Products Released from Enzymically Active Cell Wall Stimulate Ethylene Production and Ripening in Preclimacteric Tomato (Lycopersicon esculentum Mill.) Fruit

Enzymically active cell wall from ripe tomato (Lycopersicon esculentum Mill.) fruit pericarp release uronic acids through the action of wall-bound polygalacturonase. The potential involvement of products of wall hydrolysis in the induction of ethylene synthesis during tomato ripening was investigated by vacuum infiltrating preclimacteric (green) fruit with solutions containing pectin fragments enzymically released from cell wall from ripe fruit. Ripening initiation was accelerated in pectin-infiltrated fruit compared to control (buffer-infiltrated) fruit as measured by initiation of climacteric CO₂ and ethylene production and appearance of red color. The response to infiltration was maximum at a concentration of 25 micrograms pectin per fruit; higher concentrations (up to 125 micrograms per fruit) had no additional effect. When products released from isolated cell wall from ripe pericarp were separated on Bio-Gel P-2 and specific size classes infiltrated into preclimacteric fruit, ripening-promotive activity was found only in the larger (degree of polymerization >8) fragments. Products released from pectin derived from preclimacteric pericarp upon treatment with polygalacturonase from ripe pericarp did not stimulate ripening when infiltrated into preclimacteric fruit.     

Regulatory Role of Cystathionine-γ-Synthase and de novo Synthesis of Methionine in Ethylene Production during tomato Fruit Ripening

The essential amino acid methionine is a substrate for the synthesis of S-adenosyl-methionine (SAM), that donates its methyl group to numerous methylation reactions, and from which polyamines and ethylene are generated. To study the regulatory role of methionine synthesis in tomato fruit ripening, which requires a sharp increase in ethylene production, we cloned a cDNA encoding cystathionine γ-synthase (CGS) from tomato and analysed its mRNA and protein levels during tomato fruit ripening. CGS mRNA and protein levels peaked at the “turning” stage and declined as the fruit ripened. Notably, the tomato CGS mRNA level in both leaves and fruit was negatively affected by methionine feeding, a regulation that Arabidopsis, but not potato CGS mRNA is subject to. A positive correlation was found between elevated ethylene production and increased CGS mRNA levels during the ethylene burst of the climacteric ripening of tomato fruit. In addition, wounding of pericarp from tomato fruit at the mature green stage stimulated both ethylene production and CGS mRNA level. Application of exogenous methionine to pericarp of mature green fruit increased ethylene evolution, suggesting that soluble methionine may be a rate limiting metabolite for ethylene synthesis. Moreover, treatment of mature green tomato fruit with the ethylene-releasing reagent Ethephon caused an induction of CGS mRNA level, indicating that CGS gene expression is regulated by ethylene. Taken together, these results imply that in addition to recycling of the methionine moieties via the Yang pathway, operating during synthesis of ethylene, de novo synthesis of methionine may be required when high rates of ethylene production are induced.     

Fruit-localized phytochromes regulate lycopene accumulation independently of ethylene production in tomato

We show that phytochromes modulate differentially various facets of light-induced ripening of tomato fruit (Solanum lycopersicum L.). Northern analysis demonstrated that phytochrome A mRNA in fruit accumulates 11.4-fold during ripening. Spectroradiometric measurement of pericarp tissues revealed that the red to far-red ratio increases 4-fold in pericarp tissues during ripening from the immature-green to the red-ripe stage. Brief red-light treatment of harvested mature-green fruit stimulated lycopene accumulation 2. 3-fold during fruit development. This red-light-induced lycopene accumulation was reversed by subsequent treatment with far-red light, establishing that light-induced accumulation of lycopene in tomato is regulated by fruit-localized phytochromes. Red-light and red-light/far-red-light treatments during ripening did not influence ethylene production, indicating that the biosynthesis of this ripening hormone in these tissues is not regulated by fruit-localized phytochromes. Compression analysis of fruit treated with red light or red/far-red light indicated that phytochromes do not regulate the rate or extent of pericarp softening during ripening. Moreover, treatments with red or red/far-red light did not alter the concentrations of citrate, malate, fructose, glucose, or sucrose in fruit. These results are consistent with two conclusions: (a) fruit-localized phytochromes regulate light-induced lycopene accumulation independently of ethylene biosynthesis; and (b) fruit-localized phytochromes are not global regulators of ripening, but instead regulate one or more specific components of this developmental process.     

