Nitrogen storage and its interaction with carbohydrates of young apple trees in response to nitrogen supply

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1 Tree Physiology 24, Heron Publishing Victoria, Canada Nitrogen storage and its interaction with carbohydrates of young apple trees in response to nitrogen supply LAILIANG CHENG, 1,2 FENGWANG MA 1 and DAMAYANTHI RANWALA 1 1 Department of Horticulture, Cornell University, Ithaca, NY 14853, USA 2 Author to whom correspondence should be addressed (LC89@Cornell.edu) Received March 17, 2003; June 20, 2003; published online December 1, 2003 Summary Bench-grafted Fuji/M.26 apple (Malus domestica Borkh.) trees received a constant nitrogen (N) supply (10.7 mm) from bud break to the end of June, and were then fertigated with 0, 5, 10, 15 or 20 mm N in a modified Hoagland s solution for 2 months during the summer. In mid-october, half of the trees fertigated at each N concentration were sprayed twice with 3% urea, whereas the remaining trees served as controls. All trees were harvested after natural leaf fall and were stored at 2 C. Five trees from each of the N treatment combinations were destructively sampled during dormancy to determine the composition of N and total nonstructural carbohydrates (TNC). As the N supply from fertigation increased, amounts of N in both free amino acids and proteins increased, whereas C/N ratios decreased. Foliar urea applications in the fall significantly increased amounts of N in both free amino acids and proteins, but decreased their C/N ratios. Arginine, the most abundant amino acid in both free amino acids and in proteins, accounted for an increasing proportion of N in free amino acids and proteins with increasing N supply from fertigation or foliar urea application. The ratio of protein N to free amino acid N decreased from about 27.1 to 3.2 as N supply from fertigation increased from 0 to 20 mm, and decreased further to 3.0 in response to foliar urea applications in the fall. Concentrations of glucose, fructose, sucrose and TNC decreased as the N supply from fertigation increased, whereas concentrations of sorbitol and starch remained relatively unchanged. Foliar urea applications decreased the concentration of each TNC component and the TNC concentration in each N fertigation treatment. A negative linear relationship was found between carbon in TNC and N in proteins and free amino acids. The sum of carbon in TNC, proteins and free amino acids remained constant in response to N supply from fertigation. However, foliar urea applications decreased the sum of carbon in proteins, free amino acids and TNC because about 21% of the decrease in TNC carbon was not recovered in free amino acids or proteins. Young apple trees store N and carbon dynamically in response to N supply. As N supply increases, an increasing proportion of N is found in the form of free amino acids, which have a low carbon cost, although proteins remain the main form of N storage. Furthermore, part of the carbon from TNC is incorporated into amino acids and proteins, decreasing the carbon stored as TNC and increasing the carbon stored as amino acids and proteins. Key words: C/N ratio, foliar urea, free amino acids, Malus domestica, nitrogen fertigation, proteins, total nonstructural carbohydrates (TNC). Introduction Apple trees accumulate nitrogen (N) and nonstructural carbohydrates before leaf fall, store them over the winter, and remobilize them for initial growth and development the following spring (reviewed by Titus and Kang 1982, Tromp 1983, Oliveira and Priestley 1988, Loescher et al. 1990, Millard 1996). Reserve N is present in the form of proteins or free amino acids. Nonstructural carbohydrates include starch and soluble sugars (sucrose, glucose, fructose and sorbitol). Both reserve N and carbohydrates play an essential role in supporting new growth by providing structural components and energy. The initial growth of young apple trees in the spring is primarily determined by reserve N, and not by reserve carbohydrates (Tromp 1983, Cheng and Fuchigami 2002). However, whether proteins or free amino acids dominate in the storage pool of N remains controversial. In dormant young apple (Oland 1959), peach (Prunus persica L.) (Taylor and May 1967) and pear (Pyrus communis L.) (Taylor et al. 1975) trees, under conditions of high N supply, up to 50% of the N was found in soluble form, mainly as free amino acids. Considering that only part of the protein N is remobilizable in the spring, these authors concluded that free amino acids are the main form of storage N. In contrast, Kang and Titus (1980) showed that proteins accounted for 90% of total N in the bark tissue of dormant 1-year-old Golden Delicious apple trees. Based on this and other findings, Titus and Kang (1982) argued that proteins are the main storage form of N, with free amino acids of secondary importance. However, this has not been tested on a whole-tree basis over a wide range of N supply. Storing N in the form of either proteins or free amino acids requires carbon inputs to provide a carbon skeleton and energy

