Plant materials and ABA treatments
Four years old rabbiteye blueberry ‘Brightwell’ were grown in experimental base of Baima district, Nanjing city, China. The rabbiteye blueberry ‘Brightwell’ were obtained from Institute of Botany, Jiangsu Province and Chinese Academy of Sciences and identified by professor Wu Wenlong. Blueberries of same size and growing condition were selected to do the treatment. Each treatment was arranged in a randomised complete block design with 6 replications and each block replication contains 6 shrubs.
The (+)-Abscisic Acid ( Purity 95%, Coolaber company, China ) was dissolved in double distilled H2O containing 5% (v/v) ethanol and 0.1% Tween 80. When most of blueberries fruits were in a growing stage of ‘green mature’ and the top of the fruit at the top of the branch has just begun to turn red, 0, 500 and 1000 mg L−1 ABA solutions were sprayed on fruit clusters. Tiny sprayer were used to spray the ABA solutions on the peels untill the peels are wetted. The leaves and branches were carefully avoided.
In different development stage of fruits, 10 fruits of same size and weight were selected to determine fruit firmness, Brix and color and 30 fruits were immediately frozen in liquid nitrogen and stored at -80℃ for later experiments.
Physiological characterization
Fruit hardness
The fruit hardness were determined by Fruit hardness tester ( Catno.9300, Takemura Electric Works Co., Japan ). The cone type tip was used and the tip was perpendicularly applied on the side surface of blueberry fruits.The value was measured at the moment of tip intrusion to the surface.
Brix
The Brix were determined by saccharometer ( PAL-1, Atago Co., Japan ). The juice were left on the prim for 20 seconds to do the measurement.
The color of fruit peel
Fruit peel color was measured by colorimeter ( Ci64, X-Rite, US ) and shown by the International Commission on Illumination a∗ and b∗ colour space co-ordinates29. The a* value is negative for green and positive for red an the b* value is negative for blue and positive for yellow, both of the values range from -100 to 100. Due to the coloration of rabbiteye fruits starts from top to bottom, the top, side and bottom of fruit peel were separately measured in the same stage.
The total anthocyanins content
The total anthocyanins content were determined by the double pH differential method and some adjustments are made30. Absorbance of the extract was measured at 510 and 700 nm in buffers at pH 1.0 (hydrochloric acid–potassium chloride, 0.2 M) and 4.5 (acetate acid–sodium acetate, 0.2 M). Total anthocyanins content was calculated using a molar extinction coefficient of 29,600 (cyanidin-3-glucoside) and absorbance of A = [(A510 - A700)pH 1.0 -(A510 - A700)pH 4.5].
Light microscopy
Semithin section analysis was performed to observe the fruit pulp cells according the following method31. In brief, the 1mm x 3mm specimens were fixed with 0.1‐M phosphate buffer (pH 7.2) containing 2.5% (v/v) glutaraldehyde and washed thrice with 0.1‐M phosphate buffer and then soaked in 1% (v/v) osmic acid for 2 hours. Specimens were washed again using 0.1‐M phosphate buffer as above and dehydrated through a gradient ethanol series. Ultimately, the specimens were embedded and polymerized in Spurr's resin. Semithin sections (2 μm) were stained with 1% methylene blue and then photographed under a Zeiss Axio vert A1 microscope. Images are representative of at six observed samples.
Reducing power assay
Reducing power assay were adapted from the following method and the ascorbic acid (0.1 mg/ml) was used as positive control32. Briefly, 0.15g samples homogenized with 1.35 mL of phosphate buffer (0.2 M, pH 6.6) and centrifuged at 5000 × g for 10 mins. The supernatant (1mL) was mixed with 1 mL of potassium ferricyanide solution (1%, w/v). The mixtures were then reacted at 50 ℃ for 20 min. Then, 1 mL of trichloroacetic acid (10%, w/v) solution was added to stop the reaction immediately. After centrifugation, 1 mL of supernatant was mixed with 1 mL of distilled water and 0.2 mL of ferric chloride solution (0.1%, w/v). At 10 min later, the 96 plates were shaken sufficiently and measured spectrophotometrically at 700 nm.
