Abstract
Background
Blueberry (Vaccinium spp.) is a small berry with high economic value.
Although cold storage can extend the storage time of blueberry to more
than 60 days, it leads to chilling injury (CI) displaying as pedicle
pits; and the samples of 0 °C-30 days was the critical point of CI.
However, little is known about the mechanism and the molecular basis
response to cold stress in blueberry have not been explained
definitely. To comprehensively reveal the CI mechanisms in response to
cold stress, we performed high-throughput RNA Seq analysis to
investigate the gene regulation network in 0d (control) and 30d chilled
blueberry. At the same time, the pitting and decay rate, electrolyte
leakage (EL), malondialdehyde (MDA) proline content and GSH content
were measured.
Results
Two cDNA libraries from 0d (control) and 30d chilled samples were
constructed and sequenced, generating a total of 35,060 unigenes with
an N50 length of 1348 bp. Of these, 1852 were differentially expressed,
with 1167 upregulated and 685 downregulated. Forty-five cold-induced
transcription factor (TF) families containing 1023 TFs were identified.
The DEGs indicated biological processes such as stress responses; cell
wall metabolism; abscisic acid, gibberellin, membrane lipid, energy
metabolism, cellular components, and molecular functions were
significantly responsed to cold storage. The transcriptional level of
40 DEGs were verified by qRT-PCR.
Conclusions
The postharvest cold storage leads serious CI in blueberry, which
substantially decreases the quality, storability and consumer
acceptance. The MDA content, proline content, EL increased and the GSH
content decreased in this chilled process. The biological processes
such as stress responses, hormone metabolic processes were
significantly affected by CI. Overall, the results obtained here are
valuable for preventing CI under cold storage and could help to perfect
the lack of the genetic information of non-model plant species.
Keywords: Blueberry, Differentially expressed genes, Low temperature
storage, Pathways, Pitting, Transcriptome analysis
Background
Both production and consumption of blueberry (Vaccinium spp.) have
increased sharply worldwide in recent years at least partly due to
their known nutritional, economic value and health benefits. However,
it can only be stored for 5–10 days at room temperature (RT) due to the
rapid softening. Since how to prevent the decay and prolonging the
shelf-life became important topics in postharvest research. Storage at
low temperature (LT) is advantageous to preventing softening and
prolonging the postharvest life; however, cold storage triggers the
pedicle pitting, pericarp and pulp adhesion in postharvest blueberry;
which also accompanied with abnormal changes of stress-related enzymes,
destruction of outer cell wall structure, and the decrease of fiber
filaments [[39]1, [40]2]. The development of this CI reduces consumer
acceptance of the fruit, thus limiting its storage life. And it has
been confirmed that cold stress accelerated the energy consumption,
membrane lipid peroxidation and affected normal active oxygen
metabolism in blueberry.
Chilling injury is one of the main problems affecting the market value
of horticultural fruits and vegetables; which is also one of the
principal abiotic stress. Cold stress at low temperatures above zero
(0–10 °C) and freezing stress at subzero temperatures lead to decreased
cell membrane fluidity, water potential, osmotic stress causing
irreversible damage. In previous research, cold stress (4 °C) has been
found to damage the cellular structure of peppers (Capsicum annuum L.)
[[41]3–[42]5]; and the most typical CI symptom in peppers is water
immersion sunken spots [[43]6, [44]7]; in banana, the peel would pit
and discolor at low temperatures [[45]8]; in peach, CI symptoms include
lack of juiciness [[46]9], poor flavor, and the brown-red discoloration
of flesh [[47]10]; in mango, CI symptoms include scalding, softening,
internal browning and electrolyte leakage [[48]11]; in litchi, fruit
quality, membrane permeability, enzyme activities and energy changed
during cold storage [[49]12]; in pear, the aroma was less under or
after long term storage at 0 °C, seriously affecting its quality
[[50]13–[51]15]; in harvested cucumber (Cucumis sativus L.) fruit,
disorder characterized by surface pitting and dark watery patches was
confirmed when when held at 7 °C or below [[52]16]. In conclusion, it
has been identified when the fruits are subjected to cold stress, the
cell structure changed, the membranes disrupted, the malondialdehyde
(MDA) content increased, and the reactive oxygen (ROS) accumulated
[[53]17, [54]18], finally led to the decline of fruit quality. Although
the CI symptoms in different fruits and vegetables are distinct, the
nutritional and economic values are adversely affected. And the
mechanisms involved in the CI of blueberry are still not clear.
Therefore there also remains a need to study the chilling mechanism and
develop more effective techniques for blueberry fruit storage and
transport at LT. Moreover, with the rapid development of
high-throughput sequencing, great progress has been made towards the
understanding the cold-response mechanism underlying CI.
Among these, RNA-sequencing (RNA-Seq) has become a powerful technology
for characterizing molecular regulators in numerous postharvest fruits,
such as peppers, tomatoes, and ‘Nanguo’ pear [[55]14, [56]19].
Additionally, (Zhao et al. 2019) [[57]20] identified four PbBAM genes
associated with stress in pear using RNA-Seq; (Lou et al. 2018)
[[58]21] combined the isobaric tags for relative and absolute
quantification (iTRAQ) and RNA-Seq illustrated the mechanism of cold
tolerance in the loquat. Previous studies have also identified and
confirmed the expression pattern of some key genes and transcription
factor (TFs) are in response to cold stress. Among these, bZIP, MYB,
CBF, and AP2/ERF are the most well-known TFs and are regarded as
important regulators of cold-responsive genes in plants
[[59]22–[60]28]. However, CI studies on postharvest blueberry are
scarce and the genetic characterization of the underlying regulatory
mechanisms of pitting in blueberry has rarely been investigated.
In the present study, in order to characterize the CI mechanism at the
molecular level, enrich available transcriptome data, identify genes,
important pathways and regulation network potentially involved in fruit
response to cold storage, a comprehensive transcriptome profiling
analysis and physiological experiments were performed on 0d (control)
and 30d chilled blueberry (pitted). The results obtained here provide a
theoretical and a practical basis for preventing blueberry pitting; it
is also the basis for studying the functions of genes and transcription
factors affecting the blueberry fruit pitting and provide a reference
transcriptome for future studies involving blueberry fruit.
Results
Changes in phenotype and physiology at low temperature
Changes in blueberry fruit phenotype at low temperature
The physiological performance of control blueberry and 30d chilled
blueberry was characterized. The blueberry stored at 20 °C did not show
CI symptoms (Fig. [61]1a), whereas the fruits stored at 0 °C for
30 days showed typical CI symptoms as pedicle pitting (Fig. [62]1b);
these symptoms were maintained during the shelf-life.
Fig. 1.
Fig. 1
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The appearance of blueberry (a) without pitting, (b) with pitting, 30d
chilled berries
Changes in pitting rate and decaying rate at low temperature
After the measurements of the pitting rate in blueberry at shelf-life
after different storage times at 0 °C; we found no signs of pitting
were observed in the blueberry stored directly at 20 °C or for those
stored at 0 °C for 15 days beforehand (Fig. [64]2). However, signs of
pitting were observed in blueberry stored at 0 °C for 30 days
beforehand. The pitting rate was 5.2% after 30 days 0 °C storage, after
then rapidly increased to 21.9% at 2d shelf-life, and then 28.6% at 4d,
35.8% at 6d and 45.2% at 8d. For blueberry stored at 0 °C for 45 days
and 60 days, the pitting rates increased to 65.3 and 81% when the
fruits were removed from 0 °C and kept at 8d shelf-life. The decay rate
had a similar trend; both increased gradually, but the decay rate was
slightly higher in long-term low temperature stored blueberry than that
in the short-term low temperature stored (Fig. [65]2).
