Tomato Fruit Softening Research Articles

β-Galactosidase and α-L-Arabinofuranosidase in Cell Wall Modification Related with Fruit Development and Softening

Fruit softening and textural changes are two important factors of fruit quality. The loss of galactosyl and arabinosyl residues from cell wall polysaccharides is observed in many fruit species during ripening. The release of neutral sugar residues could change wall polysaccharides properties of accessibility or reactivity for other cell wall hydrolases. β-Galactosidase and α-L-arabinofuranosidase contribute to the loss of neutral sugar residues. Thus, the enzymes alter polysaccharide properties and might contribute to fruit softening or textural changes during ripening. β-Galactosidase is composed of multiple isozymes and they are clearly distinguishable by their substrate specificity and expression pattern of the related gene. A transgenic experiment revealed that a β-galactosidase isozyme plays an important role in tomato fruit softening. In addition to fruit softening, the contribution of β-galactosidase to plant development is also indicated. α-L-Arabinofuranosidase genes constitute a gene family and the isozymes are expressed in various organs and stages. Moreover, several α-L-arabinofuranosidases possess β-xylosidase activity in addition to α-L-arabinofuranosidase activity, therefore, substrate specificity against native polysaccharides is very complex. Arabinose containing polysaccharides seem to contribute to cell to cell adhesion but the crucial roles of α-L-arabinofuranosidase in fruit development or softening remain unclear. Further biochemical and physiological studies of the enzyme are required.

A Reevaluation of the Key Factors That Influence Tomato Fruit Softening and Integrity

The softening of fleshy fruits, such as tomato (Solanum lycopersicum), during ripening is generally reported to result principally from disassembly of the primary cell wall and middle lamella. However, unsuccessful attempts to prolong fruit firmness by suppressing the expression of a range of wall-modifying proteins in transgenic tomato fruits do not support such a simple model. 'Delayed Fruit Deterioration' (DFD) is a previously unreported tomato cultivar that provides a unique opportunity to assess the contribution of wall metabolism to fruit firmness, since DFD fruits exhibit minimal softening but undergo otherwise normal ripening, unlike all known nonsoftening tomato mutants reported to date. Wall disassembly, reduced intercellular adhesion, and the expression of genes associated with wall degradation were similar in DFD fruit and those of the normally softening 'Ailsa Craig'. However, ripening DFD fruit showed minimal transpirational water loss and substantially elevated cellular turgor. This allowed an evaluation of the relative contribution and timing of wall disassembly and water loss to fruit softening, which suggested that both processes have a critical influence. Biochemical and biomechanical analyses identified several unusual features of DFD cuticles and the data indicate that, as with wall metabolism, changes in cuticle composition and architecture are an integral and regulated part of the ripening program. A model is proposed in which the cuticle affects the softening of intact tomato fruit both directly, by providing a physical support, and indirectly, by regulating water status.

Isolation and characterization of a tomato non-specific lipid transfer protein involved in polygalacturonase-mediated pectin degradation

An important aspect of the ripening process of tomato fruit is softening. Softening is accompanied by hydrolysis of the pectin in the cell wall by pectinases, causing loss of cell adhesion in the middle lamella. One of the most significant pectin-degrading enzymes is polygalacturonase (PG). Previous reports have shown that PG in tomato may exist in different forms (PG1, PG2a, PG2b, and PGx) commonly referred to as PG isoenzymes. The gene product PG2 is differentially glycosylated and is thought to associate with other proteins to form PG1 and PGx. This association is thought to modulate its pectin-degrading activity in planta. An 8 kDa protein that is part of the tomato PG1 multiprotein complex has been isolated, purified, and functionally characterized. This protein, designated 'activator' (ACT), belongs to the class of non-specific lipid transfer proteins (nsLTPs). ACT is capable of 'converting' the gene product PG2 into a more active and heat-stable form, which increases PG-mediated pectin degradation in vitro and stimulates PG-mediated tissue breakdown in planta. This finding suggests a new, not previously identified, function for nsLTPs in the modification of hydrolytic enzyme activity. It is proposed that ACT plays a role in the modulation of PG activity during tomato fruit softening.

