Purification and characterization of a glucokinase from young tomato (Lycopersicon esculentum L. Mill.) fruit

In order to clearly establish the properties of the enzymes responsible for hexose phosphorylation we have undertaken the separation and characterization of these enzymes present in tomato fruit (Martinez-Barajas and Randall 1996). This report describes the partial purification and characterization of glucokinase (EC. 2.7.1.1) from young green tomato fruit. The procedure yielded a 360-fold enrichment of glucokinase. Tomato fruit glucokinase is a monomer with a molecular mass of 53 kDa. Glucokinase activity was optimal between pH 7.5 and 8.5, preferred ATP as the phosphate donor (K _m = 0.223 mM) and exhibited low activity with GTP or UTP. The tomato fruit glucokinase showed highest affinity for glucose (K _m = 65 μM). Activity observed with glucose was 4-fold greater than with mannose and 50-fold greater than with fructose. The tomato fruit glucokinase was sensitive to product inhibition by ADP (K _i = 36 μM). Little inhibition was observed with glucose 6-phosphate (up to 15 mM) at pH 8.0; however, at pH 7.0 glucokinase activity was inhibited 30–50% by physiological concentrations of glucose 6-phosphate. Received: 4 October 1997 / Accepted: 10 January 1998     

Characterization of a cDNA encoding 5-aminolevulinic acid dehydratase in tomato (Lycopersicon esculentum Mill.)

Summary A full-length cDNA clone encoding tomato (Lycopersicon esculentum Mill.) 5-aminolevulinic acid dehydratase (ALAD) was isolated and characterized. The primary structure predicts a 430-amino acid precursor which comprises a 41.7 kDa, 388-amino acid mature protein and a 47-amino acid transit sequence. The tomato primary sequence shows extensive homology to those of pea and spinach. Southern analysis indicated that 1 to 2 copies of the ALAD gene are present in the tomato genome. Northern blot analysis shows differential expression in various tomato organs, and constitutive developmental expression in tomato fruits.Abbreviations ALA 5-aminolevulinic acid - ALAD 5-aminolevulinic acid dehydratase (EC 4.2.1.24)     

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.     

Somaclonal variation versus chemically induced mutagenesis in tomato (Lycopersicon esculentum L.)

Summary A comparison was made of the type and frequency of mutational events found in the progeny of tomato plants regenerated after one passage in vitro with those induced by chemical mutagenesis with ethyl methane sulphonate. Several mutants were recovered in the progeny of regenerated and mutagenized plants of two cultivars of tomato. They can be grouped into the following categories: seedling lethality, male sterility, resistance to Verticillium, short stature, change in number of lateral shoots or in leaf shape. The results indicate that the two sources of variability differ in their effect, changing the spectrum and frequency of the mutants as well as, at least in some cases, their pattern of segregation.     

The histogenesis of somaclones from tomato (Lycopersicon esculentum Mill.) cotyledons

Summary A histological study ofin vitro cultured cotyledonary expiants of tomato (Lycopersicon esculentum) was performed in order to determine the site (differentiated tissue or developing callus) and the mode of plant regeneration.Results have shown that callus develops at the excision sites of cotyledonary expiants and that shoots are formed exclusively within the unorganized callus: excision areas are the only morphogenetic sites and the proximal excision is the preferred site for plant regeneration.Shoots differentiate by organogenesis within the superficial region of the callus. Few neocambial cells cooperate in the neoformation. Origin from a single cell is highly unlikely since rarely observed single activated cells never developed into shoots.Regenerated plants may be chimeras if invitro culture induces genetic diversity in the initial cells.Abbreviations IAA Indole-3-acetic acid - c callus - d vegetative dome - s shoot - ad adaxial - ab abaxial - t tracheid - p parenchyma - S sieve tube     

Structure and expression of a cDNA encoding zeaxanthin epoxidase, isolated from a wilt-related tomato (Lycopersicon esculentum Mill.) library

Zeaxanthin epoxldase (ZE) catalyses two early steps in the abscisicacid (ABA) biosynthetic pathway. The sequence of a cDNA cloneencoding ZE from Nicotiana plumbaginifolia was reported In 1996and represented the first DNA sequence data on an ABA biosyntheticenzyme. The N. plumbaginifolia cDNA has been used to providea heterologous probe to isolate a ZE cDNA from tomato (Lycopersiconesculentum Mill.). DNA and amino acid sequence differences areconsidered in relation to putative functional domans withinthe enzyme. The results of northern analysis in tomato are discussedin relation to the effects of water stress on ZE mRNA levels. Key words: ABA biosynthesis, zeaxanthin epoxidase, tomato     

Purification and characterization of fructokinase from developing tomato (Lycopersicon esculentum Mill.) fruits

