Abstract
This study investigated the impact of slow cooling on browning and
fruit quality at three maturity stages (early, mid and late). Slow
cooling reduced core browning in early/mid-harvest pears, as the
browning indexes of early-, middle- and late-harvested ‘Yali’ pears at
60 d were 0.13, 0 and 0.1, respectively, preserving firmness and
soluble solids. Transcriptomic analysis revealed that upregulated genes
in ‘Yali’ pears facilitated stress adaptation via enhanced catalytic
activity and phosphorylation. Mid-harvested pears exhibited activation
of phosphorus metabolism and DNA repair mechanisms to maintain cellular
homeostasis, whereas the late-harvested counterparts showed significant
suppression of photosynthesis-related pathways and pyrimidine
metabolism, which collectively accelerated senescence progression.
Universal downregulation of hormone-response pathways such as ethylene
and auxin revealed systemic stress adaptation decline. Then, the PbRAV
transcription factors’ role was also studied. EMSA confirmed that
GST-PbRAV2 binds to the PbLAC15 promoter, linking RAV2 to laccase
regulation. Overripe pears showed PbRAV2 dysregulation, impairing LAC15
suppression and accelerating browning. Findings provide a theoretical
basis for using slow cooling to mitigate browning in pear storage.
Keywords: ‘Yali’ pear, core browning, transcriptome analysis, PbRAV
transcription factors
1. Introduction
The ‘Yali’ pear (Pyrus bretschneideri Rehd.), a prominent traditional
cultivar originating from Hebei province, China, is highly valued for
its distinctive fragrance and delicate aroma [[32]1,[33]2]. However,
core browning frequently manifests during postharvest storage, as a
notable physiological affliction in pears, significantly diminishing
commodity value and impacting both export volume and revenue, remaining
undetectable via visual inspection. Prior research has established a
definitive correlation between the manifestation of core browning and
the onset of fruit senescence. The progression of senescence is
significantly accelerated by a decline in ethylene biosynthesis during
extended storage durations [[34]3]. The progression of core browning in
‘Yali’ pears can be succinctly characterized as follows: Initially, the
‘Yali’ pear core shows no browning. With prolonged storage, browning
begins in the ovary, progressing to complete core browning and
extending into the flesh. Browning consistently initiates in the core
and spreads outward into the mesocarp [[35]4].
The ‘Yali’ pear is prone to core browning during postharvest storage, a
significant postharvest physiological disorder [[36]5]. This browning
phenomenon detrimentally affects the quality attributes of ‘Yali’
pears, diminishing their palatability and, consequently, impacting
marketability. In ‘Yali’ pears, browning is primarily associated with
the modulation of specific genes and associated enzymatic activities.
Phenolic compounds serve as the principal substrates in this process.
Under aerobic conditions, these phenolic substrates are oxidized by
polyphenol oxidase, yielding quinones, which subsequently accelerate
browning [[37]6]. Core browning in ‘Yali’ pears is influenced by a
multitude of factors, including harvest maturity, fruit quality,
storage temperature, CO[2] levels, storage protocols, and exogenous
preservative applications. These variables modulate the fruit’s
sensitivity to temperature fluctuations [[38]4]. At present,
low-temperature storage is the most commonly used storage method in the
preservation of fruits and vegetables. Low-temperature storage can
delay energy consumption and reduce their metabolic rate, thus
inhibiting the reproduction of bacteria and microorganisms, delaying
senescence, effectively prolonging the storage period and slowing down
the occurrence of browning [[39]7,[40]8]. For the ‘Yali’ pear,
appropriate maturity, combined with different cooling treatment during
low-temperature storage, can effectively inhibit the occurrence of core
browning [[41]7,[42]9]. For example, postharvest ‘Yali’ pears at
advanced maturity treated with rapid cooling show that it effectively
suppresses ethylene biosynthesis and respiratory activity. This
physiological modulation subsequently retards the senescence process,
thereby preserving fruit quality attributes [[43]4]. Furthermore, this
effect is correlated with elevated activities of superoxide dismutase
(SOD), catalase (CAT), peroxidase (POD), and glutathione reductase
(GR), alongside diminished lipoxygenase (LOX) activity. Furthermore,
the slow-cooling protocol delayed membrane lipid peroxidation,
suppressed malondialdehyde (MDA) accumulation, reduced levels of
[MATH: O2− :MATH]
and H[2]O[2], and attenuated cell membrane damage [[44]7].
Initial investigations have elucidated the correlation between membrane
lipid peroxidation, ethylene metabolism, and the incidence of core
browning in ‘Yali’ pears; however, the underlying molecular mechanisms
remain obscure. Within the plant system, transcription factors (TFs)
modulate the transcriptional activity of structural genes through
interaction with cognate cis-regulatory elements located within their
promoter regions [[45]10]. Transcription factors (TFs) belonging to the
WRKY, bZIP, NAC, and MYB families have been frequently implicated in
the regulation of fruit browning [[46]11,[47]12,[48]13,[49]14]. This
study utilized ‘Yali’ pears from Xinji City, Hebei Province, as
experimental material. ‘Yali’ pears were subjected to slow-cooling
treatments at three maturity stages (early, mid, and late), determined
based on the full bloom and harvest periods to investigate their
effects on core browning and physiological quality during storage.
