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.     

Sucrose Phosphate Synthase, Sucrose Synthase, and Invertase Activities in Developing Fruit of Lycopersicon esculentum Mill. and the Sucrose Accumulating Lycopersicon hirsutum Humb. and Bonpl

The green-fruited Lycopersicon hirsutum Humb. and Bonpl. accumulated sucrose to concentrations of about 118 micromoles per gram fresh weight during the final stages of development. In comparison, Lycopersicon esculentum Mill. cultivars contained less than 15 micromoles per gram fresh weight of sucrose at the ripe stage. Glucose and fructose levels remained relatively constant throughout development in L. hirsutum at 22 to 50 micromoles per gram fresh weight each. Starch content was low even at early stages of development, and declined further with development. Soluble acid invertase (EC 3.2. 1.26) activity declined concomitant with the rise in sucrose content. Acid invertase activity, which was solubilized in 1 molar NaCl (presumably cell-wall bound), remained constant throughout development (about 3 micromoles of reducing sugars (per gram fresh weight) per hour. Sucrose phosphate synthase (EC 2.4.1.14) activity was present at about 5 micromoles of sucrose (per gram fresh weight) per hour even at early stages of development, and increased sharply to about 40 micromoles of sucrose (per gram fresh weight) per hour at the final stages of development studied, parallel to the rise in sucrose content. In comparison, sucrose phosphate synthase activity in L. esculentum remained low throughout development. The possible roles of the sucrose metabolizing enzymes in determining sucrose accumulation are discussed.     

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.     

Sucrose Phosphate Synthase and Acid Invertase as Determinants of Sucrose Concentration in Developing Muskmelon (Cucumis melo L.) Fruits

Fruits of orange-fleshed and green-fleshed muskmelon (Cucumis melo L.) were harvested at different times throughout development to evaluate changes in metabolism which lead to sucrose accumulation, and to determine the basis of differences in fruit sucrose accumulation among genotypes. Concentrations of sucrose, raffinose saccharides, hexoses and starch, as well as activities of the sucrose metabolizing enzymes sucrose phosphate synthase (SPS) (EC 2.4.1.14), sucrose synthase (EC 2.4.1.13), and acid and neutral invertases (EC 3.2.1.26) were measured. Sucrose synthase and neutral invertase activities were relatively low (1.7 ± 0.3 micromole per hour per gram fresh weight and 2.2 ± 0.2, respectively) and changed little throughout fruit development. Acid invertase activity decreased during fruit development, (from as high as 40 micromoles per hour per gram fresh weight) in unripe fruit, to undetectable activity in mature, ripened fruits, while SPS activity in the fruit increased (from 7 micromoles per hour per gram fresh weight) to as high as 32 micromoles per hour per gram fresh weight. Genotypes which accumulated different amounts of sucrose had similar acid invertase activity but differed in SPS activity. Our results indicate that both acid invertase and SPS are determinants of sucrose accumulation in melon fruit. However, the decline in acid invertase appears to be a normal function of fruit maturation, and is not the primary factor which determines sucrose accumulation. Rather, the capacity for sucrose synthesis, reflected in the activity of SPS, appears to determine sucrose accumulation, which is an important component of fruit quality.     

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.     

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.     

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.     

Sucrose metabolism and accumulation in developing fruit of Cucumis

Sucrose, reducing sugars and starch content were measured in developing cucumber (C. sativus cv Delilah), sweet melon (C. melon cv Galia and cv Noy Yizre'el) and non-sweet melon (C. melo cv Bird's Nest) fruit. Sweet melon were characterized by accumulation of sucrose in the maturing fruit, while cucumber and non-sweet melon had a low sucrose content at all stages studied. Soluble acid invertase activity (EC 3.2.1.26) dramatically decreased in sweet melon, concomitant with the onset of sucrose accumulation. Significant activity of soluble acid invertase was retained in mature cucumber and non-sweet melon. Insoluble acid invertase, determined not to be an artifact of extraction, constituted a significant portion of total invertase activity (ca 25% in young sweet melon and ca 50% in young cucumber). In sweet melon sucrose synthase activity (EC 2.4.1.13), measured in both the cleavage and synthesis direction, increased during the sucrose accumulation period. The results are discussed in terms of the roles of invertase and sucrose synthase in sucrose accumulation in Cucumis.     

