A 610 kb YAC clone harbors 7 cM of tomato (Lycopersicon esculentum) DNA that includes themale sterile 14 gene and a hotspot for recombination

Pollen development requires both sporophytic and gametophytic gene expression. We are using a map-based cloning technique to isolate sporophytic genes which, when mutant, cause pollen abortion and a male sterile (ms) phenotype in tomato (Lycopersicon esculentum). We have genetically characterized onems locus (ms14) using RFLP analysis and identified flanking markers. High-resolution genomic physical mapping indicates that thems14 locus is located in a 300 kb region. We have identified a YAC clone with an insert size of 610 kb that contains thems14-linked markers, reflects the organization of the physical map and therefore most probably contains thems14 gene. In addition, we present evidence that the relationship between physical and genetic distance in this chromosomal region changes abruptly from 105–140 kb/cM to less than 24 kb/cM, and suggest that the TG393-TG104 region is a hotspot for recombination.     

Genetic and physical analysis of a YAC contig spanning the fungal disease resistance locus Asc of tomato (Lycopersicon esculentum)

The Alternaria stem canker disease of tomato is caused by the necrotrophic fungal pathogen Alternaria alternata f. sp. lycopersici (AAL). The fungus produces AAL toxins that kill the plant tissue. Resistance to the fungus segregates as a single locus, called Asc, and has been genetically mapped on chromosome 3 of tomato. We describe here the establishment of a 1383-kb YAC contig covering the Asc locus and a series of plants selected for recombination events around the Asc locus. It was shown that the YAC contig corresponds to a genetic distance of at least 11.2 cM. Thus, the recombination rate in the Asc region is six times higher (123 kb/cM) than the average for the tomato genome. Furthermore, the Asc locus could be localised to a 91-kb fragment, thus paving the way for the cloning and identification of the Asc gene(s) by complementation. Received: 31 July 1998 / Accepted: 6 October 1998     

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.     

Cloning and characterization of the gene for phytoene desaturase (Pds) from tomato (Lycopersicon esculentum)

The gene Pds encodes phytoene desaturase, a key enzyme in carotenoid biosynthesis that converts phytoene to -carotene. We have cloned and analyzed the genomic DNA sequence of Pds from tomato. In tomato Pds is comprised of 15 exons that, together with the introns occupy over 8 kb. A putative promoter sequence has been identified by comparison with the cDNA sequence of Pds. A consensus nucleotide sequence around intron splicing sites in tomato genes was determined by compiling data on 137 introns in 34 genes. This consensus sequence generally agrees with the consensus sequence of other higher plants with only minor differences that are unique to tomato.     

DNA content of heterochromatin and euchromatin in tomato (Lycopersicon esculentum) pachytene chromosomes.

Lycopersicon esculentum (tomato) has a small genome (2C = 1.90 pg of DNA) packaged in 2n = 2x = 24 small acrocentric to metacentric chromosomes. Like the chromosomes of other members of the family Solanaceae, tomato chromosomes have pericentromeric heterochromatin. To determine the fraction of the tomato genome found in euchromatin versus heterochromatin, we stained pachytene chromosomes from primary microsporocytes with Feulgen and analyzed them by densitometry and image analysis. In association with previously published synaptonemal complex karyotype data for tomato, our results indicate that 77% of the tomato microsporocyte genome is located in heterochromatin and 23% is found in euchromatin. If heterochromatin is assumed to contain few active genes, then the functional genes of the tomato must be concentrated in an effective genome of only 0.22 pg of DNA (1C = 0.95 pg x 0.23 = 0.22 pg). The physical segregation of euchromatin and heterochromatin in tomato chromosomes coupled with the small effective genome size suggests that tomato may be a more useful subject for chromosome walking and gene mapping studies than would be predicted based on its genome size alone. Key words : tomato, Lycopersicon esculentum, genome size, heterochromatin, euchromatin, pachytene chromosomes, synaptonemal complex.     

A comparison of tomato (Lycopersicon esculentum) lectin with its deglycosylated derivative

A regime is proposed for the design of coupled enzyme assays in which auxiliary enzymes are added at concentrations proportional to their Km values. Under these conditions it is possible to calculate the complete time course of the assay including the time required for the system to approach its steady state. The consequence of increasing the number of coupling enzymes is shown to be a considerable decrease in time required to reach the steady state provided that the overall transient time remains the same. The method is extended to the general consideration of pathways and shows that pathways of the same length exhibit identical temporal responses provided that the units of concentration and time used are based on the steady-state concentration of intermediates and the transient time respectively. An unexpected finding is that increasing the number of intermediates in a pathway can decrease the time required to enter a steady state.     

Cloning and characterization of salicylic acid-induced, intracellular pathogenesis-related gene from tomato (Lycopersicon esculentum)

Intracellular pathogenesis-related gene (IPR) from tomato was cloned from a salicylic acid (SA) induced cDNA library and was designated asTSI-1 (tomato stress induced-1). The deduced amino acid sequence ofTSI-1 codes for a 178 amino acid polypeptide of molecular weight 20.4 kDa.TSI-1 is highly homologous to the potatoSTH-2 andSTH-21 IPR and tree pollen allergens which cause type I allergic reactions in humans.TSI-1 lacks a signal peptide like other IPR members. It is organized as a multigene family and is inducible by SA andFusarium oxysporum infection.     