2 92 CHENG, MA AND RANWALA supply (Taylor et al. 1975, Faust 1989). Cheng and Fuchigami (2002) showed that when tree N concentration was increased by N fertigation during the summer or by foliar urea applications in the fall, concentrations of nonstructural carbohydrates decreased. This clearly demonstrated the carbon cost associated with assimilating inorganic N. However, it is not known what proportion of the carbon that is lost from carbohydrates ends up in the amino acids and proteins. In addition, it is unclear if storing N in free amino acids versus proteins in apple trees gives the tree an advantage in terms of carbon cost. In this study, we determined the forms of stored N and nonstructural carbohydrates of 1-year-old apple trees grown under a wide range of N supply rates to understand the dynamics of N storage and its interaction with carbohydrates. Materials and methods Plant culture and nitrogen treatments Fuji apple (Malus domestica Borkh.) trees on M.26 rootstock were bench-grafted in late March. Each grafted tree was potted in a 3.8-l container in a 1:2:1 (v/v) mixture of peat moss, pumice and sandy loam soil. The trees were grown in a lath house until early June. During this period, beginning from bud break in early May, they were fertigated every 2 weeks with 300 ml of 10.7 mm N, in a 20:10:20 N,P,K water soluble fertilizer with micronutrients. When new shoots were approximately 15 cm long, the plants were selected for uniformity, and moved into full sunlight. For the following 3 weeks, they were fertigated weekly with 300 ml of 10.7 mm N as described above. Beginning on June 30, plants were randomly assigned to one of five N treatments (0, 5, 10, 15 or 20 mm N from NH 4 NO 3 ). Each N treatment was replicated five times (two plants each) in a completely randomized design, with a guard row on each of the four edges of the experimental trees. The trees were fertigated twice a week for 2 months, with 300 ml of a modified Hoagland s solution per pot (Cheng and Fuchigami 2000). Plants were subirrigated from a saucer beneath each pot. All plants had stopped shoot elongation by September 25. Five trees fertigated at each N concentration were sprayed with 3% urea twice on October 13 and 21. The other five trees at each N fertigation concentration served as unsprayed controls. Leaves of trees fertigated with the lowest concentration of N (0 mm) began to abscise on November 2, which was approximately 5 to 10 days earlier than those fertigated with medium to high N concentrations. Foliar urea applications advanced leaf abscission by about 3 days. By November 30, leaf abscission was completed in all treatments and all trees were removed from the pots and freed of rooting medium on the same day. They were stored in a cold room at 2 C with the roots buried in moist sawdust. Five trees from each of 10 treatments were destructively sampled on March 1 the following year when they were still dormant. Each tree was divided into 1-year-old stem, rootstock shank and roots. All samples were frozen at 80 C, freeze-dried, and ground to pass a 40 mesh screen. A composite sample was made for each tree, based on its dry matter distribution among different parts, to determine the chemical composition of N-containing compounds and nonstructural carbohydrates. Chemical analysis of nitrogen and carbohydrates Free amino acids were extracted from 300 mg of tissue sample with 7 ml 0.1 M sodium citrate buffer (ph 4.0) for 24 h at 4 C (Oland 1959). During extraction, samples were sonicated for 15 min. The extract was centrifuged at 13,000 g for 15 min and the supernatant was directly used for amino acid analysis. For total amino acids, each 100 mg tissue sample was hydrolyzed in 10 ml 6 M hydrochloric acid at 110 C for 22 h. After filtration, the total volume was brought to 25 ml, an aliquot of which was taken to remove HCl, then dissolved in a ph 2.2 citrate buffer. After dilution, 50 µl was injected into a Beckman 121 automatic amino acid analyzer, equipped with an FR-10 spherical cation exchange resin column (Beckman Instruments, Fullerton, CA). Individual amino acids were eluted at 54 C with a citrate buffer at ph 3.28 for 14 min, ph 3.90 for 10 min, then by elution at ph 5.26 for 14 min. The column was regenerated with 0.2 M NaOH for 5 min, then equilibrated with a citrate buffer at ph 3.28 for 6 min before the next injection. Standard amino acids