Fig. 2.
[66]Fig. 2
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Changes of pitting and decay rates at shelf-life (20 °C) after
different storage time at 0 °C. The lines indicate the pitting rate and
the column diagram indicate the decay rate. Each value is the mean of
three replicates of 150 fruits (P < 0.05). The error bars show the SD
of the means (n = 3; three biological replicates, each with three
technical replicates)
Changes in physiology at low temperature
Membrane plays a key role in fruit. Membrane lipid peroxidation often
occurs during CI and MDA is one of the products of membrane lipid
peroxidation. As shown in (Fig. [68]3a), there was an upward trend in
the MDA content, and the MDA content of blueberry fruit stored at 0 °C
for 30 days (9.2 mmol kg^− 1) was higher than that of fruit stored at
20 °C (6.5 mmol kg^− 1). Meanwhile, the permeability of the cell
membrane can reflect the stability and the injury degree of the cell
membrane; which can reflect by the cell membrane relative conductivity.
It can be seen from (Fig. [69]3b) that with the prolongation of
storage, the permeability of cell membrane of blueberry fruit increased
continuously. After 30 days storage at 0 °C, membrane structure, phase
change and permeability changed; the relative electrolytic leakage of
the blueberry stored at 0 °C for 30 days (42%) was higher than that of
blueberry stored at 20 °C (36%), then reached to 81%. Meanwhile, with
the occurrence of pedicle pitting, the relative conductivity of fruit
cell membrane increased sharply. The results showed that the GSH
content in blueberry fruit stored at 0 °C for 30 days (89.2%) was
higher than that of fruit stored at 20 °C (75%); the GSH content
accumulated with the prolongation of time, especially in the fruits
stored at 20 °C-6 days (92.4%) (Fig. [70]3c). After 30 days’ storage at
0 °C, the GSH content of blueberry fruit decreased rapidly from 89.2%,
and then maintained a low stable state (64.6%). The proline content of
blueberry fruit stored at 0 °C for 30 days (2.25 g kg^− 1) was higher
than that of fruit stored at 20 °C (0.6 g kg^− 1) (Fig. [71]3d), and
the proline content increased significantly after the fruits were
removed from cold storage and reached 3.95 g kg^− 1 at 8d shelf-life.
The results indicated that the cell membrane structure and membrane
function were seriously damaged during cold storage.
Fig. 3.
[72]Fig. 3
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Changes in (a) MDA content, (b) relative electrolyte leakage (EL), (c)
GSH content, (d) proline content at shelf-life and shelf-life after
30 days’ 0 °C storage. The error bars show the SD of the means (n = 3;
three biological replicates, each with three technical replicates)
Summary of the sequencing data
To study the effect of cold storage on postharvest blueberries, total
RNAs from six samples (control and chilled) were used for deep
sequencing. To evaluate the quality of the sequencing data,
randomization test of mRNA fragmentation saturation, length test of
insert fragment and saturation test were conducted. These tests
determined the number of obtained reads was enough to cover most of the
expressed genes. The total 50.74Gb of clean data were generated
(Table [74]1). Average clean data were above 6.25Gb and Q30 values were
obtained for more than 93.93% of the data; 76.38 to 79.53% of the clean
reads were successfully mapped to the unigene database. Meanwhile the
transcription group data detection has a high sensitivity, with the
protein encoding gene expression level FPKM (fragments per kilobase
million) having values across 10^− 2 to 10^− 4 six orders of magnitude.
The dispersion degree of sample gene expression level distribution was
average, and the overall gene expression abundance in different samples
was good (Fig. [75]4a). According to the PCA analysis, the three
biological replicates of control and chilled groups were clustered
together respectively (Fig. [76]4c), showing a Pearson correlation
above 0.94 (Fig. [77]4b).
Table 1.
Summary of RNA-Seq data and sequence assembly
Samples Base Number Clean Data Error(%) % ≥ Q30
Clean Read Mapped Reads Mapped Ratio
T01 9,135,928,834 30,590,963 23,364,104 76.38% 0.01 94.43%
T02 9,105,416,678 30,487,303 23,308,114 76.45% 0.01 93.93%
T03 6,252,371,304 20,941,667 160,042,397 76.61% 0.01 94.14%
T04 8,567,949,538 28,664,023 22,285,082 77.75% 0.01 94.29%
T05 9,096,999,984 30,453,155 24,218,167 79.53% 0.01 94.25%
T06 8,580,914,322 28,746,121 22,703,600 78.98% 0.01 94.79%
[78]Open in a new tab
Note: Read Number: Total pair-end reads in the clean data; Base Number:
Total base number in the clean data; % ≥ Q30: Percentage of bases whose
clean data mass value is greater than or equal to 30
Fig. 4.
[79]Fig. 4
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(a) Box plot representation of expression range from six libraries; (b)
Pearson correlation between samples and (c) PCA analysis among T01-T06
samples; Different colored squares represent the degree of correlation
between two samples; T01-T03 represented 0d control samples and T04-T06
represented 30d-chilled samples
Comprehensive profiling of transcript expression analysis
Number of DEGs
The analysis of DEGs revealed 561 up-regulated and 158 down-regulated
genes for FDR ≤ 0.01, and 1167 up-regulated and 685 down-regulated
genes for FDR < 0.05. To assess the diversity of DEGs, the Venn map was
constructed (Fig. [81]5).
Fig. 5.
Fig. 5
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Venn diagram of differentially expressed genes between each two samples
Heatmap of all DEGs and the selection of significantly changed DEGs
The identified DEGs were analyzed by hierarchical clustering to
organize genes with the same or similar expression behaviors to show
the different expression patterns of gene sets under different
experimental conditions (Fig. [83]6). In the blueberry kept at 20 °C,
most genes were significantly down-regulated; in blueberry kept at 0 °C
for 30 days, most of these genes were up-regulated. We clustered these
genes into 15 categories according to their functional pathways. These
included GABA receptor activity, extracellular-glutamate-gated ion
channel activity, and the G-protein coupled receptor signaling pathway.
There were 30, 27, 37, 92, 14, 351, 217, 193, 315, 1, 161, 409, 2, 1, 2
genes in each cluster via heatmap analysis, and there were more
up-regulated genes in the G4, G5, G6, G7, G8, and G12 clusters, while
the down-regulated genes mostly belonged to the G1, G2, G3, G9, G10,
G11, G13, G14 and G15 clusters (Fig. [84]6).
Fig. 6.
[85]Fig. 6
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Thermal maps of DEGs and 15 clusters produced for thermographic
analysis. Different columns represent different samples and different
rows represent different genes. Each small square represents a gene and
the color indicates the level of expression; red represents
up-regulation and green represents down-regulation
Meantime, the 10 DEGs most significantly changed among treatment groups
were selected to assess the important genes and pathways involved in CI
(Table [87]2). Among these DEGs, c127608 was up-regulated and involved
in six pathways and c120356 was down-regulated and involved in three
pathways of the blueberry kept at 0 °C for 30 days.
Table 2.
