Planta (2010) 232:511–522 DOI 10.1007/s00425-010-1194-x
ORIGINAL ARTICLE
Metabolism of organic acids, nitrogen and amino acids in chlorotic leaves of ‘Honeycrisp’ apple (Malus domestica Borkh) with excessive accumulation of carbohydrates Huicong Wang • Fangfang Ma • Lailiang Cheng
Received: 7 April 2010 / Accepted: 7 May 2010 / Published online: 20 May 2010 Ó Springer-Verlag 2010
Abstract Metabolite profiles and activities of key enzymes in the metabolism of organic acids, nitrogen and amino acids were compared between chlorotic leaves and normal leaves of ‘Honeycrisp’ apple to understand how accumulation of non-structural carbohydrates affects the metabolism of organic acids, nitrogen and amino acids. Excessive accumulation of non-structural carbohydrates and much lower CO2 assimilation were found in chlorotic leaves than in normal leaves, confirming feedback inhibition of photosynthesis in chlorotic leaves. Dark respiration and activities of several key enzymes in glycolysis and tricarboxylic acid (TCA) cycle, ATP-phosphofructokinase, pyruvate kinase, citrate synthase, aconitase and isocitrate dehydrogenase were significantly higher in chlorotic leaves than in normal leaves. However, concentrations of most organic acids including phosphoenolpyruvate (PEP), pyruvate, oxaloacetate, 2-oxoglutarate, malate and fumarate, and activities of key enzymes involved in the anapleurotic pathway including PEP carboxylase, NADmalate dehydrogenase and NAD-malic enzyme were significantly lower in chlorotic leaves than in normal leaves. Concentrations of soluble proteins and most free amino acids were significantly lower in chlorotic leaves than in normal leaves. Activities of key enzymes in nitrogen assimilation and amino acid synthesis, including nitrate reductase, glutamine synthetase, ferredoxin and NADHdependent glutamate synthase, and glutamate pyruvate H. Wang F. Ma L. Cheng (&) Department of Horticulture, Cornell University, 134A Plant Science, Ithaca, NY 14853, USA e-mail:
[email protected] H. Wang Department of Horticulture, South China Agricultural University, Guangzhou 510642, China
transaminase were significantly lower in chlorotic leaves than in normal leaves. It was concluded that, in response to excessive accumulation of non-structural carbohydrates, glycolysis and TCA cycle were up-regulated to ‘‘consume’’ the excess carbon available, whereas the anapleurotic pathway, nitrogen assimilation and amino acid synthesis were down-regulated to reduce the overall rate of amino acid and protein synthesis. Keywords Accumulation of non-structural carbohydrates Leaf chlorosis Nitrogen and amino acid metabolism Organic acid metabolism Respiration Abbreviations ATP-PFK CS DTT EDTA Fd-GOGAT F6P G6P GDH GOT GPT GS HK ICDH NADH-GOGAT NAD-MDH NAD-ME NADP-ME NR
ATP-phosphofructokinase Citrate synthase Dithiothreitol Ethylenediaminetetraacetic acid Ferredoxin-dependent glutamate synthase Fructose 6-phosphate Glucose 6-phosphate Glutamate dehydrogenase Glutamate oxaloacetate transaminase Glutamate pyruvate transaminase Glutamine synthetase Hexokinase Isocitrate dehydrogenase NADH-dependent glutamate synthase NAD-malate dehydrogenase NAD-malic enzyme NADP-malic enzyme Nitrate reductase
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OAA 2-OG PEP PEPC PK PVPP TCA
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Oxaloacetate 2-Oxoglutarate Phosphoenolpyruvate PEP carboxylase Pyruvate kinase Polyvinylpolypyrrolidone Tricarboxylic acid
Introduction When excessive accumulation of non-structural carbohydrates occurs in leaves, feedback regulation of CO2 assimilation is triggered to lower the rate of carbon acquisition. Leaves with excessive accumulation of carbohydrates often exhibit yellowing in association with lower CO2 assimilation. This has been observed in leaves of both herbaceous plants and trees grown at elevated CO2 (Cave et al. 1981; Wullschleger et al. 1992; Keutgen et al. 1997; Sicher 1998), in leaves of ‘Golden Delicious’ apple trees after deblossoming (Schupp et al. 1992), in citrus leaves after branch girdling and fruit removal (Schaffer et al. 1986), and in transgenic tobacco plants overexpressing yeast invertase in the cell wall (Stitt et al. 1991). Although the effect of excessive accumulation of nonstructural carbohydrates on leaf CO2 assimilation has been well documented, it remains unclear how the down-stream processes that utilize carbohydrates in leaves respond to excessive accumulation of carbohydrates. Carbohydrates produced in photosynthesis enter the central metabolism to generate metabolic intermediates and energy for the synthesis of other primary metabolites and secondary compounds. Organic acids derived from glycolysis, tricarboxylic acid (TCA) cycle and anapleurotic pathway combine with ammonium to form amino acids, which are building blocks for proteins. Since carbohydrates serve as substrates and also possibly as signals for the synthesis of organic acids and amino acids, it is expected that accumulation of carbohydrates would alter organic acid metabolism, nitrogen assimilation and amino acid metabolism. When tobacco plants were grown at elevated CO2 under sufficient nitrogen supply, higher levels of nonstructural carbohydrates were observed in the leaves of both young seedlings and older plants (Geiger et al. 1998). In young seedlings, the growth rate of which was significantly stimulated by elevated CO2, organic acids, nitrate assimilation and total free amino acids were increased significantly. In the older plants, nitrate assimilation and total free amino acids were largely unaffected, which corresponded to the lack of stimulation of plant growth by elevated CO2. Soybean plants grown at elevated CO2 in the
