Proteomic analysis of tomato (Lycopersicon esculentum) pollen

In flowering plants, pollen grains are produced in the anther and released to the external environment with the primary function of delivering sperm cells to the female gametophyte. This study was conducted to identify proteins in tomato pollen and to analyse their roles in relation to pollen function. Tomato is an important crop which is grown worldwide and is an excellent experimental system. Proteins were extracted from pollen, separated by two-dimensional gel electrophoresis (2-DE), and identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and peptide mass fingerprinting. Of the 960 spots observed on Colloidal Coomassie Blue (CCB)-stained 2-DE gels, 190 were selected for analysis. Of these, 158 spots, representing 133 distinct proteins, were identified by searching the NCBInr and Expressed Sequence Tag databases. The identified proteins were classified based on designated functions and the majority included those involved in defence mechanisms, energy conversions, protein synthesis and processing, cytoskeleton formation, Ca(2+) signalling, and as allergens. A number of proteins in tomato pollen were similar to those reported in the pollen of other species; however, several additional proteins with roles in defence mechanisms, metabolic processes, and hormone signalling were identified. The potential roles of the identified proteins in the survival strategy of the small, independent, two-celled pollen grain of tomato, and subsequently in pollen germination and tube growth are discussed.     

Factors influencing transformation frequency of tomato (Lycopersicon esculentum)

We developed an efficient procedure for transformation and regeneration of L. esculentum cv. Moneymaker from cotyledon explants. The effect of two parameters on the transformation frequency was investigated in detail. The use of feeder layers during cocultivation proved to be critical. In addition, it was found that Agrobacterium strains harbouring a L,L-succinamopine type helper plasmid yielded significantly higher transformation frequencies than those with octopine or nopaline type helper plasmids. The optimized protocol was used to obtain transformation frequencies averaging 9%. Of the plants produced approximately 80% proved to be diploid, of which 67% contained the transgene(s) on a single locus.Abbreviations 2,4D 2,4-dichlorophenoxy-acetic acid - gus ß-glucoronidase - IAA indoleacetic acid - LB Luria-Bertani - NPTII neomycin phosphotransferase II - ZR zeatin riboside     

Isolation of signaling mutants of tomato (Lycopersicon esculentum)

As a first step towards developing a genetic system for investigating signaling processes in plants, we have developed a screen for signaling mutants deficient in a wound response. We have isolated two mutants of tomato that lack detectable production of proteinase inhibitors induced systemically in leaves by wounding. The mutants are deficient in the induction of both proteinase Inhibitor I and proteinase Inhibitor II but can be induced to respond at near wild-type levels by methyl jasmonate, a known elicitor of inhibitor production in tomato. While completely deficient in systemic production of proteinase inhibitors, both mutants produce some proteinase inhibitor in wounded leaves. This evidence suggests the existence of two signaling pathways, one local and one systemic, that regulate the induction of proteinase inhibitor snythesis in response to wounding.     

Development and differentiation of haploid Lycopersicon esculentum (tomato)

Summary Haploid callus cultures of selected races of Lycopersicon (tomato) species can be obtained from anther culture. This is a further demonstration of a proposed general method of haploid culture developed with Arabidopsis thaliana. Differentiation of haploid callus of Lycopersicon esculentum can be controlled both in the dark and the light by hormones added to defined minimal media. Development to plantlets is achieved only in the light. Callus cells can be induced to develop into seedless pseudo-fruits. Chromosome counts on callus cells or root-tip cells establishes haploidy (n=12).Haploidy can be maintained in culture on defined minimal media for at least one year.     

Shoot regeneration from GUS-transformed tomato (Lycopersicon esculentum) hairy root

To study the influence of genetic background on the transformation and regeneration of cultivated tomato plants, hairy root lines of tomato (Lycopersicon esculentum) were obtained by inoculating the hypocotyl explants of three tomato cultivars with the Agrobacterium rhizogenes strain DCAR-2, which harbors the pBI-121 binary vector. The Ri-T-DNA transformation into the plant DNA was confirmed by both of mikimopine and GUS assay analyses. The regeneration efficiency from hairy root explants was assessed. The data indicated that white embryonic calli were formed within two weeks in the presence of 2 mgl(-1) 2, 4-D plus 0.25 mgl(-1) kinetin. Adventitious shoots emerged from the embryonic callus in the presence of 1 mgl(-1) GA3 along with 0.5 mgl(-1) NAA. The regeneration frequency was higher in the cultivar UC-97, followed by Momotaro and then Edkawi. Molecular confirmation of the integration of the GUS gene into the hairy root-derived plants genomes was done via PCR using GUS-specific primers and also using Southern blotting analysis. Our data shows that regeneration is possible from hairy roots of the cultivated tomato and this system could be used to produce transgenic tomato plants expressing the genes present in Agrobacterium rhizogenes binary vectors.     

