High hexokinase activity in tomato fruit perturbs carbon and energy metabolism and reduces fruit and seed size

Tomato (Lycopersicon esculentum var MP‐1) plants overexpressing Arabidopsis hexokinase 1 (AtHXK1) exhibited high hexokinase (HXK) activity in correlation with drastic phenotypic modifications in fruit. Transgenic fruit and seeds were reduced in size. Reduction in fruit size was due to decreased cell expansion, which could not be corrected by perfusion with sucrose (Suc). Neither could wild type (WT) fruit and seed size be obtained by grafting of transgenic flowers onto WT shoots. Starch and hexose contents were lower but organic and amino acids were higher in transgenic fruit. Lower respiratory rates measured in vitro accompanied by even lower ATP levels and ATP/ADP ratios indicated metabolic perturbations that may explain, in part, reduced fruit and seed size.     

Graft union formation in tomato plants: peroxidase and catalase involvement

BACKGROUND AND AIMS: The use of grafted plants in vegetable crop production is now being expanded greatly. However, few data are available on the formation of graft unions in vegetables. In this work, the structural development of the graft union formation in tomato plants is studied, together with the possible relationship with activities of peroxidases and catalases. METHODS: Tomato (Lycopersicon esculentum Mill.) seedlings of cultivar Fanny were grafted on the rootstock of cultivar AR-9704 using the 'tongue approach grafting' method, and were grown in a crop chamber. A study of the structural development of the graft union and the involvement of peroxidases and catalases in the process of graft formation was carried out during the first stages of the graft union (4, 8 and 15 d after grafting). KEY RESULTS: Observation of the structure of the graft union showed formation of xylem and phloem vessels through the graft union 8 d after grafting. In addition, root hydraulic conductance, L0, indicate that the graft union is fully functional 8 d after grafting, which coincided with an increase of peroxidase and catalase activities. CONCLUSIONS: These results suggest that increased peroxidase and catalase activities might be implicated in graft development in tomato plants.     

Effect of sulphate on photosynthesis in greenhouse–grown tomato plants

Sulphate accumulates in the rhizosphere of plants grown in hydroponic systems. To avoid such sulphate accumulation and promote the use of environmentally sound hydroponic systems, we examined the effects of four sulphate concentrations (0.1, 5,2, 10.4 and 20.8 m M ) on photosynthesis, ribulose-l,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) activities and related physiological processes in greenhouse–grown tomato plants ( Lycopersicon esculentum Mill. cv. Trust). The lowest sulphate concentration (0.1 m M ) significantly decreased photosynthetic capacity (P_c) and Rubisco activities on a leaf area basis. This result was supported by our data for dry matter per plant, which was low for plants in the 0.1 m M treatment. The photosynthesis-related variables such as leaf conductance, chlorophyll and soluble protein were lowest for the 0.1 m M treatment. Both total Rubisco activity and the activated ratio were reduced with this treatment. However, Rubisco activities expressed per g of protein or per g of chlorophyll were not significantly affected. These results suggest that sulphur deficiency depressed P_c– by reducing the amount of both Rubisco and chlorophyll and by causing an inactivation of Rubisco. The ratio of organic sulphur vs organic nitrogen (S/N) in plants of the 0.1 m M treatment was far below the normal values. This low S/N ratio might be accountable for the negative effect of low sulphate on P_c and plant growth. P_c and dry matter were not affected until sulphate concentration in the nutrient solution reached a high level of 20.8 m M .     

Developmental effects of 24-epibrassinolide in excised roots of tomato grown in vitro

The effects of 1 p M –10 μ M 24-epibrassinolide presented to apical and basal regions of excised roots of tomato ( Lycopersicon esculentum Mill. cv. Best of All) grown aseptically in a two-well culture vessel have been investigated. Only inhibitory effects were observed and only at 0. 1 μ M or greater. At 10 μ M basally- and apically-applied epibrassinolide inhibited growth in apical regions, but not in basal regions. Lower concentrations (1 and 0. 1 μ M ) also inhibited growth, again only in apical regions and usually only when presented directly to those regions. Cultured tomato roots therefore appear to transport epibrassinolide acropetally, but whether they do so basipetally is not yet clear. The reduced responsiveness to epibrassinolide observed in roots grown by this method is thought to be due to the larger inoculum used rather than the physiological age of the roots. There was some evidence that the sensitivity of cultured roots to epibrassinolide is directly related to growth rate     

