Activity of sucrose hydrolysing enzymes and sugar accumulation during tomato fruit development

Relationships between the assimilate import rate and the activity of acid invertase and/or sucrose synthase have been investigated in the pericarp, locule and placenta of tomato fruit during development to establish the possible role of sucrose cleavage as the control step for the import of sucrose into these sink tissues. The rate of sucrose cleavage was estimated from the activities of these two enzymes as well as the ratio of hexoses to sucrose (i.e. the sucrose degradation index, SDI) in the tissues of the fruit, based on the assumption that the accumulation of hexoses is the consequence of imported sucrose being degraded by either or both of these two enzymes. The results showed that the change of sucrose synthase activity during fruit development was positively related to both the rate of dry matter accumulation in the fruit tissue and SDI. Although the role of acid invertase in regulating the rate of import during development remains uncertain, the actions of sucrose synthase on sucrose cleavage may regulate the import and compartmentation of sucrose in the early stage of tomato fruit development.     

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.     

Endogenous cytokinin in developing kiwifruit is implicated in maintaining fruit flesh chlorophyll levels

Background and Aims

Green kiwifruit (Actinidia deliciosa) retain high concentrations of chlorophyll in the fruit flesh, whereas in gold-fleshed kiwifruit (A. chinensis) chlorophyll is degraded to colourless catabolites during fruit development, leaving yellow carotenoids visible. The plant hormone group the cytokinins has been implicated in the delay of senescence, and so the aim of this work was to investigate the link between cytokinin levels in ripening fruit and chlorophyll de-greening.

Methods

The expression of genes related to cytokinin metabolism and signal transduction and the concentration of cytokinin metabolites were measured. The regulation of gene expression was assayed using transient activation of the promoter of STAY-GREEN2 (SGR2) by cytokinin response regulators.

Key Results

While the total amount of cytokinin increased in fruit of both species during maturation and ripening, a high level of expression of two cytokinin biosynthetic gene family members, adenylate isopentenyltransferases, was only detected in green kiwifruit fruit during ripening. Additionally, high levels of O-glucosylated cytokinins were detected only in green kiwifruit, as was the expression of the gene for zeatin O-glucosyltransferase, the enzyme responsible for glucosylating cytokinin into a storage form. Season to season variation in gene expression was seen, and some de-greening of the green kiwifruit fruit occurred in the second season, suggesting environmental effects on the chlorophyll degradation pathway. Two cytokinin-related response regulators, RRA17 and RRB120, showed activity against the promoter of kiwifruit SGR2.

Conclusions

The results show that in kiwifruit, levels of cytokinin increase markedly during fruit ripening, and that cytokinin metabolism is differentially regulated in the fruit of the green and gold species. However, the causal factor(s) associated with the maintenance or loss of chlorophyll in kiwifruit during ripening remains obscure.     

网纹甜瓜发育果实糖分积累与蔗糖代谢参与酶的关系

随着网纹甜瓜果实的发育,果实中葡萄糖和果糖的含量增加,蔗糖的快速积累发生在果实发育的中后期,高蔗糖积累型果实中蔗糖积累速率明显快于低蔗糖积累型.蔗糖磷酸合成酶活性在果实发育的前期短暂下降, 而后稳步上升,在果实发育的中后期高蔗糖积累型果实中该酶的活性显著高于低蔗糖积累型果实;随着果实发育,蔗糖合成酶的分解活性降低而合成活性升高.酸性和中性转化酶在未成熟果实中活性较高,而在成熟果实中很低; 高蔗糖积累型果实中酸性转化酶活性显著低于同期低蔗糖积累型果实.合成蔗糖的酶活性小于分解蔗糖的酶活性时蔗糖几乎没有积累.根据这些结果推测,转化酶活性的下降、蔗糖磷酸合成酶活性的增加以及蔗糖合成酶分解活性的下降和合成活性的增加,是引起果实蔗糖积累的主要内在因子.     

Change in susceptibility of satsuma mandarin fruit to sour rot pathogen (Geotrichum candidum citrus race) with relation to biochemical changes during maturation and storage

Change in susceptibility of satsuma mandarin fruit (Citrus unshiu) cultivar “Miyagawawase” to sour rot pathogen was studied with relation to biochemical changes during maturation and storage. The susceptibility of the fruit decreased with the advancement of maturity and was relatively constant during storage at 25°C for 4 wk. The young, green fruit that contained less total soluble solid, sugars and polyphenol, but more citric acid and water contents than mature, yellow fruit was more susceptible to sour rot pathogen. The susceptibility was correlated with total soluble solid, citric acid, sugars and water contents, but not with polyphenol. The results suggested that the difference in susceptibility during maturation was influenced to some extent by several constituents of fruit, although they may not be the only factors involved in susceptibility.     

