Inheritance and genetic mapping of fruit sucrose accumulation in Lycopersicon chmielewskii

Fruit of the domestic tomato (Lycopersicon esculentum Mill.) accumulate soluble sugars primarily in the form of the hexoses, glucose and fructose. In contrast, the predominant sugar in fruit of the wild tomato relative, L. chmielewskii, is sucrose. In the present study, the inheritance and linkage relations of sucrose accumulation were examined in interspecific L. esculentum x L. chmielewskii populations. In backcrosses to either the wild or domestic tomato, segregation for sucrose accumulation permitted qualitative analysis of the trait and indicated monogenic recessive control, although deviations from Mendelian inheritance were observed in some populations. This major gene, designated sucr, was mapped in F₂, F₃, and BC₁F₂ populations using a set of 95 informative RFLP and isozyme markers covering the tomato genome. A map location near the centromere of chromosome 3 was established, with tight linkage to the genomic clone TG102. Association of sucrose accumulation with yellow fruit, encoded by an allele of the r gene, permitted alignment with the classical map, thereby confirming the map location of sucr. A linkage map of the region surrounding sucr was obtained by monitoring recombination between flanking markers in the back-crosses to tomato. A cDNA clone of tomato fruit acid invertase, TIV1, was mapped to TG102 and sucr, with no recombination between the two RFLP markers observed in over 1700 meiotic products. Despite the tight linkage, TG102 and TIV1 hybridize to distinct restriction fragments, hence do not represent the same gene. The genetic data strongly suggest that sucr is an allele of the invertase gene and thus support previous biochemical studies that demonstrated low invertase activity in sucrose-accumulating fruit. L. hisutum, another low-invertase, sucrose-accumulating species, was hybridized with L. chmielewskii and the resulting F₁ plants accumulated sucrose, indicating that genetic control of soluble sugar composition is conserved in these two species.     

Enzymic Components of Sucrose Accumulation in the Wild Tomato Species Lycopersicon peruvianum

Sugar and soluble solids content and invertase (EC 3.2.1.26), sucrose synthase (EC 2.4.1.13), and sucrose phosphate synthase (EC 2.4.1.14) enzyme activities were measured throughout fruit development in tomato (Lycopersicon esculentum Mill.) and the green fruited species Lycopersicon peruvianum. Fruit of L. peruvianum accumulated predominantly sucrose, in contrast with hexose accumulation, which is characteristic of L. esculentum. The percentage of soluble solids in ripe L. peruvianum fruit was more than twice that present in L. esculentum and attributed primarily to the high level of sucrose accumulated in L. peruvianum. Low levels of invertase and sucrose synthase activity were associated with the period of significant sucrose accumulation and storage in L. peruvianum. Increased sucrose phosphate synthase activity was observed during the latter stages of fruit development in sucrose-accumulating fruit but was not coincident with maximum rates of sucrose accumulation.     

Reduction in Pectin Methylesterase Activity Modifies Tissue Integrity and Cation Levels in Ripening Tomato (Lycopersicon esculentum Mill.) Fruits

Pectin methylesterase (PME, EC 3.1.1.11) is an ubiquitous enzyme in the plant kingdom; however, its role in plant growth and development is not yet understood. Using transgenic tomato (Lycopersicon esculentum Mill.) fruits that show more than 10-fold reduction in PME activity because of expression of an antisense PME gene, we have investigated the role of PME in tomato fruit ripening. Our results show that reduced PME activity causes an almost complete loss of tissue integrity during fruit senescence but shows little effect on fruit firmness during ripening. Low PME activity in the transgenic fruit pericarp modified both accumulation and partitioning of cations between soluble and bound forms and selectively impaired accumulation of Mg2+ over other major cations. Decreased PME activity was associated with a 30 to 70% decrease in bound Ca2+ and Mg2+ in transgenic pericarp. Levels of soluble Ca2+ increase 10 to 60%, whereas levels of soluble Mg2+ and Na+ are reduced by 20 to 60% in transgenic pericarp. Changes in cation levels associated with lowered PME activity do not affect the rate of respiration or membrane integrity of fruit during ripening. Overall, these results suggest that PME plays a role in determining tissue integrity during fruit senescence, perhaps by regulating cation binding to the cell wall.     

Antisense acid invertase (TIV1) gene alters soluble sugar composition and size in transgenic tomato fruit.