CHARACTERIZATION OF DFD (DELAYED FRUIT DETERIORATION): A NEW TOMATO MUTANT

Fruit softening is a complex physiological event and a major determinant of postharvest fruit quality. Attempts to understand the molecular basis of softening have focused on cell wall metabolism and it is now generally believed that fleshy fruits soften as a consequence of polysaccharide degradation in the primary cell wall and middle lamella. Tomato has provided the principal model system for these studies and yet efforts over the last ten years to substantially reduce softening in transgenic tomato lines by suppressing the expression of genes encoding cell wall degrading proteins, such as polygalacturonase (PG), pectin methylesterase (PME) and expansin, have been largely unsuccessful. We have recently identified a tomato line that we named the DFD (delayed fruit deterioration) mutant, which promises to provide a new perspective of the key molecular determinants of softening and other postharvest quality traits. The two major characteristics of DFD are substantially reduced fruit softening and complete resistance to postharvest disease, which collectively result in a remarkable shelf life. Unlike other well known ripening-related tomato mutants such as rin (ripening inhibitor) and nor (non-ripening), DFD fruits undergo essentially normal climacteric ripening while attached to the plant, with all the organoleptic characteristics and quality properties that are essential for commercialization, such as color, aroma and accumulation of sugars. However, the DFD fruits also maintain a firm texture for a dramatically extended period of time after harvesting and can be stored with no further treatment at room temperature for more than seven months with any signs off deterioration. Moreover, after more than one year, intact and untreated DFD tomatoes show no signs of microbial infection. These data suggest the need for a new model to understand the key features that underlay tomato fruit softening and tissue disintegration.

Application of CP-MAS and liquid-like solid-state NMR experiments for the study of the ripening-associated cell wall changes in tomato

13C and 1H NMR spectra of an ethanol insoluble material (EIM) prepared from the pericarp of mature-green (MG) and red-ripe (RR) tomato fruits were acquired in ‘liquid-like’ and cross-polarisation with dipolar decoupling and magic angle spinning (CPMAS) conditions using the same triple resonance probe. Such a strategy allowed acquisitions of various NMR experiments aimed at detecting compositional differences as well as distinguishing differences in molecular mobility for various constituent polysaccharides related with the two ripening stages. Increase of the proton dipolar decoupling power levels from 3 to 50–55 kHz during single pulse 13C acquisition, led to more intense signals for pectic and hemicellulosic polysaccharides. This behaviour was interpreted as reflecting motional restrictions of these polysaccharides inside the porous cell wall network. Measurements of the proton rotating frame relaxation times T 1ρ in the ‘liquid-like’ conditions and of the proton transverse relaxation times T 2 from CPMAS spectra, revealed changes in mobilities for some pectic polysaccharides in relation with ripening, particularly for the H1 and H5 protons of α-1,5 arabinan (Ara) side chains of rhamnogalacturonans. These data are discussed in relation with known pectic modifications occurring during ripening and associated with the tomato fruit softening.

Characterisation of an endo-(1,4)-β-mannanase ( LeMAN4) expressed in ripening tomato fruit

A cDNA, LeMAN4, containing an open-reading frame of 1185 bp and putatively encoding an endo-(1,4)-β-mannanase (EC 3.2.1.78) was isolated from red tomato ( Lycopersicon esculentum Mill.) fruit. Native PAGE IEF of fruit protein extracts revealed a single mannanase of p I 9, close to the predicted p I of 8.8 for the mature protein ( M r of 42,400) encoded by LeMAN4. In mature green fruit LeMAN4 mRNA was detectable, increased markedly at the breaker stage and remained at high levels thereafter. endo-(1,4)-β-Mannanase activity, however, was almost undetectable in the early stages of ripening and did not increase significantly until the pink and red stages of ripeness, possibly reflecting post-transcriptional regulation of the enzyme. Cell wall analysis revealed no net change in non-cellulosic mannosyl residues during ripening, suggesting cell wall mannans may not be the in vivo substrate of this enzyme. Southern analysis indicated that LeMAN4 is a single copy gene although several weakly hybridising bands imply the presence of other related genes. LeMAN4 is not fruit-specific, being expressed in flowers but not in leaves, and is probably not rate-limiting in the ripening-related softening of tomato fruit.

Ethylene and Glycosidase Promotion in GA3- and IAA-treated Tomato Fruit (Lycopersicon esculentum Mill.)