Transcriptome analysis of the fruit core was then performed to identify
key transcription factors regulating browning, and to systematically
characterize the transcriptional regulatory network of RAV
transcription factors in the core browning pathogenesis. The aim was to
elucidate the molecular mechanisms of cooling-mediated browning
inhibition, providing a theoretical basis for optimizing postharvest
preservation strategies.
2. Materials and Methods
2.1. Plant Material and Postharvest Treatment
‘Yali’ pears sourced from Xinji City, Hebei Province, served as the
experimental material. Fruits were harvested 145 days after bloom
(early-maturity), 155 days after bloom (mid-maturity), and 165 days
after bloom (late-maturity). Uniformly sized fruits (about 90 mm),
devoid of pest infestation and mechanical injury, were selected. For
each maturity stage, 3 replicates, each comprising approximately 80
fruits, were utilized. Following harvest, the fruits were immediately
transported to the cold storage facility at Tianjin Agricultural
College. Pre-cooling was initiated for 24 h, succeeded by a controlled
cooling regime. The slow-cooling protocol comprised pre-cooling at 12
°C for 24 h, followed by a progressive decrease to 0 ± 0.5 °C over 30
d, with a 2 °C reduction every 5 d. Relative humidity was sustained at
80–85%. Core browning and the physiological quality of ‘Yali’ pears
were assessed at 30 d intervals for each maturity group.
Simultaneously, core tissue samples were excised and immediately
subjected to flash-freezing in liquid nitrogen. These samples were
subsequently stored at −80 °C until further analysis.
2.2. Determination of Core Browning and Physiological Quality of ‘Yali’ Pears
After Slow-Cooling Treatment
The core browning index and physiological quality of ‘Yali’ pears was
assessed according to the methodology described by Li et al. [[50]7]
and Zhang et al. [[51]4], with some modification.
2.2.1. Determination of Core Browning Index of ‘Yali’ Pear
A total of 30 pears were randomly selected at each maturity stage.
These were subsequently divided into three replicates, each comprising
10 pears. Core browning in ‘Yali’ pears was monitored and documented at
intervals of 0, 30, 60, 90, 120, 150, 180, and 210 d of storage, and
the core browning index was computed as detailed below:
[MATH: core browning index=∑(Browning grade×The number of fruits of this grade)The highest browning grade×Check the number of fruits
:MATH]
2.2.2. Determination of Soluble Solids (SSC) Content
A total of 15 fruits were randomly sampled from each treatment group,
with every 5 fruits forming one group, and 3 replications were
performed. After peeling 5 fruits with a knife, the pulp portion was
cut into small pieces, mixed and juiced, and a saccharimeter was used
to determine the soluble solids content of the ‘Yali’ pear.
2.2.3. Firmness Determination
A total of 15 fruits were randomly selected for the experiment. The
firmness of ‘Yali’ pear fruits was assessed utilizing a TA-XT plus
texture analyzer, with measurements expressed in kg cm^−2.
2.2.4. Determination of Respiratory Intensity
A total of 12 ‘Yali’ pears were randomly selected and apportioned into
three treatment groups. These were designated and reserved for
subsequent analyses. Subsequently, the grouped ‘Yali’ pears were housed
within modified Lekou boxes and sealed for 1 h. Gas samples were
extracted from each box using a 1 mL medical syringe, with four needles
per box (three for replication and one backup). The respiratory
intensity of the ‘Yali’ pears was assessed using a respiration
detector. Determination of respiratory intensity was performed using a
CA-10 CO[2] analyzer and calculated according to the formula in Zhang
et al. [[52]4].
2.2.5. Determination of Ethylene Release
The gas was collected in accordance with the procedure used for
measuring respiratory intensity, and the samples obtained were
determined using a Shimadzu GC-14 gas chromatograph. The experimental
parameters were set as follows: the GDX-502 stainless-steel-packed
column was the chromatographic column; the detector was a
hydrogen-flame ionization detector, and the carrier gas was N2; the
temperature of the inlet was 60 °C, the temperature of the column
temperature box was 60 °C, and the temperature of the detector was 60
°C. The calculation formula is shown below, and the unit is expressed
in μL kg^−1·h^−1 µL.
[MATH: Ethylene release=C×VM×T×1000 :MATH]
* in which C: ethylene content in the sample gas with unit of µL
L^−1;
* V: volume of the enclosed space, unit is mL;
* M: the mass of fruits and vegetables, the unit is kg;
* T: smothering time; the unit is h.
2.2.6. Determination of Weight Loss
We measured the mass of fixed ‘Yali’ pears every 30 d with an
electronic balance and then calculated the change in fruit weight. The
calculation formula was as follows:
[MATH: Weight loss rate (%)=weight of the initial sample−weight of the current sampleweight of the initial sample×100 :MATH]
2.3. Transcriptome Sequencing
The samples fruit core samples were frozen in liquid nitrogen stored in
an ultra-low-temperature refrigerator at −80 °C. The frozen samples
were sent to Beijing Novozymes for transcriptome sequencing.
2.4. Analysis of Cis-Acting Elements
The analysis of cis-acting elements in Pyrus bretschneideri RAV (PbRAV)
was performed in PlantCARE
([53]https://bioinformatics.psb.ugent.be/webtools/plantcare/html/)
accessed on 8 September 2022.