Ripening-related occurrence of phosphoenolpyruvate carboxykinase in tomato fruit

Phosphoenolpyruvate carboxykinase (PEPCK) is present in ripening tomato fruits. A cDNA encoding PEPCK was identified from a PCR-based screen of a cDNA library from ripe tomato fruit. The sequence of the tomato PEPCK cDNA and a cloned portion of the genomic DNA shows that the complete cDNA sequence contains an open reading frame encoding a peptide of 662 amino acid residues in length and predicts a polypeptide with a molecular mass of 73.5 kDa, which corresponds to that detected by western blotting. Only one PEPCK gene was identified in the tomato genome. PEPCK is shown to be present in the pericarp of ripening tomato fruits by activity measurements, western blotting and mRNA analysis. PEPCK abundance and activity both increased during fruit ripening, from an undetectable amount in immature green fruit to a high amount in ripening fruit. PEPCK mRNA, protein and activity were also detected in germinating seeds and, in lower amounts, in roots and stems of tomato. The possible role of PEPCK in the pericarp of tomato fruit during ripening is discussed.     

Effect of tunicamycin on in vitro ripening of tomato pericarp tissue

Ripening of pericarp tissue from mature green, early breaker and late breaker stages of tomato ( Lycopersicon esculentum Mill. cv. Dombito) fruit development was inhibitied by tunicamycin. Ripening was evaluated by lycopene accumulation, chlorophyll degradation, rate of ethylene production and cell wall-bound polygalacturonase (EC 3.2.1.15) activity. Maximum inhibition of these ripening parameters occurred at a treatment of 240 μ M tunicamycin for 2 h except for cell wall-bound polygalacturonase activity, which was greatly inhibited by concentrations of 12 μ tunicamycin or higher. Tunicamycin treatment at 120 μ M for 2 h inhibited the incorporation of [³H]-mannose into macromolecules (about 70%) and pronase-sensitive material (about 65%) and the incorporation of [³H]-leucine into proteins (about 20%). Our results indicate that protein glycosylation plays an important role in the ripening of tomato pericarp tissue.     

Changes in the protein and activity levels of the plastid NADH–plastoquinone–oxidoreductase complex during fruit development

Plastids contain an NADH dehydrogenase complex (Ndh complex) homologous to the mitochondrial complex I (EC 1.6.5.3). In this work, we have analysed the changes in the Ndh complex during ripening of pepper (Capsicum annum L., cv. Maor) and tomato (Lycopersicon esculentum Mill., cv. Marglobe) fruits. The Ndh complex was mainly present in the outer pericarp of tomato fruits, whereas it was evenly distributed in the pericarp of pepper. In both kinds of fruit we observed a decrease in the total amount of Ndh complex from the green to the red stage of development. This decrease corresponds to parallel decreases in the content and activity of the complex in plastids during the transition from chloroplasts to chromoplasts. Levels of plastidial quinol peroxidase activity were also higher during the first stages of tomato fruit development than during the latter stages of ripening. However, when referred to total plastid protein, the amount and activity of the Ndh complex in chloroplasts isolated from green fruits was higher than in chloroplasts isolated from leaves. These results strongly suggest that function of the Ndh complex, probably related to a plastidial electron transport chain, can be important during the first stages of fruit development.     

Characterization of tomato endo-β-1,4-glucanase Cel1 protein in fruit during ripening and after fungal infection

The tomato (Lycopersicon esculentum Mill.) endo--1,4-glucanase (EGase) Cel1 protein was characterized in fruit using specific antibodies. Two polypeptides ranging between 51 and 52 kDa were detected in the pericarp, and polypeptides ranging between 49 and 51 kDa were detected in locules. The polypeptides recognized by Cel1 antiserum in fruit are within the size range predicted for Cel1 protein and could be derived from heterogeneous glycosylation. Cel1 protein accumulation was examined throughout fruit ripening. Cel1 protein appears in the pericarp at the stage in which many ripening-related changes start, and remains present throughout fruit ripening. In locules, Cel1 protein is already present at the onset of fruit ripening and remains constant during fruit ripening. This pattern of expression supports a possible role for this EGase in the softening of pericarp tissue and in the liquefaction of locules that takes place during ripening. The accumulation of Cel1 protein was also analyzed after fungal infection. Cel1 protein and mRNA levels are down-regulated in pericarp after Botrytis cinerea infection but are not affected in locular tissue. The same behavior was observed when fruits were infected with Penicillium expansum, another fungal pathogen. Cel1 protein and mRNA levels do not respond to wounding. These results support the idea that the tomato Cel1 EGase responds to pathogen infection and supports a relationship between EGases, plant defense responses and fruit ripening.This revised version was published online in August 2004 with corrections to Fig. 1 and Fig. 5.     