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.     

Effects of maturity status on biochemical content,polyphenol composition and antioxidant capacity of pomegranate fruit arils (cv. ‘Bhagwa’)

Pomegranate fruit (Punica granatum L.) has gained commercial importance in recent years in the food and health industries due to increasing scientific evidence linking its consumption to better health outcomes. In the present study, the evolution of some chemical contents, individual organic acids and sugars, phenolic composition and antioxidant capacity of pomegranate (cv. ‘Bhagwa’) during maturation was investigated. The results showed significant (P < 0.05) increases in sugar content, ascorbic acid and total anthocyanins during fruit maturation, while significant decreases occurred in titratable acidity (TA), organic acids and total phenolic contents (TPCs). The significant increase in TSS/TA ratio, which plays a significant role in juice flavor, peaked at 140 DAFB, while the highest accumulation of anthocyanin content occurred at the full-ripe stage (165 DAFB). Total antioxidant capacity (both DPPH and FRAP) declined during fruit maturation, suggesting a decrease in antioxidant power of fruit juice. Strong correlations between TPC and antioxidant capacity measured by the DPPH (r² = 0.99) and FRAP (r² = 0.96) methods were observed. Fructose and glucose were established to be the major sugars in the fruit cultivar while tartaric acid was the predominant organic acid. Principal component analysis (PCA) showed that harvest maturity of ‘Bhagwa’ pomegranate fruit is dependent on time from full bloom hence could be fixed around 165 DAFB, where fruits were characterized by intense fruit and aril pigmentation as well as high juice quality parameters. This information provided could help pomegranate juice producers to assess and optimize juice quality and antioxidant value of ‘Bhagwa’ pomegranate cultivar through maturity.     

Cucumber fruit sucrose synthase isozymes

Two forms of sucrose synthase (SSI and SSII) were resolved from cucumber (Cucumis sativus) fruit pericarp and fruit peduncle tissue using DEAE-cell     

Glyoxylate metabolism and fatty acid oxidation in mango fruit during development and ripening

Throughout the development (maturation) of mango fruit the contents of citric and glyoxylic acids increased steadily. As the fruit matured the levels of isocitrate lyase, malate lyase and alanine: glyoxylate aminotransferase increased and reached maximum values prior to the time of harvesting. At and after harvest the levels of malate lyase and alanine : glyoxylate aminotransferase began to decrease but that of isocitrate lyase remained high until after the harvest when it decreased. The level of glyoxylate reductase was highest in the early developmental stage but declined as the fruit matured and ripened. As the fruit ripened, after harvest, the amounts of citric and glyoxylic acids decreased concomitant with a considerable increase in the levels of isocitrate dehydrogenase, malic dehydrogenase, malic enzyme and glyoxylate dehydrogenase.Fatty acid oxidizing capacity of mitochondria isolated from immature (developing) and postclimacteric fruit pulps was much less than that observed with mitochondria from preclimacteric and climacteric fruit. Glyoxylate stimulated the oxidation of caprylic, lauric, myristic and palmitic acids and inhibited the activity of isocitrate dehydrogenase in vitro.     

Biochemical characterisation of MdCXE1, a carboxylesterase from apple that is expressed during fruit ripening

Esters are an important component of apple (Malus × domestica) flavour. Their biosynthesis increases in response to the ripening hormone ethylene, but their metabolism by carboxylesterases (CXEs) is poorly understood. We have identified 16 members of the CXE multigene family from the commercial apple cultivar, ‘Royal Gala’, that contain all the conserved features associated with CXE members of the α/β hydrolase fold superfamily. The expression of two genes, MdCXE1 and MdCXE16 was characterised in an apple fruit development series and in a transgenic line of ‘Royal Gala’ (AO3) that is unable to synthesise ethylene in fruit. In wild-type MdCXE1 is expressed at low levels during early stages of fruit development, rising to a peak of expression in apple fruit at harvest maturity. It is not significantly up-regulated by ethylene in the skin of AO3 fruit. MdCXE16 is expressed constitutively in wild-type throughout fruit development, and is up-regulated by ethylene in skin of AO3 fruit. Semi-purified recombinant MdCXE1 was able to hydrolyse a range of 4-methyl umbelliferyl ester substrates that included those containing acyl moieties that are found in esters produced by apple fruit. Kinetic characterisation of MdCXE1 revealed that the enzyme could be inhibited by organophosphates and that its ability to hydrolyse esters showed increasing affinity (K_m) but decreasing turnover (k_cat) as substrate acyl carbon length increases from C2 to C16. Our results suggest that MdCXE1 may have an impact on apple flavour through its ability to hydrolyse relevant flavour esters in ripe apple fruit.     

