Introduction
Anthocyanins are a group of water-soluble
flavonoid compounds and widely distributed in various organs of plants, which makes plants blue, purple or red color to perform different biological functions, such as
attracting pollinators, seed dispersal, enhancing stress resistance, avoiding UV light damage
and regulation of auxin transport (Zhang et al. 2014; Lloyd et al.
2017). Additionally, anthocyanins have
antioxidant properties, help scavenging free radicals, anti-aging,
anti-cancer and boost immunity in human body (Holton
and Cornish 1995). Therefore, the studies on the mechanism of anthocyanin accumulation are triggering strong
interests in scientists. The basic
chemical structure of anthocyanins is phenyl-2-benzopyran, which is composed of
A and B rings. Generally, there were substituted hydroxyl groups at 3-, 5-, 7-carbon
sites of A ring, and the different
substituents in the B ring form different kinds of anthocyanins (Fang and Ni
2001). The structure of anthocyanins monomer is not stable and usually coupled
with monosaccharide or
polysaccharide to form stable glycosides (Tsuda
2012). More than 20 anthocyanins have been found, mainly
derived from six anthocyanins: delphinidin (Dp), petunidin (Pt), cyanidin (Cy), pelargonidin (Pg), peonidin (Pn) and malvidin (Mv) (Kong et al. 2003). The type and content of anthocyanins determine
the color of flower and fruits.
In recent years, a well-studied pathway of anthocyanin
synthesis is constructed with a series of enzymes, including anthocyanidin
synthase (ANS), chalcone synthase (CHS), flavanone 3-hydroxylase
(F3H), chalcone isomerase (CHI), dihydroflavonol 4-reductase (DFR), UDP-glucosyltransferase (UFGT),
flavanone 3`-hydroxylase (F3`H), flavanone 3`5`-hydroxylase (F3`5`H), flavonol
synthase (FLS), dihydroflavonol-4-reductase (DFR), leucoanthocyanidin
reductase (LAR) and anthocyanidinreductase
(ANR) (Zhang et al. 2014), which
contribute to coloration in plant parts. MYB-bHLH-WD repeat (MBW) MBW ternary complex, which comprises one R2R3-MYB protein, one bHLH (helix-loop-helix)
protein and one WD-40 protein, regulated the genes coding for anthocyanin
biosynthesis enzyme from transcription level in some plant species (Xu et al. 2014a). R2R3-MYB is considered as one of the
key regulators of anthocyanins biosynthesis. The N-terminal of bHLH has a highly
conserved domain, which can bind to MYB to form dimerization, and then to
initiate or regulate the expression of genes responsible for anthocyanin
biosynthesis (Zimmermann et al. 2004). WD40 can promote the interaction between bHLH transcription factors and MYB
transcription factors, and enhance the stability of MBW protein complex during
anthocyanin synthesis. The MBW complex activates the transcription of late biosynthetic genes (DFR,
ANR, UFGT, LAR and ANR) involved anthocyanin
pathway (Liu et al. 2018). ANT1 (MYB) (Mathews 2003), MdMYBl0 (Espley et al. 2007), PpMYB9, PpMYB10 (Zhou et al. 2016), VvMYB5a (Deluc et al. 2006), FaMYB10 and FvMYB10 (Lin et al. 2010) have been identified and proved to be
involved in the synthesis of anthocyanins. It has been reported that the production of
pigment in maize (Zea mays) needs the existence of bHLH (Ludwig et al. 1989). In strawberry, FvbHLH
promoted anthocyanins accumulation under light by co-regulated with FvHY5 (Li
et al. 2020). In apple, the induction of anthocyanins by MdMYB10 mainly depends on the co-expression of MdbHLH3 and MdbHLH33 (Espley et al. 2007), and FvMYB10 and FvbHLH33
may form a complex to promote transcription of structural genes in strawberry (Lin-Wang
et al. 2014). More
recently, it was reported that the WRKY transcription factors (TTG2/PH3)
cooperated with the MBW complex to regulate anthocyanin accumulation (Gonzalez
et al. 2016; Verweij et al. 2016). MdWRKY40
was essential for wound-induced anthocyanin accumulation in association with MdMYB1.