Invertase (beta-fructosidase, EC 3.2.1.26) hydrolyzes sucrose to hexose sugars and thus plays a fundamental role in the energy requirements for plant growth and maintenance. Transgenic plants with altered extracellular acid invertase have highly disturbed growth habits. We investigated the role of intracellular soluble acid invertase in plant and fruit development. Transgenic tomato (Lycopersicon esculentum Mill.) plants expressing a constitutive antisense invertase transgene grew identically to wild-type plants. Several lines of transgenic fruit expressing a constitutive antisense invertase gene had increased sucrose and decreased hexose sugar concentrations. Each transgenic line with fruit that had increased sucrose concentrations also had greatly reduced levels of acid invertase in ripe fruit. Sucrose-accumulating fruit were approximately 30% smaller than control fruit, and this differential growth correlated with high rates of sugar accumulation during the last stage of development. These data suggest that soluble acid invertase controls sugar composition in tomato fruit and that this change in composition contributes to alterations in fruit size. In addition, sucrose-accumulating fruit have elevated rates of ethylene evolution relative to control fruit, perhaps as a result of the smaller fruit size of the sucrose-accumulating transgenic lines.     

Sink Metabolism in Tomato Fruit : IV. Genetic and Biochemical Analysis of Sucrose Accumulation

Fruit of domesticated tomato (Lycopersicon esculentum) accumulate primarily glucose and fructose, whereas some wild tomato species, including Lycopersicon chmielewskii, accumulate sucrose. Genetic analysis of progeny resulting from a cross between L. chmielewskii and L. esculentum indicated that the sucrose-accumulating trait could be stably transferred and that the trait was controlled by the action of one or two recessive genes. Biochemical analysis of progeny resulting from this cross indicated that the sucrose-accumulating trait was associated with greatly reduced levels of acid invertase, but normal levels of sucrose synthase. Invertase from hexose-accumulating fruit was purified and could be resolved into three isoforms by chromatofocusing, each with isoelectric points between 5.1 and 5.5. The invertase isoforms showed identical polypeptide profiles on sodium dodecyl sulfate polyacrylamide gel electrophoresis, consisting of a primary 52 kilodalton polypeptide and two lower molecular mass polypeptides that appear to be degradation products of the 52 kilodalton polypeptide. The three invertase isoforms were indistinguishable based on pH, temperature, and substrate concentration dependence. Immunological detection of invertase indicated that the low level of invertase in sucrose-accumulating fruit was due to low levels of invertase protein rather than the presence of an invertase inhibitor. Based on comparison of genetic and biochemical data we speculate that a gene either encoding tomato fruit acid invertase or one required for its expression, plays an important role in determining sucrose accumulation.     

Isolation and characterization of fruit vacuolar invertase genes from two tomato species and temporal differences in mRNA levels during fruit ripening

To determine the relationship between invertase gene expression and glucose and fructose accumulation in ripening tomato fruit, fruit vacuolar invertase cDNA and genomic clones from the cultivated species, Lycopersicon esculentum cv. UC82B, and a wild species, Lycopersicon pimpinellifolium, were isolated and characterized. The coding sequences of all cDNA clones examined are identical. By comparison to the known amino acid sequence of mature L. esculentum fruit vacuolar invertase, a putative signal sequence and putative amino-terminal and carboxy-terminal propeptides were identified in the derived amino acid sequence. Of the residues 42% are identical with those of carrot cell wall invertase. A putative catalytic site and a five-residue motif found in carrot, yeast, and bacterial invertases are also present in the tomato sequence. Minor differences between the nucleotide sequences of the genomic clones from the two tomato species were found in one intron and in the putative regulatory region. The gene appears to be present in one copy per haploid genome. Northern analysis suggests a different temporal pattern of vacuolar invertase mRNA levels during fruit development in the two species, with the invertase mRNA appearing at an earlier stage of fruit development in the wild species. Nucleotide differences found in the putative regulatory regions may be involved in species differences in temporal regulation of this gene, which in turn may contribute to observed differences in hexose accumulation in ripening fruit.     