A procedure is described which allows the purification of fructokinase (EC 2.7.1.4) from young tomato fruit. The procedure yielded a 400-fold purification and two isoenzymes designated fructokinase I and II (FKI and FKII) were separated by anion-exchange chromatography. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) the molecular mass was estimated to be 35 kDa. Gel filtration on Sepharose-12 indicated that for both fructokinases the functional form is a dimer. Two dimensional isoelectric focusing/SDS-PAGE combined with immunoblotting showed that FKI has two components with isoelectric points (pIs) of 6.42 and 6.55, while four components with pIs from 6.07 to 6.55 were detected for FKII. A mixture of both fructokinases showed that the components of FKI match the more alkaline components of FKII. The activity of both fructokinases increased with increasing pH to around 8.0 and equal activity was observed from 8.0 to 9.5. Both fructokinases were specific for fructose with K _m values for fructose of 0.131 and 0.201 mM for FKI and FKII, respectively. At high concentrations (> 0.5 mM), fructose was also a strong inhibitor with inhibition constants (K _i) of 1.82 and 1.39 mM for FKI and FKII, respectively. The preferred phosphate donor for both isoforms was ATP, and K _m values of 0.11 and 0.15 mM were observed for FKI and FKII. At low concentrations (0.05–0.2 mM), fructose exhibited noncompetitive inhibition with respect to ATP for both fructokinases. This inhibition pattern changed to uncompetitive when higher fructose concentrations (0.5–10 mM) were used. These data indicated that substrate addition is ordered, with ATP adding first. Inhibition by ADP was also affected by the fructose concentrations. At 0.5 mM fructose, FKI showed non-competitive inhibition by ADP with respect to ATP and this inhibition changed to uncompetitive when 3 mM fructose was used. The isoform FKII showed a competitive inhibition pattern for ADP at 0.5 mM fructose which also changed to uncompetitive when 3 mM fructose was used. The features of the regulation of both fructokinases suggest that this enzyme might have a relevant role in carbon metabolism during tomato fruit development.     

Callose accumulation during treatment of tomato (Lycopersicon esculentum L.) cells with biotic elicitors

Time-course of induced accumulation of callose in tomato cells has been studied. Localization of callose in L. esculenthum cells was investigated by fluorescent microscopy technique, and the optimal time for its determination was found. Callose accumulation in tomato cells treated with different biotic elicitors was determined. Nonlinear dependence between callose accumulation and concentration of chitin oligomers (with 3-5 N-acetylglucosamine fragments) was established. Increasing of callose accumulation in tomato cells was proportional to the increase of concentration ofchitin dimer and chitosan in the culture medium.     

Allotriploid somatic hybrids of diploid tomato (Lycopersicon esculentum Mill.) and monoploid potato (Solanum tuberosum L.)

Allotriploid somatic hybrids were obtained from fusions between protoplasts of diploid tomato and monohaploid potato. The selection of fusion products was carried out in two different ways: (1) The fusion of nitrate reductase-deficient tomato with potato gave rise only to hybrid calli if selection was performed on media lacking ammonium. Parental microcalli were rarely obtained and did not regenerate. (2) The fusion of cytoplasmic albino tomato with potato gave rise to albino and green hybrid calli and plants. Allotriploids were identified from the two somatic hybrid populations by counting chloroplast numbers in leaf guard cells and by flow cytometry of leaf tissue. Although some pollen fertility of allotriploids and pollen-tube growth of tomato, potato andLycopersicon pennellii into the allotriploid style were observed, no progeny could be obtained. The relevance of allotriploid somatic hybrids in facilitating limited gene transfer from potato to tomato is discussed.     

Transient and stable expression of hepatitis B surface antigen in tomato (Lycopersicon esculentum L.)

Cotyledonary leaves of tomato cv. Megha were transformed with the hepatitis B virus ‘s’ gene, which encodes surface antigen. Six plant expression cassettes (pHBS, pHER, pEFEHBS, pEFEHER, pSHER and pEFESHER) were used to assay the possible expression levels by agroinfiltration. The maximum transient expression level of 489.5 ng/g D.W. was noted in pEFEHER-infiltrated cotyledonary leaves. Transgenic tomato plants with pEFEHBS and pEFEHER expression cassettes were regenerated and characterized by molecular analysis. The expression of the antigen in the fruits was confirmed by RT-PCR and ELISA analysis. This is the first report on the expression of hepatitis B surface antigen in tomato.     

Identification and distribution of profilin in tomato (Lycopersicon esculentum Mill.)

