Chromoplast-Targeted Proteins in Tomato (Lycopersicon esculentum Mill.) Fruit

The chloroplast to chromoplast transition during tomato (Lycopersicon esculentum Mill.) fruit ripening is characterized by a dramatic change in plastid structure and function. We have asked whether this process is mediated by an increase in the steady-state level of RNA for plastid targeted proteins. Assays for import of radiolabeled translation products into isolated pea (Pisum sativum L.) chloroplasts were used to monitor levels of chromoplast-targeted proteins at four stages of tomato fruit development. We have found striking increases during development in levels of translatable RNA for two such proteins. Additionally, the import of in vitro translation products was examined for seven individual cDNA clones known to encode RNA that increase during fruit ripening. Three of these clones produced in vitro translation products that were imported into pea chloroplasts. This implies that there is synthesis and import of new proteins during the transition from chloroplast to chromoplast and that the plastid conversion is an active developmental program rather than a simple decline in synthesis of the photosynthetic apparatus. Furthermore, our results demonstrate the utility of this method for identification of structural genes involved in plastid morphogenesis.     

Cloning of tomato (Lycopersicon esculentum Mill.) arginine decarboxylase gene and its expression during fruit ripening.

Extracts of soybean (Glycine max) root nodules and greening etiolated leaves catalyzed radiolabeled delta-aminolevulinic acid (ALA) formation from 3,4-[3H]glutamate but not from 1-[14C]glutamate. Nevertheless, those tissue extracts expressed the activity of glutamate 1-semialdehyde (GSA) aminotransferase, the C5 pathway enzyme that catalyzes ALA synthesis from GSA for tetrapyrrole formation. A soybean nodule cDNA clone that conferred ALA prototrophy, GSA aminotransferase activity, and glutamate-dependent ALA formation activity on an Escherichia coli GSA aminotransferase mutant was isolated. The deduced product of the nodule cDNA shared 79% identity with the GSA aminotransferase expressed in barley leaves, providing, along with the complementation data, strong evidence that the cDNA encodes GSA aminotransferase. GSA aminotransferase mRNA and enzyme activity were expressed in nodules but not in uninfected roots, indicating that the Gsa gene is induced in the symbiotic tissue. The Gsa gene was strongly expressed in leaves of etiolated plantlets independently of light treatment and, to a much lesser extent, in leaves of mature plants. We conclude that GSA aminotransferase, and possibly the C5 pathway, is expressed in a nonphotosynthetic plant organ for nodule heme synthesis and that Gsa is a regulated gene in soybean.     

Mannosyl- and Xylosyl-Containing Glycans Promote Tomato (Lycopersicon esculentum Mill.) Fruit Ripening

The oligosaccharide glycans mannosylα1-6(mannosylα1-3)mannosylα1-6(mannosylα1-3) mannosylβ1-4-N-acetylglucosamine and mannosylα1-6(mannosylα1-3)(xylosylβ1-2) mannosylβ1-4-N-acetylglucosaminyl(fucosylα1-3) N-acetylglucosamine were infiltrated into mature green tomato fruit (Lycopersicon esculentum Mill., cv Rutgers). Coinfiltration of 1 nanogram per gram fresh weight of the glycans with 40 micrograms per gram fresh weight galactose, a level of galactose insufficient to promote ripening, stimulated ripening as measured by red coloration and ethylene production.     

Growth and Water Relations of Wilty Mutants of Tomato (Lycopersicon esculentum Mill.)

