欧李果实发育期糖和酸组分及其含量的动态变化特性

以农大3号、农大4号、农大5号3个欧李品种为材料,测定果实发育过程中各组分糖、酸及总糖、总酸的含量,以明确欧李果实糖酸积累的动态变化特性.结果显示:(1)3个欧李品种果实成熟期糖含量、酸含量及糖酸比存在明显差异,其中农大3号品种的总糖含量最高,总酸含量最低,糖酸比值最高.(2)成熟期3品种各糖组分中均以果糖含量较高,葡萄糖和蔗糖含量较低,山梨醇含量微量;酸组分中均以苹果酸为主,柠檬酸少量.(3)3个品种果糖、蔗糖、葡萄糖、山梨醇含量在整个果实发育期均呈持续增加态势,并以果糖积累为主;农大3号果糖含量在8周后增幅明显高于另2个品种,且一直保持到果实成熟;3个品种蔗糖含量的变化趋势相近,在前期和中期增加缓慢,接近成熟的2～3周则迅速增加并占整个发育期积累量的70%以上.(4)3个品种酸含量的变化趋势较为相似,苹果酸和柠檬酸在果实发育的前期和中期含量均较低,在果实发育后期迅速增加,但接近果实成熟时又大幅下降.研究表明,果糖与苹果酸的含量及其动态变化是影响欧李果实糖酸比、决定果实风味的主要因素.     

Regulation of climacteric respiration in ripening avocado fruit

Ripening of avocado fruit is associated with a dramatic increase in respiration. In vivo³¹P nuclear magnetic resonance spectroscopy revealed large increases in ATP levels accompanying the increase in respiration. Both glycolytic enzymes, phosphofructokinase, and pyrophosphate: fructose-6-phosphate phosphotransferase were present in avocado fruit with the latter activity being highly stimulated by fructose 2,6-bisphosphate. Fructose 2,6-bisphosphate levels increased approximately 90% at the onset of ripening, suggesting that the respiratory increase in ripening avocado fruit may be regulated by the activation of pyrophosphate:fructose-6-phosphate phosphotransferase by an increase in fructose 2,6-bisphosphate.     

Polyamine metabolism in ripening tomato fruit : I. Identification of metabolites of putrescine and spermidine

The metabolism of [1,4-¹⁴C]putrescine and [terminal methylene-³H]spermidine was studied in the fruit pericarp (breaker stage) discs of tomato (Lycopersicon esculentum Mill.) cv Rutgers, and the metabolites identified by high performance liquid chromatography and gas chromatography-mass spectrometry. The metabolism of both putrescine and spermidine was relatively slow; in 24 hours about 25% of each amine was metabolized. The ¹⁴C label from putrescine was incorporated into spermidine, γ-aminobutyric acid (GABA), glutamic acid, and a polar fraction eluting with sugars and organic acids. In the presence of gabaculine, a specific inhibitor of GABA:pyruvate transaminase, the label going into glutamic acid, sugars and organic acids decreased by 80% while that in GABA increased about twofold, indicating that the transamination reaction is probably a major fate of GABA produced from putrescine in vivo. [³H]Spermidine was catabolized into putrescine and β-alanine. The conversion of putrescine into GABA, and that of spermidine into putrescine, suggests the presence of polyamine oxidizing enzymes in tomato pericarp tissues. The possible pathways of putrescine and spermidine metabolism are discussed.     

Upregulation of pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) activity in strawberry

Pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) is a cytosolic enzyme catalyzing the first committed step in glycolysis by reversibly phosphorylating fructose-6-phosphate to fructose-1,6-bisphosphate. The position of PFP in glycolytic and gluconeogenic metabolism, as well as activity patterns in ripening strawberry, suggest that the enzyme may influence carbohydrate allocation to sugars and organic acids. Fructose-2,6-bisphosphate activates and tightly regulates PFP activity in plants and has hampered attempts to increase PFP activity through overexpression. Heterologous expression of a homodimeric isoform from Giardia lamblia, not regulated by fructose-2,6-bisphosphate, was therefore employed to ensure in vivo increases in PFP activity. The coding sequence was placed into a constitutive expression cassette under control of the cauliflower mosaic virus 35S promoter and introduced into strawberry by Agrobacterium tumefaciens-mediated transformation. Heterologous expression of PFP resulted in an up to eightfold increase in total activity in ripe berries collected over two consecutive growing seasons. Total sugar and organic acid content of transgenic berries harvested during the first season were not affected when compared to the wild type, however, fructose content increased at the expense of sucrose. In the second season, total sugar content and composition remained unchanged while the citrate content increased slightly. Considering that PFP catalyses a reversible reaction, PFP activity appears to shift between gluconeogenic and glycolytic metabolism, depending on the metabolic status of the cell.     

Composition of ethanol-insoluble polysaccharides in water extracts of ripening tomatoes

The carbohydrate composition of the 80% ethanol-insoluble polysaccharides (EIP) from water extracts of ‘Rutgers,’ rin (ripening inhibitor) and nor (non-ripening) tomatoes has been determined. The amount of EIP extracted from ‘Rutgers’ fruit increased from 0.34 to 0.61 mg/g fr. wt during ripening little change occurred in rin or nor fruit. The carbohydrate composition (μg/g fr. wt) of EIP from mature green fruit was: galacturonic acid (48); rhamnose (3); arabinose (20); xylose (48); mannose (31); glucose (139); galactose (51). The most obvious changes that accompanied ripening were a 7.4-fold and 4-fold increase in galacturonic acid and rhamnose content, respectively. These changes were attenuated in the ripening mutants. EIP was fractionated into three major peaks by using DEAE-cellulose ion exchange chromatography. The first peak, which was not retained by the column, contained predominantly glucose and mannose, with lower amounts of galacturonic acid and galactose. The two retained peaks which eluted at 0.1 and 0.2 M sodium chloride contained primarily galacturonic acid, xylose, galactose and arabinose. The galacturonic acid content of these two fractions increased substantially during ripening, whereas the other components decreased. No changes were evident in the ripening mutants. No increase in water-soluble polysaccharides high in galactose content was observed during ripening.     

Stimulation of ethylene production by a cell wall component from mature green tomato fruit

Pectic (carbonate-soluble, covalently-bound pectin, CBP) material stimulated increased ethylene production when vacuum-infiltrated into whole, mature green tomato ( Lycopersicon esculentum Mill. cv. Rutgers) fruit. Activity was greatest if CBP was extracted from mature green tomatoes with jellied locules. CBP extracted from mature green tomatoes with immature seeds had no elicitor activity, while CBP from turning or red ripe tomatoes was only moderately active. Infiltration of CBP from normal mature green fruit into ripening inhibitor ( rin ) mutant tomato fruit stimulated ethylene production and attenuated red pigmentation in these fruits. Partial purification of the active material was accomplished using DEAE-Sephadex and BioGel P-100 chromatography. The most highly purified fraction is comprised of neutral carbohydrate (95%) with a relatively low content of amino acids (1%) and a uronic acid content of less than 5%. This material may be an endogenous trigger of ethylene production and ripening.     

Metabolism of sugars and organic acids in immature grape berries

Individual intact excised immature Sultana berries were supplied through the cut pedicel with ¹⁴C-sugars and organic acids. When ¹⁴C-hexoses were supplied malic and tartaric acids accounted for 25% and 10% of the total activity extracted after 24 hours, and sucrose was synthesized. It is proposed that the changes in the levels of organic acids during ripening are related to changes in the ability of the berry to synthesize them. Although administration of uniformly labeled sucrose resulted in the unequal labeling of glucose and fructose, the results indicate breakdown of sucrose by invertase. It is suggested that the route of entry of the pedicel-fed sugars into the berry may be different from the route taken by sugar translocated from the leaf.     

