Materials and methods
2.1 E-2-Hexenal treatment and
sampling
Tomato (solanum lycopersicum )
fruits were harvested in Jinan. The developmental status was strict
controlled, only the tomatoes with slight reddish colour were selected.
Tomato fruits were transported to
the laboratory within 1 hour,
sterilization with 2% sodium hypochlorite for 2 min. The tomatoes were
divided into different groups and storage at 20oC,
each group contained no less than 30 fruits.
When tomatoes were treated with E -2-Hexenal (Aladdin, Shanghai,
China), they were placed in 50 L plastic sealed boxes. Different volumes
of E -2-Hexenal liquid were added to filter paper in the petri
dish and placed in boxes to achieve environmental concentrations at 0.5,
2.5, 12.5 and 25 μL L-1 as liquid volume / gas volume.
The treatments were named as E2H (0.5), E2H (2.5), E2H (12.5) and E2H
(25) separately.
When tomatoes were treated with combined E -2-Hexenal and
ethylene, tomatoes were treated in the same 50 L plastic sealed boxes
with E -2-Hexenal and ethylene (12 mg L-1)
sequentially [ET (12 h) and then E2H (12 h) or E2H (12 h) and then ET
(12 h)] or simultaneously.
The pericarp tissues of the tomatoes were sampled at 0, 6, 18, 36 and 72
h, then quickly frozen with liquid nitrogen and
stored at -80 ° C.
2.2 Transmission electron
microscopy
(TEM)
The tomatoes were treated with E2H for 24 h at the concentration of 2.5
μL L-1. After 72 h, tissues of the pericarp close to
the skins were collected. All tissues were cut with a razor blade to
sizes of about 0.5 mm × 2.0 mm2 and immediately
treated with 5% glutaraldehyde (0.1 M sodium phosphate buffer, pH 7.4)
for 1 day at 4°C. The sodium cacodylate buffer (0.1 M) was used for
washing the tissues three times for 20 min. The post fixing was
performed with 1% osmium tetraoxide for 1 h at 4 °C. The tissues
after incubation for 1 h with 1% uranyl acetate at 4 °C were
dehydrated in graded dimethyl ketone and then transferred into Epon 812
(Plano, Marburg, Germany). A rotating microtome blade was used to cut
65 nm thin sections which were mounted subsequently onto the copper
grids. The section staining and counter staining was performed using
uranyl acetate (5%) and Reynold’s lead citrate, respectively. The
section examination was carried out under Jeol-Jem 1011 transmission
electron microscope (Jeol, Tokyo, Japan)
(Schweiggert et al. , 2011).
2.3 Pigment
contents
The 3nh Colorimeter (3nh, Shenzhen, China) were used to detect the a*
values of the tomatoes. Data was obtained by detecting the four sides of
the fruit equator and a* values were calculated as the average of the
four data. All values were in triplicate.
The fruit tissues (0.2 g) were extracted in 80% ice-cold acetone. Total
chlorophyll contents were measured spectrophotometrically by reading
absorbance at 663 (A645) and 645 nm
(A663). The total chlorophyll quantities were calculated
as (Arnon, 1949):
Total chlorophyll contents = 20.29
A645+8.05A663
The fruit tissues (1.0 g) were ground and homogenized with 2 mL ice-cold
acetone and 2 mL dichloromethane. The extraction was repeated until the
supernatant appeared colorless. Combined the supernatants and diluted to
25 mL and then filtered. The extractions should avoid the lights all the
time.
Carotenoids were separated by HPLC (Shimadzo, Tokyo, Japan) using a
Zorbax SB-C18 column (silica 5 μm,4.6 nm x 250nm) (Agilent, Santa Clara,
USA).
The mobile phase consisted of acetonitrile:H2O (9:1)
(solvent A) and 100% ethyl acetate (solvent B), which were used in a
linear gradient between A and B for 30 min, at a flow of 1 mL
min-1. The carotenoids were detected using UV detector
at the wavelength of 475nm. The carotenoids were confirmed by their
retention times compared to the lycopene and β‐carotene standard
compounds. Peak areas of the standard compounds were used to
quantitative analysis the carotenoids in the samples
(Ronen, Cohen, Zamir & Hirschberg,
1999).
2.4 LXOC and HPL gene
expression
Total RNA was isolated using the Plant Total RNA Isolation Kit Plus
(Foregene, Chengdu, China) according to the manufacturer’s instructions.
cDNA was synthesized using M5 Superfast qPCR RT Kit (Juhemei, Beijing,
China) (Yang, Wang, Tan, Fu & Sun,
2019). Quantitative PCR was performed using the SYBR Green PCR kit
(Juhemei, Beijing, China) on a PTC-200 real time PCR system: 95 °C for
10 min followed by 30 cycles of 95 °C for 15 s, and 60 °C for 60 s.
Gene-specific primers were listed in Table 1. The Actin was used
as a housekeeping gene. Calculation of the relative quantification was
performed by the comparative 2-ΔΔCTmethod (Livak & Schmittgen, 2001). All
reactions were in triplicate.
2.5 The titratable acids, ascorbic
acids, soluble solids and malondialdehyde
contents
The titratable acidity (TA) and soluble solid contents (SSC) were
analyzed according to the previously reported method
(Sun, Zhang, Guo, Yu & Chen, 2013). Nine
fruits were selected to determine the TA and SSC. The pericarp tissues
were homogenized in a mortar, and then homogenates were filtered.
Obtained juice was used to determine TA and SSC. The TA was analyzed by
0.1 mol·L−1 NaOH adding dropwise to the juices until the solution
reached a pH of 8.1. The SSC was analyzed with a held-held refractometer
(Atago, Tokyo, Japan).
The ascorbic acids (Vc) and malondialdehyde (MDA) contents were analyzed
according to the previously reported method
(Wei, Lv, Xia, Tan, Sun, Yu, Jia & Cheng,
2017). Tomato pericarp tissues (5 g) were homogenized with 10 mL of 5%
(w/v) metaphosphoric acid using a T25 homogenizer (IKA, German) at speed
6 (highest). The supernatant was used for ascorbic acid measurements
with the 2, 4-dinitrophenylhydrazine method. After a sulfuric acid
(85%, v/v)-assisted colour reaction, spectrophotometry was used to
determine the ascorbic acid content at 500 nm. For MDA, the absorbencies
of the aqueous phase at 450 nm, 532 nm and 600 nm were measured. The MDA
content in the aqueous phase was calculated according to the following
formula (Wei et al. , 2017):
MDA
(mol L−1) = [6.45 × (A532 − A600) − 0.56 × A450] × 10−6.
2.6 Statistical
analysis
Data were subjected to Duncan’s Multiple Range Test to determine
significant differences (P < 0.05)
(Wei et al. , 2017).