Soluble and membrane symbiosis-related polypeptides associated with the development of arbuscular mycorrhizas in tomato (Lycopersicon esculentum)

To analyse the effect of arbuscular mycorrhizal (AM) colonization on tomato gene expression, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) patterns of crude extracts, soluble and membrane proteins of tomato roots, either mycorrhizal and the AM fungus Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe or non-mycorrhizal, have been compared. In the three fractions analysed, AM colonization induced up-regulation with down-regulation of the synthesis of polypeptides already present in tomato roots and induction of some new polypeptides. Separation of root extracts into soluble and membrane fractions allowed us to identify two soluble, and five membrane-bound, newly induced polypeptides in AM roots. Comparison of the protein patterns of AM roots with those of the external mycelium of G. mosseae showed that one of the newly induced polypeptides might correspond to a fungal polypeptide. By using this experimental approach, we have been able to detect 44 polypeptides that are differentially displayed in tomato roots as a consequence of the establishment of the AM symbiosis.     

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.     

Isolation and characterization of endophytic Streptomyces strains from surface-sterilized tomato (Lycopersicon esculentum) roots

AIMS: To isolate endophytic Streptomyces strains from tomato and examine their antimicrobial activity. METHODS: Endophytic Streptomyces strains were isolated using surface-sterilization methods and identified by morphological characteristics. Antimicrobial activities were measured by the agar plate sensitivity method. Antifungal activity in vivo was measured by seedling mortality in infested soils. RESULTS: Twenty-one per cent of endophytic streptomycete isolates produced antibacterial metabolites and 41% produced antifungal metabolites in S medium. Sixty-five per cent of the most frequently isolated strains inhibited the growth of Rhizoctonia solani by the antibiosis assay but only 32% produced metabolites active against R. solani in S medium. Growth promotion and enhanced disease resistance of seedlings inoculated with Streptomyces sp. strain S30 were observed in tomato but not in cucumber seedlings. CONCLUSIONS: Endophytic Streptomyces spp. strains were successfully isolated using stringent methods and strain S30 promoted growth and enhanced resistance to R. solani in tomato seedlings. SIGNIFICANCE AND IMPACT OF THE STUDY: Endophytic streptomycetes showing antifungal activity in vitro and in vivo may indicate the potential for their use as biocontrol agents particularly of R. solani disease of tomato seedlings.     

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.     

Relationships between fruit exocarp antioxidants in the tomato (Lycopersicon esculentum) high pigment-1 mutant during development

Development-dependent changes in fruit antioxidants were examined in the exocarp (epidermal and hypodermal tissues) of the monogenic recessive tomato (Lycopersicon esculentum L.) mutant high pigment (hp-1) and its wild-type parent 'Rutgers' grown under non-stress conditions in a greenhouse. The hp-1 mutant was chosen for this study because the reportedly higher lycopene and ascorbic acid (AsA) contents of the fruit may alter its tolerance to photooxidative stress. Throughout most of fruit development, reduced AsA concentrations in the exocarp of hp-1 were 1.5 to 2.0 times higher than in 'Rutgers', but total glutathione concentrations were similar in both genotypes. Only in ripe red fruit were reduced AsA and total glutathione concentrations lower in hp-1 than in 'Rutgers'. The redox ratios (reduced : reduced + oxidized) of AsA in hp-1 and 'Rutgers' exocarps were similar and usually > 0.9, however, the redox ratio of glutathione was lower in hp-1 than in 'Rutgers' throughout development. Lycopene concentrations in ripe red fruit were about 5 times higher in hp-1 than in 'Rutgers'. Large increases in the specific enzyme activities of superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11), and monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) occurred during fruit development in both genotypes, with an inverse relationship between the activities of these enzymes and chlorophyll content. Glutathione reductase (EC 1.6.4.2) and MDHAR-specific activities were higher in hp-1 than 'Rutgers' only at the later stages of fruit development. Dehydroascorbate reductase (EC 1.8.5.1) activities, however, were usually higher in 'Rugters' than in hp-1. Catalase (CAT, EC 1.11.1.6) activities increased with fruit development until the fruit were orange/light red, when CAT was higher in 'Rutgers' than in hp-1, but then declined in the ripe red fruit of both genotypes. These results suggest that elevated AsA in the exocarp of hp-1 fruit early in fruit development may increase the tolerance of hp-1 fruit to photooxidative injury at that time, but the increasing activities of antioxidant enzymes appear to be developmentally associated with fruit ripening.     

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.     

