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To determine the cause of zonal chlorosis of `Honeycrisp' apple leaves, we compared CO₂ assimilation, carbohydrate metabolism, xanthophyll cycle and the antioxidant system between chlorotic leaves and normal leaves. Chlorotic leaves accumulated higher levels of non-structural carbohydrates, particularly starch, sorbitol, sucrose, and fructose at both dusk and predawn, and no difference was found in total non-structural carbohydrates between predawn and dusk. CO₂ assimilation and the key enzymes in the Calvin cycle, ribulose 1,5-bisphosphate carboxylase/oxygenase, NADP-glyceraldehyde-3-phosphate dehydrogenase, phosphoribulokinase, stromal fructose-1,6-bisphosphatase, and enzymes in starch and sorbitol synthesis, ADP-glucose pyrophosphorylase, cytosolic fructose-1,6-bisphosphatase, and aldose 6-phosphate reductase were significantly lower in chlorotic leaves than in normal leaves. However, sucrose phosphate synthase activity was higher in chlorotic leaves. Thermal dissipation of excitation energy was enhanced in chlorotic leaves under full sun, lowering the efficiency of excitation energy transfer to PSII reaction centers. This was accompanied by a corresponding increase in both xanthophyll cycle pool size (on a chlorophyll basis) and conversion of violaxanthin to antheraxanthin and zeaxanthin. The antioxidant system was up-regulated in chlorotic leaves in response to the increased generation of reactive oxygen species. These findings support the hypothesis that phloem loading and/or transport is partially or completely blocked in chlorotic leaves, and that excessive accumulation of non-structural carbohydrates may cause feedback suppression of CO₂ assimilation via direct interference with chloroplast function and/or indirect repression of photosynthetic enzymes.

About 80 days after full bloom, well-exposed fruit on the south part of the canopy of mature Liberty/M.9 apple trees were randomly assigned to one of the following two treatments. Some fruit were turned about 180 degrees to expose the original shaded side to full sun whereas the rest served as untreated controls. On day 0, 1, 2, 4, 7, and 10 after treatment, fruit peel samples were taken from the original shaded side of the treated fruit and both the sun-exposed side and the shaded side of the control fruit at midday to determine photosynthetic pigments and enzymatic and non-enzymatic antioxidants. Maximum photosystem II efficiency of the original shaded side decreased sharply after 1 day exposure to full sun, and then gradually recovered to a similar value of the sun-exposed side of the control fruit by day 10. The shaded side of the control fruit had much lower xanthophyll cycle pool size and conversion and antioxidant enzymes and soluble antioxidants of the ascorbate-glutathione cycle than the sun-exposed side. In response to full sun exposure, xanthophyll cycle pool size of the original shaded side increased, reaching a similar value of the sun-exposed side by day 10. Ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase and total pool size and reduction state of both ascorbate and glutathione of the original shaded side all increased to the corresponding values found in the sun-exposed side of the control fruit over a 10-day period. It is concluded that both xanthophyll cycle and the ascorbate-glutathione cycle in the original shaded side are up-regulated in response to fullsun exposure to minimize photo-oxidative damage and contributes to its re-acclimation to full sun.

Sorbitol is the primary photosynthetic end product in the leaves of many tree fruit species in the Rosaceae family, but its physiological role remains unclear. In this study, we determined the effect of decreased sorbitol synthesis on the antioxidant system that scavenges reactive oxygen species (ROS) in apple leaves. Sorbitol synthesis was decreased in apple leaves by antisense inhibition of aldose-6-phosphate reductase activity. Dehydroascorbate reductase (DHAR), glutathione reductase, and catalase (CAT) activities increased in the leaves of the transgenic plants with decreased sorbitol synthesis, whereas superoxide dismutase, ascorbate peroxidase, NADH dependent and NADPH dependent monodehydroascorbate reductase activity did not show significant changes. Ascorbate and glutathione concentrations were higher in leaves of the transgenic plants compared with the control. The effect of decreased sorbitol synthesis on the antioxidant enzyme activity was dependent on leaf developmental stages. Larger changes in the enzyme activities of CAT, DHAR, and GR were observed in the old leaves than in the young leaves. These results suggest that sorbitol may play a role in ROS scavenging in apple leaves.

