Genes from Lycopersicon chmielewskii affecting tomato quality during fruit ripening

Three chromosomal segments from the wild tomato, L. chmielewskii, introgressed into the L. esculentum genome have been previously mapped to the middle and terminal regions of chromosome 7 (7M, 7T respectively), and to the terminal region of chromosome 10 (10T). The present study was designed to investigate the physiological mechanisms controlled by the 7M and 7T segments on tomato soluble solids (SS) and pH, and their genetic regulation during fruit development. The effects of 7M and 7T were studied in 64 BC₂F₅ backcross inbred lines (BILs) developed from a cross between LA 1501 (an L. esculentum line containing the 7M and 7T fragments from L. chmielewskii), and VF145B-7879 (a processing cultivar). BILs were classified into four homozygous genotypes with respect to the introgressed segments based on RFLP analysis, and evaluated for fruit chemical characteristics at different harvest stages. Gene(s) in the 7M fragment reduce fruit water uptake during ripening increasing pH, sugars, and SS concentration. Gene(s) in the 7T fragment were found to be associated with higher mature green fruit starch concentration and red ripe fruit weight. Comparisons between tomatoes ripened on or off the vine suggest that the physiological mechanisms influenced by the L. chmielewskii alleles are dependent on the translocation of photosynthates and water during fruit ripening.     

Wound ethylene and 1-aminocyclopropane-1-carboxylate synthase in ripening tomato fruit

Ethylene production, 1-aminocyclopropane-1-carboxylic acid (ACC) levels and ACC-synthase activity were compared in intact and wounded tomato fruits (Lycopersicon esculentum Mill.) at different ripening stages. Freshly cut and wounded pericarp discs produced relatively little ethylene and had low levels of ACC and of ACC-synthase activity. The rate of ethylene synthesis, the level of ACC and the activity of ACC synthase all increased manyfold within 2 h after wounding. The rate of wound-ethylene formation and the activity of wound-induced ACC synthase were positively correlated with the rate of ethylene production in the intact fruit. When pericarp discs were incubated overnight, wound ethylene synthesis subsided, but the activity of ACC synthase remained high, and ACC accumulated, especially in discs from ripe fruits. In freshly harvested tomato fruits, the level of ACC and the activity of ACC synthase were higher in the inside parts of the fruit than in the pericarp. When wounded pericarp tissue of green tomato fruits was treated with cycloheximide, the activity of ACC synthase declined with an apparent half life of 30–40 in. The activity of ACC synthase in cycloheximide-treated, wounded pericarp of ripening tomatoes declined more slowly.Abbreviation ACC 1-aminocyclopropane-1-carboxylic acid     

Expression of phytoene synthase gene (Psy) is enhanced during fruit ripening of Cara Cara navel orange (Citrus sinensis Osbeck)

Citrus is an important fruit crop as regards accumulation of carotenoids. In plant carotenoid biosynthesis, phytoene synthase gene (Psy) plays a key role in catalyzing the head-to-head condensation of geranylgeranyl diphosphate molecules to produce colorless phytoene. In the present paper, we reported the phytoene contents determination and characterization of Psy during fruit ripening of “Washington” navel orange and its red-fleshed mutant “Cara Cara”. Results showed that phytoene was exclusively accumulated in peel and pulp of “Cara Cara”. Although phytoene was observed accumulating with fruit ripening of “Cara Cara”, the contents in pulp were 10 times higher than those in peel. The isolated two Psy cDNAs were both 1520 bp in full length, containing 436 deduced amino acid residues, with a different amino acid at 412th. Genomic hybridization results showed that one or two copies might be present in “Cara Cara” and “Washington” genomes. During “Cara Cara” and “Washington” fruit coloration, expression of Psy was observed to be up-regulated, as revealed by tissue specific profiles in the flavedo, albedo, segment membrane and juice sacs. However, Psy expression in albedo of “Cara Cara” was higher than that in “Washington”, as evidenced by phytoene accumulation in the peel.     