Identification of cytokinins in primary crown gall tumours of tomato

Abstract. Identification of the cytokinin complex of primary crown gall tumours of tomato ( Lycopersicon esculentum L.) by combined gas chromatography-mass spectrometry has been described. Several cytokinins have been identified which included zeatin, dihydrozeatin, isopentenyladenine, and their respective riboside and nucleotide derivatives. In addition, 6-benzylaminopurine, its riboside and the corresponding nucleotide have also been identified as major endogenous compounds in this tissue. This would appear to be the first report on the identification of cytokinins from a primary crown gall tumour tissue using unequivocal methods.     

Inhibition of brassinosteroid-induced epinasty in tomato plants by aminooxyacetic acid and Co2+

The inhibitory effects of aminooxyacetic acid (AOA) and cobalt chloride (CoCl₂) on brassinosteroid (BR)-induced epinasty in tomato plants ( Lycopersicon esculentum Mill. cv. Heinz 1350) are evaluated. CoCl₂ dramatically decreases petiole bending and ethylene production as the concentration increases from 50 to 200 μ M. The content of 1-aminocyclopropane-1-carboxylic acid (ACC) in the petiole, instead of accumulating, is reduced and does not change over the concentration range tested. Inhibition of BR-induced epinasty by AOA results from inhibition of ACC synthesis. There are dramatic reductions in petiole bending, ethylene and ACC production as the concentration of AOA is increased from 50 to 200 μ M. Maximum inhibition occurs when the plants are pretreated with the inhibitors. The degree of inhibition increases as the length of pretreatment increases from 1 to 4 h. The response of BR-treated plants to AOA and CoCl₂ is similar to the effect of auxin, indicating the integral relationship between BR and auxin.     

Physiological effects of peracetic acid on hydroponic tomato plants

Peracetic acid (PAA) has potential as a disinfectant of low environmental impact for glasshouse hydroponic systems and other horticultural applications, but can have phytotoxic effects. This study examined the physiological effects of PAA when applied hydroponically to tomato plants. Plants treated with 0.5–5 μg ml^?1 PAA over several weeks exhibited a reduction in size of all vegetative organs. During the first 2 h of PAA treatment, plants also exhibited a transient wilting, with increased stomatal resistance, and reductions in transpiration and CO2 assimilation. The toxicity of PAA to roots was apparent from increased leakage of root electrolytes, reduced oxygen consumption, death of root tips, and collapse of the internal tissues. The shrivelling of PAA‐treated roots resulted from loss of water to the shoot in the transpiration stream, as the effect could be eliminated by removal of the shoot and sealing of the cut stump. HgCl2, a reagent known to reduce the hydraulic conductivity of root systems, caused the same root shrivelling effects as PAA. Long‐term growth of PAA‐treated plants was dependent upon the replacement of taproot systems by adventitious roots, which, initially at least, displayed greater tolerance of PAA. In aqueous solution, PAA exists in equilibrium with H2O2 and acetic acid, both of which were individually toxic, but acetic acid exhibited a syndrome of effects distinct from those of PAA, while the effects of H2O2 paralleled those of PAA more closely, suggesting that oxidative rather than acidic mechanisms were primarily responsible for the phytotoxicity of PAA solutions.     

Tropic responses of hypocotyls from normal tomato plants and the gravitropic mutant Lazy-1

Abstract Etiolated hypocotyls from normal tomato plants show a negative gravitropic response within 20 min of stimulation. In contrast, etiolated hypocotyls from the gravitropic mutant Lazy-l do not reorientate after gravistimulation. Etiolated hypocotyls from both types of plant are positively phototropic, however, Lazy-l seedlings achieve a greater final angle of bending following phototropic stimulation compared to normal plants. Anatomical studies reveal that etiolated hypocotyls from normal plants contain sedimenting amyloplasts located within the endodermal cells. Such sedimenting amyloplasts are absent in Lazy-l tissue. It is hypothesized that the hypocotyl of Lazy-l is agravitropic since it is unable to perceive a gravistimulus.     