Effect of Sodium Chloride on Fruit Ripening of the Nonripening Tomato Mutants nor and rin

Tomato (Lycopersicon esculentum Mill) plants of the nonripening mutant nor, the ripening-inhibited mutant rin, and the normal cultivar `Rutgers' were grown in nutrient solution supplemented with 3 grams per liter NaCl from the time of anthesis. In plants treated with NaCl, all the ripening parameters of the fruits of the nor mutant increased, but those of the rin mutant did not. The ripening of the fruits of the NaCl-treated nor plants was characterized by the development of a red color and taste, increased pectolytic activity, and increased evolution of CO₂ and ethylene. These changes do not normally take place in nor under control conditions. The values of these ripening parameters in nor were lower than those of the normal Rutgers fruits. In addition, both in nor and rin and in the normal variety, exposure of the plants to NaCl shortened the developmental period of the fruit, decreased the fruit size, and increased the concentrations of total soluble solids, Na⁺, Cl⁻, reducing sugars, and titratable acids in the fruit. The role of NaCl in overcoming the inability of nor to ripen is discussed.     

Carbohydrate Metabolism and Activity of Pyrophosphate: Fructose-6-Phosphate Phosphotransferase in Photosynthetic Soybean (Glycine max, Merr.) Suspension Cells

Activity of pyrophosphate:fructose-6-phosphate phosphotransferase (PFP) was investigated in relation to carbohydrate metabolism and physiological growth stage in mixotrophic soybean (Glycine max Merr.) suspension cells. In the presence of exogenous sugars, log phase growth occurred and the cells displayed mixotrophic metabolism. During this stage, photosynthetic oxygen evolution was depressed and sugars were assimilated from the medium. Upon depletion of medium sugar, oxygen evolution and chlorophyll content increased, and cells entered stationary phase. Activities of various enzymes of glycolysis and sucrose metabolism, including PFP, sucrose synthase, fructokinase, glucokinase, UDP-glucose pyrophosphorylase, and fructose-1,6-bisphosphatase, changed as the cells went from log to stationary phases of growth. The largest change occurred in the activity of PFP, which was three-fold higher in log phase cells. PFP activity increased in cells grown on media initially containing sucrose, glucose, or fructose and began to decline when sugar in the medium was depleted. Western blots probed with antibody specific to the -subunit of potato PFP revealed a single 56 kilodalton immunoreactive band that changed in intensity during the growth cycle in association with changes in total PFP activity. The level of fructose-2,6-bisphosphate, an activator of the soybean PFP, increased during the first 24 hours after cell transfer and returned to the stationary phase level prior to the increase in PFP activity. Throughout the growth cycle, the calculated in vivo cytosolic concentration of fructose-2,6-bisphosphate exceeded by more than two orders of magnitude the previously reported activation coefficient (K_a) for soybean PFP. These results indicate that metabolism of exogenously supplied sugars by these cells involves a PFP-dependent step that is not coupled directly to sucrose utilization. Activity of this pathway appears to be controlled by changes in the level of PFP, rather than changes in the total cytosolic level of fructose-2,6-bisphosphate.     

Utilization of exogenously supplied 14C-saccharose into primary metabolites and alkaloid production in Catharanthus roseus

Partitioning of exogenously supplied U-¹⁴C-saccharose into primary metabolic pool as sugars, amino acids, and organic acids was analyzed and simultaneous utilization for production of alkaloid by leaf, stem, and root in twigs and rooted plants of Catharanthus roseus grown in hydroponic culture medium was determined. Twigs revealed comparable distribution of total ¹⁴C label in leaf and stem. Stems contained significantly higher ¹⁴C label in sugar fraction and in alkaloids [47 kBq kg⁻¹(DM)] than leaf. In rooted plants, label in ¹⁴C in metabolic fractions in root such as ethanol-soluble, ethanol-insoluble, and chloroform-soluble fractions and in components such as sugars, amino acids, and organic acids were significantly higher than in stems and leaves. This was related with significantly higher content of ¹⁴C in alkaloids in stems and leaves. ¹⁴C contents in sugars, amino acids, and organic acids increased from leaf to stem and roots. Roots are the major accumulators of metabolites accompanied by higher biosynthetic utilization for alkaloid accumulation.     