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.     

宁夏枸杞果实糖积累和蔗糖代谢相关酶活性的关系

通过对枸杞果实发育过程中果实生长模式、蔗糖、果糖、葡萄糖和淀粉含量及糖代谢相关酶活性的测定,研究了宁夏枸杞果实生长发育过程中糖的代谢积累与相关酶活性的关系.结果表明:(1)宁夏枸杞果实发育呈双S"曲线,果实主要以积累己糖为主.(2)蔗糖磷酸合成酶(SPS)活性在果实发育初期处于下降的趋势,在花后19d开始上升,果实转色后又逐渐下降;蔗糖合成酶(SS)活性总体表现为SS分解方向的活性大于SS合成方向的活性,说明枸杞果实发育过程中,SS的活性主要以分解方向的为主;酸性转化酶(AI)和中性转化酶(NI)的活性随果实发育呈上升趋势,但在果实成熟后期有所下降,且AI和NI活性高于合成酶类的活性,较高的转化酶类活性促进了果实内部己糖的积累.(3)在枸杞果实生长发育中,葡萄糖和果糖含量与AI和NI均呈极显著正相关,而与其它酶不具有相关性.说明AI和NI在宁夏枸杞果实的糖代谢中起着主要的调控作用.     

Differences in sucrose metabolism relative to accumulation of bird-deterrent sucrose levels in fruits of wild and domestic Vaccinium species

We examined variability in sucrose levels and metabolism in ripe fruits of wild and domestic Vaccinium species and in developing fruits of cultivated blueberry (V. ashei and V. corymbosum). The objective was to determine if sufficient variability for fruit sucrose accumulation was present in existing populations to warrant attempts to breed for high-sucrose fruit, which potentially would be less subject to bird predation. Threefold differences in fruit sucrose concentration were found among Vaccinium species, ranging from 19 to 24 mg (g fresh weight)^?1 in V. stamineum and V. arboreum to approximately 7 mg (g fresh weight)^?1 in cultivated blueberry (V. ashei and V. corymbosum) and V. darrowi. Hexose levels were similar among species, ranging from 90 to 110 mg (g fresh weight)^–1, and glucose and fructose were present in equal amounts. Soluble acid invertase (EC 3.2.1.26) activity was negatively correlated with fruit sucrose concentration. There was no apparent correlation between fruit sugar concentration and either sucrose synthase (EC 2.4.1.13) or sucrose phosphate synthase (EC 2.4.1.14) activities, both of which were low for all species studied. Developmental increases in fruit sugar levels of cultivated blueberry followed a pattern similar to that observed in fruit fresh weight accumulation. Hexose concentrations ranged from 6 to 30 mg (g fresh weight)^?1 during the first 60 days after anthesis. Between 60 days and fruit ripening (80 days), hexose levels rose from 30 to 80 mg (g fresh weight)^?1. Sucrose was not detected in fruits until ripening, when low levels were found. Insoluble acid invertase activity was relatively high early in fruit development, decreasing as soluble acid invertase activity increased. Between 60 days and fruit ripening, soluble acid invertase activity increased from 3 to 55 μmol (g fresh weight)^–1 h^–1. Both sucrose synthase and sucrose phosphate synthase activities were low throughout development. The extent of sucrose accumulation in fruits and the degree of variability for this trait among Vaccinium species support the feasibility of developing high sucrose fruits, which would be a potentially valuable addition to current strategies of minimizing crop losses to birds.     

