Cytosolic fructose-1,6-bisphosphatase (cytoFBPase) (EC 22.214.171.124) occupies a strategic site in sucrose synthesis and has been demonstrated to play a key role in carbon partitioning between sucrose and starch in non-sorbitol forming plants. In addition to sucrose and starch, Sorbitol is the primary photosynthetic end product in the leaves of many tree fruit species in the Rosaceae family. To understand the biochemical regulation of photosynthetic carbon partitioning between sorbitol, sucrose and starch in sorbitol synthesizing species, we purified cytoFB-Pase to apparent homogeneity from apple leaves. The enzyme was a homotetramer with a subunit mass of 37 kDa. It was highly specific for fructose-1,6-bisphosphate with a Km of 3.1 μm and a Vmax of 48 units/mg protein. Either Mg2+ or Mn2+ was required for its activity with a Km of 0.59 mm and 62 μM, respectively. Li+, Ca2+, Zn2+, Cu2+ and Hg2+ inhibited whereas Mn2+ enhanced the Mg2+-activated enzyme activity. Fructose-6-phosphate was found to be a mixed type inhibitor with a Ki of 0.47 mm. Fructose 2,6-bisphosphate (F2,6BP) competitively inhibited the enzyme activity and changed the substrate saturation curve from hyperbolic to sigmoidal. Adenosine monophosphate (AMP) was a non-competitive inhibitor for the enzyme. F2,6BP interacted with AMP to inhibit the enzyme in a synergistic way. Dihydroxyacetone phosphate did not have inhibitory effect on apple leaf cytosolic FBPase activity. Sorbitol increased the susceptibility of the enzyme to the inhibition by F1,6BP. The presence of sorbitol in the reaction mixture led to a reduction in the enzyme activity.
One-year-old `Concord' grapevines (Vitis labrusca L.) were fertigated twice weekly for 11 weeks with a complete nutrient solution containing 1, 10, 20, 50 or 100 μmol iron (Fe) from ferric ethylenediamine di (o-hydroxyphenylacetic) acid (Fe-EDDHA). Leaf total Fe content did not increase in response to Fe supply, however both “active” Fe (extracted with 2, 2'-dipyridyl) and chlorophyll (Chl) content increased as applied Fe increased. At the lowest active Fe level, leaf absorptance and maximum PSII efficiency (Fv/Fm) were slightly decreased, and non-photochemical quenching was significantly greater. PSII quantum efficiency decreased curvilinearly as active Fe content decreased. On a Chl basis, the xanthophyll cycle pool size, lutein, and beta-carotene increased curvilinearly as active Fe decreased, and neoxanthin increased at the lowest Fe level. Activities of antioxidant enzymes superoxide dismutase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase followed a similar trend and increased under Fe deficiency, when expressed on a Chl basis. Antioxidant metabolites also increased in response to Fe limitation. On a Chl basis, ascorbate (AsA), dehydroascorbate (DAsA), reduced glutathione (GSH) and oxidized glutathione (GSSG) content was greater at the lowest active Fe levels. We did not find a difference in the ratio of AsA to DAsA or GSH to GSSG. In conclusion, both photoprotective mechanisms, xanthophyll cyle-dependent thermal dissipation and the ascorbate-glutatione antioxidant system, are enhanced in response to iron deficiency to cope with excess absorbed light.
One-year-old `Concord' vines were fertigated with 0, 5, 10, 15, or 20 mm N in a modified Hoagland's solution for 8 weeks during summer. Half of the vines fertigated at each N concentration were sprayed with 3% foliar urea twice in late September while the rest served as controls. Four vines from each treatment combination were destructively sampled during dormancy to determine the levels and forms of N and carbohydrates. Nitrogen fertigation during the summer only slightly increased vine N concentration whereas foliar urea application in the fall significantly increased vine N concentration. In response to foliar urea application, concentrations of both free amino acid-N and protein-N increased, but the ratio of protein N to amino acid N decreased. Arginine was the most abundant amino acid in free amino acids and proteins, and its concentration was linearly correlated with vine N concentration. Concentrations of total non-structural carbohydrates (TNC) decreased slightly in response to N supply from fertigation. Foliar urea application in the fall significantly decreased TNC concentration at each N fertigation level. Starch, glucose and fructose decreased in response to foliar urea applications, but sucrose concentration remained unaffected. Approximately 60% of the carbon decrease in TNC caused by foliar urea application was recovered in proteins and free amino acids. We conclude that free amino acids account for a larger proportion of the N in vines sprayed with foliar urea, but proteins remain as the main form of N storage. In response to foliar urea application, part of the carbon from TNC is incorporated into proteins and free amino acids, leading to a decrease in the carbon stored in TNC and an increase in the carbon stored in proteins and free amino acids.