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field had higher levels of non-structural carbohydrates in leaves throughout the growing season (Rogers et al. 2006). These plants had lower levels of foliar nitrogen and amino acids early in the growing season when the plants were largely dependent on uptake of nitrogen from soil, but were able to overcome the N limitation in the latter part of the season when symbiotic nitrogen fixation became the main nitrogen source. It appears that the effect of elevated CO2 on the metabolism of organic acids, nitrogen and amino acids is dependent on plant developmental stages. In both cases, however, plants only accumulated moderate levels of non-structural carbohydrates, which were probably still below the threshold level that could trigger significant inhibition of photosynthesis. To date, the metabolism of organic acid, nitrogen and amino acids has not been fully examined in situations where excessive accumulation of carbohydrates and significant inhibition of photosynthesis occur. In ‘Honeycrisp’ apple, leaf chlorosis develops in late June or early July when shoot growth slows down (Rosenberger et al. 2001) and trees bearing a light crop have a larger proportion of leaves developing chlorosis symptoms than those with a heavy crop (Schupp 2003; Robinson and Watkins 2003). These chlorotic leaves are characterized by excessive accumulation of non-structural carbohydrates, which leads to feedback repression of the key enzymes in CO2 assimilation (Chen and Cheng 2004). Our previous study suggests that phloem transport of carbohydrates is partially or completely blocked in the chlorotic leaves (Chen and Cheng 2004), given that both sucrose and sorbitol diffuse into the phloem from mesophyll cells via plasmodesmata in apple leaves (Reidel et al. 2009). It appears that the chlorotic leaves versus normal leaves of ‘Honeycrisp’ apple provide an interesting experimental system for studying the relationship between excessive accumulation of carbohydrates and metabolism of organic acids, nitrogen and amino acids. In this study, metabolite profiles and related enzyme activities were compared between chlorotic leaves and normal leaves to understand how the metabolism of organic acids, nitrogen assimilation and amino acid synthesis responds to excessive accumulation of non-structural carbohydrates. Materials and methods Plant materials Nine-year-old ‘Honeycrisp’ apple trees on Malling 9 rootstocks were used in this study. They were grown at a spacing of 1.2 9 4.2 m in the field at Cornell Orchards in Ithaca, NY, USA. The trees received standard horticultural practices, and disease and insect control. When the
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diameter of the largest fruit in a cluster was 10 mm, crop load of the trees was adjusted to either 2 fruit/cm2 trunk cross-sectional area (light crop treatment) or 9 fruit/cm2 trunk cross-sectional area (heavy crop treatment) by hand thinning. Each treatment was repeated eight times with three trees per replicate in a completely randomized design. On 24 July (about 4 weeks after the initial appearance of leaf disorder symptoms) when leaf samples were taken, about 15–20% of the leaves had developed chlorotic symptoms on trees in the light crop treatment, whereas it was less than 3% on trees in the heavy crop treatment. Well-exposed, recently fully expanded normal leaves and chlorotic leaves on extension shoots in the southern part of the tree canopy were selected from heavy crop and light crop treatments, respectively. Gas exchange measurements were made on three leaves per replicate (1 leaf per tree, 3 trees per replicate). After gas exchange measurements, leaf discs were taken from normal leaves and chlorotic leaves, both at noon under full sun and 12 h later at midnight (to cover two respresentative situations), frozen immediately in liquid N2, and stored at -80°C to measure metabolites and enzyme activities. Each sample consisted of nine leaves (3 leaves per tree, 3 trees per replicate) at each sampling point. Gas exchange measurements Leaf CO2 assimilation and respiration were measured with a CIRAS-1 portable photosynthesis system (PP systems, Herts, UK) using a standard broadleaf cuvette on both normal leaves and chlorotic leaves. CO2 assimilation was measured at noon under ambient CO2 concentration of 360 lmol mol-1, photon flux density (PFD) of 1,650 ± 50 lmol m-2 s-1 and air temperature of 23 ± 1°C. Respiration was measured both at noon and midnight after the leaves were dark adapted for 30 min at an air temperature of 23 ± 1°C. Measurements of soluble sugars, starch, organic acids and total phenolics Soluble sugars, hexose phosphates and most organic acids including shikimic acid, quinic acid and chlorogenic acid were extracted and derivatized following the protocol of Lisec et al. (2006) with minor modifications. Three leaf discs (about 100 mg) were extracted in 1.4 ml 75% methanol with ribitol added as internal standard. We found in our preliminary experiments that 75% methanol gave better extraction of organic acids than anhydrous methanol. Due to the wide range of concentrations of primary metabolites in apple leaves, two vials containing different volumes of extract were prepared for each sample, one with a small volume (5 ll) for highly abundant metabolites
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(sorbitol, sucrose, glucose, fructose galactose, malate, quinate, etc.) and the other with a large volume (100 ll) for less abundant metabolites. Briefly, after fractionation of non-polar metabolites into chloroform, 5 and 100 ll of the polar phase of each sample were taken and transferred into two 2.0 ml Eppendorf vials, respectively. They were dried under vacuum without heating and then derivatized with methoxyamine hydrochloride and N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) sequentially (Lisec et al. 2006). After derivatization, metabolites were analyzed with an Agilent 7890A GC/5795C MS (Agilent Technology, Palo Alto, CA, USA). Injection of 1 ll sample was performed at 230°C in splitless mode with helium carrier gas flow set to 1 ml/min. Chromatography was performed on a DB-5MS capillary column (20 m 9 0.18 mm 9 0.18 lm) with a 5 m Duraguard column (Agilent Technology). The temperature program was isothermal at 70°C for 2.471 min, followed by a 10.119°C/min ramp to 330°C and a final 2.471 min heating at 330°C. Cooling was performed as fast as possible. The system was then temperature equilibrated at 70°C for 5 min before the next injection. Mass spectra were collected at 5.6 scans s-1 over an m/z 50–600 scanning range. The transfer line temperature and the ion source temperature were set to 250 and 230°C, respectively. Metabolites were identified by comparing fragmentation patterns with those in a mass spectral library generated on our GC/MS system and an annotated quadrupole GC–MS spectral library downloaded from the Golm Metabolome Database (http://csbdb.mpimp-golm. mpg.de/csbdb/gmd/msri/gmd_msri.html) and quantified based on standard curves generated for each metabolite and internal standard. The tissue residue after 75% methanol extraction for GC/MS analysis was re-extracted with 80% (v/v) ethanol at 80°C three times, and the pellet was retained for determination of starch. After digesting the residue with 30 units (U) of amyloglucosidase (EC 3.2.1.3) at pH 4.5 overnight, starch was determined enzymatically as glucose equivalents (Chen et al. 2002). Extraction of phosphoenolpyruvate (PEP), pyruvate, and oxaloacetate followed the protocol of Chen et al. (2002). PEP was measured according to Pistelli et al. (1987) with some modifications. The reaction mixture (1 ml) contained 100 mM Hepes–KOH (pH 7.6), 10 mM MgSO4, 100 mM KCl, 1 mM ADP, 1.5 mM EDTA, 0.2 mM NADH, 14 U lactate dehydrogenase, 2 U pyruvate kinase and 50 ll extract. Before and after adding pyruvate kinase, the absorbance at 340 nm was recorded. Pyruvate and oxaloacetate were measured according to Du et al. (1998) and Wahlefeld (1974), respectively. The Folin–Ciocalteu reagent assay was used to determine leaf total phenolics (Singleton and Rossi, 1965). Two leaf discs were extracted with 70% (v/v) methanol. An