Except genetic factors, environmental elements, sugar and phytohormones can
affect anthocyanin biosynthesis in plant. As an important plant hormone, abscisic acid (ABA) has been
shown to regulate plant development and growth, and stress-response signaling (Hirayama and Shinozaki 2007). Recently, it has been shown that ABA
regulates the ripening of nonclimateric fruit as a key factor (Coombe 1992; Davies et al. 1997; Giovannoni 2001) and enhanced anthocyanin synthesis in grape and strawberry (Jia et al. 2013). ABA and sugar undergo extensive crosstalk (Finkelstein
and Gibson 2002; Carrari et al. 2004). It has been reported that sucrose
is involved in ABA signaling pathway and co-regulated fruit growth and ripening
related genes expression in grapes (Cakir et
al. 2003; Jia et al. 2017). In fleshy fruits, sugar accumulation and
metabolism were affected by ABA (Kobashi et al. 1999; Pan et al. 2005). Meanwhile, sucrose can also affect the content of
ABA in strawberry fruit (Luo et al.
2019). Recently, it has been reported that sucrose can regulate strawberry fruit ripening and anthocyanin accumulation by ABA-independent and ABA-dependent pathways (Jia et al. 2013). The complexity of the
multiple and synergistic signals may meet
the need for the orderly development and ripening of fruits. Except ABA, auxin
also plays an important role in regulation of anthocyanin accumulation in
plants (Tsukasa et al. 1994; Daminato et al. 2013).
Color is an important index to
evaluate the maturity and quality of fruit, which is not only regulated by
genetic factors, but also by external factors, such as light, water,
temperature, ABA and sucrose (Azuma et
al. 2011; Jaakola 2013). Changes in environmental factors cause poor fruit
color, which affects fruit quality and reduce commodity
value. Therefore, it is a convenient and quick approach to take plant
growth regulators and sucrose to manipulate the color of fruits.
Strawberry (Fragaria ×
ananassa) is a model plant for studying non-climacteric fruit (Li et al. 2011; Cherian
et al. 2014). It has been proven that
ABA and sucrose participate in
strawberry fruits ripening
and coloring (Chai et al. 2011), and ABA + sucrose treatment had the greatest effect in our
previous study (Luo et al. 2019; Ling
et al. 2019). However, the molecular mechanism of ABA, sucrose
and ABA/sucrose interaction in anthocyanin accumulation in strawberry fruit is still unclear. As a
typical non-climacteric fruit, strawberry is favored by consumers for its unique flavor,
rich nutrients and bright color. Among the fruit
quality attributes, bright color may affect consumers' purchasing psychology and decision. Interestingly, our
previous results showed that exogenous ABA and sucrose could accelerate the
synthesis of anthocyanins during strawberry fruit development and ripening,
while not changing the final anthocyanin concentration of full-red strawberry
(Ling et al. 2019). Therefore, there
is a need for further research on how ABA and sucrose affect
anthocyanin accumulation in strawberry fruit at molecular level. Based on our
previous study, 95 μM
ABA, 100 mM sucrose and 95 μM ABA + 100 mM sucrose were
selected to spay strawberry fruit at the de-greening stage. Subsequently, the
treated strawberry fruits were used for transcriptome sequencing and analysis and the regulatory and structural genes involved in
anthocyanin metabolism were assessed. The objectives of this present
study is not only to understand the functions of ABA and sucrose in anthocyanin
biosynthesis of strawberry, but also to divulge promising genes candidate for
manipulating strawberry anthocyanin biosynthesis, which might have a potential suggestion for improving commodity
value and quality of strawberry.