Fluctuations in sugar content are not determinant in explaining variations in vitamin C in tomato fruit

The present study aimed to clarify the relationship between sugars and vitamin C in fruit. The objective was to determine whether vitamin C content was regulated by sugar content due to the role of sugar as a precursor for vitamin C. During summer, maximal content in sugar and vitamin C were found in both genotypes tested Solanum lycopersicon ‘Cervil’ and ‘Levovil’. During autumn, fruit pruning increased fruit size and hexose content but fruit vitamin C content did not increase. Therefore sugar substrate was not limiting for vitamin C synthesis during autumn. We demonstrated for two cultivars, ‘Cervil’ and ‘Levovil’, with different sugar accumulation profiles during ripening, that sugar content was not determinant in the regulation of vitamin C content. The strong correlation observed between sugars and vitamin C in ‘Cervil’ was due to their concomitant increase during fruit ripening.     

Regulation of tomato fruit ascorbate content is more highly dependent on fruit irradiance than leaf irradiance

Background and Aims

The mechanisms involving light control of vitamin C content in fruits are not yet fully understood. The present study aimed to evaluate the impact of fruit and leaf shading on ascorbate (AsA) accumulation in tomato fruit and to determine how fruit sugar content (as an AsA precursor) affected AsA content.

Methods

Cherry tomato plants were grown in a glasshouse. The control treatment (normally irradiated fruits and irradiated leaves) was compared with the whole-plant shading treatment and with leaf or fruit shading treatments in fruits harvested at breaker stage. In a second experiment, the correlation between sugars and AsA was studied during ripening.

Key Results

Fruit shading was the most effective treatment in reducing fruit AsA content. Under normal conditions, AsA and sugar content were correlated and increased with the ripening stage. Reducing fruit irradiance strongly decreased the reduced AsA content (−74 %), without affecting sugars, so that sugar and reduced AsA were no longer correlated. Leaf shading delayed fruit ripening: it increased the accumulation of oxidized AsA in green fruits (+98 %), whereas it decreased the reduced AsA content in orange fruits (−19 %), suggesting that fruit AsA metabolism also depends on leaf irradiance.

Conclusions

Under fruit shading only, the absence of a correlation between sugars and reduced AsA content indicated that fruit AsA content was not limited by leaf photosynthesis or sugar substrate, but strongly depended on fruit irradiance. Leaf shading most probably affected fruit AsA content by delaying fruit ripening, and suggested a complex regulation of AsA metabolism which depends on both fruit and leaf irradiance and fruit ripening stage.Key words: Ascorbate, fruit quality, irradiance, shading, Solanum lycopersicon, sugars, tomato, vitamin C     

Water Permeability during Tomato Fruit Development in Normal and rin Nonripening Mutant

This work tested one aspect of the relations between membrane permeability and fruit ripening. Membrane permeability was measured as [³H]water efflux rate from preloaded fruit pericarp disks. Different stages of fruit development were compared between two tomato (Lycopersicon esculentum Mill) strains: the normal Rutgers and the isogenic nonripening rin strain. The first significant increase in permeability was measured in Rutgers tissue at 110% of development, after fruit ripening had already begun as indicated by ethylene and CO₂ evolution and lycopene synthesis. The rin did not show any increase in tissue permeability during fruit development or maturation.     

Synthesis of amide and sulfonamide substituted N-aryl 6-aminoquinoxalines as PFKFB3 inhibitors with improved physicochemical properties

In oncology, the “Warburg effect” describes the elevated production of energy by glycolysis in cancer cells. The ubiquitous and hypoxia-induced 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) plays a noteworthy role in the regulation of glycolysis by producing fructose-2,6-biphosphate (F-2,6-BP), a potent activator of the glycolysis rate-limiting phosphofructokinase PFK-1. Series of amides and sulfonamides derivatives based on a N-aryl 6-aminoquinoxaline scaffold were synthesized and tested for their inhibition of PFKFB3 in vitro in a biochemical assay as well as in HCT116 cells. The carboxamide series displayed satisfactory kinetic solubility and metabolic stability, and within this class, potent lead compounds with low nanomolar activity have been identified with a suitable profile for further in vivo evaluation.     