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aliquot of the extract (20 ll) was mixed with 1.8 ml of Folin–Ciocalteu reagent pre-diluted with distilled water (1:10). The mixture was allowed to stand at 25°C for 5 min before adding 1.2 ml of 15% sodium carbonate solution in distilled water. The absorbance at 765 nm was read after initial mixing and up to 90 min until it reached a plateau. Gallic acid was used as a standard for the calibration curve. The amount of total phenolic compounds was calculated and expressed as gallic acid equivalent (GAE). Measurements of free amino acids Two leaf discs (2 cm2) were ground in 1.0 ml 20 mM HCl with norleucine added as an internal standard. The extract was centrifuged at 13,000g for 10 min, and the supernatant was passed through a 0.2 lm syringe filter into an Eppendorf vial. Derivatization of free amino acids was carried out using Waters AccQFluorTM reagent kit following the manufacturer’s instructions (Waters Corp., Milford, MA, USA). Individual amino acids were separated and quantified according to Cohen and Michaud (1993) with some modifications, using an Agilent 1100 Liquid Chromatograph equipped with an Agilent 1200 fluorescence detector (Agilent Technology). The Waters AccQTagTM amino acid analysis column (Nova-PakTM C18 column, 4.0 lm particle size, 3.9 mm 9 150 mm) was used in the separation, preceded by a Waters SentryTM guard column (Waters Corp.) at 38°C with a flow rate of 1.0 ml/min. A volume of 5 ll of the derivatized mixture was injected into HPLC. Mobile phase A consisted of 140 mM sodium acetate and 17 mM triethanolamine (pH 5.05, adjusted with phosphoric acid). Mobile phase B was 60% acetonitrile in water (v/v). The gradient was 0% B (0 min), 2% B (0.5 min), 7% B (15 min), 10% B (19 min), 33% B (32–33 min) and 100% B (34–40 min). Post-run time was 10 min. Excitation at 250 nm and emission at 395 nm were used for fluorescence detection. Each peak was identified by comparing retention time with those of authentic standards, and concentrations of individual amino acids was quantified based on peak area and calibration curves derived from authentic standards. Assays of key enzymes in organic acid metabolism, nitrogen assimilation and amino acid metabolism Hexokinase (HK, EC 2.7.1.1), ATP-dependent phosphofructokinase (ATP-PFK, EC 2.7.1.11), pyruvate kinase (PK, EC 2.7.1.40), citrate synthase (CS, EC 4.1.3.7), aconitase (EC 4.2.1.3), NAD-malate dehydrogenase (NAD-MDH, EC 1.1.1.37), NAD-malic enzyme (NAD-ME, EC 1.1.1.38), NADP-malic enzyme (NADP-ME, EC 1.1.1.40) and nitrate reductase (NR, EC 1.6.6.1) were extracted according to Ha¨usler et al. (2000) with some modifications. Three leaf
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discs (3 cm2) were homogenized in 1.5 ml extraction buffer containing 100 mM Hepes (pH 7.5), 30% glycerol, 5 mM MgCl2, 1 mM ethylenediaminetetraacetic acid (EDTA), 5 mM dithiothreitol (DTT) and 0.3% Triton-100, 5% insoluble polyvinylpolypyrrolidone (PPVP) and a pinch of sand. After centrifugation at 16,000g for 10 min at 4°C, 1 ml of the supernatant was desalted at 4°C in a SephadexTM G-25 M PD-10 column (GE Healthcare, UK) equilibrated with 25 mM Hepes (pH 7.5) containing 30% glycerol, 5 mM MgCl2 and1 mM EDTA to a final volume of 2 ml. HK, ATP-PFK, PK, aconitase, NAD-ME and NADP-ME were measured by standard procedures described in Bergmeyer et al. (1983). Citrate synthase activity was determined according to Chen et al. (2009). NR activity was measured in 1 ml assay mixture containing 50 mM Hepes– KOH buffer (pH 7.5), 10 mM NaNO3, 0.25 mM NADH and 200 ll desalted enzyme extract. The mixture was incubated at 30°C for 30 min. Colorimetric determination of nitrite was carried out as described in Hageman et al. (1980). A standard curve was generated using authentic KNO2. Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) was extracted with 1.5 ml extraction buffer containing 100 mM Tris–H2SO4 (pH 8.2), 1 mM EDTA, 7 mM mercaptoethanol, 5% glycerol and 3% (w/v) PVPP. After centrifugation, 1 ml of supernatant was adjusted to 40% ammonium sulfate, kept at 4°C for 2 h and centrifuged at 16,000g for 15 min. The precipitate was re-suspended in 0.2 ml extraction buffer. PEPC activity was measured spectrophotometrically at 340 nm in 1 ml of assay mixture containing 50 mM Tris–HCl (pH 9.2), 4 mM PEP, 10 mM MgSO4, 10 mM NaHCO3, 0.1 mM NADH and 5 U malate dehydrogenase (EC 1.1.1.37). NADH-dependent glutamate 2-oxoglutarate aminotransferase (NADH-GOGAT, EC 1.4.7.14), glutamine synthetase (GS, EC 6.3.1.2), glutamate dehydrogenase (GDH, EC 1.4.1.2), isocitrate dehydrogenase (ICDH, EC 1.1.1.42), glutamate pyruvate transaminase (GPT, EC 2.6.1.2) and glutamate oxaloacetate transaminase (GOT, EC 2.6.1.1) were extracted according to Ha¨usler et al. (2001) with some modifications. Five leaf discs (5 cm2) were homogenized in 2 ml extraction buffer containing 100 mM Tris–HCl (pH 7.5), 1 mM EDTA, 1 mM mercaptoethanol, 0.3% Triton X-100 and 5% PVPP. After centrifugation at 16,000g for 10 min at 4°C, 1 ml of the supernatant was desalted at 4°C in a PD-10 column equilibrated with extraction buffer (without Triton X-100 and PVPP) to a final volume of 2 ml. Ferredoxin-dependent glutamate 2-oxoglutarate aminotransferase (FdGOGAT, EC 1.4.7.1) was extracted from five leaf discs (5 cm2) in 1.5 ml of extraction buffer containing 100 mM KH2PO4–KOH (PH 7.5), 10 mM MgCl2, 2 mM EDTA, 10 mM DTT, 10% glycerol, 1% BSA, 1% Triton X-100
Statistical analysis Data were analyzed via analysis of variance followed by Duncan’s multiple range test for means separation at P = 0.05 by using SAS 9.1 (SAS Institute, Cary, NC, USA).