2.5. RNA Isolation and cDNA Synthesis
The improved CTAB method was used to extract total RNA from each
sampling point of ‘Yali’ pear fruit. The quality of RNA extraction was
detected by 1% agarose gel electrophoresis, the concentration and
purity of RNA were detected using a NanoDrop 2000 spectrophotometer
(Thermo, Waltham, MA, USA). Approximately 2 μL RNA was used to produce
cDNA with an HiScript^®Ⅱ 1st Strand cDNA synthesis kit (R212, vazyme,
Nanjing, China).
2.6. Quantitative Real-Time PCR (RT-qPCR)
In RT-qPCR analyses, cDNA was diluted with ddH[2]O at a ratio of 1:10.
Gene-specific primers were designed using Primer 5.0 software
([54]Table S1). RT-qPCR was performed on the QuantageneQ225 Real-time
PCR system (Q225, Novegene, Beijing, China) with ChamQ SYBR Color qPCR
Master Mix (Q421, vazyme, Nanjing, China). The RT-qPCR analyses were
performed with three replicates to ensure accurate results. The Pyrus
bretschneideri actin ([55]GU830958.1) was used as reference gene, and
the relation gene expression level of each gene was calculated using
the 2^−ΔΔCT method.
2.7. Identification of Downstream Target Genes of PbRAV Across the Entire
Genome
The binding sites of the PbRAV transcription factor were predicted
using the JASPAR online database. Subsequently, the complete Pyrus
bretschneideri genome data was downloaded from the NCBI database. The
upstream 2000 bp promoter sequences of all protein-coding genes in P.
bretschneideri were extracted using TBtoolsV1.098 software. Finally,
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment
analysis was performed on the putative downstream target genes of the
PbRAV transcription factor using the OmicShare tools [[56]15]. Gene
Ontology (GO) enrichment analysis was performed using the R package
clusterProfiler (v4.6.2). The hypergeometric test was applied with all
protein-coding genes detected in RNA-seq as the background gene set.
Terms with FDR-adjusted p-values < 0.05 were defined as significantly
enriched.
2.8. Protein Expression and Purification and Electrophoretic Mobility Shift
Assays (EMSA)
The methodology adhered to the protocols described by Fan et al.
[[57]16] and Tan et al. [[58]17], alongside the operational guidelines
provided by the Beyotime EMSA Chemiluminescence Kit (GS009).
2.9. Statistical Analysis
Statistical analysis was performed using Excel 2016. Origin was used
for drawing. Values are represented as the mean of three independent
biological replicates. IBM 22 (IBM Inc., Armonk, NY, USA) statistics
software was used for the variance analysis of data, and p ≤ 0.05 was
considered significant.
3. Results
3.1. Effects of Slow-Cooling Treatment on Core Browning Index and
Physiological Indicators of Different Maturity ‘Yali’ Pear
The browning index of ‘Yali’ pears at different maturity stages
gradually increased with prolonged storage time ([59]Figure 1A). During
the early storage period (0–30 d), there was no core browning in the
‘Yali’ pears. Early- and late-harvested ‘Yali’ pears first began to
show browning after 60 d of storage, and the browning indexes were 0.13
and 0.1, respectively. The browning index of middle-harvested ‘Yali’
pears was 0.2 when it first appeared, at 90 d of storage. At the end of
storage (180–210 d), it was found that the core browning index of
different maturity ‘Yali’ pear was as follows: late-harvest >
early-harvest > middle-harvest. The results showed that the ‘Yali’
pears with either higher or lower harvest maturity were not suitable
for slow-cooling treatment.
Figure 1.
[60]Figure 1
[61]Open in a new tab
Effect of slow-cooling treatment on core browning index (A), ethylene
production (B), respiration intensity (C), hardness (D), soluble solids
(E), the weight loss rate (F) of ‘Yali’ pears at different maturity
stages, and the core browning of ‘Yali’ pears at different maturity
stages at 210 d of storage (G). Variations in lowercase letters
indicate a statistically significant difference (p < 0.05) among five
groups simultaneously. Y-bars represent SD.
The overall trend in ethylene release in fruits first rose to a peak
and then decreased ([62]Figure 1B). The ethylene peak timing varied
with fruit maturity: late-harvested ‘Yali’ pears exhibited the first
peak at 30 days (223.95 μL·kg^−1·h^−1), while at 90 days both early-
and middle-harvested fruits showed peaks at 195.02 and 154.64
μL·kg^−1·h^−1, respectively. From 180 to 210 days, ethylene release
increased, correlating with accelerated senescence and browning, as
reflected in the browning index. Throughout storage, late-harvested
pears consistently released more ethylene, indicating that higher
maturity adversely affects long-term preservation post-slow cooling.
During the storage process of ‘Yali’ pear, the respiratory intensity of
‘Yali’ pears harvested at different maturity stages changed as shown in
[63]Figure 1C. Overall, the trend showed an initial decline, then a
rise, and finally a decline again. At 60 d of storage, the respiratory
peak of late-harvested ‘Yali’ pears first appeared, reaching 577.63 mg
CO[2] kg^−1·h^−1; at 90 d of storage, the respiratory peak of
early-harvested ‘Yali’ pears occurred, reaching 213.9 mg CO[2]
kg^−1·h^−1; at 120 d, the respiratory peak of middle-harvested ‘Yali’
pears appeared, reaching 239.47 mg CO[2] kg^−1·h^−1. During the whole
storage period, the late-harvested ‘Yali’ pears were the first to
exhibit the respiratory peak, indicating that the fruit with higher
maturity was not suitable for long-term preservation under the
slow-cooling treatment. Based on the timing of the respiratory peak and
the sudden increase in the core browning index in ‘Yali’ pear, the
earlier the respiratory peak occurred, the more prone ‘Yali’ pear was
to core browning.
