Three soluble invertase isoforms from Lilium longiflorum flower buds that had been separated by DEAE-Sephacel chromatography were purified to near homogeneity by further chromatography on hydroxylapetite, Con-A sepharose, phenyl agarose, and Sephacryl S-200 gel filtration. Nondenaturing polyacrylamide gel electrophoresis (PAGE) gave a single band in all three invertases that corresponded to a band of invertase activity in a duplicate gel. The SDS-PAGE of the purified invertase I resulted in a single band with apparent relative molecular mass of 78 kDa. Invertase II and III were resolved to a similar polypeptide pattern by SDS-PAGE with three bands of 54, 52, and 24 kDa. Antiserum of tomato acid invertase cross-reacted with all three invertase protein bands. Antiserum of wheat coleoptile acid invertase cross-reacted only with 54 and 52 kDa bands of invertase II and III but did not recognize invertase I protein. Con-A peroxidase was bound to invertase I protein and all three protein bands of invertase II and III, suggesting that all proteins were glycosylated. Invertase I protein could be completely deglycosylated by incubating with peptide-N-glycosidase F to result in a peptide of 75 kDa. Invertase II and III were partially deglycosylated by peptide-N-glycosidase F resulting proteins bands of 53, 51, 50, and 22 kDa.
Anil P. Ranwala and William B. Miller
Takaya Moriguchi, Tetsuro Sanada, and Shohei Yamaki
Sugar levels and composition were determined in developing `Hakuto' peach (Prunus persica Batsch var. vulgaris Maxim.) fruit. Glucose and fructose in nearly equal amounts were the predominant sugars detected during the early stage of development. Sucrose subsequently began to accumulate and was the predominant sugar in mature fruit. Sorbitol remained at a low level throughout development. The large increase in the amount of sucrose was accompanied by a rapid increase in sucrose synthase (EC 18.104.22.168) activity. Sucrose phosphate synthase (EC 22.214.171.124) was also detected in flesh extracts, but the activities were low throughout development. Acid invertase (EC 126.96.36.199) activity was highest in young fruit and declined with development. Activity, however, increased again at a later stage of development. Peach fruit contained appreciable sorbitol oxidase activity, while other sorbitol-related enzymes were barely detectable, suggesting that transported sorbitol was predominantly converted to glucose. These results suggest that the supply of glucose and fructose depends on acid invertase and sorbitol oxidase, and that accumulation of sucrose depends on-sucrose synthase.
Md. Shahidul Islam, S. Khan, and T. Matsui
Sucrose metabolism was followed in developing fruit of domesticated cherry tomato (Lycopersicon esculentum var. cerasiforme Alef.). The high amounts of reducing sugars were consistently linked to high soluble acid invertase (EC 188.8.131.52), whereas sucrose synthase (EC 184.108.40.206) followed the same pattern of sucrose levels and reached a peak of activity during early stage of maturation and then decreased to near nil. In comparison, sucrose phosphate synthase (EC 220.127.116.11) activity remain relatively constant throughout development. Thus, sucrose synthase and acid invertase, rather than sucrose phosphate synthase, are the critical enzymes regulating sucrose accumulation in tomatoes. Cultivated cherry tomato sucrose synthase (UDP-glucose: D-fructose 2-glucosyltransferase) was purified to homogeneity by ammonium sulfate precipitation, anion exchange chromatography on DEAE-Toyopreal 650, and gel filtration on Sephadex G-200. Further purification to homogeneity resulted from a single band from SDS-PAGE. The enzyme was identified as a homotetramer with a total molecular mass of 370 kDa and subunits of 92 kDa. The enzyme showed maximum activity for the cleavage and synthesis of sucrose was at pH 7.0 and 8.0, respectively, and the optimum temperature was 40°C in both directions for HEPES-KOH buffer. The enzymatic reaction followed typical Michaelis–Menten kinetics, with the following parameters: Km (fructose),7.4; Km (UDP-glucose), 0.2612; Km (sucrose), 33.24; Km (UDP), 0.0946. The enzyme was very sensitive to inhibition by heavy metals.