In contrast to some previous reports on the growth of the ABA-deficientwilty mutants of tomato, growth was at least as rapid in themutants as in the wild type, as long as an adequate plant waterstatus was maintained by growing the plants under mist. Moreover,shoot extension was greater and the rate of leaf productionmore rapid in the mutants. Stomatal changes in response to environmentand to time in the light-dark cycle were generally similar inboth wilty mutants and the wild type, though the wild-type weregenerally more closed. Grafting experiments confirmed that thegenotype of the shoot was dominant in determining stomatal aperture,though wild-type rootstocks could cause a slight reduction inthe stomatal conductance of mutant leaves. The effect on plantwater relations of draughting only part of the root system wasinvestigated in a ‘split-root’ experiment. Withholdingwater from only part of the root system was found to lower significantlythe mean leaf water potential, even though the potential evaporationrate was kept very small. Key words: Abscisic acid, stomata, tomato     

Changes in indole-3-acetic acid and abscisic acid levels during tomato (Lycopersicon esculentum Mill.) fruit development and ripening

Changes in the levels of indole-3-acetic acid (IAA) and abscisic acid (ABA) in tomato (Lycopersicon esculentum Mill.) fruit pericarp tissue during development through ripening were measured by GC-SIM-MS using d₃-ABA and ¹³C₆-IAA internal standards. In the two cultivars of fieldgrown tomatoes analyzed, the highest IAA levels (8–24 ng/g fw) were found at the earliest stage of development (7 days after anthesis) followed by a rapid decline in levels of the hormone. ABA levels of 40–60 ng/g fw were found at the earliest stages of development followed by a decline in levels until ripening occurred when elevated ABA levels (125 ng/g fw) were measured.     

Characterization of expression, and cloning, of beta-D-xylosidase and alpha-L-arabinofuranosidase in developing and ripening tomato (Lycopersicon esculentum Mill.) fruit

Modifications to the cell wall of developing and ripening tomato fruit are mediated by cell wall-degrading enzymes, including a beta-d-xylosidase or alpha-l-arabinofuranosidase, which participate in the breakdown of xylans and/or arabinoxylans. The activity of both enzymes was highest during early fruit growth, before decreasing during later development and ripening. Two beta-d-xylosidase cDNAs, designated LeXYL1 and LeXYL2, and an alpha-l-arabinofuranosidase cDNA, designated LeARF1, were obtained. Accumulation of mRNAs for beta-d-xylosidase and alpha-l-arabinofuranosidase was examined during fruit development and ripening. LeARF1 and LeXYL2 genes were relatively highly expressed during fruit development and decreased after the onset of ripening. By contrast, LeXYL1 was not expressed during fruit development, but was expressed later, particularly during over-ripening. The expression of all three genes was also followed in ripening-impaired mutants, Nr, Nr2, nor, and rin of cv. Ailsa Craig fruit. LeXYL2 mRNA was detected in the ripe fruits of all the mutants and its abundance was similar to that in mature green wild-type fruit. By contrast, LEXYL1 mRNA was expressed only in the ripe fruits of the Nr mutant, suggesting that the two beta-d-xylosidase genes are subject to distinct regulatory control during fruit development and ripening. LeARF1 mRNA was detected in ripe fruits of Nr2, nor and rin, and not in ripe fruit of the Nr mutant. The accumulation of LeARF1 in ripe fruit was restored by 1-methylcyclopropene (1-MCP), an inhibitor of ethylene action, while 1-MCP had no effect on the expression of LeXYL1 or LeXYL2. This suggests that LeARF1 expression is subject to negative regulation by ethylene and that the two beta-d-xylosidase genes are independent of ethylene action.     

Compartments and Permeability for Potassium in Developing Fruits of Tomato (Lycopersicon esculentum Mill.)

Permeabilities of plasmalemma and tonoplast, and the distributionof potassium between free space, cytoplasm, and vacuole, wereestimated from the kinetics of the efflux of potassium fromslices of tomato pericarp. The permeabilities of plasmalemmaand tonoplast did not change during development, but the proportionof potassium in the cytoplasm increased during ripening. Itis suggested that the effects of ethylene, and the changes takingplace during ripening are not caused by changing permeabilitiesof membranes. Increasing activities of ions in the cytoplasm,rather than increasing membrane permeabilities, may explainprevious observations that the efflux of solutes from fruittissues increases during ripening.     

