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.     

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.     

Sink Metabolism in Tomato Fruit : III. Analysis of Carbohydrate Assimilation in a Wild Species

Carbohydrate composition and key enzymes involved in carbohydrate metabolism were assayed throughout development of Lycopersicon esculentum and L. chmielewskii fruit. Translocation and assimilation of asymmetric sucrose and total soluble solids content was also determined in both species. The data showed that L. chmielewskii accumulated less starch than L. esculentum, and this was related to a lower level of ADPglucose pyrophosphorylase and a higher level of phosphorylase in L. chmielewskii. L. chmielewskii accumulated sucrose throughout fruit development rather than glucose and fructose which were accumulated by L. esculentum. A low level of invertase and nondetectable levels of sucrose synthase were associated with the high level of sucrose in L. chmielewskii. Translocation and assimilation of asymmetrically labeled sucrose indicated that sucrose accumulated in L. chmielewskii fruit was imported and stored directly in the fruit without intervening metabolism along the translocation path. In contrast, the relatively low level of radioactive sucrose found in L. esculentum fruit appeared to arise from hydrolysis and resynthesis of sucrose. The possible relationship between the level of soluble solids and differences in carbohydrate metabolism in sink tissue of the two species is discussed.     

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.     

Introgression into tomato (Lycopersicon esculentum) of the L. chmielewskii sucrose accumulator gene (sucr) controlling fruit sugar composition

High sucrose concentration in fruit of Lycopersicon chmielewskii is governed by the recessive sucrose accumulator gene (sucr) that is situated in the pericentromeric region of chromosome 3. The sucr gene was introgressed into the genetic background of the hexose-accumulating cultivated tomato (L. esculentum cv Hunt 100) by marker-assisted selection using tightly linked RFLP markers and a tomato acid invertase cDNA as probes for sucr. RFLP mapping indicated that the segment containing sucr comprised over 43.2 cM in the BC₁F₂ generation, representing over one-third of the total length of chromosome 3. By selecting for crossovers between sucr and the flanking visual marker r (yellow fruit flesh) and RFLP marker TG288, we were able to reduce the size of the sucr introgression fragment to 0.8–7.1 cM by the BC₅ generation. Smaller recombinant fragments were not obtained despite screening a large BC₆F₂ population. The smallest sucr introgression reduced recombination between the flanking visual markers sy (sunny) and bls (baby lea syndrome) by 38%. To facilitate future introgression and recombination experiments, a PCR-based test for the sucr gene was developed using primers specific to the tomato invertase gene. This assay takes advantage of a small deletion that maps to the second intron of the L. chmielewskii nvertase gene. The assay detected significant allelic variation both within and between hexose- and sucrose-accumulating Lycopersicon spp.     

PCR-generated molecular markers for the invertase gene and sucrose accumulation in tomato

The green-fruited tomato species, Lycopersicon hirsutum, unlike the domesticated red-fruited species, L. esculentum, accumulates sucrose during the final stages of fruit development, concomitant with the loss of soluble acid invertase activity. In order to study the genetic linkage of sucrose accumulation to the invertase gene, part of the invertase gene from L. hirsutum was cloned, sequenced and the sequence compared with the invertase sequence of the red-fruited L. esculentum. Several base changes were found in the coding region of the two invertase genes. Based on these base -pair differences, we developed a species-specific PCR assay capable of determining, in a single PCR reaction, the origin of the invertase gene in segregating seedlings of an interspecific cross. Our results indicate that the invertase gene is genetically linked to sucrose accumulation in the green-fruited L. hirsutum.     

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.     

Overexpression of sucrose phosphate synthase increases sucrose unloading in transformed tomato fruit

Sucrose unloading and sink activity were examined in tomato plants (Lycopersicon esculentum) overexpression sucrose phosphate synthase (SPS; EC 2.3.1.14). Like the leaves, the fruit of the transformed tomato plants had elevated (2.4-fold) SPS activity. SPS over-expression in tomato fruit did not significantly change acid invertase, and only slightly reduced ADPglc ppase activity, but enhanced sucrose synthase activity by 27%. More importantly, the amount of sucrose unloaded into the fruit was considerably increased. Using [³H]- (fructosyl)-sucrose in in vitro unloading experiments with harvested 20-d-old fruit, 70% more sucrose was unloaded into the transformed fruits compared to the untransformed controls. Furthermore, the turnover of the sucrose unloaded into the fruit of transformed plants was 60% higher than that observed in the untransformed controls. Taken together, these results demonstrate that SPS overexpression increases the sink strength of transformed tomato fruit.     

Invertase Activity, Soluble Carbohydrates and Inflorescence Development in the Tomato (Lycopersicon esculentum Mill.)