were added to tissue samples during extraction to determine the recovery of individual amino acids following the same extraction procedures. The recovery of free arginine and arginine after protein hydrolysis was 89.7 and 85.4%, respectively. The recovery of free amino acids and total amino acids after protein hydrolysis was approximately 90 and 80%, respectively. Tissue N concentration was determined colorimetrically with an autoanalyzer after micro-kjeldahl digestion (Schuman et al. 1973), which did not account for nitrate-n. For soluble carbohydrates, 50 mg composite samples, with xylitol added as an internal standard, were extracted three times at 70 C with 80% ethanol (3 ml each, 30 min per extraction). Tissue suspensions were centrifuged at 4000 g for 10 min after each extraction, and the supernatants were combined. The extract was passed through ion exchange columns consisting of 1 ml Amberlite IRA-67 (acetate form) (Sigma) and 1 ml Dowex 50W (hydrogen form) (Sigma) to remove charged material. The extract was then evaporated to dryness at 55 C and dissolved in 10 ml of water. After appropriate dilution, 25 µl was injected into a Dionex DX-500 series chromatograph, equipped with a Carbopac PA-1 column, a pulsed amperometric detector and a gold electrode (Dionex, Sunnyvale, CA). Carbohydrates were eluted at a flow rate of 1.0 ml min 1 with 200 mm NaOH for 15 min. The concentration of individual soluble carbohydrates was determined based on peak area and the calibration curve derived from the corresponding standard authentic sugar. Adjustments were made based on the recovery of the internal standard for each sample. After soluble sugar extraction, the tissue residue was dried and digested with amyloglucosidase at 55 C in a sodium acetate buffer (ph 4.5) overnight to convert starch to glucose. The concentration of glucose was quantified by HPLC. Total nonstructural carbohydrates (TNC) were the sum of starch and soluble sugars. TREE PHYSIOLOGY VOLUME 24, 2004

3 NITROGEN STORAGE AND ITS INTERACTION WITH CARBOHYDRATES 93 Calculations and statistical analysis Nitrogen in free amino acids or total amino acids after protein hydrolysis was the sum of N from each individual amino acid adjusted for recovery. The difference between total N in amino acids after protein hydrolysis and that in free amino acids was considered as protein N. Carbon in nonstructural carbohydrates was calculated as the sum of carbon in sorbitol, glucose, fructose, sucrose and starch. Analysis of variance (ANOVA) for a 5 (N fertigation) 2 (foliar N) factorial design was used for most variables measured in this experiment. Linear regression analysis was used for the total carbon in TNC in relation to total N in proteins and free amino acids, free arginine or total arginine in relation to tissue N, and the increase in carbon in proteins and free amino acids in relation to the decrease in carbon in TNC caused by foliar urea applications. Results Dry mass of dormant Fuji/M.26 trees Whole-tree dry mass at destructive sampling in response to N fertigation has been reported previously (Cheng and Fuchigami 2002). Plant dry mass increased almost linearly up to 10 mm N, then leveled off with further increases in N supply. Nitrogen stored as free amino acids and proteins The tissue concentration of free amino acid N increased as a consequence of increasing N supply from fertigation (Figure 1A). Foliar urea application in the fall increased the concentration of free amino acid N at each N fertigation concentration, with the increase being larger in trees fertigated with low N concentrations than those fertigated with high N concentrations. As N fertigation increased, the C/N ratio of free amino acids decreased, reaching minimum values at N concentrations of 15 to 20 mm (Figure 1B). Foliar urea application in the fall significantly decreased the C/N ratio of free amino acids of trees fertigated with 0 and 5 mm N. Arginine was the most abundant amino acid in the free amino acid pool. Free arginine concentration showed a similar response to N fertigation and foliar urea application as the N in free amino acids (data not shown). The ratio of N in free arginine to total N in free amino acids increased curvilinearly from 0.5 to 0.8 as N fertigation concentration increased from 0 to 20 mm (Figure 1C). Foliar urea application significantly increased this N ratio in trees fertigated with 0 and 5 mm N. The concentration of protein N increased linearly as N supplied from fertigation increased (Figure 2A). Foliar urea application in the fall increased protein N at each N fertigation concentration, but trees fertigated with low N concentrations were more responsive than those fertigated with high N concentrations. All trees that received