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CO2 Assimilation, Carbohydrate Metabolism, Xanthophyll Cycle, and the Antioxidant System of `Honeycrisp' Apple Leaves with Zonal Chlorosis
| Content Provider | Semantic Scholar |
|---|---|
| Author | Chen, Li-Song Cheng, Lailiang |
| Copyright Year | 2004 |
| Abstract | To determine the cause of a characteristic zonal chlorosis of ʻHoneycrisp ̓apple (Malus ×domestica Borkh.) leaves, we compared CO2 assimilation, carbohydrate metabolism, the xanthophyll cycle and the antioxidant system between chlorotic leaves and normal leaves. Chlorotic leaves accumulated higher levels of nonstructural carbohydrates, particularly starch, sorbitol, sucrose, and fructose at both dusk and predawn, and no difference was found in total nonstructural carbohydrates between predawn and dusk. This indicates that carbon export was inhibited in chlorotic leaves. CO2 assimilation and the key enzymes in the Calvin cycle, ribulose 1,5-bisphosphate carboxylase/oxygenase, NADP-glyceraldehyde-3-phosphate dehydrogenase, phosphoribulokinase, stromal fructose-1,6-bisphosphatase, and the key enzymes in starch and sorbitol synthesis, ADP-glucose pyrophosphorylase, cytosolic fructose-1,6-bisphosphatase, and aldose 6-phosphate reductase were signifi cantly lower in chlorotic leaves than in normal leaves. However, sucrose phosphate synthase activity was higher in chlorotic leaves. In response to a reduced demand for photosynthetic electron transport, thermal dissipation of excitation energy (measured as nonphotochemical quenching of chlorophyll fl uorescence) was enhanced in chlorotic leaves under full sun, lowering the effi ciency of excitation energy transfer to PSII reaction centers. This was accompanied by a corresponding increase in both xanthophyll cycle pool size (on a chlorophyll basis) and conversion of violaxanthin to antheraxanthin and zeaxanthin. The antioxidant system, including superoxide dismutase and ascorbate peroxidase and the ascorbate pool and glutathione pool, was up-regulated in chlorotic leaves in response to the increased generation of reactive oxygen species via photoreduction of oxygen. These fi ndings support the hypothesis that phloem loading and/or transport is partially or completely blocked in chlorotic leaves, and that excessive accumulation of nonstructural carbohydrates may cause feedback suppression of CO2 assimilation via direct interference with chloroplast function and/or indirect repression of photosynthetic enzymes. ʻHoneycrisp ̓is a new apple cultivar that is being extensively planted in the cooler apple-producing areas due to its unique fruit quality. A persistent problem in ̒ Honeycrisp ̓is a leaf disorder that often develops in late June or early July when shoot growth slows down or stops (Rosenberger et al., 2001). The initial symptom of zonal chlorosis appears randomly in part (toward the edges) of a leaf confi ned by secondary or tertiary veins, and then gradually spreads to other parts of the leaf. The chlorotic area becomes thicker, leathery, and brittle, and turns brown later in the season. The symptoms occur on almost every tree, but trees bearing a light crop have a larger number of leaves developing symptoms compared with those with a heavy crop (Robinson and Watkins, 2003; Schupp, 2003). It is not known what causes this disorder and how it affects tree carbon supply to sink organs. The symptoms of this disorder are similar to the damage that potato leafhoppers [Empoasca fabae (Harris)] cause on apple leaves (Rosenberger et al., 2001). However, trees that are protected Received on 2 Dec. 2003. Accepted for publication 23 Apr. 2004. This work was supported in part by New York Apple Research and Development Program and Hatch funds. The authors thank Dr. Jim Schupp for helpful discussions on zonal chlorosis of ̒ Honeycrisp ̓leaves, Drs. Anil Ranwala and Bill Miller for assistance with carbohydrate analysis, and Drs. Ian Merwin and Chris Watkins for critical reading of the manuscript. 1Present address: Department of Horticulture, Fujian Agriculture and Forestry University, Fuzhou 350002, P.R. China. 2To whom reprint requests should be addressed: fax: 607-255-4355; e-mail: LC89@Cornell.edu. L. Cheng and L.S. Chen contributed equally to this work. J. AMER. SOC. HORT. SCI. 129(5):729–737. 2004. SeptJournal.indb 729 8/15/04 5:04:41 PM 730 J. AMER. SOC. HORT. SCI. 129(5):729–737. 