Four-year-old `Gala'/M.26 trees were grown under low (2.5 mm), medium (12.5 mm), or high (25 mm) N supply with balanced nutrients in sand culture and the cropload was adjusted to 5 fruit/cm2 trunk cross-sectional area at 10 mm king fruit. At about 100 days after bloom, exposed fruit on the south side of the canopy were chosen for monitoring chlorophyll fluorescence and fruit peel samples were taken for measuring xanthophyll cycle pigments, antioxidant enzymes, and metabolites. At noon, the efficiency of excitation transfer (Fv'/Fm') of the sun-exposed peel was higher in the low N treatment than in the medium or high N treatments. Photochemical quenching coefficient did not differ between fruits in different N treatments. The Photosystem II operating efficiency was higher in the peel of low N fruit compared with medium N or high N fruit. However, maximum quantum efficiency (Fv/Fm) of fruit peel after overnight dark adaptation was similar across the N treatments. The xanthophyll cycle pool size expressed on peel area basis was larger in the high N fruit than in the low N fruit. This corresponds well with the thermal dissipation capacity, as indicated by efficiency of excitation transfer. Over 95% of the xanthophyll cycle pool in the sun-exposed side was present in the form of zeaxanthin and antheraxanthin at noon regardless of N treatments. Activities of superoxide dismutase and all the antioxidant enzymes and metabolites in the ascorbate-glutathione cycle were higher in the high N fruit than in low N fruit. The results indicate that apple fruit with a good N status have a higher photoprotective capacity in terms of xanthophyll cycle-dependent thermal dissipation and detoxification of reactive oxygen species compared with low N fruit.
Four-year-old `Gala'/M.26 trees were grown under low (2.5 mm), medium (12.5 mm), or high (25 mm) N supply with balanced nutrients in sand culture and the cropload was adjusted to 5 fruit/cm2 trunk cross-sectional area at 10 mm king fruit. After harvesting, half of the trees in each N treatment were sprayed twice with 3% urea a week apart in late September. Before budbreak the following spring, four trees from each treatment combination were destructively sampled for reserve nitrogen and carbohydrate analysis. Foliar urea application significantly increased tree N concentration and concentrations of both free amino acids and proteins, but decreased the concentration of total nonstructural carbohydrates (TNC) at each soil N supply level. When the carbon in free amino acids and proteins are taken into account, trees sprayed with foliar urea had similar levels of total sum of carbon in TNC, free amino acids and proteins. On a whole tree basis, trees sprayed with foliar urea had more N and less TNC. During the second year of the experiment, all the trees received normal N supply. Trees sprayed with foliar urea the previous fall had a significantly larger total leaf area and higher fruit set, fruit number, and total yield than those unsprayed. We conclude that fruit set and early fruit development as well as vegetative growth in spring is mainly determined by reserve nitrogen, not by reserve carbohydrates. Conversion of a portion of TNC to amino acids and proteins leads to better growth and fruiting of apple trees.