Production of phytoene, a carotenoid, and induction of connexin 26 in transgenic mice carrying the phytoene synthase gene crtB

Carotenoids have been recognized as chemopreventive agents against human diseases, such as cancer and cardiovascular disease. Mammalians utilize carotenoids supplied from their food since they are unable to perform the de novo synthesis of carotenoids. We previously created mammalian cultured cells producing phytoene, a type of carotenoid, and showed that these cells acquired resistance against oxidative stress and oncogenic transformation. In the present study, we established a transgenic mouse line, carrying the crtB gene encoding phytoene synthase, which could produce phytoene endogenously. It was found that connexin 26 was induced in these phytoene-producing mice. Since it is known that carotenoids enhance gap junctional communication by inducing the expression of connexin genes, the present data suggest that the induction of connexin 26 in phytoene-producing mice may play a role in controlling cell-to-cell communication. Phytoene-producing mice provide a useful system in which to investigate the in vivo function of the carotenoid phytoene.     

Transient increase in locular pressure and occlusion of endocarpic apertures in ripening tomato fruit

Locular pressure was monitored during ripening of tomato (Lycopersicon esculentum Mill.) fruit and the anatomy of the endocarp surface examined using scanning electron microscopy. The manometric pressure of the locule tissue increased from 0 in mature-green fruit to 10 to 50 Pa at the turning or pink stages, and then subsided in ripe fruit. Nonclimacteric fruit containing the ripening inhibitor (rin) mutation showed a similar pattern of internal pressure accumulation during senescence. Build-up of locular tissue pressure occurred in fruit ripening, on or off the plant, as well as in fruit with different susceptibility to cuticle cracking. Apertures ranging from 18-31 μm in width and 33-41 μm in length, with densities ranging from 6.7 to 47.9 apertures · mm⁻² were observed in the endocarp of mature-green fruit. These apertures were progressively occluded during early ripening and were absent in late ripening fruit. Aperture occlusion might result in reduced gas exchange between the locule and external fruit atmosphere, resulting in modification of the locular gas composition.     

Synthesis of polygalacturonase during tomato fruit ripening

The cell wall degrading enzyme polygalacturonase (E.C. 3.2.1.15) is not detectable in green tomatoes (Lycopersicon esculentum Mill). Activity appears at the onset of ripening and in ripe fruit it is one of the major cell-wall-bound proteins. Radioimmunoassay results, employing an antibody against purified polygalacturonase, suggest that during ripening the enzyme is synthesised de novo. Radioimmunoassay data also show that the low level of polygalacturonase in Never ripe mutants and the lack of activity in ripening inhibitor mutants can be correlated to the levels of immunologically detectable polygalacturonase protein.Abbreviations PG polygalacturonase - Nr Never ripe mutation - rin ripening inhibitor mutation     

The effect of reduced activity of phytoene synthase on isoprenoid levels in tomato pericarp during fruit development and ripening

Carotenoids, gibberellins (GAs), sterols, abscisic acid and -amyrins were analysed in tomato (Lycopersicon esculentum Mill.) pericarp during fruit development and ripening. The contents of these isoprenoids in wild-type (cv. Ailsa Craig) fruit were compared with those in fruit of the carotenoid-deficient R-mutant and a transgenic plant containing antisense RNA to a phytoene synthase gene. In both carotenoid-deficient genotypes, a 14-fold reduction in carotene and twofold decrease in xanthophyll content, compared to the wild type, was found in ripe fruit. Immature green fruit from wild type and R-mutant plants contained similar amounts of the C₁₉-GAs, GA₁, and GA₂₀, and their C₂₀ precursor, GA₁₉. Immature fruit from the transgenic plants contained three- to fivefold higher contents of these GAs. In wild-type fruit at the mature green stage the contents of these GAs had decreased to < 10% of the levels in immature fruit. A similar decrease in GA₁₉ content occurred in the other genotypes. However, the contents of GA₁ and GA₂₀ in fruit from phytoene synthase antisense plants decreased only to 30% between the immature and mature green stages and did not decrease at all in R-mutant fruit. At the breaker and ripe stages, the contents of each GA were much reduced for all genotypes. The amount of abscisic acid was the same in immature fruit from all three genotypes, but, on ripening, the levels of this hormone in antisense and R-mutant fruit were ca. 50% of those in the wild type. Quantitative differences in the amounts of the triterpenoid -amyrins, total sterols, as well as individual sterols, such as campesterol, stigmasterol and sitosterol, were apparent between all three genotypes during development. Amounts of free sterols of wild type and antisense fruit were greatest during development and decreased during ripening, whereas the opposite was found in the R-mutant. This genotype also possessed less free sterol and more bound sterol in comparison to the other varieties. These data provide experimental evidence to support the concept of an integrated metabolic relationship amongst the isoprenoids.Abbreviations ABA abscisic acid - dpb days post breaker - FDP farnesyl diphosphate - GA gibberellin - GGDP geranyl-geranyl diphosphate We thank Mr. Paul Gaskin (Long Ashton Research Station) for the qualitative GC-MS of triterpenoids and Dr. R. Horgan (University of Wales, Aberystwyth) for a gift of [6-³H₂]ABA. The work was supported by a research grant (No. PG111/617) to P.M.B. from the Agricultural and Food Research Council to whom we express our thanks.     