Results CO2 assimilation and respiration CO2 assimilation was much lower in chlorotic leaves than in normal leaves (Fig. 1a). However, dark respiration rate was significantly higher in chlorotic leaves than in normal leaves at both midday and midnight (Fig. 1b). Non-structural carbohydrates There was no significant difference in leaf sorbitol concentration between chlorotic leaves and normal leaves at midday, but slightly higher concentration was detected in chlorotic leaves at midnight (Fig. 2a). Concentrations of
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and 5% PVPP. The homogenate was centrifuged and the supernatant was desalted the same way as described above. NADH-GOGAT activity was measured in a 1-ml assay mixture containing 100 mM KH2PO4–KOH (pH 7.5), 0.1 mM NADH, 10 mM glutamine, 10 mM 2-oxoglutarate and 200 ll desalted enzyme extract. Fd-GOGAT activity was assayed by measuring glutamate formation using reduced methyl viologen as the electron donor as described by Lea et al. (1990). The assay mixture contained 100 mM KH2PO4–KOH (PH 7.5), 3 mM EDTA, 10 mM glutamine, 10 mM 2-oxoglutarate, 15 mM reduced methyl viologen, 1 mM O-(carboxymethyl)hydroxylamine hemihydrochloride, and 200 ll desalted enzyme extract. After pre-incubation at 30°C for 5 min, the reaction was started by addition of 50 mg NaHCO3 and 50 mg Na2S2O4. The reaction mixture was shaken in an Eppendorf thermomixer at 30°C for 30 min with cap open. The reaction was terminated at the end of 30 min by adding ethanol, vortexed and then filtered through a 0.2-lm filter. As described above for free amino acid analysis, 5 ll of the filtered reaction mixture was derivatized and analyzed via HPLC. GS activity was assayed as described by Scheible et al. (1997). GDH activity was measured according to Lutts et al. (1999). Activities of GOT, GPT and NADP-dependent ICDH were measured following standard procedures described in Bergmeyer et al. (1983). Leaf-soluble protein concentrations were measured according to Bradford (1976) using BSA as standard.
515 CO2 assimilation
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Chlorotic
Fig. 1 CO2 assimilation (a) and respiration (b) of chlorotic leaves and normal leaves of ‘Honeycrisp’ apple. Bars represent mean ± SE of eight replicates. Different letters above the bars indicate significant difference using Duncan’s multiple range test at P \ 0.05, respectively
sucrose, fructose, starch, maltose, maltitol (4-O-a-D-glucopyranosyl-D-glucitol) and trehalose were significantly higher in chlorotic leaves than in normal leaves at both midday and midnight (Fig. 2b, c, f, g, h, i). Concentrations of glucose, galactose and rhamnose were higher in chlorotic leaves at midday, but no significant difference was detected between the two leaf types at midnight (Fig. 2d, e, j). Of all the non-structural carbohydrates, starch showed the largest difference between chlorotic leaves and normal leaves, followed by maltitol, maltose, fructose, trehalose and sucrose. Compared with normal leaves, chlorotic leaves had lower concentrations of both G6P and F6P at midday, but higher concentrations of both G6P and F6P at midnight (Fig. 2k, l). Concentrations of both G6P and F6P were higher at midnight than at midday in chlorotic leaves, which is just opposite to that in normal leaves. Organic acids and activities of key enzymes in organic acid metabolism Concentrations of most organic acids, including phosphoenolpyruvate (PEP), pyruvate, oxaloacetate (OAA), malate, 2-oxoglutarate (2-OG), succinate, fumarate, glycerate, shikimate and quinate were lower in chlorotic leaves than in normal leaves at both midday and midnight (Fig. 3a–d, f–k). However, concentrations of both citrate and chlorogenate were higher in chlorotic leaves than in normal leaves at both midday and midnight (Fig. 3e, l). In addition, the concentration of total phenolics was 45% (midday) to 47% (midnight) higher in chlorotic leaves than in normal leaves, respectively (the actual concentration in normal leaves was 5.22 and 5.58 g m-2 at midday and midnight, respectively). No difference in hexokinase activity was detected between chlorotic leaves and normal leaves at either midday or midnight (Fig. 4a). Activities of ATP-phosphofructokinase (ATP-PFK), pyruvate kinase (PK), citrate synthase (CS), aconitase, and isocitrate dehydrogenase (ICDH) were significantly higher in chlorotic leaves than in normal leaves (Fig. 4b–f). In contrast, activities of PEP
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Fig. 2 Concentrations of sorbitol (a), sucrose (b), fructose (c), glucose (d), galactose (e), starch (f), maltose (g), maltitol (h), trehalose (i), rhamnose (j), glucose 6-phosphate (k) and fructose 6-phosphate (l) in chlorotic leaves and normal leaves of ‘Honeycrisp’ apple. Bars represent mean ± SE of eight replicates. Different letters above the bars indicate significant difference using Duncan’s multiple range test at P \ 0.05, respectively
carboxylase (PEPC) and NAD-malate dehydrogenase (NAD-MDH) (Fig. 4g, h) were significantly lower in chlorotic leaves than in normal leaves. Interestingly, chlorotic leaves had lower NAD-malic enzyme (NAD-ME) activity (Fig. 4i), but higher NADP-malic enzyme (NADPME) activity (Fig. 4j). Amino acids, soluble proteins and activities of key enzymes in nitrogen assimilation and amino acid metabolism Concentrations of Glu, Ala,Val, Met, Ile, Leu and Phe were significantly lower in chlorotic leaves than in normal leaves at both midday and midnight (Fig. 5b, i, m, n, r, s, t).
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Concentrations of Asp, Asn, Gln, Ser, Gly and Thr were significantly lower in chlorotic leaves than in normal leaves at midday, but no significant difference was detected between the two leaf types at midnight (Fig. 5a, c, d, e, f, h). Concentrations of Tyr, Lys and Arg were significantly lower in chlorotic leaves than in normal leaves at midnight, but no difference was found at midday (Fig. 5l, p, q). There was no difference in the concentration of His, GABA and Orn between chlorotic and normal leaves at either midday or midnight (Fig. 5g, k, o) . However, the concentration of Pro was significantly higher in chlorotic leaves than in normal leaves at midday (Fig. 5j). The concentration of total free amino acids was about 27.2 and 28.8% lower in
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Fig. 4 Activities of hexokinase (HK, a), ATP-dependent phosphofructokinase (ATP-PFK, b), pyruvate kinase (PK, c), citrate synthase (CS, d), aconitase (e), isocitrate dehydrogenase (ICDH, f), phosphoenolpyruvate carboxylase (PEPC, g), NAD-malate dehydrogenase (NAD-MDH, h), NAD-malic enzyme (NAD-ME, i) and NADP-malic enzyme (NADP-ME, j) in chlorotic leaves and normal leaves of ‘Honeycrisp’ apple. Bars represent mean ± SE of eight replicates. Different letters above the bars indicate significant difference using Duncan’s multiple range test at P \ 0.05, respectively
chlorotic leaves than in normal leaves at midday and midnight, respectively. In normal leaves, concentrations of Asn, Gln, Ser, Gly and His were significantly higher at midday than at midnight (Fig. 5c–g), whereas concentrations of Lys, Arg, Leu and Phe were significantly lower at midday than at midnight (Fig. 5p, q, s, t). In chlorotic leaves, however, no significant difference was found between midday and midnight for these amino acids. Soluble protein concentration and activities of nitrate reductase (NR), glutamine synthetase (GS), ferridoxin- and NADH-dependent glutamate synthase (Fd-GOGAT and NAD-GOGAT) and glutamate pyruvate transaminase (GPT) were significantly lower in chlorotic leaves than in normal leaves (Fig. 6a–e, h). However, activities of glutamate dehydrogenase (GDH) and glutamate oxaloacetate transaminase (GOT) were significantly higher in chlorotic leaves than in normal leaves (Fig. 6f, g).