Endogenous Gibberellin Profile During Christmas Rose (Helleborus niger L.) Flower and Fruit Development

Gibberellins (GAs) were identified and quantified during flower and fruit development in the Christmas rose (Helleborus niger L.), a native of southeastern Europe with a long international horticultural tradition. Physiologically, the plant differs from popular model species in two major respects: (1) following anthesis, the initially white or rose perianth (formed in this species by the sepals) turns green and persists until fruit ripening, and (2) the seed is shed with an immature embryo, a miniature endosperm, and a prominent perisperm as the main storage tissue. GA₁ and GA₄ were identified by full-scan mass spectra as the major bioactive GAs in sepals and fruit. LC-MS/MS system in accord with previously verified protocols also afforded analytical data on 12 precursors and metabolites of GAs. In the fruit, GA₄ peaked during rapid pericarp growth and embryo development and GA₁ peaked during the subsequent period of rapid nutrient accumulation in the seeds and continued pericarp enlargement. In the sepals, the flux through the GA biosynthetic pathway was highest prior to the light green stage when the photosynthetic system was induced. Unfertilized, depistillated, and deseeded flowers became less green than the seed-bearing controls; chlorophyll accumulation could be restored by applying GA₁, GA₄, and, less efficiently, GA₃ to the deseeded fruit. The sepals of unfertilized and depistillated flowers indeed contained very low levels of GA₄ and gradually decreasing levels of GA₁. However, the concentrations of their precursors and metabolites were less affected. These data suggest that a signal(s) from the fruit stimulates GA biosynthesis in the sepals resulting in greening. The fruit-derived GAs appear to be mainly involved in pericarp growth and seed development.     

Differentiation and development of bundle sheath and mesophyll thylakoids in maize. Thylakoid polypeptide composition, phosphorylation, and organization of photosystem II

Photosynthetic electron flow, polypeptide pattern, presence of chlorophyll-protein complexes, and phosphorylation of thylakoid polypeptides have been investigated in differentiated mesophyll (M) and bundle sheath (B) thylakoids of the C4 plant Zea mays. The polypeptide pattern of M thylakoids and their photosynthetic electron flow are comparable to those of other green plants. B thylakoids exhibit only photosystem I (PSI) activity, contain only traces of the PSII light harvesting (LHCII) polypeptide, do not bind [3H] diuron, and lack polypeptides of the water-oxidation complex of PSII and the herbicide binding 32-kDa polypeptide, as detected by specific antibodies. However, B thylakoids possess a partially active PSII reaction center, as demonstrated by light-dependent reduction of silicomolybdate with 1,5-diphenylcarbazide (DPC) as an electron donor, and the presence of the PSII reaction center polypeptides of 44-47 kDa. Only one chlorophyll a-protein complex, corresponding to the PSI reaction center-core antenna, was detectable in B thylakoids, as opposed to chlorophyll a and chlorophyll a,b-protein complexes present in M thylakoids. The light-dependent, membrane-bound kinase activity present in M thylakoids could not be detected in B thylakoids which, nevertheless, contain a protein kinase able to phosphorylate casein. A total of 19 differences between the electrophoretic pattern of B and M thylakoid polypeptides were observed. The mRNA coding for the LHCII polypeptide is primarily, if not exclusively, localized in M cells. The development of PSII complex precedes that of PSI during the differentiation of B and M chloroplasts in expanding leaves of light-grown plants and during the greening of dark-grown etiolated seedlings. The differentiation of the maize leaf into cells programmed to form B or M chloroplasts does not require light. In light-grown plants, the differentiation of B and M thylakoids occurred progressively from the base of the leaf and was completed at 4-5 cm from the leaf base.     

Photosynthetic activity of the calyx, green shoulder, pericarp, and locular parenchyma of tomato fruit