Genes significantly up-regulated and down-regulated in 30d-chilled
blueberry
Number Name Route and name FC (up)/ (down)
c118333 HSPA1s ko3040/ko04141/ko04144 protein processing in endoplasmic
reticulum 3.428
c76944 Synthase ko00904 diterpenoid biosynthesis 3.407
c122315 8′-hydroxylas ko00906 carotenoid biosynthesis 2.582
c120652 2.501
c100356 AGXT2 ko00250/ko00260alanine aspartate and glutamate metabolism
2.485
c122874 MIOX ko00053/ko00562 ascorbate and aldarate metabolism 2.417
c111137 GH3 ko04075 plant hormone signal transduction 2.404
c59127 INV ko00052/ko00500 galactose metabolism 2.400
c127608 ALDO ko00010/ko00030/ko00051/ko00710/ ko01200/ko01230
glycolysis/gluconeogenesis 2.321
c115136 CYP85A2,BR6OX2 ko00905 brassinosteroid biosynthesis 2.305
c120356 HIBCH ko00280/ko00410/ko00640 valine leucine and isoleucine
degradation −5.716
c89494 CYP82G1 ko00904 diterpenoid biosynthesis −5.190
c110095 LAR ko00941 flavonoid biosynthesis −4.842
c121415 INV ko00052 galactose metabolism −4.740
c97853 psbY ko00195 photosynthesis −4.640
c111548 CKX ko00908 zeatin biosynthesis −4.554
c99806 crtZ ko00906 carotenoid biosynthesis −4.201
c129827 G6PDf ko00480 glutathione metabolism −3.828
c127697 RP-L5e,RPL5 ko03010 ribsome −3.807
c111628 RP-L9,MRPL9 −3.787
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These genes were selected with an FDR adjusted P-value< 0.05
Functional annotations and classifications
In our research, the N50 was 1348 bp, and there were 21,649 unigenes
longer than 1Kb. The assembly integrity was high among the 84,260
unigenes. To predict and analyze the functions of the assembled
unigenes, the NR, Swiss-Prot, GO, COG, KOG, Pfam, KEGG, KOG, and eggNOG
databases were used as the basic local alignment search tool: 1142
(18.4%) unigenes were annotated in NR; 834 (13.4%) in Swiss-Prot; 685
(11.2%) in GO; 394 (6.4%) unigenes in COG; 922 (14.8%) in Pfam; 413
(6.7%) in KEGG; 638 (10.4%) in KOG; and 1099 (17.7%) in eggNOG.
Then the GO enrichment analysis was performed to obtain functional
information for the DEGs mentioned above. Significantly enriched GO
terms, identified based on corrected P-value< 0.05, were found for DEGs
involved in biological processes, cellular components, and molecular
functions (Fig. [89]7a). The DEGs found in chilled blueberry were
mainly enriched in biological processes, metabolic processes, cellular
processes, single organism processes, responses to stimulus, biological
regulation, localization, cellular components, developmental processes,
and 12 other different biological processes. The DEGs included in the
metabolic process, cellular process, and single-organism process
categories were not expressed in blueberry that did not pit.
Fig. 7.
[90]Fig. 7
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Functional annotations and classifications of unigenes in 30d-chilled
blueberry according to (a) GO, (b) KEGG and (c) COG databases
To further identify the functions of DEGs in response to cold stress,
the KEGG database was used to classify and characterize the DEGs into
corresponding pathways. By comparing the samples, we found that 413
DEGs were enriched into 50 pathways; three of these 50 pathways were
related to cellular processes, two pathways belonged to environmental
information processing, 12 pathways contained 98 DEGs involved in
genetic information processing, 32 pathways included 174 DEGs involved
in metabolism, and the remaining pathway was related to organism
systems (Fig. [92]7b). The results of the KEGG analysis showed that, in
blueberry under cold stress, 26 DEGs were involved in plant hormone
signal transduction pathways, including auxin (Aux), cytokinin (CK),
gibberellin (GA), abscisic acid (ABA), ethylene (ET), brassinosteroid
(BR), jasmonic acid (JA), and salicylic acid (SA) signaling pathways,
each gathering different numbers of DEGs. Moreover, carotenoid
biosynthesis, which is involved in the biosynthesis of ABA, comprised
five DEGs. These results indicated that not only fatty acid metabolism,
but also multiple plant hormone signal transduction pathways were
activated under cold stress leading to blueberry fruit pitting. Based
on enrichment results, the six most significantly changed pathways in
response to cold stress were plant hormone transduction, carotenoid
biosynthesis, GSH metabolism, starch and sucrose metabolism, protein
processing in endoplasmic reticulum, and chlorophyll metabolism
(Table [93]3).
Table 3.
The top 6 enriched pathways of DEGs in 30d-chilled blueberry
Pathway Type KO_ID DEGs in pathway All genes in pathway P-value
Plant hormone transduction Environmental information ko04075 21 232
5.23e-05
Carotenoid biosynthesis Metabolism ko00906 5 34 0.006
Glutathione metabolism Metabolism ko00480 11 134 0.007
Starch and sucrose metabolism Metabolism ko00500 15 218 0.009
Protein processing in endoplasmic Genetic information ko04141 22 410
0.03
Porphyrin and chlorophyll metabolism Metabolism ko00860 6 67 0.03
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These were selected with an FDR adjusted P-value< 0.05
In addition, 394 DEGs (25classes) were annotated in the COG database
(Fig. [95]7c). The top three COG terms were: general function
prediction only (95DEGs); replication, recombination and repair (50
DEGs). Energy production and conversion, amino acid transport and
metabolism of carbohydrate transport, inorganic ion transport, and
metabolic biosynthesis transport and catabolism were also important.
Identification and analysis of the DEGs under cold stress
Cold-response DEGs involved in membrane lipid and energy metabolism
In this study, six highly differentially expressed pathways of the 129
pathways including membrane lipid metabolism, including glycerolipid
metabolism (Ko00561), glycerophospholipid metabolism (Ko00564), ether
lipid metabolism (Ko00565), α-linolenic acid metabolism (Ko00592),
sphingolipid metabolism (Ko00600), and phosphatidylinositol signaling
system (Ko04070) were significantly changed in chilled blueberry. There
was one down-regulated DEG in Ko00561; and the Ko00564; Ko00592,
Ko00565, Ko00564, Ko00600, and Ko04070 comprised one, one, four, two,
and one highly differentially up-regulated expressed structural genes,
respectively. Among these six pathways, Ko00564 and Ko00592 were
strongly induced in plants under cold stress;
c107765.graphc0(Phosphocholine CytidylyltransferaseA) was
down-regulated; the c100748.graphc0 (glycerol-3-phosphate
dehydrogenase1), c131228.graphc1 (phospholipaseD1/2), c125240.graphc0
(N-myristoyltransferase), c126471.graphc0 (Triacylglycerol lipase4),
and c130733.graphc0 (phosphatidylserine synthase2) were up-regulated in
Ko00564, while c130733.graphc0 was up-regulated in Ko00592.
The changes in cell membrane lipid compositions are closely related to
fatty acid metabolism under cold stress. Six of the 129 pathways
significantly changed in chilled blueberry were related to fatty acid
metabolism, which included fatty acid biosynthesis (Ko00061), fatty
acid elongation (Ko00062), fatty acid degradation (Ko00071), linoleic
acid metabolism (Ko00591), biosynthesis of unsaturated fatty acids
(Ko01040), and fatty acid metabolism (Ko01212). There were three, one,
and two DEGs down-regulated in Ko00061, Ko00071, and Ko01040,
respectively; there were one, one, five, one, and two DEGs up-regulated
in Ko00062, Ko00071, Ko00591, Ko01040, and Ko01212, respectively. Genes
c126515.graphc0 (Fas-associated protein with death domain),
c109637.graphc0 (fatty acyl-ACP thioesteraseB), c126471.graphc0
(doxycycline) were down-regulated in Ko00061 (Fig. [96]8a).
