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One-year-old `Concord' grapevines (Vitis labruscana Bailey) were fertigated with 0, 5, 10, 15, or 20 mm nitrogen by using a modified Hoagland's solution for 8 weeks during active vine growth in summer. Half of the vines at each N concentration were sprayed with 3% foliar urea twice in late September while the rest served as controls. After natural leaf fall, all the vines were overwintered in a cold room (2 to 4 °C). Four vines from each treatment were destructively sampled before budbreak for reserve N and carbohydrate analysis. The remaining vines were supplied with either no N or sufficient N (10 mm N) from 2 weeks before bloom to 1 month after bloom. All the vines were destructively harvested at 1 month after bloom. Total amount of N in dormant vines increased with increasing N fertigation concentration. Total nonstructural carbohydrates (TNC) increased with increasing N fertigation concentration from 0 to 10 mm, and then leveled off with further rises in N supply. Foliar urea application increased total N but decreased TNC of dormant vines at each given N fertigation level. When no N was provided during the regrowth period, vine total leaf area, fruit yield, and total dry weight increased with increasing N supply from fertigation the previous year. Vines sprayed with foliar urea the previous fall produced a larger total leaf area, a higher yield, and a higher total vine dry weight at each given N fertigation concentration. Providing vines with sufficient N during the regrowth period significantly increased total leaf area, fruit yield, and vine total dry weight across the previous N fertigation concentrations, but vines sprayed with foliar urea still had a larger leaf area, a higher yield, and a higher total vine dry weight at each given N fertigation concentration. Therefore, we conclude that both vegetative growth and fruiting of young `Concord' vines are largely determined by reserve nitrogen, not by reserve carbohydrates, and that current-season N supply plays a very important role in sustaining vine growth and development, especially fruit growth.

To determine the cause of a characteristic zonal chlorosis of `Honeycrisp' apple (Malus ×domestica Borkh.) leaves, we compared CO₂ assimilation, carbohydrate metabolism, the xanthophyll cycle and the antioxidant system between chlorotic leaves and normal leaves. Chlorotic leaves accumulated higher levels of nonstructural carbohydrates, particularly starch, sorbitol, sucrose, and fructose at both dusk and predawn, and no difference was found in total nonstructural carbohydrates between predawn and dusk. This indicates that carbon export was inhibited in chlorotic leaves. 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 the key 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. In response to a reduced demand for photosynthetic electron transport, thermal dissipation of excitation energy (measured as nonphotochemical quenching of chlorophyll fluorescence) 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, including superoxide dismutase and ascorbate peroxidase and the ascorbate pool and glutathione pool, was up-regulated in chlorotic leaves in response to the increased generation of reactive oxygen species via photoreduction of oxygen. These findings support the hypothesis that phloem loading and/or transport is partially or completely blocked in chlorotic leaves, and that excessive accumulation of nonstructural carbohydrates may cause feedback suppression of CO₂ assimilation via direct interference with chloroplast function and/or indirect repression of photosynthetic enzymes.

One-year-old grapevines (Vitis labrusca L. `Concord') were supplied twice weekly for 5 weeks with 0, 5, 10, 15, or 20 mm nitrogen (N) in a modified Hoagland's solution to generate a wide range of leaf N status. Both light-saturated CO₂ assimilation at ambient CO₂ and at saturating CO₂ increased curvilinearly as leaf N increased. Although stomatal conductance showed a similar response to leaf N as CO₂ assimilation, calculated intercellular CO₂ concentrations decreased. On a leaf area basis, activities of key enzymes in the Calvin cycle, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), NADP-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoribulokinase (PRK), and key enzymes in sucrose and starch synthesis, fructose-1,6-bisphosphatase (FBPase), sucrose phosphate synthase (SPS), and ADP-glucose pyrophosphorylase (AGPase), increased linearly with increasing leaf N content. When expressed on a leaf N basis, activities of the Calvin cycle enzymes increased with increasing leaf N, whereas activities of FBPase, SPS, and AGPase did not show significant change. As leaf N increased, concentrations of glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), and 3-phosphoglycerate (PGA) increased curvilinearly. The ratio of G6P/F6P remained unchanged over the leaf N range except for a significant drop at the lowest leaf N. Concentrations of glucose, fructose, and sucrose at dusk increased linearly with increasing leaf N, and there was no difference between predawn and dusk measurements. As leaf N increased, starch concentration increased linearly at dusk, but decreased linearly at predawn. The calculated carbon export from starch degradation during the night increased with increasing leaf N. These results showed that 1) grapes leaves accumulated less soluble carbohydrates under N-limitation; 2) the elevated starch level in low N leaves at predawn was the result of the reduced carbon export from starch degradation during the night; and 3) the reduced capacity of CO₂ assimilation in low N leaves was caused by the coordinated decreases in the activities of key enzymes involved in CO₂ assimilation as a result of direct N limitation, not by the indirect feedback repression of CO₂ assimilation via sugar accumulation.

