Effects of exogenous quercetin on the levels of carbohydrates and amino acids in fruits ofLycopersicon esculentum

Low doses of the exogenous flavonoid quercetin increased the content of sugars in tomato fruits of various cultivars. The content of glucose in tomato fruits of cv.Ukrainskii teplichnyi increased from 3.62 to 11.24% per unit dry weight. Increases in the content of glucose were found in all tomato cultivars examined. An analysis of qualitative and quantitative compositions of amino acids showed that their levels were markedly decreased in fruits of quercetin-treated plants. Our studies and data found in the literature suggest that this effect is due to the synthesis of sugars from amino acids by gluconeogenesis. The reverse process of sugar hydrolysis does not occur because exogenous quercetin inhibits the activity of pyruvate kinase.     

Biomechanics and anatomy of Lycopersicon esculentum fruit peels and enzyme-treated samples

We report the biomechanics and anatomy of fruit wall peels (before and after cellulase/pectinase treatment) from two Lycopersicon esculentum cultivars (i.e., Inbred 10 and Sweet 100 cherry tomatoes). Samples were tested before and after enzyme treatment in uniaxial tension to determine their rate of creep, plastic and instantaneous elastic strains, breaking stress (strength), and work of fracture. The fruit peels of both cultivars exhibited pronounced viscoelastic and strain-hardening behavior, but differed significantly in their rheological behavior and magnitudes of material properties, e.g., Inbred 10 peels crept less rapidly and accumulated more plastic strains (but less rapidly), were stiffer and stronger, and had a larger work of fracture than Sweet 100 peels. The cuticular membrane (CM) also differed; e.g., Sweet 100 CM strain-softened at forces that caused Inbred 10 to strain-harden. The mechanical behavior of peels and their CM correlated with anatomical differences. The Inbred 10 CM develops in subepidermal cell layers, whereas the Sweet 100 CM is poorly developed below the epidermis. Based on these and other observations, we posit that strain-hardening involves the realignment of CM fibrillar elements and that this phenomenon is less pronounced for Sweet 100 because fewer cell walls contribute to its CM compared to Inbred 10.     

Sucrose uptake,invertase localization and gene expression in developing fruit of Lycopersicon esculentum and the sucrose-accumulating Lycopersicon hirsutum

By using immunolocalization and differential extraction methods we show that only apoplastic invertase, but not vacuolar invertase, was present in the mature, sucrose-accumulating L. hirsutum pericarp. In contrast, in the hexose-accumulating L. esculentum fruit, both the apoplastic and vacuolar invertase activities and protein content increase in the mature fruit. Quantitative expression studies of the soluble invertase gene (TIV1) and the apoplastic invertase genes (LINs) showed that only TIV1 gene expression could account for the species and developmental differences of both soluble and insoluble enzyme activity of the pericarp. The expression of the LIN genes encoding for apoplastic tomato invertases was unrelated to the differences in bound enzyme activity and could not account for the rise in bound invertase activity in the mature L. esculentum fruit. Evidence is presented that the bound invertase activity of tomato fruit is also the TIV1 gene product. The presence of apoplastic invertase in the mature sucrose-accumulating L. hirsutum fruit suggests a hydrolysis-resynthesis mechanism of sucrose uptake. In order to test this hypothesis, we studied short- and long-term uptakes of asymmetrically labelled ³H-fructosyl-sucrose accompanied by compartmental analysis of the sugars in attached whole fruits of L. hirsutum and L. esculentum. The results indicate that hydrolysis-resynthesis is slow in the sucrose-accumulating fruit but is not an integral part of an uptake and compartmentation mechanism.     

Effect of exogenous amino acids on the intracellular content of proline and other amino acids in Daucus carota cells

The effect of tryptophan on the biosynthesis of proline has been investigated. Cells of Daucus carota grown in B5 medium supplemented with 5×10^–4M tryptophan acquired the ability to grow in the presence of inhibitory concentrations of azetidine-2-carboxylic acid, an analog of proline. When trp was added to carrot cell cultures at sub-growth inhibiting concentrations, overproduction of intracellular free proline was observed. An increase was also observed for lys, his, ala, leu and phe. Likewise, the addition of asparagine, glutamic acid and phenylalanine to the medium stimulated the intracellular increase of free proline and other amino acids.Abbreviations A2CA azetidine-2-carboxylic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 5MT 5-methyltryptophan - P5C pyrroline-5-carboxylic acid - f.wt. fresh weight - d.wt. dry weight     

