Red Ripe Tomato Research Articles

Developmental gene regulation during tomato fruit ripening and in-vitro sepal morphogenesis

BackgroundRed ripe tomatoes are the result of numerous physiological changes controlled by hormonal and developmental signals, causing maturation or differentiation of various fruit tissues simultaneously. These physiological changes affect visual, textural, flavor, and aroma characteristics, making the fruit more appealing to potential consumers for seed dispersal. Developmental regulation of tomato fruit ripening has, until recently, been lacking in rigorous investigation. We previously indicated the presence of up-regulated transcription factors in ripening tomato fruit by data mining in TIGR Tomato Gene Index. In our in-vitro system, green tomato sepals cultured at 16 to 22°C turn red and swell like ripening tomato fruit while those at 28°C remain green.ResultsHere, we have further examined regulation of putative developmental genes possibly involved in tomato fruit ripening and development. Using molecular biological methods, we have determined the relative abundance of various transcripts of genes during in vitro sepal ripening and in tomato fruit pericarp at three stages of development. A number of transcripts show similar expression in fruits to RIN and PSY1, ripening-associated genes, and others show quite different expression.ConclusionsOur investigation has resulted in confirmation of some of our previous database mining results and has revealed differences in gene expression that may be important for tomato cultivar variation. We present new and intriguing information on genes that should now be studied in a more focused fashion.

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.

Cell wall metabolism in ripening fruit. VIII. Cell wall composition and synthetic capacity of two regions of the outer pericarp of mature green and red ripe cv. Jackpot tomatoes

Pericarp discs were excised from mature green and red ripe tomato (Lycopersicon esculentum Mill. cv. Jackpot) fruit and kept in sterile tissue culture plates for 4 days, including 2 days of incubation with D‐[U‐13C]‐glucose. Cell walls were prepared and differentially extracted with dimethylsulfoxide (DMSO), trans‐1,2‐diaminocyclohexane‐N,N,N′,N′‐tetraacetic acid (CDTA). Na2CO3, 4 M KOH and 8 M KOH. Cell wall noncellulosic neutral sugar (NS) composition and cell wall synthetic capacity (i.e. incorporation of density label into cell wall sugars) were determined by using a gas chromatograph coupled to a flame ionization detector and a mass spectrometer, respectively. In the crude cell wall, there was significantly less galactose (Gal) and glucose (Glc) in the “outer”2‐mm pericarp region, including the cuticle, compared to the “inner”2‐mm region immediately below it (closer to the locules). In the CDTA‐soluble pectin, rhamnose (Rha), arabinose (Ara) and Gal accounted for approximately 90% of the total NS. The ratios of these sugars were very similar in the total (12C plus 13C) sugars, and also in the newly synthesized ([13C]‐labeled) sugars, suggesting that newly synthesized NS associated with the chelator‐extractable pectic fraction has a composition very similar to that of preexisting NS. In the 4 M KOH‐soluble material, xylose (Xyl) and Glc accounted for approximately 70% of the total NS. The ratio of these sugars was very similar in the total sugars, but much lower in the newly synthesized portion. This suggests that the hemicellulosic polymers synthesized during the ripening process are different in type and/or proportion from those present in the developing fruit. Because the outer pericarp of tomatoes contains at least two distinct tissue types and these have a distinct cell wall composition, analysis of tomato cell wall polysaccharide composition by homogenization of the entire outer pericarp will obscure subtle changes associated with ripening/softening within specific tissue types.

Heat Stress and Chilling Sensitivity of Tomato Fruit

Mature green `Sunbeam' tomato fruit (Lycopersicon esculentum Mill.) were treated in water for 1 hr at 27 (ambient), 39, 42, 45, or 48°C, and then either ripened at 20°C (nonchilled) or stored at 2°C (chilled) for 14 days before ripening at 20°C. The most-effective heat treatment was 42°C, which reduced decay 67% in chilled fruit and 53% in nonchilled fruit. Heat treatment had no effect on time required to ripen the fruit. Red-ripe tomatoes had higher respiration rates and evolved more ethylene following nonchilling storage, but heat treatment had no effect on respiration or ethylene evolution. Red color development was enhanced by heat treatment, and inhibited by chilling. At red ripe, fruit were firmer as a result of storage at the chilling temperature, while heat treatment had no effect on firmness. Heat-treated fruit were preferred in terms of taste and texture over nontreated fruit in informal taste tests, with the exception of the 45°C treatment. With increasing temperature of heat treatment, there was increased electrolyte leakage following chilling storage. Of the 15 flavor volatiles analyzed, the levels of five were decreased with increasing temperature of heat treatment. Storage at the chilling temperature reduced the levels of six flavor volatiles. Prestorage heat treatments can reduce decay with only minimal adverse effects on tomato fruit quality.