The effect of growth regulators on tomato (Lycopersicon esculentum) fertility

The aim of the study was to determine the effect of 2,4 D (2,4dichlorophenoxyacetic acid) used separately and in a mixture with BAP (benzylaminopurine) on fertility and some features of tomato fruit. The plant flowers were immersed only once in water solutions of the growth regulators (2,4 D 0.001 %; 0.005 %; 2,4 D 0.001 % + BAP 0.001 %; 2,4 D 0.005 % + BAP 0.005 %). The mean fruit weight, fruit volume, specific weight, and the jelly weight were determined. The number of seeds collected from the fruit was used as a criterion of fertility estimation. No statistically significant differences were found in the mean fruit weight, fruit volume and specific weight. The fruits derived from the plants which were not exposed to the action of growth regulators were characterized by the smallest amount of jelly while the fruits set under the influence of 0.001 % 2,4 D + 0.001 % BAP had the largest jelly amount. The greatest differentiation was found in fertility which ranged from 7.5 seeds from the fruit derived from the plants treated with 0.005 % 2,4 D, to 75.7 seeds from the non-treated plants’ fruit. The BAP addition caused the limitation of fertility reduction.     

A major QTL introgressed from wild Lycopersicon hirsutum confers chilling tolerance to cultivated tomato (Lycopersicon esculentum)

Many plants of tropical or subtropical origin, such as tomato, suffer damage under chilling temperatures (under 10°C but above 0°C). An earlier study identified several quantitative trait loci (QTLs) for shoot turgor maintenance (stm) under root chilling in an interspecific backcross population derived from crossing chilling-susceptible cultivated tomato (Lycopersicon esculentum) and chilling-tolerant wild L. hirsutum. The QTL with the greatest phenotypic effect on stm was located in a 28 cM region on chromosome 9 (designated stm9), and enhanced chilling-tolerance was conferred by the presence of the Lycopersicon hirsutum allele at this QTL. Here, near-isogenic lines (NILs) were used to verify the effect of stm9, and recombinant sub-NILs were used to fine map its position. Replicated experiments were performed with NILs and sub-NILs in a refrigerated hydroponic tank in the greenhouse. Sub-NIL data was analyzed using least square means separations, marker-genotype mean t-tests, and composite interval mapping. A dominant QTL controlling shoot turgor maintenance under root chilling was confirmed on chromosome 9 using both NILs and sub-NILs. Furthermore, sub-NILs permitted localization of stm9 to a 2.7 cM interval within the original 28 cM QTL region. If the presence of the L. hirsutum allele at stm9 also confers chilling-tolerance in L. esculentum plants grown under field conditions, it has the potential to expand the geographic areas in which cultivated tomato can be grown for commercial production.     

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.     

A comparison of the chilling-stress response in two differentially tolerant cultivars of tomato (Lycopersicon esculentum).

The chilling responses of two differentially cold tolerant cultivars of tomato were monitored through in vivo labelling of polypeptides with [35S]methionine, both during a gradual temperature decrease (2 degrees C/day) and also during a rapid cold shock (4 degrees C). The polypeptides were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and revealed by fluorography. Both cultivars showed changes in the polypeptide profiles resulting from either chilling treatment. During the gradual temperature decrease, there were few differences exhibited between the two cultivars. However, during cold shock both cultivars showed the altered synthesis of several unique polypeptides. Both cultivars showed the appearance of a 35-kDa polypeptide during the gradual temperature decrease and also during the cold shock. The appearance of three high relative mass polypeptides was found in both cultivars only during the gradual temperature decrease. Treatments with cycloheximide and chloramphenicol suggested that cold-shock polypeptides are both nuclear and organelle encoded. The cold-shock response in roots was different from the response in leaves and between cultivars. A comparison of the two cultivars showed a number of differences in polypeptide synthesis which may be related to increased cold tolerance.     

Cadmium toxicity in tomato (Lycopersicon esculentum) plants grown in hydroponics

The effects of Cd have been investigated in tomato (Lycopersicon esculentum) plants grown in a controlled environment in hydroponics, using Cd concentrations of 10 and 100 μM. Cadmium treatment led to major effects in shoots and roots of tomato. Plant growth was reduced in both Cd treatments, leaves showed chlorosis symptoms when grown at 10 μM Cd and necrotic spots when grown at 100 μM Cd, and root browning was observed in both treatments. An increase in the activity of phosphoenolpyruvate carboxylase, involved in anaplerotic fixation of CO₂ into organic acids, was measured in root extracts of Cd-exposed plants. Also, significant increases in the activities of several enzymes from the Krebs cycle were measured in root extracts of tomato plants grown with Cd. In leaf extracts, significant increases in citrate synthase, isocitrate dehydrogenase and malate dehydrogenase activities were also found at 100 μM Cd, whereas fumarase activity decreased. These data suggest that at low Cd supply (10 μM) tomato plants accumulate Cd in roots and this mechanism may be associated to an increased activity in the PEPC–MDH–CS metabolic pathway involved in citric acid synthesis in roots. Also, at low Cd supply some symptoms associated with a moderate Fe deficiency could be observed, whereas at high Cd supply (100 μM) effects on growth overrule any nutrient interaction caused by excess Cd. Cadmium excess also caused alterations on photosynthetic rates, photosynthetic pigment concentrations and chlorophyll fluorescence, as well as in nutrient homeostasis.     