Fig. 8.
[97]Fig. 8
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Transcripts involved in (a) liquid related pathways, (b) proline,
glutathione, and flavonoid metabolism, (c) brassinosteroid
biosynthesis, carotenoid biosynthesis, and zeatin biosynthesis in
chilled blueberry
In the Ko00230 purine metabolism pathway, the DEGs related to energy
metabolism affected by cold stress. Gene c121781.graphc0, which was
up-regulated, participates in the pentose phosphate pathway. This is
involved in several biosynthesis pathways of microbial metabolism in
diverse environments and in carbon metabolism biosynthesis;
c123543.graphc0 was expressed in chilled blueberry and its expression
was lower in this group than control fruits. In the Ko00280 valine,
leucine, and isoleucine metabolism pathway, c122233graphc0 was
up-regulated; 3-Hydroxyisobutyryl-CoA Hydrolase was co-regulated by two
genes, c120356.graph and c122037.graphc0, which were significantly
down-regulated (fold-change = − 5.71) and are involved in the
beta-alanine metabolic and carbon metabolism pathways.
Cold-response genes involved in proline, glutathione, and flavonoid
metabolism
Additionally, dehydration stress is often accompanied by osmotic
adjustment. Plants need to accumulate or reduce organic or inorganic
substances such as proline, glutathione, and flavonoid to maintain
cellular moisture holding capacity. According to pathway enrichment
analysis, arginine and proline metabolism is activated by cold-induced
dehydration. In our study, c126860.graphc0 (NADP-specific glutamate
dehydrogenase) also participates in alanine, aspartate, glutamate
metabolism, D-glutamate metabolism, and carbon metabolism, was
up-regulated; c123304.graphc0 (polyamine oxidase2/3/4) was up-regulated
during spermidine synthesis and it is also involved in beta-alanine
metabolism. In our validation experiments, we found that the relative
expression of c126860.graphc0 in chilled blueberry was significantly
up-regulated. The expression of c123304.graphc0 was also significantly
up-regulated in chilled blueberry.
In the present study, c123222.graphc0 [Isocitrate dehydrogenase1/2
(IDH1/2)] plays a role in intermediary metabolism and energy
production, might tightly associate or interact with the pyruvate
dehydrogenase complex in the citric acid cycle (TCA cycle). Some genes
related to glutathione were also identified. GPX and glutathione
transferase (GST) are also important scavengers of ROS that participate
in many kinds of environmental stress responses to adverse conditions.
Glutathione peroxidase participates in arachidonic acid metabolism and,
together with c112252.graphc0 [glutathione s-transferase (GST)],
c119253.graphc0 (GST), c102135.graphc0 (GST), c105671.graphc0 (GST),
c105671.graphc1 (GST), c122443.graphc0 (GST), c110117.graphc0, and
c120425.graphc0 coordinates the GST metabolism of xenobiotics by
cytochrome P450. In our validation, GST and IDH1 were down-regulated in
chilled blueberry compared to the control blueberry. The expression of
GST1 was significantly higher in chilled blueberry; the expressions of
GST2 and GST3 were significantly lower in chilled blueberry
(Fig. [99]8b).
The gene c116187.graphc0 [Hydroxycinnamoyl-CoA shikimate (HCT)] encodes
shikimate O-hydroxycinn amoyltransferase (EC:2.3.1.133), which
participates in metabolic pathways and secondary metabolite
biosynthesis; the leucoanthocyanidin reductases (LAR) encoded by
c119207.graphc1 and c110095.graphc0 participate in secondary metabolite
biosynthesis. Our qRT-PCR results showed that the expressions of HCT
and LAR (c110095.graphc0) were down-regulated and that of LAR
(c119207.graphc1) was up-regulated in chilled blueberry.
(Fig. [100]8b).
Cold-response genes involved in hormone biosynthesis and signal transduction
Brassinosteroid biosynthesis (Ko00905), carotenoid biosynthesis (Ko00906),
and zeatin biosynthesis (Ko00908).
In the zeatin biosynthesis pathway, c111548.graphc0 (Cytokinin
oxidase/dehydrogenase) was significantly down-regulated to − 4.5 in
chilled blueberry; c126830.graphc0 (tRNA isopentenyltransferase1) was
up-regulated during the production of uridine phosphorylase and
cis-zeatin glucoside. Genes c112010.graphc0
[Cytochrome-P450-90A1-(CYP90A1)] and c115136.graphc0
[Cytochrome-P450-85A2-(CYP85A2)] participate in secondary metabolite
biosynthesis and metabolic pathways, including BR biosynthesis. These
two genes had higher expression in chilled blueberry (Fig. [101]8c).
In the carotenoid biosynthesis pathway, c99806.graphc0 (beta-carotene
3H-hydroxylase), the precursor of ABA synthesis, was significantly
down-regulated, while the synthesis and metabolism of ABA, also
included in this pathway, and related genes were significantly
up-regulated in chilled blueberry. This finding was in agreement with
the fact that stress can increase ABA biosynthesis and accumulation as
part of the plant defense response. 9-Cis-epoxycarotenoid dioxygenase
[NCED; c126206.graphc0) is the most critical rate-limiting enzyme in
ABA synthesis and it was significantly up-regulated after storage at
0 °C. Xanthoxin dehydrogenase/ABA2 (c125901.graphc0) is another
rate-limiting enzyme in the ABA synthesis pathway, and genes
c120652.graphc1 and c122315.graphc1 co-regulate ABA 8′-hydroxylase. The
results of our qRT-PCR showed that the expression of beta-carotene
3-hydroxylase was up-regulated in chilled blueberry. The ABA2 genes
were significantly up-regulated in chilled blueberry. Expression of
Cytochrome P450 707A (CYP707A), which encodes a cytochrome P450 that
participates in plant metabolism of terpenoids and polyketides in the
ABA metabolic pathway, was significantly up-regulated in chilled
blueberry and was 25 times that measured in control blueberry. Thus,
cold storage stimulated the expression of genes that encode ABA
synthase-related enzymes and are involved in ABA synthesis, but also
promoted its ABA metabolism to dihydroxy phaseic acid. Pitting in
blueberry after a 30 days’ storage at 0 °C might be affected by these
genes; however, the specific mechanism needs further study
(Fig. [102]8c).
Plant hormone signal transduction analysis
The results of transcriptome analysis indicated a large number of DEGs
were enriched in the plant hormone signal transduction pathways,
especially in IAA signal transduction, ABA signal transduction, SA
signal transduction and GA3 signal transduction (Fig. [103]9).
Fig. 9.
[104]Fig. 9
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Transcripts involved in hormone related pathways in 30d-chilled
blueberry
Our results indicated that stress-response pathways interact with auxin
regulation through the transcription of the Aux/IAA, IAA5, IAA6, and
IAA19 are required for stress tolerance; one gene was down-regulated
and two genes were up-regulated in the auxin signaling pathway in
chilled blueberries. Gene c113524.graphc0 encodes transport inhibitor
response1 was down-regulated in chilled blueberry. Genes
c128076.graphc0 (IAA), c122133.graphc1 (IAA), and c97770graphc0
coordinate the regulation of IAA, an auxin-responsive protein that
participates in plant hormone signal transduction; c111137.graphc1, a
member of the GH3 gene family, showed higher expression in chilled
blueberry. Genes c102439.graphc0 and c113051.graphc0 belong to the
small auxin-upregulated RNA gene family, and are regulated by auxin and
environmental factors; these genes were similarly expressed with IAA,
which was significantly up-regulated in chilled blueberry. Pyrabactin
resistance 1-like (PYL) is an ABA receptor, PYL1, PYL4, PYL5, PYL6,
PYL7, PYL10, PYL11, PYL13, and PYL15 have been isolated and identified
from Arabidopsis thaliana. In the present study, the expression of
c121430.graphc0, an ABA receptor of the PYR/PYL family involved in
mitogen-activated protein kinase signaling, was significantly higher in
chilled blueberry fruits. The upregulation of both genes indicated that
ABA biosynthesis and catabolism were activated by low temperature. The
ABA response element binding factor gene (c112990.graphc0) was
down-regulated in our transcriptome results, while the brassinosteroid-
signaling kinase (c122496.graphc0) was up-regulated in chilled
blueberry fruits which was eight times higher than that in control
blueberry.