Reserve N and carbohydrate levels of bench-grafted Fuji/M26 plants were altered by fertigation with seven N concentrations from 30 June to 1 Sept. in combination with or without 3% foliar urea application in mid-October. The plants were harvested after natural leaf fall and stored at 2 °C. One set of plants were destructively sampled in January for reserve N and carbohydrates analysis, and the remaining plants were transplanted into a N-free medium in the spring and supplied with or without 5 mM ¹⁵N-ammonium nitrate in a Hoagland solution for 60 days after budbreak. Plants fertigated with higher N concentrations had higher reserve N content and lower carbohydrate concentrations. Foliar urea application increased whole plant N content and decreased reserve carbohydrate concentration at each given N concentration used in fertigation. Regardless of N supply in the spring, total new shoot and leaf growth of plants fertigated with N was closely related to the amount of reserve N but not reserve carbohydrates. Plants treated with foliar urea had more new shoot and leaf growth than the fertigated controls. By pooling all the data concerning reserve N used for growth regardless of the spring N supply, a linear relationship was found between the amount of reserve N used for new shoot and leaf growth and the total amount of N. We conclude that the growth of apple nursery plants in the spring is mainly determined by reserve N, not reserve carbohydrates. The amount of reserve N used for new shoot and leaf growth in the spring is dependent on the total amount of reserve and is not affected by the current N supply.

`Concord' grapevines (Vitis labruscana Bailey) can readily develop iron deficiency-induced leaf chlorosis when grown on calcareous or high pH soils. Iron (Fe) chelates are often applied to the soil to remedy chlorosis but can vary in their stability and effectiveness at high pH. We transplanted own-rooted 1-year-old `Concord' grapevines into a peat-based medium adjusted to pH 7.5 and fertigated them with 0, 0.5, 1.0, 2.0, or 4mg·L^–1 Fe from Fe-EDDHA [ferric ethylenediamine di (o-hydroxyphenylacetic) acid] to determine the effectiveness of this Fe chelate for alleviating Fe deficiency-induced chlorosis at high pH. Vines were sampled midseason for iron, chlorophyll, CO₂ assimilation, and photosystem II quantum efficiency (PSII) and at the end of the season for leaf area, dry weight, and cane length. We found that leaf total Fe concentration was similar across all treatments, but active Fe (extracted with 0.1 n HCl) concentration increased as the rate of Fe-EDDHA increased. Chlorophyll concentration increased curvilinearly as applied Fe increased and was highly correlated with active Fe concentration. CO₂ assimilation, stomatal conductance, and PSII were very low without any supplemental Fe and increased rapidly in response to Fe application. Total leaf area, foliar dry weight, and cane length all increased as Fe application increased to 1 mg·L^–1 Fe, but above this rate, a further increase in Fe did not significantly increase growth. Our results demonstrate that Fe-EDDHA is very effective in alleviating Fe deficiency-induced leaf chlorosis in `Concord' grapevines grown at high pH, which provides a foundation for continuing research related to the optimum rate and timing of application of Fe-EDDHA in `Concord' vineyards on calcareous soils. Compared with total Fe, leaf “active Fe” better indicates the actual Fe status of `Concord' vines.

