Photosynthetic enzymes and carbohydrate metabolism of apple ...

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Department of Horticulture, Cornell University, Ithaca, NY14853, USA. (e-mail: [email protected]). (Accepted 15 July 2004). SUMMARY. One-year-old apple ...
Journal of Horticultural Science & Biotechnology (2004) 79 (6) 923–929

Photosynthetic enzymes and carbohydrate metabolism of apple leaves in response to nitrogen limitation By LI-SONG CHEN1 and LAILIANG CHENG* Department of Horticulture, Cornell University, Ithaca, NY14853, USA (e-mail: [email protected])

(Accepted 15 July 2004)

SUMMARY One-year-old apple (Malus domestica Borkh. cv. Gala) trees were supplied twice weekly for 5 weeks with 500 ml of a modified Hoagland’s solution at a nitrogen (N) concentration of 0, 5, 10, or 15 mM. Both CO2 assimilation and stomatal conductance decreased with decreasing leaf N. However, the calculated intercellular CO2 concentration increased as leaf N decreased. The chlorophyll a/b ratio remained unchanged as N supply decreased, except for a slight drop at 0 mM N. On a leaf area basis, the activities of key enzymes in the Calvin cycle [ribulose-1,5bisphosphate carboxylase/oxygenase (Rubisco), NADP-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoribulokinase (PRK), stromal fructose-1,6-bisphosphatase (FBPase)] and those in end-product synthesis [cytosolic FBPase, aldose-6-phosphate reductase (A6PR), sucrose phosphate synthase (SPS), and ADP-glucose pyrophosphorylase (AGPase)] decreased linearly with decreasing leaf N. Contents of glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P) increased slightly as leaf N decreased from 2.39 g m–2 to 1.31 g m–2, then decreased in the lowest N leaves. The ratio of G6P/F6P remained unchanged over the leaf N range examined. The content of 3-phosphoglycerate (PGA) decreased linearly with decreasing leaf N. Starch content increased with decreasing leaf N both at dusk and pre-dawn. However, the contents of sorbitol, glucose, fructose, and sucrose decreased or remained unchanged as leaf N decreased. In conclusion, N limitation leads to accumulation of starch, but not soluble carbohydrates in apple leaves. Our data are consistent with the notion that N limitation restricts CO2 assimilation by directly limiting the activities of Rubisco and other enzymes, not by indirect feedback repression via sugar accumulation.

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ecause CO2 assimilation depends on the function of many proteins and enzymes in the photosynthetic system, N limitation leads to a decrease in CO2 assimilation capacity (Evans, 1989; Chen and Cheng, 2003). The decrease in CO2 assimilation is accompanied by decreases in the activities of ribulose-1,5bisphosphate carboxylase/oxygenase (Rubisco, EC4.1.1.39) and other enzymes involved in photosynthesis (Robinson and Baysdorfer, 1985; Terashima and Evans, 1988; Reddy et al., 1996; Chen and Cheng, 2003). However, the mechanism by which the activities of the enzymes are decreased remains unclear. Nitrogen limitation may directly reduce the amount of Rubisco and other photosynthetic enzymes. This action has been suggested in studies where decreases of CO2 assimilation and photosynthetic enzymes were not associated with increases in soluble carbohydrates (Reddy et al., 1996; Nakano et al., 1997; Chen and Cheng, 2003). Alternatively, N limitation leads to significant accumulation of soluble carbohydrates in leaves (Rufty et al., 1988; Paul and Driscoll, 1997; Geiger et al., 1999; Logan et al., 1999), which, in turn, may repress the expression of Rubisco and other enzymes in CO2 assimilation. Most studies of N limitation on CO2 assimilation have been conducted on plant species that synthesize sucrose *Author for correspondence. 1 Current address: Department of Horticulture, Fujian Agriculture and Forestry University, Fuzhou 350002, P. R. China.

and starch as the main photosynthetic end-products. In addition to sucrose and starch, many commercially important deciduous tree fruits in the Rosaceae family synthesize sorbitol, which serves as the major photosynthetic end-product and the primary translocatable carbohydrate and storage carbohydrate in these plants (Bieleski, 1982). Although it has been demonstrated that CO2 assimilation and Rubisco activity of apple leaves decrease in response to N limitation (Cheng and Fuchigami, 2000a; 2000b), the activities of other key enzymes in the Calvin cycle and carbohydrate metabolism, and their relationships with levels of non-structural carbohydrates, have not been determined. The objective of this study was to determine how N limitation affects CO2 assimilation, key enzymes in the Calvin cycle and end-product synthesis, metabolites, and levels of non-structural carbohydrates in apple leaves to better understand the mechanism by which N limitation leads to a decrease in CO2 assimilation in sorbitol synthesizing species.