Hardness is a key indicator of fruit quality. The hardness of ‘Yali’
pear showed a decreasing trend during the whole storage period. In 0 d
of storage, the hardness of ‘Yali’ pear was 9.72, 7.76, and 6.90 kg
cm^−2 in the early, middle, and late harvesting stages, respectively
([64]Figure 1D). At 30 d of storage, the hardness of ‘Yali’ pear was as
follows: early harvesting > middle harvesting > late harvesting, which
indicated that the higher the maturity, the lower the initial hardness
of the fruits. With the prolongation of storage time, the fruit
gradually senesced. At the end of storage (180–210 d), the firmness of
‘Yali’ pear fruit was in the following order: middle harvesting > early
harvesting > late harvesting, indicating that the hardness of the
middle-harvested ‘Yali’ pear fruit declined the slowest after
slow-cooling treatment, and that it can better maintain the fruit
quality of ‘Yali’ pear.
With the prolongation of storage time, the soluble solids content (SSC)
of ‘Yali’ pear fruits of three maturity levels showed a trend of first
increasing and then decreasing ([65]Figure 1E). At 0 d of storage, the
SSC content of early-, middle-, and late-harvested ‘Yali’ pears was
9.05%, 9.06%, and 10.23%, respectively. The SSC content of
late-harvested ‘Yali’ pears was higher in the pre-storage period, and
in the late storage period (180–210 d), the soluble solids content of
late-harvested ‘Yali’ pears was slightly lower than that of the
early-harvested and middle-harvested pears.
The weight loss of ‘Yali’ pears exhibited a progressive increase with
prolonged storage duration ([66]Figure 1F). Within the initial 30 days
of storage, the weight loss, contingent upon the maturity stage at
harvest, followed this order: fruit harvested at the middle stage >
early stage > late stage (early stage: 1.03%; middle stage: 1.41%; late
stage: 0.86%). Over the entire storage period (180–210 days), the
weight loss rate of the fruit was ordered as follows: middle stage >
late stage > early stage.
3.2. Expression Trend Analysis of Total DEGs
[67]Figure 2 depicted differential gene expression (|log[2]FC| > 0,
padj ≤ 0.05) in ‘Yali’ pears during early, middle, and late maturity.
Module 9 shows upregulation, with DEG counts of 6644, 7209, and 8382,
respectively, increasing with storage time. Conversely, module 0
exhibits downregulation, with 6169, 5951, and 5460 DEGs, respectively,
decreasing over time.
Figure 2.
[68]Figure 2
[69]Open in a new tab
Impact of slow cooling on DEG expression trends in ‘Yali’ pears
harvested at different maturity stages. DEG dynamics during storage (0,
30, 120, and 210 days) in early-harvested (A), middle-harvested (B),
and late-harvested (C) ‘Yali’ pears.
3.3. GO Enrichment Analysis of Different Expression Trend Modules
The top 20 GO terms enriched by the DEGs included in the upregulated
expression trend Profile 9 of the three maturity levels of ‘Yali’ pears
after the slow-cooling treatment ([70]Figure 3A–C). Subsequently, the
logical linkage analysis of the top 20 GO terms for the three maturity
levels was carried out ([71]Figure 3D,E), and three maturity stages of
‘Yali’ pears were found to be commonly enriched in 10 GO terms, where
the catalytic activity is closely related to core browning in ‘Yali’
pears. Between maturity stages, one GO term was shared between early
and late-harvested pears, while mid-harvested pears shared four terms
with early and two terms with late-harvested fruits. Compared to the
other two maturity stages, the mid-harvested ‘Yali’ pears were
primarily enriched in GO terms, such as sequence-specific DNA binding,
phosphorus metabolic process, and phosphate-containing compound
metabolic process. This may be one of the reasons why the mid-harvested
‘Yali’ pears exhibited a lower degree of core browning under
slow-cooling treatment.
Figure 3.
[72]Figure 3
[73]Open in a new tab
GO enrichment of upregulated genes (Profile 9) in ‘Yali’ pears at
different maturities after slow-cooling treatment. (A–C) GO terms for
early-, middle-, and late-maturity samples. (D) Venn diagram showing
shared and unique enriched GO terms among the three maturity stages.
(E) Heatmap displaying enrichment status: blue indicates enriched,
while white indicates not enriched.
The top 20 GO terms enriched by the DEGs included in the downregulated
expression trend Profile 0 across the three maturity levels of ‘Yali’
pears after the slow-cooling treatment ([74]Figure 4A–C). [75]Figure 4D
shows that the three maturity stages of ‘Yali’ pears were co-enriched
to six GO terms, and eight, six, and eight GO terms were enriched,
respectively. The late-harvested ‘Yali’ pears exhibited significant
enrichment in terms related to zinc ion binding, photosynthesis,
pyrimidine nucleotide metabolic process, pyrimidine nucleotide
biosynthetic process, thylakoid part, negative regulation of the cell
cycle, photosynthesis, light reaction, and photosynthetic membrane
([76]Figure 4E). This suggested that these processes are inhibited in
late-harvested ‘Yali’ pears, as storage time increases. This enrichment
may contribute to the increased core browning observed in
late-harvested ‘Yali’ pears subjected to slow-cooling treatment.