Effects of increasing inorganic carbon supply to roots on net nitrate uptake and assimilation in tomato seedlings

We investigated the influence of an increased inorganic carbon supply in the root medium on NO^?₃ uptake and assimilation in seedlings of Lycopersicon esculentum (L.) Mill. cv. F144. The seedlings were pre-grown for 4 to 7 days with 0 or 100 mM NaCl in hydroponic culture using 0.2 mM NO^?₃ (group A) or 0.2 mM NH⁺₄ (group B) as nitrogen source. The nutrient solution for group A plants was aerated with air or with air containing 4 800 μumol mol^?1 CO₂. Nitrate uptake rate and root and leaf malate contents in these plants were determined. The plants of group B were subdivided into two sets. Plants of one set were transferred either to N-free solution containing 0 or 5 mM NaHCO₃, or to a medium containing 2 mM NO^?₃ and 5 mM NaHCO₃. Both sets of group B plants were grown for 12 h in darkness prior to 2 h of illumination, and were assayed for malate content and NO^?₃ uptake rate (only for plants grown in N-free solution). The second set of group B plants was labeled with ¹⁴C by a 1-h pulse of H¹⁴CO^?₃ which was added to a 5 mM NaHCO₃ solution containing 0 or 100 mM NaCl and 0 or 2 mM NO^?₃, and ¹⁴C-assimilates were extracted and fractionated. The roots of group B plants growing in carbonated medium accumulated twice as much malate as did control plants. This malate was accumulated only when NO^?₃ was absent from the root medium. Both a high level of root malate and aeration with CO₂-enriched air stimulated NO^?₃ uptake. Analysis of ¹⁴C-assimilates indicated that with no NO^?₃ in the medium, the ¹⁴C was present mainly in organic acids, whereas with NO^?₃, a large proportion of ¹⁴C was incorporated into amino acids. Transport of root-incorporated ¹⁴C to the shoot was enhanced by NO^?₃, while the amino acid fraction was the major ¹⁴C-assimilates in the shoot. It is concluded that inorganic carbon fixed through phosphoenolpyruvate carboxylase (EC 4.1.1.31) in roots of tomato plants may have two fates: (a) as a carbon skeleton for amino acid synthesis; and (b) to accumulate, mainly as malate, in the roots, in the absence of a demand for the carbon skeleton. Inorganic carbon fixation in the root provides carbon skeletons for the assimilation of the NH⁺₄ resulting from NO₃ reduction, and the subsequent removal of amino acids through the xylem. This ‘removal’ of NO^?₃ from the cytoplasm of the root cells may in turn increase NO^?₃ uptake.     

Salt stress increases ferredoxin-dependent glutamate synthase activity and protein level in the leaves of tomato

Ferredoxin-dependent glutamate synthase (EC 1.4.7.1) catalyzes an essential step in the pathway of glutamate biosynthesis. Exposing detached tomato ( Lycopersicon esculentum ) leaves for 6 h to 12 g l⁻¹ NaCl resulted in a significant two-fold increase in the activity of ferredoxin-dependent glutamate synthase extracted from the leaves. Western blot studies demonstrated that salt treatment also increased the ferredoxin-dependent glutamate synthase content of the leaves. A similar effect of salt on the concentration of this enzyme was found in the leaves of hydroponically-grown tomato plants. The induction of ferredoxin-dependent glutamate synthase under salt stress may provide the glutamate required for the proline synthesis which is a common response to salt stress.     

Inhibition of hypocotyl elongation by ultraviolet-B radiation in de-etiolating tomato seedlings. II. Time-course, comparison with flavonoid responses and adaptive significance

UV-B radiation inhibits hypocotyl elongation in etiolated tomato (Lycopersicon esculentum Mill. cv. Alisa Craig) seedlings acting through a photoreceptor system with peak apparent effectiveness around 300 nm. In order lo further characterize the response and gain insight into its potential ecological significance, the time-course of inhibition was measured and compared with the time-course of flavonoid accumulation in the same seedlings. When a background of strong (> 620 μmol m^?2 s^?1) white light (WL) was supplemented with low irradiance UV-B (～ 3 μmol m^?2 s^?1). substantial (～ 50%) inhibition of elongation occurred within 3 h of the light treatment. The magnitude of UV-B-induced elongation inhibition was similar in wild type (WT) and au-mutant seedlings, in spite of the large differences between genotypes in rate and temporal pattern of elongation. In comparison to the effect of UV-B on elongation, induction of flavonoid accumulation in WT and au seedlings undergoing de-etiolation was a much slower response. Several UV-absorbing compounds appeared to be specifically induced by light, and some of them accumulated faster under the WL + UV-B treatment than under WL alone. However, there was little or no delectable effect of WL on flavonoid levels until up to 3 h of treatment, and the specific UV-B effect was measurable only after 6 h of continuous treatment. Indeed. UV-B-screening properties of crude alcoholic extracts were not different between WL and WL + UV-B treatments until after 9 or 24 h. When the light treatments were applied to seedlings that were just breaking through the soil surface. UV-B was found to consistently retard seedling emergence. These results suggest that the rapid inhibition of elongation in de-etiolating seedlings is an evolved response lo UV-B, which may serve to minimize seedling exposure to sunlight until protective pigmentation responses (triggered by WL and UV-B) have taken place in the seedlings epidermis.     