Photocontrol of anthocyanin biosynthesis in tomato

Juvenile anthocyanin biosynthesis has been studied in dark-grown seedlings of tomato (Lycopersicon esculentum Mill.) wild types (WTs) and photomorphogenic mutants. During a subsequent 24-hr period of monochromatic irradiation at different fluence rates of red light (R) the fluence-rate response relationships for induction of anthocyanin in all the WTs are similar, yet complex, showing a response at low fluence rates (LFRR) followed by a fluence rate-dependent high irradiance response (HIR). In the hypocotyl this response is restricted to the sub-epidermal layer of cells. The high-pigment-1 (hp-1) mutant exhibits a strong amplification of both response components. Theatroviolacea (atv) mutant shows strongest amplification of the HIR component. In contrast, a transgenic line overexpressing an oat phytochrome A gene (PHYA3 ⁺) shows a most dramatic amplification of the LFRR component. The far-red light (FR)-insensitive (fri) mutant, deficient in phytochrome A (phyA), lacks the LFRR component whilst retaining a normal HIR. The temporarily R-insensitive (tri) mutant, deficient in phytochrome B1 (phyB1) retains the LFRR, but lacks the HIR. Thehp-1,fri andhp-1,tri double mutant, exhibit amplified, yet qualitatively similar responses to the monogenicfri andtri mutants. Thefri,tri double mutant lacks both response components in R, but a residual response to blue light (B) remains. Similarly, theaurea (au) mutant deficient in phytochrome chromophore biosynthesis and presumably all phytochromes, lacks both response components in the R and FR regions of the spectrum. Experiments at other wavelengths demonstrate that while there is only a small response in the FR spectral region (729 nm) in tomato, there is an appreciable HIR response in the near FR at 704 nm, which is retained in thetri mutant. This suggests that the labile phyA pool participates in the HIR at this wavelength. The intense pigmentation (Ip) mutant appears to be specifically deficient in the B1 induced anthocyanin biosynthesis. Adult plants, grown under fluorescent light/dark cycles, show a reduction of anthocyanin content of young developing leaves upon application of supplemtary or end-of-day FR. The involvement of different phytochrome species in anthocyanin biosynthesis based on micro-injection studies into theau mutant and studies using type specific phytochrome mutants is discussed.     

A histidine decarboxylase-like mRNA is involved in tomato fruit ripening

DNA sequencing of a tomato ripening-related cDNA, TOM 92, revealed an open reading frame with homology to several pyridoxal 5-phosphate histidine decarboxylases, containing the conserved amino acid residues known to bind pyridoxal phosphate and -fluoromethylhistidine, an inhibitor of enzyme activity. TOM 92 mRNA accumulated during early fruit ripening and then declined. Fruit of the ripeningimpaired tomato mutant, ripening inhibitor (rin), did not accumulate TOM 92 mRNA, and its accumulation was not restored by treatment of fruit with ethylene. The TOM 92 mRNA was not detected in tomato leaves and unripe fruit.     