Profilin is a G-actin monomer-binding protein which has been shown to participate in actin-based tipgrowth of animal cells. The abundance of profilin in pollen and its occurrence in several vegetable foods raises the question of the role of profilin in plants. First, its distribution throughout various parts of the plant needs to be determined. This paper describes observations on the presence of profilin in the tomato plant (Lycopersicon esculentum Mill.). The distribution of profilin in flower buds, stems, leaves, roots, and fruits of tomato was determined by immunoblotting and by tissue printing, showing that profilin is present in most if not all parts of the tomato plant.We gratefully acknowledge the help provided by Dr. A.T. Jagendorf and the donation of tomato seeds and maize pollen by N. Eanetta and Dr. M. Smith, respectively. The use of Dr. R. Wayne's SZH ILLD dissecting microscope is gratefully acknowledged. This work was aided by helpful discussions with C.S. Combs, Dr. C.A. Conley, and Dr. J. Andersland. This work was supported by a Hatch grant and NRI Competitive Grants Program/USDA 94-37304-1046 to MVP. This material is based upon work supported under a National Science Foundation Graduate Research Fellowship to DWD.     

Phosphorus use efficiency in anthocyanin-free tomato (Lycopersicon esculentum Mill.)

An anthocyanin-free tomato plant, H957, and its parental wild type, H883, were hydroponically grown to test for tolerance to a low phosphorus (P) in H957. The tolerance was evaluated by comparing growth and metabolism of H957 vs. H883 at different P concentrations ranging 25–400 μM. Fresh weights were measured weekly. Dry weight, mineral contents, photosynthetic rate, and P utilization ratios of the plants were measured after five weeks of growth in the hydroponic culture. Although the growth of both varieties was severely impaired at 25 μM P, H957 showed a greater fresh weight and dry weight at 50–400 μM P. H957 showed a higher net photosynthetic rate on older leaves while both varieties showed similar photosynthetic rate on young leaves. H957 tissue contains an overall lower P concentration in its tissue than H883. These observations together indicate that the anthocyaninless mutant H957 tolerate to lower P concentration. It does so by utilizing internal P with better efficiency rather than by absorbing external P better.     

An immunochemical testing of pathophysiological reactions of several PSTVinfected tomato (Lycopersicon esculentum L.) cultivars

Several tomato cultivars were infected with a severe strain of PSTV and a pathophysiological reaction was characterized by means of enzyme-linked immunosorbent assay with serum containing IgGs to disease-associated host-specific leaf proteins. A strong expression of disease-associated symptoms (stunting, epinasty, leaf blade malformation and rugosity) and strong immunochemical reaction was found for cultivars Bizon, Linia, Revermun and Rutgers. The immunochemical assay revealed appearence of a major antigenic protein having a molecular mass of about 70 kD in these cultivars. The immunochemical reaction with disease-associated proteins reached a maximum four weeks after inoculation of PSTV. A weak immunochemical reaction was observed, if proteins from cvs. Sonato, Harzfeuer and Karlik were analyzed. Cvs. Sonato and Harzfeuer did not show characteristic symptoms such as stunting, leaf blade malformation and rugosity. Except for stunting, the same was true for cv. Karlik. On the other hand, no significant difference in PSTV accumulation was observed among the cultivars analyzed. Reciprocal hybrids obtained by crossing cvs. Revermun and Karlik showed strong symptoms of the disease (Revermun-type) and increased activity of nuclease due to PSTV infection. On the contrary, an immunochemical analysis revealed a low level of the disease-associated antigens in these hybrid tomatoes, suggesting rather recessive genetic background determining their expression during PSTV-caused pathogenesis.     

Mapping of ripening-related or -specific cDNA clones of tomato (Lycopersicon esculentum)

Summary Nineteen ripening-related or -specific clones from Lycopersicon esculentum were mapped via RFLP analysis using an F₂ population from the cross L. esculentum x L. pennellii and cDNA or genomic clones of known map location. The map produced using cDNA and genomic clones of known map location corresponded well with previously published maps of tomato. The number of loci detected for each ripening-related or-specific clone varied from one to seven. These loci were located on all 12 chromosomes of the tomato genome. There was no significant clustering of ripening-related or-specific genes. Regions of very low recombination were observed. The clone for polygalacturonase (TOM6) mapped to a single region on chromosome 10, the same chromosome as the nor and alc ripening mutants. To fine map this chromosome, two backcross populations were produced from the cross of L. esculentum x L. pimpenillifolium, in which the esculentum parents used were homozygous for either the alc or the nor. The coding region for polygalacturonase is functionally unlinked to either of these two ripening mutants.     

Plant Regeneration from Cotyledon Protoplasts of Tomato (Lycopersicon esculentum L.)

otyledon protoplasts of tomato (Lycopersicon esculentum L.) isolated from 2–3 week grown seedlings were cultured in MS liquid medium (2,4-D 1, 6-BA 0.1 mg/l) and fresh medium added subsequently. After 6 weeks culture, the cell clusters were transferred to semisolid medium (the additive same as in liquid medium, agar 0.3%). When the calli grew to 0.5 cm in diameter, transfer them to MS medium (6-BA 2, IAA 0.2 mg/l) for differentiation. The regenerated plants were obtained. After comparing different culture methods, tomato protoplasts grew better in double layers than in agar plate and hanging drops.