Gene expression profiling during peach ripening and novel
transcripts annotation
To reveal the
molecular
responses to HT and CT in post-harvest peach fruits, samples with two
biological replicates from 6 time points were processed to construct 24
RNA libraries. A total of 684 M reads were generated with an average
length of 100 bp, ranging from 10.18 to 21.57 M raw reads per library
(Supplementary Table S2 ). The reads data were pre-processed by
trimming adapters and filtering out short reads (< 36 bp) and
low-quality reads (Q < 30). After pre-processing, 562 Mb
(82.22%) high-quality sequences were retained. The remaining paired
reads were mapped to the Prunus persica reference genome, and the
total mapped rate was approximately 92.26% in average
(Supplementary
Table S3 ).
Relative abundance of genes was calculated and normalized as FPKM, and
the robustness and reproducibility of data were indicated by high
Pearson correlation coefficients (> 0.97) among biological
replicates for all comparison pairs
(Supplementary
Table S4 ). Besides, we identified 1,281 novel transcripts which were
aligned to the reference genome but not included in the 26,873
protein-coding genes
(Supplementary Data ). The
lengths of those transcripts ranged from 46 to 215,643 bp, with an
average length of 3,045 bp. The biological functions of novel
transcripts were annotated with Gene Ontology (GO) terms by successively
using BLASTx (against the NCBI database) and Blast2GO; they were also
subjected to KAAS to obtain the KEGG Orthology (KO) identifiers.
Finally, 353 transcripts were classified into 3 GO categories and 176
transcripts have KO annotations
(Supplementary
Figure S1 & Table S5 ).
Analysis
of DEGs
To identify DEGs between HT and
CT conditions, the samples of each time point (day) were compared and
the DEGs were detected. Firstly, we counted the expressed genes in each
sample and found that peach fruits at CT underwent a more substantial
decrease in the number of expressed genes
compared
with HT (Figure 2a ). A total of 7,245 DEGs (P< 0.05 and | Log2 ratio | ≥
1) were identified in all comparison pairs between HT and CT. The
numbers of up- and down-regulated DEGs were shown inFigure
2b . Interestingly, there is a strong decline in the numbers of both
up-regulated and down-regulated genes at ripening stage. The number of
up-regulated DEGs reached summit on day 2 with 2,778 genes, showing the
most significant response to
HT
in transcriptome.
Several groups of transcription factors (TFs) were identified involved
in plant responses to diverse abiotic stresses including temperature and
drought. Here, 301 TFs from all DEGs were identified belonging to 49 TF
families. Among all the TF families, ethylene response factors (ERFs)
showed the highest numbers (44) compared with others (Figure
2c ). These data suggest the important role of ERFs in high-temperature
response.
GO analysis was performed to dissect the functions of DEGs. As a result,
2,665 up-regulated DEGs and 1,990 down-regulated DEGs were classified
into 3 main GO categories: cellular component (CC), biological process
(BP) and molecular function (MF). In up-regulated DEGs, the most
abundant BP subcategories were ‘nucleobase-containing compound metabolic
process’, ‘gene expression’ and ‘nucleic acid metabolic process’
(Figure
3a ). In down-regulated DEGs, the major BP subcategories were ‘organic
substance biosynthetic process’, ‘cellular macromolecule biosynthetic
process’ and ‘cellular nitrogen compound biosynthetic process’
(Figure
3b ). Besides, only down-regulated DEGs had enriched groups in CC and MF
which are ‘cytoplasm’, ‘structural constituent of ribosome’
(Figure 3b ), etc.
Mitogen–activated
protein kinase (MAPK) cascades response to HT stress
HT
induces changes of cytoplasmic calcium signaling, which exerts
regulatory effects on plant development and stress responses
(Berridge et al., 2003;
Perochon et al., 2011). Among the 7 genes
of calmodulin (CALM),
Prupe.3G266000
and Prupe.4G269100 showed higher transcript abundance in CT fruits,
while Prupe.1G089100 was on the
contrary
(Figure
4a ).
The MAPK cascades are one of the major signaling pathways and highly
conserved in evolution for the transformation of
extracellular stimuli into
intracellular
responses in eukaryotic cells (Hirt,
1997; Tena et al., 2001;
Zhang and Klessig, 2001;
Nakagami et al., 2005). The MAPK cascades
consist of three interconnected protein kinase modules:
MAPK
kinase kinase (MEKK), MAPK kinase (MKK), MAPK. In MAPK gene families, we
found that MEKK1-MKK2-MPK4 and MPK6 had high
expression levels responding to HT
(Figure 4b ), which were also detected in A. thalianaresponding to salt, drought and cold
(Teige et al., 2004). Therefore,
MEKK1-MKK2-MPK4/6
module is the most likely signaling pathway activated during HT stress.