Dynamic characteristics of sugar accumulation and related enzyme activities in sweet and non-sweet watermelon fruits

Sugar content largely determines watermelon fruit quality. We compared changes in sugar accumulation and activities of carbohydrate enzymes in the flesh (central portion) and mesocarp of elite sweet watermelon line 97103 (Citrullus lanatus subsp. vulgaris) and exotic non-sweet line PI296341-FR (C. lanatus subsp. lanatus) to elucidate the physiological and biochemical mechanisms of sugar accumulation in watermelon fruit. The major translocated sugars, raffinose and stachyose, were more unloaded into sweet watermelon fruit than non-sweet fruit. During the fruit development, acid α-galactosidase activity was much higher in flesh of 97103 than in mesocarp of 97103, in flesh and mesocarp of PI296341-FR fruit. Insoluble acid invertase activity was higher in 97103 flesh than in 97103 mesocarp, PI296341-FR flesh or mesocarp from 18 days after pollination (DAP) to 34 DAP. Changes in soluble acid invertase activity in 97103 flesh were similar to those in PI296341-FR flesh and mesocarp from 18 DAP to full ripening. Sucrose synthase and sucrose phosphate synthase activities in 97103 flesh were significantly higher than those in 97103 mesocarp and PI296341-FR fruits from 18 to 34 DAP. Only insoluble acid invertase and sucrose phosphate synthase activities were significantly positively correlated with sucrose content in 97103 flesh. Therefore, phloem loading, distribution and metabolism of major translocated sugars, which are controlled by key sugar metabolism enzymes, determine fruit sugar accumulation in sweet and non-sweet watermelon and reflect the distribution diversity of translocated sugars between subspecies.     

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。     

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.     

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.     

Sucrose phosphate synthase and other sucrose metabolizing enzymes in fruits of various species

Recent reports have suggested that sucrose phosphate synthase (EC 2.4.1.14), a key enzyme in sucrose biosynthesis in photosynthetic “source” tissues, may also be important in some sucrose accumulating “sink” tissues. These experiments were conducted to determine if sucrose phosphate synthase is involved in sucrose accumulation in fruits of several species. Peach (Prunus persica NCT 516) and strawberry (Fragaria x ananassa cv. Chandler) fruits were harvested directly from the plant at various stages of fruit development. Kiwi (Actinidia chinensis), papaya (Carica papaya), pineapple (Ananas comosus) and mango (Mangifera indica) were sampled in postharvest storage over a period of several days. Carbohydrate concentrations and activities of sucrose phosphate synthase, sucrose synthase (EC 2.4.1.13), and acid and neutral invertases (EC 3.2.1.26) were measured. All fruits contained significant activities of sucrose phosphate synthase. Moreover, in fruits from all species except pineapple and papaya, there was an increase in sucrose phosphate synthase activity associated with the accumulation of sucrose in situ. The increase in sucrose concentration in peaches was also associated with an increase in sucrose synthase activity and, in strawberries, with increased activity of both sucrose synthase and neutral invertase. The hexose pools in all fruits were comprised of equimolar concentrations of fructose and glucose, except in the mango. In mango, the fructose to glucose ratio increased from 2 to 41 during ripening as sucrose concentration more than doubled. The results of this study indicate that activities of the sucrose metabolizing enzymes, including sucrose phosphate synthase, within the fruit itself, are important in determining the soluble sugar content of fruits of many species. This appears to be true for fruits which sweeten from a starch reserve and in fruits from sorbitol translocating species, raffinose saccharide translocating species, and sucrose translocating species.     

The role of sucrose-metabolizing enzymes in pear fruit that differ in sucrose accumulation

In the paper, the soluble sugar composition and activities of enzymes metabolizing sucrose: invertase (β-fructosidase, EC 3.2.1.26), sucrose synthase (SS; EC 2.4.1.13) and sucrose-phosphate synthase (SPS; EC 2.4.1.14) were investigated during fruit development of two pear species: Pyrus bretschneideri Rehd. cv. ‘Yali’ and P. pyrifolia Nakai cv. ‘Aikansui’, characterized as low and high sucrose types, respectively. It was found that, at the end of fruit development of ‘Aikansui’, the level of sucrose was five times higher than in ‘Yali’ in the same period. It was coincident with the significantly higher activities of SS (synthesis) and SPS and lower activities of invertase (vacuolar and cell wall-bound acid invertase and neutral invertase). The high correlation was found between sucrose level and SS (synthesis) and SPS activities in ‘Aikansui’ pears.     

