Food Bioscience 54 (2023) 102892

Contents lists available at ScienceDirect

Food Bioscience
journal homepage: www.elsevier.com/locate/fbio

Variation in antioxidant enzyme activity and key gene expression during

fruit development of blackberry and blackberry–raspberry hybrids
Xin Huang a, Yaqiong Wu a, *, Shanshan Zhang a, Hao Yang b, Wenlong Wu a, Lianfei Lyu a,
Weilin Li b, **
a
Institute of Botany, Jiangsu Province and Chinese Academy of Sciences (Nanjing Botanical Garden Mem. Sun Yat-Sen), Jiangsu Key Laboratory for the Research and
Utilization of Plant Resources, Qian Hu Hou Cun No. 1, Nanjing, 210014, China
b
Co-innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, 159 Longpan Road, Nanjing, 210037, China

A R T I C L E I N F O A B S T R A C T

Keywords: Blackberry (Rubus spp.) belongs to the Rubus genus in the Rosaceae family, and its fruits are rich in nutrients and
Blackberry (Rubus spp.) function in exerting antioxidant effects. Based on this, the fruits of ’Chester’ blackberry, ’Hull’ blackberry and the
Antioxidant ability hybrid cultivars ’Boysen’ and ’Young’ at different developmental stages were selected to evaluate antioxidant-
Fruit ripening
related indexes, antioxidant enzyme activity and the expression patterns of genes encoding antioxidant en­
Expression
zymes. The results showed that the total antioxidant capacity of the hybrid fruits was higher than that of the pure
blackberry fruits. PCA (principal component analysis) and correlation analysis showed that the antioxidant ca­
pacity was related to anthocyanin, ASA (ascorbic acid), GSH (glutathione), MDA contents and O-2 production
rate. However, the correlations between antioxidant capacity and metabolites were different among the cultivars.
The O-2 production rate and MDA (malondialdehyde) content increased with fruit development. The activities of
CAT (catalase) and POD (peroxidase) were maintained at a relatively low level, while SOD (superoxide) activity
was maintained at a relatively high level. This study was the first to analyze the content of antioxidant substances
in different developmental stages of blackberry fruits and blackberry-raspberry hybrids fruits, which provided
some suggestion for the extraction of specific antioxidant substances in different developmental stages. Based on
the transcriptome data, the role of antioxidant enzyme genes in antioxidant system was concerned, providing a
practical basis for the expression characteristics of key genes.

1. Introduction The extracts of blackberry fruits have many pharmacological benefits,

Blackberry (Rubus spp.) is a small shrub that belongs to the Rubus
genus (Rosaceae), and its fruits are polymeric berries (Kim et al., 2016).
The fruits of blackberry, a third-generation fruit species, have a unique
flavor and high nutritional and medicinal value; as a result, they are
popular with consumers. Hybrid cultivars obtained by crossing black­
berry and raspberry parents not only produce fruits with the excellent
flavor of blackberry fruits but also overcome the problem involving
difficult germination of seeds (Lim & Knight, 2000). Therefore, hybrid
cultivars have broader research importance and application markets.
such as protecting against liver damage, treating diabetes and protecting
the cardiovascular system (Dou et al., 2021; Park et al., 2019; Serino
et al., 2020). In addition, blackberry fruits contain vitamins, poly­
phenols, flavonoids, anthocyanins and other substances (Betta et al.,
2018) that delay aging, improve immunity and increase antioxidant
capacity (Betta et al., 2018).
Flavones, flavonols, flavanones, flavanonols, anthocyanidins, flava­
nols and isoflavones are the most common flavonoids in nature. They all
have a C6-C3-C6 ring structure, ensuring that flavonoids have the ability
to scavenge free radicals, provide hydrogen atoms or electrons, and

Abbreviations: MDA, malondialdehyde; PAL, phenylalanine ammonia-lyase; FLS, flavonol synthase; LAR, leucoanthocyanidin reductase; ANR, anthocyanin
reductase; UFGT, UDP-glucose: flavonoid 3-O-glucosyltransferase; ROS, reactive oxygen species; SOD, superoxide; POD, peroxidase; CAT, catalase; SA, salicylic acid;
GSH, glutathione; ASA, ascorbic acid; NBT, nitroblue tetrazolium; PTM, posttranslational modification.
* Corresponding author.
** Corresponding author.
E-mail addresses: HuangX19980601@163.com (X. Huang), ya_qiong@126.com (Y. Wu), 15943097183@163.com (S. Zhang), yanghao_19940720@163.com
(H. Yang), 1964wwl@163.com (W. Wu), njbglq@163.com (L. Lyu), wlli@njfu.edu.cn (W. Li).