Molecular cloning of a ripening-specific lipoxygenase and its expression during wild-type and mutant tomato fruit development.

A 94-kD protein that accumulates predominately in tomato (Ly-copersicon esculentum) fruit during ripening was purified, and antibodies specific for the purified protein were used to isolate cDNA clones from a red-ripe fruit cDNA library. A sequence analysis of these cDNAs and cross-reactivity of the 94-kD-specific antibodies to the soybean lipoxygenase (LOX) L-1, L-2, and L-3 proteins and soybean LOX L-1-specific antibodies to the 94-kD protein identified it as a member of the LOX gene family. Maximum levels of the 94-kD LOX mRNA and protein are present in breaker to ripe and red-ripe stages, respectively. Expression of 94-kD LOX in different tissues from mature green and red-ripe tomato fruits was found to be greatest in the radial walls of ripe fruit, but immunocytolocalization using tissue printing suggests that the highest accumulation of its protein occurs in locular jelly. None of 94-kD LOX is expressed in nonripening mutant fruits of any age. Never-ripe mutant fruit accumulate the 94-kD LOX mRNA to levels similar to those obtained in wild-type fruit, but fail to accumulate the 94-kD LOX protein. Collectively, the results show that expression of 94-kD LOX is regulated by the ripening process, and ethylene may play a role in its protein accumulation.

Polymer mobility in cell walls of transgenic tomatoes with reduced polygalacturonase activity

Cell walls were prepared from unripe and red-ripe tomato fruit, cv. Ailsa Craig, and from ripe transgenic fruit carrying antisense genes downregulating the ripening-related polygalacturonase activity. The cell walls were examined by 13C NMR using a cross-polarization/magic-angle spinning experiment with variable contact time to estimate the proton magnetic relaxation parameter T 1 p , which is sensitive to molecular motions on the kHz timescale and hence to the rigidity of the polymer network of the cell walls; and the cross-polarization time constant T CH which is sensitive to similar, and more local, motional effects. The proton T 1 p values for the 13C resonances from each cell wall preparation were separated into two groups. One of these showed slow relaxation, implying low mobility, and included the 13C resonances characteristic of cellulose. The other, corresponding to resonances characteristic of pectins only, showed two-component relaxation behaviour with one component relaxing much faster than the previous group, and hence corresponding to the pectic polymers in the middle lamella and perhaps also between the microfibrils. The T 1 p values for the first group shortened on ripening as polymer mobility increased within the cell wall. This was still evident, but less so, in the PG-antisense material, showing that the antisense genes reduced the softening of the cell wall, but did not do so as much as might be expected from the almost complete loss of polygalacturonase activity. The T CH was separated into very fast and slower components corresponding to the times required for local and larger-scale magnetization transfer.

TOMATO POLYGALACTURONASE EXTRACTABILITY

The extractability of polygalacturonase (PG) activity from alcohol-insoluble solids (AIS) prepared from red-ripe tomato pericarp tissue was investigated using acetate (pH 4.5), citrate (pH 4.5) and MES (pH 6.0) buffers. The acetate buffer was also investigated with increasing concentrations of NaCl, CaCl 2 , MgCl 2 , ethylenediaminetetraacetic acid (EDTA) or ethyleneglycol-bis(β-aminoethylether)-N,N'-tetraacetic acid (EGTA). Heat treatment of AIS to prepare control material Suppressed PG extractability but not in situ PG activity, as evidenced by the release of significant quantities of soluble uronides into extraction media. With only a single extraction, acetate buffer containing > 1 M NaCl yielded the greatest amount of PG activity (> 50% of maximum extractability), with MES buffer being only marginally less efficient. However, 3 successive extractions over a 2 h period yielded significantly greater amounts of PG activity. The greatest yields of PG activity were obtained by successive extraction with parent MES and acetate buffers. There appeared to be little benefit in adding NaCl or a chelating agent to the extraction medium. Use of these extractants is suggested to have led to losses of PG activity during dialysis, via coprecipitation of PG protein with otherwise soluble uronide material that was released in greater quantities when these extractants were used. Increasing CaCl 2 and MgCl 2 concentrations reduced the amount of extracted PG activity similarly, to about 50% of maximum levels with successive extractions.