Transient (1 h) treatment of breaker tomatoes (Lycopersicon esculentum Mill, cv. Bonanza) with exogenous GA 3 or IAA at a high concentration (20 mM) resulted in a two- to four-fold increase compared with the control in ethylene biosynthesis during a 9-day experiment. This sharp increase in ethylene emission is characteristic of a stress response. Both phytohormones promoted the activity of 1-aminocyclopropane-I-carboxylate synthase that probably accounts for most of the enhanced ethylene synthesis. GA 3 and IAA also stimulated total α- and (3-galactosidase and α-L-arabinofuranosidase activity but showed some potential to delay ripening parameters, among them, fruit softening, chlorophyll loss, and total carotenoid synthesis. GA 3 - or IAA-treated fruit did not respond to exogenous 100 ppm C 2 H 4 with an increase of autocatalytic ethylene production. Moreover, GA 3 or IAA applied alone showed a faster increase in ethylene biosynthesis than that achieved by exogenous C 2 H 4 . The combination of GA 3 and C 2 H 4 -supplemented atmosphere did not result in synergistic effects on glycosidase activity except for a few cases. IAA-treated fruit exposed to C 2 H 4 -supplemented atmosphere did not promote additional glycosidase activity but rather seemed to have antagonistic effects on β-galactosidase during the first few days of the experiment. Glycosidase response to GA 3 and IAA treatments did not correlate with changes in tomato pericarp firmness, thus suggesting that some isoforms may have no role in tomato fruit softening.

A simple non-destructive method for laboratory evaluation of fruit firmness

Summary. Devices which offer simple, inexpensive, reliable and non-destructive objective measurement of fruit firmness assist in the monitoring of quality. For the present study, the Analogue CSIRO Tomato Firmness Meter (AFM), which measures fruit deformation under a 500 g load applied for 30 s was modified by replacing the analogue displacement gauge with a digital gauge and by using a laboratory jack for positioning the fruit in the vertical dimension. Non-destructive measurements of tomato fruit softening during ripening and determined with the Digital Firmness Meter (DFM) were strongly correlated with both firmness measured with the AFM (r2 = 0.96, n = 19) and with firmness determined subjectively by hand pressure (r2 = 0.93, n = 19). Similarly, mango fruit softening during ripening was monitored and DFM and hand firmness measurements were well correlated (r2 = 0.95, n = 10). The firmness of individual fruit could be measured around 20% faster with the DFM than with the AFM, and displacement was easier to read from the digital than from the analogue display. The DFM proved to be a suitable device for measuring fruit firmness in postharvest laboratory studies and warrants evaluation under commercial packing and handling conditions.

Chilling‐Associated Softening of Tomato Fruit is Related to Increased Pectinmethylesterase Activity

ABSTRACTMature‐green tomatoes chilled 15 days (5°C; RH >85%) were softer than nonchilled during subsequent ripening (22°C) by both whole fruit and pericarp tissue puncture (p<0.05), but not by flat‐plate compression. No differences in total polygalactnronase (PG) or PG isozyme activity were evident although total activity was greater in nonchilled after 10 days ripening. Softening of nonchilled fruit correlated (p<0.05) with extracted PGI activity, while chilling‐associated softening correlated (p<0.05) with higher initial extracted pectinmethylesterase (PME) activity. Extracted peroxidase remained constant throughout ripening but was greater (p<0.05) in pre‐chilled fruit consistent with chill‐induced membrane dysfunction. Transmission electron microscopy showed the middle lamella from pre‐chilled tomatoes was swollen and less defined. Loss of turgor from translocation of water to the PME‐modified cell wall was suggested to be responsible for softening as a consequence of chilling.

THE IMPORTANCE OF α AND β-GALACTOSIDASE IN TOMATO FRUIT RIPENING

Antisense technology has shown that neither polygalacturonase nor pectin methylesterase alone are responsible for tomato fruit softening, leading to the likelihood that other enzymes or factors are important. Our laboratory recently found that α and β-galactosidase from avocado fruit solubilized tomato fruit pectin in vitro. Previously, Pressey (Plant Physiol. 1983,71:132) found that the activity of one of three α-galactosidase isozymes from tomato fruit increased during ripening and was capable of degrading cell wall galactan, suggesting a role for the enzyme in fruit softening. Increased β-galactosidase activity was observed in a number of other fruit during ripening. In the present study, NaCl extraction of tomato pericarp yielded relatively high levels of cc- and β-galactosidase activity. At least two isozymes of each were resolved during Mono-Q HPLC α-Galactosidase was further purified by additional Mono Q and Superose 12 gel filtration HPLC. Gel filtration and SDS-PAGE yielded an apparent molecular weight of 44 kD. The partially pure α-galactosidase had a specific activity of 294 μmol product/min per mg protein, a Km of 317 μm, a pl of 5.0, and a pH optimum of 5.5. Activity was inhibited 67% by α-d-galactose. Preliminary results show that β-galactosidase can also be purified by the same techniques. Following further purification, the isozymes will be sequenced and cloned. A second approach being used in an attempt to identify cDNA clones for the α- and β-galactosidase genes from tomato fruit involves using heterologous cDNA clones from guar (Overbeeke et al., 1989; Plant Molecular Biology 13:541-550) and carnation (Raghothama et al., 1991; Plant Molecular Biology 17:61-71), respectively, to screen a ripening tomato fruit cDNA library. Basic molecularbiological techniques will be used to elucidate the role of these enzymes in tomato fruit ripening.