Materials and Methods
Plant materials and treatments
The strawberry (Fragaria
× ananassa cv. Benihoppe) plants were
grown in a plastic greenhouse in a farm under natural culture conditions in
Chengdu, China. A total of about 500 strawberry plants were selected, and 2000
secondary flowers were tagged. Subsequently, fruits at the de-greening stage
(18 days after flowering, DG) were treated with water, 95 μM ABA, 100 mM
sucrose, or 95 μM ABA+100 mM
Sucrose, respectively. Based on the coloring difference in field, the fruits of
0 and 8 days after treatment were picked, frozen in liquid nitrogen and then
stored at -80℃ for RNA extraction.
RNA extraction and sequencing
Total RNA was extracted from the fruit at 0 and 8 days after treatment by using a
RNeasy Plant Mini Kit (Qiagen, Dusseldorf, Germany), and DNA was removed with
RNase-Free DNase (Qiagen). Final extracted RNA samples concentration and
integrity were determined by Nanodrop 1000 spectrophotometer and Agilent 2100
Bioanalyzer. The samples were named CK0, CK8, ABA8, Scu8 and AS8, respectively.
Sequencing of five cDNA libraries (CK0,
CK8, ABA8, Scu8 and AS8) was performed using
the Illumina HiSeq 2000 platform (Novogene
Biotechnology Company, Beijing, China). All
analyses were conducted using replicates.
Transcripts assembly and expression quantification
Considering the incompleteness of
the genome of cultivated strawberry (Fragaria × ananassa), all clean reads were de novo assembled by
Trinity software version 2.4.0 (Grabherr et al. 2011). To obtain protein-coding transcripts, the assembled non-redundant transcripts were compared
with protein databases including Swiss-Prot (http://www.uniprot.org) and NCBI non-redundant (NR) by using Diamond BLASTx (Buchfink
et al. 2015). RNA-Seq gene expression levels were quantified and normalized
by calculating the fragments per
kilobase million (FPKM) values using RSEM (Li and Dewey 2011). Pearson correlation coefficient was
analyzed by Minitab 15, which could be denoted by rx,y and calculated
as follows:
%201487-1496_files/image002.png)
Where n was the number of samples from different
experiments and Xi and Yi were expression
profile values of probe sets X and Y in the ith sample,
respectively. DESeq2 R package (Love
et al. 2014) was applied for determining
differential expression analysis.
Significantly differentially expressed genes (DEGs) were defined as genes
with the absolute value of False
Discovery Rate (FDR) at < 0.05 and log2
fold change (log2 FC) ≥1.
Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment
analysis
DEGs were subjected to GO and KEGG enrichment analysis implemented by Topgo R software
package (Alexa et al. 2006) and KOBAS 3.0
software (Xie et al. 2011), respectively. The application of R ggplot
function visualized the data of enrichment analysis of DEGs. Values for heat maps were created using each of the normalized sample FPKM expression values. Subsequently, TBtools software was used to draw heat
map.
Results
Summary of sequencing data quality and gene
mapping
In order to fully understand the
transcriptional responses of strawberry fruit to ABA and sucrose, the fruit
of 0 and 8 days after treatments were selected as time point of RNA-seq, and
named as CK0, CK8, ABA8, Suc8 and AS8. The original data were submitted to
Sequence Read Archive (SRA) database in NCBI website (Accession ID:
PRJNA565646; Link: https://www.ncbi.nlm.nih.gov/sra/PRJNA565646). Five samples were sequenced to obtain over 41
million high-quality clean reads, which accounted for 87.48, 96.39,
96.50, 96.4 and 96.58% of the raw reads, respectively (Table 1). In addition, 65.03, 69.90, 70.90, 71.63 and
71.65% of the five samples (CK0, CK8, ABA8, Suc8 and AS8) sequences were mapped to the strawberry (Fragaria vesca) reference genome. The mean GC content in the transcripts was about 47%.
More than 96.82 and 92.21% of the reads had a quality score over Q20 and Q30, respectively. These results indicated that the assembly quality of high throughput RNA
sequencing was good and the
mismatch rate was low.