DPPH radical scavenging activity assay
DPPH radical scavenging activity were measured by the following method and the ascorbic acid (0.5 mg/ml) was used as positive control33. DDPH(0.1 mM) was dissolved in ethanol. Sample slurry was added to 3 DPPH reaction systems. Absorbance was measured at 515 nm after 30 min to reach a steady state.
The scavenging activity of the DPPH radical was calculated as follows:
See formula 1 in the supplementary files.
The reaction system: Ai, 1mL ethanol + 1mL DDPH solution; Aj, 1mL sample slurry + 1 mL DDPH ; Ao, 1mL sample slurry + 1mL ethanol.
Metabolite analysis
Metabolite extraction
About 5g blueberry fruits of each treatment were thoroughly grounded with liquid nitrogen. Then, 100mg of homogenate was taken and resuspended with prechilled 80% methanol and 0.1% formic acid by well vortexing. The samples were incubated on ice for 5 min and then were centrifuged at 15000 rpm, 4°C for 5 min. Equal supernatant was diluted to final concentration containing 60% methanol by LC-MS grade water. The samples were subsequently transferred to a fresh Eppendorf tube with 0.22 μm filter and then were centrifuged at 15000 g, 4°C for 10 min. Finally, the filtrate was injected into the LC-MS/MS system analysis.
UHPLC-MS/MS Analysis
LC-MS/MS analyses were performed using a Vanquish UHPLC system (Thermo Fisher) coupled
with an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher). Samples were injected
onto an Hyperil Gold column (100×2.1 mm, 1.9μm) using a 16-min linear gradient at a flow rate
of 0.2 mL/min. The eluents for the positive polarity mode were eluent A (0.1% FA in Water) and
eluent B (Methanol). The eluents for the negative polarity mode were eluent A (5 mM ammonium
acetate, pH 9.0) and eluent B (Methanol). The solvent gradient was set as follows: 2% B, 1.5 min;
2-100% B, 12.0 min; 100% B, 14.0 min;100-2% B, 14.1 min;2% B, 16 min. Q Exactive HF-X
mass spectrometer was operated in positive/negative polarity mode with spray voltage of 3.2 kV,
capillary temperature of 320°C, sheath gas flow rate of 35 arb and aux gas flow rate of 10 arb.
Metabolite obtainment
The raw data files generated by UHPLC-MS/MS were processed using the Compound Discoverer 3.0 (CD 3.0, Thermo Fisher) to perform peak alignment, peak picking, and quantitation for each metabolite. The main parameters were set as follows: retention time tolerance, 0.2 minutes; actual mass tolerance, 5ppm; signal intensity tolerance, 30%; signal/noiseratio, 3; and minimum intensity ,100000. After that, peak intensities were normalized to the total spectral intensity. The normalized data was used to predict the molecular formula based on additive ions, molecular ion peaks and fragment ions. And then peaks were matched with the mzCloud (https://www.mzcloud.org/) and ChemSpider (http://www.chemspider.com/) database to obtained the accurate qualitative and relative quantitative results.
Differential Analysis
Significant differences were calculated by one-way ANOVA tests or T-test in SPSS 19 (IBM, USA). Different letters and asterisk (*) in figure indicate statistical significance (P< 0.05). Unsupervised principal component analyses (PCA) and partial leastsquares discriminant analysis (PLS-DA) clustering method were performed by MetaboAnalyst (http://www.metaboanalyst.ca/). Variable importance in projection (VIP) is the weighted sum of squares of the PLS-DA analysis, which represent the contribution rate of metabolites difference in different groups; Fold Change (FC) is the ratio of the mean of all replicate quantitative values for each metabolite in the comparison group. Combined with T-test, differential metabolites are screened by the following criteria: VIP > 1.0, FC > 1.2 or FC < 0.833 and P value < 0.05.