In the SA pathway, gene expression of the plant-pathogen interaction
genes c129050.graphc0 (Pathogenesis related protein1) and
c126145.graphc0 (TGA) were down-regulated in chilled blueberries. The
expression pattern of both genes was similar. Moreover, the treatment
of horticultural crops and fruits with GA3 is known to alleviate CI
symptoms after treatments at 0 °C [[106]29]. In the GA3 pathway, the
expression of DELLA, a negative regulator of GA3 signaling, was
up-regulated after 30 days’ storage at 0 °C. Three genes were
up-regulated in the CK pathway, a CK receptor and the 2-component
response regulators ARR-B or ARR-A. The expression levels of
c107755.graphc0 (ARR-B) and c107605.graphc0 (ARR-A) in chilled
blueberries were significantly up-regulated, and 18 and 59 times that
in control blueberries, respectively. Therefore, genes in hormone
signal transduction pathways were significantly affected by cold
storage at 0 °C, especially those involved in CK and Aux regulation and
metabolism.
TFs in response to cold stress
The different gene expression patterns across the 30d-chilled blueberry
indicated that multiple structural genes have contributed to blueberry
fruit pitting. In the present study, we screened our assembled
transcripts and predicted a total of 1023 TFs from 45 families and
identified 738 protein kinase, and 327 transcriptional regulators
(TRs); the expressions of most of them in chilled blueberry fruits were
changed. The 1023 TFs comprised 42 categories of TFs including 92 C2H2,
87 MYB 68, 74 Ap2/erf-erf, 56 bHLH, 53 C2C2, 51 bZIP, 51 C3H, 45 FAR1,
43WRKY, 39 NAC (Fig. [107]10).
Fig. 10.
[108]Fig. 10
[109]Open in a new tab
The numbers and classification of differentially expressed TFs in
chilled blueberries. Categories of transcription factors less than 1%
of the total are not marked on the pie chart
Validation of the RNA-Seq results by qRT-PCR
To ensure the reliability of the RNA-Seq data, the expression patterns
of 40 random DEGs were evaluated by qRT-PCR. (Figs. [110]11 and
[111]12). The genes represented various functional categories or
pathways, including liquid related, defense systems, flavonoid
metabolism, brassinosteroid biosynthesis, carotenoid biosynthesis,
zeatin biosynthesis and hormone signal transduction pathways. The
linear regression showed that the results from RNA-Seq and qRT-PCR were
highly relevant (Pearson’s r = 0.8624), despite the difference in the
absolute FC between the two methods. The results showed the expression
patterns derived by both methods were consistent, confirming the
reliability of the transcriptome results. These consistent expression
patterns further confirmed the transcriptome data reported in this
study constitutes a valuable supplement to the available blueberry
genomic and transcriptome information.
Fig. 11.
[112]Fig. 11
[113]Open in a new tab
The correlation analysis between qRT-PCR of RNA-Seq results of 40
randomly genes
Fig. 12.
[114]Fig. 12
[115]Open in a new tab
qRT-PCR verification of expression pattern obtained by RNA sequencing.
The relative transcription level was calculated according to the
2^−ΔΔCT method with actin reference genes as control. Each value is the
mean ± SD; P < 0.05 (n = 3, three biological replicates, each with
three technical replicates)
Discussion
Although LT storage is the major method to prevent most fruits and
vegetables from decaying, it may cause CI symptoms, such as the surface
pitting and dark watery patches in harvested cucumber [[116]16], peel
pitting in banana [[117]8], and less aroma in pear [[118]13]. In our
present study, the blueberry were stored at low temperature
(0 ± 0.5 °C) for 15, 30, 45, and 60 days. This method can preserve the
fruit quality and extend their postharvest life. However, pedicle
pitting developed due to LT storage and the cold stress; the longer the
blueberries are stored, the more serious the pitting is. And the
0 °C-30 days storage was the critical point of pitting. With the
occurance of pitting, the phenotype of fruit changed accompanied with
the wrinkled cell membrane and the destroyed structure [[119]30].
Meantime, the physiology indices of EL, MDA, proline content, and GSH
content, which were considered to reflect physiological state of plant
exposure to cold stress [[120]31], were also determined in our study.
And the significant increase in EL, content of MDA and proline, and the
decrease in GSH content in pitted blueberry indicated that membrane
lipid metabolism plays an important role in cold stress response.
However, this is only the changes in physiological.
Although cold stress responses and physiological changes have been
extensively studied [[121]6, [122]7, [123]9, [124]10], the its
molecular and genetic mechanisms remain poorly characterized in
blueberry. In order to explore the direct cause of fruit pitting and
the gene regulation net under cold stress at the molecular level, we
performed deep transcriptome sequencing of the chilled blueberry and
control blueberry samples. According to the RNA-Seq data, the three
independent biological replicates were highly consistent
(0.9462 < R^2 < 0.9998), which indicated that the RNA-Seq data was
reliable. The transcriptome sequences were assembled into 35,060
transcripts, of which 73.7% were annotated. The annotations provide a
valuable resource for investigating specific processes, functions, and
pathways during blueberry research. For further verification, we used
qRT-PCR, and the expression patterns of 40 significantly changed DEGs
corresponded (Pearson’s r = 0.8624) in both methods. Under cold stress,
numerous DEGs participating in diverse cell, molecular, and biological
pathways were identified as putatively involved in pitting. We mainly
focused on the genes involved in lipid metabolism, energy metabolism,
antioxidant defense system, hormone signaling networks and TFs based on
enrichment results (Tables. [125]4, [126]5, [127]6 and
Figs. [128]8, [129]9 and S1). These results demonstrate that our
transcriptome database is a rich resource for mining cold
stress-responsive genes. These transcriptome analysis results provide
key points to explain the mechanism underlying the metabolic changes in
pitted blueberry in response to cold stress at the molecular level.
Table 4.
Genes involved in lipid related pathways in in response to low
temperature
Name Gene ID FC Definition Name Gene ID FC Definition
PCYT1 c107765.graphc0 −1.3 choline-phosphate TGL4 c126471.graphc0 1.12
triacylglycerol lipase SDP1-like
GPD1 c100748.graphc0 1.14 [NAD(+)] PTDS2 c130733.graphc0 1.17
Phosphatidyl serine synthase
PLD1/2 c131228.graphc1 1.33 Phospholipase-D-p1 fadD c109637.graphc0
2.22 palmitoyl-acyl carrier protein
NMT c125240.graphc0 1.45 N-methyltransferase1 FATB c126515.graphc0 −2.5
acyl-CoA synthetase1
[130]Open in a new tab
These genes were selected with an FDR adjusted P-value< 0.05
Table 5.