Novel Technique for Measuring Tissue Firmness within Tomato (Lycopersicon esculentum Mill.) Fruit

Developmental changes of tomato (Lycopersicon esculentum) fruit tissues during maturation were analyzed by a physically defined method (stress-relaxation analysis). The tip of a conical probe connected to a load sensor was positioned on the cut surface of a sliced tomato fruit, and the decay of the imposed stress was monitored. Stress-relaxation data thus obtained were used for the calculation of three stress-relaxation parameters. Different zones within tomato fruit harvested at six different ripening stages were analyzed. One of the stress-relaxation parameters, minimum stress-relaxation time (T₀), decreased as the fruits matured. The decrease in T₀ was first found in the core of the carpel junction within the endopericarp at the blossom end during the breaker stage. The decrease in T₀ progressed from the blossom end, through the equatorial region and finally throughout the shoulder, as the fruit matured. In mature green fruit, T₀ values within the placenta and the proximal carpel junction were lower than those by other parts of the fruit. For all measurements the maximum stress-relaxation time was not substantially changed during maturation, nor were their changes observed in different regions of the fruit. The observed relaxation rate was therefore correlated with softening. The results indicate that fruit softening may be physically associated with the stress-relaxation parameter, T₀, and the extent of softening is a function of position within the fruit. Decreases in T₀ value appear to be correlated with the reported regional variation in the appearance of polygalacturonase.     

Gibberellins and the Early Disease Syndrome of Aspermy Virus in Tomato (Lycopersicon esculentum Mill.)

A quantitative examination of the development of the diseasesyndrome created by as permyvirus in tomato (Lycopersicon esculentumMill., cultivar Potentate) revealed an early reduction in subapicalmitotic activity which led to the development of smaller internodes.Later symptoms were associated with necrosis of young axillarybuds and the failure of a new vegetative apex to develop onceflower initiation had begun in the primary apical bud. Application of gibberellic acid (GA₃) to the growing point tendedto reverse virus-induced stunting by increasing cell expansionbut the response was less than that in healthy plants. Thiswas due to a reduction in the number of mitoses associated withvirus infection. No consistent difference was found betweenthe endogenous gibberellin levels of healthy and infected plants.It is suggested that the most important single factor in earlysymptom development is a virus-induced reduction in cell divisionwhich cannot be overcome by applying GA₃.     

Activation of the Cell Cycle in Tomato (Lycopersicon esculentum Mill.) Seeds during Osmoconditioning as Related to Temperature and Oxygen

Using flow cytometric analyses of the nuclear DNA content, westudied the effects of various conditions of osmopriming onthe activation of the cell cycle in embryo root tips of tomato(Lycopersicon esculentum‘Elko’) seeds. In dry untreatedseeds, 90.7% of the nuclei revealed 2C signals. Priming of seedsin polyethylene glycol-8000 (PEG) improved the germination rateof seeds transferred onto water at 15 °C. This was associatedwith an increase in 4C signals when priming was carried outat -1.0 and -1.5 MPa. Priming at -2.0 MPa enhanced subsequentgermination but had no effect on DNA replication. For temperaturesduring priming up to 25 °C, a positive linear correlationexisted between the efficiency of the treatment, evaluated bythe reciprocal of time to obtain 50% germination at 15 °C,and the frequency of 4C nuclei or the 4C/2C values. Such a correlationdid not exist when priming was performed at higher temperatures.At least 5% oxygen in the atmosphere was required during primingfor the induction of DNA replication and for the enhancementof subsequent germination. In the presence of 5x10^-4M and 10^-3MNaN₃during priming, most of the cells were maintained with 2CDNA levels and the treatment had no stimulatory effect on germination.The results show a positive linear relationship between thefrequency of 4C DNA nuclei or the 4C/2C ratio and the improvingeffect of priming. However, in suboptimal conditions of priming(-2.0 MPa or temperatures higher than 25 °C), the improvementof seed germination was not associated with the onset of DNAreplication.Copyright 1999 Annals of Botany Company Cell cycle, germination, osmopriming, oxygen, temperature, Lycopersicon esculentum, tomato.     

Invertase Activity, Soluble Carbohydrates and Inflorescence Development in the Tomato (Lycopersicon esculentum Mill.)