Apple leaf ADP-glucose pyrophosphorylase was purified over 1400-fold to apparent homogeneity with a specific activity of 58.9 units per mg of protein. The enzyme was activated by 3-phosphoglycerate (PGA) and inhibited by inorganic phosphate (Pi) in the ADPG synthesis direction. In the pyrophosphorolysis direction, however, high concentrations of PGA (>2.5 mm) inhibited the enzyme activity. The enzyme was resistant to thermal inactivation with a T0.5 (temperature at which 50% of the enzyme activity is lost after 5 min of incubation) of 52 °C. Incubation with 2 mm PGA or 2 mm Pi increased T0.5 to 68 °C. Incubation with 2 mm dithiothreitol (DTT) decreased T0.5 to 42 °C, whereas inclusion of 2 mm PGA in the DTT incubation maintained T0.5 at 52 °C. DTT-induced decrease in thermal stability was accompanied by monomerization of the small subunits. Presence of PGA in the DTT incubation did not alter the monomerization of the small subunits of the enzyme induced by DTT. These findings indicate that the binding of PGA may have dual functions in regulating apple leaf AGPase activity—activating the enzyme and rendering the enzyme with a conformation more stable to thermal inactivation.
Apple maturity is often assessed by starch hydrolysis index, skin color, soluble solids, flesh firmness, and the rate of ethylene evolution. In red-fruited apple cultivars, the intensity and extent of coloration is an important consideration in determining the time of fruit harvest. Negative relationships have been found between tree nitrogen (N) status and fruit skin pigmentation, but how N affects flesh starch breakdown has not been examined in detail. The objective of this study was to determine how N supply affects flesh starch breakdown relative to skin color development. Seven-year-old ‘Gala’/M.26 trees were provided with four levels of N (8.8, 26.4, 52.7, and 105.4 g N per tree) in a modified Hoagland's solution. The effects of N supply on yield, fruit quality, and fruit maturation were evaluated. At harvest, fruit in the lowest N treatment was significantly smaller and had lower soluble solids but higher starch concentration, better color, and higher firmness than those grown at higher N supplies. Increasing N supply decreased both anthocyanin synthesis and chlorophyll degradation in fruit skin. Flesh starch concentration was higher at higher N supply at 38 days before harvest but was lower at higher N supply at harvest. Starch degradation was completed earlier during cold storage with increasing N supply. These results indicate that increasing N supply delays skin red color development but accelerates flesh starch degradation in ‘Gala’ apples. These differential effects of N supply should be taken into account when assessing fruit maturity for optimizing harvest time.
Xanthophyll cycle conversion and the antioxidant system in the peel of apple fruit (Malus ×domestica Borkh. `Liberty') were monitored in the field over a diurnal course at about 3 months after full bloom. Compared with leaves, sun-exposed peel of apple fruit had much lower photosystem II operating efficiency at any given photon flux density (PFD) and a larger xanthophyll cycle pool size on a chlorophyll basis. Zeaxanthin (Z) level increased with rising PFD in the morning, reached the highest level during midday, and then decreased with falling PFD for the rest of the day. At noon, Z accounted for >90% of the xanthophyll cycle pool in the fruit peel compared with only 53% in leaves. Efficiency of excitation transfer to PSII reaction centers (Fv′/Fm′) was negatively related to the level of Z in fruit peel and leaves throughout the day. In fruit peel, the antioxidant enzymes in the ascorbate-glutathione cycle, ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) showed a diurnal pattern similar to that of incident PFD. In contrast, the activities of APX and GR in leaves did not change significantly during the day although activities of both MDAR and DHAR were higher in the afternoon than in the morning. In both fruit peel and leaves, superoxide dismutase activity did not change significantly during the day; catalase activity showed a diurnal pattern opposite to that of PFD with much lower activity in fruit peel than in leaves. The total ascorbate pool was much smaller in fruit peel than in leaves on an area basis, but the ratio of reduced ascorbate to oxidized ascorbate reached a maximum in the early afternoon in both fruit peel and leaves. The total glutathione pool, reduced glutathione and the ratio of reduced glutathione to oxidized glutathione in both fruit peel and leaves also peaked in the early afternoon. We conclude that the antioxidant system as well as the xanthophyll cycle responds to changing PFD over the course of a day to protect fruit peel from photooxidative damage.