乙酰水杨酸在番茄果实发育期间对蔗糖代谢相关酶活性的影响

研究不同浓度乙酰水杨酸(ASA)对番茄品种‘辽园多丽’果实发育期间蔗糖代谢相关酶影响的结果表明:ASA可抑制果实的维管束和胶质胎座中酸性转化酶(AI)和中性转化酶(NI)活性,而提高蔗糖合成酶(SS)与蔗糖磷酸合成酶(SPS)活性;心室隔壁和中果肉中ASA的作用与此相反。ASA促进果实维管束中可溶性糖积累主要通过调控AI和NI活性实现,而在胶质胎座中主要通过调控SS活性实现;在中果肉和心室隔壁中主要通过调控SS和AI活性实现。     

Sugar uptake by protoplasts isolated from tomato fruit tissues during various stages of fruit growth

The uptake of radioactive glucose and sucrose by protoplasts isolated from pericarp and placenta tissues of tomato ( Lycopersicon esculentum cv. Counter) fruit was investigated in relation to the dry matter accumulation rates of these tissues. Uptake of glucose by protoplasts isolated from pericarp tissue was highest in fruit of around 20 g fresh weight or 25 days after anthesis. Sucrose uptake by pericarp protoplasts was lower than that of glucose and did not show a peak of uptake. The maximum rate of glucose uptake by protoplasts from the pericarp was at the time when the tomato fruit was accumulating dry matter at the highest rate. Glucose uptake by placenta protoplasts was lower and at a similar level as sucrose.
Protoplast uptake of glucose, but not of sucrose, was partially inhibited by (1) p -chloromercuribenzene sulphonic acid, a sulphydryl group modifier; (2) erythrosin B, an H⁺-ATPase inhibitor; and (3) valinomycin, a K⁺-ionophore, suggesting that membrane transport of glucose by tomato fruit sink cells may be a carrier-mediated, energy-dependent process.
The main route of carbohydrate accumulation by tomato fruit during the period of rapid fruit growth may be by cleavage of sucrose by apoplastic acid invertase prior to hexose transport across the plasma membrane.     

Evidence for the involvement of sucrose phosphate synthase in the pathway of sugar accumulation in sucrose-accumulating tomato fruits

To better understand the mechanism of sugar unloading and sugar concentration in hexose- and sucrose-accumulating tomato fruits (Lycopersicon chmielewskii and L. esculentum, respectively) and to determine the causes of the late accumulation of sucrose present in sucrose-accumulating tomato fruits, the assimilation of [³H](fructosyl)-sucrose was studied. Key enzymes involved in carbohydrate metabolism were also assayed. The results demonstrated that the low level of sucrose present in young fruits accumulates directly without undergoing hydrolysis, suggesting a symplastic pathway for sucrose unloading. By contrast, the large quantity of the sucrose present in ripe sucrose-accumulating fruits originates from hydrolysis and resynthesis, suggesting an apoplastic pathway for sucrose unloading. The increase in sucrose level observed in sucrose-accumulating fruits is associated with a gradual decline in invertase activity and an increase in sucrose phosphate synthase activity. This latter enzyme seems to play a key biochemical role in the accumulation of sucrose and the establishment of a high sugar content in tomato fruits.     

Genetic control and evolution of sesquiterpene biosynthesis in Lycopersicon esculentum and L. hirsutum

Segregation analysis between Lysopersicon esculentum (cultivated tomato) and L. hirsutum (wild form) in conjunction with positional verification by using near-isogenic lines demonstrated that biosynthesis of two structurally different classes of sesquiterpenes in these species is controlled by loci on two different chromosomes. A locus on chromosome 6, Sesquiterpene synthase1 (Sst1), was identified for which the L. esculentum allele is associated with the biosynthesis of beta-caryophyllene and alpha-humulene. At this same locus, the L. hirsutum allele is associated with biosynthesis of germacrene B, germacrene D, and an unidentified sesquiterpene. Genomic mapping, cDNA isolation, and heterologous expression of putative sesquiterpene synthases from both L. esculentum and L. hirsutum revealed that Sst1 is composed of two gene clusters 24 centimorgans apart, Sst1-A and Sst1-B, and that only the genes in the Sst1-A cluster are responsible for accumulation of chromosome 6-associated sesquiterpenes. At a second locus, Sst2, on chromosome 8, the L. hirsutum allele specified accumulation of alpha-santalene, alpha-bergamotene, and beta-bergamotene. Surprisingly, the L. esculentum allele for Sst2 is not associated with the expression of any sesquiterpenes, which suggests that cultivated tomato may have a nonfunctional allele. Sesquiterpene synthase cDNA clones on chromosome 6 do not cross-hybridize on genomic DNA gel blots with putative sesquiterpene synthases on chromosome 8, an indication that the genes in Sst1 and Sst2 are highly diverged, each being responsible for the biosynthesis of structurally different sets of sesquiterpenes.     