Banana ripening: implications of changes in glycolytic intermediate concentrations, glycolytic and gluconeogenic carbon flux, and fructose 2,6-bisphosphate concentration

In ripening banana (Musa sp. [AAA group, Cavendish subgroup] cv Valery) fruit, the concentration of glycolytic intermediates increased in response to the rapid conversion of starch to sugars and CO₂. Glucose 6-phosphate (G-6-P), fructose 6-phosphate (Fru 6-P), and pyruvate (Pyr) levels changed in synchrony, increasing to a maximum one day past the peak in ethylene synthesis and declining rapidly thereafter. Fructose 1,6-bisphosphate (Fru 1,6-P₂) and phosphoenolpyruvate (PEP) levels underwent changes dissimilar to those of G 6-P, Fru 6-P, and Pyr, indicating that carbon was regulated at the PEP/Pyr and Fru 6-P/Fru 1,6-P₂ interconversion sites. During the climacteric respiratory rise, gluconeogenic carbon flux increased 50- to 100-fold while glycolytic carbon flux increased only 4- to 5-fold. After the climacteric peak in CO₂ production, gluconeogenic carbon flux dropped dramatically while glycolytic carbon flux remained elevated. The steady-state fructose 2,6-bisphosphate (Fru 2,6-P₂) concentration decreased to ½ that of preclimacteric fruit during the period coinciding with the rapid increase in gluconeogenesis. Fru 2,6-P₂ concentration increased thereafter as glycolytic carbon flux increased relative to gluconeogenic carbon flux. It appears likely that the initial increase in respiration in ripening banana fruit is due to the rapid influx of carbon into the cytosol as starch is degraded. As starch reserves are depleted and the levels of intermediates decline, the continued enhancement of respiration may, in part, be maintained by an increased steady-state Fru 2,6-P₂ concentration acting to promote glycolytic carbon flux at the step responsible for the interconversion of Fru 6-P and Fru 1,6-P₂.     

Polyamine Metabolism in Ripening Tomato Fruit : II. Polyamine Metabolism and Synthesis in Relation to Enhanced Putrescine Content and Storage Life of a/c Tomato Fruit

The fruit of the Alcobaca landrace of tomato (Lycopersicon esculentum Mill.) have prolonged keeping qualities (determined by the allele a/c) and contain three times as much putrescine as the standard Rutgers variety (A/c) at the ripe stage (ARG Dibble, PJ Davies, MA Mutschler [1988] Plant Physiol 86: 338-340). Polyamine metabolism and biosynthesis were compared in fruit from Rutgers and Rutgers-a/c—a near isogenic line possessing the allele a/c, at four different stages of ripening. The levels of soluble polyamine conjugates as well as wall bound polyamines in the pericarp tissue and jelly were very low or nondetectable in both genotypes. The increase in putrescine content in a/c pericarp is not related to normal ripening as it occurred with time and whether or not the fruit ripened. Pericarp discs of both normal and a/c fruit showed a decrease in the metabolism of [1,4-¹⁴C]putrescine and [terminal labeled-³H]spermidine with ripening, but there were no significant differences between the two genotypes. The activity of ornithine decarboxylase was similar in the fruit pericarp of the two lines. Arginine decarboxylase activity decreased during ripening in Rutgers but decreased and rose again in Rutgers-a/c fruit, and as a result it was significantly higher in a/c fruit than in the normal fruit at the ripe stage. The elevated putrescine levels in a/c fruit appear, therefore, to be due to an increase in the activity of arginine decarboxylase.     

