5.1 Experimental Site Description and Experimental Materials
The research site was located in Jianning County, Fujian Province (116° 35’~117° 04’E, 26° 30’~27° 06’N), where the elevation ranges from 280 to 858 m, the annual average temperature is 16.5 to 17.5°C, the average rainfall is 1800 mm-2100 mm, the average sunshine duration is 1721 h per year and the relative humidity is 84% [3, 21]. The soil parent material is feldspar sandstone, and the soil texture is mostly heavy loam (Cartesian) to light clay (soil layer thickness > 100 cm) [68, 69].
The experimental forest (approximately 5200 ha) was planted by Yuan Hua Forestry Biotechnology Co. in 2008. The experimental plot was located in the Fengyuan Demonstration Zone. The study trees were planted in 2009 with 2-year-old nursery stock, which came from Tiantai, Zhejiang Province, using level terraces, and the planting density was 675-750 plants/ha. Management measures included pruning once per year, fertilization once per year, and disease and insect pest prevention 3-5 times per year.
Thirty healthy trees that had 3 scaffold branches with an angle of 60° and 16-18 fruiting branches per unit area that had grown for 8 years were selected. The planting holes were 50 × 40 × 40 cm, the plant spacing was 4 × 4 m, the tree height was 5.9 ± 0.64 m, the tree basal diameter was 16.14 ± 0.51 cm, and the tree crown width was 4.2 ± 0.66 m.
5.2 Field Experimental Measurements
5.2.1 Spraying Treatments, 13C Labelling and Sampling
This experiment was conducted to evaluate the effects of exogenous sucrose application at two stages: 20 days before blooming (DBB stage) and the second fruit abscission stage (DBFA stage); the dates of these two stages were defined according to the research of Gao et al.  from 2013-2015. The blooming stage began on 18th May ± 2 days, and the abscission stage began on 28th August ± 2 days (Fig. 8). Sucrose solutions of 6 different concentrations, 0% (control), 1%, 1.5%, 3%, 5% and 7%, were applied by spraying three times every seven days [7, 70] until 5 days before blooming and abscission, for a total of 6 times during these two different stages. The experiment was conducted with a completely randomized block design with 5 replications (n= one tree per replicate) and 6 treatments. Each tree was sprayed with 5 L of water containing dissolved sucrose, which made the surface wet to the point of dripping. The experiment was carried out under temperatures of 27 and 21°C (maximum and minimum, respectively), and the light ratio percentage under the canopy was in the range of approximately 30-40%. The samples were randomly selected; 8 soapberry inflorescences and infructescences from outer branches growing in different directions at the middle-canopy height were selected from each treatment after 7 days of spraying. The leaves were selected from the 4th-8th leaf positions from the shoot tip.
13CO2 labelling experiments were carried out under sunny conditions from 11:00 to 13:00 after 3 sprayings during the two stages. Four inflorescences and infructescences were selected from branches with different orientations for each treatment group at the middle-canopy height for 13C labelling. Transparent polyethylene (PE) plastic bags (40 × 50 cm) were used to cover the inflorescences and infructescences.
Twenty millilitres of 13CO2 (99 atom%) (purchased from SRICI) was injected into the bags every hour a total of 4 times to maintain the 13CO2 density at 500 μmol/mol. The samples were taken 24, 48, 72 and 96 h after 13C isotope labelling; 5 soapberry leaves, flowers or fruits were randomly selected from the labelled inflorescences and infructescences. The consumption trends in these four samples were compared for the same time periods, and there was no significant consumption at 96 h, so 24 and 48 h were chosen for analysis of the results.
All sampled material was converted to CO2 and then analysed for δ13C by isotope ratio mass spectrometry (DELTA V Advantage) with an accuracy better than 0.1‰, which was performed by Thermo Fisher Scientific, Inc., USA. 13C (mg/g) was calculated by the formula below:
13C(mg/g) = mDW*C%*(Atom%13Clabelled- Atom%13Cunlabelled) .
5.2.2 Determination of Sucrose, Glucose and Fructose
One-gram samples were dissolved in 5 mL of ultra-pure water and subjected to ultrasonic bath extraction for 10 min. The samples were centrifuged for 10 min at 12000 r/min and 4°C and then filtered through 0.45-μm hydrophobic membranes.