The concentration of reducing sugars in the developing firstinflorescence of the tomato (Lycopersicon esculentum Mill.)increased steadily between the macroscopic appearance of theflower buds and the initial stages of fruit expansion. Overthis period sucrose concentrations remained relatively constant.The rise in reducing sugar concentration was accompanied byan increase in the activity of an acid invertase. In individualflower buds invertase activity rose to a maximum shortly beforeanthesis and declined sharply as the anthers dehisced. Increased planting densities and removal of source leaves reducedthe rate of dry matter accumulation by the first inflorescenceand increased the incidence of flower bud abortion. These changeswere correlated with reductions in reducing sugar concentrations,in reducing sugar/sucrose ratios and in acid invertase levels.Removal of young leaves at the shoot apex significantly increasedthe relative growth rate of the inflorescence and led to a substantialincrease in its invertase content. These treatments had relativelylittle effect on sucrose concentration in the inflorescence. The data are consistent with the operation of an invertase-mediatedunloading mechanism for transported sucrose at sinks in theflower buds. It is suggested that the retarded development ofthe first inflorescence and the high incidence of flower budabortion observed under conditions of reduced photoassimilateavailability are causally related to the decline in invertaseproduction in the flower buds. Possible mechanisms for the regulationof invertase synthesis in the flowers are discussed. Lycopersicon esculentum Mill, tomato, inflorescence development, invertase, sink activity     

Sucrose Synthase in Wild Tomato, Lycopersicon chmielewskii, and Tomato Fruit Sink Strength

Here it is reported that sucrose synthase can be readily measured in growing wild tomato fruits (Lycopersicon chmielewskii) when suitable methods are adopted during fruit extraction. The enzyme also was present in fruit pericarp tissues, in seeds, and in flowers. To check for novel characteristics, the wild tomato fruit sucrose synthase was purified, by (NH₄)₂SO₄ fraction and chromatography with DE-32, Sephadex G-200, and PBA-60, to one major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The following characteristics were obtained: native protein relative molecular weight 380,000; subunit relative molecular weight 89,000; K_m values with: sucrose 53 millimolar, UDP 18.9 micromolar, UDP-glucose 88 micromolar, fructose 8.4 millimolar; pH optima between 6.2 to 7.3 for sucrose breakdown and 7 to 9 for synthesis; and temperature optima near 50°C. The enzyme exhibited a high affinity and a preference for uridylates. The enzyme showed more sensitivity to divalent cations in the synthesis of sucrose than in its breakdown. Sink strength in tomato fruits also was investigated in regard to sucrose breakdown enzyme activities versus fruit weight gain. Sucrose synthase activity was consistently related to increases in fruit weight (sink strength) in both wild and commercial tomatoes. Acid and neutral invertases were not, because the published invertase activity values were too variable for quantitative analyses regarding the roles of invertases in tomato fruit development. In rapidly growing fruits of both wild and commercially developed tomato plants, the activity of sucrose synthase per growing fruit, i.e. sucrose synthase peak activity X fruit size, was linearly related to final fruit size; and the activity exceeded fruit growth and carbon import rates by at least 10-fold. In mature, nongrowing fruits, sucrose synthase activities approached nil values. Therefore, sucrose synthase can serve as an indicator of sink strength in growing tomato fruits.     

Analysis of mRNAs that Accumulate in Response to Low Temperature Identifies a Thiol Protease Gene in Tomato

We have studied the induction of gene expression at low temperature by cloning mRNAs that accumulate when unripe tomato (Lycopersicon esculentum) fruit are incubated at 4°C. Two cloned mRNAs, C14 and C17, accumulate relatively rapidly in response to cold treatment, while a third, C19, displays a delayed response. Significant levels of these mRNAs were not detected during fruit ripening at normal temperature. We have analyzed gene expression at different temperatures and detect half-maximal accumulation of the C14 and C17 mRNAs at 16°C and 11°C, respectively, and have observed that sustained gene expression requires continuous cold treatment. Furthermore, the level of C14 and C17 gene expression in cold-tolerant (hybrid L. esculentum/Lycopersicon pimpinellifolium) fruit is different from that in cold-sensitive (L. esculentum) fruit. DNA sequence analysis indicates that the C14 mRNA encodes a polypeptide with a region that is homologous to the plant thiol proteases actinidin and papain and to the animal thiol protease cathepsin H. We conclude from these experiments that low temperature selectively induces the expression of specific genes and that one such gene encodes a thiol protease.     