Lili Zhou and Robert E. Paull
This study examined the relationship between the activity of fruit enzymes involved in metabolizing sucrose and sugar accumulation during fruit development, to clarify the role of these key enzymes in sugar accumulation in papaya fruit. Papaya fruit (Carica papaya L. cv. Sunset) were harvested from 14 to 140 days after anthesis (DAA). Fruit dry matter persent, total soluble solids (TSS), and sugar composition and the activity of enzymes: sucrose phosphate synthetase (SPS), sucrose synthetase (SS), and acid invertase were measured. `Sunset' papaya matured 140 days after anthesis during the Hawaii summer season and in about 180 days in cool season on the same plant. Fruit flesh dry matter persent, TSS, and total sugar did not significantly increase until 30 days before harvest. Sucrose synthetase was very high 2 weeks post-anthesis, then decreased to less than one-third in 42 to 56 DAA, then remained relatively low during the rest of fruit development. Seven to 14 days before fruit maturation, SS increased about 30% at the same time as sucrose accumulation in the fruit. Acid invertase activity was very low in the young fruit and increased more than 10-fold 42 to 14 days before maturation. SPS activity remained very low throughout the fruit development and was about 40% higher in mature-green fruit. The potential roles of invertase and sucrose synthetase in sugar accumulation will be discussed.
Lili Zhou and Robert E. Paull
Papaya (Carica papaya L.) fruit flesh and seed growth, fruit respiration, sugar accumulation, and the activities of sucrose phosphate synthase (SPS), sucrose synthase (SS), and acid invertase (AI) were determined from anthesis for ≈150 days after anthesis (DAA), the full ripe stage. Sugar began to accumulate in the fruit flesh between 100 and 140 DAA, after seed maturation had occurred. SPS activity remained low throughout fruit development. The activity of SS was high 14 DAA and decreased to less than one-fourth within 56 DAA, then remained constant during the remainder of fruit development. AI activity was low in young fruit and began to increase 90 DAA and reached a peak more than 10-fold higher, 125 DAA, as sugar accumulated in the flesh. Results suggest that SS and AI are two major enzymes that may determine papaya fruit sink strength in the early and late fruit development phases, respectively. AI activity paralleled sugar accumulation and may be involved in phloem sugar unloading.
Md. Shahidul Islam, S. Khan, and T. Matsui
Seasonal fluctuations of carbohydrate levels and compositions and the activities of related enzymes of three cultivated tomato (Lycopersicon esculentum Mill. cv. Lady First, Momotaro, and Minicarol) cultivars were examined at 45-days interval with seven different sowing in the relatively warm climate of Japan. Fruits picked on early winter to spring seasons had higher sugar concentrations compared to hot season. Fructose and glucose in nearly equal amounts were the predominant sugar in all the seasons. Sucrose was present in trace quantities, but cherry cultivar Minicarol accumulated higher levels than the other two large-fruited types.
Acid invertase (EC 18.104.22.168) was highest at red stage during December to April, while fruit matured during May to August had lowest activity. The activity levels of soluble invertase were predominant compared to cell wall-bound fraction. The sucrose synthase (EC 22.214.171.124) showed highest activity in rapidly growing fruits followed by a very low activity with fruit maturation. Sucrose synthase showed the higher activity during November to February, and almost low activity during all the experimental periods. The sucrose phosphate synthase (EC 126.96.36.199) also showed higher activity during October to February, but the activity levels did not change drastically throughout the fruit development. The results substantiate the conclusion that, in all the planting seasons, acid invertase is a principal enzyme in the process of tomato fruit ripening and during early stage of tomato fruit development, sucrose synthase is the dominant enzyme, which, in turn, plays a part in regulating the translocation of sucrose into the fruit.