Discussion Accumulation of non-structural carbohydrates in chlorotic leaves Compared with normal leaves, chlorotic leaves accumulated much higher levels of non-structural carbohydrates, especially starch (Fig. 2f), maltose (Fig. 2 g), maltitol (Fig. 2 h), fructose (Fig. 2c) and sucrose (Fig. 2b) at both midday and midnight, and consequently had a much lower rate of CO2 assimilation (Fig. 1a). The leaf chlorosis observed in this study does not appear to be related to early senescence, because the chlorotic leaves stayed on the trees for another 3–4 months after developing chlorosis and dropped from the trees at approximately the same time as the normal leaves in the autumn. The excessive accumulation of non-structural carbohydrates and extremely low CO2 assimilation measured in the chlorotic leaves are consistent with our previous findings (Chen and Cheng 2004), confirming feedback inhibition of photosynthesis by excessive accumulation of carbohydrates in the chlorotic leaves. Excessive accumulation of non-structural carbohydrates and consequent inhibition of photosynthesis have also been observed in the leaves of apple (Schupp et al. 1992; Zhou and Quebedeaux 2003), citrus (Schaffer et al. 1986) and many other plants (Goldschmidt and Huber 1992; Krapp et al. 1993; Krapp and Stitt 1995; Morcuende et al. 1996) when phloem transport was blocked via girdling or the major sink of the plant was removed. Although the signal transduction pathway that leads to feedback inhibition of photosynthesis by excessive accumulation of carbohydrates remains unclear, sugar-mediated repression of the expression of photosynthetic genes plays a major role (Krapp et al. 1993; Krapp and Stitt 1995) and physical damage of starch grains to chloroplasts may also contribute especially at the late stage. Our data also suggest that maltose, in addition to starch, could serve as a sensitive indicator of excessive accumulation of non-structural carbohydrates. Both glycolysis and TCA cycle were up-regulated, while anapleurotic pathway was down-regulated in chlorotic leaves Both higher rates of dark respiration (Fig. 1b) and higher activities of several key enzymes in glycolysis and TCA cycle, including ATP-PFK, PK, citrate synthase, aconitase and ICDH (Fig. 4b–f), clearly indicate that both glycolysis and TCA cycle were up-regulated in chlorotic leaves in response to excessive accumulation of non-structural carbohydrates to ‘‘consume’’ the extra carbon available. These results are consistent with stimulation of nighttime respiration of soybean plants by additional carbohydrates
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Fig. 5 Concentrations of aspartic acid (Asp, a), glutamic acid (Glu, b), asparagine (Asn, c), glutamine (Gln, d), serine (Ser, e), glycine (Gly, f), histidine (His, g), threonine (Thr, h), alanine (Ala, i), proline (Pro, j), c-aminobutyric acid (GABA, k), tyrosine (Tyr, l), valine (Val, m), methionine (Met, n), ornithine (Orn, o), lysine (Lys, p),
arginine (Arg, q), isoleucine (Ilu, r), leucine (Leu, s) and phenylalanine (Phe, t) in chlorotic leaves and normal leaves of ‘Honeycrisp’ apple. Bars represent mean ± SE of eight replicates. Different letters above the bars indicate significant difference using Duncan’s multiple range test at P \ 0.05, respectively
available from enhanced photosynthesis at elevated CO2 via increased abundance of transcripts encoding enzymes throughout the respiratory pathway (Leakey et al. 2009). When more glucose became available due to cytosolic expression of a yeast invertase in potato tubers, a large increase in the activity of several key enzymes in glycolysis and a dramatic increase in the levels of glycolytic intermediates led to a higher rate of respiration (Trethewey et al. 1998, 1999). Although no difference in hexose kinase activity was detected between chlorotic leaves and normal leaves (Fig. 4a), concentrations of both G6P and F6P were higher in chlorotic leaves than in normal leaves at midnight (Fig. 2k, l), which is consistent with a higher rate of dark respiration in the chlorotic leaves. The lower concentrations of G6P and F6P in chlorotic leaves at midday are most likely caused by the lower photosynthesis in chlorotic leaves (Fig. 1a) as CO2 assimilation is the main source of hexose phosphates measured in leaves during the day. Compared with normal leaves, concentrations of most intermediates in glycolysis and TCA cycle, including PEP, pyruvate, OAA, malate, 2-OG, succinate and fumarate, were significantly lower in chlorotic leaves (Fig. 3a–d, f–h).