Cell wall metabolism in ripening fruit. VIII. Cell wall composition and synthetic capacity of two regions of the outer pericarp of mature green and red ripe cv. Jackpot tomatoes

Pericarp discs were excised from mature green and red ripe tomato (Lycopersicon esculentum Mill. cv. Jackpot) fruit and kept in sterile tissue culture plates for 4 days, including 2 days of incubation with D-[U-¹³C]-glucose. Cell walls were prepared and differentially extracted with dimethylsulfoxide (DMSO), trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CDTA). Na₂CO₃, 4 M KOH and 8 M KOH. Cell wall noncellulosic neutral sugar (NS) composition and cell wall synthetic capacity (i.e. incorporation of density label into cell wall sugars) were determined by using a gas chromatograph coupled to a flame ionization detector and a mass spectrometer, respectively. In the crude cell wall, there was significantly less galactose (Gal) and glucose (Glc) in the “outer”2-mm pericarp region, including the cuticle, compared to the “inner”2-mm region immediately below it (closer to the locules). In the CDTA-soluble pectin, rhamnose (Rha), arabinose (Ara) and Gal accounted for approximately 90% of the total NS. The ratios of these sugars were very similar in the total (¹²C plus ¹³C) sugars, and also in the newly synthesized ([¹³C]-labeled) sugars, suggesting that newly synthesized NS associated with the chelator-extractable pectic fraction has a composition very similar to that of preexisting NS. In the 4 M KOH-soluble material, xylose (Xyl) and Glc accounted for approximately 70% of the total NS. The ratio of these sugars was very similar in the total sugars, but much lower in the newly synthesized portion. This suggests that the hemicellulosic polymers synthesized during the ripening process are different in type and/or proportion from those present in the developing fruit. Because the outer pericarp of tomatoes contains at least two distinct tissue types and these have a distinct cell wall composition, analysis of tomato cell wall polysaccharide composition by homogenization of the entire outer pericarp will obscure subtle changes associated with ripening/softening within specific tissue types.     

Dark fixation of CO2 during gluconeogenesis by the cotyledons of Cucurbita pepo L.

We did this work to discover the pathway of CO₂ fixation into sugars in the dark during gluconeogenesis by the cotyledons of 5-day-old seedlings of Cucurbita pepo L. We paid particular attention to the possibility of a contribution from ribulosebisphosphate carboxylase. The detailed distribution of ¹⁴C after exposure of excised cotyledons to ¹⁴CO₂ in the dark was determined in a series of pulse and chase experiments. After 4s in ¹⁴CO₂, 89% of the ¹⁴C fixed was in malate and aspartate. In longer exposures, and in chases in ¹²CO₂, label appeared in alanine, phosphoenolpyruvate, 3-phosphoglycerate and sugar phosphates, and accumulated in sugars. The transfer of label from C-4 acids to sugars was restricted by inhibition of phosphoenolpyruvate carboxykinase in vivo by 3-mercaptopicolinic acid. We conclude as follows. Initial fixation of CO₂ in the dark is almost entirely into phosphoenolpyruvate, probably via phosphoenolpyruvate carboxylase (EC 4.1.1.31) which we showed to be present in appreciable amounts. Incorporation into sugars occurs chiefly, if not completely, as a result of randomization of the carboxyl groups of the C-4 acids and subsequent conversion of the oxaloacetate to sugars via the accepted sequence for gluconeogenesis. Ribulosebisphosphate carboxylase appears to make very little contribution to sugar synthesis from fat.     

Subcellular Distributions of Isoenzymes in Fruits of a Normal Cultivar of Tomato and of the rin Mutant at Two Stages of Development

Fruits of tomato (Lycopersicon esculentum Mill.) cv. Rutgers and of a nearly isogenic stock containing the ripening inhibitor gene rin harvested at green (66% mature) and ripe (107% mature) stages were studied for the subcellular distribution of isoenzymes using isoelectric focusing. The enzymes studied were peroxidases, esterases, phosphatases, phosphorylase, malate dehydrogenases, and IAA oxidases. During ripening of normal fruit the activities in the supernatant fraction of all of these enzymes, except malate dehydrogenase, decreased. In the particulate fractions some enzymes decreased while others increased in activity. The rin gene inhibited only some of the changes which occurred during ripening of normal fruit. It is postulated that changes in the degree to which enzymes are bound to membranes comprise one of the mechanisms by which the activities of enzymes are controlled in tomato pericarp, and that these membranes remain intact during ripening.