Effect of P-deficiency on photoassimilate partitioning and rhythmic changes in fruit and stem diameter of tomato (Lycopersicon esculentum) during fruit growth

Tomato (Lycopersicon esculentum) plants were grown in liquid culture inside the greenhouse of Hiroshima University, Japan. At the first fruiting stage, P was withdrawn from the rooting medium for a period of 19 d and its effect was studied on photosynthesis, stomatal conductance, transpiration, partitioning of 13C and 15N, P contents of various organs, and changes in stem and fruit diameter of the plant in order to identify the mechanism of resource management on the part of the plant at low P. Compared to the control, P-deficiency treatment decreased biomass growth of all organs except the roots. The treatment also depressed leaf photosynthesis, stomatal conductance and diameter of fruit and stem after a lag period of about 1 week. The stem diameter of the plant shrank during daytime and expanded during the night; the adverse effect of P-deficiency on stem diameter change was more evident during the night than the day. The circadian rhythm in fluctuations of diameter was less manifested in the fruit compared with the stem. P-deficiency induced daytime shrinkage and reduced night expansion of fruit. However, within the plant, P-deficiency encouraged partitioning of 13C, 15N and P into the fruit at the cost of autotrophic organs such as leaves and the upper parts of the stem. The results were discussed in the light of a plausible effect of P-deficiency on water relations of the plant. It is concluded that, in spite of the preference in partitioning of C and N received within the plant parts, assimilate flow into the fruit is limited at low-P compared with the control, owing to the restriction in fruit expansion.     

Effect of exogenous amino acids on Cu uptake and translocation in maize seedlings

This study aimed to assess the effects of four contrasting proteinogenic amino acids on copper (Cu) uptake and translocation in maize (Zea mays L.) seedlings grown in a modified Hoagland solution. Glycine, aspartic acid and lysine at three concentrations (10, 25 and 100 μM) did not have any significant effect on Cu uptake and translocation in maize seedlings over a two-day experimental period. However, cysteine (a reductive amino acid) at the three concentrations increased very significantly (P < 0.01) Cu accumulations in the root symplast and the shoots of maize seedlings in comparison to the control. Cu uptake in the whole plant and Cu translocation from root to shoot were also increased in the cysteine treatments. In the 25 μM cysteine treatment, where cysteine was in moderate excess, the Cu uptake in the whole plant and Cu translocation from root to shoot were significantly (P < 0.01) higher than those of the 10 or 100 μM cysteine treatments, where the concentration of cysteine was equivalent to that of Cu(II) or in great excess according to the stoichiometry of the redox reaction of cysteine with Cu(II). It is hypothesized that the cysteine-induced oxidation state alteration from Cu(II) to Cu(I) could be responsible for the increased Cu uptake and Cu translocation, on the ground that Cu(I), as free cuprous ion or cysteine cuprous complex, may be more available to maize roots than Cu(II).     

Polyamine levels in pollinated and auxin-induced fruit of tomato (Lycopersicon esculentum) during development

The changes taking place during fruit development in the concentration of the 3 polyamine fractions, i.e. free, perchloric acid-soluble conjugates and perchloric acid-insoluble bound polyamines, were analyzed in tomato fruits ( Lycopersicon esculentum Mill, cv. F121) induced to set by either pollination or auxin application. Before the onset of cell division, total polyamines were 50% higher in auxin-treated fruits than in pollinated ones, most of the polyamines being found as perchloric acid-soluble conjugates in both fruit set treatments. At the onset the level of polyamines in both fruit types was 100 times higher than during cell expansion or ripening. The highest polyamine found during cell division was perchloric acid-soluble conjugated spermidine in both fruit set treatments. After cell division, polyamine concentration was similar in both fruit set treatments. The concentration of polyamines in the jelly was similar in pollinated and auxin-induced fruits during cell expansion but not during ripening. At the onset of ripening there was an increase of one order of magnitude in the concentration of perchloric acid-insoluble bound putrescine in the jelly of pollinated fruits. Polyamines were more than 5-fold higher in unpollinated ovaries than in fruits induced to set by either pollination or auxins. It is suggested that pollinated and parthenocarpic fruits differ in their polyamine metabolism during the initial stages of development, but not after cell division. It is also suggested that polyamines undergo rapid turnover during cell division. Perchloric acid-insoluble bound putrescine might play a role in seed formation in tomatoes.     

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.     

