomato mutants. The TBG4-encoded protein, known to correspond to beta-galactosidase II, was expressed in yeast and exo-galactanase activity was confirmed via a quantified release of galactosyl residues from cell wall fractions containing beta(1-->4)-D-galactan purified from tomato fruit.

Exo-galactanase/beta-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/beta-galactosidase mRNA were generated to test this hypothesis and to investigate the role of the enzyme in fruit softening. A previously identified tomato beta-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 beta-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 beta-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.

Transcript abundance of the gene encoding beta-galactosidase II, a beta-galactosidase/exo-galactanase (EC 3.2.1.23) present during tomato (Lycopersicon esculentum) fruit ripening, was suppressed by expression of an antisense tomato beta-galactosidase 4 (TBG4) cDNA driven by the cauliflower mosaic virus 35S promoter. RNA gel-blot analysis was used to evaluate TBG4 mRNA levels in transgenic fruit. All of the antisense lines had attenuated TBG4 mRNA levels in turning stage fruit; however, TBG4 mRNA suppression was unstable, and mRNA levels varied in red-ripe fruit among the lines. Suppression of TBG4 mRNA levels in antisense fruit was correlated with a reduction in extractable exo-galactanase activity against a lupin galactan. All of the antisense lines had reduced free galactose levels at mature green stage 4, but levels comparable with controls during ripening. Total cell wall galactosyl contents in the antisense fruit were not significantly different from control fruit. Whole-fruit firmness was measured using a texture analyzer and the means of the peak force measurements for four of six antisense lines were significantly higher than control fruit. One antisense line had red-ripe fruit that were 40% firmer than controls. Fruit from this antisense line also had the lowest TBG4 mRNA and exo-galactanase levels and the highest wall galactosyl content during the early stages of ripening, implicating an involvement of this gene product in cell wall modification leading to fruit softening.

Antisense suppression of a tomato beta-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.

Tomatoes (Lycopersicon esculentum L.) contained a high level of beta-galactosidase activity which was due to three forms of the enzyme. During tomato ripening, the sum of their activities remained relatively constant, but the levels of the individual forms of beta-galactosidase changed markedly. The three enzymes were separated by a combination of chromatography of DEAE-Sephadex A-50 and Sephadex G-100. During ripening of tomatoes, beta-galactosidases I and III levels decreased but the beta-galactosidase II level increased more than 3-fold. The three enzymes were optimally active near pH 4, and all were inhibited by galactose and galactonolactone. However, the enzymes differed in molecular weight, K(m) value with p-nitrophenyl-beta-galactoside, and stability with respect to pH and temperature. beta-Galactosidase II was the only enzyme capable of hydrolyzing a polysaccharide that was isolated from tomatoes and that consisted primarily of beta-1, 4-linked galactose. The ability of beta-galactosidase II to degrade the galactan and the increase in its activity during tomato ripening suggest a possible role for this enzyme in tomato softening.