Based on the curvilinear relationship between carboxylation efficiency and leaf N in apple leaves, we hypothesized that deactivation of Rubisco accounts for the lack of response of photosynthesis to increasing leaf N under high N supply. A wide range of leaf N content (from 1.0 to 5.0 g·m^–2) was achieved by fertigating bench-grafted Fuji/M26 apple trees for 6 weeks with different N concentrations using a modified Hoagland solution. Analysis of photosynthesis in response to intercellular CO₂ under both 21% and 2% O₂ indicated that photosynthesis at ambient CO₂ was mainly determined by the activity of Rubisco. Measurements of Rubisco activity revealed that initial Rubisco activity increased with leaf N up to 3.0 g·m^–2, then leveled off with further rise in leaf N, whereas total Rubisco activity increased linearly with increasing leaf N throughout the leaf N range. As a result, Rubisco activation state decreased with increasing leaf N. Photosynthesis at ambient CO₂ and carboxylation efficiency were both linearly correlated with initial Rubisco activity, but showed curvilinear relationships with total Rubisco activity and leaf N. As leaf N increased, photosynthetic nitrogen use efficiency declined with decreasing Rubisco activation state.

Ribulose bisphosphate carboxylase/oxygenase (Rubisco) initiates the photosynthetic carbon metabolism;therefore, its activity has been measured in many physiological studies. However, information on in vitro Rubisco activity from leaves of deciduous fruit crops is very limited and the reported activities are suspiciously low. We measured Rubisco activity in crude extracts of leaves of apple, pear, peach, cherry, and grape by using a photometric method in which RuBP carboxylation was enzymically coupled to NADH oxidation. Replacing polyvinylpyrrolidone with polyvinylpolypyrrolidone in the extraction solution significantly increased extractable Rubisco activity. Depending on species, freezing leaf discs in liquid nitrogen followed by storage at –80°C for only 24 hr reduced both initial and total Rubisco activity to 5% to 50% of that obtained from fresh leaves. Initial Rubisco activity from fresh leaf tissues of all species was well correlated with maximum Rubisco activity (V_cmax) estimated from gas exchange; an exception was pear, where initial Rubisco activity was higher than V_cmax. In most cases, initial Rubisco activity was approximately two to three times higher than net photosynthesis.

‘Concord’ grapevines (Vitis labruscana Bailey) are susceptible to lime-induced chlorosis, which decreases growth and productivity. In two separate experiments, we grew own-rooted vines in a peat–perlite medium adjusted to different pHs with CaCO₃ to characterize how lime-induced Fe deficiency affects root and leaf ferric chelate reductase (FCR) and key enzymes and metabolites involved with glycolysis and the tricarboxylic acid (TCA) cycle in leaves. In addition, we measured the pH of the xylem sap as well as Fe, citrate, and malate concentrations. For both experiments, foliar levels of total Fe, active Fe (extracted in 0.1N HCl), and chlorophyll decreased as lime rate increased. An increase in root-medium pH from 5.8 to 7.5 resulted in a 10-fold increase in root FCR activity, whereas leaf FCR activity decreased 10-fold. An increase in root-medium pH did not raise xylem sap pH but decreased Fe and citrate to some extent. Xylem malate was highest at pH 6.6 and decreased both above and below this pH. Foliar data were evaluated in relation to active Fe content, because it is a better indicator of Fe nutritional status. Lower active Fe decreased midday CO₂ assimilation and PSII quantum efficiency as well as night respiration. As active Fe decreased, aconitase activity decreased linearly, whereas the activity of glucose-6-phosphate dehydrogenase, NAD(P)-isocitrate dehydrogenase, NAD(P)-malic enzyme, malate dehydrogenase, phosphoenolpyruvate (PEP) carboxylase, PEP phosphatase, and pyruvate kinase increased curvilinearly. Glucose-6-phosphate, fructose-6-phosphate, and 3-phosphoglycerate content decreased curvilinearly as active Fe decreased. Malate content increased as active Fe increased to 1.0 mg·m⁻² and then decreased above this level. Citrate increased linearly as active Fe decreased and was an order of magnitude lower than malate content. Our results suggest that leaf FCR activity may limit Fe assimilation to a greater extent than root FCR activity. The decreased leaf aconitase activity under Fe deficiency is the most likely cause of the increase in citrate levels. Greater activity of the other glycolytic and TCA enzymes under Fe deficiency may help to funnel carbon into the mitochondria and enhance NAD(P) reduction. Citrate levels (and the citrate:malate ratios) in the xylem exudate and leaf were much lower when compared with other species and may be linked to Fe inefficiency of ‘Concord’.