Figure 4.
[77]Figure 4
[78]Open in a new tab
GO enrichment of downregulated genes (Profile 0) in ‘Yali’ pears at
different maturity stages after slow-cooling treatment. (A–C) GO terms
for early-, middle-, and late-maturity samples. (D) Venn diagram
showing shared and unique enriched GO terms among the three maturity
stages. (E) Heatmap displaying enrichment status: blue indicates
enriched, while white indicates not enriched.
3.4. KEGG Enrichment Analysis of Different Expression Trend Modules
The KEGG pathway analysis depicted in [79]Figure 5 indicated that the
genes upregulated during the slow-cooling treatment across three ‘Yali’
pear maturity stages are significantly enriched in metabolic pathways,
secondary metabolite biosynthesis, terpenoid backbone biosynthesis,
sesquiterpenoid and triterpenoid biosynthesis, and steroid
biosynthesis. Early- and mid-harvested pears share enrichment in eight
KEGG pathways, including glycerophospholipid metabolism and plant
hormone signal transduction, suggesting delayed core browning. Mid- and
late-harvested pears share enrichment in amino sugar and nucleotide
sugar metabolism. Additionally, mid-harvested pears are predominantly
enriched in glycerolipid metabolism, pentose and glucuronate
interconversions, nucleotide sugar biosynthesis, fatty acid
degradation, mismatch repair, and homologous recombination pathways,
contributing to delayed core browning onset.
Figure 5.
[80]Figure 5
[81]Open in a new tab
KEGG enrichment of upregulated genes (Profile 9) in ‘Yali’ pears at
different maturity stages after slow-cooling treatment. (A–C) KEGG
pathway for early-, middle-, and late-maturity samples. (D) Venn
diagram showing shared and unique enriched KEGG pathways among the
three maturity stages. (E) Heatmap displaying enrichment status: blue
indicates enriched, while white indicates not enriched.
The KEGG enrichment analysis indicated that the downregulated genes
across three maturity stages of ‘Yali’ pears post-slow cooling were
predominantly enriched in metabolic pathways, glyoxylate and
dicarboxylate metabolism, starch and sucrose metabolism, and tyrosine
metabolism ([82]Figure 6). Additionally, the genes downregulated in
early- and mid-harvested pears were commonly enriched in purine
metabolism and base excision repair pathways. The downregulated genes
in mid- and late-harvested pears were enriched in four KEGG pathways,
while the upregulated genes across early, mid-, and late-harvested
stages were enriched in 14, 10, and 12 pathways, respectively.
Late-harvested pears showed significant enrichment in pyruvate
metabolism, GPI-anchor biosynthesis, pentose phosphate pathway,
butanoate metabolism, and amino acid degradation pathways, indicating
that suppression of these pathways may underlie core browning in ‘Yali’
pears.
Figure 6.
[83]Figure 6
[84]Open in a new tab
KEGG enrichment of downregulated genes (Profile 0) in ‘Yali’ pears at
different maturity stages after slow-cooling treatment. (A–C) KEGG
pathway for early-, middle-, and late-maturity samples. (D) Venn
diagram showing shared and unique enriched KEGG pathways among the
three maturity stages. (E) Heatmap displaying enrichment status: blue
indicates enriched, while white indicates not enriched.
3.5. Analysis of Expression Patterns and Regulatory Mechanisms of PbRAV in
Core Browning of ‘Yali’ Pear
3.5.1. Identification of PbRAV Transcription Factors
Multiple RAV transcription factors were identified as key regulators in
the core browning of ‘Yali’ pear, with four genes (RAV1-4) showing
differential expression. A cluster analysis revealed stage-specific
expression patterns during storage; RAV1 and RAV2 were upregulated at
mid-harvest maturity, while RAV4 was downregulated at this stage
([85]Figure S1).
3.5.2. Analysis of the PbRAV Gene Promoter Sequence
[86]Table S2 lists the identified cis-acting elements associated with
growth, development, abiotic stress, and hormonal regulation, such as
CAT-box, TC-rich, LTR, MBS, ABRE, GARE-motif, P-box, TATC-box,
CGTCA-motif, and AuxRR, indicating PbRAV’s potential role in hormone
metabolism and stress response modulation.
3.5.3. Relative Expression of PbRAV Genes in the Core of ‘Yali’ Pears at
Varying Maturity Stages Following Slow-Cooling Treatment
The relative expression of PbRAV1 and PbRAV2 genes in the ‘Yali’ pear
core exhibited an initial increase, followed by a decrease, then a
subsequent rise during storage ([87]Figure 7). Specifically, both genes
showed rapid upregulation early in storage, declining at 120 days, then
increasing again from 180 to 240 days. The increase in PbRAV2
expression was significantly greater than that of PbRAV1 throughout. In
mid-harvested fruit, both genes peaked at 90 days (p < 0.05), with
lower levels in early and late harvests. PbRAV1 was downregulated at
30, 150, and 180 days in early-harvested fruit, and at 120 and 150 days
in late-harvested fruit; PbRAV2 was downregulated at 150 days in
late-harvested fruit.