STUDIES ON RHAMNOGALACTURONASE IN FRUIT

Rhamnogalacturonase (RGase) is a new fungal enzyme which degrades the highly branched regions of apple fruit cell wall pectin by cleaving the glycosyl linkage between rhamnosyl and galacturonosyl residues (Schols et al., 1990. Carhohydr. Res. 206:105.). This enzyme, if present in fruit, could play a significant role in fruit softening. Partial purification of RGase was accomplished from a fungal enzyme preparation (Pectinex Ultra SP-L, NOVO Ferment) produced from Aspergillus niger. The crude enzyme hydrolyzed chelator-soluble pectin from red ripe tomato fruit. Methylation linkage analysis of the product suggested that an increase in terminal-rhamnosyl residues accompanied pectin hydrolysis, indicative of RGase activity. Cross-linked alginate, hydroxyapatite, and DEAE-Sephadex chromatography were used to partially purify RGase. Polygalacturonase was efficiently removed using the alginate column. Crude pectin obtained from mature-green tomato fruit cell wall by extracting with 0.5 M imidazole buffer (pH 7) and 50 mM Na-carbonate was incubated with pure polygalacturonase and the residue hydrolyzed with 0.1 N trifluoroacetic acid. This modified pectin was used as a substrate to investigate the presence of RGase in tomato and other fruit.

Controlling Relative Humidity in Modified Atmosphere Packages of Tomato Fruit

The feasibility of controlling relative humidity in modified atmosphere packages using compounds possessing Type III sorption isotherm behavior was studied. Ten grams each of dry sorbitol, xylitol, NaCl, KCl, or CaCl2 sealed with one maturegreen tomato (Lycopersicon esculentum L.) fruit at 20C in simulated packages for 48 days resulted in stable relative humidities of ≈75%, 80%, 75%, 85%, and 35%, respectively. Relative humidity was a function of the ratio of chemical to fruit mass. Relative humidities within control packages were in the range of 96% to 100% throughout the experiments. A simple system that uses spunbonded polyethylene pouches for the application of this humidity control method to packages is described. The storage life of packaged red-ripe tomato fruit at 20C was extended from 5 days using no pouch to 15 to 17 days with a pouch containing NaCl, mainly by retardation of surface mold development.

Effect of food preparation on qualitative and quantitative distribution of major carotenoid constituents of tomatoes and several green vegetables

The major carotenoid constituents of extracts from several raw and cooked green vegetables (broccoli, green beans, spinach), red ripe tomatoes, and tomato paste have been identified and quantified by high-performance liquid chromatography on a C18 reversed-phase column. The predominant carotenoids in raw green vegetables were neoxanthin, violaxanthin, lutein epoxide, lutein, alpha-carotene, and beta-carotene. The carotenoids in tomatoes and tomato paste were lutein, 5,6-dihydroxy-5,6-dihydrolycopene, lycopene 1,2-epoxide, lycopene 5,6-epoxide, lycopene, neurosporene, gamma-carotene, zeta-carotene, beta-carotene, phytofluene, and phytoene. The effect of various means of cooking on the levels of carotenoids in raw and cooked (microwaved, boiled, steamed, stewed) green vegetables and tomatoes has been extensively studied. It was shown that while the epoxycarotenoids were somewhat sensitive to heat treatment, lutein and hydrocarbon carotenoids such as neurosporene, alpha- and beta-carotene, lycopene, zeta-carotene, phytofluene, and phytoene survived the heat treatments.

Stimulation of ethylene production by a cell wall component from mature green tomato fruit

Pectic (carbonate‐soluble, covalently‐bound pectin, CBP) material stimulated increased ethylene production when vacuum‐infiltrated into whole, mature green tomato (Lycopersicon esculentum Mill. cv. Rutgers) fruit. Activity was greatest if CBP was extracted from mature green tomatoes with jellied locules. CBP extracted from mature green tomatoes with immature seeds had no elicitor activity, while CBP from turning or red ripe tomatoes was only moderately active. Infiltration of CBP from normal mature green fruit into ripening inhibitor (rin) mutant tomato fruit stimulated ethylene production and attenuated red pigmentation in these fruits. Partial purification of the active material was accomplished using DEAE‐Sephadex and BioGel P‐100 chromatography. The most highly purified fraction is comprised of neutral carbohydrate (95%) with a relatively low content of amino acids (1%) and a uronic acid content of less than 5%. This material may be an endogenous trigger of ethylene production and ripening.