Oligosaccharides that induce proteinase inhibitor activity in tomato plants cause depolarization of tomato leaf cells

Abstract. Two size ranges of oligosaccharide elicitors of pectic origin have been investigated for their effects on tomato plants. Both size ranges, with degrees of polymerization of 1–7 and 10–20 respectively, induced the accumulation of proteinase inhibitor (PI) activity in excised plants, and also induced changes in membrane potential of leaf mesophyll cells. The depolarizations were substantial, rapid, and reversible on removal of the elicitors. The effects are discussed in the context of early events in the signal transduction pathway linking oligosaccharides to changes in PI gene expression.     

The role of mineral nutrients in the increased growth response of tomato plants in solarized soil

Soil solarization is a non-chemical disinfestation technique that frequently promotes plant growth in the absence of known major pathogens, a phenomenon termed increased growth response (IGR). The effect of solarization on plant nutrients and their role in the IGR was studied with tomato plants grown in solarized or non-solarized (control) sandy soil, under controlled conditions. Solarization considerably increased the soil concentrations of water extractable N, K, Ca, Mg and Na at most sites, whereas Cl and DTPA extractable Mn, Zn, Fe and Cu were decreased by the treatment. Plant growth and specific leaf area were enhanced in solarized as well as in N-supplemented control soil. In tomato plants grown in solarized soil, concentrations of most nutrients in the xylem sap, including N, were increased compared to the control, whereas Cl and SO4 levels decreased. The most significant increase in leaf nutrient concentration caused by soil solarization was recorded for N. Furthermore, leaf N concentration was highly and positively correlated with shoot growth. The concentration of Cu increased in leaves from the solarization vs. the control treatment, whereas that of SO4 and Cl decreased, the latter presumably below the critical toxicity level. The correlation between shoot growth and leaf concentration was positive for Cu and inverse for Cl and SO4. In conclusion, we found that soil solarization significantly affects nutrient composition in tomato plants, and provided strong evidence that N, and eventually also Cl, play a major role in IGR.     

An experimental assessment of the factors influencing Agrobacterium-mediated transformation in tomato

Agrobacterium tumefaciens strain LBA4404 carrying a binary vector pTOK233, which contained the GUS reporter gene and a kanamycin-resistance gene nptII, was employed for optimizing the transformation efficiency evaluated by a GUS gene transient expression level. Eight factors including explant types, explant size and source, the concentration of cytokinin, inoculation time, pH of inoculation and cocultivation media, bacterial concentration, acetosyringone concentration, and cocultivation duration were investigated in detail. This optimized protocol was then adopted to obtain transgenic tomato plants resistant to cucumber mosaic virus (CMV) mediated by Agrobacterium tumefaciens, strain LBA4404, carrying a binary vector pR-ΔGDD containing the kanamy cin-resistance gene and CMV replicase gene with GDD deletion. The presence of the CMV-RNA2 gene was confirmed by genomic DNA Southern blot analysis in all transformants analyzed. Field spray test showed that the transgenic tomato plants were resistant to 100 mg/l kanamycin. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 2, pp. 280–284. The text was submitted by the authors in English.     