https://doi.org/10.1016/j.fbio.2023.102892
Received 13 April 2023; Received in revised form 17 June 2023; Accepted 26 June 2023
Available online 28 June 2023
2212-4292/© 2023 Elsevier Ltd. All rights reserved.
X. Huang et al.
chelate metal ions, which equates to antioxidant activity (Dias et al.,
2021). In recent years, research and utilization of flavonoids has been a
popular topic worldwide and has been reported in a variety of plant
species. Chalcone in licorice can significantly reduce the content of
malondialdehyde (MDA) and inhibit lipid peroxidation to reduce the
rate of apoptosis (Wu, Wang, et al., 2022). The antioxidant activity in
citrus peel and pulp is positively correlated with some flavonoids, such
as diosgenin and naringin (Zhu et al., 2020). Therefore, the content and
type of flavonoids in plants can greatly affect their antioxidant capacity.
However, the biosynthesis of flavonoids and the contents of their com­
ponents are regulated by complex networks. Biosynthesis begins with
L-phenylalanine and is regulated by both structural genes and tran­
scription factors (Naik et al., 2022). Structural genes encode enzymes
involved in the biosynthesis of flavonoids and anthocyanins, which
include phenylalanine ammonia-lyase (PAL), flavonol synthase (FLS),
leucoanthocyanidin reductase (LAR), anthocyanin reductase (ANR) and
flavonoid 3-O-glucosyltransferase (UFGT). Downregulation of the
PuPAL gene in pear decreases the contents of flavonoids and indirectly
affects the scavenging capacity of reactive oxygen species (ROS) (Sun
et al., 2022). Heterologous expression of the RuFLS2 gene was shown to
increase the flavonoid content significantly in tobacco leaves (Huang
et al., 2022a). The expression of both CsUFGT genes in purple tea was
positively correlated with anthocyanin accumulation (Chen et al.,
2020). Transcription factors indirectly regulate the synthesis of flavo­
noids and anthocyanins through the regulation of the expression of
structural genes (Qi et al., 2020). There are currently many transcription
factor families studied, including the MYB, bHLH and WRKY families.
The PpMYB17 gene can upregulate the expression of the PpFLS gene and
ultimately regulate the synthesis of flavonoids (Premathilake et al.,
2020). Different members of the same transcription factor family in the
same species tend to regulate flavonoids in different directions. The
cherry PabHLH3 gene enhances MYB-induced anthocyanin synthesis,
whereas PabHLH33 appears to have a repressive effect (Starkevič et al.,
2015). MYB, bHLH, and WD40 also commonly bind to form W-B-M
complexes that coregulate flavonoid and anthocyanin accumulation,
and the expression patterns of blueberry MYB, bHLH, and WD40 genes
are positively correlated with anthocyanin accumulation (Zhao et al.,
2019).
There are other enzymes involved in antioxidation in plants, such as
superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD).
Scavenging of intracellular excess O-2 is the main function of SOD, which
can convert O-2 to H2O2 (Che et al., 2022); both CAT and POD can
effectively scavenge H2O2 (Duarte et al., 2021). It has been found that
the activities of antioxidant enzymes are affected by numerous factors,
and exogenous NO can significantly increase the activities of CAT, POD
and SOD (Madebo et al., 2022), while cytokinin and salicylic acid (SA)
can also induce an increase in the activities of these enzymes (Cavusoglu
et al., 2021; Hanif et al., 2020). Changes in antioxidant enzyme activity
are also associated with cultivar differences. Sweet potato (Dioscorea
esculenta (Lour.) Burkill cv. Xushu 32) had significantly higher antioxi­
dant activity than cultivar ’Yanshu 25′ under the same storage condi­
tions (Wang et al., 2019). The POD enzyme activity of dates (Phoenix
dactylifera L. cv. Piarom) was significantly higher than that of cultivar
’Zahedi’ and cultivar ’Deiri’ under the same radiation treatment (Zar­
bakhsh & Rastegar, 2019). At the molecular level, the activity of anti­
oxidant enzymes is also linked to the genes that encode them. An
increase in CAT and SOD activity was associated with an increase in the
expression level of related genes in senescence control experiments in
apple (Malus domestica Borkh cv. Golden delicious) (Wei et al., 2019).
The expression levels of POD3, POD6 and POD63 also increased signif­
icantly with increasing POD activity in strawberry (Fragaria × ananassa
cv. Falandi) (Zhang, Wang, et al., 2019).
At present, research on the antioxidants of blackberry has mainly
focused on the extraction, purification and pharmacological effects of
active substances (Wu et al., 2022c), but there is a lack of research on the
dynamic changes in the antioxidant system during fruit growth and
2
Food Bioscience 54 (2023) 102892
development. To explore the changes in antioxidant enzyme activity, the
variety and content of antioxidant active substances and the molecular
mechanism through which their synthesis and accumulation are regu­
lated during the development of blackberry fruit were examined. In this
study, we used the fruits of pure blackberry cultivars (Rubus spp. cv.
’Chester’; Rubus spp. cv. ’Hull’) and two hybrid blackberry cultivars:
’Young’ (Rubus ursinus derivative) which hybridized of Rubus, raspberry
and blackberry; ’Boysen’ (Rubus ursinus Chamisso & Schlenhtendal)
which may hybridized of Rubus ursinus and Rubus idaeus or originate
from Rubus ursinus var. Loganobaccus, a hybrid of loganberry, blackberry
and raspberry, as plant materials to (I) reveal the change trend of anti­
oxidant capacity at different fruit developmental stages, (II) identify
which indicators represent the major antioxidant capacity via principal
component analysis (PCA), (III) evaluate the levels of antioxidant
enzyme synthesis and flavonoid biosynthesis gene expression during
fruit development, and (IV) further understand the affinity of antioxi­
dant enzymes and related genes via correlation analysis. This study was
the first to compare the weight, color parameters and the content of
antioxidative substances in different developmental stages of pure and
hybrid blackberry fruits, providing a basis for the directional selection of
active substances in blackberry fruits and blackberry-raspberry hybrid
fruits, and lays a foundation for the production and marketing of fruits
and the development and utilization of medicinal value of their fruits.
2. Materials and methods
2.1. Plant materials and growth environment
The fruits of blackberry (Rubus spp.) cultivars ’Chester’ (Genealogy:
SIUS 47 × Thornfree) and ’Hull’ (Genealogy: (US 1487 × Darrow) ×
Thornfree) and the hybrid blackberry cultivars ’Boysen’ (Genealogy:
(Rubus ursinus (female) × Rubus idaeus (male)) or Rubus ursinus var.
loganobaccus) and ’Young’ (Genealogy: (Rubus × raspberry) × Mayes
Dewberry) were used as plant materials (Fig. 1). These four cultivars
were grown in acidic clay soil at the Baima experimental station in
Lishui District, Nanjing Province, China (119◦ 11′E, 31◦ 36 N). The period
from the beginning of fruit development to full maturity spans from May
to July. The fruits of all cultivars were grown under natural conditions
without any special treatment. We distinguished the sampling period
according to the color of the fruits. There were five stages: green fruit
was defined as stage one (S1), green to red fruit was defined as stage two
(S2), red fruit was defined as stage three (S3), red to purple fruit was
defined as stage four (S4), and purple fruit was defined as stage five (S5)
(Fig. 1a–d). The fruits used to take the pictures were repeated three
times per stage for each cultivar. The fruits used for the determination of
dry weight, fresh weight and moisture content were repeated six times
per group. Once the fruits were picked, they are brought back to the
laboratory in an ice box and tested on the same day. The other experi­
ments were biologically repeated at least three times. The samples used
in these experiments were stored in liquid nitrogen after collection and
brought back to the laboratory for storage in a − 80 ◦ C refrigerator for
later use.
2.2. Determination of fresh weight (FW), dry weight and color of fruits
The weight of fresh fruits was measured by an electronic balance
with a precision of 0.001 g. Then, a constant-temperature blast dryer
was used to dry these fruits, and the dry weight was determined. The
drying temperature was 80 ◦ C. All fresh fruits were imaged under the
same conditions and with sufficient light. The color, lightness and
saturation of the fruits of the 4 cultivars at 5 different developmental
stages were analyzed using Adobe Photoshop 2020 software (Wu,
Zhang, et al., 2022). The Color Picker tool in the software was used to
identify the color parameter of upper, lower, left, right and middle parts
of each fruit, and the color parameters (brightness (L*), red‒green phase
(a*) and yellow‒blue phase (b*)) of each color were recorded. The
X. Huang et al. Food Bioscience 54 (2023) 102892

Fig. 1. (a) ’Chester’, (b) ’Hull’, (c) ’Boysen’, and (d) ’Young’ fruit phenotypes; (e) ’Chester’, (f) ’Hull’, (g) ’Boysen’, and (h) ’Young’ fruit color parameters; and (i)
’Chester’, (j) ’Hull’, (k) ’Boysen’, and (l) ’Young’ fruit moisture contents at different fruit developmental stages. The different letters indicate significant differences
across the different developmental stages of harvested fruits (p<0.05). The different colors represent different color parameters. L* indicates the brightness of color,
where positive values indicate whiteness and negative values indicate blackness; a* represents red‒green preferences, where positive values indicate red and
negative values indicate green; b* represents yellow‒blue preferences, where positive values indicate yellow and negative values indicate blue; C* indicates the ratio
of colored components to white components, and the higher the value is, the brighter the color. (For interpretation of the references to color in this figure legend, the
reader is referred to the Web version of this article.)

biology was repeated three times, and the saturation value (C*) refers to was separated into a clean centrifuge tube with a pipette. After that, 0.3
the value resulting from the calculation method of Voss (1992). mol L-1 acetic acid-sodium acetate buffer (pH 3.6), 0.01 mol L-1 TPTZ
solution and 0.02 mol L-1 FeCl3 were prepared and added to a working
solution at a volumetric ratio of 10:1. After preheating 5 μL of working
2.3. Determination of total antioxidant capacity
solution at 37 ◦ C, 180 μL of the supernatant was added. The solution was
subsequently mixed and incubated at 37 ◦ C for 5 min. The absorption
The total antioxidant capacity was determined by the ferric ion
value at 593 nm was measured by an enzyme-labeled instrument (model
reducing antioxidant power (FRAP) method and ABTS (2.2′-azino-bis (3-
Scientific Multiskan Sky, Thermo Fisher Technology (China) Co., Ltd.,
ethylbenzothiazoline-6-sulfonate)) method. The principle is that the
Shanghai, China). Each sample was detected three times. A linear
antioxidant can reduce ferric-tri-pyridyl-tria-azine (Fe3+-TPTZ) to pro­
regression equation between the concentration and absorbance of FeS­
duce blue Fe2+-TPTZ under acidic conditions. The total antioxidant
O4⋅7H2O was established with distilled water as the control and different
capacity can be calculated by measuring the absorbance at a wavelength
concentrations of FeSO4⋅7H2O as the standards. The absorbance of the
of 593 nm. A total of 0.5 g of fruit tissue was added to 2 mL 0.1 mol L-1
sample was introduced into the equation to calculate the antioxidant
phosphate-buffered saline (PBS) buffer (pH = 7.4). Afterward, the tissue
capacity of the sample. The results were expressed as mmol (FeS­
was milled with a precooled mortar in an ice water bath and centrifuged
O4⋅7H2O equivalent) of FeSO4⋅7H2O per gram of tissue protein.
at 6150×g (4000 rpm in an F-34-6-38 rotor, model 5804 R centrifuge,
The principle of ABTS method is that ABTS can be oxidized to green
Eppendorf Co., Hamburg, Germany) at 4 ◦ C for 10 min. The supernatant