foliar urea application reached a similar concentration of protein N. The C/N ratio of proteins decreased from 4 to 3.5 as N fertigation increased from 0 to 20 mm (Figure 2B). Foliar urea application in the fall decreased the C/N ratio of proteins at each N fertigation concentration. All trees treated with foliar urea had a C/N ratio Figure 1. Nitrogen (N) concentration (A), C/N ratio (B), and the proportion of N present as arginine (C) in free amino acids of dormant Fuji/M.26 trees in response to N fertigation during the growing season and foliar urea application in the fall. Each point is the mean ± SE, n = 5. All three indices were analyzed by ANOVA for a 5 2 factorial design. P-Values for N fertigation, foliar urea, and the interaction between the two are all < of proteins of approximately 3.3. Compared with free amino acids, proteins had a much higher C/N ratio at each N fertigation concentration (Figure 1B and 2B). The arginine set free by hydrolysis of proteins responded to N fertigation and foliar urea application in a similar way as protein N (data not shown). Arginine accounted for an increased proportion of protein N as N supply from fertigation increased (Figure 2C). Foliar urea application further increased the proportion of protein N present in arginine to a similar value at all N fertigation concentrations. Trees fertigated with low N concentrations responded more to foliar urea application than those fertigated with high N concentrations. The sum of N in proteins and free amino acids increased as N supply from fertigation increased (Figure 3A). Foliar urea application increased the N in proteins and free amino acids to a similar value in all N fertigation treatments. The C/N ratio of proteins plus free amino acids showed a similar response to N TREE PHYSIOLOGY ONLINE at

4 94 CHENG, MA AND RANWALA supply as that of proteins alone, but with a smaller value at each N supply rate (Figure 3B). The ratio of N in proteins to that in free amino acids decreased curvilinearly from 27.1 to 3.2 as N supply from fertigation increased from 0 to 20 mm (Figure 3C). Foliar urea application in the fall decreased the N ratio to about 3.0 across N fertigation concentrations, with the decrease being larger in trees fertigated with low N concentrations than in those fertigated with high N concentrations. Both free arginine N and the total arginine N (N from free arginine plus N from arginine in proteins) were closely correlated with tissue N concentration across all treatments (Figure 4). Nonstructural carbohydrates Sorbitol concentration decreased only slightly at the two highest N fertigation rates (Figure 5A). Foliar urea application in the fall decreased sorbitol concentration at each N fertigation concentration, with the decrease being larger at the lower N fertigation concentrations. Glucose, fructose and sucrose concentrations responded similarly to N fertigation and foliar urea applications (Figure 5B D), decreasing significantly as N supply from fertigation increased. Foliar urea application in the fall further decreased glucose, fructose and sucrose concentrations at each N fertigation rate, with the decrease being larger in trees fertigated with low N concentrations. Starch concentration did not change significantly in response to N fertigation (Figure 5E). However, foliar urea application in the fall significantly decreased starch concentration in each N fertigation treatment. Figure 2. Nitrogen (N) concentration (A), C/N ratio (B) and the proportion of N present as arginine (C) in proteins of dormant Fuji/M.26 trees in response to N fertigation during the growing season and foliar urea application in the fall. Each point is a mean ± SE, n=5. All three indices were analyzed by ANOVA for a 5 2 factorial design. P-Values for N fertigation, foliar urea and the interaction between the two are all < Figure 3. The sum of nitrogen (N) concentration in proteins and free amino acids (A), C/N ratio of proteins plus free amino acids (B), and the ratio of protein N to free amino acid N (C) of dormant Fuji/M.26 trees in response to N fertigation during the growing season and foliar urea application in the fall. Each point is a mean ± SE, n = 5. All three indices were analyzed by ANOVA for a 5 2 factorial design. P-Values for N fertigation, foliar urea and the interaction between the two are all < TREE PHYSIOLOGY VOLUME 24, 2004