This is consistent with lower concentrations of many individual free amino acids (Fig. 5), for example, lower 2-OG concentration corresponded with lower concentrations of Glu family amino acids and lower pyruvate concentration was in parallel with lower concentrations of Ala, Val and Leu. The lower concentrations of these glycolytic and TCA cycle intermediates in chlorotic leaves are most likely caused by a combination of the following three factors. First, higher carbon fluxes going through glycolysis and TCA cycle as indicated by higher dark respiration rates and higher activities of several key enzymes involved (as discussed above) might have lowered the concentrations of some intermediates. Second, lower concentrations of shikimate (Fig. 3j), quinate (Fig. 3k) and phenylalanine (Fig. 5t), but much higher concentrations of chlorogenic acid (Fig. 3l) and total phenolics (see ‘‘Results’’) found in chlorotic leaves, suggest that the phenylpropanoid pathway was up-regulated in chlorotic leaves in response to excessive accumulation of carbohydrates, which might have diverted some PEP away from glycolysis. Finally, the decreased demand for organic acids by nitrogen assimilation and amino acid synthesis and corresponding
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Fig. 6 Soluble protein concentration (a) and activities of nitrate reductase (NR, b), glutamine synthetase (GS, c), ferridoxin-dependent glutamate synthase (Fd-GOGAT, d), NADH-dependent glutamate synthase (NADH-GOGAT, e), glutamate dehydrogenase (GDH, f), glutamate oxaloacetate transaminase (GOT, g), and glutamate pyruvate transaminase (GPT, h) in chlorotic leaves and normal leaves of ‘Honeycrisp’ apple. Bars represent mean ± SE of eight replicates. Different letters above the bars indicate significant difference using Duncan’s multiple range test at P \ 0.05, respectively
down-regulation of the anapleurotic pathway in organic acid metabolism in chlorotic leaves as discussed below might have resulted in lower concentrations of some organic acids. In addition to direct entry into the mitochondria via pyruvate, PEP generated in glycolysis can be converted to OAA by PEP carboxylase and then to malate by NADMDH in the cytosol, both of which can enter the mitochondria to supplement the pool of C4 acids (Hill 1997). In leaves of C3 plants, PEPC and NAD-MDH are involved in the anapleurotic replenishment of TCA cycle intermediates for several biosynthetic pathways including biosynthesis of amino acids and phenolic compounds (Andrews 1986; Melzer and O’Leary 1987; Noguchi and Yoshida 2008). Malate can also be decarboxylated in the cytosol by NADP-ME or in the mitochondrial matrix by NAD-ME to produce pyruvate, which is then oxidized in the TCA cycle. We found that activities of PEPC, NAD-MDH and NADME were significantly lower in chlorotic leaves than in normal leaves (Fig. 4g–i), which is similar to the finding of Sawada et al. (2002) that activities of PEPC, NAD-ME and NAD-MDH were all decreased in sink-limited Amaranthus (C4) leaves. Although an increase in cytosolic NADP-ME
was observed in chlorotic leaves (Fig. 4j), this would not alter the overall decrease of malate decarboxylation into pyruvate because NADP-ME activity was much lower than NAD-ME (Fig. 4i). These results indicate that the anapleurotic pathway was down-regulated as a result of excessive accumulation of non-structural carbohydrates in chlorotic leaves. The higher citrate concentration in chlorotic leaves (Fig. 3e) is most likely related to their lower malate concentration (Fig. 3d). Most malate and citrate in leaves are present in vacuoles, and tonoplast transporters are involved in their accumulation (Rentsch and Martinola 1991; Emmerlich et al. 2003; Hurth et al. 2005). Citrate transport into the vacuole is competitively inhibited by malate and vice versa (Rentsch and Martinola 1991; Emmerlich et al. 2003). The lower malate concentration in chlorotic leaves might have released some of the inhibition on citrate transport, leading to more accumulation of citrate in the vacuole. Nitrogen assimilation and amino acid synthesis was down-regulated in chlorotic leaves We found that concentrations of most free amino acids were significantly lower in chlorotic leaves than in normal leaves (Fig. 5), which resulted in significantly lower total free amino acids. Lower concentrations of free amino acids were also obtained in barley primary leaves under CO2 enrichment (Sicher 2008). The decreased levels of free amino acids in chlorotic leaves were closely related to the activities of several key enzymes in nitrogen assimilation and amino acid synthesis. Compared with normal leaves, chlorotic leaves had significantly lower activities of NR, GS, Fd-GOGAT and NADH-GOGAT (Fig. 6b–e). In Festuca arundinacea leaves under high temperature stress, the decrease in total free amino acids was found to parallel with the decrease in activities of NR, GS and NADHGOGAT (Cui et al. 2006). Both lower concentrations of free amino acids and lower activities of key enzymes in nitrogen metabolism found in chlorotic leaves indicate that nitrogen assimilation and amino acid synthesis were downregulated in chlorotic leaves in response to excessive accumulation of non-structural carbohydrates. The lack of significant differences in the concentrations of several amino acids (Asn, Gln, Ser, Gly and His) observed in chlorotic leaves between midday and midnight (Fig. 5) may be related to the extremely low rate of CO2 assimilation measured in chlorotic leaves (Fig. 1a). This is because photosynthesis provides the reducing power necessary for the reduction of nitrate into ammonium and carbon skeletons necessary for amino acid synthesis, and photorespiratory nitrogen metabolism accounts for a significant portion of the nitrogen flux in leaves. In the leaves
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of tobacco plants grown at elevated CO2 where photorespiration was inhibited, Gly, Ser and Glu accumulated to lower levels during the photoperiod relative to those grown at ambient CO2 (Geiger et al. 1998). In addition, light stimulation of nitrate reductase activity was completely abolished as a result of excessive accumulation of nonstructural carbohydrates in chlorotic leaves (Fig. 6b). In contrast to NR, GS and GOGAT, GDH activity was significantly higher in chlorotic leaves than in normal leaves (Fig. 6f). GDH catalyzes the reversible conversion between 2-OG and glutamate involving ammonium (Kumar et al. 2000), but it has been confirmed in transgenic tobacco plants that the primary function of GDH is to deaminate glutamate (Labboun et al. 2009). The activity of GDH has been found to increase with leaf age (Thomas 1978; Kar and Feierabend 1984) or in response to stresses (Ramajulu et al. 1994; Lutts et al. 1999; Cui et al. 2006), which is in general agreement with increased protein degradation and catabolism of amino acids under those conditions. The higher activity of GDH found in chlorotic leaves suggests that deamination of glutamate was enhanced in response to excessive accumulation of nonstructural carbohydrates, which also contributed to the decreased levels of free amino acids in chlorotic leaves. Lower GPT activity, but higher GOT activity, was found in chlorotic leaves than in normal leaves (Fig. 6g, h). The differential responses of GPT and GOT to excessive accumulation of non-structural carbohydrates might be related to lower GS activity in chlorotic leaves (Fig. 6c). Singh and Verma (1996) found that low GS activity in the wild-type cells of Anacysis nidulans under high light stress was accompanied by low GPT activity and high GOT activity and vice versa in the high light-tolerant mutant cells. The activity of GOT was found to be dependent on the energy and carbon supplied by respiration, whereas the activity of GPT was more sensitive to the metabolic inhibitors of photosynthesis (Singh and Verma 1996). So, the differential responses of GPT and GOT are in line with the lower photosynthesis and higher dark respiration observed in chlorotic leaves. GOT plays an important role in the synthesis of the aspartate family of amino acids (Bryan 1980). The concentration of aspartate was comparable between chlorotic and normal leaves, though the majority of amino acids decreased significantly in chlorotic leaves (Fig. 5). The lower GPT activity is consistent with the decreased Ala concentration in chlorotic leaves. Higher Pro concentration was found in chlorotic leaves during the day (Fig. 5j). Pro accumulation is often considered to be involved in stress resistance mechanisms (Lutts et al. 1999). Our previous study indicated that chlorotic leaves underwent oxidative stress and that the antioxidant system was up-regulated to protect the leaves against photooxidative damage (Chen and Cheng 2004). Stress-induced
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Pro accumulation in plants occurs predominantly through the enhanced biosynthesis of Pro from Glu (Lutts et al. 1999; Hayashi et al. 2000). Accumulation of Pro has been found to be associated with an increase in GDH and a decrease in GS under salinity stress (Lutts et al. 1999; Wang et al. 2007). This is also the case in chlorotic leaves with excessive accumulation of carbohydrates (Fig. 6c, f). In conclusion, in response to excessive accumulation of non-structural carbohydrates in chlorotic leaves of ‘Honeycrisp’ apple, glycolysis and tricarboxylic acid cycle are up-regulated to ‘‘consume’’ the excess carbon available, whereas the anapleurotic pathway, nitrogen assimilation and amino acid synthesis are down-regulated to reduce the overall rate of amino acid and protein synthesis. Acknowledgments The Agilent GC/MS system used in this work was generously donated by Dr. David Zimerman, Cornell Pomology Ph.D. 1954. The authors would like to thank Drs. Alisdair Fernie and Maria Ines at Max Planck Institute of Molecular Plant Physiology in Germany for their valuable advice and help on metabolite profiling using GC/MS.