Riccardo Lo Bianco, Brunella Morandi, and Mark Rieger
Along with sucrose, sorbitol represents the major photosynthetic product and the main form of translocated carbon in peach. The objective of the present study was to determine whether in peach fruit, sorbitol and sucrose enzyme activities are source-regulated, and more specifically modulated by sorbitol or sucrose availability. In two separate trials, peach fruit relative growth rate (RGR), enzyme activities, and carbohydrates were measured 1) at cell division stage before and after girdling of the shoot subtending the fruit; and 2) on 14 shoots with different leaf to fruit ratio (L:F) at cell division and cell expansion stages. Fruit RGR and sorbitol dehydrogenase (SDH) activity were significantly reduced by girdling, whereas sucrose synthase (SS), acid invertase (AI), and neutral invertase (NI) where equally active in girdled and control fruits on the fourth day after girdling. All major carbohydrates (sorbitol, sucrose, glucose, fructose and starch) were reduced on the fourth day after girdling. SDH activity was the only enzyme activity proportional to L:F in both fruit developmental stages. Peach fruit incubation in sorbitol for 24 hours also resulted in SDH activities higher than those of fruits incubated in buffer and similar to those of freshly extracted samples. Overall, our data provide some evidence for regulation of sorbitol metabolism, but not sucrose metabolism, by photoassimilate availability in peach fruit. In particular, sorbitol translocated to the fruit may function as a signal for modulating SDH activity.
R.L. Darnell, R. Cano-Medrano, and K.E. Koch
Variability in sucrose levels and metabolism in ripe fruit of several Vaccinium species were examined. 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. Three-fold differences in fruit sucrose concentration were found among species, ranging from 19 to 24 mg·(g fw)-1 in V. stamineum and V. arboreum to about 7 mg·(g fw)-1 in cultivated blueberry (V. ashei and V. corymbosum) and V. darrowi. Soluble acid invertase activity was negatively correlated with fruit sucrose concentration. There was no apparent correlation between fruit sugar concentration and either sucrose phosphate synthase or sucrose synthase activities, both of which were low for all species studied. The degree of variability in fruit sucrose accumulation among Vaccinium species supports the feasibility of developing high sucrose fruit, which would be a potentially valuable addition to current strategies of minimizing crop losses to birds.
Y.H. Huang, David H. Picha, and Charles E. Johnson
The glucose-6-phosphate dehydrogenase (G-6-PDH) and glucose oxidase methods are commonly adapted for plant invertase assay. A disadvantage of the G-6-PDH assay is the relatively high cost of the coupling enzymes and cofactors. A disadvantage of the glucose oxidase method, which uses a glucose kit (Sigma, 510-A), is the presence of high activities of acid invertase and alkaline invertase in the PGO enzyme formula (peroxidase and glucose oxidase), which gives a falsely high invertase activity value. An alternative and inexpensive coupled assay was developed for enzymatic assay of plant invertases. In this assay, ADP produced from phosphorylation of glucose and fructose (hydrolysis products of invertases) is coupled to oxidation of NADH by the enzymes pyruvate kinase and lactate dehydrogenase in presence of phosphoenolpyruvate and NADH. This method was compared with the glucose-6-phosphate dehydrogenase method by using protein preparations derived from plant materials of three different species. Statistical analysis indicated that the alternative assay was similar in accuracy to the glucose-6-phosphate dehydrogenase method, with an advantage of reducing the cost from $0.85 to $0.35 per assay.
Riccardo Lo Bianco, Mark Rieger, and She-Jean S. Sung
Terminal portions of `Flordaguard' peach roots [Prunus persica (L.) Batsch] were divided into six segments and the activities of NAD+-dependent sorbitol dehydrogenase (SDH), sorbitol oxidase (SOX), sucrose synthase (SS), soluble acid invertase (AI), and soluble neutral invertase (NI) were measured in each segment 10, 15, and 20 days after seed germination. The same type of experiment was conducted with terminal portions of `Flordaguard' and `Nemaguard' peach shoots except that one of the six segments consisted of the leaflets surrounding the apex. Independent of the age of individual roots, activities of SDH and AI were consistently highest in the meristematic portion and decreased with tissue maturation. In shoots, AI was the most active enzyme in the elongating portion subtending the apex, whereas SDH was primarily associated with meristematic tissues. A positive correlation between SDH and AI activities was found in various developmental zones of roots (r = 0.96) and shoots (r = 0.90). Sorbitol and sucrose contents were low in roots regardless of distance from tip, while sucrose showed a decreasing trend with distance and sorbitol, fructose, and glucose increased with distance from the meristem in shoots. Activity of SDH in internodes, but not apices, correlated with shoot elongation rate of both cultivars, whereas activities of other enzymes did not correlate with shoot elongation rate. We conclude that AI and SDH are the predominant enzymes of carbohydrate catabolism and the best indicators of sink growth and development in vegetative sinks of peach.