DEGs and
KEGG enrichment
In order to ensure the accuracy and
reliability of the expression data, Pearson correlation coefficient was applied
to calculate gene expression
correlation. According to the
data, all correlation coefficients (R2 values) between biological
replicates were larger than those outside biological replicates (Fig. S1). DEGs were
analyzed by using Deseq R package (v.1.10.1) based on the negative binomial
distribution (Padj < 0.05). The results were shown through the Venn diagram (Fig. 1). There were 346, 623 and 3676 DEGs specific to the ABA, sucrose and ABA +
Sucrose treatments, respectively, whereas 1003 DEGs were common to the three
different comparisons, suggesting that the gene expression patterns showed
different response to ABA, sucrose, and ABA + Sucrose treatments. Subsequently,
we subjected the five samples to hierarchical clustering analysis. The results
showed that samples of Suc8 and
AS8 clustered together, and ABA8
and CK8 clustered together (Fig. 2B), indicating these genes after sucrose or ABA +
sucrose treatment had similar expression profiles, and ABA8 had similar
expression patterns to CK8. The potential
effects of ABA, sucrose and ABA +
Sucrose on the altered biological pathways needed to be investigated. KEGG (http://www.genome.jp/kegg/) enrichment analysis of
DEGs was carried out (Fig. 2). A total of 20028 DEGs were mapped to the KEGG
database, and the top 20 enriched pathways in differenct
treatments were revealed in the present study. In CK8 vs ABA8 (Fig. 2A), “Linoleic acid metabolism”, “alpha-Linolenic
acid metabolism” and “Flavonoid biosynthesis” were the most enriched KEGG pathway terms, which included 11 DEGs, 19 DEGs and 11 DEGs,
respectively. Meanwhile, the “flavonoid biosynthesis”, “pantothenate
and CoA biosynthesis”, and “glutathione metabolism” were enriched in CK8 vs
Suc8 (Fig. 2B). In CK8 vs AS8 (Fig. 2C), the most
enriched pathway terms were “photosynthesis - antenna proteins”, “oxidative
phosphorylation”, and pyruvate metabolism” (Fig. 2C). Anthocyanins were the
main pigment in strawberry fruit coloration. Therefore, we focused on the flavonoid biosynthesis pathway and anthocyanin biosynthesis
pathway.
Table 1: Summary statistics of the sequencing data
|
Sample |
Raw Reads |
Clean Reads |
Clean Reads/Raw Reads (%) |
Clean Bases |
Error Rate (%) |
Q20 (%) |
Q30 (%) |
GC (%） |
Total mapped (%) |
|
CK0 |
47958757 |
41954921 |
87.48 |
6.31G |
0.02 |
96.82 |
92.21 |
47.92 |
65.03 |
|
CK8 |
48498646 |
46746930 |
96.39 |
7.01G |
0.01 |
98.10 |
95.20 |
47.06 |
69.90 |
|
ABA8 |
48357580 |
46663692 |
96.50 |
7.00G |
0.01 |
98.12 |
95.23 |
46.94 |
70.90 |
|
Suc8 |
50404051 |
48601499 |
96.42 |
7.29G |
0.01 |
96.99 |
92.57 |
46.81 |
71.63 |
|
AS8 |
48325528 |
46675098 |
96.58 |
7.00G |
0.01 |
98.10 |
95.11 |
46.82 |
71.65 |
Q20, Q30: The percentage of bases
with a Phred value of >20 or 30, Phred = -10log10(e); CK0, CK8; ABA8, Suc8, AS8
represent three biological
replicates for control, ABA, sucrose and
ABA + Sucrose treatment, respectively
%201487-1496_files/image004.png)
Fig. 1: Differentially expressed genes
expression in response to ABA, sucrose, and ABA + Sucrose treatment. (A)Venn
diagram showing DEGs distributions; and (B) expression profile clustering. Red
and blue colors mean up-expression and down-regulated expression of genes,
respectively. CK0, CK8, ABA8, Suc8 and AS8 indicate the 0 day and the 8th
samples treated with water, ABA, sucrose and ABA + Sucrose, respectively
Transcription factors
It has been reported that MYB, bHLH, WD40 and WRKY are critical transcription factors for anthocyanin biosynthesis. In this study, 12 MYB, 3 bHLH and 12 WRKY were identified (Fig. 3). Compared with CK8, ABA treatment significantly
changed the expression level of MYB and WRKY. The expression level of LHY-like
(Cluster-12337.69059), MYB44-like (Cluster-12337.54712), WRKY40
(cluster-12337.60739), WRKY24 (cluster-12337.25453), WRKY11
(cluster-12337.97104) and WRKY46 (cluster-12337.104131) were
significantly down-regulated by 0.99, 1.11, 4.39, 2.62, 2.14 and 2.95 times,
respectively. Meanwhile, the expression level of MYB114-like (Cluster-12337.67445)
and WRKY71 (Cluster-12337.81062) were up-regulated by 2.02 time and 0.70
time, respectively (Fig. 3A, B).