One-year-old `Concord' grapevines (Vitis labruscana Bailey) were fertigated with 0, 5, 10, 15, or 20 mm N in a modified Hoagland's solution for 8 weeks during summer. Half of the vines fertigated at each N concentration were sprayed with 3% foliar urea twice in late September while the rest served as controls. Four vines from each treatment combination were destructively sampled during dormancy to determine the levels and forms of N and carbohydrates. Nitrogen fertigation during the summer did not significantly alter vine N concentration whereas foliar urea application in the fall significantly increased vine N concentration. In response to foliar urea application, concentrations of both free amino acid-N and protein-N increased, but the ratio of protein-N to free amino acid-N decreased. Arginine was the most abundant amino acid in free amino acids and proteins, and its concentration was linearly correlated with vine N concentration. Concentrations of total nonstructural carbohydrates (TNC) decreased slightly in response to N supply from fertigation. Foliar urea application in the fall significantly decreased TNC concentration at each N fertigation level. Starch, glucose, and fructose decreased in response to foliar urea applications, but sucrose concentration remained unaffected. Approximately 60% of the carbon decrease in TNC caused by foliar urea application was recovered in proteins and free amino acids. We conclude that free amino acids account for a larger proportion of the N in vines sprayed with foliar urea compared with the unsprayed vines, but proteins remain as the main form of N storage. In response to foliar urea application, part of the carbon from TNC is incorporated into proteins and free amino acids, leading to a decrease in the carbon stored in TNC and an increase in the carbon stored in proteins and free amino acids.
Six-year-old ‘Gala’/‘Malling26’ (‘M.26’) apple (Malus ×domestica Borkh.) trees grown in sand culture were provided with a total of 30 g of N per tree as enriched 15N-NH4NO3 in Hoagland's solution via fertigation to determine the magnitude and seasonal patterns of accumulation of macro- and micronutrients and the demand-supply relationship of N. Crop load was adjusted to 8.2 fruit/cm2 trunk cross-sectional area, at king fruit diameter of 10 mm by hand-thinning. At each of seven key developmental stages throughout one annual growth cycle, four trees were excavated and destructively sampled for complete nutrient analysis. Nutrient concentrations in leaves and fruit fell within the recommended optimal range, and the fruit yield was 18.8 kg/tree (equivalent to 52.45 t·ha−1) with an average fruit weight of 181 g. The net accumulation of N, P, K, Ca, Mg, and S from budbreak to fruit harvest was 19.8, 3.3, 36.0, 14.2, 4.4, and 1.6 g/tree, respectively, and that for B, Zn, Cu, Mn, and Fe was 93.6, 60.9, 46.5, 184.8, and 148.7 mg/tree, respectively. Nutrient accumulation by new growth (fruit plus shoots and leaves) accounted for over 90% of the net gain for N, P, K, Mg, S, and B in the whole tree and a large proportion of the net gain for Ca, Zn, Mn, and Fe (from 58.1% for Zn to 87.2% of Fe) from budbreak to fruit harvest. Differential nutrient accumulation patterns were found in shoots and leaves and fruit. The most rapid accumulation of all nutrients in shoots and leaves took place during active shoot growth from bloom to the end of shoot growth. The accumulation pattern of most nutrients corresponded well with the accumulation of dry matter, with continued accumulation observed only in total Ca and Mn in shoots and leaves after the end of shoot growth. Nutrient accumulation in fruit largely followed its dry matter accumulation, and a large proportion of the nutrient accumulation (from 58.1% for Zn to 77.4% of K) occurred from the end of shoot growth to fruit harvest. At harvest, fruit contained more P, K, B, and Fe, whereas shoots and leaves had more N, Ca, Mg, S, Zn, and Mn. Most of the N demand by new growth at bloom was provided by tree reserve N. Remobilization of N from perennial parts of the tree was found to support rapid fruit expansion from the end of shoot growth to fruit harvest. The most rapid uptake from current season's N supply occurred from bloom to the end of shoot growth, corresponding to the highest tree N demand. At harvest, 62.4% of the total N in new growth was in shoots and leaves, with the balance in fruit. Reserve N and current season's N uptake each contributed about 50% to the total N in the whole tree at harvest.