Photosynthesis of tomato fruit was studied using green fruit from six heritage cultivars of Lycopersicon esculentum Mill. and one of Lycopersicon pimpinellifolium. Chlorophyll concentrations in the green shoulder, pericarp and locular parenchyma of the fruit were determined and the apparent photosynthetic electron transport activity (ETR) and chlorophyll fluorescence quenching characteristics of these tissues and the calyx were compared. In all cultivars, green shoulder formation, apparent as intense pigmentation of the proximal pericarp shoulder, was positively related to the degree of shading of the fruit during development. Appearing as a photosynthetic adaptive trait for increasing the photoautotrophic capacity of fruit grown under low light, the green shoulder contained 17-57% of the total pericarp chlorophyll content. The pericarp below the green shoulder had lower chlorophyll a+b. At a photon flux density (PFD) of 1200 mol m^-2 s^-1, different fruit tissues were found to have different levels of ETR. In 'Yellow Pear', the upper surface of the calyx had an ETR of 154 mol m^-2 s^-1, while the lower surface had an ETR of 88 mol m^-2 s^-1. On the green shoulder, ETR was 203 mol m^-2 s^-1, whereas in the pericarp distal to the green shoulder, ETR was 97 mol m^-2 s^-1. In the locular parenchyma, ETR was 66 mol m^-2 s^-1. This trend towards a lower ETR in distal and internal fruit tissues appeared to indicate a shift towards a more shade-type photosynthesis. Concomitant with this shift were changes in chlorophyll fluorescence quenching characteristics. Generally when tissues displayed reduced levels of ETR they also displayed a faster decrease in the photochemical quenching coefficient qp and a more rapid diversion of absorbed photon energy to non-photosynthetic activity found in the calyx, green shoulder, pericarp, and locular parenchyma suggest that all of these tissues have significant roles in CO2 scavenging and the provision of carbon assimilates. The potential role of fruit photosynthesis in influencing the fruit acid to sugar ratio and hence fruit quality is discussed.     

Endogenous Levels of Phenolics in Tomato Fruit during Growth and Maturation

Changes in the metabolism of several types of phenolics in the pulp and pericarp of tomato (Lycopersicon esculentum) fruit var. Ailsa Craig and Pik-Red were related to the stage of development. The highest levels of chlorogenic acid were found in the pulp and pericarp at the earliest stage of fruit development, and quantities declined rapidly during fruit ripening. Levels of rutin, found only in the pericarp, followed a similar pattern of change. The p-coumaric acid conjugate of rutin was found in low levels through fruit growth and ripening. High levels of p-coumaric acid glucoside were detected in the pulp only as the fruit matured with no rapid decline in levels during ripening. The decline of chlorogenic acid and rutin levels during fruit ripening paralleled the decline in indole-3-acetic acid levels measured previously in the pericarp tissues of these two varieties of tomato fruit during maturation. These phenolics are among those that have been suggested as regulants of auxin metabolism. Received April 30, 1996; accepted December 26, 1996     

Developmental Regulation of the (1,3)-beta-Glucan (Callose) Synthase from Tomato : Possible Role of Endogenous Phospholipases

Activity levels of UDP-glucose: (1,3)-β-glucan (callose) synthase in microsomal membranes of pericarp tissue from tomato fruit (Lycoperisicon esculentum Mill, cv Rutgers) were determined during development and ripening. Addition of the phospholipase inhibitors O-phosphorylcholine and glycerol-1-phosphate to homogenization buffers was necessary to preserve enzyme activity during homogenization and membrane isolation. Enzyme activity declined 90% from the immature green to the red ripe stage. The polypeptide composition of the membranes did not change significantly during ripening. The enzyme from immature fruit was inactivated by exogenously added phospholipases A₂, C, and D. These results suggest that the decline in callose synthase activity during ontogeny may be a secondary effect of endogenous lipase action.     

Tissue Specific Localization of Pectin–Ca2+ Cross-Linkages and Pectin Methyl-Esterification during Fruit Ripening in Tomato (Solanum lycopersicum)

Fruit ripening is one of the developmental processes accompanying seed development. The tomato is a well-known model for studying fruit ripening and development, and the disassembly of primary cell walls and the middle lamella, such as through pectin de-methylesterified by pectin methylesterase (PE) and depolymerization by polygalacturonase (PG), is generally accepted to be one of the major changes that occur during ripening. Although many reports of the changes in pectin during tomato fruit ripening are focused on the relation to softening of the pericarp or the Blossom-end rot by calcium (Ca²⁺) deficiency disorder, the changes in pectin structure and localization in each tissues during tomato fruit ripening is not well known. In this study, to elucidate the tissue-specific role of pectin during fruit development and ripening, we examined gene expression, the enzymatic activities involved in pectin synthesis and depolymerisation in fruit using biochemical and immunohistochemical analyses, and uronic acids and calcium (Ca)-bound pectin were determined by secondary ion-microprobe mass spectrometry. These results show that changes in pectin properties during fruit development and ripening have tissue-specific patterns. In particular, differential control of pectin methyl-esterification occurs in each tissue. Variations in the cell walls of the pericarp are quite different from that of locular tissues. The Ca-binding pectin and hairy pectin in skin cell layers are important for intercellular and tissue–tissue adhesion. Maintenance of the globular form and softening of tomato fruit may be regulated by the arrangement of pectin structures in each tissue.     