5 NITROGEN STORAGE AND ITS INTERACTION WITH CARBOHYDRATES 95 Figure 4. Free arginine ( ) and total arginine ( ), including free arginine and protein arginine, in relation to nitrogen (N) concentration of dormant Fuji/M.26 apple trees. Regression equations: for free arginine, y = x (r 2 = 0.97, P < ), and for total arginine, y = x (r 2 = 0.98, P < ). The concentration of TNC showed a curvilinear response to N supply from fertigation (Figure 5F). Trees fertigated without N had the highest TNC concentration. Total nonstructural carbohydrates decreased with increasing N supply from fertigation, reaching the lowest concentration when N fertigation concentration was highest. Foliar urea application in the fall decreased TNC concentration for each N fertigation regime, bringing the TNC concentration to a similar lower concentration in all N fertigation treatments. Interaction between nitrogen and carbohydrates When data from all treatments were pooled, a negative linear relationship was found between N in total amino acids (free amino acids plus protein amino acids) and carbon in nonstructural carbohydrates (Figure 6). As N supplied from fertigation increased, the carbon in TNC decreased, whereas the carbon in proteins and free amino acids increased (Figures 7A and 7B). Foliar urea application in the fall decreased the amount of carbon in TNC, but increased the amount of carbon in proteins and free amino acids across the N fertigation treatments. As a result, the sum of carbon in proteins, free amino acids and nonstructural carbohydrates did not change significantly in response to N fertigation (Figure 7C). Foliar urea application in the fall slightly decreased the sum of carbon in total proteins, free amino acids and TNC. The increase in carbon in proteins and free amino acids caused by foliar urea application was proportional to the decrease in carbon in TNC (Figure 8). Approximately 79% of the decrease in carbon in TNC caused by foliar urea application was recovered in proteins and amino acids. Discussion Our data showed that young apple trees store N dynamically in response to N supply. At low N supply, most of the N was in proteins, with only a small proportion in free amino acids. However, as N supply increased through N fertigation or foliar urea application, the proportion of free amino acid N increased Figure 5. Concentrations of sorbitol (A), glucose (B), fructose (C), sucrose (D), starch (E) and total nonstructural carbohydrates (TNC) (F) of dormant Fuji/M.26 apple trees in response to nitrogen (N) fertigation during the growing season and foliar urea application in the fall. Each point is a mean with standard error of five replicates. Solid circles are trees that were only fertigated with five N concentrations during the growing season, whereas the open circles are trees that also received foliar urea applications. All the indices were analyzed by ANOVA for a 5 2 factorial design. For sorbitol, P-values are 0.005, < and < for N fertigation, foliar urea and the interaction between the two. For glucose, fructose and sucrose, P-values are all < for N fertigation, foliar urea, and the interaction between the two. For starch, P-value for foliar urea is < , with no effect of N fertigation or interaction between N fertigation and foliar urea. For TNC, P-values are , < and < for N fertigation, foliar urea and the interaction between the two. TREE PHYSIOLOGY ONLINE at

6 96 CHENG, MA AND RANWALA Figure 6. Carbon in total nonstructural carbohydrates (TNC) in relation to nitrogen (N) in proteins and free amino acids of dormant Fuji/M.26 apple trees. Symbols: = trees that were only fertigated with five N concentrations during the growing season, and = trees that also received fall applications of foliar urea. Regression equations: y = x (r 2 = 0.83, P < ). significantly (Figure 3C). This trend is consistent with that found previously in dormant tissues of young apple (Oland 1959, Tromp 1970), peach (Taylor and May 1967) and pear (Taylor et al. 1975) trees. Tromp (1970) suggested that protein synthesis might not keep up with a high N supply, thus leading to an accumulation of free amino acids. We found that the C/N ratio of free amino acids was much lower than that of proteins due to the higher proportion of arginine present in free amino acids (Figures 1 and 2). This indicates that, in terms of carbon cost, storing N in free amino acids is more efficient than storing N in proteins. These free amino acids are readily available when growth resumes in the spring. Kang et al. (1982) suggested that the immediate demand for N in early growth in the spring is met by the transport of free amino acids present in adjacent bark tissues and those redistributed from wood tissues. As growth proceeds, hydrolysis of proteins of the aerial parts of the tree and translocation of amino acids from roots become more important for supplying N to the growing tissues (Kang et al. 1982, Khemira 1995, Malaguti et al. 2001). Although N in the form of free amino acids accounts for an increasing proportion of total N in apple trees as N supply increases, proteins are still the main