The reaction catalyzed by ADP-glucose pyrophosphorylase (AGPase) to form ADP-glucose is a regulatory and rate-limiting step in starch synthesis in plants. In response to decreased sorbitol synthesis, starch synthesis was up-regulated in the transgenic apple plants. In this study, we examined both redox and metabolite regulation of AGPase to understand the mechanism responsible for the up-regulation of starch synthesis. No difference in the monomerization/dimerization of apple leaf AGPase small subunits was observed between the transgenic plants and the untransformed control. NADP-dependent malate dehydrogenase, indicative of chloroplastic redox status, did not show significant change in the transgenic plants either. Determination of key metabolites with nonaqueous fractionation indicated that concentrations of hexose phosphates (mainly glucose-6-phosphate and fructose-6-phosphate) were higher in both the cytosol and chloroplasts of the transgenic plants than in the control, whereas 3-phosphoglycerate (PGA) concentration in the chloroplast was not higher in the transgenic plants. We conclude that accumulation of hexose-phosphates results in a decrease in inorganic phosphate (Pi) concentration and an increase in PGA/Pi ratio in the chloroplast, leading to up-regulation of starch synthesis via activating AGPase.

Considering starch synthesis was enhanced in leaves of transgenic apple trees with decreased sorbitol synthesis, we hypothesized that starch degradation must be up-regulated correspondingly to maintain carbon supply to sink tissues. Compared with the untransformed control, mature leaves of the transgenic plants had a larger drop in starch concentration between dusk and pre-dawn, higher maltose concentration, and higher activities of two key enzymes in starch degradation: -amylase and cytosolic glucosyltransferase during the day and night. ¹⁴C-maltose and ¹⁴C-glucose were fed to the apple leaves to study the fate of starch breakdown products in the synthesis of sorbitol and sucrose. Under light, a larger proportion of both ¹⁴C-maltose and ¹⁴C-glucose were converted to sorbitol than to sucrose in the untransformed control, whereas conversion of ¹⁴C-maltose and ¹⁴C-glucose to sucrose predominated over that to sorbitol in the transgenic apple leaves. The leaf samples fed with ¹⁴C-maltose and ¹⁴C-glucose in the dark are still being analyzed, but it appears that sucrose is the main product in both the untransformed control and the transgenic plants. These results support the hypothesis that starch degradation is up-regulated in the transgenic plants.

Plants grown on calcareous soils often exhibit symptoms of Fe-deficiency induced chlorosis despite a high content of total Fe in the leaf tissue. Iron is transported in the xylem primarily as the ferric citrate (Fe-Citr) chelate, and changes in pH, HCO ^- ₃, and Citr can lead to the formation of different Fe-Citr species. Understanding how Fe dissociates from these chelates may help explain why Fe is immobilized in the leaves. The goal was to quantify Fe mobilization (Fe-Mob) from Fe-Citr in an assay system buffered at pH 5, 6, or 7 when: 1) the molar ratio of HCO ^- ₃ to Fe increased in a 1 Fe: 1 Citr system; 2) the molar ratio of Citr increased in a 1 Fe: 3 HCO ^- ₃ system; and 3) solutions were photoreduced (PR) or left in the dark. For non-PR solutions, Fe-Mob from Fe-Citr using 500 μmol NADH was the greatest at the 1 Fe: 0 HCO ^- ₃-level, and decreased as HCO ^- ₃ increased. Fe-Mob also decreased as buffer pH increased from 5 to 7. Increasing the Citr ratio was effective in increasing Fe-Mob, but the effect decreased as buffer pH increased from 5 to 7. PR solutions behaved quite differently. In the 1 Fe: 1 Citr system, little to no Fe-Mob was detected at any buffer pH. However, there were already large pools of Fe²⁺ in solution, which decreased as HCO ^- ₃ increased, irrespective of buffer pH. Increasing the Citr ratio greatly increased Fe-Mob in the 1 Fe: 3 HCO ^- ₃ system, and mobilization decreased as buffer pH increased. Increasing Citr did not increase the amount of Fe²⁺ in solution. This work illustrates that increasing the HCO ^- ₃: Fe ratio can lead to an immobilization of Fe, and that increasing the Citr ratio can aid in Fe-Mob from Fe-Citr when the HCO ^- ₃: Fe ratio is high. Increasing the Citr ratio, however, does not increase the amount of PR Fe²⁺.