Phloem unloading follows an extensive apoplasmic pathway in cucumber (Cucumis sativus L.) fruit from anthesis to marketable maturing stage

The phloem unloading pathway remains unclear in fruits of Cucurbitaceae, a classical stachyose-transporting species with bicollateral phloem. Using a combination of electron microscopy, transport of phloem-mobile symplasmic tracer carboxyfluorescein, assays of acid invertase and sucrose transporter, and [(14)C]sugar uptake, the phloem unloading pathway was studied in cucumber (Cucumis sativus) fruit from anthesis to the marketable maturing stage. Structural investigations showed that the sieve element-companion cell (SE-CC) complex of the vascular bundles feeding fruit flesh is apparently symplasmically restricted. Imaging of carboxyfluorescein unloading showed that the dye remained confined to the phloem strands of the vascular bundles in the whole fruit throughout the stages examined. A 37 kDa acid invertase was located predominantly in the cell walls of SE-CC complexes and parenchyma cells. Studies of [(14)C]sugar uptake suggested that energy-driven transporters may be functional in sugar trans-membrane transport within symplasmically restricted SE-CC complex, which was further confirmed by the existence of a functional plasma membrane sucrose transporter (CsSUT4) in cucumber fruit. These data provide a clear evidence for an apoplasmic phloem unloading pathway in cucumber fruit. A presumption that putative raffinose or stachyose transporters may be involved in soluble sugars unloading was discussed.     

Changes in Sugar Content and Activities of Sucrose Metabolizing Enzymes in Roots and Nodules of Lentil

Activities of acid and alkaline invertases and sucrose synthase were determined in roots and nodules of lentil at various stages of development. Alkaline invertase and sucrose synthase were both involved in sucrose metabolism in the nodule cytosol, but there was only a small amount of acid invertase present. Activity of sucrose metabolizing enzymes in roots was significantly less than that observed in the nodules. Amongst sugars, sucrose was found to be the main component in the host cytosol. Lentil neutral invertase (LNI) was partially purified from nodules at 50 days after sowing (DAS). Two forms of invertase were identified, i.e., a major form of 71 kDa which was taken for enzyme characterization and a minor form of 270 kDa which was not used for further studies. The purified enzyme exhibited typical hyperbolic saturation kinetics for sucrose hydrolysis. It had a Km of 11.0 to 14.0 mM for sucrose depending upon the temperature, a pH optimum of 6.8 and an optimum temperature of 40 °C. Compared with raffinose and stachyose, sucrose was better substrate for LNI. The enzyme showed no significant hydrolysis of maltose and p-nitrophenyl--D-glucopyranoside, showing its true -fructosidase nature. LNI is completely inhibited by HgCl₂, MnCl₂ and iodoacetamide but not by CaCl₂, MgCl₂ or BaCl₂.     

Sugar regulation of plastid interconversions in epicarp of citrus fruit

Seasonal transformations between chloroplasts and chromoplasts, as measured by changes in chlorophyll content, in the epicarp of degreening and regreening Citrus sinensis (L.) Osbeck cv Valencia fruit closely parallelled the accumulation and later loss of soluble sugars. At any stage of development, reversing the relative soluble sugar content in the epicarp by culturing pericarp segments on agar media with low (15 millimolar) or high (150 millimolar) sucrose concentrations reversed the direction of change in chlorophyll content. Fruit of C. madurensis Lour., which mature year around and do not regreen, also accumulated soluble sugars in the pericarp as degreening was initiated.

The epicarp of C. sinensis fruit accumulated nitrogen, but total nitrogen concentrations and amino acid concentrations changed little, during degreening and regreening of C. sinensis fruit. Cessation of nitrogen fertilization reduced the tendency of pericarp segments to regreen in vitro during subsequent years, but regreening tendency was restored by inclusion of KNO₃ in the media.