Changes in indole-3-acetic acid levels during tomato (Lycopersicon esculentum Mill.) seed development

Summary The changes in the level of indole-3-acetic acid (IAA) were investigated in seeds and fruit tissues-placenta and mesocarp-during tomato (Lycopersicon esculentum Mill.) zygotic embryogenesis, which was characterized through eight morphological embryo stages [from globular (stage 1) to mature embryo (stage 8)]. In whole seeds, IAA levels increased mainly at stage 3 (young torpedo) and at stage 5 (late torpedo stage). As the seed matured and dehydrated, IAA levels decreased and showed a new distribution pattern within seed structures, preferentially in endosperm tissue. IAA contents in fruit tissues were lower but followed the same pattern as those of seeds. These data support the hypothesis of IAA biosynthesis in seeds with a transient role of the endosperm at the end of embryo development and suggest a role of IAA in fruit and seed growth. Moreover a comparison of IAA and ABA changes suggests that IAA could be especially necessary for the beginning of embryo growth, whereas ABA could act mainly at the end of the growth phase.Abbreviations ABA abscisic acid - ABTS 2,2-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) - BHT butylhydroxytoluene - DW dry weight - ELISA enzyme linked immunosorbent assay - HPLC high performance liquid chromatography - IAA indole-3-acetic acid. PGRs: plant growth regulators     

The response of tomato roots (Lycopersicon esculentum Mill.) to iron deficiency stress: alterations in the pattern of protein synthesis

Dicotyledon plants adapt to iron (Fe) deficiency through a seriesof reactions that increase the ability of the plant to assimilateFe and to increase the efficiency of Fe utilization. In an attemptto gain an insight into these adaptive processes, the specificchanges in protein synthesis associated with the onset of theFe deficiency response in tomato roots (Lycopersicon esculentumMill cv. Rutgers) have been investigated. Roots were grown underFe—sufficient and —deficient conditions, and thepattern of protein synthesis was analysed by in vitro translationof root mRNA and by in vivo labelling of root proteins. Polypeptideswere resolved by two—dimensional polyacrylamide gel elec—trophoresis.Seven polypeptides were identified by in vitro translation,whose synthesis was significantly increased during Fe deficiency.The increase was probably specific to Fe deficiency in thatthe polypep—tide synthesis was not increased during phosphatedeficiency stress, was less prominent following prolonged Fedeficiency and was decreased following re—supply of Feto the hydroponic medium. The pattern of in vitro translation of mRNA isolated from Fe—deficientroots was compared to the results obtainedin vivo followingradiolabelling of proteins. In these analyses, eight polypeptideswere identified, tentatively including the seven polypeptidespreviously identified by in vitro translation. All polypeptideswere characterized with regard to molecular mass and pl andtheir localization in the cell, whether being membrane boundor soluble. It is suggested that members of this group of polypeptidesare involved in the response of the root to Fe deficiency: althoughtheir functions remain to be identified. Key words: In vitro protein synthesis, iron, iron deficiency, root, 2-dimensional PAGE     

Expression of SYMRK affects the development of arbuscular mycorrhiza in tobacco roots

SYMRK is a plant receptor-like kinase with a role in root endosymbiosis. Heterologous expression of SYMRK from non-legumes can complement the loss-of-function effects of the mutant symrk in legumes. However, it is unclear whether the development of arbuscular mycorrhiza (AM) is affected along with the enhanced expression of SYMRK. In the present study, the full-length LsSYMRK gene was cloned from Lathyrus sativus. Overexpression of LsSYMRK in tobacco roots was essential for AM development, and affected the expression of genes which are involved in the potential signaling pathway of AM. In addition, the pattern of hyphal penetration changed from radial to longitudinal when hyphae reached the root cortex. Furthermore, overexpression of LsSYMRK increased tobacco biomass in the presence of AM fungi. These results suggest that increased expression of SYMRK in roots of AM-infected tobacco can increase the colonization and biomass.     

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.     

Abscisic acid determines arbuscule development and functionality in the tomato arbuscular mycorrhiza

The role of abscisic acid (ABA) during the establishment of the arbuscular mycorrhiza (AM) was studied using ABA sitiens tomato (Lycopersicon esculentum) mutants with reduced ABA concentrations. Sitiens plants and wild-type (WT) plants were colonized by Glomus intraradices. Trypan blue and alkaline phosphatase histochemical staining procedures were used to determine both root colonization and fungal efficiency. Exogenous ABA and silver thiosulfate (STS) were applied to establish the role of ABA and putative antagonistic cross-talk between ABA and ethylene during AM formation, respectively. Sitiens plants were less susceptible to the AM fungus than WT plants. Microscopic observations and arbuscule quantification showed differences in arbuscule morphology between WT and sitiens plants. Both ABA and STS increased susceptibility to the AM fungus in WT and sitiens plants. Fungal alkaline phosphate activity in sitiens mutants was completely restored by ABA application. * The results demonstrate that ABA contributes to the susceptibility of tomato to infection by AM fungi, and that it seems to play an important role in the development of the complete arbuscule and its functionality. Ethylene perception is crucial to AM regulation, and the impairment of mycorrhiza development in ABA-deficient plants is at least partly attributable to ethylene.     