Figure 7.
[88]Figure 7
[89]Open in a new tab
Relative expression of PbRAV genes in the core of ‘Yali’ pears at
different maturity stages following slow-cooling treatment (Note: (A):
PbRAV1; (B): PbRAV2; (C): PbRAV3; (D): PbRAV4). Variations in lowercase
letters indicate a statistically significant difference (p < 0.05)
among the five groups.
This study indicated that PbRAV3 was generally upregulated during
storage in ‘Yali’ pears across all harvest stages, except at 30 days in
the mid-harvested fruit, with peak expression at 240 days (p < 0.05).
Conversely, PbRAV4 was downregulated at multiple time points in
early-harvested fruit and at 60 days in mid-harvested fruit, with the
highest expression at 210 days (13.66) in mid-harvested samples. In the
late-harvested pears, PbRAV4 was upregulated at 60 days and
downregulated thereafter. These expression patterns correlated with
core browning indices (late-harvested > early-harvested >
mid-harvested), suggesting that PbRAV gene upregulation may inhibit
core browning in mid-harvested fruit, while PbRAV4 downregulation may
promote core browning in late-harvested pears.
3.5.4. Prediction and Analysis of Downstream Target Genes of PbRAV Based on
the Complete Genome
Binding sites prediction revealed that PbRAV can specifically bind to
DNA sequences with the motifs 5′-CAACA-3′ and 5′-CACCTG-3′. A
cis-element analysis of the promoter sequences of all protein-coding
genes in the genomic data identified a potential presence of 140,798
CAACA binding sites and 10,980 CACCTG binding sites for the PbRAV
transcription factor. This suggested a substantial number of potential
downstream target genes for the PbRAV transcription factor within the
Pyrus bretschneideri genome. A KEGG enrichment analysis was performed
on the potential downstream target genes of the PbRAV transcription
factor. The results indicated that the PbRAV transcription factor may
regulate the synthesis and accumulation of metabolites, lipid
metabolism, and signal transduction pathways. Laccase is one of the
potential downstream target genes of the PbRAV transcription factor
([90]Figure S2). This study selected the LAC15 gene (containing two LTR
elements) to initiate an investigation into the regulation of target
genes by the PbRAV2 transcription factor.
3.5.5. Validating the Binding of the PbRAV2 Transcription Factor with the
PbLAC15 Gene Promoter
The purified GST-PbRAV2 protein displayed a band size matching the
predicted molecular weight, confirming its suitability for EMSA. The
EMSA results showed a single probe band without protein, which shifted
upon GST-PbRAV2 addition, indicating binding. The binding was
competitively inhibited by excess unlabeled probe but was unaffected by
mutated probe, demonstrating specific interaction with the PbLAC15
promoter ([91]Figure 8).
Figure 8.
[92]Figure 8
[93]Open in a new tab
Purification of GST-PbRAV2 protein (A) and EMSA verifies that PbRAV2
binds to the LAC15 promoter (B).
4. Discussion
During extended cold storage of ‘Yali’ pears (up to 1 year), the fruit
is prone to core browning. Controlling the cooling rate of postharvest
storage of ‘Yali’ pear can effectively inhibit the occurrence of core
browning, and the most significant effect of inhibiting core browning
of postharvest ‘Yali’ pear is slow cooling combined with
low-temperature storage [[94]7]. To explore which maturity stage was
more appropriate for storage under slow-cooling treatment, ‘Yali’ pears
at early, middle, and late maturity stages were harvested and stored
under slow-cooling treatment, then their physiological indexes were
measured during storage, and the changes in physiological metabolism
were observed. It was found that the appropriate maturity stage of
‘Yali’ pear combined with slow-cooling treatment could reduce the
browning phenomenon. Early-harvested ‘Yali’ pears exhibited browning at
60 days post-storage, while middle-harvested pears browned at 90 days.
Core browning was most severe in late-harvested pears, consistent with
the findings of Yan [[95]18]. Slow cooling is unsuitable for all mature
‘Yali’ pears; lower internal browning incidence in middle-harvested
fruits [[96]19] suggests that cooling rate and fruit maturity should be
optimized to mitigate core browning.
Core browning correlates with fruit senescence onset, influenced by
declining ethylene synthesis during storage [[97]3]. ‘Yali’ pears
exhibit a peak in ethylene production, with timing varying by harvest
maturity—earliest in late-harvested, delayed in early- and
mid-harvested. A secondary increase in ethylene production during
180–210 days of storage accelerates senescence and core browning.
Elevated ethylene in late-harvested pears indicates reduced storability
under slow cooling. Respiratory activity shows an initial decline, then
an increase, then a decline, with earlier peaks in late-harvested
pears. The timing of respiratory peaks aligns with browning onset,
highlighting that earlier respiratory surges and higher maturity impair
long-term storage. Flesh browning disorder correlates with nutrient
composition in mesocarp tissues [[98]20]. Firmness declines
progressively during storage, inversely related to harvest maturity;
mid-stage harvest retains firmness better, indicating delayed
senescence. Rising demand for high-quality fruit emphasizes titratable
acidity (TA) and soluble solids content (SSC) as quality indicators
[[99]5,[100]21]. The SSC initially increases and then decreases, with
late-harvested pears showing higher early SSC but lower later values
compared to earlier harvests. Weight loss accumulates over time,
peaking in mid-stage harvest, reflecting maturity-dependent water
permeability. Results suggest late harvest accelerates ethylene-induced
senescence and browning, while mid-stage harvest balances firmness
retention and physiological activity, favoring extended storage under
slow cooling.