糖代谢相关酶在灵武长枣叶片和果柄糖积累中的作用

以不同发育时期灵武长枣为试材,测定果实生长发育过程中叶片、果柄可溶性糖含量及蔗糖代谢相关酶活性的变化,探讨果实生长发育过程中叶片、果柄糖的积累与蔗糖代谢相关酶活性的关系。结果表明:(1)灵武长枣叶片、果柄均主要以积累蔗糖为主,叶片、果柄中葡萄糖和果糖含量的变化平缓且随果实发育略有上升,蔗糖含量则呈先下降后迅速上升的趋势,且蔗糖含量始终高于葡萄糖和果糖的含量。(2)在果实的整个发育期,叶片和果柄的酸性转化酶(AI)活性均远高于中性转化酶(NI),AI在前期升高后变化较平稳,而蔗糖合成酶(SS)和蔗糖磷酸合成酶(SPS)活性的变化各不相同。(3)SS分解方向酶活性(SSd)对叶片和果柄蔗糖的积累具有重要的调节作用。研究认为,蔗糖合成酶分解方向酶活性(SSd)对灵武长枣叶片和果柄蔗糖的积累起主要的调控作用。     

Sucrose metabolism and fruit growth in parthenocarpic vs seeded blueberry (Vaccinium ashei) fruits

Although fruit set and development are induced by applications of gibberellins, final fruit weight of gibberellin-induced parthenocarpic fruit is often less than that of pollinated fruit. We examined changes in the activities of sucrose-metabolizing enzymes and sugar accumulation in developing fruits of cultivated blueberry (Vaccinium ashei Reade) and their correlation with fruit growth upon pollination or exogenous applications of gibberellic acid (GA₃). The objective was to determine if differences in fruit growth could be attributed to differences in enzyme activities and subsequent sugar accumulation in fruits. The fruit development period of GA₃-treated fruits was 15 days longer than that of pollinated fruits. At maturity, GA₃-treated fruit accumulated an average of 180 mg dry weight while pollinated fruit accumulated 390 mg dry weight. Dry weight accumulation in nonpollinated fruits was negligible and these fruits abscised by 45 days after bloom (DAB). The total carbon (C) cost (dry weight C + respiratory C) for fruit development was 109 and 244 mg C fruit^-1 for GA₃-treated and pollinated fruits, respectively. Hexose concentration increased to 100 mg (g fresh weight)^-1 at ripening in both GA₃-treated and pollinated fruits. Nonpollinated fruits reached a maximum hexose concentration at 45 DAB. Sucrose phosphate synthase (EC 2.4.1.14) and sucrose synthase (EC 2.4.1.13) activities reached a maximum of ≤5.0 μmol (g fresh weight)^-1 h^-1 in both GA₃-treated and pollinated fruits. Soluble acid invertase (EC 3.2.1.26) activity increased to about 60 μmol (g fresh weight)^-1 h^-1 in both GA₃-treated and pollinated fruits at ripening, while in nonpollinated fruits, a maximum soluble acid invertase activity of 0.12 μmol (g fresh weight)^-1 h^-1 was measured at 24 DAB. Insoluble acid invertase activity declined during the early stages of fruit growth and remained relatively low throughout fruit development. Neutral invertase activity was low throughout development, increasing to 5 μmol (g fresh weight)^-1 h^-1 at ripening in GA₃-treated and pollinated fruits. Our studies demonstrate that blueberry fruit development does not appear to be limited by sucrose metabolizing enzyme activity and/or the ability to accumulate sugars in either GA₃-treated or pollinated fruits.