3
X. Huang et al.
ABTS+, which can be inhibited by the antioxidants (Cano et al., 2023). It
has absorption peak at 405 nm. The antioxidation ability can be calcu­
lated by measuring the absorbance of the reaction fluid. A commercial
total antioxidant capacity (ABTS method) kit (Nanjing Jiancheng Insti­
tute of Biological Engineering) was used for the determination. A total of
0.5 g of fruit tissue was added to 4.5 mL 0.1 mol/L PBS buffer (pH = 7.4).
Afterward, the tissue was milled with a pre-cooled mortar in an ice water
bath and centrifuged at 18450×g (12000 rpm in a F-34-6-38 rotor,
model 5804 R centrifuge, Eppendorf Co., Hamburg, Germany) at 4 ◦ C for
5 min. Then the experiment is carried out according to the instruction in
the kit. Preparing trolox solutions of 0.1, 0.2, 0.4, 0.8, 1 mM, repeated
the operation, and the results were plotted as a standard curve. The
absorbance values at 405 nm were determined by enzyme-labeled in­
strument (model Scientific Multiskan Sky, Thermo Fisher Technology
(China) Co. , Ltd., Shanghai, China). Each sample was detected three
times. The absorbance of the sample was introduced into the curve to
calculate the antioxidant capacity of the sample. Besides, the antioxi­
dant capacity of plant tissue should be calculated by dividing by the
protein concentration of the homogenate. The results were expressed as
mmol (trolox equivalent) of trolox per gram of tissue protein.
The protein concentration of homogenate used in the calculation was
determined by Coomassie Brilliant Blue method (Huang et al., 2022b). A
total of 0.5 g of fruit tissue was added to 4.5 mL 0.1 mol/L PBS buffer
(pH = 7.4). Afterward, the tissue was milled with a pre-cooled mortar in
an ice water bath and centrifuged at 6150×g (4000 rpm in a F-34-6-38
rotor, model 5804 R centrifuge, Eppendorf Co., Hamburg, Germany) at
4 ◦ C for 10 min. The supernatant and Coomassie G-250 were combined
and incubated at room temperature for 5 min. The absorbance values at
595 nm were determined by enzyme-labeled instrument (model Scien­
tific Multiskan Sky, Thermo Fisher Technology (China) Co. , Ltd.,
Shanghai, China). The same experimental procedure was performed on
the protein standards in the kit.The protein content in the sample was
calculated according to the following formula.
(AS − A0 ) × Cck × n
Cprot (mg / g FW) =
Ack − A0
Where AS, A0, and Ack are the absorbance of the sample, blank control
and the standard separately; Cck is the concentration of protein standard;
n is the dilution factor.
2.4. Determination of MDA content
The basic principle of MDA content determination is that MDA can
react with thiobarbituric acid (TBA) at low pH and elevated tempera­
ture, generating a red adduct (Janero, 1990). The adduct has absorption
peak at 532 nm and low peak at 600 nm. It should be noted that sugars in
plants can interfere with the detection results, and the absorption peak
at 450 nm needs to be detected to eliminate the interference. The
experimental procedures is modified with reference to Zeng, Guo, Xiao,
Cao, and Peng’s (2018) method. The specific operation steps are as
follows. A total of 0.5 g of fruit tissue was added to 5 mL of 0.05 mol L-1
PBS solution. Afterward, the mixture was homogenized in an ice water
bath and then centrifuged at 6150×g (4000 rpm in an F-34-6-38 rotor,
model 5804 R centrifuge, Eppendorf Co., Hamburg, Germany) at 4 ◦ C for
10 min. One milliliter of the supernatant was added to 1 mL of 10%
tricarboxylic acid (TCA) (containing 0.5% TBA), mixed in a centrifuge
tube, and allowed to react at 100 ◦ C for 30 min. Washing the outer
surface of the tube with cold water for rapid cooling, then the super­
natant was centrifuged at 6150×g (4000 rpm in an F-34-6-38 rotor,
model 5804 R centrifuge, Eppendorf Co., Hamburg, Germany) at room
temperature (24–26 ◦ C) for 10 min, and its absorbance values at 450 nm,
532 nm and 600 nm were determined by an enzyme-labeled instrument
(model Scientific Multiskan Sky, Thermo Fisher Technology (China) Co.,
Ltd., Shanghai, China). Each sample was detected three times. Then, the
MDA content in the sample was calculated according to the following
4
Food Bioscience 54 (2023) 102892
formula. The results were expressed as μmol of MDA per gram of FW.
[6.45 × (A532 − A600 ) − 0.56 × A450 ]×Vt
CMDA (μmol / g FW) =
Vs × W
A532、A600、A450 refer to the absorbance of the reaction solution at
450, 532 and 600 nm, respectively. Vt is the volume of the extraction
solution, Vs is the sample size, and W is the weight of the sample.
2.5. Determination of ROS content
The superoxide anion (O-2) production rate was determined using the
hydroxylamine oxidation method (Wei et al., 2021). A total of 0.5 g of
fruit tissue was added to 1 mL of 0.05 mol⋅L-1 precooled PBS solution and
placed in a precooled mortar. After grinding the sample on ice, PBS was
added until the final volume reached 5 mL. Afterward, the mixture was
homogenized in an ice water bath and then centrifuged at 15400×g
(10000 rpm in an F-34-6-38 rotor, model 5804 R centrifuge, Eppendorf
Co., Hamburg, Germany) at 4 ◦ C for 10 min. Then, 0.5 mL of supernatant
was added to 0.5 mL of 50 mmol/L PBS buffer and 1 mL of 10 mmol/L
NH4Cl solution. The liquid was mixed and reacted at 25 ◦ C for 20 min.
After adding 1 mL of 58 mmol/L sulfa solution and 1 mL of 7 mmol/L
α-naphthylamine solution, the mixture reacted at 25 ◦ C for 20 min. The
absorbance was measured at 530 nm, and each sample was detected
three times. Standard solutions of 0.5, 1, 2, 3, 4, and 5 μg/mL were
prepared, the operation was repeated, and the results were plotted as
standard curves. The concentration of NO2- was calculated by the
standard curve. The reaction time between hydrogen amine chloride
and O-2 was used to calculate the formation rate of superoxide anion. The
results were expressed as micromoles of O-2 produced per gram of FW per
minute.
H2O2 reacts with molybdic acid to form a complex with an absorp­
tion peak at 405 nm. A commercial hydrogen peroxide test kit (Nanjing
Jiancheng Institute of Biological Engineering) was used to determine the
hydrogen peroxide content in the sample. One gram of fresh fruit was
homogenized in an ice bath with 9 mL of 0.1 mol⋅L-1 PBS and then
centrifuged at 15400×g (10000 rpm in an F-34-6-38 rotor, model 5804 R
centrifuge, Eppendorf Co., Hamburg, Germany) at room temperature for
10 min. The reagent was added according to the requirements of the kit
and mixed thoroughly, and the absorbance was measured at 405 nm.
The absorbency was determined with double distilled water used as a
blank control and 163 mmol⋅L-1 H2O2 used as a standard control. Each
sample was detected three times. The results were expressed as μmol of
H2O2 per gram of FW.
2.6. Determination of glutathione (GSH) and ascorbic acid (AsA)
contents
The glutathione and ascorbic acid (AsA) contents were determined in
reference to the methods of Aguilera et al. (2020). In total, 0.5 g of fruit
tissue was mixed with 5 mL of 5% TCA and centrifuged at 12300×g
(4000 rpm in an F-34-6-38 rotor, model 5804 R centrifuge, Eppendorf
Co., Hamburg, Germany) at room temperature for 10 min. The contents
of GSH and AsA in the supernatant were subsequently determined.
Under neutral conditions, glutathione can catalyze colorless 5,
5′-dithio-bis (2-nitrobenzoic acid) (DTNB) to form yellow 5-mercap­
to-2-nitrobenzoic acid, which has a maximum absorption peak at 412
nm. A total of 0.2 mL of the supernatant was mixed with 2.6 mL of 0.15
mol L-1 NaH2PO4 and 0.2 mL DTNB in a 37 ◦ C water bath for 5 min. The
absorbance value at 412 nm was measured by an enzyme-labeled in­
strument (model Scientific Multiskan Sky, Thermo Fisher Technology
(China) Co., Ltd., Shanghai, China). Each sample was detected three
times. GSH standard solutions with concentration gradients of 0, 10, 20,
30, 40, and 50 mg mL-1 were prepared. The GSH content in the sample
was calculated according to the standard curve. The results were
expressed as milligrams of GSH per gram of FW.
AsA can reduce iron ions to ferrous ions, and ferrous ions and red
X. Huang et al.
phenanthroline form a red chelate with an absorption peak at 525 nm.
The AsA content was determined by adding 0.2 mL of supernatant to 0.2
mL of 0.15 mol⋅L-1 NaH2PO4 and 0.2 mL of water. After 30 s of the re­
action occurring at room temperature, 0.4 mL of 10% TCA and 0.4 mL of
44% H3PO4 were added successively, followed by 0.4 mL of 44%
bipyridine (MSDS) and 0.2 mL of 3% FeCl3, in a water bath for 60 min.
The absorption value at 525 nm was measured by an enzyme-labeled
instrument (model Scientific Multiskan Sky, Thermo Fisher Technol­
ogy (China) Co., Ltd., Shanghai, China). The samples were replaced by
TCA at different concentrations, the other operations were consistent,
and standard curves were subsequently constructed. Each sample was
detected three times. The AsA content in the sample was calculated
according to the standard curve. The results were expressed as milli­
grams of AsA per gram of FW.
2.7. Determination of antioxidant enzyme activity
A commercial peroxidase kit (Nanjing Institute of Biological Engi­
neering) was used to determine the activity of POD. Take 0.1 mL of the
supernatant (the preparation of the supernatant reference to 2.5 H2O2
extraction method) in a tube, and reagents were added according to the
instructions of the kit. The liquid was mixed and reacted in a 37 ◦ C water
bath for 30 min, and then reagent IV was added immediately to stop the
reaction. The absorption value at 593 nm was measured by an enzyme-
labeled instrument (model Scientific Multiskan Sky, Thermo Fisher
Technology (China) Co., Ltd., Shanghai, China). The standard POD was
diluted to different concentrations to replace the supernatant of the
sample for the same operation, and the standard curve was drawn with
the results obtained. Each sample was detected three times. The results
were expressed as units of POD activity per gram of FW.
Based on ammonium molybdate colorimetry (Chandlee & Scandal­
ios, 1984), the decomposition of H2O2 by CAT can be terminated by
adding ammonium molybdate because the remaining H2O2 can react
with ammonium molybdate to form a yellowish complex. The activity of
CAT was determined using a commercial peroxidase kit (Nanjing Jian­
cheng Institute of Biological Engineering). The method of extracting
supernatant was described in section 2.3. The experimental operation
and the result calculation were carried out according to the instructions
and the formula provided by the manual. Each sample was detected
three times. The results were expressed as units of enzyme activity per
milligram of tissue protein.
The activity of SOD was determined by the nitro blue tetrazolium
(NBT) method (Puchoňová et al., 2018). The method of extracting su­
pernatant was described in section 2.5 (O-2 production rate determina­
tion). 50 μL of supernatant was added to 1.5 mL of PBS (0.05 mol/L),
300 μL of Met (130 mmol/L), 300 μL of NBT (750 μmol/L), 300 μL of
EDTA-NA2 (100 μmol/L), 300 μL of riboflavin (20 μmol/L), and 250 μL
of distilled water. At the same time, 0.05 mol/L PBS was used instead of
enzyme solution as two control groups: one group was placed in the
dark, and the other group was placed in the assay tube under sunlight for
color reaction. The control group in the dark was used to zero the
spectrophotometer (model 759 photometer, Shanghai Jinghua Tech­
nology Instrument Co., Ltd. Shanghai, China). Each sample was detected
three times. The results were expressed as the number of active units per
gram of FW.
2.8. Screening and quantitative analysis of antioxidant-related genes
Based on the transcriptome expression statistics of previous studies
conducted by our research group (Wu, Zhang, et al., 2022), genes with
significant differences in expression levels before and after fruit ripening
were screened and named according to the annotated information to
investigate the expression of genes in blackberry fruits. Genes related to
antioxidant activity, including 12 antioxidant enzyme-related genes, 6
flavonoid synthesis pathway structural genes and 13 transcription
factor-encoding genes, were selected. Primers were designed using Oligo
5
Food Bioscience 54 (2023) 102892
6 software (Table S1).
Total RNA extraction was performed using a universal plant total
RNA rapid extraction kit (Bioteke, Beijing, China), followed by reverse
transcription of total RNA for cDNA synthesis by a PrimeScript RT
Master Mix (Perfect Real Time) Kit (Takara, Dalian, China). TB Green
Premix Taq II (TLI RNASEH Plus) (Takara, Dalian, China) was used for
RT‒qPCR. A 15 mL reaction system was used, which consisted of 7.5 mL
of TB Green Premix Taq II fluorescent dye, 1 mL of cDNA template, 0.6 μl
of upstream and downstream primers and 5.3 μl of distilled water. The
qRT‒PCR procedure was as follows: 95 ◦ C for 2 min, 95 ◦ C for 10 s, 60 ◦ C
for 10 s, 72 ◦ C for 15 s, and melt for 6 s for 40 cycles. Each gene in each
sample was detected three times.
2.9. Statistical methods for data analysis
All the experimental data were analyzed for comparisons using the
SPSS 24.0 statistical software program (SPSS, Chicago, IL). The mean
values were compared by one-way ANOVA, and Duncan’s multiple
comparisons were used to calculate the significant differences (p<0.05)
in the substances measured in the fruits of each cultivar at different
stages. Pearson correlation analysis and bivariate correlation analysis
were used to analyze the mean and standard deviation of antioxidant-
related indexes of all the stages in each cultivar, and two-tailed test
was used to analyze the correlation of all indexes. The positive value
represents the positive correlation, the negative value represents the
negative correlation. Principal component analysis (PCA) reduces the
dimensionality of the original data of these indexes by factor reduction,
and SPSS automatically calculates the eigenvector matrix and principal
component matrix.
3. Results
3.1. Analysis of fruit color, weight and moisture content
With fruit development, the fruit brightness (L*) of the four cultivars
decreased, and the red‒green phase (a*) first increased and then
decreased, with the lowest value recorded at S1 (negative) and the
highest value recorded at S3. The yellow‒blue phase (b*) values of the
four cultivars were all positive, and the ’Chester’ b* values decreased
gradually with fruit development. The other three cultivars showed a
slight increase from S2 to S3 and a decreasing trend overall. Interest­
ingly, the b* values were higher than the a* values in the green period
and the first color-changing period, but the b* values were lower than
the a* values in the last three periods (Fig. 1e–h), which was consistent
with the results shown in the image (Fig. 1a–d). The C* values of the 4
cultivars showed a trend of first decreasing, then increasing and then
decreasing again; the C* values in S3 were the highest. The C* values of
’Young’ mature fruits were higher than those of the other three culti­
vars; as such, the ’Young’ mature fruits were more brightly colored,
while the fruits of the other three cultivars were darker.
By analyzing the changes in weight and moisture content during the
fruit development of blackberry and blackberry hybrid cultivars, we
found that the moisture content of small fruits of the four cultivars
increased gradually with fruit development (Fig. 1i-l). The cultivar
whose fruits had the highest moisture content was ’Chester’, the content
of which reached 90.41% (Fig. 1i). The fruit weights of ’Chester’, ’Hull’
and ’Boysen’ were higher than those of ’Young’. We also did correlation
analysis among dry weight, fresh weight and moisture content during
fruit development. The results showed that they were significantly
positively correlated in all the 4 cultivars (Table S3).
3.2. Analysis of the antioxidant capacity of fruits at different
developmental stages
We chose FRAP method and ABTS method to determine the antiox­
idant capacity of blackberry fruits at different developmental stages. The
X. Huang et al. Food Bioscience 54 (2023) 102892