MATERIALS AND METHODS Plant culture and N treatments One-year-old dormant apple (Malus domestica Borkh. cv. Gala) trees were pruned to two buds and transplanted into 7.6 l pots containing sand. The trees were grown under natural conditions at Cornell Orchards in Ithaca, New York, USA. Beginning from budbreak, extra shoots were removed, and only one shoot was allowed to grow

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Photosynthesis in N-limited apple leaves

on each plant. Starting from the second week after budbreak, each tree was supplied twice weekly with 500 ml of 10 mM N in a modified Hoagland’s solution (Cheng and Fuchigami, 2000a). When new shoots were approximately 30 cm long, uniform plants were selected for N treatments. Thereafter, they were supplied twice weekly with 500 ml of the modified Hoagland’s solution at an N concentration of 0, 5, 10, or 15 mM (from NH4NO3) to each pot. There were five replications for each N treatment with four trees per replication arranged in a completely randomized design. After 5 weeks, recent fully expanded leaves were chosen to measure gas exchange, chlorophyll (Chl) content, photosynthetic enzyme activities, metabolites, nonstructural carbohydrates and N. Gas exchange measurements Measurements were made with a CIRAS-1 portable photosynthesis system (PP systems, Herts, UK) at ambient CO2 (360 µmol mol–1). For all measurements, photon flux density (PFD), air temperature, and ambient water vapour pressure were kept at 1600 ± 50 µmol m–2 s–1, 28.0 ± 1.0°C, and 2.3 ± 0.1 kPa, respectively. Assay of key enzymes in the Calvin cycle and carbohydrate metabolism Leaf discs (1 cm2 in size) were taken at noon under full sun (PFD of 1600 µmol m–2 s–1), frozen in liquid N2 and stored at –80°C. Rubisco, NADP-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC1.2.1.12), phosphoribulokinase (PRK, EC2.7.1.19), fructose-1,6-bisphosphatase (FBPase, EC3.1.3.11), and sucrose phosphate synthase (SPS, EC2.4.1.14) were extracted according to Chen and Cheng (2003). Three (total of 3 cm2) frozen leaf discs were ground with a pre-cooled mortar and pestle in 1.5 ml extraction buffer containing 50 mM HEPES (pH 7.5), 10 mM MgCl2, 2 mM ethylenediaminetetraacetic acid (EDTA), 10 mM dithiothreitol (DTT), 1% (v/v) Triton X-100, 5% (w/v) insoluble polyvinylpolypyrrolidone (PVPP), 1% (w/v) bovine serum albumin (BSA), and 10% (v/v) glycerol. The extract was centrifuged at 13,000  g for 5 min in a microcentrifuge, and the supernatant was used immediately for enzyme activity assays. Total Rubisco activity was measured according to Cheng and Fuchigami (2000a). GAPDH activity was determined in a mixture (1 ml) of 100 mM Tricine (pH 8.0), 4 mM 3-phosphoglycerate (PGA), 5 mM ATP, 10 mM MgCl2, 0.2 mM NADPH and 20 units 3-phosphoglyceric phosphokinase (PCK, EC2.7.2.3). The reaction was initiated by adding the enzyme extract (Leegood, 1990). PRK activity was assayed in a mixture (1 ml) of 100 mM Tricine (pH 8.0), 0.5 mM ribose-5-phosphate (R5P), 1 mM ATP, 10 mM MgCl2, 50 mM KCl, 5 mM phosphoenolpyruvate (PEP), 0.4 mM NADH, 7 units pyruvate kinase (EC2.4.1.40), 10 units lactate dehydrogenase (LDH, EC1.1.1.27), and 1 unit R5P isomerase (EC5.1.3.4). The reaction was initiated by adding the enzyme extract (Leegood, 1990). Stromal FBPase activity was measured in a mixture (1 ml) of 50 mM Tris-HCl (pH 8.2), 10 mM MgCl2, 1 mM EDTA, 0.1 mM fructose-1,6-bisphosphate (FBP), 0.5 mM NADP, 4 units of phosphoglucoisomerase (PGI,