Characterisation of the ACC oxidase activity in transgenic auxin overproducing tomato during ripening

As part of our studies on the role of auxin in regulating the ethylenebiosynthesis during fruit ripening, in this paper we describe the functionalproperties of the ACC oxidase activity extracted from transgenic tomato(Lycopersicum esculentum Mill. cv. Ailsa craig)overexpressing the tryptophan monooxygenase or iaaM protein fromAgrobacterium tumefaciens that increases the auxin levels.Maximal activity was recovered by extracting the enzyme at pH 8.0 from fruitspicked three days after the onset of the colour change. The enzyme exhibits ahalf-life of 85 min, two relative maxima at 30 and 38°C, an optimum pH of 7.9 and an apparent Km forACC of 118 M. Our results also show the first evidence of anallosteric type kinetic of the ACC oxidase activity with respect to itscosubstrate ascorbate, with an apparent Km of 12.5mM,estimated as the concentration which gave 50% Vmax.     

A defective pollen-pistil interaction contributes to hamper seed set in the<Emphasis Type="Italic"> parthenocarpic fruit</Emphasis> tomato mutant

In the parthenocarpic fruit (pat) tomato mutant, parthenocarpy is associated with partial aberrations of stamens (shortness and carpelloidy) and ovules (defective integument growth) that contribute to impair seed set. However, these do not seem to be the only reasons for seed infertility because hand-pollination fails to restore seed set in ovaries where a fraction of the ovules are still morphologically normal. Therefore, it is conceivable that other unreported defects occur during the reproductive process in the mutant. In this research, we show that the mutation does not affect pollen or embryo sac development and viability, but generates sporophytic effects that reduce seed production and seed size. While pollen germination and stylar growth were normal in mutant pistils, fertilization does not take place because of abnormalities in the pollen tube-ovary interaction in this genotype. Inside the ovary of pat plants, pollen tubes appeared to be disorientated; they wandered about in the ovarian cavity and often lost their adherence to the placental surface. Interestingly, in pat ovaries fertilization was strongly impaired even in those ovules that appeared normal. It may be that apparently 'normal' ovules cannot guide pollen tubes to their micropyle in the altered pat ovary because adhesion molecules are not properly arrayed on a placenta that is already preparing for cell division or, alternatively, chemotropic signals in the pat ovary may be altered by the presence of aberrant ovules, which are not simply devoid of attractivity, but disrupt pollen tube guidance overall.     

Changes in ligno-suberization of cell walls of tomato hairy roots produced by salt treatment: the relationship with the release of a basic peroxidase

A highly basic peroxidase isoenzyme was shown to be released to the culture medium of tomato (Lycopersicon esculentum) hairy roots grown in Murashige-Skoog (MS) liquid medium when it was supplemented with 100 mM NaCl. In this paper we demonstrate that this enzyme is ionically bound to cell walls and that the release was a consequence of the continuous agitation of the tissue in a high ionic strength medium with salt addition. In order to establish the physiological role of this isoenzyme we partially purified it, and we analysed its kinetic properties as coniferyl alcohol peroxidase. The peroxidase isoenzyme showed a high catalytic efficiency for this substrate, which suggests that it would be associated with the ligno-suberization process. To confirm the involvement of this isoenzyme in that process, we studied the pattern of ligno-suberization of the tissue under different conditions of growth. Our results suggest that this basic peroxidase would be indeed involved in ligno-suberization since its leakage from cell walls, induced by 100 mM NaCl in liquid MS, caused less ligno-suberization of exo and endodermis. On the contrary, more ligno-suberization was seen in cell walls when the hairy roots were grown in a salt-supplemented MS solid medium without contact with it, a condition in which the release of the isoenzyme would be avoided. Thus, through the changes produced by the release of the enzyme from its site of action, we could demonstrate the physiological role of this peroxidase in the processing of root cell walls, being part of control mechanisms of ion and water fluxes through the root.     

