2.1 Changes in phenotype and physiology at low temperature
2.1.1 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. 1a), whereas the fruits stored at 0°C for 30 days showed typical CI symptoms as pedicle pitting; these symptoms were maintained during the shelf-life.
2.1.2 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. 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. 2).
2.1.3. 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.3a), there was an upward trend in the MDA content, and the MDA content of blueberry fruit stored at °C for 30 days (9.2mmol kg-1) was higher than that of fruit stored at 20°C (6.5mmol 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.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 °C for 30 days (42%) was higher than that of blueberry stored at 2°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. 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 (2.25g kg-1) was higher than that of fruit stored at 20°C (0.6g kg-1) (Fig. 3d), and the proline content increased significantly after the fruits were removed from cold storage and reached 3.95g kg-1 at 8d shelf-life. The results indicated that the cell membrane structure and membrane function were seriously damaged during cold storage.
2.2. Sequence assessment
The reproducibility of the RNA-Seq data was assessed for blueberry fruit samples (T01-T03 were defined as 0d samples and T04-T06 were defined as 30d-chilled samples). The results were highly consistent among the three biological replicates with 0.9462< R2< 0.9998 (Fig.4), which indicated that the reproducibility of RNA-Seq data was reliable. 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.
2.3. Transcriptome sequencing and read assembly
The total 50.74 Gb of clean data were generated (Table1). 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.
Table1 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%
|
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.
2.4. Comprehensive profiling of transcript expression analysis
2.4.1. Number of DEGs
The analysis of DEGs revealed 561 up-regulated and 158 down-regulated genes for FDR≤ 0.01, and 1,167 up-regulated and 685 down-regulated genes for FDR0.05. To assess the diversity of DEGs, the Venn map was constructed (Fig.5).
2.4.2. 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.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.6).
Meantime, the 10 DEGs most significantly changed among treatment groups were selected to assess the important genes and pathways involved in CI (Table2). 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.
Table2 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
|
These genes were selected with an FDR adjusted P-value< 0.05
2.5. Functional annotations and classifications
In our research, the N50 was 1,348bp, 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: 1,142 (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 1,099 (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.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.
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.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 (Table3)
Table3 The top 6 enriched pathways of DEGs in 30d-chilled blueberry
These were selected with an FDR adjusted P-value< 0.05
In addition, 394 DEGs (25classes) were annotated in the COG database (Fig.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.
2.6. Identification and analysis of the DEGs under cold stress
2.6.1. 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. 8a).
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.
Table4 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
|
These genes were selected with an FDR adjusted P-value< 0.05
2.6.2. 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.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 LAR (c119207.graphc1) was up-regulated in chilled blueberry. (Fig. 8b).
Table5 Genes involved in proline, glutathione, and flavonoid in response to low temperature
These genes were selected with an FDR adjusted P-value< 0.05
2.6.3 Cold-response genes involved in hormone biosynthesis and signal transduction
2.6.3.1 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. 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. 8c).
2.6.3.2 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.
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 [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.
Table6 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
|
These genes were selected with an FDR adjusted P-value< 0.05
2.7. 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 1,023 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 1,023 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.10).
2.8. 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. (Fig.11&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 positively correlated (Pearson’s r = 0.86244), 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 dataset reported in this study constitutes a valuable supplement to the available blueberry genomic and transcriptome information.