Interaction of Auxin-TIBA in Lycopersicon esculentum

[5-³H] indoleacetic acid (IAA) was applied to the apical budof young tomato plants (Lycopersicon esculentum Mill.) treatedor not with 2,3,5-triiodobenzoic acid (TIBA). Chromatographicanalysis showed that treatment with TIBA increased the quantityof breakdown products in the apical 1.5 mm segment of the stem.Autoradiographs of ultra-thin sections of this segment wereprepared after treatment with glutaraldehyde. The inhibitionof polar auxin transport by TIBA caused an increase in the densityof labelling of all the cellular types in the shoot apex. TheTIBA treatment provoked a modification of the structure of theshoot apex in which the greatest increases in density of labellingwere detected in the superficial tunical layer and in the pithmeristem. The increase in radioactivity of the superficial tunicallayer could be explained by the presence of polyphenols in thevacuoles. For the pith meristem, the TIBA treatment intensifiedthe high labelling previously observed at this level in thecontrol plants. This could indicate a role for auxin in cellularelongation. It could also point to the importance of the pithmeristem in the functioning of the shoot apex. Key words: [³H] IAA, Lycopersicon esculentum, TIBA     

Effect of temperature on biomass allocation in tomato (Lycopersicon esculentum)

Temperature may influence dry matter partitioning between fruits and vegetative plant parts either directly or indirectly through its influence on development, flower and/or fruit abortion. The objective of the present work was to investigate whether there is any direct effect of temperature on dry matter partitioning between fruits and vegetative plant parts in tomato. A greenhouse experiment was conducted, with alternating 3-week periods of high (23°C) and low (18°C) temperature setpoint. Dry matter partitioning during these 3-week periods was determined from destructive plant harvests at two levels of fruit pruning (3 and 7 fruits per truss). Indirect temperature effects on dry matter partitioning were excluded by fruit pruning.
On average, the fraction of dry matter distributed to the fruits during a 12-week period, starting with the flowering of the fifth truss (28 days after planting), was 0.53 (3 fruits per truss) and 0.70 (7 fruits per truss). These ratios were also calculated for every 3-week period separately and did not depend on the average temperature (18–24°C) during that period.
It is concluded that dry matter distribution in tomato is not significantly affected by temperature directly, which means that the temperature effect (18–24°C) on the generative sink strength is not much different from the temperature effect on the vegetative sink strength.     

Analysis of amino acids and carbohydrates in green coffee

The analysis of carbohydrates and amino acids in green coffee is of the utmost importance since these two classes of compounds act as precursors of the Maillard reaction during which the colour and aroma are formed. During the course of the Maillard reaction potentially harmful substances like acrylamide or 5-hydroxymethyl-furfural accrue as well. The carbohydrates were analysed by anion-exchange chromatography with pulsed amperometric detection and the amino acids by reversed phase chromatography after derivatization with 6-amino-quinolyl-N-hydroxysuccinimidyl carbamate and fluorescence detection. Both methods had to be optimized to obtain a sufficient resolution of the analytes for identification and quantification. Sucrose is the dominant carbohydrate in green coffee with a concentration of up to 90 mg/g (mean = 73 mg/g) in arabica beans and significantly lower amounts in robusta beans (mean=45 mg/g). Alanine is the amino acid with the highest concentration (mean = 1200 microg/g) followed by asparagine (mean = 680 microg/g) in robusta and 800 microg/g and 360 microg/g in arabica respectively. In general, the concentration of amino acids is higher in robusta than in arabica.     

Properties of proton and sugar transport at the tonoplast of tomato (Lycopersicon esculentum) fruit

Tonoplast vesicles were isolated from tomato (Lycopersicon esculentum Mill.) fruit pericarp and purified on a discontinuous sucrose gradient. ATPase activity was inhibited by nitrate and bafilomycin A₁ but was insensitive to vanadate and azide. PPase hydrolytic activity was inhibited by NaF but was insensitive to nitrate, bafilomycin A₁ vanadate and azide. Kimetic studies of PPase activity gave an apparent K_m, for PP₃ of 18 μM. Identical distributions of bafilomycin- and NO₃-sensitive ATPase activities within continuous sucrose density gradients, confirmed that bafilomycin-sensitive ATPase activity is a suitable marker for the tonoplast. By comparing the distribution of bafilomycin-sensitive ATPase activity with that of PPase activity, it was possible to locate the PPase enzyme exclusively at the tonoplast. The apparent density of the tonoplast did not change during fruit development. Measurements of tonoplast PPase and ATPase activities during fruit development over a 35-day period revealed an 80% reduction in PPase specific activity and a small decrease in ATPase specific activity. ATP- and PP₁-dependent ΔpH generation was measured by the quenching of quinacrine fluorescence in tonoplast vesicles prepared on a discontinuous Dextran gradient. No H⁺ efflux was detected on the addition of sucrose to energized vesicles. Therefore a H⁺/sucrose antiport may not be the mechanism of sucrose uptake at the tomato fruit tonoplast. Similar results were obtained with glucose, fructose and sorbitol. The lack of ATP (or PP₁) stimulation of [¹⁴C]-sucrose uptake also suggested that an antiport was not involved. Initial uptake rates of radiolabelled glucose and fructose were almost double that for sucrose. The inhibition of hexose uptake by p-chloromercuribenzene sulphonate (PCMBS) implicated the involvement of a carrier. Therefore storage of hexose in the tomato fruit vacuole and maintenance of a downhill sucrose concentration gradient into sink cells is likely to be regulated by the activity of sucrose metabolizing enzymes, rather than by energy-requiring uptake mechanisms at the tonoplast.     