Molecular cloning of tomato fruit polygalacturonase: Analysis of polygalacturonase mRNA levels during ripening

The expression of a gene encoding the cell wall-degrading enzyme polygalacturonase [poly(1,4-alpha-D-galacturonide) glucanohydrolase, EC 3.2.1.15] was characterized during tomato fruit ripening. Polygalacturonase was purified from ripe tomato fruit and used to produce highly specific antiserum. Immunoblot analyses detected a 45- and a 46-kDa protein in ripe fruit but immunoprecipitation of in vitro translation products of mRNA from ripe tomato fruit yielded a single 54-kDa polypeptide, suggesting post-translational processing. A plasmid cDNA library was prepared from poly(A)(+) RNA isolated from ripe tomato fruit. The cDNA library was inserted into a lambda-based expression vector, and polygalacturonase cDNA clones were identified by immunological screening. Hybrid-select translation experiments indicated that the cDNAs encode a 54-kDa in vitro translation product that is specifically immunoprecipitated with polygalacturonase antiserum. RNA-blot analysis indicated that the 1.9-kilobase polygalacturonase mRNA was virtually absent from immature-green fruit, accumulated steadily during the ripening process, and was at its highest level in red-ripe fruit. There was at least a 2000-fold increase in the level of polygalacturonase mRNA between immature-green and red-ripe tomato fruit. These studies show that the levels of polygalacturonase mRNA are developmentally regulated during tomato fruit ripening.

Near Infrared Reflectance of Colored Fruits

The near ir (NIR) spectral properties of colored fruits were measured and showed a wide variation between species. Within a species, NIR reflectance was found to vary with the degree of ripeness of the fruit, particularly for the softer fruits. Extreme values of NIR reflectances were 75% for unripe pears and 21% for the red-ripe tomatoes. This variation is considerably larger than would have been expected from similar studies with leaves and grains. Solar reflectances were calculated for the NIR region for possible use in subsequent energy balance studies. Results from this study indicate that soft fruits will have a solar energy input 10-20% greater than for hard fruits, due to a 20-40% difference in NIR spectral properties. Softening accompanying ripening of tomatoes increases solar heat input by up to 10%.

Precursors of volatile components in tomato fruit-III. Enzymatic reaction products

The volatile components produced by enzyme extracts from tomato fruit using amino acid substrates were analysed by gas and thin-layer chromatography. Alanine, leucine, and valine were among the best substrates. Extracts from red-ripe tomatoes were more active than those from green fruit. The products differed somewhat with each preparation and with the particular substrate used. Propanal was present in most reaction products especially when alanine was used as substrate. With enzymes from red-ripe fruit, alanine gave mainly carbonyl compounds while leucine and valine gave mainly alcohols. Mixed amino acids sometimes resulted in new volatile components. The mechanism appears to involve transamination.

Gas chromatography of the field-, glass- Greenhouse-grown, and artificially ripened tomatoes.: Lycopersicon esculentum mill.

Fifteen distinct peaks were resolved from red-ripe tomato flavor extract by gas Chromatographic techniques. Except isobutanol, n-hexanol, and n-hexanal, concentrations of the volatile components in the field-grown tomatoes were higher than those in the greenhouse-grown tomatoes. Artificially ripened tomatoes contained less volatile components.

Pectolytic Enzyme Activity in Various Parts of the Cucumber Plant and Fruit

1. The cucumber plant and fruit (Cucumis sativus) have been found to be a source of a pectolytic enzyme as measured by a loss in viscosity of a pectin solution. 2. The enzyme of the cucumber was strongly active in the seeds, staminate and pollinated pistillate flowers, and ripe fruit but was not found in the unpollinated flowers, leaves, petioles, and stems. 3. Enzyme activity was weak to negative in the eight stages of cucumber development of immature fruit. 4. Pectolytic enzyme activity was absent in the green tomato (Lycopersicon esculentum) including the embryo with flowers and six stages of green-fruit development. 5. High activity of the enzyme was found in the red ripe tomato fruit.