Changes in free polyamine levels induced by salt stress in leaves of cultivated and wild tomato species

The effects of N_aCl on endogenous free levels of the poluamines putrescine, spermi dine and spermine, and the relationships between polyamines, K⁺ levels and Na⁺ accumulation were determined in leaves of the cultivated tomato ( Lycopersicon esculentum Mill.) and its wild, salt-tolerant relative L. pennellii (Correll) D' Arcy at different exposure times during a 32-day period. Both stress treatments (100 and 200 m M NaCl) decreased the levels of putrescine and spermidine, although to a different degree for the cultivated and wild tomato species. The spermine levels did not decrease with salinity in L. pennellii over the salinization period, whereas they decreased in L. esculentum , except at the first application of the 100m M NaCl treatment. In both species, the changes induced by salinity in total polyamines and K⁺ were very similar, with the accumulation of Na⁺ in the leaf being concomitant with a decrease in both total polyamines and K⁺. This suggests that the main role of the polyamines in the leaf tissues. In this sense, a direct relationship between total polyamines and K⁺, and inverse relationship between polyamines and Na⁺ and between K⁺ and Na⁺ were found for both species. In the short term (up to 4 days) a peculiar physiological behavior was found in L. pennellii , as the total polyamine and K⁺ levels decreased at 100 m M but not at 200 m M NaCl, while after this time the latter plants had values lower than those of the 100 m M NaCl-treated plants at day 11.     

Characterization of phytochelatin synthase from tomato

The enzyme that synthesizes Cd-binding phytochelatins (PCs), PC synthase, has been studied in tomato ( Lycopersicon esculentum ) cell cultures and plants. This enzyme transfers γ-GluCys from GSH or PC to either GSH or an existing polymer of (γ-GluCys)_nGly. PC synthase from tomato requires GSH or PCs as substrates but cannot utilise γ-GluCys or GSSG. PC synthase is activated both in vivo and in vitro by a variety of heavy metal ions, including Cd²⁺, Ag⁺, Cu²⁺, Au⁺, Zn²⁺, Fe²⁺, Hg²⁺ and Pb²⁺. In crude protein extracts from tomato cells the enzyme has an apparent K_m of 7.7 m M for GSH in the presence of 0.5 m M Cd²⁺, and exhibits maximum activity at pH 8.0 and 35°C. PC synthase is present in tomato cells grown in the absence of Cd. The level of enzyme activity is regulated during the cell culture cycle, with the highest activity occurring 3 days after subculture. Cadmium-resistant tomato cells growing in medium containing 6 m M CdCl₂ have a 65% increase in PC synthase activity compared to unselected cells. PC synthase is also present in roots and stems of tomato plants, but not in leaves or fruits. The distribution of the enzyme in tomato plants and regulation of PC synthase activity in tomato cells indicate that PC synthase, and PCs, may have additional functions in plant metabolism that are not directly related to the formation of Cd-PC complexes in response to cadmium.     

Determination of the surface area of fine tomato roots via cation uptake experiments

A method is described by which the surface area of a root is estimated from cation uptake data. ²⁴Na⁺ was supplied to excised roots of tomato ( Lycopersicon esculentum Mill, cv. Tiny Tim) seedlings at 3 μ M in unstirred solution. Coarse roots, for which external surface area and specific gravity could be measured accurately, were used to estimate the thickness (d_N) of the Nernst boundary layer at the root surface. ²⁴Na⁺ uptake (J₁) was measured by γ-ray spectroscopy. J_t and d_N were used to calculate the total surface area for ion absorption in fine roots, assuming that Na uptake rate was diffusion-limited. The results were compared to data obtained by conventional methods and indicated the usefulness of the cation uptake technique for quantitative estimates of root surfaces.     

Fate of benzyladenine metabolites extracted from tomato shoots in biological systems

The metabolism of radioactive metabolites of BA, obtained from tomato shoots, and purified by chromatographic techniques, was studied in rootless tomato shoots and soybean callus. There is apparently a great deal of interconversion between BA, BAR and BARMP. From the present results it is not possible to determine whether BARMP is formed directly from BA or whether BAR serves as an intermediate in its production. Irrespective of the metabolite applied, the principal metabolite formed in all instances had a retention time of 51 min following HPLC separation. This metabolite was itself not metabolized to any great extent by either tomato shoots or soybean callus. In contrast to tomato shoots this metabolite was not formed when a radioactive metabolite tentatively identified as BAR, was applied to soybean callus.Abbreviations AAW acetic acid: water - BA benzyladenine - BAR benzyladenosine - BARMP benzyladenosine-5-mono-phosphate - TEA triethylamine