EC5.3.1.9) and 2 units of glucose-6-phosphate dehydrogenase (G6PDH, EC1.1.1.49). The reaction was initiated by adding the enzyme extract (Leegood, 1990; Holaday et al., 1992). Cytosolic FBPase activity was assayed according to Holaday et al. (1992) with some modifications. The enzyme activity was assayed in 1 ml reaction mixture containing 50 mM HEPES (pH 7.0), 2 mM MgCl2, 0.1 mM FBP, 0.5 mM NADP, 4 units of PGI, and 2 units of G6PDH. The reaction was initiated by adding the enzyme extract. SPS was assayed according to Grof et al. (1998). Leaf extract (60 µl) was incubated for 15 min at 30°C with 100 mM HEPES (pH 7.5), 100 mM KCl, 6 mM EDTA, 30 mM uridine-5’-diphosphoglucose (UDPG), 10 mM fructose-6-phosphate (F6P), and 40 mM glucose-6phosphate (G6P) in a total volume of 100 µl. At the end of the 15 min incubation period, the reaction was stopped by adding 100 µl ice-cold 1.2 M HClO4 and held on ice for another 15 min. The reaction mixture was neutralized by adding 60 µl of 2 M KHCO3, held on ice for 15 min, then centrifuged at 13,000  g for 1 min. A proportion (130 µl) of the supernatant was assayed for uridine-5’-diphosphate (UDP) by coupling to oxidation of NADH with LDH and pyruvate kinase. The reaction mixture (1 ml) contained 50 mM HEPES (pH 7.0), 5 mM MgCl2, 0.3 mM NADH, 0.8 mM PEP, 14 units LDH, and 4 units pyruvate kinase. The reaction was started by adding pyruvate kinase (Stitt et al., 1988). Controls without F6P and G6P were carried through for all the samples. ADP-glucose pyrophosphorylase (AGPase, EC2.7.7.27) was extracted and assayed as described previously in Chen and Cheng (2003). The only modification was that reduced glutathione (GSH) was not included in the extraction buffer. Aldose-6-phosphate reductase (A6PR, EC1.1.1.200) was extracted according to Negm and Loescher (1981) with some modifications. Three frozen leaf discs were ground with a pre-cooled mortar and pestle in 1.5 ml extraction buffer containing 100 mM Tris-HCl (pH 8.0), 5 mM DTT, 0.3% (v/v) Triton X-100, 5% insoluble PVPP, and 6% (v/v) glycerol. The extract was then centrifuged at 13,000  g for 5 min in a microcentrifuge, and the supernatant was used immediately for the assay. A6PR was assayed in the direction of sorbitol-6-phosphate synthesis by following the oxidation of NADPH in the presence of G6P as described by Negm and Loescher (1981). The reaction mixture (1 ml) contained 100 mM Tris-HCl (pH 9.0), 0.11 mM NADPH, and 50 mM G6P. The reaction was initiated by adding 25 µl of the enzyme extract. Determination of G6P, F6P and PGA Leaf tissues (40 cm2) were taken at noon under full sun (PFD of 1600 µmol m–2 s–1), frozen in liquid N2, and stored at –80°C until analysis. Metabolites were extracted and measured according to Chen et al. (2002) with some modifications (Chen and Cheng, 2003). Extraction and quantification of non-structural carbohydrates Three leaf discs (total of 3 cm2) were taken at dusk and at pre-dawn from the same leaf, frozen in liquid N2 and stored at –80°C. Sorbitol, sucrose, glucose, and fructose

LI-SONG CHEN and LAILANG CHENG were extracted with 80% (v/v) ethanol at 80°C and determined using a Dionex DX-500 series chromatograph, equipped with a Carbopac PA-1 column, a pulsed amperometric detector and a gold electrode (Dionex, Sunnyvale, CA) as previously described in Cheng and Fuchigami (2002). The tissue residue, after soluble sugar extraction, was retained for determination of starch. After digesting the residue with amyloglucosidase (EC3.2.1.3), starch was determined enzymatically as glucose equivalents using G6PDH and hexokinase (EC2.7.1.1) (Chen et al., 2002). Leaf N and Chl analysis Leaf N content was determined by a modified Kjeldahl method (Schuman et al., 1973). Leaf Chl was measured according to Arnon (1949).

linearly (Figure 1A, B). The Chl a/b ratio remained unchanged over the range of N supply, except for a slight drop at 0 mM N (Figure 1C). Both CO2 assimilation and stomatal conductance decreased with decreasing leaf N (Figure 2A, B), whereas the calculated intercellular CO2 concentration increased as leaf N decreased (Figure 2C). Activities of key enzymes in the Calvin cycle and carbohydrate metabolism On a leaf area basis, activities of all key enzymes in the Calvin cycle, Rubisco, GAPDH, PRK, and stromal FBPase, and those involved in end-product synthesis, cytosolic FBPase, A6PR, AGPase and SPS, all decreased linearly with decreasing leaf N (Figure 3A–H). When expressed on a leaf N basis, the activities of Rubisco, GAPDH, cytosolic FBPase, A6PR and AGPase decreased with decreasing leaf N (Figure 3I, J, M–O). The activities of PRK and stromal FBPase remained unchanged as leaf N decreased from 2.39 g m–2 to 1.31 g m–2, then dropped significantly in the lowest N leaves

Chl a/b ratio

RESULTS Leaf N, Chl and gas exchange As N supply decreased, leaf N and Chl decreased

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Leaf N content (g m2) FIG. 1 Effects of nitrogen (N) supply levels (mM) on N content (A); chlorophyll content (B); and the Chl a/b ratio (C) in apple leaves. Each point is mean ± standard error (n=5). Regression equations: (A) y = 0.871 + 0.107x (r2 = 0.971, P