The number and expression
characteristic of MYB identified in Suc8 was similar to that in ABA8.
However, there was a greater change in gene expression level in Suc8 compared
with ABA8. Compared with CK8, the expression level of WRKY40, WRKY11,
WRKY46 and WRKY33 were significantly down-regulated by 3.25,
1.86, 2.37 and 2.18 times, and that of WRKY31 (Cluster-12337.98154) and WRKY71
were significantly up-regulated by 1.55 times and 2.26 times, respectively. Furthermore, there was no significant
difference in the expression of bHLH after ABA or sucrose treatment
(Fig. 3C).
ABA, sucrose or ABA + sucrose
treatments had similar effect on the expression level of MYB, but MYB1R1
(Cluster-12337.57122), bHLH48 (Cluster-12337.58812), bHLH51 (Cluster-12337.38826)
and bHLH93 (Cluster-12337.103164) were identified in AS8, the expression
level of which was significantly down-regulated compared with CK8. Furthermore,
ABA + sucrose treatment significantly reduced the expression level of WRKY2 (Cluster-12337.112247),
WRKY53 (Cluster-12337.102451) and WRKY24 (Cluster-12337.25453),
and increased the expression of WRKY31 and WRKY71, which was
higher in AS8 treatment compared with that of Suc8 treatment. It is interesting
to note, LHY-like and MYB44-like in significantly down-regulated
genes, and MYB114-like and WKRY71 in significantly up-regulated
genes were quite common in ABA8, Suc8 and AS8 treatments (Fig. 3).
Structural genes involved in the anthocyanin pathway
Based on RNA-Seq data, we identified
36 anthocyanin biosynthesis structural genes related to PAL, C4H, CHS, F3H, F3`H, F3`5`H, DFR, FLS, UFGT and ANR genes, respectively. Most of PAL, C4H, CHS, F3H, DFR, ANR and UFGT were up-regulated with the development of strawberry
fruit (Fig. 4). Compared with CK8,
most of ‘early’ flavonoid
biosynthetic step genes, such as CHS,
CHI and F3H, with enhanced expression
levels were up-regulated in ABA8, Suc8 and AS8 treatments: especially in
Suc8 and AS8 treatments. Meanwhile, of the
‘late’ flavonoid biosynthetic steps genes, two members of eight DFR (Cluster-12337.70165 and
cluster-12337.64510), one member of three UFGT (Cluster-12337.64199)
and all members of ANS were significantly up-regulated in ABA8,
Suc8 and AS8 treatments, especially in AS8 treatment. Meanwhile, two members of
eight DFR (Cluster-12337.97275
and Cluster-12337.61538), one member of three UFGT (Cluster-12337.80836)
and one member of three ANR (Cluster-12337.70357) were significantly
up-regulated in Suc8 and AS8 treatments, contrasting with their having
lower expression level in CK8. Additionally, there was one member of eight DFR
(Cluster-12337.34755) only in Suc8
treatment and one member of three ANR
(Cluster-12337.71858) only in AS8 treatments were significantly up-regulated compared with
CK8. Moreover, one member of three UFGT (Cluster-12337.61103) was significantly inhibited in ABA8 and Suc8 treatments (Fig.