It is concluded that chloroplasts become chromoplasts and citrus fruit degreen partially in response to the accumulation of sugars in the epicarp and that the reverse transformation accompanying regreening of certain citrus species occurs when accumulated sugars disappear. Change in nitrogen flux to the fruit is probably not a factor in regulating seasonal transformations, but an abundance of nitrogen in the epicarp diminishes the effects of high sugar concentrations in inducing transformation of chloroplasts to chromoplasts, thereby retarding degreening and promoting regreening.

     

Post-translational inhibitory regulation of acid invertase induced by fructose and glucose in developing apple fruit

Acid invertase (EC 3.2.1.26) is one of the key enzymes involved in the carbohydrate sinkorgan development and the sink strength modulation in crops. The experiment conducted with ‘Starkrimson’ apple (Malus domestica Borkh) fruit showed that, during the fruit development, the activity of acid invertase gradually declined concomitantly with the progressive accumulation of fructose, glucose and sucrose, while Western blotting assay of acid invertase detected a 30 ku peptide of which the immuno-signal intensity increased during the fruit development. The immunolocalization via immunogold electron microscopy showed that, on the one hand, acid invertase was mainly located on the flesh cell wall with numbers of the immunosignals present in the vacuole at the late stage of fruit development; and on the other hand, the amount of acid invertase increased during fruit development, which was consistent with the results of Western blotting. The in vivo preincubation of fruit discs with soluble sugars showed that the activity of extractible acid invertase was inhibited by fructose or glucose, while Western blotting did not detect any changes in apparent quantity of the enzyme nor other peptides than 30 ku one. So it is considered that fructose and glucose induced the post-translational or translocational inhibitory regulation of acid invertase in developing apple fruit. The mechanism of the post-translational inhibition was shown different from both the two previously reported ones that proposed either the inhibition by hexose products in the in vitro chemical reaction equilibrium system or the inhibition by the proteinaceous inhibitors. It was hypothesized that fructose and glucose might induce acid invertase inhibition by modulating the expression of some inhibition-related genes or some structural modification of acid invertase.     

Quantification of ABA and its metabolites in sweet cherries usingdeuterium-labeled internal standards

Abscisic acid (ABA), phaseic acid (PA), dihydrophaseic acid (DPA), and epi-dihydrophaseic acid (epi-DPA) were quantified in developing fruit and seeds of sweet cherry using each deuterium-labeled internal standard. ABA concentrations in the pulp were low at the early stage of fruit development, reached to the maximum before maturation, and subsequently declined during maturation. The significant increase of ABA after 29 days after full bloom (DAFB) coincides with the softening suggests that ABA may play a role to induce fruit maturation in sweet cherries. ABA metabolite levels were high at the immature stage and decreased with fruit maturation. This fact suggests that fruit may not need ABA in the early stage of fruit development. It was considered that DPA may be the major metabolite of ABA since the concentrations were higher than PA and epi-DPA at all stages of fruit development. ABA concentrations increased at the beginning of seed maturation and then decreased toward harvest. This decrease may be necessary to end seed dormancy. DPA in seeds changed similarly with ABA but its concentrations were always higher than those of ABA.     

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.     

Carbohydrate metabolism during fruit development in sweet pepper (Capsicum annuum) plants

Growth, accumulation of sugars and starch, and the activity of enzymes involved in sucrose mobilization were determined throughout the development of sweet pepper fruits. Fruit development was roughly divided into three phases: (1) an initial phase with high relative growth rate and hexose accumulation, (2) a phase with declining growth rate and accumulation of sucrose and starch, and (3) a ripening phase with no further fresh weight increase and with accumulation of hexoses, while sucrose and starch were degraded. Acid and neutral invertase (EC 3.2.1.26) were closely correlated to relative growth rate until ripening and inversly correlated to the accumulation of sucrose. Acid invertase specifically increased during ripening, concurrently with the accumulation of hexoses. Sucrose synthase (EC 2.4.1.13) showed little correlation to fruit development, and in periods of rapid growth the activity of sucrose synthase was low compared to the invertases. However, during late fruit growth sucose synthase was more active than the invertases. We conclude that invertase activities determine the accumulation of assimilates in the very young fruits, and a reactivation of acid invertase is responsible for the accumulation of hexoses during ripening. During late fruit growth, before ripening, sucrose synthase is transiently responsible for the sucrose breakdown in the fruit tissue. Results also indicate that pyrophosphate-dependent phosphofructokinase (EC 2.7.1.90) and its activator fructose-2,6-bisphosphate (Fru2,6bisP) are involved in the regulation of the sink metabolism of the fruit tissue.