References Andrews M (1986) The partitioning of nitrate assimilation between root and shoot of higher plants. Plant Cell Environ 9:511–519 Bergmeyer H, Grassl M, Walter H (1983) In: Bergmeyer HU (ed) Methods in enzymatic analysis, vol 2. VCH, Weinheim, pp 273– 274 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254 Bryan JK (1980) Synthesis of the aspartate family and branched-chain amino acids. In: Miflin BJ (ed) The biochemistry of plants: a comprehensive treatise, vol 5. Academic Press, New York, pp 403–452 Cave G, Tolley LC, Strain BR (1981) Effect of carbon dioxide enrichment on chlorophyll content, starch content and starch grain structure in Trifolium subterraneum leaves. Physiol Plant 51:171–174 Chen L, Cheng L (2004) CO2 assimilation, carbohydrate metabolism, xanthophyll cycle, and antioxidant system of ‘Honeycrisp’ apple leaves with zonal chlorosis. J Amer Soc Hort Sci 129:729–737 Chen LS, Lin Q, Nose A (2002) A comparative study on diurnal changes in metabolite levels in the leaves of three crassulacean acid metabolism (CAM) species, Ananas somosus, Kalanchoe¨ daigremontiana and K. pinnata. J Exp Bot 53:341–350 Chen F, Liu X, Chen L (2009) Developmental changes in pulp organic acid concentration and activities of two loquat (Eriobotrya japonica Lindl) cultivars differing in fruit acidity. Food Chem 114:657–664 Cohen SA, Michaud DP (1993) Synthesis of a fluorescent derivatizing reagent, 6-amino-quinolyl-N-hydroxysuccinimidyl carbamate and its application for the analysis of hydrolysate amino acids via high-performance liquid chromatography. Anal Biochem 211:279–287 Cui L, Cao R, Li J, Zhang L, Wang J (2006) High temperature effects on ammonium assimilation in leaves of two Festuca arundinacea cultivars with different heat susceptibility. Plant Growth Regul 49:127–136
Planta (2010) 232:511–522 Du YC, Nose A, Wasano K, Uchida Y (1998) Responses to water stress of enzyme activities and metabolite levels in relation to sucrose and starch synthesis, the Calvin cycle and the C4 pathway in sugarcane (Saccharum sp.) leaves. Aust J Plant Physiol 25:253–260 Emmerlich V, Linka N, Reinhold T, Hurth MA, Traub M, Martinoia E, Neuhaus HE (2003) The plant homolog to the human sodium/ dicarboxylic cotransporter is the vacuolar malate carrier. Proc Natl Acad Sci USA 100:11122–11126 Geiger M, Walch-Liu P, Engels C, Harnecker J, Shulze ED, Ludewig F, Sonnewald U, Scheible WR, Stittt M (1998) Enhanced carbon dioxide leads to a modified diurnal rhythm of nitrate reductase activity in older plants, and a large stimulation of nitrate reductase activity and higher levels of amino acids in young tobacco plants. Plant Cell Environ 21:253–268 Goldschmidt EE, Huber SC (1992) Regulation of photosynthesis by end-product accumulation in leaves of plants storing starch, sucrose, and hexose sugars. Plant Physiol 99:1443–1448 Hageman RH, Reed AJ, Femmer RA, Sherrard JH, Dalling MJ (1980) Some new aspects of the in vivo assay for nitrate reductase in wheat (Triticum aestivum L.) leaves, 1: re-evaluation of nitrate pool sizes. Plant Physiol 65:27–32 Ha¨usler RE, Fischer KL, Flu¨gge UI (2000) Determination of lowabundant metabolites in plant extracts by NAD(P)H fluorescence with a microtiter plate reader. Anal Biochem 281:1–8 Ha¨usler RE, Thomas R, Li J, Lipka V, Fischer KL, Schuber S, Kreuzaler F, Hirsch H (2001) Single and double overexpression of C4-cycle genes had differential effects on the pattern of endogenous enzymes, attenuation of photorespiration and on contents of UV protectants in transgenic potato and tobacco plants. J Exp Bot 52:1785–1803 Hayashi F, Ichino T, Osanai M, Wada K (2000) Oscillation and regulation of proline content by P5CS and ProDH gene expressions in the light/dark cycles in Arabidopsis thaliana L. Plant Cell Physiol 41:1096–1101 Hill SA (1997) Carbon metabolism in mitochondria. In: Dennis DT, Turpin DH, Lefebvre DD, Layzell DB (eds) Plant metabolism. Addison Welsey Longman, London, pp 181–199 Hurth MA, Suh SJ, Kretzschmar T, Geis T, Bregante M, Gambale F, Martinoia E, Neuhaus HE (2005) Impaired pH homeostasis in Arabidopsis lacking the vacuolar dicarboxylate transporter and analysis of carboxylic acid transport across the tonoplast. Plant Physiol 137:901–910 Kar M, Feierabend J (1984) Changes in the activities of enzymes involved in amino acid metabolism during senescence of detached wheat leaves. Physiol Plant 62:39–44 Keutgen N, Chen K, Lenz F (1997) Responses of strawberry leaf photosynthesis, chlorophyll fluorescence and macronutrient contents to elevated CO2. J Plant Physiol 150:395–400 Krapp A, Stitt M (1995) An evaluation of direct and indirect mechanisms for the ‘‘sink-regulation’’ of photosynthesis in spinach: changes in gas exchange, carbohydrates, metabolites, enzyme activities and steady-state transcript levels after coldgirdling source leaves. Planta 195:313–323 Krapp A, Hofmann B, Scha¨fer C, Stitt M (1993) Regulation of the expression of rbcS and other photosynthetic genes by carbohydrates: a mechanism for the ‘sink regulation’ of photosynthesis? Plant J 3:817–826 Kumar RG, Shanh K, Dubey RS (2000) Salinity induced behavioral changes in malate dehydrogenase and glutamate dehydrogenase activities in rice seedlings of differing salt tolerance. Plant Sci 156:23–24 Labboun S, Terce´-Laforgue T, Roscher A, Bedu M, Restivo FM, Velanis CN, Skopelitis DS, Moshou PN, Roubelakis-Angelakis KA, Suzuku A, Hirel B (2009) Resolving the role of plant glutamate dehydrogenase, I: in vivo real time nuclear magnetic