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.     

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.     

Flower development schedule in tomato Lycopersicon esculentum cv. sweet cherry

The ontogeny of tomato (Lycopersicon esculentum cv. sweet cherry) flowers was subdivided into 20 stages using a series of landmark events. Stamen primordia emergence and carpel initiation occur at stage 4; archesporial and parietal tissue differentiate at stage 6 and meiosis in anthers begins at stage 9. Subepidermal meristematic ovule primordia are formed on the placenta at stage 9; megasporogenesis begins at stage 11-12 and embryo sac differentiation and ovule curvature take place at stage 14, once the pollen is maturing. We established a correlation between the characteristic cellular events in carpels and stamens and morphological markers of the perianth. The model of tomato flower development schedule was then used to analyse the spatial, temporal and tissue-specific expression of gene(s) involved in the regulation of floral organ development. As an example, the expression pattern of ORFX, a gene controlling cell size in tomato fruits, shows that expression starts very early during the ontogeny of reproductive organs.     

Epibrassinolide-inhibition of development of excised, adventitious and intact roots of tomato (Lycopersicon esculentum): comparison with the effects of steroidal estrogens

The brassinosteroid, 24-epibrassinolide, caused observable inhibition of the growth of aseptically-cultured excised tomato ( Lycopersicon esculentum Mill. cv. Best of All) roots at a concentration of 0.01 μ M . Root formation and outgrowth in tomato shoot cuttings and root biomass in intact tomato seedlings were also depressed by epibrassinolide, but only at a higher concentration (0.1μ M ). Concentrations of the steroidal estrogens, estrone and estradiol (as sulphate derivatives), in excess of 1.0 μ M , reduced cultured root growth as well as root number and total (but not mean) root length in shoot cuttings. Seedling root growth was only slightly inhibited by estradiol but not by estrone. Both epibrassinolide and estrogens caused morphological abnormalities, such as epinasty and leaf-inrolling in cuttings and seedlings. Epibrassinolide stimulated extension growth of the hypocotyl and epicotyl in cuttings and of the hypocotyl in seedlings, but estrogens exerted no effects on shoot growth.     

Response of superoxide dismutase isoenzymes in tomato plants (Lycopersicon esculentum) during thermo-acclimation of the photosynthetic apparatus

Seedlings of Lycopersicon esculentum Mill. var. Amalia were grown in a growth chamber under a photoperiod of 16 h light at 25 degrees C and 8 h dark at 20 degrees C. Five different treatments were applied to 30-day-old plants: Control treatment (plants maintained in the normal growth conditions throughout the experimental time), heat acclimation (plants exposed to 35 degrees C for 4 h in dark for 3 days), dark treatment (plants exposed to 25 degrees C for 4 h in dark for 3 days), heat acclimation plus heat shock (plants that previously received the heat acclimation treatment were exposed to 45 degrees C air temperature for 3 h in the light) and dark treatment plus heat shock (plants that previously received the dark treatment were exposed to 45 degrees C air temperature for 3 h in the light). Only the heat acclimation treatment increased the thermotolerance of the photosynthesis apparatus when the heat shock (45 degrees C) was imposed. In these plants, the CO(2) assimilation rate was not affected by heat shock and there was a slight and non-significant reduction in maximum carboxylation velocity of Rubisco (V(cmax)) and maximum electron transport rate contributing to Rubisco regeneration (J(max)). However, the plants exposed to dark treatment plus heat shock showed a significant reduction in the CO(2) assimilation rate and also in the values of V(cmax) and J(max). Chlorophyll fluorescence measurements showed increased thermotolerance in heat-acclimated plants. The values of maximum chlorophyll fluorescence (F(m)) were not modified by heat shock in these plants, while in the dark-treated plants that received the heat shock, the F(m) values were reduced, which provoked a significant reduction in the efficiency of photosystem II. A slight rise in the total superoxide dismutase (SOD) activity was found in the plants that had been subjected to both heat acclimation and heat shock, and this SOD activity was significantly higher than that found in the plants subjected to dark treatment plus heat shock. The activity of Fe-SOD isoenzymes was most enhanced in heat-acclimated plants but was unaltered in the plants that received the dark treatment. Total CuZn-SOD activity was reduced in all treatments. Darkness had an inhibitory effect on the Mn-SOD isoenzyme activity, which was compensated by the effect of a rise in air temperature to 35 degrees C. These results show that the heat tolerance of tomatoplants may be increased by the previous imposition of a moderately high temperature and could be related with the thermal stability in the photochemical reactions and a readjustment of V(cmax) and J(max). Some isoenzymes, such as the Fe-SODs, may also play a role in the development of heat-shock tolerance through heat acclimation. In fact, the pattern found for these isoenzymes in heat-acclimated Amalia plants was similar to that previously described in other heat-tolerant tomato genotypes.