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.     

Characterization of mitochondria-located small heat shock protein from tomato (Lycopersicon esculentum)

We cloned and sequenced a full-length cDNA encoding the precursor of the mitochondria-located small heat shock protein (MT-sHSP) gene (LeHSP23.8) from tomato (Lycopersicon esculentum). The deduced protein precursor with a calculated molecular weight of 23.8 kDa was predicted to target mitochondria and was classified as a plant MT-sHSP. A single copy of LeHSP23.8 was found in tomato genomic DNA by southern-blot analysis. Northern-blot analysis revealed the heat inducible character of LeHSP23.8 mRNA. The LeHSP23.8 mRNA was hardly detectable at about 36 degrees C but accumulated markedly at 40 degrees C. The molecular chaperone function of LeHSP23.8 was confirmed in vitro. The recombinant LeHSP23.8 was able to enhance the renaturation of chemically denatured citrate synthase (CS). Moreover, the recombinant LeHSP23.8 protected CS from thermal inactivation and also promoted the renaturation of thermally inactivated citrate synthase.     

Mapping salt-tolerance genes in tomato (Lycopersicon esculentum) using trait-based marker analysis

The germination responsiveness of an F₂ population derived from the cross Lycopersicon esculentum (UCT5) x L. pennellii (LA716) was evaluated for salt tolerance at two stress levels, 150 mM NaCl + 15 mM CaCl₂ and 200 mM NaCl + 20 mM CaCl₂. Individuals were selected at both tails of the response distribution. The salt-tolerant and salt-sensitive individuals were genotyped at 16 isozyme loci located on 9 of the 12 tomato chromosomes. In addition, an unselected (control) F₂ population was genotyped at the same marker loci, and gene frequencies were estimated in both selected and unselected populations. Trait-based marker analysis was effective in identifying genomic locations (quantitative trait loci, QTLs) affecting salt tolerance in the tomato. Three genomic locations marked by Est-3 on chromosome 1, Prx-7 on chromosome 3, and 6Pgdh-2 and Pgi-1 on chromosome 12 showed significant positive effects, while 2 locations associated with Got-2 on chromosome 7 and Aps-2 on chromosome 8 showed significant negative effects. The identification of genomic locations with both positive and negative effects on this trait suggests the likelihood of recovering transgressive segregants in progeny derived from these parental lines. Similar genomic locations were identified when selection was made either for salt tolerance or salt sensitivity and at both salt-stress treatments. Comparable results were obtained in uni- and bidirectional selection experiments. However, when marker allele gene frequencies in a control population are unknown, bidirectional selection may be more efficient than unidirectional selection in identifying marker-QTL associations. Results from this study are discussed in relationship to the use of molecular markers in developing salt-tolerant tomatoes.     

A genetic analysis of a tomato (Lycopersicon esculentum) genotype with a high frequency of twin spots

Among pale-green tomato plants heterozygous for the xanthophyllic2 (xa-2) mutation that were transformed with a T-DNA harbouring the NPTII and GUS gene, a plant with a high frequency of green/white twin spots was found. The genetic analysis of this plant indicated that the occurrence of these twin spots was caused by a genetic defect located at the distal end of chromosome 10S, where xa-2 also is located. The genetic analysis of green plants regenerated from leaf expiants of this twin-spot plant revealed that the green sectors derive from non-disjunction of the xa-2 ⁺ allele. In an analysis of mitotic chromosome behaviour bridges were observed in approximately 5% of the anaphases, providing arguments that a breakage-fusion-bridge cycle caused by a tissue culture-induced genomic instability is the most likely cause of this aberrant behaviour of chromosome 10.     

Nitrogen fixation in tomato (Lycopersicon esculentum Mill ‘Pusa Ruby’)

Nitrogen fixation (acetylene reduction) was found in intact tomato (Lycopersicon esculentum Mill ‘Pusa Ruby’) plants in the field, in pots and also in aseptic cultures. The unsterilized as well as sterilized rhizoplane and phylloplane of the plant when assayed separately also responded to the test. From root bits of tomato sterilized upto 20 minutes with 0.1% mercuric chloride, growth of the bacterium from the interior of the root into the medium was observed thereby indicating their presence within the endorhizosphere. Phase contrast and electron microscopic studies of the root system of tomato revealed the presence of bacterial colonies in the epidermis, cortex and vascular bundles. Bacterial numbers in the endosphere, of root and leaf were 30×10⁴ and 12×10⁴, respectively, per gram fresh weight of tissue. The bacteria were predominantly rod-shaped 1.4–4.8×0.9–1.95 μm in 24-h-old cultures, pleomorphic, polar or bipolary flagellated having β-hydroxy butyrate granules. The bacterium has been identified as a new species of Azospirillum.