Genes involved in proline, glutathione, and flavonoid in response to
low temperature
Name Gene ID FC Definition Name Gene ID FC Definition
allB c123543.graphc0 −1.4 allantoinase IDH1 c123222.graphc0 −1.44
isocitrate dehydrogenase
HIBCH c120356.graphc0 −5.7 3-hydroxyisobutyryl-CoA hydrolase gpx
c112498.graphc0 1.26 glutathione peroxidase
BCKDHA c122233.graphc0 1.14 2-oxoisovalerate dehydrogenase E1 GST
c112252.graphc0 1.61 glutathione S-transferase
gdhA c126860.graphc0 1.12 NAD(P)+ c119253.graphc0 1.50
P5CS c122210.graphc0 −1.7 delta-1-pyrroline-5-carboxylate
c102135.graphc0 −1.68
PAO2/3/4 c123304.graphc0 1.03 polyamine oxidase c105671.graphc0 −1.71
PRPS c121781.graphc0 1.41 ribose-phosphate pyrophosphokinase
c105671.graphc1 −1.72
G6PD c129827.graphc0 −3.81 glucose-6-phosphate 1-dehydrogenase
c122443.graphc0 −1.54
[131]Open in a new tab
These genes were selected with an FDR adjusted P-value< 0.05
Table 6.
Genes involved in plant hormone signal transduction in response to low
temperature
Name Gene ID FC Definition Name Gene ID FC Definition
CYP85A2 c115136.graphc0 2.31 brassinosteroid-6-oxidase 2 GH3
c111137.graphc1 2.41 auxin responsive family
CYP90A1 c112010.graphc0 1.32 cytochrome P450 family 90 TIR1
c113524.graphc0 −1.7 transport inhibitor response 1
crtZ c99806.graphc0 −4.2 beta-carotene3-hydroxylase SAUR
c102439.graphc0 1.41 SAUR family protein
NCED c126206.graphc0 1.43 9-cis-epoxycarotenoid AHK2/3/4
c122606.graphc0 1.38 cytokinin receptor
ABA2 c125901.graphc0 1.12 Xanthoxin dehydrogenase ARR-B c107755.graphc0
1.43 ARR-B family
E1.14.13.93 c120652.graphc1 2.51 (+)-abscisic acid 8′-hydroxylase ARR-A
c107605.graphc0 1.18 ARR-A family
CKX c111548.graphc0 −4.5 cytokinin dehydrogenase c116928.graphc0 2.17
LAR c110095.graphc0 1.39 leucoanthocyanidin reductase PYL
c121430.graphc1 1.02 ABA receptor PYR/PYL
c119207.graphc1 −4.8 BSK c122496.graphc0 1.71 BR-signaling kinase
HCT c116187.graphc0 −1.1 O-hydroxycinnamoyltransferase c129050.graphc0
−1.4
IAA c128076.graphc2 1.13 auxin-responsive protein IAA TGA
c126145.graphc0 −2.45 transcription factor TGA
c122133.graphc1 −1.71 PR1 c119719.graphc1 1.14 pathogenesis-protein 1
[132]Open in a new tab
These genes were selected with an FDR adjusted P-value< 0.05
Lipid metabolism and energy metabolism under cold stress
When the environmental temperature decreases, physical phase and cell
arrangement change first; then membrane permeability, lipids’
composition and content begin to degrade. Electrolyte leakage is an
important indicator of cell membrane permeability and is the most
frequently used method to evaluate plant tissue injury under severe
stress conditions. The substantial increase in EL is commonly caused by
changes in membrane structure and lipid composition and considered as
an index reflecting cell membrane function and integrity [[133]32,
[134]33]. In our present study, the severity of pitting in blueberries
was related to relatively higher EL. In addition to electrical leakage,
the lipid peroxidation are also evaluated in studies of fruit
mechanisms under cold stress [[135]34]. Since MDA content is a direct
measure of lipid peroxidation, it can be evaluated to assess the degree
of cell damage [[136]35]. In the present study, we observed a
significant increase in MDA content during cold storage, especially
during the shelf life after 0 °C storage. These clearly indicated that
blueberry membrane lipid peroxidation occurs during LT storage and it
is intensified during shelf life after 0 °C storage.
Meantime, the accumulation of proline is also related to the cold
tolerance in most plant [[137]36]. And the proline has been confirmed
to accumulate when plants are exposed to salt or low temperature stress
[[138]37]. In our present study, the proline content of blueberries
after 30 days’ cold storage was significantly higher than that of other
blueberries during the 8-days shelf life; additionally, the proline
content of blueberry fruits after 30 days’ cold storage was more than 2
times higher than the blueberries in the other group. Obviously, the
increase of proline provided strong evidence that LT storage caused
cold stress and blueberries responded to cold stress. In the meantime,
we also observed that the GSH in the cold storage group had a downward
trend while that in the other group had an upward trend. In conclusion,
membrane lipid metabolism plays an important role in blueberry response
to cold stress and may be crucially related to the tolerance of fruit
to cold stress. This was consistent with some studies in other fruits.
Li et al. (2012) [[139]38] confirmed relative EL had a remarkable
increase in the mango fruit stored at 5 °C. Kong et al. (2017) [[140]4]
also indicated that LT (4 °C) caused serious membrane damage in peppers
and the MGDG, PC, PE and PA changed in response to cold. Wang et al.
(2019) [[141]30] also identified the blueberries had a higher level of
DGDG after LT storage. Additionally, changes of fatty acids were also
involved in membrane lipid metabolism. And the change of the lipid not
only occurs to the fruits and vegetables in the post-harvest
low-temperature storage process, but also occur to the fruits and
vegetables or the plants during the cold acclimation [[142]39].
Additionally, genetic and molecular evidence shows that pitting is a
complex phenomenon involving the alteration of metabolism with
synthesis of specific metabolites, lipids, energy and other pathways.
And the changes in gene expression underlie some of the biochemical and
physiological changes that occur during cold storage. In the present
study, the genes regulating membrane lipid components and fatty acids
also significantly changed under cold stress. Six highly differentially
expressed pathways of membrane lipid metabolism, including glycerolipid
metabolism (Ko00561), glycerophospholipid metabolism (Ko00564), ether
lipid metabolism (Ko00565), α-linolenic acid metabolism (Ko00592),
sphingolipid metabolism (Ko00600), and phosphatidylinositol signaling
system (Ko04070) were significantly changed in chilled blueberries.
Additionally, six highly differentially expressed pathways related to
fatty acid metabolism including fatty acid biosynthesis (Ko00061),
fatty acid elongation (Ko00062), fatty acid degradation (Ko00071),
linoleic acid metabolism (Ko00591), biosynthesis of unsaturated fatty
acids (Ko01040), and fatty acid metabolism (Ko01212) were also
significantly changed in chilled blueberries. Among these genes, the
fadD, c109637.graph_c0 encoding palmitoyl-acyl carrier protein was the
most obvious up-regulated gene, its expression was 2.2 times higher
than that in the control group; the FATB, c126515.graph_c0 encoding
acyl-CoA synthetase 1 was the most obvious down-regulated gene, its
expression was 2.5 times lower than that the control group. These
results suggested the pathways related to membrane lipid had a strong
response to cold stress, which was consistent withe the results in
loquat [[143]21] and these two genes may be the most ideal genes to
further study the regulation of membrane lipid metabolism response to
fruit pitting. The study of Die and Rowland. 2014 [[144]40] also
confirmed the pathways associated with lipid metabolism, especially
regarding enzymes involved in fatty acid elongation,
glycerophospholipid metabolism and -linolenic acid metabolism changed
significantly in response to cold stress. Moreover, Die and Rowland.