results of FRAP showed that the fruits of ’Chester’, ’Boysen’ and ’Young’
fruits had the lowest total antioxidant capacity in S3. ’Hull’ fruits had
the lowest antioxidant capacity except in S3, the value of which was
slightly higher than that of ’Chester’ fruits. The mature fruits of ’Boysen’
had the greatest antioxidant capacity (Fig. 2a). The results of ABTS
showed the similiar trends. The fruits of ’Hull’, ’Boysen’ and ’Young’
had the lowest total antioxidant capacity in S3 (Fig. 2b). Both methods
showed that the antioxidant capacity of blackberry-raspberry hybrids
was higher than that of pure blackberry cultivars at S1, S2, S3 and S5.
The content of H2O2 in the ’Chester’, ’Hull’ and ’Boysen’ fruits
decreased first and then increased, while the content of H2O2 in the
’Young’ fruits increased and then decreased sharply from S4 to S5.
Overall, the content of H2O2 in green fruits was significantly higher than
that in fruits at other developmental stages, and the content of H2O2 in
red fruits was the lowest (Fig. 2c).The changes in O-2 were very similar to
those in MDA content. The rate of O-2 production was the slowest in S1.
Conversely, the O-2 production rate was the fastest at S5 except in
’Chester’ fruits, in which the fastest was in S4. Among that in the fruits of
the other three cultivars, the O-2 production rate in the fruits of ’Boysen’
was the fastest and that of ’Young’ was the slowest; the concentrations
were 15.64 μmol min-1⋅g-1 and 3.74 μmol min-1⋅g-1, respectively
(Fig. 2d).
The MDA content in the four cultivars was the lowest in S1 (Fig. 2e).
The MDA content in ’Chester’ peaked at S4. The MDA content of the
other 3 cultivars increased gradually with growth and was the highest in
the fully mature fruits. The results showed that the MDA content in
’Boysen’ fruits was the highest, reaching 71.36 μmol/g FW (Fig. 2e).
The variation trend of AsA content in the fruits of different black­
berry cultivars was very different. The AsA content in the second color-
changing period was nearly the same in all the cultivars. ’Boysen’ and
’Young’ fruits had significantly higher AsA contents at S1, S3 and S5
than at the other stages. ’Hull’ fruits had very high AsA contents at S1
and S5, and the highest AsA content was at S1, reaching 17.17 mg/g FW
(Fig. 2f). The changes in GSH content in the fruits of the 4 cultivars
tended to become smooth in S1, S2, and S3 and increased in S4 and S5
(Fig. 2g). The increasing trend of ’Hull’ was the most intense, and the
GSH content of mature fruit was the highest, reaching 15.10 mg/g FW.
3.3. Analysis of antioxidant enzyme activities in fruits in different stages
We measured the activities of SOD, POD and CAT and found that the
activity of SOD was lower than that of POD and similar to that of CAT in
green fruits. Then, the activity of SOD increased, and the activities of
POD and CAT decreased. The activities of SOD in ’Boysen’ and ’Young’
were higher than those of POD and CAT during the five fruit develop­
mental stages. The SOD activity of ’Hull’ and ’Young’ showed a trend of
increasing first and then decreasing. The SOD activity of ’Boysen’
increased gradually with the growth of the fruits, and there was no