The RNA-Seq analysis of ‘Yali’ pears at various maturity stages
revealed conserved GO terms—catalytic activity, defense response, and
phosphorylation—among upregulated DEGs, indicating shared stress
response and signaling pathway activation during core browning under
slow cooling. The correlation between mineral nutrient concentrations
and the incidence of physiological disorders in postharvest fruit is
well established. Phosphorus (P), a key component of energy metabolism,
has been implicated in the development of internal browning in ‘Rocha’
and ‘Conference’ pears, and the results of Wang indicated that the P
content was lower in the browning tissue compared to the healthy fruit
[[101]20]. Notably, mid-harvested pears exhibited unique enrichment in
phosphorus metabolism and DNA-binding processes (e.g.,
sequence-specific DNA binding), potentially enhancing phosphorus
metabolism, which may contribute to their reduced core browning
susceptibility. Conversely, downregulated DEGs revealed distinct
maturity-specific suppression patterns. Late-harvested pears
demonstrated significant inhibition of photosynthesis-related pathways
(e.g., thylakoid function, light reactions) and pyrimidine nucleotide
metabolism, likely reflecting accelerated senescence and energy
deficits during extended storage. The study by Peng also revealed that
with increasingly serious internal browning in Nane plum fruit, genes
related to photosynthesis were downregulated, while genes related to
senescence were upregulated [[102]22]. This suppression correlated with
the observed increase in core browning, as impaired metabolic processes
may exacerbate cellular dysfunction. Furthermore, shared downregulation
of hormone-responsive pathways (e.g., auxin, endogenous stimuli) across
all stages underscores a systemic decline in stress adaptation capacity
during storage.
The KEGG pathway analysis of ‘Yali’ pears under slow cooling revealed
conserved stress response mechanisms, with genes upregulated across all
maturity stages enriched in metabolic pathways, including secondary
metabolite biosynthesis, terpenoid backbone, sesquiterpenoid,
triterpenoid biosynthesis, and steroid biosynthesis. Notably, early-
and mid-harvested pears shared enrichment in glycerophospholipid
metabolism, plant hormone signal transduction, and MAPK signaling
pathways, which likely contributed to delayed core browning via
enhanced stress adaptation and membrane stability [[103]23].
Mid-harvested pears uniquely exhibited enrichment in glycerolipid
metabolism, nucleotide sugar biosynthesis, and DNA repair pathways,
further supporting their resilience. Conversely, late-harvested pears
demonstrated significant downregulation in pyruvate metabolism,
GPI-anchor biosynthesis, and amino acid degradation pathways, implying
that suppressed energy metabolism and membrane integrity maintenance
exacerbated browning. Downregulated genes across all stages were linked
to metabolic pathways, glyoxylate/dicarboxylate metabolism, and
starch/sucrose metabolism, indicating a general metabolic slowdown.
These findings collectively suggest that core browning in ‘Yali’ pears
is influenced by both upregulated stress-adaptation pathways and
downregulated metabolic processes, with mid-harvested pears exhibiting
the most robust protective mechanisms.
Transcription factors (TFs) regulate gene expression by specifically
binding to cis-acting elements, thereby constructing complex regulatory
networks. These networks play a crucial role in plant responses to
environmental stresses and the maintenance of developmental homeostasis
[[104]24]. Of particular interest was those associated with the RAV
family transcription factors. The RAV family is a member of the B3
superfamily, which also encompasses the ARF, LAV, and REM families. The
B3 superfamily is characterized by the presence of the B3 domain, a
conserved region comprising approximately 110 amino acids [[105]25].
RAV family transcription factors are implicated in the regulation of
diverse plant physiological processes, including leaf senescence,
floral development, organogenesis, and hormone signaling pathways
[[106]25]. The cis-element analysis in this study provided critical
insights into the regulatory potential of the PbRAV transcription
factor. The presence of growth-related motifs (e.g., CAT-box), abiotic
stress-responsive elements (e.g., LTR, MBS), and hormone-associated
sequences (e.g., ABRE, GARE-motif) in the promoter regions suggests
that PbRAV integrates diverse signaling pathways to modulate stress
adaptation and developmental processes. Notably, the abundance of
hormone-responsive elements, particularly those linked to abscisic acid
(ABRE) and gibberellin (P-box, TATC-box), implies that PbRAV may act as
a nexus for hormonal regulation, potentially coordinating stress
responses with growth dynamics during fruit storage. Zhao also found
that the RAV family members mediate plant growth and the developmental
process, and that these proteins are responsive to diverse
hormonal/pathogenic bacterial stimuli [[107]25]. The temporal
expression profiles of PbRAV genes within ‘Yali’ pear cores further
substantiate their functional significance. The biphasic upregulation
of PbRAV1 and PbRAV2 in mid-harvested fruit, with peak expression at 90
d, is consistent with their putative role in attenuating core browning,
as demonstrated by the reduced browning index observed in these
samples. The significantly higher fold increase in PbRAV2 expression
compared to PbRAV1 suggested a dominant regulatory role for PbRAV2 in
suppressing oxidative or enzymatic pathways driving browning.