Bench-grafted Fuji/M26 trees were fertigated with seven nitrogen concentrations (0, 2.5, 5.0, 7.5, 10, 15, and 20 mm) by using a modified Hoagland solution from 30 June to 1 Sept. In Mid-October, plants in each N treatment were divided into three groups. One group was destructively sampled to determine background tree N status before foliar urea application. The second group was painted with 3% ¹⁵N-urea solution twice at weekly interval on both sides of all leaves while the third group was left as controls. All the fallen leaves from both the ¹⁵N-treated and control trees were collected during the leaf senescence process and the trees were harvested after natural leaf fall. Nitrogen fertigation resulted in a wide range of tree N status in the fall. The percentage of whole tree N partitioned into the foliage in the fall increased linearly with increasing leaf N content up to 2.2 g·m^–2, reaching a plateau of 50% to 55% with further rise in leaf N. ¹⁵N uptake and mobilization per unit leaf area and the percentage of ¹⁵N mobilized from leaves decreased with increasing leaf N content. Of the ¹⁵N mobilized back to the tree, the percentage of ¹⁵N partitioned into the root system decreased with increasing tree N status. Foliar ¹⁵N-urea application reduced the mobilization of existing N in the leaves regardless of leaf N status. More ¹⁵N was mobilized on a leaf area basis than that from existing N in the leaves with the low N trees showing the largest difference. On a whole-tree basis, the increase in the amount of reserve N caused by foliar urea treatment was similar. We conclude that low N trees are more effective in utilizing N from foliar urea than high N trees in the fall.

New roots of Malus domestica Borkh MM106 apple rootstock were divided into two categories, 1) feeder roots and 2) extension roots based on morphology and their ability to take up NH₄ ⁺, were studied. The roots were harvested in August from 1-year-old potted plants growing under natural conditions in Corvallis, Ore. Extension roots were thicker and longer than feeder roots. Average diameter and length were 0.89 and 45.29 mm for extension roots and 0.27 and 5.36 mm for feeder roots. Root special length (cm/g FW) and surface area (cm²/g FW) were 11.94 and 33.17 for extension roots and 108.97 and 93.38 for feeder roots. Maximum uptake rate, I_max, K_m, an_d root absorption power, α (α = I_max•1/K_m), for NH₄ ⁺ absorption were 6.875, 0.721, and 9.48 for extension roots and 4.32, 0.276, and 15.63 for feeder roots. Feeder roots had stronger affinity to NH₄ ⁺ (low K_m) and higher NH₄ ⁺ absorption power (high α value) than extension roots. The feeder roots were better able to uptake NH₄ ⁺ at lower external solution concentrations than extension roots according to the nutrient depletion curve, which indicates feeder roots being more efficient than extension roots in nutrient absorption when NH₄ ⁺ availability was low.

The nutrient uptake kinetics by new roots of 1-year-old potted clonal apple rootstocks (M7, M9, M26, M27, MM106, and MM111) were determined by the ion depletion technique at the stable development stage of trees in August. The total roots of five of the rootstocks (except MM111) consisted of more than 60% feeder roots and less than 12% extension roots. MM111, the most vigorous rootstocks tested, had 60.7% feeder roots and 24.5% extension roots. Root: top ratio was negatively related to the growth inhibiting character of the rootstock. Nutrient uptake by excised new roots was found to fit into Michaelis-Menton kinetic model for all rootstocks tested. The kinetic characteristics (maximum uptake rate, I_max, apparent Michaelis-Menton constant, K_m, and root absorption power, (α = I_max•1/K_m) between rootstocks differed significantly. MM111 had the highest I_max for NH₄ ⁺ absorption and M9 for NO₃ ^-. Root affinity to ions was highest with MM106 for NH₄ ⁺ and with M26 for NO₃ ^-. Root absorption power (α = I_max•1/K_m) was greatest in MM106 for NH₄ ⁺ and M9 for NO₃ ^-. At this developmental stage the data suggest no relationship between nutrient uptake and dwarfing character of the rootstocks.

Sorbitol is the predominant phloem-translocated carbohydrate in apple. The pathway—either apoplastic or symplastic—of sugar transport from photosynthetic cells to the phloem is not established. Furthermore, the presence of absence of phloem loading has not been tested. This study characterized the morphology and physiology of sugar movement to the phloem in apple leaves. An electron micrographic survey of apple leaf minor vein morphology was performed. Plasmodesmata were abundant and found at the interfaces of each cell type from mesophyll to sieve elements, indicating a symplastic sugar pathway. We also tested for a phloem loading mechanism. First, ¹⁴C-labeled sorbitol and sucrose were introduced exogenously to leaf discs to determine if they are loaded into veins from the apoplast. Although leaf discs floated on a solution containing either sugar actively accumulated label, the labeling pattern was diffuse, with no accumulation in minor veins. The addition of the sulfhydryl reagent PCMBS to the leaf disc assay inhibited sugar uptake. We also attempted plasmolysis of apple leaf sections to measure the solute concentration difference between photosynthetic mesophyll cells and cells of the minor vein phloem. Apple leaf pieces fixed in a solution containing 1.5 mol/kg osmoticum did not plasmolyze. We conclude that although active uptake of both sorbitol and sucrose takes place in apple leaves, apoplastic phloem-loading is absent. Considering the high sugar concentration and the symplastic connectivity among leaf cell types, we propose that sugars are instead enter the phloem after moving down—rather than against—a concentration gradient.