Acclimation of Two Tomato Species to High Atmospheric CO(2): I. Sugar and Starch Concentrations

Lycopersicon esculentum Mill. cv Vedettos and Lycopersicon chmielewskii Rick, LA 1028, were exposed to two CO₂ concentrations (330 or 900 microliters per liter) for 10 weeks. Tomato plants grown at 900 microliters per liter contained more starch and more sugars than the control. However, we found no significant accumulation of starch and sugars in the young leaves of L. esculentum exposed to high CO₂. Carbon exchange rates were significantly higher in CO₂-enriched plants for the first few weeks of treatment but thereafter decreased as tomato plants acclimated to high atmospheric CO₂. This indicates that the long-term decline of photosynthetic efficiency of leaf 5 cannot be attributed to an accumulation of sugar and/or starch. The average concentration of starch in leaves 5 and 9 was always higher in L. esculentum than in L. chmielewskii (151.7% higher). A higher proportion of photosynthates was directed into starch for L. esculentum than for L. chmielewskii. However, these characteristics did not improve the long-term photosynthetic efficiency of L. chmielewskii grown at high CO₂ when compared with L. esculentum. The chloroplasts of tomato plants exposed to the higher CO₂ concentration exhibited a marked accumulation of starch. The results reported here suggest that starch and/or sugar accumulation under high CO₂ cannot entirely explain the loss of photosynthetic efficiency of high CO₂-grown plants.     

Distribution of acid invertase in the tomato plant

Acid invertase activity in Lycopersicon esculentum was highest in the locular wall of ripe fruit and lowest in roots. Activity was greater in leaf laminae than in petiole tissue and increased with leaf age, whereas there was more invertase in the upper part of the stem compared with the older portion. Activity in whole fruit increased with increasing ripeness and was greatest in overripe fruit. Of various tissues from a number of wild tomato species examined, the fruit of L. pimpinellifolium were particularly rich in the enzyme, in contrast to the fruit of L. hirsutum, L. hirsutum, var. glabratum and L. peruvianum which had low activity.     

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.     

Sucrose uptake,invertase localization and gene expression in developing fruit of Lycopersicon esculentum and the sucrose-accumulating Lycopersicon hirsutum

By using immunolocalization and differential extraction methods we show that only apoplastic invertase, but not vacuolar invertase, was present in the mature, sucrose-accumulating L. hirsutum pericarp. In contrast, in the hexose-accumulating L. esculentum fruit, both the apoplastic and vacuolar invertase activities and protein content increase in the mature fruit. Quantitative expression studies of the soluble invertase gene (TIV1) and the apoplastic invertase genes (LINs) showed that only TIV1 gene expression could account for the species and developmental differences of both soluble and insoluble enzyme activity of the pericarp. The expression of the LIN genes encoding for apoplastic tomato invertases was unrelated to the differences in bound enzyme activity and could not account for the rise in bound invertase activity in the mature L. esculentum fruit. Evidence is presented that the bound invertase activity of tomato fruit is also the TIV1 gene product. The presence of apoplastic invertase in the mature sucrose-accumulating L. hirsutum fruit suggests a hydrolysis-resynthesis mechanism of sucrose uptake. In order to test this hypothesis, we studied short- and long-term uptakes of asymmetrically labelled ³H-fructosyl-sucrose accompanied by compartmental analysis of the sugars in attached whole fruits of L. hirsutum and L. esculentum. The results indicate that hydrolysis-resynthesis is slow in the sucrose-accumulating fruit but is not an integral part of an uptake and compartmentation mechanism.     

Effects of the Lycopersicon chmielewskii sucrose accumulator gene (sucr) on fruit yield and quality parameters following introgression into tomato

A gene controlling fruit sucrose accumulation, sucr, was introgressed from the wild tomato species Lycopersicon chmielewskii into the genetic background of a hexose-accumulating cultivated tomato, L. esculentum. During introgression, the size of the L. chmielewskii chromosomal segment containing sucr was reduced by selection for recombination between RFLP markers for the sucr gene and flanking loci. The effects of sucr on soluble solids content, fruit size, yield and other fruit parameters were studied in the genetic background of the processing tomato cultivar Huntl00. In a segregating BC₅F₂ generation, the smallest introgression containing sucr-associated markers was necessary and sufficient to confer high-level sucrose accumulation, the effects of which were completely recessive. Fruit of sucr/sucr genotypes were smaller than those of +/sucr or +/+ genotypes at all stages of development. The timing of sugar accumulation and total sugar concentration were unaffected by sugar composition. No differences in total fruit biomass (fresh weight of red and green fruit) at harvest were observed between the genotypes, and sucrose accumulators produced greater numbers of fruit than hexose accumulators in one family. However, the proportion of ripe fruit at harvest, and hence yield of ripe fruit, as well as average ripe fruit weight and seed set were reduced in sucr/sucr genotypes. Sucrose accumulation was also associated with increased soluble solids content, consistency, serum viscosity, predicted paste yield and acidity, and decreased color rating. In the first backcross to L. chmielewskii, hexose accumulators (+/sucr) had larger fruit than sucrose accumulators (sucr/sucr), while no difference in soluble solids was detected.     