Plants of Fragaria chiloensis cv. RCP-37 were grown with their root system split between two separate containers. Water was withheld from one container of each pair (drought side), while the other was subirrigated. Control plants were subirrigated in both containers. Over several days, a drought-side leaf exhibited reductions in stomatal conductance (g_s) and transpiration rate (T), while a subirrigated side leaf showed no change in either parameter. However, foliar water relations components (water, osmotic, and pressure potential) did not differ between the two leaves. The leaf on the subirrigated side exhibited g_s, T, and water relations components similar to leaves on control plants. The abscisic acid (ABA) content of xylem exudate, collected from a stolon emerging from the axils of the measured leaves, was highest from the drought side and was negatively correlated to g_s and T at some sampling dates. A root-derived drought stress signal, perhaps ABA, although other factors cannot be discounted, was limited within the plant to the drought side, even though water relations components indicated that water from the subirrigated side was allocated to all parts of the plant.

The objective of this study was to quantify how photoprotective mechanisms in the leaves of `Concord' grapevines (Vitis labruscana Bailey) respond to a range of iron (Fe) supply. Own-rooted, 1-year-old container-grown vines were fertigated twice weekly for 11 weeks with a complete nutrient solution containing 1, 10, 20, 50, or 100 μm Fe from ferric ethylenediamine di (o-hydroxyphenylacetic) acid (Fe-EDDHA). Leaf total Fe content did not increase in response to Fe supply; however, “active” Fe (extracted with 2,2′-dipyridyl) and chlorophyll (Chl) increased on a leaf area basis as applied Fe increased. At the lowest active Fe level, leaf absorptance and the efficiency of excitation transfer (F_v′/F_m′) was lower, and nonphotochemical quenching (NPQ) was significantly greater. Photosystem II (PSII) quantum efficiency decreased curvilinearly, and the proportion of PSII reaction centers in the open state (qP) decreased linearly as active Fe content decreased. On a Chl basis, the xanthophyll cycle pool size [violaxanthin (V) + antheraxanthin (A) + zeaxanthin (Z)], lutein, and β-carotene increased curvilinearly as active Fe decreased, and neoxanthin (Neo) increased at the lowest Fe level. On a leaf area basis, as active Fe decreased, V+A+Z and β-carotene decreased curvilinearly, and lutein and Neo decreased linearly. At noon, conversion of V to A and Z increased as active Fe decreased. On a Chl basis, activities of antioxidant enzymes superoxide dismutase (SOD), monodehydroascorbate reductase (MDAR), and dehydroascorbate reductase (DHAR) increased curvilinearly, and glutathione reductase (GR) activity increased linearly as active Fe levels declined. Ascorbate peroxidase (APX) and catalase (CAT), on a Chl basis, were relatively constant. On a leaf area basis, a decrease in active Fe increased SOD and MDAR activity, whereas APX, CAT, DHAR and GR activity decreased. Antioxidant metabolites ascorbate (AsA), dehydroascorbate (DAsA), reduced glutathione (GSH) and oxidized glutathione (GSSG) also increased in response to Fe limitation when expressed on a Chl basis, whereas on a leaf area basis AsA and DAsA decreased and GSH increased curvilinearly. The GSH:GSSG ratio increased as active Fe declined, whereas the AsA:DAsA ratio did not change. In conclusion, both photoprotective mechanisms, xanthophyll cycle-dependent thermal dissipation and the ascorbate-glutathione antioxidant system, are enhanced in response to Fe deficiency to cope with excess absorbed light. In a low soil pH tolerant species such as V. labruscana, the foliar antioxidant system was upregulated in response to excess absorbed light from Fe deficiency-induced chlorosis, and there was no evidence of an increase in oxidative stress from high rates of applied Fe-EDDHA.