Apoplastic pH and inorganic ion levels in tomato fruit: A potential means for regulation of cell wall metabolism during ripening

Apoplastic pH and ionic conditions exert strong influence on cell wall metabolism of many plant tissues; however, the nature of the apoplastic environment of ripening fruit has been the subject of relatively few studies. In this report, a pressure-bomb technique was used to extract apoplastic fluid from tomato fruit ( Lycopersicon esculentum Mill.) pericarp at several developmental stages. pH and the levels of K⁺, Na⁺, Ca²⁺, Mg²⁺, Cl⁻ and P were determined and compared with the values for the bulk pericarp and locule tissues. The pH of the apoplastic fluid from pericarp tissue decreased from 6.7 in immature and mature-green fruits to 4.4 in fully-ripe fruit. During the same period, the K⁺ concentration increased from 13 to 37 m M . The levels of Na⁺ and divalent cations did not change, whereas the anions P and Cl⁻ increased in ripe fruit. Ca²⁺ levels remained relatively constant during ripening at 4–5 m M , concentrations that effectively limit pectin solubilization. The electrical conductivity of the apoplastic liquid increased 3-fold during ripening, whereas osmotically active solutes increased 2-fold. Pressure-treated fruit retained the capacity to ripen. The decline in apoplastic pH and increase in ionic strength during tomato fruit ripening may regulate the activity of cell wall hydrolases. The potential role of apoplastic changes in fruit ripening and softening is discussed.     

Fine mapping of quantitative trait loci for improved fruit characteristics from Lycopersicon chmielewskii chromosome 1.

The near-isogenic line (NIL) TA1150 contains a 56-cM introgression from Lycopersicon chmielewskii chromosome 1 and has several interesting phenotypic characteristics including fruit with orange color, high levels of soluble solids, thick pericarp, small stem scars, and good firmness. A set of overlapping recombinant lines (subNILs) was developed and field tested to fine map the quantitative trait loci (QTL) controlling these traits. The results indicated that the solids, pericarp thickness, and firmness QTL are distinct from the color locus. Several of the QTL mapped in this study, including the soluble-solids QTL, probably correspond to QTL mapped in other wild species of tomato. However, analysis of a set of TA523 subNILs containing complementary introgressions from Lycopesicon hirsutum chromosome 1 suggests that this wild species may contain a different locus for improved soluble solids. Thus, it might be possible to combine the L. chmielewskii and L. hirsutum alleles for these loci in a single line with the potential for extremely highly soluble solids. The TA1150 subNIL TA1688 contains the smallest introgression of the solids locus (approximately 19 cM), as well as the pericarp thickness and firmness QTL, with a yield that was equivalent to two of the three control lines. Isolation of recombinant subNILs from TA1688 should break the linkage between orange color and high solids and provide a small introgressed segment for marker-assisted breeding and genetic improvement of processing tomato.     

Sucrose Synthase,Starch Accumulation,and Tomato Fruit Sink Strength

Contrasting evidence has accumulated regarding the role of acid invertase and sucrose synthase in tomato fruit sink establishment and maintenance. In this work the relationships among the activities of sucrose synthase and acid invertase, Lycopersicon esculentum Mill cv UC-82B fruit growth, and starch accumulation were analyzed in fruit at 0 to 39 d after anthesis. Sucrose synthase, but not acid invertase, was found to be positively correlated with tomato fruit relative growth rate and with starch content in the pericarp tissue. A similar association between sucrose synthase activity and starch accumulation was also evident in the basal portion of the stem. Heat-shock treatments, which inhibited the increase in sucrose synthase activity at the beginning of the light period and had no effect on acid invertase activity, were used to examine the importance of sucrose synthase in relation to sucrose metabolism and starch synthesis. After the heat-shock treatment, concomitantly with the suppressed sucrose synthase activity relative to the controls, there was a reduction in sucrose cleavage and starch accumulation. These data substantiate the conclusion that, during the early phases of tomato fruit development, sucrose synthase rather than acid invertase is the dominant enzyme in metabolizing imported sucrose, which in turn plays a part in regulating the import of sucrose into the fruit.     