521 resonance spectroscopy experiments. Plant Cell Physiol 50:1761–1773 Lea PJ, Robinson SA, Stewart GR (1990) The enzymology and metabolism of glutamine, glutamate, and asparagine. In: Miflin BJ, Lea PJ (eds) The biochemistry of plants: intermediary nitrogen metabolism. Academic Press, New York, pp 121–159 Leakey ADB, Xu F, Gillespie KM, McGrath JM, Ainsworth EA, Ort DR (2009) Genomic basis for stimulated respiration by plants growing under elevated carbon dioxide. Proc Natl Acad Sci USA 106:3597–3602 Lisec J, Schauer N, Kopka J, Willmitzer L, Fernie AR (2006) Gas chromatography mass spectrometry-based metabolite profiling in plants. Nat Protoc 1:387–396 Lutts S, Majerus V, Kinet JM (1999) NaCl effects on proline metabolism in rice (Oryza sativa) seedlings. Physiol Plant 105:450–458 Melzer E, O’Leary M (1987) Anapleurotic CO2 fixation by phosphoenolpyruvate carboxylase in C3 plants. Plant Physiol 84:58–60 Morcuende R, Perez P, Martinez-carrasco R, Molino IM, Puente LS (1996) Long- and short-term effects of decreased sink demand on carbohydrate levels and photosynthesis in wheat leaves. Plant Cell Environ 19:1203–1209 Noguchi K, Yoshida K (2008) Interaction between photosynthesis and respiration in illuminated leaves. Mitochondrion 8:87–99 Pistelli G, Marigo G, Ball E, Lu¨ttge U (1987) Day–night changes in the levels of adenine nucleotides, phosphoenolpyruvate and inorganic pyrophosphate in leaves of plants having Crassulacean acid metabolism. Planta 172:479–486 Ramajulu S, Vlerajaniyulu K, Sudhakar C (1994) Short-term shifts in nitrogen metabolism in mulberry Morus alba under salt shock. Phytochemistry 35:991–995 Reidel RJ, Rennie EA, Amiard V, Cheng L, Turgeon R (2009) Phloem loading strategies in three plant species that transport sugar alcohols. Plant Physiol 149:1601–1608 Rentsch D, Martinola E (1991) Citrate transport into barley mesophyll vacuoles–comparison with malate-uptake activity. Planta 184:532–537 Robinson TL, Watkins CB (2003) Cropload of ‘Honeycrisp’ affects not only fruit size but many quality attributes. New York Fruit Q 11(3):7–10 Rogers A, Gibon Y, Stitt M, Morgan PB, Bernacchi CJ, Ort DR, Long SP (2006) Increased C availability at elevated carbon dioxide concentration improves N assimilation in a legume. Plant Cell Environ 29:1651–1658 Rosenberger DA, Schupp JR, Watkins CB, Iungerman K, Hoying S, Straub D, Cheng L (2001) ‘Honeycrisp’: a darling new apple variety or just another problem child? New York Fruit Q 9(3): 9–13 Sawada S, Sakamoto T, Sato M, Kasai M, Usuda H (2002) Photosynthesis with single-rooted Amaranthus leaves. II. Regulation of ribulose-1, 5-bisphosphate carboxylase, phosphoenolpyruvate carboxylase, NAD-malic enzyme and NAD-malate dehydrogenase and coordination between PCR and C4 photosynthetic metabolism in response to changes in the source–sink balance. Plant Cell Physiol 43:1293–1301 Schaffer AA, Liu KC, Goldschmidt EE, Boyer CD, Goren R (1986) Citrus leaf chlorosis induced by sink removal: starch, nitrogen, and chloroplast ultrastructure. J Plant Physiol 124:111–121 Scheible WR, Gonzales-Fontes A, Lauerer M, Mu¨ller-Ro¨ber B, Caboche M, Stitt M (1997) Nitrate acts as a signal to induce organic acid metabolism and repress starch metabolism in tobacco. Plant Cell 9:783–798 Schupp JR (2003) Effects of chemical thinners on fruit set, yield, fruit size, and fruit quality of ‘Honeycrisp’ apple. New York Fruit Q 11(3):3–5
123
522 Schupp JR, Ferree DC, Warrington IJ (1992) Interaction of root pruning and deblossoming on growth, development and yield of ‘Golden Delicious’ apple. J Hortic Sci 67:465–480 Sicher RC (1998) Yellowing and photosynthetic decline of barley primary leaves in response to atmospheric CO2 enrichment. Physiol Plant 103:193–200 Sicher RC (2008) Effects of CO2 enrichment on soluble amino acids and organic acids in barley primary leaves as a function of age, photoperiod and chlorosis. Plant Sci 174:576–582 Singh DP, Verma K (1996) Differential regulation of nitrogen metabolism in the wild type and the high-light-tolerant mutant cells of Anacysis nidulans. Curr Microbiol 32:272–278 Singleton VL, Rossi JA (1965) Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am J Enol Vitic 16:144–158 Stitt M, von Schaewen A, Willmitzer L (1991) ‘Sink’ regulation of photosynthetic metabolism in transgenic tobacco plants expressing yeast invertase of glycolytic enzymes. Planta 183:40–50 Thomas H (1978) Enzymes of nitrogen mobilization in detached leaves of Lolium temulentum during senescence. Planta 142:161– 169 Trethewey RN, Geigenberger P, Riedel K, Hajirezaei MR, Sonnewald U, Stitt M, Riesmeier JW, Willmitzer L (1998) Combined expression of glucokinase and invertase in potato tubers leads to a dramatic reduction in starch accumulation and a stimulation of glycolysis. Plant J 15:109–118
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Planta (2010) 232:511–522 Trethewey RN, Riesmeier JW, Willmitzer L, Stitt M, Geigenberger P (1999) Tuber-specific expression of a yeast invertase and a bacterial glucokinase in potato leads to an activation of sucrose phosphate synthase and the creation of a futile cycle. Planta 208:227–238 Wahlefeld AW (1974) Oxalacetate: UV spectrophotometric determination. In: Bergmeyer HU (ed) Methods of enzymatic analysis, vol 3. Academic Press, New York, pp 1604–1608 Wang ZQ, Yuan YZ, Ou YQ, Lin QH, Zhang CF (2007) Glutamine synthetase and glutamate dehydrogenase contribute differentially to proline accumulation in leaves of wheat (Triticum aestivum) seedlings exposed to different salinity. J Plant Physiol 164:695– 701 Wullschleger SD, Borby RJ, Hendrix DL (1992) Carbon exchange rates, chlorophyll content, and carbohydrate status of two forest tree species exposed to carbon dioxide enrichment. Tree Physiol 10:21–31 Zhou R, Quebedeaux B (2003) Changes in photosynthesis and carbohydrate metabolism in mature apple leaves in response to whole plant source–sink manipulation. J Am Soc Hort Sci 128:113–119