4). Therefore, strong signals for transcripts of DFR, ANS, ANR and UFGT were clearly seen in sucrose and
ABA + sucrose treatment, which was consistent with the higher level of
anthocyanins in strawberry fruit under these treatments in our previous study
(Ling et al. 2018).
Expression pattern of glutathione S-transferase
Glutathione S-transferase (GST) played an important role
in plant primary and secondary metabolism, hormone response, oxidative stress
and herbicide detoxification. A total of 54 GSTs were screened in this
study, and the analysis of differential gene expression showed that 6 GSTs
(Cluster-12337.70402, Cluster-12337.74163, Cluster-12337.67740,
Cluster-12337.80939, Cluster-12337.64226 and Cluster-12337.63035) were
significantly up-regulated by ABA8, Suc8 and AS8 treatments compared with CK8,
especially in Suc8 (Fig. 5), suggesting that GST
proteins might be involved in ABA and sucrose induced anthocyanin
accumulation.
%201487-1496_files/image006.png)
Fig. 2: Top 20 enriched KEGG pathways
of DEGs in three pairwise comparisons. (A) CK8 vs ABA8; (B)
CK8 vs Suc8; (C) CK8 vs
AS8. The vertical axis on the left represents KEGG pathways,
the horizontal axis indicates the Rich factor. Low qvalue
are shown in red, and high qvalue are depicted in
purple. Qvalue < 0.05 are significantly enriched.
The size of the spot reflects the number of DEGs, and the color of the spot
corresponds to different qvalue ranges. CK8, ABA8,
Suc8 and AS8 indicate the 8th samples treated by water, ABA, sucrose
and ABA + sucrose, respectively
%201487-1496_files/image008.png)
Fig. 3: Expression profile clustering
of transcription factors related to anthocyanin biosynthesis. (A)
MYB; (B) WRKY; (C) bHLH,
basic helix-loop-helix. The color
scale at the right represents the re-processed log10 (FPKM+1) using Pheatmap, representing the relative expression level. The
expression variance for each gene is indicated by colors ranging from low
(blue) to high (red). CK0, CK8, ABA8, Suc8 and AS8
indicates samples of 0 and 8 days after water, ABA, sucrose and ABA + Sucrose treatments, respectively
%201487-1496_files/image010.png)
Fig. 4: Effect of ABA, sucrose and ABA
+ Suc on the expression of genes encoding flavonoid
and anthocyanins biosynthetic enzymes in strawberry. The expression pattern of each structural gene in CK0, CK8, ABA8, Suc8 and AS8 isarranged from left to right., CK0, CK8, ABA8, Suc8 and AS8
indicates the samples of 0 and
8 days after water, ABA, sucrose and ABA + Sucrose treatments, respectively. The color ratio
represents log10 (FPKM + 1) reprocessed with Pheatmap,
representing the relative expression level. The expression of each gene varies
from low (blue) to high (red) color. PAL, phenylalanine
ammonia-lyase; 4CL, 4-coumarate-CoA ligase; C4H, cinnamate 4-hydroxylase; CHS, chalcone synthase; F3`H, flavanone 3`-hydroxylase; F3`5`H, flavanone 3`5`-hydroxylase FLS, flavonol synthase; DFR, dihydroflavonol-4-reductase;
ANR, anthocyanidinreductase;
LAR, leucoanthocyanidinreductase; UFGT, flavonoid
3-O-glucosyltransferase
%201487-1496_files/image012.png)
Fig. 5: Effect
of ABA, sucrose and ABA + Suc on the expression of
genes encoding glutathione S-transferase (GST). The color scale at the right represents the re-processed log10
(FPKM+1) using Pheatmap, representing the relative
expression level. The expression variance for each gene is indicated by colors
ranging from low (blue) to high (red). CK0, CK8, ABA8, Suc8 and AS8 indicates samples of 0 and 8 days after water, ABA, sucrose and ABA + Sucrose treatments, respectively
%201487-1496_files/image014.png)