Trichomes of Lycopersicon spp. and their effect on Myzus persicae (Sulzer) (Hemiptera: Aphididae)

Abstract The green peach aphid ( Myzus persicae ) is becoming resistant to many of the pesticides used in the tomato ( Lycopersicon esculentum ) industry. A potential alternative method of control is host-plant resistance, which may be mediated by trichomes. Resistance to the green peach aphid was assessed for six accessions of L. hirsutum, three of L. pennellii and a L. esculentum control. Mortality, entrapment and number of aphids unaffected by trichomes were recorded 24, 48 and 72 h after placement. Using multiple regression against counts of glandular and non-glandular trichomes, the role of foliar pubescence in resistance to the aphid was established. Lycopersicon pennellii possessed the highest level of resistance after 72 h and type IV trichome density accounted for 26.5% of variance in mortality ( P  < 0.001). Low densities of type IV and high densities of type VII trichomes increased the number of aphids 'unaffected' by trichomes, a relationship accounting for 29% of variance ( P  < 0.001). Information on which types of trichomes most strongly affect resistance to insect pests will aid future breeding and L. pennellii shows potential for use in breeding programs to increase levels of host-plant resistance in L. esculentum.     

Regulation and tissue-specific distribution of mRNAs for three extracellular invertase isoenzymes of tomato suggests an important function in establishing and maintaining sink metabolism.

The aim of the present study was to gain insight into the contribution of extracellular invertases for sink metabolism in tomato (Lycopersicon esculentum L.). The present study shows that extracellular invertase isoenzymes are encoded by a gene family comprising four members: Lin5, Lin6, Lin7, and Lin8. The regulation of mRNA levels by internal and external signals and the distribution in sink and source tissues has been determined and compared with mRNA levels of the intracellular sucrose (Suc)-cleaving enzymes Suc synthase and vacuolar invertase. The specific regulation of Lin5, Lin6, and Lin7 suggests an important function of apoplastic cleavage of Suc by cell wall-bound invertase in establishing and maintaining sink metabolism. Lin6 is expressed under conditions that require a high carbohydrate supply. The corresponding mRNA shows a sink tissue-specific distribution and the concentration is elevated by stress-related stimuli, by the growth-promoting phytohormone zeatin, and in response to the induction of heterotrophic metabolism. The expression of Lin5 and Lin7 in gynoecia and stamens, respectively, suggests an important function in supplying carbohydrates to these flower organs, whereas the Lin7 mRNA was found to be present exclusively in this specific sink organ.     

Leucine uptake by tomato leaf tissue under low temperature: Preincubation dependence of uptake reduction

The uptake of ³H-leucine by leaf fragments of Lycopersicon esculentum Mill. cv. Rutgers and L. hirsutum Humb. & Bonpl., a wild tomato, was studied. Two altitudinal races of L. hirsutum were used which differed in chilling tolerance. The temperature dependence of uptake was initially similar for all plant varieties. However, at temperatures below about 11°C, uptake progressively decreased in the more chilling-sensitive varieties ( L. esculentum , Low-altitude L. hirsutum ), but not in the more chilling-tolerant (high-altitude L. hirsutum ) with increasing preincubation time. More than 60 min preincubation was required for this effect, and it was greatest at the lower temperatures. When leaf fragments, chilled for short periods of time (>22 h), were returned to 22°C, initial rates of uptake were recovered within 2 h. The relationship between membrane lipid changes and membrane protein activity under chill stress is discussed.     

Resistance of wild Lycopersicon species to the potato moth, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae)

Abstract  Trichome-based host plant resistance of Lycopersicon species to potato moth, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae), was examined in a laboratory bioassay. Neonate P. operculella were placed on the foliage of accessions of L. hirsutum , L. hirsutum f. glabratum , L. pennellii , L. cheesmanii f. minor and L. esculentum for 48 h . At the end of this period, larval mortality, the numbers of larvae emigrating from, and mining into, the leaf surface were recorded as were leaf area and the densities of trichomes. Analysis by categorical logistic regression found accessions differed in levels of resistance to P. operculella ( P  < 0.001). Further analysis using generalised linear models showed mortality was associated with high densities of type VI and low densities of type V trichomes; emigration from the leaf was associated with high densities of type I and type VII trichomes; and the numbers of mines associated with low densities of type I and type VI trichomes and increased leaf area. Although results indicate that certain accessions of L. hirsutum and L. hirsutum f. glabratum may be most appropriate for inclusion in a breeding program aimed at introducing trichome-based host plant resistance of wild Lycopersicon species into the tomato, further research is necessary to fully understand the mechanisms.