Light regulation of sink metabolism in tomato fruit : I. Growth and sugar accumulation

Light/dark effects on growth and sugar accumulation in tomato (Lycopersicon esculentum) fruit during early development were studied on intact plants (in vivo) and in tissue culture (in vitro). Through the use of an in vitro culture of tomato fruit, it was possible to investigate the direct effects of light on sink metabolism by eliminating the source tissue. Similar growth patterns were found in vivo and in vitro. Fruit growth in different sugars indicated that sucrose was the best source of carbon for in vitro fruit growth. Fruit growth increased as sucrose concentration increased up to 8%. Darkening the fruit decreased fruit dry weight about 40% in vivo and in vitro. The differences in the CO₂ exchange rate between light and dark grown fruit indicated that light stimulation of fruit growth was due to mechanisms other than photosynthesis. Supporting this conclusion was the fact that light intensities ranging from 40 to 160 micromoles per square meter per second had no significant influence on fruit growth, and light did not increase growth of fruit cultured with glucose or fructose as a carbon source. However, light stimulated fruit growth significantly when sucrose was used as the carbon source. Light-grown fruit took up 30% more sucrose from the same source and accumulated almost twice as much hexose and starch as dark-grown fruit. A possible expansion of an additional sink for carbon by light stimulation of starch synthesis during early development will be discussed.     

Light Regulation of Sink Metabolism in Tomato Fruit : II. Carbohydrate Metabolizing Enzymes

Effects of light on carbohydrate levels and certain carbon metabolizing enzyme activities were studied during the early development of tomato (Lycopersicon esculentum) fruit. Sucrose levels were low and continued to decline during development and were unaffected by light. Starch was significantly greater in light. Invertase activity was similar in both light- and dark-grown fruit. Sucrose synthase activity was much lower than invertase and showed a slight decrease in light-grown fruit between days 21 and 28. Light-grown fruit also had higher ADP glucose pyrophosphorylase activity than dark-grown fruit, which was correlated with higher starch levels. The rapidly decreasing activity of ADP glucose pyrophosphorylase during early fruit development in the dark in conjunction with reduced starch levels and rates of accumulation indicates that ADP glucose pyrophosphorylase is crucial for carbon import and storage in tomato. The differential stimulation of ADP glucose pyrophosphorylase activity from light- and dark-grown tissue by 3-phosphoglycerate suggests that this enzyme may be allosterically altered by light.     

Carbohydrate supplements to the callus culture medium modify the growth of potato tuber moth larvae feeding on Lycopersicon chmielewskii callus

Lycopersicon esculentum and L. chmielewskii are respectively susceptible and resistant to the potato tuber moth (Phthorimaea operculella Zeller) in the field. Feeding bioassays were conducted with the herbivore caterpillars reared on callus derived from both tomato species and grown in vitro, and the influence of carbohydrate supplements to the callus culture medium, on the insect's feeding behavior was investigated. Newly-hatched larvae fed with L. esculentum or L. chmielewskii callus raised on a medium with 88 mM sucrose, reached a weight of 12–15 mg and 1.5–3.0 mg, respectively, within 9 days. Restriction of larval weight increase in insects reared on L. chmielewskii callus, disappeared when the host tissue was transferred 24 h prior to the callus-insect assay to a medium supplemented with 264 mM of either sucrose, glucose, fructose or mannose. The capability of L. chmielewskii callus to restrict growth of larvae was restored in host tissue retransferred from a medium with 264 mM sucrose to a 24-h incubation on one supplemented with 264 mM of either mannitol, sorbitol, glycerol or myo-inositol, before the callus-insect bioassay. The larval growth response remained unaltered by callus incubated on a medium with 264 mM xylose. The ameliorating effect on insect growth of high sucrose in the callus medium was not due to sucrose as an ingredient of the insect's diet. The diverse response of L. chmielewskii callus, and its dependence on the type of carbohydrate in the medium, rule out effects of these substances as nonspecific medium osmotica. The swift callus responses to carbohydrates (within hours of a change in medium composition), as reflected in the insect's growth, were not accompanied by visible morphological variations in the host tissue. We suggest that suppression by high levels of exogenously applied saccharides and derepression by exogenous polyols and myo-inositol of the impedement to growth of the potato tuber moth larva, reflect the existence in L. chmielewskii of a carbon metabolic control mechanism of gene expression whose products affect insect growth.