The concentrations of sucrose, glucose, and fructose were quantified by high-performance liquid chromatography (HPLC). An Agilent Zorbax carbohydrate column (4.6×150 mm, 5 μm) was used for the analysis and kept at 35°C. The mobile phase was 80% acetonitrile + 20% ultra-pure water. The sample injection volume was 10 μl, the flow velocity was 1 mL/min, and the injection time was 17 min. Prior to carrying out the determination of TNCs in the sample, standard solutions of sucrose, fructose and glucose were prepared. Calibration curves were made for each of the sugars using these solutions [34, 72, 73].
5.2.3 Determination of Endogenous Hormones
In this study, extraction of plant hormones was performed by HPLC as previously described [74, 75], with some modifications as detailed below. Approximately 1 g of fresh leaves was frozen in liquid nitrogen and ground into powder. Five millilitres of 80% ice-methanol (4°C, kept in the dark) was used to soak the samples for 10 h. Then, the samples were centrifuged at 12000 r/min at 4°C for 30 min and transferred to a bottle, to which 1-2 drops of ammonium hydroxide were added. The bottles were rotary evaporated at 38°C until reaching 1/3 of their original volume. Then, 2 mL of distilled water was used to wash the samples into 10-mL tubes; the samples were centrifuged at 12000 r/min at 4°C for 30 min again and adjusted to a pH of 2.5-3.0 (for IAA, gibberellic acid (GA3) and ABA) or 7.5-8.0 (for ZT). IAA, GA3 and ABA were extracted by ethyl acetate, ZT was extracted by water-saturated n-butyl alcohol, and the extractions were repeated 3 times. The samples were rotary evaporated to dryness and finally dissolved in 1 mL of methanol-0.1 mol/L acetic acid (1:4, v/v), except for ZT, which was dissolved in 3% methanol-97% ultra-pure water with a pH=7. Prior to HPLC analysis, the solution was filtered using a 0.45-µm microfilter. A 4.6 × 250-mm 5-micron Zorbax Eclipse×DB-C18 analytical column was used for analysis, with a sample injection volume of 5 μL, a flow velocity of 1 mL/min, and a temperature of 30°C. The mobile phase for IAA, GA3 and ABA was 20% methanol + 80% 1N acetic acid and for ZT was 40% methanol+60% ultra-pure water with a pH=7.
5.2.4 Determination of Mineral Elements
The leaves were cleaned, separated, and dried in a 70°C oven, and the dry plant samples were ground and analysed for their nutrient contents (N, P, and K). H2SO4-H2O2 was used for digestion, and then the N (total nitrogen), P (total phosphorus), and K (total potassium) concentrations were measured using a Kjeldahl nitrogen meter (Beijing, China), the vanadium molybdenum yellow colorimetric method, and ﬂame photometry, respectively . Samples were wet digested in a mixture of nitric acid-perchloric acid (HNO3:HClO4 (4:1), and the K, Ca, and Mg concentrations in the digest were quantified by atomic absorption spectrophotometry (Varian Model Spectra-400 Plus) .
5.3 Data Analysis
The data were statistically analysed using Student’s t-test (P < 0.05). Five biological replicates were used in the TNC, endogenous hormone, and mineral element analyses, and data were analysed with SPSS Statistics 20.0 software (SPSS Inc., Chicago, IL, USA) at the P ≤ 0.05 significance level using Duncan’s multiple range test. To identify significant differences, repeated measurements were statistically compared between the treatments using one-way analysis of variance (ANOVA). Correlation and linear regression analyses were performed to identify and evaluate the relationships among the lengths of shoots, inflorescences, and infructescences and the 13C content of leaves, fruits and seeds.
The relationships between fruit and leaf nutrients were investigated using canonical correlation analysis (CCA). Five groups were created (1. Leaf sugar: LS, LG, and LF; 2. Fruit sugar: FS, FG, and FF; 3. Leaf hormones: L-GA3, L-IAA, L-ABA, and L-ZT; 4. Fruit hormones: F-GA3, F-IAA, F-ABA, and F-ZT; and 5. Leaf mineral elements: LN, LP, LK, LCa, and LMg) to examine the correlation between a linear combination of the fruit nutrient variables (X-set), designated canonical variable U, and a linear combination of the leaf nutrient variables (Y-set), designated canonical variable V. All the data were standardized. Ten repeated comparisons were used in the analysis. All computations used to examine the relationships between the two sets of traits were performed with R software [78, 79]. Excel 2016 and Origin 2017 were used to create the charts.