Fig. 6: Effect of ABA, sucrose and ABA + Suc on the expression of genes related to auxin singaling pathway. (A) auxin response factor (ARF); (B) Auxin responsive protein (Aux/IAA). The color scale at the
right represents the re-processed log10 (FPKM+1) using Pheatmap,
representing the relative expression level. The expression variance for each
gene is indicated by colors ranging from low (blue) to high (red). CK0, CK8,
ABA8, Suc8 and AS8 indicates samples of 0 and 8 days after water, ABA, sucrose
and ABA + Sucrose treatments, respectively
Auxin signaling
transduction related to anthocyanin biosynthesis
Auxin response factor (ARF) and auxin responsive
protein (Aux/IAA) are related to auxin
signaling transduction, which were involved in
anthocyanin accumulation as negative regulators. In this study, 61 ARFs and 31 Aux/IAAs were screened in the result. Among them, the expression level of ARF5 (Cluster-12337.73750), Aux/IAA4
(Cluster-12337.76254) and Aux/IAA16 (Cluster-12337.73689) was significantly
down-regulated in ABA8, Suc8 and AS8 treatments, especially in AS8 treatment
(Fig. 6). Other AFRs and Aux/IAAs showed no significant changes in
the three treatments. These results suggested that AFR5, Aux/IAA4 and Aux/IAA16 might play roles
in anthocyanin accumulation.
Discussion
In plants,
many studies have demonstrated that anthocyanin biosynthesis is coordinately
regulated by intricate regulatory networks of developmental signals and
environmental factors (Zimmermann et al.
2004; Hichri et al. 2011). However,
whether ABA and sucrose promotes anthocyanin biosynthesis remains elusive and
exactly how they stimulation promotes anthocyanin biosynthesis remains unclear.
In this study, RNA-seq analysis enabled comparative analysis of differential transcriptional
genes between different treatments. Approximately 79% of DEGs in AS8 were not
found in the DEGs of ABA8 or Suc8 (Fig. 1A), suggesting the synergistic interaction may happen between ABA
and sucrose. Meanwhile, the DEGs of ABA, sucrose
and ABA + sucrose treatment were all enriched in flavonoid
biosynthesis pathways (Fig. 2), which is related
to anthocyanin
biosynthesis, suggesting that ABA and sucrose treatment affect anthocyanin biosynthesis of strawberry fruit
at the molecular level.
It
is well known that MBW complex regulates the flavonoid pigment biosynthetic
genes in many plants whereby its diversity of combination and interaction with
related genes (Xu et al. 2015) and is
modulated by light, sugar and hormones (Das et
al. 2012). The MYB transcription factors have been shown to interact
closely with bHLH and WD40 to regulate anthocyanin synthesis (de Vetten et al. 1997; Nesi et al. 2001; Zimmermann et
al. 2004; Hichri et al. 2011).
However, the role of bHLH and WD40 on anthocyanin biosynthesis and accumulation
in strawberry fruit under ABA regulation is unknown. Meanwhile, it has been
reported that ABA and sucrose mainly affects anthocyanin level via regulation
of the transcription levels of AtWRKY40 and AtWRKY60 (Liu et al. 2012) as well as AtMYB44 (Li
et al. 2014) in Arabidopsis.
In our study, most of the MYB, bHLH and WRKY were down-regulated after ABA, sucrose and ABA
+ Sucrose treatments, suggesting that these genes may act as negative
regulatory factors for the regulation of anthocyanin synthesis. Zhang et
al. (2018) reported that blue light promotes anthocyanin accumulation of
strawberry fruit, while most of MYB, bHLH and WRKY were inhibited at
the level of transcription. In
addition, MYB114-like genes, WRKY71 and
WRKY31 genes were identified in present study, which transcript level were up-regulated in ABA8, Suc8 and AS8
treatments, especially in AS8 treatment. MYB114 has been
reported to be associated with anthocyanin biosynthesis in Arabidopsis and pear (Gonzalez et al.