Differential effects of senescence on the molecular organization of membranes in ripening tomato fruit

Changes in the molecular organization of membranes in pericarp cells of ripening tomato fruit were examined by fluorescence depolarization after labeling with fluorescent lipid-soluble probes. The fluorescent labels were partitioned into isolated protoplasts and purified plastids from fruit at various stages of senescence. Values for steady-state anisotropy (r_ss) of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labeled protoplasts rose progressively during the early stages of ripening over a time frame that overlapped the climacteric rise in ethylene production. This can be interpreted as reflecting a decrease in the lipid fluidity of primarily plasma membrane. By contrast, there was no significant change during ripening in r_ss for plastid membranes labeled with DPH, 1-[4-trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), and cis- or trans-parinaric acid. Nor was there any change during ripening in the limiting fluorescence anisotropy (r_oo) and order parameter (S) for plastids labeled with DPH or TMA-DPH, parameters that are corrected for any differences in lifetime. Some degree of lifetime heterogeneity, possibly reflecting structurally distinct domains, was discerned in both young and senescent plastids that had been labeled with DPH or TMA-DPH, but this also did not change as ripening progressed. Thus membranes of the pericarp cells sustain different fates as the tomato fruit ripens, implying that there are distinguishable mechanisms of membrane deterioration in senescing tissues.     

Cell Wall Metabolism in Ripening Fruit (VII. Biologically Active Pectin Oligomers in Ripening Tomato (Lycopersicon esculentum Mill.) Fruits)

A water-soluble, ethanol-insoluble extract of autolytically inactive tomato (Lycopersicon esculentum Mill.) pericarp tissue contains a series of galacturonic acid-containing (pectic) oligosaccharides that will elicit a transient increase in ethylene biosynthesis when applied to pericarp discs cut from mature green fruit. The concentration of these oligosaccharides in extracts (2.2 [mu]g/g fresh weight) is in excess of that required to promote ethylene synthesis. Oligomers in extracts of ripening fruits were partially purified by preparative high-performance liquid chromatography, and their compositions are described. Pectins were extracted from cell walls prepared from mature green fruit using chelator and Na2CO3 solutions. These pectins are not active in eliciting ethylene synthesis. However, treatment of the Na2CO3-soluble, but not the chelator-soluble, pectin with pure tomato polygalacturonase 1 generates oligomers that are similar to those extracted from ripening fruit (according to high-performance liquid chromatography analysis) and are active as elicitors. The possibility that pectin-derived oligomers are endogenous regulators of ripening is discussed.     

Degradation of isolated tomato cell walls by purified polygalacturonase in vitro

Cell wall preparations from green pericarp of normal and mutant Neverripe (Nr) and ripening inhibitor (rin) tomato (Lycopersicon esculentum Mill.) fruit were all equally degraded in vitro by a cell wall-bound protein extract from ripe normal tomatoes.     

Photosynthetic characteristics of two Cylindrospermopsis raciborskii strains differing in their toxicity

We studied the growth and photosynthetic characteristics of a toxic (CS506) and a nontoxic strain (CS509) of the bloom‐forming cyanobacterium Cylindrospermopsis raciborskii grown under identical experimental conditions. When exposed to light‐saturating growth conditions (100 μmol photons · m^?2 · s^?1), values for maximal photosynthetic capacity (P_max) and maximum quantum yield (F_v/F_m) indicated that both strains had an equal ability to process captured photons and deliver them to PSII reaction centers. However, CS506 grew faster than CS509. This was consistent with its higher light requirement for saturation of photosynthesis (I_k). Greater shade tolerance of CS509 was indicated by its higher ability to harvest light (α), lower photosynthetic light compensation point (I_c), and higher chlorophyll a to biovolume ratio. Strain‐specific differences were found in relation to non‐photochemical quenching, effective absorption cross‐sectional area of PSIIα‐centers (σPSIIα), and the antenna connectivity parameter of PSIIα (J_conPSIIα). These findings highlighted differences in the transfer of excitation from phycobilisome/PSII to PSI, on the dependence on different pigments for light harvesting and on the functioning of the PSII reaction centers between the two strains. The results of this study showed that both performance and composition of the photosynthetic apparatus are different between these strains, though with only two strains examined we cannot attribute the performance of strain 506 to its ability to produce cylindrospermopsins. The emphasis on a strain‐specific light adaptation/acclimation is crucial to our understanding of how different light conditions (both quantity and quality) can trigger the occurrence of different C. raciborskii strains and control their competition and/or dominance in natural ecosystems.