The concentration of reducing sugars in the developing firstinflorescence of the tomato (Lycopersicon esculentum Mill.)increased steadily between the macroscopic appearance of theflower buds and the initial stages of fruit expansion. Overthis period sucrose concentrations remained relatively constant.The rise in reducing sugar concentration was accompanied byan increase in the activity of an acid invertase. In individualflower buds invertase activity rose to a maximum shortly beforeanthesis and declined sharply as the anthers dehisced. Increased planting densities and removal of source leaves reducedthe rate of dry matter accumulation by the first inflorescenceand increased the incidence of flower bud abortion. These changeswere correlated with reductions in reducing sugar concentrations,in reducing sugar/sucrose ratios and in acid invertase levels.Removal of young leaves at the shoot apex significantly increasedthe relative growth rate of the inflorescence and led to a substantialincrease in its invertase content. These treatments had relativelylittle effect on sucrose concentration in the inflorescence. The data are consistent with the operation of an invertase-mediatedunloading mechanism for transported sucrose at sinks in theflower buds. It is suggested that the retarded development ofthe first inflorescence and the high incidence of flower budabortion observed under conditions of reduced photoassimilateavailability are causally related to the decline in invertaseproduction in the flower buds. Possible mechanisms for the regulationof invertase synthesis in the flowers are discussed. Lycopersicon esculentum Mill, tomato, inflorescence development, invertase, sink activity     

Studies on two Gibberellin-like Substances in Young Shoots of Tomato (Lycopersicon esculentum Mill.)

Two gibberellin-like substances were found in the acidic fractionof shoot extracts of the tomato (Lycopersicon esculentum Mill.,cultivar Potentate). These were resolved by paper chromotographywith iso-propanol/ammonia/water (10:1:1) as the developing solventbut not with n-butanol/1.5 N ammonia (3:1). Both substanceswere active in the dwarf maize bioassay on mutants d-1, d-2,d-3, and d-5, and appeared to be more active on d-5 than d-1.Neither was active in the Meteor Pea assay. Neutral and basicfractions were inactive. The relative amounts of these two substances varied accordingto the age of the tissues from which they were extracted andthis feature is discussed in relation to future studies on thephysiology of gibberellin-like substances in vivo.     

Identification of cDNA clones for tomato (Lycopersicon esculentum Mill.) mRNAs that accumulate during fruit ripening and leaf senescence in response to ethylene

Changes in gene expression during foliar senescence and fruit ripening in tomato (Lycopersicon esculentum Mill.) were examined using in-vitro translation of isolated RNA and hybridization against cDNA clones.During the period of chlorophyll loss in leaves, changes occurred in mRNA in-vitro translation products, with some being reduced in prevalence, whilst others increased. Some of the translation products which changed in abundance had similar molecular weights to those known to increase during tomato fruit ripening. By testing RNA from senescing leaves against a tomato fruit ripening-related cDNA library, seven cDNA clones were identified for mRNAs whose prevalence increased during both ripening and leaf senescence. Using dot hybridization, the pattern of expression of the mRNAs corresponding to the seven clones was examined. Maximal expression of the majority of the mRNAs coincided with the time of greatest ethylene production, in both leaves and fruit. Treatment of mature green leaves or unripe fruit with the ethylene antagonist silver thiosulphate prevented the onset of senescence or ripening, and the expression of five of the seven ripening- and senescence-related genes.The results indicate that senescence and ripening in tomato involve the expression of related genes, and that ethylene may be an important factor in controlling their expression.Abbreviations cDNA copy-DNA - MW molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate     

Role of extensin peroxidase in tomato (Lycopersicon esculentum Mill.) seedling growth