Fig. 2. Variation in antioxidant activities. (a) FRAP; (b) ABTS; (c) MDA content; (d) H2O2 content; (e) production rate of O-2; (f) AsA content; (g) GSH content. The
different letters indicate significant differences between the cultivars (p<0.05). The different colors represent the different developmental stages of the harvested
fruits. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

6
X. Huang et al.
significant difference at S4 and S5 of development. The SOD activity of
Chester continued to increase during the whole fruit growth stage. The
activities of SOD in mature fruits were significantly higher than those of
the other two antioxidant enzymes. The CAT activity of ’Chester’ fruits
was the highest at S5, and that of ’Hull’ fruits was the highest at S4
(Fig. 3). The changes in POD activity in ’Chester’ and ’Hull’ were
similar. The POD activity decreased rapidly from S1 to S3 and then
increased slightly. In contrast, the changes in POD enzymes in ’Boysen’
and ’Young’ fruits were less drastic. The activity of POD in Chester fruits
was the highest (132.85 U⋅g-1 FW), while that in ’Young’ fruits was the
lowest (59.85 U⋅g-1 FW). Overall, only the CAT activity in ’Young’ fruit
was slightly higher than the POD activity at S2, and the CAT activity was
lower than the POD activity at the other stages in the four cultivars.
During fruit development, the CAT activity decreased gradually and
continuously.
3.4. PCA
To understand the relationships between members of the antioxidant
system and antioxidant capacity in blackberry fruits, the following were
analyzed by PCA: the activities of CAT, SOD, and POD; the contents of
flavonoids, anthocyanins, total phenolics, total antioxidant capacity,
AsA, GSH, H2O2, and MDA; and the O-2 production rate of each cultivar
(Table S2). What’s more, we drew PCA diagrams of the four cultivars
(Fig. 4). The figure showed that the indicators of ’Chester’, ’Hull’ and
’Boysen’ could be divided into two principal components. The first
principal component had a negative load on SOD, GSH, MDA, TAC and
the O-2 production rate. The second principal component had a positive
load on most indexes (Fig. 4a, b, c). The indicators were divided into
Fig. 3. Variation in antioxidant enzyme activity. (a) ’Chester’; (b) ’Hull’; (c) ’Boysen’; (d) ’Young’.
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Food Bioscience 54 (2023) 102892
three principal components in ’Young’ (Fig. 4d). The first principal
component had a positive load on CAT, POD, H2O2, TFC and TPC while
the second principal component had a negative load on CAT and SOD.
The third principal component had a negative load on H2O2, MDA and
the O-2 production rate (Table S2). The location of the samples showed
that the antioxidant indicators of the fruits in ’Chester’ at S4 and S5 were
very similar (Fig. 4a).
3.5. Correlation analysis of antioxidant contents
The correlations between the antioxidant capacity and antioxidant
enzyme activity of the fruits of the 4 cultivars at different stages were
analyzed. FRAP was always highly positively correlated with ABTS. It
means that both FRAP and ABTS can explain the antioxidant capacity of
blackberry fruits well. In ’Chester’, ABTS was positively correlated with
O-2 production rates and MDA content. FRAP was positively correlated
with the CAT and POD activities of ’Hull’ and negatively correlated with
SOD activity (Table 1). FRAP in ’Young’ was also significantly nega­
tively correlated with SOD activity, whereas in ’Boysen’, the opposite
was true. The O-2 production rates and MDA content were negatively
correlated with CAT activity, as was POD activity (Tables 1 and 2). The
AsA and H2O2 contents were positively correlated with the activities of
CAT and POD in pure blackberry cultivars (Table 1). Notably, the cor­
relations between these four indexes and SOD enzyme activity are
opposite those of the other two enzymes (POD and CAT). In ’Chester’,
’Hull’ and ’Boysen’, the enzyme activity was significantly or extremely
significantly correlated with the production rate of H2O2, O-2 and MDA.
Because flavonoids, anthocyanins and total phenolics have anti­
oxidation abilities and based on the results of the determination of
X. Huang et al.
Fig. 4. Principal component analysis diagram of (a) ’Chester’; (b) ’Hull’; (c) ’Boysen’; (d) ’Young’.
flavonoid, anthocyanin and total phenolic contents in the fruits of Rubus
L. at different stages (Huang et al., 2022b), the relationships between
antioxidant capacity and the three factors were analyzed. The results
showed that the total flavonoid content (TFC) and total phenolic content
(TPC) were positively correlated with the H2O2 content in all cultivars
except ’Young’. Conversely, there was a significant negative correlation
between TFC and O-2 production rate and between TPC and MDA content
in ’Chester’ and ’Hull’. In ’Chester’, TAC was significantly positively
correlated with the O-2 production rate and GSH content. In the other
three cultivars, the total anthocyanin content (TAC) was significantly
positively correlated with the O-2 production rate, GSH and MDA con­
tent. In general, TAC was positively correlated with GSH content and O-2
production rate in blackberries. TFC and TPC were positively correlated
with H2O2 content and negatively correlated with MDA content and O-2
production rate.
3.6. Analysis of antioxidant enzyme-encoding gene expression patterns in
fruits at different stages
Based on previous transcriptome data, the expression patterns of 12
genes encoding antioxidant enzymes (namely, 1 CAT gene, 4 SOD genes
and 7 POD genes) were analyzed. The largest change in RuSOD1
expression was detected in ’Hull’ fruits at different stages of develop­
ment, and the smallest was in ’Young’ fruits (Fig. 5a). The expression
level of RuSOD2 in ’Chester’ and ’Hull’ fruits was similar to that of
RuSOD1 at different developmental stages (Fig. 5b). The peak of
RuSOD2 expression occurred at S5 of fruits. RuSODCP.2 tended to
decrease first, increase rapidly and then decrease rapidly during the
development of ’Hull’ fruits and peaked at S3 (Fig. 5c). RuFSD was
highly expressed in the fruits of ’Chester’ at S2, while the other three
cultivars presented the highest expression level at S1 (Fig. 5d). The
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Food Bioscience 54 (2023) 102892
expression levels of the RuPOD, RuPER5, RuPER17, RuPER25, RuPER29
and RuPER45 genes significantly increased in the fruits of ’Chester’ at
the S2 stage (Fig. 5e–i). During the development of ’Young’ fruits, the
expression level of RuPER45 changed greatly, especially at S2 (Fig. 5j).
The expression of RuPRDX6 changed greatly during the development of
’Chester’ and ’Young’ fruits (Fig. 5k). Unlike in ’Chester’, RuCAT had
the highest expression level in S2, S3 and S5 of fruit development, and
the highest expression level was detected in S5 of ’Chester’ fruit
development. The other three cultivars presented little difference across
fruit development (Fig. 5l).
3.7. Correlation analysis between antioxidant enzyme activity and
antioxidant enzyme-encoding genes
To understand the relationship between antioxidant enzymes and the
expression levels of related genes in blackberry, the activities of CAT,
SOD, POD and 12 genes in 4 cultivars were analyzed (Table 3). The
results showed that CAT activity was negatively correlated with the
RuCAT gene in 4 cultivars and was significant only in ’Hull’. RuSOD1
and RuSOD2 were positively or significantly correlated with SOD ac­
tivity in ’Chester’ and ’Hull’ and negatively or significantly correlated
with SOD activity in ’Boysen’. RuSODCP.2 and RuFSD were negatively
correlated with SOD activity in 4 cultivars and reached an extremely
significant level in ’Young’. There was no significant correlation be­
tween the seven genes encoding POD and POD activity in ’Chester’, but
in ’Boysen’, POD activity was positively correlated with the seven genes
encoding POD. POD activity was positively correlated with ’Hull’ POD
activity and negatively correlated with ’Young’ POD activity. However,
there was a significant negative correlation between RuPER29 and ’Hull’
POD activity and a significant positive correlation between RuPER29
and ’Young’ POD activity.
 X. Huang et al.