2004. Materials and Methods PLANT MATERIALS. Four-year-old ʻHoneycrisp ̓ apple trees on Malling 9 rootstocks were used in this study. The trees were grown at a spacing of 1.2 × 4.2 m in the fi eld at Cornell Orchards in Ithaca, N.Y. They received standard horticultural practices, and disease and insect control. The cropload of trees was hand-thinned to four fruit/cm2 trunk cross-sectional area when the diameter of the largest fruit was 10 mm. On 6 July (2 weeks after the initial appearance of leaf disorder symptoms), recent fully expanded normal and chlorotic leaves were chosen at random on single-tree replicates to measure gas exchange, enzyme activities, metabolites, carbohydrates, chlorophyll (Chl) fl uorescence, pigments, and antioxidant enzymes and antioxidant metabolites. GAS EXCHANGE MEASUREMENTS. Carbon dioxide assimilation and stomatal conductance were measured with a CIRAS-1 portable photosynthesis system (PP systems, Herts, U.K.) using a standard broadleaf cuvette on both normal leaves and the chlorotic area of symptomatic leaves, at noon under ambient CO2 (360 μmol·mol–1), a photon fl ux density (PFD) of 1650 ± 30 μmol·m–2·s–1, air temperature of 22.0 ± 0.5 °C, and ambient water vapor pressure of 1.6 ± 0.2 kPa. EXTRACTION AND ASSAY OF KEY ENZYMES IN THE CALVIN CYCLE AND CARBOHYDRATE METABOLISM. Leaf disks (1 cm2 in size) were taken from both normal leaves and chlorotic areas of symptomatic leaves at noon under full sun (PFD of 1650 μmol·m–2·s–1), frozen in liquid N2, and stored at –80 °C until assay. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC4.1.1.39), NADP-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC1.2.1.12), phosphoribulokinase (PRK, EC2.7.1.19), both stromal and cytosolic 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 (2003a). Two or three frozen leaf disks were ground with a precooled mortar and pestle in 1.5 mL extraction buffer containing 50 mM Hepes-KOH (pH7.5), 10 mM MgCl2, 2 mM ethylenediaminetetraacetic acid (EDTA), 10 mM dithiothreitol (DDT), 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 gn for 5 min in an Eppendorf microcentrifuge at 2 °C, and the supernatant was used immediately for enzyme assays. Total Rubisco activity was measured after incubating the leaf extract in the assay solution for 15 min at room temperature as described previously (Cheng and Fuchigami, 2000). GAPDH activity was determined in a mixture (1 mL) of 100 mM Tricine-KOH (pH8.0), 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-KOH (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, seven units pyruvate kinase (EC2.7.1.40), 10 units lactate dehydrogenase (LDH, EC1.1.1.27), and one unit R5P isomerase (EC5.1.3.6). The reaction was initiated by adding the enzyme extract (Leegood, 1990). Stromal FBPase was assayed in a mixture (1 mL) of 50 mM Tris-HCl (pH8.2), 10 mM MgCl2, 1 mM EDTA, 0.1 mM fructose 1,6-bisphosphate (FBP), 0.5 mM NADP, four units of phosphoglucoisomerase (PGI, EC5.3.1.9), and two units of glucose-6phosphate dehydrogenase (G6PDH, EC1.1.1.49). The reaction was initiated by adding the enzyme extract (Holaday et al., 1992; Leegood, 1990). Cytosolic FBPase was assayed according to Holaday et al. (1992) with some modifi cations. The enzyme was assayed in 1 mL reaction mixture containing 50 mM Hepes-NaOH (pH 7.0), 2 mM MgCl2, 0.1 mM FBP, 0.5 mM NADP, four units of PGI and two units of G6PDH. The reaction was initiated by adding the enzyme extract. SPS was assayed according to Grof et al. (1998) with some modifi cations. Sixty microliters of enzyme extract were incubated for 15 min at 30 °C with 100 mM Hepes-KOH (pH7.5), 100 mM KCl, 6 mM EDTA, 30 mM uridine 5 ́-diphosphoglucose (UDPG), 10 mM fructose-6-phosphate (F6P), and 40 mM glucose6-phosphate (G6P) in a total volume of 100 μL. At the end of the incubation period, the reaction was stopped by adding 100 μL ice-cold 1.2 N 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 gn for 1 min. An aliquot (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-NaOH (pH 7.0), 5 mM MgCl2, 0.3 mM NADH, 0.8 mM PEP, 14 units LDH, and four 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 according to Chen and Cheng (2003a) without including reduced glutathione (GSH) in the extraction buffer. Aldose-6-phosphate reductase (A6PR, EC1.1.1.200) was extracted according to Negm and Loescher (1981) with some modifi cations. Three frozen leaf disks were ground with a precooled 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 gn for 5 min in an Eppendorf 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 NADPH at 340 nm. The reaction mixture (1 mL) contained 0.1 M Tris-HCl (pH 9.0), 0.11 mM NADPH, 50 mM G6P, and 25 μL of the extract. Reaction was ini |
| Starting Page | 886 |
| Ending Page | 887 |
| Page Count | 2 |
| File Format | PDF HTM / HTML |
| DOI | 10.21273/hortsci.39.4.886e |
| Alternate Webpage(s) | https://journals.ashs.org/jashs/view/journals/jashs/129/5/article-p729.pdf |
| Alternate Webpage(s) | https://doi.org/10.21273/hortsci.39.4.886e |
| Volume Number | 39 |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