 It is proposed that inhibition of extensin peroxidase activity leads to a less rigid cell wall and thus promotes cell expansion and plant growth. A low-molecular-weight inhibitor derived from the cell walls of suspension-cultured tomato cells was found to completely inhibit extensin peroxidase-mediated extensin cross-linking in vitro at a concentration of 260 μg/ml. The inhibitor had no effect upon guaiacol oxidation catalyzed by extensin peroxidase or horseradish peroxidase. We have demonstrated that the light-irradiated inhibition of plant growth may be partially offset by inhibition of endogenous extensin peroxidase activity. Overall plant growth was enhanced by up to 15% in the presence of inhibitor relative to control plants. Inhibitor-treated and illuminated tomato hypocotyls grew up to 15% taller than untreated controls. The inhibitor had no effect upon etiolated plants over a 15-d period, suggesting that only low levels of peroxidase-mediated cross-linking can be found in the cell walls of etiolated plants. SDS-PAGE/Western blots of ionically bound protein from both etiolated and illuminated hypocotyls identified a doublet at 57/58.5 kDa which is immuno-reactive with antibodies raised to tomato extensin peroxidase. Levels of the 58.5-kDa protein, determined by SDS-PAGE, were at least threefold higher in illuminated tomato hypocotyls than in etiolated hypocotyls. Three fold higher levels of extensin peroxidase, elevated in-vitro extensin cross-linking activity and 15% higher levels of cross-linked, non-extractable extensin were observed in illuminated tomato hypocotyls compared with etiolated tomato hypocotyls. This suggests that white-light inhibition of tomato hypocotyl growth appears to be mediated, at least partially, by deposition of cell wall extensin, a process regulated by M_r-58,500 extensin peroxidase. Our results indicate that the contribution of peroxidase-mediated extensin deposition to plant cell wall architecture may have an important role in plant growth. Received: 22 July 1999 / Accepted: 11 October 1999     

Characterization of the Stimulation of Ethylene Production by Galactose in Tomato (Lycopersicon esculentum Mill.) Fruit

We have characterized the stimulation of ethylene production by galactose in tomatoes (Lycopersicon esculentum Mill.). The effect of concentration was studied by infiltrating 0, 4, 40, 100, 200, 400, or 800 micrograms galactose for each gram of fresh fruit weight into mature green `Rutgers' fruit. Both 400 and 800 micrograms per gram fresh weight consistently stimulated a transient increase in ethylene approximately 25 hours after infiltration; the lower concentrations did not. Carbon dioxide evolution of fruit infiltrated with 400 to 800 micrograms per gram fresh weight was greater than that of lower concentrations. The ripening mutants, rin and nor, also showed the transient increase in ethylene and elevated CO₂ evolution by 400 micrograms per gram fresh weight galactose. 1-Aminocyclopropane-1-carboxylic acid (ACC) content and ACC-synthase activity increased concurrently with ethylene production. However, galactose did not stimulate ACC-synthase activity in vitro. The infiltrated galactose in pericarp tissue was rapidly metabolized, decreasing to endogenous levels within 50 hours. Infiltrated galacturonic acid, dulcitol, and mannose stimulated transient increases in ethylene production similar to that of galactose. The following sugars produced no response: sucrose, fructose, glucose, rhamnose, arabinose, xylose, raffinose, lactose, and sorbitol.     

Enzymic properties and capacities of developing tomato (Lycopersicon esculentum L.) fruit plastids

To characterize the developmental stage of tomato fruits, chlorophyll content, photosynthetic O2 evolution and CO2 fixation of pericarp slices were determined. During the first developmental stages a higher expression level of the triose phosphate translocator was detected. Transport measurements revealed that both the hexose phosphate and the triose phosphate translocator are very likely to be active at this time. Plastidic and cytosolic fructose-1,6-bisphosphatase are active in green fruit pericarp, whereas in red pericarp only the cytosolic form is present. Tomato fruit chloroplasts are able to synthesize starch from Glc6P. Starch synthesis is strongly dependent on the addition of 3PGA and ATP and on plastid illumination. Fruit chloroplasts exhibit very low CO2 fixation rates and so the capacities of green pericarp slices were investigated. In relation to chlorophyll content, pericarp slices show the same capacity of starch synthesis as spinach or potato leaves. To investigate the presence of further reactions consuming the products of photosynthetic electron transport, the GOGAT activity was measured. In the light, glutamine/2-oxoglutarate-dependent formation of glutamate occurred with a high activity. In the presence of Glc6P only 18% of the light activity was obtained. Since the Glc6P-dependent activity is rather low, the release of ¹⁴CO2 from labelled [1-¹⁴C]-Glc6P was also measured. In the dark, the formation of glutamate and oxidation of Glc6P are very tightly coupled to each other in fruit chloroplasts.     