Table 1
Correlations of antioxidant enzyme activity with antioxidant capacity and relative expression level of antioxidant enzyme-encoding genes in blackberry.
CAT SOD POD H2O2 O-2 MDA ASA GSH TFC TAC TPC FRAP ABTS

Chester CAT 1 -0.918** 0.938** 0.882** -0.840** -0.882** 0.454 -0.502 0.936** -0.657** 0.898** 0.855** 0.555* CAT Hull
SOD -0.944** 1 -0.797** -0.797** 0.848** 0.854** -0.399 0.516* -0.890** 0.649** -0.808** -0.730** -0.311 SOD
POD 0.951** -0.837** 1 0.862** -0.640* -0.716** 0.598* -0.231 0.953** -0.406 0.851** 0.739** 0.596* POD
H2O2 0.788** -0.623* 0.903** 1 -0.571* -0.626* 0.756** -0.209 0.913** -0.389 0.993** 0.884** 0.771** H2O2
O-2 -0.808** 0.862** -0.736** -0.397 1 0.985** 0.058 0.867** -0.642** 0.939** -0.628* -0.740** -0.227 O-2
MDA -0.810** 0.820** -0.795** -0.496 0.979** 1 -0.008 0.822** -0.693** 0.912** -0.681** -0.784** -0.325 MDA
ASA 0.943** -0.806** 0.930** 0.833** -0.629* -0.655** 1 0.454 0.691** 0.299 0.692** 0.409 0.606* ASA
GSH -0.563* 0.717** -0.382 -0.01 0.860** 0.760** -0.336 1 -0.209 0.969** -0.302 -0.548* -0.06 GSH
TFC 0.994** -0.914** 0.962** 0.829** -0.760** -0.772** 0.966** -0.494 1 -0.377 0.895** 0.732** 0.528* TFC
TAC -0.597* 0.774** -0.333 -0.06 0.640* 0.49 -0.429 0.800** -0.539* 1 -0.471 -0.691** -0.208 TAC
TPC 0.939** -0.803** 0.905** 0.772** -0.659** -0.671** 0.988** -0.387 0.957** -0.463 1 0.926** 0.787** TPC
FRAP 0.28 -0.249 0.244 0.511 0.255 0.271 0.353 0.295 0.316 -0.188 0.271 1 0.797** FRAP
ABTS -0.092 0.068 -0.21 0.041 0.533* 0.610* 0.004 0.359 -0.062 -0.189 -0.038 0.855** 1 ABTS

Note: ’*’ denotes significant differences between various indicators by Duncan’s multiple range test (p<0.05). ’**’ denotes significant differences between various indicators by Duncan’s multiple range test (p<0.01).

9
Table 2
Correlations of antioxidant enzyme activity with antioxidant capacity and relative expression level of antioxidant enzyme-encoding genes in blackberry–raspberry hybrids.
CAT SOD POD H2O2 O-2 MDA ASA GSH TFC TAC TPC FRAP ABTS

Boysen CAT 1 -0.542* 0.699** 0.499 -0.927** -0.918** -0.06 -0.518* 0.657** -0.791** 0.639* 0.188 0.797** CAT Young
SOD -0.769** 1 -0.484 -0.818** 0.251 0.223 0.227 -0.198 -0.801** 0.196 -0.846** -0.698** -0.884** SOD
POD 0.916** -0.556* 1 0.239 -0.593* -0.567* 0.477 -0.131 0.862** -0.541* 0.818** -0.182 0.504 POD
H2O2 0.930** -0.563* 0.947** 1 -0.238 -0.231 -0.591* -0.116 0.513 -0.435 0.554* 0.612* 0.807** H2O2
O-2 -0.760** 0.881** -0.559* -0.499 1 0.995** 0.003 0.685** -0.441 0.821** -0.402 0.022 -0.592* O-2
MDA -0.837** 0.941** -0.631* -0.613* 0.973** 1 0.014 0.699** -0.406 0.823** -0.368 0.03 -0.572* MDA
ASA 0.37 -0.492 0.418 0.421 -0.102 -0.267 1 0.256 0.266 0.193 0.227 -0.512 -0.291 ASA
GSH -0.719** 0.919** -0.516* -0.47 0.950** 0.932** -0.244 1 0.165 0.840** 0.202 0.361 -0.092 GSH
TFC 0.807** -0.358 0.968** 0.905** -0.372 -0.44 0.389 -0.318 1 -0.308 0.994** 0.285 0.767** TFC
TAC -0.728** 0.884** -0.530* -0.458 0.997** 0.966** -0.12 0.955** -0.339 1 -0.266 0.226 -0.454 TAC
TPC 0.977** -0.835** 0.895** 0.890** -0.793** -0.873** 0.439 -0.763** 0.764** -0.767** 1 0.365 0.794** TPC

FRAP -0.14 0.653** 0.074 0.188 0.699** 0.626* -0.114 0.729** 0.248 0.738** -0.235 1 0.673** FRAP
ABTS 0.17 0.462 0.385 0.445 0.345 0.312 -0.247 0.444 0.532* 0.4 0.065 0.876** 1 ABTS

Note: ’*’ denotes significant differences between various indicators by Duncan’s multiple range test (p<0.05). ’**’ denotes significant differences between various indicators by Duncan’s multiple range test (p<0.01).
Food Bioscience 54 (2023) 102892
X. Huang et al. Food Bioscience 54 (2023) 102892

Fig. 5. Relative expression levels of antioxidant enzyme-encoding genes of Rubus fruits at different developmental stages. (a) RuCAT; (b) RuSOD1; (c) RuSOD2; (d)
RuSODCP.2; (e) RuFSD; (f) RuPOD; (g) RuPER5; (h) RuPER17; (i) RuPER25; (j) RuPER29; (k) RuPER45; (l) RuPRDX6. The different letters indicate significant dif­
ferences between different developmental stages (p<0.05). The different colors represent different developmental stages. (For interpretation of the references to color
in this figure legend, the reader is referred to the Web version of this article.)

Table 3
Correlations of antioxidant enzyme activity and relative expression level of antioxidant enzyme-encoding genes.
Chester Hull Boysen Young

CAT SOD POD CAT SOD POD CAT SOD POD CAT SOD POD

RuCAT -0.446 0.44 -0.258 -0.804** 0.886** -.632* -0.169 -0.431 -0.433 -0.35 0.432 -0.401
RuSOD1 -0.397 0.596* -0.114 -0.793** 0.721** -.618* 0.05 -0.644** -0.255 0.518* 0.258 -0.005
RuSOD2 -0.459 0.633* -0.176 -0.819** 0.783** -0.642** 0.338 -0.599* 0.3 -0.116 0.734** -0.081
RuSODCP.2 0.535* -0.336 0.748** -0.152 -0.145 -0.442 0.622* -0.164 .828** 0.924** -0.734** 0.777**
RuFSD 0.46 -0.641* 0.424 0.387 -0.345 0.201 0.833** -0.394 .949** 0.610* -0.859** 0.760**
RuPOD 0.338 -0.522* 0.324 0.741** -0.888** 0.580* 0.875** -0.404 .957** -0.570* 0.550* -0.900**
RuPER5 0.368 -0.547* 0.358 0.597* -0.571* 0.425 0.835** -0.372 .927** -0.526* 0.313 0.124
RuPER17 0.471 -0.640* 0.448 0.061 -0.287 -0.259 0.954** -0.600* 0.984** 0.206 -0.681** 0.653**
RuPER25 0.336 -0.522* 0.329 0.367 -0.163 0.264 0.499 0.104 .638* 0.143 -0.761** 0.358
RuPER29 0.374 -0.523* 0.4 -0.285 0.082 -0.579* 0.761** -0.346 .921** 0.760** -0.837** 0.839**
RuPER45 0.404 -0.575* 0.396 0.688** -0.747** 0.549* 0.574* -0.458 0.699** 0.101 0.472 -0.439
RuPRDX6 -0.642** 0.794** -0.406 -0.192 -0.014 -0.46 0.603* -0.547* .670** -0.258 0.235 -0.431

Note: ’*’ denotes significant differences between various indicators by Duncan’s multiple range test (p<0.05). ’**’ denotes significant differences between various
indicators by Duncan’s multiple range test (p<0.01).

