## 4 Materials and Methods
### Determination of Activities of Enzymes Involved in C Metabolism in Roots and Leaves  
Sucrose phosphate synthase activity (SPS, EC 2.4.1.14), sucrose synthase activity (SUS, EC 2.4.1.13), and hexokinase activity (HxK, EC 2.7.1.1) in roots and leaves were measured according to the instructions provided by the manufacturer of the assay kit (JCbio, Nanjing, China). For SPS, approximately 1 g of frozen material was ground to a fine powder in an icebath with 5 mL of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-NaOH buffer (50 mM, pH 7.5) containing 50 mM MgCl\({}_{2}\), 2 mM EDTA, 0.2% (\(w/v\)) bovine serum albumin, and 2% polyvinyl pyrrolidone (PVP). After centrifugation (10,000\(\times\)\(g\), 10 min), 50 \(\upmu\)L of supernatant was mixed with 50 \(\upmu\)L of HEPES-NaOH buffer, 20 \(\upmu\)L of 50 mM MgCl\({}_{2}\), 20 \(\upmu\)L of 100 mM uridine diphosphglucose (UDPG), and 20 \(\upmu\)L of 100 mM fructose. The mixture was incubated at 30 \({}^{\circ}\)C for 30 min, and the reaction was stopped by the addition of 200 \(\upmu\)L of 2 mM NaOH and boiling for 10 min. The solution was then cooled to room temperature. Next, 1.5 mL of 30% HCl and 0.5 mL of 0.1% resorcin were added and mixed thoroughly. Then, the mixture was incubated at 80 \({}^{\circ}\)C for 10 min. The solution was cooled to room temperature again and light absorption was measured spectrophotometrically at 480 nm. The sucrose concentration was calculated from a standard curve of known sucrose concentrations. For SUS, 0.5 g of the frozen material was ground to a fine powder in 5 mL of ice cold extraction buffer containing 100 mM Tris-HCl (pH 7.0), 5 mM MgCl\({}_{2}\), 2 mM EDTA-Na\({}_{2}\), 2% glycol, 0.2% bovine serum protein (BSP), 2% PVP, and 5 mM dithiothreitol (DTT). The extract was centrifuged at 10,000\(\times\)\(g\) for 20 min at 2 \({}^{\circ}\)C. Then, 3 mL of the supernatant was dialyzed overnight at 4 \({}^{\circ}\)C. The dialyaste (25 mM Tris-HCl (pH 7.0), 2.5 mM MgCl\({}_{2}\), 1 mM EDTA-Na\({}_{2}\), 1% glycol, 1 mM DTT) was refreshed every 4 h. Next, 0.1 mL of enzyme extract, 0.1 mL of 10 mM UDPG, 0.4 mL H\({}_{2}\)O, and 0.4 mL of reaction solution (100 mM Tris-HCl (pH 7.0), 10 mM fructose, 2 mM EDTA-Na\({}_{2}\), 5 mM magnesium acetate, 5 mM DTT) were incubated at 30 \({}^{\circ}\)C for 10 min. The reaction was stopped by boiling for 3 min. Then, 0.1 mL of 2 mM NaOH was added to the mixture, followed by boiling for 10 min. After cooling, 3.5 mL of 30% HCl and 1 mL of 0.1% resorcin were added, and the mixture was incubated at 80 \({}^{\circ}\)C for 10 min. The mixture was then cooled to room temperature again and the sucrose concentration was measured at 480 nm using the standard sucrose curve. For HxK, crude extracts were made by grinding frozen material (0.5 g) to a fine powder in liquid nitrogen. The fine powder was homogenized with 5 mL of ice- cold extraction buffer containing 20 mM KH\({}_{2}\)PO\({}_{4}\) (pH 7.5), 0.5 mM EDTA, and 5 mM DTT. After centrifugation (12,000\(\times\)\(g\), 20 min), the crude extract was desalted with Sepahdex G25 columns. HxK activity was quantified spectrophotometrically at 340 nm in 1 mL of reaction solution containing 50 mM Tris-HCl (pH 8.5), 10 mM MgCl\({}_{2}\), 110 mM glucose, 0.2 mM NADH, 0.5 mM ATP, two units of glucose-6-phosphate dehydrogenase (EC 1.1.1.49), and 33 \(\upmu\)L of crude extract. The activities of cell wall and vacuolar invertase (EC 3.2.1.26) were analysed according to the methods described by [PERSON] et al. [50]. Briefly, approximately 1 g of tissue was ground to a fine powder in liquid nitrogen and homogenized in 2 mL of extraction buffer (30 mM 3-(\(N\)-morpholino) propanesulfonic acid, 250 mM sorbitol, 10 mM MgCl\({}_{2}\), 10 mM KCl, and 1 mM phenylmethanesulfonyl fluoride, pH 6). The mixture was centrifuged at 3500\(\times\)\(g\) for 10 min. The pellet was washed twice with extraction buffer and resuspended in 1 mL of assay buffer (20 mM triethanol amine, 7 mM citric acid, and 1 mM PMSF, pH 4.6), and used for the determination of cell wall invertase activity.