Changes in physical properties and cell wall polysaccharides of tomato (Lycoperskon esculentum) pericarp tissues

Green and red tomato pericarp tissues were subjected to stress‐relaxation analyses to evaluate their physical properties. Significant decreases in the initial stress, minimum stress‐relaxation and maximum stress‐relaxation times in the red tissues predict the losses of both viscosity and elasticity in the tissue. Cell walls of red fruit yielded more water‐soluble polysaccharides and less pectin, hemicelluloses and cellulose. Average molecular mass of pectin determined by gel filtration chromatography was similar in the green and red, but molecular mass of hemicellulose of red fruit walls was reduced to 50% of that of the green fruit. The decreases in the amount of hemicellulose B and in the average molecular mass were associated primarily with the degradation of xylo‐glucans. These data demonstrate that pectin solubilization, depolymerization of xyloglucans and over‐all changes in the quantity of cell wall polysaccharide fractions contribute to tomato fruit softening.

Inhibition of Cell Wall Degradation by Silver (I) Ions during Ripening of Tomato Fruit

Summary Infiltration of silver (I) ions into tomato fruit, after the onset of ripening, prevented the normal changes in the total uronic acid and neutral sugar contents of the pericarp tissue. This treatment also inhibited the increased solubilisation of polyuronide which normally accompanies ripening. The mechanism for these inhibitions is not known. However, the results described here and those of a previous report (Tucker and Brady, 1987) are consistent with the proposal that continued polygalacturonase (EC 3.2.1.15) synthesis is necessary for the maintenance of pectin degradation, and hence tomato fruit softening, during ripening.

Effects of NaCl, pH and Ca2+ on autolysis of isolated tomato fruit cell walls

Cell walls isolated from ripening tomato (Lycopersicon esculentum Mill. cv. Rutgers) fruit released pectic polymers when incubated under conditions that allow activity of wall‐bound polygalacturonase (EC 3.2.1.15). Autolysis was optimally stimulated by 150–300 mM NaCl at either pH 2.5 or 4.5. This stimulation was negated by exposure to pH 6.5 or higher and by pretreatment of walls with boiling 80% ethanol. Five mM CaCl2 did not affect autolysis at pH 2.5, but significantly inhibited at pH 4.5 or higher. Inclusion of 1 M NaCl at selected steps in the extraction scheme did not inhibit subsequent autolysis of isolated walls. Exposure of isolated walls to 1 M NaCl at pH 2.5–8.5 also did not inhibit autolytic activity compared to walls that received no ionic treatment. These data support the concept that cell wall hydrolysis during tomato fruit softening is regulated by pH, Ca2+ levels and ionic strength of the apoplast.

In Vitro Characterization of Tomato Fruit Softening

Cell wall isolated from pericarp of normal tomato (Lycopersicon esculentum Mill. cv ;Rutgers') fruit released pectic polymers in a reaction apparently mediated by wall-bound polygalacturonase that appears with the onset of ripening. Release was negligible in wall preparations from normal green and the ripening mutant rin fruit. Pectin solubilization was most extensive at pH 2.5 with a less significant peak at 5.5. Brief exposure to low (1.5) or high (7.5) pH resulted in reduction of autolytic activity, which was also inhibited by high temperature, Ca(2+), and treatments employed to dissociate protein from cell wall. Uronic acid solubilization was significantly enhanced by 150 millimolar NaCl and by increasing temperature within the physiological range. These data indicate that the release of polyuronide from isolated cell walls is enzymic and may provide a convenient and reliable system for the study of softening metabolism.

Pectic enzymes associated with the softening of tomato fruit

Some degree of softening of maturing tomato fruit takes place before the usually accepted mechanism for the loss of firmness, involving the sequential action of pectin esterase and polygalacturonase, is operative to any extent. The possibility that pectin and pectic acid transeliminases were implicated, providing an alternative pathway for the breakdown of pectin in the tomato, was investigated. Direct spectrophotometric determination of these enzymes proved unreliable and the thiobarbituric acid test, which should give a specific colour reaction with the products, was negative. It is concluded that transeliminases do not play a significant part in the softening of tomato fruit either prior to or during ripening.