3.8. Expression pattern analysis of genes related to flavonoid and
anthocyanin pathways
The expression levels of RuPAL and RuFLS peaked in the second color
transition period of ’Chester’, ’Hull’ and ’Boysen’ (Fig. 6a and b). The
expression of RuANR was different in different fruit developmental
stages. It increased at S4 to S5 of blackberry fruit and peaked at S3 of
hybrid fruits. The expression of RuANR in ’Chester’ was significantly
higher than that in the other cultivars (Fig. 6c). The expression patterns
of RuLAR were different in different cultivars during fruit development
(Fig. 6d). The changing trend of the RuUFGT1 expression level was
different in different cultivars. In ’Chester’, the expression level was
highest in S3, and ’Hull’ and ’Boysen’ mature fruits had the highest
expression level. The expression level of RuUFGT1 in ’Young’ fruit was
the highest (Fig. 6e). The expression of RuUFGT2 increased suddenly in
the second color transition period of ’Chester’ and ’Hull’ and then
decreased sharply. In ’Boysen’ and ’Young’, the expression of RuUFGT2
was highest in S1 and decreased later (Fig. 6f).
The transcription factor RubHLH9 was not detected in Boysen, and its
expression level varied greatly across ’Chester’ fruit development, with
the highest expression level in mature fruit (Fig. 7a). The expression
level of RubHLH13 in S1 was the highest across all the cultivars (Fig. 7b).
The highest expression level of RubHLH48 in ’Chester’ and ’Young’ was
found in S1, while the highest expression level was found in S3 of ’Hull’

10
X. Huang et al.
Fig. 6. Relative expression of flavonoid biosynthesis genes of Rubus fruits in different fruit developmental stages. (a) RuPAL; (b) RuFLS; (c) RuLAR; (d) RuANR; (e)
RuUFGT1; (f) RuUFGT2. The different letters indicate significant differences between the fruits harvested at different developmental stages (p<0.05). The different
colors represent different developmental stages. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of
this article.)
and ’Boysen’ (Fig. 7c).
RuMYB1 was highly expressed in ’Young’ fruits, with the peak
occurring at S3 and the highest expression level occurring in S1 of the
other three cultivars (Fig. 7d). The expression levels of RuMYB6 and
RuMYB8 were different in the different cultivars during fruit develop­
ment (Fig. 7e and f). RuMYB33 and RuMYB306 expression levels were
different during the development of ’Chester’ fruits, especially that of
RuMYB306, and the difference was up to 360-fold (Fig. 7g and h).
Among the five WRKY genes, the expression levels of RuWRKY6 and
RuWRKY75 were relatively low during fruit development (Fig. 7i and
m). The expression level of RuWRKY6 was higher in the ’Chester’, ’Hull’
and ’Boysen’ fruit across developmental stages (Fig. 7i). RuWRKY24
expression fluctuated significantly during fruit development in ’Chester’
and ’Young’ fruits (Fig. 7j), while RuWRKY69 showed significant dif­
ferences during fruit development in ’Chester’ and ’Hull’ fruits (Fig. 7k).
RuWRKY70 exhibited the highest expression level in S3 in ’Chester’,
’Hull’ and ’Young’ (Fig. 7l). RuWRKY75 had the highest expression level
in the fruit of ’Young’ at S2 and the highest expression level in the fruit
of the other three cultivars at S1 (Fig. 7m).
4. Discussion
Both blackberry and raspberry belong to the genus Rubus; these
species are distinguished by the fact that the aggregate fruit does not
separate from the receptacle when blackberry fruits mature, whereas
raspberry fruits appear to separate from the receptacle (Edees &
Newton, 1988). ’Young’ is a hybrid offspring of blackberry and rasp­
berry. The shape of ’Young’ is partial to that of raspberry, and the fruits
are hollow when mature, which is also the reason why ’Young’ fruits
weigh less than do those of the other three cultivars. The correlation
analysis among dry weight, fresh weight and moisture content during
fruit development indicated that the weight increase of blackberry fruits
was caused by the increase of both moisture and nutrients. Blackberry
contains a wide cultivarof nutrients such as protein, sugars, organic
acids, fatty acids and essential vitamins and minerals (Mayara &
Josiane, 2019). Therefore, we can do further research on the cultivarand
the changing rule of the nutrient composition of blackberry. The color
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Food Bioscience 54 (2023) 102892
parameters of mature fruits of ’Chester’, ’Hull’ and ’Boysen’ showed
that the values of a* and C* were smaller than those of ’Young’
(Fig. 1e–h). The mature fruits of ’Chester’, ’Hull’ and ’Boysen’ were
darker and blacker than those of ’Young’ (Fig. 1a–d). Previous studies
showed that the anthocyanin contents of ’Chester’, ’Hull’ and ’Boysen’
fruits were significantly higher than those of ’Young’ fruits, which
indicated that anthocyanin content was one of the factors causing fruit
color change. The increase in anthocyanin content during the ripening
process also changed the color of jujube fruit (Shi et al., 2018). The
market generally favors large, plump fruits, and anthocyanin content is
also an important quality of blackberry fruit. Therefore, ’Young’ mature
fruit may not be suitable for extension planting.
Different cultivars often differ in terms of their antioxidant capacity
and metabolites (Choi et al., 2022). The total antioxidant capacity of
’Boysen’ was significantly positively correlated with the flavonoids and
GSH contents. The total antioxidant capacity of ’Hull’ was positively
correlated with the content of phenols, suggesting that the correlation
between antioxidant capacity and bioactive substances is related to
plant cultivar. A similar finding was made for Tropaeolum tuberosum Ruiz
& Pavón, showing that the antioxidant capacity of purple cultivars was
related to total phenols or flavan3-alcohols, whereas the antioxidant
capacity of yellow cultivars was related only to total phenols (Chirinos
et al., 2007). In many fruit species, such as citrus (Deng et al., 2022) and
strawberry (Hwang et al., 2019), flavonoids have been found to be
significantly associated with antioxidant activity, and the biosynthesis
of flavonoids is regulated by multiple genes, including both structural
genes and transcription factors (Liu et al., 2021; Zhao et al., 2019). The
expression levels of RuPAL and RuFLS in ’Chester’, ’Hull’ and ’Boysen’
peaked at S4. The accumulation rate of anthocyanins peaked at S5, that
is, after the expression peak of RuPAL occurred, suggesting that the
expression of RuPAL and RuFLS is a prerequisite for the synthesis of
anthocyanins catalyzed by key enzymes. Relevant studies have shown
that bHLH transcription factors typically bind to MYB and WD40 pro­
teins to form MBW complexes that activate the expression of genes
downstream of the flavonoid synthesis pathway, leading to the synthesis
of anthocyanins and proanthocyanidins (Chen, Li, et al., 2019). For
example, there is an interaction between VcbHLHL1 and VcMYBL1
 X. Huang et al.
12

Fig. 7. Relative expression of transcription factors in Rubus fruits at different developmental stages. (a) RubHLH9; (b) RubHLH13; (c) RubHLH48; (d) RuMYB1; (e) RuMYB6; (f) RuMYB8; (g) RuMYB33; (h) RuMYB306; (i)
RuWRKY6; (j) RuWRKY24; (k) RuWRKY69; (l) RuWRKY70; (m) RuWRKY75. The different letters indicate significant differences between fruits harvested at different developmental stages (p<0.05). The different colors