2014 [[145]40] identified the genes encoding phospholipaseC, histidine
kinase, 5 lipid-transfer protein (LTP) genes and 2 fatty acid
desaturases with potential roles in altering the composition of
proteins and lipids.
In fact, the occurrence of CI is not just the results in detrimental
changes in membrane structure but also related to the changes of energy
status [[146]2, [147]41]. Published data on other fruits also suggest a
close association between membrane integrity regulation is cellular
energy status. It has been suggested that the damage of cell membranes
was associated with lack of energy status, and ATP played important
roles in synthesis of fatty acid and repair of membranes [[148]42].
Recently, many evidences have shown that development of CI in
postharvest fruit is partly attributed to limited availability of
energy or low energy production, whereas acquaintance of higher levels
of ATP and energy charge alleviate CI [[149]16, [150]43]. Moreover,
energy metabolism is important to sustain plant life and plant
resistance to environmental stresses [[151]14, [152]44, [153]45]. In
the present study, there were numerous down-regulated DEGs involved in
energy metabolism, which may suggest that the available energy status
and the stable enzymatic system in blueberry collectively contribute to
improve chilling tolerance by alleviating pitting and maintaining the
quality of blueberry fruits during storage at 0 °C [[154]1]. Energy
metabolism is linked to adenosine triphosphate (ATP) production and it
is the major determinant of cell function and viability, as indicated
by plants with higher energy and better maintenance of biological
membrane structure being less susceptible to CI [[155]46, [156]47].
Conversely, long-term storage at 0 °C could result in the decrease of
ATP levels in postharvest fruit, causing a disruption in their energy
metabolism [[157]38]. In particular, it has been shown that ATP and
energy content in papaya decreased after fruit storage at 0 °C,
indicating that CI has a close relationship with energy metabolism
[[158]45]. Consistent with these findings, our results suggest that
energy metabolism might be a main reason underlying the pitting
symptoms and therefore exogenous ATP treatment might reduce pitting of
blueberry fruits. And it has been proved that the high ATP level can be
maintained by increasing the D-pyrroline-5-carboxylatesynthetase (P5CS)
activity and then reduce the CI [[159]44]. And suppressing reactive
oxygen species [[160]16] modulating proline accumulation [[161]48] may
can also enhance the chilling tolerance.
Hormone signals under cold stress
Previous studies have demonstrated that several plant hormones are
involved in modulating response and adaptation to a changing
environment in plants [[162]49]. As secondary signals, they can
initiate a series of signal events (cascade reactions) that ultimately
induce stress responsive genes. They may highly correlated with stress
responses under LT after harvest and the changes in plant hormone
contents can also affect fruit quality. BRs can confer resistance of
plants to various abiotic and biotic stresses [[163]50]. Mango fruits
treated with Br had higher cold stress tolerance through the regulation
of lipids [[164]44]. And exogenous BL can markedly decreased CI
incidence of mango fruit. In addition, other plant hormones, including
ABA, GA3, JA and ethylene [[165]51, [166]52], have been also implicated
to modulate abiotic and biotic stresses. MeJA treatment can effectively
inhibit the CI in postharvest loquat fruit [[167]53, [168]54] and
exhibit higher tolerance to cold stress in postharvest peach fruit by
inducing enzyme activities related to energy metabolism and maintaining
high levels of ATP and energy charge [[169]55]. Recent studies revealed
that exogenous application of SA was able to significantly remit
chilling symptoms in harvested loquat fruit [[170]56] and mango fruit.
And treatment with ETH or BR, at an effective concentration, could also
alleviate CI of tomato fruit [[171]57, [172]58]. And the role of ABA in
the stress-resistant process of the plant is also essential. The
content of ABA increases during plant defense responses and this
phytohormone plays multiple roles in plant stress responses to drought
and cold [[173]59]. ABA is also involved in regulation of cold
acclimation. The ABA-insensitive 5 (ABI5) protein was one of the 13
functionally and structurally-related bZIP proteins found to play
crucial roles in mediating changes in stress responses [[174]60]. Thus,
plant hormones have an important role in low temperature tolerance in
postharvest fruit.
In the present study, we found numerous plant-hormone-related genes
were responsive to CI. The numbers and expression patterns of these
genes are shown in (Fig. [175]9); in particular, genes involved in BR,
CK, Aux, and SA pathways were up-regulated while those involved in ET
and methyl jasmonates pathways were not affected by CI. But it may be
not consistent with Ding et al., 2015 [[176]51], they revealed that,
during cold storage, endogenous levels of GA3 and the expression level
of the key GA metabolic genes were lower in chilled fruit compared to
that of fruit stored at room temperature. Perhaps because of the
different species, the trend of hormone content change under low
temperature stress is also different. Additionally, genes involved in
Aux and SA regulation were similarly affected CI, probably because the
expression of receptor genes decreased, but the expression of PR1 and
GH3 increased. Overall, ABA, GA3, BRs, and CK played important roles in
pitting caused by cold storage by regulating the expression of numerous
downstream genes in blueberries. These results indicate that hormone
signaling pathways may play unique functions under cold stress
conditions, and may be forming a complex antioxidant defense system in
blueberry. However, whether and how ABA, GA3, BRs, or CK mediated
signaling participates in cold stress responses in blueberry need to be
further determined.
TFs involved in blueberry pitting responses to cold stress
In many biological processes, defense responses also require the
regulation of specific TFs, which comprise one of the complex
regulatory networks in plants [[177]14]. TFs may play essential roles
in stress responses by regulating their target genes through specific
binding to cis-acting elements in their promoters. Although the TFs
families play diverse roles in plant developmental processes and
environmental responses, most of them have been reported to be linked
to cold stress resistance in plants. For example, six BrbZIP genes have
been identified as putative key factors in cold stress response
[[178]23]. NAC genes are also reported to be induced by cold in
Arabidopsis [[179]61], chrysanthemum [[180]62], rice [[181]63] and
sugarcane [[182]64]. Additionally, the NAC TFs can also interact with
CBF1 to regulate cold tolerance in apples [[183]65] and bananas
[[184]66]. The bHLH family, the second largest family [[185]67], can
also increase cold tolerance in plants by regulating the expression of
ROS clearance-related and stress-responsive genes. Meanwhile, the first
WRKY transcription factor was found in sweet potato under cold stress
[[186]68]; in cucumber, CsWRKY46 confers cold tolerance and positively
regulates ABA-dependent cold signaling pathways [[187]69].
Some less common TFs like C2H2-zinc finger proteins are also essential
in plant stress responses, although their transcriptional regulatory
mechanisms remain largely unclear [[188]70]. Overall, our results
revealed that the 1023 TFs comprised 42 categories of TFs including 92
C2H2, 87 MYB 68, 74 Ap2/erf-erf, 56 bHLH, 53 C2C2, 51 bZIP, 51 C3H, 45
FAR1, 43WRKY, 39 NAC were changed in the pitting process; this was a
little similarity with Die and Rowland (2014) [[189]40]; they found the
seven most highly represented TF families in response to cold stress
were WRKY, ARF, C3H, AP2/ERF, bHLH, C2H2, and NAC. Based on our current
results, the bHLH family was significantly up-regulated in chilled
blueberries, but Ap2, ZIP, and WRKY families were significantly
down-regulated under this condition. In addition, Plant hormones and
transcription factors may also interact during cold stress. For
instance, NAC TFs have been demonstrated to regulate the expression of
ABA-related genes during abiotic stress responses [[190]71]. And
ethylene was shown to negatively regulate cold signaling at least
partially through the direct transcriptional control of CBFs [[191]52].