串番茄果实货架期间与耐贮性有关的生理特性的变化

串番茄品种随着货架期延长,果实硬度、果肉硬度、原果胶含量逐渐下降;可溶性果胶含量逐渐上升;多聚半乳糖醛酸酶(PG)活性呈峰值变化,变化幅度小于普通番茄.     

Patterns of Assimilate Distribution and Source--sink Relationships in the Young Reproductive Tomato Plant (Lycopersicon esculentum Mill.)

Patterns of distribution of ¹⁴C were determined in 47-day-oldtomato plants (Lycopersicon esculentum Mill.) 24 h after theapplication of [¹⁴C]sucrose to individual source leaves fromleaves 1–10 (leaf 1 being the first leaf produced abovethe cotyledons). The first inflorescence of these plants wasbetween the ‘buds visible’ and the ‘firstanthesis’ stages of development. The predominant sink organs in these plants were the root system,the stem, the developing first inflorescence and the shoot ‘apex’(all tissues above node 10). The contribution made by individualsource leaves to the assimilate reaching these organs dependedupon the vertical position of the leaf on the main-stem axisand upon its position with respect to the phyllotactic arrangementof the leaves about this axis. The root system received assimilateprincipally from leaf 5 and higher leaves, and the stem apexfrom the four lowest leaves. The developing first inflorescencereceived assimilates mainly from leaves in the two orthostichiesadjacent to the radial position of the inflorescence on thevertical axis of the plant; these included leaves which weremajor contributors of ¹⁴C to the root system (leaves 6 and 8)and to the shoot apex (leaves 1 and 3). This pattern of distributionof assimilate may explain why root-restriction treatments andremoval of young leaves at the shoot apex can reduce the extentof flower bud abortion in the first inflorescence under conditionsof reduced photoassimilate availability. Lycopersicon esculentum Mill, tomato, assimilate distribution, source-sink relationships     

Physiological and biochemical responses of fruit exocarp of tomato (Lycopersicon esculentum Mill.) mutants to natural photo-oxidative conditions

Photo-oxidative stress was imposed under natural solar radiation on exposed and shaded sections of detached fruit of immature green tomato (Lycopersicon esculentum Miller = Solanum lycopersicum L.) mutants (anthocyanin absent, beta-carotene, Delta, and high pigment-1) and their nearly isogenic parents ('Ailsa Craig' and 'Rutgers'). After 5 h exposure to high solar irradiance, either with or without ultraviolet (UV) radiation, surface colour changes, pigment composition, photosynthetic efficiency, antioxidant metabolites and enzyme activities, and selected flavonoids and antioxidant proteins in exocarp tissue were evaluated. The imposed photo-oxidative stress reproduced the symptoms observed on attached fruit. Both high temperature and solar irradiance caused fruit surface discoloration with faster degradation of chlorophyll (Chl) than carotenoids (Car), leading to an increase in the Car/Chl ratio. Surface bleaching was mostly caused by visible light, whereas elevated temperatures were mostly responsible for the inactivation of photosynthesis, measured as decreased F(v)/F(m). Ascorbate, glutathione, and total soluble protein concentrations decreased in the exocarp as the duration of exposure increased. Specific activities of superoxide dismutase, ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase (MDHAR), glutathione reductase (GR), and catalase increased with exposure, suggesting that these proteins were conserved during the imposed stress. GR protein expression remained stable during the imposed stress, whereas, MDHAR protein expression increased. Quercetin and kaempferol concentrations increased rapidly upon exposure, but not to UV radiation, suggesting rapid photo-protection in response to visible light; however, naringenin synthesis was not induced. The apparent increased tolerance of hp-1 fruit is discussed.