Food Bioscience 54 (2023) 102892

represent different developmental stages. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
X. Huang et al.
proteins in blueberry, and the change trend of the expression levels of
the genes encoding the two proteins also tends to be consistent (Zhao
et al., 2019). In ’Chester’ and ’Hull’, the expression patterns of
RubHLH13 and RuMYB1 were found to be similar, and similar changes in
transcript levels may indicate that they are involved in the metabolic
regulation of flavonoids. WRKY genes are involved in plant metabolic
pathways in a cultivar of ways, and they can directly bind to and
regulate the expression of the promoters of genes involved in the
flavonoid synthesis pathway (Yue et al., 2022); they can also bind to
their own promoters and activate their own expression (Xiao et al.,
2023). Members of the WRKY gene family can also interact to form a
sequential transcriptional regulatory cascade, which is involved in plant
stress responses and growth (Chen, Li, et al., 2019). MdWRKY11 over­
expression promoted the expression of F3H, FLS, DFR, ANS, and UFGT,
increasing the accumulation of flavonoids and anthocyanins in apple
calli (Wang et al., 2018). VvWRKY26 was identified in Vitis Vinifera and
transiently expressed in grape leaves, and the expression of genes
encoding both F3Hs was found to be upregulated, promoting procya­
nidin synthesis (Amato et al., 2017). The expression patterns of
RuWRKY6 and RuWRKY75 were different among the four cultivars, but
the trends of the expression levels of RuWRKY6 and RuWRKY75 in the
same cultivar were very similar, suggesting that RuWRKY6 and
RuWRKY75 may synergistically regulate flavonoid biosynthesis.
In this study, we found that the total antioxidant capacity of black­
berry decreased first and then increased with fruit development. The
highest total antioxidant capacity was found in ’Boysen’, followed by
’Young’, and the hybrid cultivars presented better antioxidant activity
than did the pure blackberry cultivars. Therefore, hybrid cultivars may
be more suitable for related research on the antioxidant activity. In
addition to anthocyanins, the antioxidant capacity of blackberry fruits
was correlated with the AsA content, GSH content, O-2 production rate
and MDA content during fruit development. Our previous experiments
showed that the DPPH free radical scavenging ability of blackberry is
significantly correlated with vitamin C, also known as ASA (Huang et al.,
2022b), which has been suggested to have antioxidant activity in many
kinds of fruits to help plants resist oxidative stress. For example, the
content of AsA in Citrus limon (L.) decreased gradually with fruit
development, and the antioxidant capacity of the fruits also decreased
(Di Matteo et al., 2021). The MDA content was positively correlated with
the GSH content and O-2 production rate. MDA is an important indicator
of membrane lipid peroxidation, indicating that the increased rate of O-2
production results in oxidative damage to plant cells (Zhang, Liu, et al.,
2019). Overall, the MDA content increased with the development of
blackberry fruit, which indicated that the degree of oxidative damage of
blackberry fruit was increasing. Therefore, blackberry fruits should be
picked as soon as possible after ripening. GSH biosynthesis is activated
by stress (Zhu et al., 2021), and given the increased lipid peroxidation
during blackberry fruit development, it is possible that GSH production
may be affected by stress, leading to increased levels of GSH. GSH can
also form an AsA-GSH cycle with AsA, and an increase in ROS can
activate the AsA-GSH cycle in plants (Hasanuzzaman et al., 2019).
However, there was no significant correlation between AsA content and
GSH content in blackberry fruits, indicating that during blackberry
growth, the AsA-GSH cycle may not be activated.
Antioxidant enzymes are also ROS-scavenging enzymes in plants.
The results showed that the activity of SOD in blackberry and the
blackberry hybrid cultivars changed most sharply, and the activity of
SOD increased with the development of fruits. However, the activities of
CAT and POD decreased with fruit development. In contrast to that in
blackberry, SOD activity in strawberry decreased with fruit develop­
ment, and its activity changed more than that of CAT and POD enzymes
(López et al., 2010), which was also the same in blackberry. This result
indicated that SOD activity might be more affected by fruit growth and
development. Correlation analysis showed that the total antioxidant
capacity of ’Boysen’ fruits was positively correlated with SOD activity,
while the total antioxidant capacity of Hull and Young fruits was
13
Food Bioscience 54 (2023) 102892
negatively correlated with SOD activity. The activities of all antioxidant
enzymes in pepper were shown to increase under drought stress (Kopta
et al., 2020). The response intensity of different cultivars was different,
the GPX response intensity of the gene-sensitive type was higher, and the
response of APX and CAT of the gene-tolerant type was stronger. Thus,
the main antioxidant enzymes involved in different blackberry cultivars
are different. In addition to cultivar, there are numerous factors related
to enzyme activity, such as the expression levels of antioxidant
enzyme-related genes, temperature, hormone levels, and metal ion
content (Abbas et al., 2020; Liu, Wang, et al., 2021; Miłek, 2020).
The CAT activity decreased gradually in the 4 cultivars, the trend of
which was different from the trend of gene expression, indicating that
high expression of RuCAT could not improve CAT activity. CAT activity
is generally regulated by posttranslational modifications (PTMs), such as
phosphorylation, glycosylation, and acetylation (Zhou et al., 2018). In
addition, signaling molecules such as NO and H2S can also regulate CAT
activity (Palma et al., 2020), so further studies on the activity of
blackberry CAT should consider protein structure modification,
signaling molecules and so forth. Correlation analysis showed that the
SOD activity of ’Chester’ and ’Hull’ was positively correlated with the
expression levels of RuSOD1 and RuSOD2, indicating that both the
RuSOD1 and RuSOD2 genes are involved in the positive regulation of
SOD activity. SOD is a kind of metal-containing protein. According to
the different metal cofactors binding to the active site, SOD can be
divided into four types: Cu/ZnSOD, FeSOD, MnSOD and NiSOD
(Broering et al., 2012). RuSOD1 belongs to the Cu/ZnSOD type, and
RuSOD2 belongs to the MnSOD type. Although the crystal structures and
catalytic mechanisms of MnSOD and Cu/ZnSOD are different, these
enzymes have the same role in scavenging ROS (Vuleta et al., 2016). The
correlations between the genes encoding POD enzymes and POD enzyme
activity were different in the different cultivars. The seven genes in
’Chester’ had no significant correlation with POD enzyme activity. In
’Boysen’, the seven genes showed significant or extremely significant
positive correlations with POD activity. POD activity in ’Hull’ was
positively correlated with RuPOD, while POD activity in ’Young’ was
negatively correlated. In these two cultivars, the correlation between
RuPER29 and POD enzyme activity was opposite that of RuPOD. In
addition to the differences in cultivars, the diversity of POD gene func­
tions may also be the cause of their differential expression. According to
previous studies, the POD gene is also associated with fruit color traits
(Ring et al., 2013). Therefore, seven POD genes were identified in this
study, and their specific functions need to be further studied.
5. Conclusions
The O-2 production rate and MDA content of the fruits of the 4 cul­
tivars increased gradually during fruit development, which indicated
that the development of blackberry fruits was accompanied by oxidative
damage. As a whole, the activity of SOD increased with fruit develop­
ment, while the activities of POD and CAT decreased and remained at a
lower level throughout development. Correlation analysis showed that
antioxidant capacity was correlated with anthocyanin content, AsA
content, GSH content, O-2 production rate and MDA content during
blackberry fruit development. However, the correlation between anti­
oxidant capacity and metabolites was different in the different black­
berry cultivars. The total antioxidant capacity of ’Boysen’ was
significantly positively correlated with the flavonoid and GSH contents.
The total antioxidant capacity of ’Hull’ was positively correlated with
the phenol content. ’Boysen’ exhibited the highest total antioxidant
activity at all stages, followed by ’Young’, and the hybrid showed
greater antioxidant activity than the pure blackberry. RuSOD1 and
RuSOD2 positively regulated SOD activity in pure blackberry fruits, and
RuPOD and RuPER29 showed different functions in different cultivars.
This study was the first to compare the changes in antioxidant ac­
tivity and antioxidant capacity during fruit development between
blackberry and blackberry–raspberry hybrids fruits, providing some
X. Huang et al.
suggestions and data support for the production, cultivation and utili­
zation of blackberry fruits. Moreover, based on the transcriptome data
and the activity of antioxidant enzymes, this study provides a scientific
basis for the further research on the antioxidant mechanism and regu­
lation of the biosynthesis of related enzymes in blackberry fruits.
Funding
The research was supported by the National Natural Science Foun­
dation of China (32101566), the Natural Science Foundation of Jiangsu
Province (BK20210165), the “JBGS” Project of Seed Industry Revitali­
zation in Jiangsu Province (JBGS[2021]021), the earmarked fund for
Jiangsu Agricultural Industry Technology System (JATS[2022]510) and
the Jiangsu Province Key Project of R&D Plan (Modern Agriculture)
(BE2020344).
Role of the funding source
The funding bodies had no role in the design of the study; the
collection, analysis, and interpretation of data; or the writing of the
manuscript.
Author statement
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper. We believe that this manuscript is
appropriate for publication in Plant Science because it fits the aims and
scope of this journal. All coauthors have reviewed the final version of
this manuscript and approve it for publication. This manuscript has been
neither published nor submitted in whole or in part elsewhere.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
The data that has been used is confidential.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.fbio.2023.102892.
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