Since our current research focus on further confirming whether the
C2H2, MYB, and NAC TFs play an important role in the response to LT,
and how they regulate the expression of downstream genes in pitted
blueberry.
Conclusion
Overall, the harvest longterm cold storage leads serious CI in
blueberries, which substantially decreases the quality, storability and
consumer acceptance. Our results identified the occurrence of pitting,
and proved 30 days’ cold storage is the critical point of pitting. We
found higher MDA content, proline content, EL and lower GSH content in
30d-chilled berries. Additionally, a comprehensive transcriptome
profile of blueberry under cold stress were explored by RNA-seq this
time in order to characterize the CI mechanism at the molecular level.
We identified and summarized the genes in response to cold stress as
follows: (1) transcription factors; (2) membrane lipid and energy
metabolism; (3) defensive; and (4) hormone biosynthesis and signal
transduction, including the genes involved in membrane lipids, proline,
glutathione, flavonoids, brassinosteroid, carotenoid, and zeatin
biosynthesis pathway. These DEGs and the biological processes related
to the stress responses, lipid and hormone metabolic processes almost
all accumulated and up-regulated in the CI process. And may play a
crucial role in the response to cold stress in blueberry. These results
provides novel insights into a series of molecular mechanisms
underlying physiological metabolism and defense. Further, the enormous
amount of blueberry transcriptome data generated here will serve as the
foundation in finding available effective way in alleviating CI and
also help to perfect the lack of the genetic information of non-model
plant species. In future work, we aim to focus on the function
verification of special structural genes fadD (c109637.graph_c0)
related to membrane lipid and fatty acid metabolism and 8′-hydroxylas
(c122315.graph_c0) related to plant hormone and explore how they work
in chilling process.
Methods
Plant materials and temperature treatments
‘Duke’ blueberry (Vaccinium corymbosum L.) fruits were harvested at
commercial maturity from an blueberry base in Shenyang (N41°39′55.82″,
E123°05′12.46″), Liaoning Province, China, from April to July in 2016
to 2018. The blueberries were manually picked to prevent any mechanical
damage. After 2 h pre-cooling at 20 °C, blueberry fruits without
physical injury, disease, or rot were enclosed in plastic boxes; then
stored at different storage temperatures (20 °C and 0 °C). Each box
contained 125 g blueberry fruits and the relative humidity was kept at
85%. The fleshy tissues were collected, quickly frozen in liquid
nitrogen, and stored at − 80 °C [[192]72]. The blueberry samples
non-pitted (T01, T02, and T03) were defined as control blueberry and
the pitted blueberry stored at 0 °C for 30 days (T04, T05, and T06)
were defined as 30d-chilled blueberry. They were collected for RNA-Seq
and the RNA-Seq was did by Biomarker Technologies (Beijing, China).
During this period, we ground 20 different blueberry fruits of each
samples (T01–06), and in each samples (T01–06), we also perform three
technical replicates.
Evaluation of pitting rate, decay rate, electrolyte leakage, MDA and proline
Pitting and decay rate were calculated for fruits at 0, 2, 4, 6, and
8 days shelf-life, with or without previous storage at 0 °C, using the
following equations:
[MATH: Pitting rate%=An/A
m×100 :MATH]
1
[MATH: Decay rate%=Ai/Am
×100 :MATH]
2
In these equations, An is the number of blueberry fruits with pitting,
Ai is the number of blueberry fruits cankered, juicy, or mildew, and Am
is the total number of blueberry fruits. Three independent measurements
were performed at each time point for 150 fruits per replicate sample.
The cell membrane permeability determined by electrical conductivity
meter according to the method described in Mao et al. (2007) [[193]73].
MDA content was determined according to the method described in Zhao et
al. (2005) [[194]74]. The proline (Pro) content and reduced glutathione
(GSH) content were determined according to the method described in Zhao
et al. (2009) [[195]75].
Total RNA isolation, cDNA library construction, and sequencing
Total RNAs from all samples were isolated using the OminiPlant RNA kit
(CWBio, Beijing, China). The integrity was monitored on 1% agarose gels
and the purity was checked using the NanoDrop spectrophotometer (Thermo
Fisher Scientific, Inc.) according to Niu et al. (2017) [[196]76].
Sequencing libraries were generated using NEBNext®Ultra™ RNA Library
Prep Kit (Illumina®NEB, USA) and index codes were added to attribute
sequences to each sample. The library quality was assessed on the
Agilent Bioanalyzer 2100 system. Finally, the cDNA libraries were
sequenced on the Illumina® HiSeq 2000 platform (Illumina Inc.) and
paired-end reads were generated at Beijing Biomarker Technology Company
(Beijing, China) [[197]77]. All the downstream analyses were based on
clean data with high quality. And all the sequencing data generated in
this study have been deposited in the NCBI with the link of
[198]http://www.ncbi.nlm.nih.gov/bioproject/578101, under the accession
number SAMN13049031, the Temporary Submission ID SUB643610 and the
BioProject ID PRJNA578101.
Functional annotation and differential expression analysis of genes
Because of the absence of a accurate reference-grade genome for
blueberry, a transcriptome was de novo assembled from this RNAseq data
and other publicly available transcriptome data from blueberry
downloaded from the National Center for Biotechnology Information
(NCBI). And the non-redundant unigenes were annotated based on NCBI
non-redundant (NR); Protein family (Pfam); Swiss-Prot, Clusters of
Orthologous Groups (KOG/COG/eggNOG); Kyoto Encyclopedia of Genes and
Genomes (KEGG) and Gene Ontology (GO) databases according to Liu et al.
(2015) [[199]77].
DESeq R package (1.10.1) was used to identify the DEGs between control
and chilled groups with the standard P-value< 0.05 and |log2 (fold
change)| ≥ 1. The GO enrichment and KEGG analysis of the DEGs was
performed to identify significantly enriched biological pathways of
DEGs, with an FDR adjusted P-value< 0.05.
Validation of the RNA-Seq data and expression analysis by qRT-PCR
Quantitative real-time PCR was performed to validate the expression
patterns obtained from the RNA-Seq data. Gene specific primers were
designed in Primer 5.0 and synthesized by Genewiz Biotechnology
Synthesis Lab (Jiangsu, China) ([200]Table S1 Primers used in qRT-PCR).
The program and reaction conditions for the 2 × ULtraSYBR Mixture (Low
ROX) Kit (ComWin Biotech, Beijing, China) using QuantStudio 6 Flex
(Life Technologies) were as described in the manufacturer protocols.
Template cDNA was diluted five-fold, and 2 μl of the diluted cDNA was
then added to each 20-μl PCR mixture. The qRT-PCR was performed 2-step
method. Each sample was carried out with three independent biological
replicates and three technical replicates. Relative expression levels
were calculated by the comparative 2^−ΔΔCT method using the expression
of actins as the internal control.
Statistical analyses
One-way analysis of variance (ANOVA) with the least significant
difference (LSD) was performed for all data using SPSS 20.0 software
(BM Corp, Armonk, NY, USA). Each experiment was repeated three times
independently and the represent significance was considered to
P < 0.05. Origin 8.1 ([201]https://www.originlab.com/) and R3.5.1
([202]https://www.r-project.org/) were used to display the data
obtained from the experiments.
Supplementary information
[203]12870_2020_2281_MOESM1_ESM.docx^ (95.9KB, docx)
Additional file 1: Table S1. Primers used in qRT-PCR. Figure S1.
Graphical Abstract.
Acknowledgements