form in which N is stored, even at the highest N supply (Figure 3C). This is in general agreement with the result of Kang and Titus (1980) with dormant apple bark tissues. Proteins in a dormant apple tree can be divided into those that can be remobilized to support new growth the following spring and those that cannot. Only the former serve as reserve N. Approximately 50% of the total N in a dormant young apple tree is reserve N (Neilsen et al. 2001, Cheng and Fuchigami 2002). Assuming that this 50% rule applies to protein N, whereas all the N in free amino acids is reserve N, the ratio of protein N to free amino acid N (Figure 3C) indicates that reserve N in proteins accounts for about 60% of the N storage pool on a whole-tree basis even at the highest N supply rate. This contrasts with some previous findings on dormant young apple (Oland 1959), peach (Taylor and May 1967) and pear (Taylor et al. 1975) trees that indicate about Figure 7. Carbon (C) in total nonstructural carbohydrates (TNC) (A), carbon in proteins and free amino acids (B), and sum of carbon in proteins, free amino acids and TNC (C) of dormant Fuji/M.26 trees in response to nitrogen (N) fertigation during the growing season and foliar urea application in the fall. Each point is a mean ± SE, n=5. All three indices were analyzed by ANOVA for a 5 2 factorial design. In (A), P-values for N fertigation, foliar urea and the interaction between the two are , < and < , respectively. In (B), P-values for N fertigation, foliar urea and the interaction between the two are all < In (C), P-value for foliar urea is , with no significant effect of N fertigation or interaction between N fertigation and foliar urea. 50% of the total N is present in soluble form, mainly as amino acids, when N supply is high. The reason for the contrasting results is unclear, but variation in timing of sampling may have contributed to the discrepancy. It is clear from the data of Oland (1959), Tromp (1970) and O Kennedy et al. (1975) that the closer to bud break, the higher the proportion of free amino acids present in the total N pool as a result of protein hydrolysis. Oland (1959) sampled apple trees for reserve N analysis in the middle of May, which must be close to bud break even in Norway, a high latitude country. Analysis of protein amino acids enabled us to calculate the total carbon and the C/N ratio of proteins, but it did not allow TREE PHYSIOLOGY VOLUME 24, 2004

7 NITROGEN STORAGE AND ITS INTERACTION WITH CARBOHYDRATES 97 Figure 8. Relationship between increase in carbon in proteins and amino acids and decrease in carbon in total nonstructural carbohydrates (TNC) caused by foliar urea application. Each value represents the difference between means with and without foliar urea application at each of five nitrogen fertigation concentrations. Regression equation: y = x (r 2 = 0.92, P < 0.01). us to identify which proteins are the major players in N storage. O Kennedy and Titus (1979) separated total proteins extracted from apple bark tissues into three working groups, designated as peak I, II and III proteins. Predominant accumulation of arginine-rich peak III proteins in the bark takes place in the later stages of leaf senescence (Kang and Titus 1980). All three groups of proteins break down when growth resumes in the spring, but they show differential rates of hydrolysis, and considerable variations were observed among experiments for the same group of proteins (O Kennedy and Titus 1979, Kang et al. 1982). Nonetheless, several proteins have been identified in apple bark tissues that meet the criteria set forth by O Kennedy and Titus (1979), i.e., storage proteins are prominent proteins in dormant tissues that disappear or at least decrease as growth resumes. These include proteins with molecular masses of 16, 17, 30, 38 and 56 kda (Kang et al. 1982, Khemira 1995). However, each of these individual storage proteins accounts for only a small proportion of the protein N. This is quite different from what was found in poplar (Populus spp.) trees where a single 32 kda bark storage protein accounted for a large proportion of protein N (Coleman et al. 1991). Theoretically, any protein that is rich in high N-containing amino acids could have a potential role in N storage. As N supply increased, arginine N accounted for an increasing proportion of the protein N in our experiment (Figure 2C). As a result, the C/N ratio of proteins decreased with increasing N supply, making N storage more efficient in terms of carbon investment. Tromp and Ovaa (1973) also found that arginine accounted for a larger proportion of protein N in trees with a high N status than in trees with a low N status. The close correlation of arginine concentration with tissue N