Isolation and characterization of a gene encoding a drought-induced cysteine protease in tomato (Lycopersicon esculentum).

In a previous study, a 65 kDa protein, TDI-65, was found to be accumulated in the leaves of drought-stressed tomato (Lycopersicon esculentum cv. Starfire) plants. The protein level returns to control level when the drought-stressed plants are rewatered. Antibodies raised against the purified protein were used to elucidate the subcellular localization of the protein. The protein was found to be mainly localized in the nuclei and chloroplasts of drought-stressed leaf cells. To identify the nature of the protein, a cDNA library was constructed and screened by the purified anti-TDI-65 antibody. A cDNA clone designated tdi-65 was isolated and characterized. The deduced amino acid sequences of tdi-65 protein has extensive homology with known cysteine proteases such as actinidin and papain. Northern blot analysis revealed that tdi-65 mRNA is 10-fold higher in drought-stressed plants as compared to control and rewatered plants. Similar results were observed in the tomato cultivar Ailsa and its near isogenic abscisic acid (ABA)-deficient mutant line, flacca, suggesting that the gene does not require ABA for its expression under drought conditions. Based on the previous immunolocalization findings we suggest that tdi-65 encoded cysteine protease functions in relation to drought-induced senescence and programmed cell death.     

Comparison of carotenoid content,gene expression and enzyme levels in tomato (Lycopersicon esculentum) leaves

Physiological conditions which lead to changes in total carotenoid content in tomato plantlets were identified. Carotenoid levels were found to increase after the onset of a dark period during a normal 24 h cycle. This rapid initial increase is followed by a steady decrease in carotenoid content throughout the night. A decrease in the expression of several carotenogenic genes, namely pds, zds (carotenoid desaturases) and ptox (plastid terminal oxidase), was observed following the removal of the light (when carotenoid content is at its highest). An increase in gene expression was observed before the return to light for pds and zds (when carotenoid levels were at their lowest), or following the return to light for ptox. The phytoene desaturation inhibitor norflurazon leads to a decrease coloured carotenoid content and, in the light, this correlated with pds and zds gene induction. In the dark, norflurazon treatment led to only a weak decrease in carotenoid content and only a small increase in pds and zds gene expression. The striking absence of phytoene accumulation under norflurazon treatment in the dark suggests a down-regulation of carotenoid formation in darkness However, prolonged dark conditions, or treatment with photosynthetic inhibitors, surprisingly led to higher carotenoid levels, which correlated with decreased expression of most examined genes. In addition to light, which acts in a complex way on carotenoid accumulation and gene expression, our results are best explained by a regulatory effect of carotenoid levels on the expression of several biosynthetic genes. In addition, monitoring of protein amounts for phytoene desaturase and plastid terminal oxidase (which sometimes do not correlate with gene expression) indicate an even more complex regulatory pattern.     

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.     

Evidence that a part of cellular uridine of a tomato (Lycopersicon esculentum) cell suspension culture is located in the vacuoles

Cells, protoplasts and isolated vacuoles of a tomato (Lycopersicon esculentum) cell suspension culture were analyzed by high pressure liquid chromatography (HPLC) for the presence of uridine. It was found that the uridine content in 10⁸ cells or protoplasts varied between 70 and 150 nmol for different growth stages. The vacuolar location of a part of cellular uridine was evidenced by its co-migration (i) with α-mannosidase, a soluble vacuolar marker, in the gradient used for the purification of vacuoles and (ii) with α-mannosidase and vacuoles (counted microscopically) during repeated centrifugation of isolated vacuoles. Quantitatively, vacuoles sequestered about 13–35% of the amount of uridine present in protoplasts of different culture age. The possible origin of uridine in the vacuoles is discussed.     

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.     

Cloning, expression, and characterization of tomato (Lycopersicon esculentum) aminopeptidase P.

A cDNA (LeAPP2) was cloned from tomato coding for a 654 amino acid protein of 72.7 kDa. The deduced amino acid sequence was >40% identical with that of mammalian aminopeptidase P, a metalloexopeptidase. All amino acids reported to be important for binding of the active site metals and catalytic activity, respectively, were conserved between LeAPP2 and its mammalian homologues. LeAPP2 was expressed in Escherichia coli in N-terminal fusion with glutathione S-transferase and was purified from bacterial extracts. LeAPP2 was verified as an aminopeptidase P, hydrolyzing the amino-terminal Xaa-Pro bonds of bradykinin and substance P. LeAPP2 also exhibited endoproteolytic activity cleaving, albeit at a reduced rate, the internal -Phe-Gly bond of substance P. Apparent K(m) (15.2 +/- 2.4 microm) and K(m)/k(cat) (0.94 +/- 0.11 mm(-1) x s(-1)) values were obtained for H-Lys(Abz)-Pro-Pro-pNA as the substrate. LeAPP2 activity was maximally stimulated by addition of 4 mm MnCl(2) and to some extent also by Mg(2+), Ca(2+), and Co(2+), whereas other divalent metal ions (Cu(2+), Zn(2+)) were inhibitory. Chelating agents and thiol-modifying reagents inhibited the enzyme. The data are consistent with LeAPP2 being a Mn(II)-dependent metalloprotease. This is the first characterization of a plant aminopeptidase P.