Effect of trypsin inhibitor from Crotalaria pallida seeds on Callosobruchus maculatus (cowpea weevil) and Ceratitis capitata (fruit fly)

A proteinaceous trypsin inhibitor was purified from Crotalaria pallida seeds by ammonium sulfate precipitation, affinity chromatography on immobilized trypsin-Sepharose and TCA precipitation. The trypsin inhibitor, named CpaTI, had M(r) of 32.5 kDa as determined by SDS-PAGE and was composed of two subunits with 27.7 and 5.6 kDa linked by disulfide bridges. CpaTI was stable at 50 degrees C and lost 40% of activity at 100 degrees C. CpaTI was also stable from pH 2 to 12 at 37 degrees C. CpaTI weakly inhibited chymotrypsin and elastase and its inhibition of papain, a cysteine proteinase, were indicative of its bi-functionality. CpaTI inhibited, in different degrees, digestive enzymes from Spodoptera frugiperda, Alabama argillacea, Plodiainterpunctella, Anthonomus grandis and Zabrotes subfasciatus guts. In vitro and in vivo susceptibility of Callosobruchus maculatus and Ceratitis capitata to CpaTI was evaluated. C. maculatus and C. capitata enzymes were strongly susceptible, 74.4+/-15.8% and 100.0+/-7.3%, respectively, to CpaTI. When CpaTI was added to artificial diets and offered to both insect larvae, the results showed that C. maculatus was more susceptible to CpaTI with an LD(50) of 3.0 and ED(50) of 2.17%. C. capitata larvae were more resistant to CpaTI, in disagreement with the in vitro effects. The larvae were more affected at lower concentrations, causing 27% mortality and 44.4% mass decrease. The action was constant at 2-4% (w/w) with 15% mortality and 38% mass decrease.     

Increases in cell elongation,plastid compartment size and phytoene synthase activity underlie the phenotype of the<Emphasis Type="Italic"> high pigment-1</Emphasis> mutant of tomato

A characteristic trait of the high pigment-1 ( hp-1) mutant phenotype of tomato ( Lycopersicon esculentum Mill.) is increased pigmentation resulting in darker green leaves and a deeper red fruit. In order to determine the basis for changes in pigmentation in this mutant, cellular and plastid development was analysed during leaf and fruit development, as well as the expression of carotenogenic genes and phytoene synthase enzyme activity. The hp-1 mutation dramatically increases the periclinal elongation of leaf palisade mesophyll cells, which results in increased leaf thickness. In addition, in both palisade and spongy mesophyll cells, the total plan area of chloroplasts per cell is increased compared to the wild type. These two perturbations in leaf development are the primary cause of the darker green hp-1 leaf. In the hp-1 tomato fruit, the total chromoplast area per cell in the pericarp cells of the ripe fruit is also increased. In addition, although expression of phytoene synthase and desaturase is not changed in hp-1 compared to the wild type, the activity of phytoene synthase in ripe fruit is 1.9-fold higher, indicating translational or post-translational control of carotenoid gene expression. The increased plastid compartment size in leaf and fruit cells of hp-1 is novel and provides evidence that the normally tightly controlled relationship between cell expansion and the replication and expansion of plastids can be perturbed and thus could be targeted by genetic manipulation.     

A butenolide, isolated from smoke, can overcome the detrimental effects of extreme temperatures during tomato seed germination

The butenolide, 3-methyl-2H-furo[2, 3-c]pyran-2-one, is an highly active compound isolated from plant-derived smoke. This compound is known to stimulate seed germination in a wide range of plants akin to smoke or aqueous extracts of smoke. The present study attempted to elucidate the role of the butenolide in overcoming detrimental effects of low and high temperatures on tomato seed germination and seedling growth. The germination percentage followed a parabolic curve for temperatures ranging from 10 to 40°C, with 25°C being the optimum for all treatments. Control seeds showed radicle emergence at two extreme temperatures (10 and 40°C) and seedlings failed to develop further, even upon prolonged incubation. By comparison the butenolide-treated seeds grew into phenotypically normal seedlings at these non-optimum temperatures. The smoke–water-treated seeds had an intermediate response as only a fraction of germinated seed developed into normal seedlings. Seedling vigour indices as well as seedling weight were significantly higher (p ≤ 0.05) for butenolide-treated seeds at all temperatures. Furthermore, seedlings developed in the presence of the butenolide had about a 1:1 correspondence between root and shoot length. Butenolide-treated seeds grew better than the control seeds in the temperature shift experiments. A gradual decline in the vigour index values was recorded with an increased duration of incubation at the extreme temperatures. Results of the present study are very important from an horticultural point of view as they indicate the potential use of the butenolide compound in restoring normal seed germination and seedling establishment in tomato below and above optimum temperatures.