Conversely, the sustained downregulation of PbRAV4 in late-harvested
fruit correlates with accelerated browning, indicating that PbRAV4
suppression may disrupt protective metabolic pathways, exacerbating
oxidative damage.
While low-temperature storage effectively inhibits fruit senescence,
this study employed a gradual cooling method to delay core browning in
optimally mature ‘Yali’ pears. However, prolonged cold stress during
storage can induce metabolic disturbances, leading to quality
deterioration, including phenolic oxidation browning, cell wall
structural alterations, and the loss of secondary metabolites
[[108]26], ultimately triggering core browning in the fruit. RAV
transcription factors are implicated in modulating plant responses to
both biotic and abiotic stressors [[109]27]. However, the downstream
regulatory network requires further investigation. The JASPAR-based
prediction of PbRAV-binding motifs (5′-CAACA-3′ and 5′-CACCTG-3′) and
the subsequent identification of 140,798 CAACA and 10,980 CACCTG sites
within the P. bretschneideri genome underscore the expansive regulatory
network orchestrated by PbRAV. KEGG enrichment analysis, which
associates PbRAV with lipid metabolism and signal transduction
pathways, supports its putative role in maintaining membrane integrity
and the synthesis of stress-responsive metabolites. Previous studies
also have reported that RAV transcription factors have a regulatory
function in response to drought stress, salt stress, hormonal stress,
and virus infection [[110]25,[111]27].
The lychee fruit-derived laccase (LAC), designated as LcADE/LAC,
implicated in anthocyanin degradation, was identified as a key factor
in pericarp browning [[112]28]. Specifically, ADE/LAC-mediated
flavonoid polymerization significantly contributes to the observed
pericarp browning phenotype [[113]29]. In this study, the specific
binding of GST-PbRAV2 to the PbLAC15 promoter, as validated by EMSA,
mechanistically links PbRAV2 to the regulation of laccase, a key enzyme
involved in phenolic oxidation and browning. Competitive binding assays
further confirm the specificity of this interaction, suggesting that it
may participate in the core browning process of Yali pear fruit by
regulating the upregulation of the LAC15 gene through the
phenylpropanoid metabolic pathway.
5. Conclusions
This study investigates core browning mechanisms in postharvest ‘Yali’
pears under slow-cooling treatment. The analysis of three maturity
stages revealed that slow cooling effectively reduced browning in
early/mid-harvest fruit by preserving firmness and soluble solids.
Transcriptomic profiling demonstrated conserved stress adaptation, as
evidenced by the upregulation of specific genes across various maturity
stages in ‘Yali’ pears subjected to slow-cooling treatment. The
mid-harvest fruit uniquely exhibited the activation of phosphorus
metabolic pathways and DNA repair mechanisms, thereby promoting
cellular homeostasis and consequently delaying core browning.
Conversely, the late-harvest fruit displayed suppressed photosynthetic
activity and pyrimidine metabolism, leading to energy deficits and
accelerated senescence. This maturity-dependent core browning mechanism
was orchestrated by differential metabolic reprogramming: the
mid-maturity pears maintained membrane integrity via enhanced lipid
metabolism, whereas the late-maturity fruit showed downregulation of
energy production pathways (e.g., pyruvate metabolism) and
hormone-response pathways, collectively exacerbating browning
progression. Key RAV transcription factors (e.g., PbRAV2) were linked
to browning via regulation of downstream LAC15 (laccase gene). EMSA
confirmed PbRAV2 binds the PbLAC15 promoter, with dysregulation in
overripe fruit impairing LAC15 suppression, accelerating browning.
Future work will employ in vivo ChIP-qPCR on chilled pear tissue and
transient expression assays in ‘Yali’ pear fruit to confirm regulatory
functionality during browning progression. These findings provide
mechanistic insights for optimizing slow-cooling strategies to mitigate
postharvest losses.
Supplementary Materials
The following supporting information can be downloaded at:
[114]https://www.mdpi.com/article/10.3390/foods14122132/s1, Figure S1:
The heat map for cluster analysis of RAV differentially expressed genes
in the core of harvested ‘Yali’ pear; Figure S2: KEGG enrichment
analysis of PbRAV downstream target gene (Note: Figure A: Contains
CAACA binding site; Figure B: Contains CACCTG binding site). Table S1:
Primer sequences used for qPCR validation; Table S2: PbRAV promoter
sequence analysis.
[115]foods-14-02132-s001.zip^ (381KB, zip)
Author Contributions
Conceptualization, B.D.; methodology, B.D. and X.Z.; software, H.Z.;
validation, L.L.; formal analysis, B.D.; investigation, Q.L.;
resources, H.Z.; data curation, H.Z.; writing—original draft
preparation, B.D. and X.Z.; writing—review and editing, H.Z.;
visualization, Q.L.; supervision, L.L.; project administration, B.D.;
funding acquisition B.D. and H.Z. All authors have read and agreed to
the published version of the manuscript.
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
The original contributions presented in the study are included in the
article/[116]Supplementary Material, further inquiries can be directed
to the corresponding author.
Conflicts of Interest
The authors declare no conflicts of interest.
Funding Statement
This research was funded by the National Nature Science Foundation
Project of China, Grant/Award Number: 32072278 and Key Laboratory of
Storage of Agricultural Products, Ministry of Agriculture and Rural
Affairs (kt202409).
Footnotes
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References