concentration (Figure 4) also makes arginine a good indicator of tree N status. The negative relationship between N in free amino acids and proteins, and carbon in nonstructural carbohydrates (Figure 6), indicates that increased amino acid and protein synthesis with increasing N supply comes at the expense of nonstructural carbohydrates. Nonstructural carbohydrates provide the carbon skeleton and energy supply for synthesis of amino acids and proteins (Taylor et al. 1975, Faust 1989). Among the nonstructural carbohydrates, glucose, fructose, and sucrose concentrations decreased significantly in response to increasing N fertigation, whereas sorbitol and starch concentrations remained relatively unchanged (Figures 5A E). This suggests that glucose, fructose and sucrose are more easily metabolized than sorbitol and starch. When the demand for nonstructural carbohydrates was dramatically increased by foliar urea applications in the fall, all components of nonstructural carbohydrates decreased (Figures 5A E). Of the carbon decrease in TNC caused by foliar urea application in the fall, 79% was recovered in proteins and free amino acids (Figure 8). The rest (21%) may have been used in respiration and other processes. As a result, the sum of carbon in proteins, free amino acids and nonstructural carbohydrates was slightly lower in the trees sprayed with foliar urea than in trees fertigated only (Figure 7C). In contrast, increasing N supply via N fertigation during the summer did not decrease the sum of carbon in TNC and total amino acids (Figure 7C), although it decreased carbon in TNC (Figure 7A). Considering that both CO 2 assimilation capacity and leaf area duration increased with increasing N supply from fertigation (Cheng and Fuchigami 2000), the respiratory carbon loss may have been compensated for by the increased carbon availability at higher N fertigation concentrations. We did not determine how different parts of the tree respond to N fertigation during the summer and during the foliar urea application in the fall as N storage and its interaction with carbohydrates of the whole tree was the focus of this study. If transport of reserve N becomes a limiting factor during remobilization the following spring, changes in its distribution within the tree could have an impact on its reutilization. However, this does not seem to be the case as the amount of N remobilized for new shoot and leaf growth is linearly related to the total amount of N in dormant young apple trees (Cheng and Fuchigami 2002). In addition, the N derived from foliar urea application late in the season is translocated to the root system of young apple (Han et al. 1989, Cheng et al. 2002, Dong et al. 2002), young nectarine (Tagliavini et al. 1998) and fieldgrown peach and nectarine (Rosecrance et al. 1998) trees. Because the current study was performed on 1-year-old vegetative apple trees, our findings may be more applicable to apple nursery trees than apple bearing trees in orchards, as bearing trees have a much larger storage capacity for both N and carbohydrates, which may buffer significant changes in N and TNC caused by alteration in N supply. However, young apple (Neilsen et al. 2001, Cheng and Fuchigami 2002), 5-year-old pear (Sanchez et al. 1991), mature almond (Prunus dulcis (Mill.) D.A. Webb) (Weinbaum et al. 1987) and walnut (Juglans regia L.) (Weinbaum and Kessel 1998) trees are similar in that they all remobilize approximately 50% of the total tree N to support new growth the following year. In conclusion, N storage in young apple trees is a dynamic process that interacts with carbohydrate metabolism in response to N supply. Under low N supply, most N is stored in TREE PHYSIOLOGY ONLINE at

8 98 CHENG, MA AND RANWALA proteins, with only a small fraction stored in free amino acids, and high concentrations of nonstructural carbohydrates accumulate. As N supply increases, part of the carbon from TNC is incorporated into free amino acids and proteins, leading to a decrease in the carbon stored in TNC. The proportion of N in the form of free amino acids increases to make N storage more cost-effective, but proteins remain the main form of storage N, even at very high N supply. Acknowledgments This work was supported, in part, by the Washington Tree Fruit Research Commission. We gratefully acknowledge the help of Drs. Anil Ranwala and Bill Miller with carbohydrate analysis. Reference Cheng, L. and L.H. Fuchigami Rubisco activation state decreases with increasing nitrogen content in apple leaves. J. Exp. Bot. 51: Cheng, L. and L.H. Fuchigami Growth of young apple trees in relation to reserve nitrogen and carbohydrates. Tree Physiol. 22: Cheng, L., S. Dong and L.H. 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