Placental transport and utilization of amino acids and carbohydrates

Uteroplacental tissues have been shown to have a high rate of metabolism under in vivo steady-state conditions. Fully two-thirds of the glucose and one-half of the oxygen consumed by the uterus are utilized by these tissues rather than by the fetus. Its high metabolic rate must be borne in mind in any analysis of tracer kinetics, which prohibits the exclusion of these tissues and the use of a two-compartment model for analysis of carbohydrate and amino acid metabolism. Current techniques permit determination of utilization rates of nutrients in all three compartments (fetal, uteroplacental, and maternal) with considerable precision. Using tracer amino acids one can determine rates of protein synthesis and protein breakdown as well as rates of amino acid oxidation. These techniques should prove useful in investigating the role of various trophic factors in fetal life and in assessing the impact of changes in placental function or maternal nutritional state on fetal growth and metabolism.     

Effects of metals on analytical procedures for amino acids and carbohydrates

Low levels of some metals, particularly Cu(II) and Fe(III), prevent the accurate determination of certain amino acids and carbohydrates. The degree of interference depends upon the procedure used and on the nature of the sample. No completely satisfactory method has been found to relieve this interference except to remove the metal ions prior to analysis.     

Tomato (Lycopersicon esculentum Mill.) fruit growth and ripening as related to the biomechanical properties of fruit skin and isolated cuticle

The control of growth rate and the mechanical integrity of the tomato (Lycopersicon esculentum Mill.) fruit has been attributed to the exocarp. This study focused on the biomechanics of the fruit skin (FS) comprising cuticle, epidermis and a few subdermal cell layers, and the enzymatically isolated cuticular membrane (CM) during fruit growth and ripening. Morphology and mechanical properties of the FS and the CM of three cultivars were analysed separately at three distinct ripening stages by scanning electron microscopy (SEM) and one-dimensional tension testing, respectively. Both were subject to significant cultivar-specific changes. Thickness of the CM increased during ripening from 7.8-8.6 to 9.9-15.7 microm and exceeded by far that of the epidermal cell wall. The mechanical properties, such as modulus of elasticity, strength, and failure strain, were highest in the FS for all cultivars at any stage, with only one exception; however, the cuticle largely mirrored these properties throughout fruit maturation. Stiffness of both isolated CM and FS increased from immature to fully ripe fruits for all cultivars, while failure stress and failure strain displayed a tendency to decrease for two of them. Stress-strain behaviour of the CM could be described as strain softening, mostly linear elastic throughout, and strain hardening, and was subject to growth-related changes. The FS displayed strain hardening throughout. The results indicate evidence for the cuticle to become increasingly important as a structural component for the integrity of the tomato fruit in addition to the epidermis. A supplementary putative model for tomato fruit growth is proposed.     

Identification and quantitation of gibberellins in fruits of Lycopersicon esculentum, and their relationship to fruit size in L. esculentum and L. pimpinellfolium

GA₁, GA₈, GA₁₇, GA₁₉, GA₂₀ and GA₂₉ were identified by combined gas chromatography-mass spectrometgry (GC-MS) in immature seeds and pericarp of Lycopersicon esculentum Mill. (tomato). Higher levels of these GAs were present in the seeds than in the pericarp; seeds in addition contained GA₁₅, GA₂₄, GA₂₅, and GA₄₄. Fruits of the Lycopersicon pimpinellifolium Mill. mutant I were smaller and contained lower GA₁ concentrations, but higher GA₂₀ concentrations, than those of mutants III and IV. In contrast, differences in fruit size in L. esculentum due to position on the truss did not correlate with GA₁ concentration in either the pericarp or seeds.