2008; Yao et al. 2017). The expression level of TaWRKY71 was 3-folds higher in ABA treated-wheat than that of the control (Xu et al. 2014b). Therefore,
our results indicated that MYB114-like, WRKY71 and WRKY31 were
responsive to ABA and/or sucrose, and then further involved in activating anthocyanin biosynthesis.
Based on the structural genes of anthocyanin biosynthesis regulated by MBW
complexes, the ‘late’ flavonoid biosynthetic steps depend on these complexes
and ‘early’ steps that are not (Martin et
al. 1991; Shirley et al. 1995;
Pelletier and Shirley 1996; Pelletier et
al. 1997; Zhang et al. 2003). DFR, ANS and UFGT genes play critical roles in anthocyanin biosynthesis (Zhao
et al. 2012; Li et al. 2013; Gao et al. 2019). In
our study, the transcripts level of DFR (Cluster-12337.70165),
UFGT (Cluster-12337.64199) and ANS
(Cluster-12337.69226 and cluster-12337.70413) were significantly
up-regulated in ABA8, Suc8 and AS8 treatments,
especially AS8 treatment. The transcripts level of UFGT
(Cluster-12337.80836) was significantly up-regulated in Suc8 and AS8
treatments, and the transcripts level of UFGT (Cluster-12337.61103) was
down-regulated in ABA8 and Suc8 treatments. Ban et al. (2003) reported that ABA
treatment increased the anthocyanin content in grape skin with
the upregulation of DFR and UFGT genes. In red-leaf peach, 0.3%
sucrose could increase the expression level of UFGT (Wen et al. 2016). ABA/or sucrose treatments
enhanced anthocyanin accumulation in increasing expression level of VvUFGT
in grapes (Olivares et al. 2017). Our
results suggested that these genes might be important structural genes involved
in rapid accumulation of anthocyanins induced by ABA and sucrose in strawberry
fruit. Glutathione S-transferase (GST) was commonly
considered to be the detoxification of endogenous and xeno-biotic compounds in
plants (Marrs 1996) and its role in response to biotic and abiotic stress has
been clarified in Arabidopsis (Wagner et al. 2002). Overexpression of ThGSTZ1 enhanced the salt
and drought tolerance in Tamarix hispida (Gao et al. 2016). Meanwhile, Shi et
al. (2014) and Kitamura et al.
(2012) reported that GST protein was a positive regulator in the
accumulation (Kitamura et al.
2012) and transport (Shi et al. 2014)
of anthocyanins in cyclamen and Paeonia delavayi, respectively.
The highest transcripts level of GST was detected as the anthocyanin
content reached maximum in chili pepper (Aza-Gonzalez et al. 2013). Furthermore, ABA could induce the expression of LcGST
in litchi (Hu et al. 2016) and GmGST26A
in maize (Alfenito et al. 1997). In
this study, the transcripts of 6 GSTs were up-regulated by ABA8, Suc8
and AS8 treatments, especially Suc8 treatment. This suggested that GST gene
was involved in ABA and sucrose induced anthocyanin accumulation.
Auxin could inhibit ABA accumulation
and fruit ripening related genes expression (Jia et al. 2017). In raspberry, auxin decreased the content of
anthocyanins by down-regulating the expression of MYB10 and ANS (Moro
et al. 2017). Similar results
were reported in carrot (Ozeki
and Komamine 1986). Auxin responsive
protein (Aux/IAA) and auxin response factor (ARF) were
involved in auxin signaling pathway (Wang et
al. 2018). When there was no auxin, Aux / IAA and ARF formed dimer to
prevent ARF from binding with auxin response element (ARE), and then inhibited
the transcription of auxin response genes. In the presence of auxin, Aux/IAA
was degraded by ubiquitin, and then ARF and ARE bonded
to each other, which led to the transcription of auxin responsive genes (Leyser
2002). In apple, auxin treatment upregulated the transcripts of 9 Aux/IAAs
and 7 ARFs while the transcripts of some MYB and bHLH
and anthocyanins content were down-regulated (Ji et al. 2015). Overexpressed MdARF5 decreased anthocyanin
content by direct inhibition of MdMYB1 expression and indirect
inhibition of anthocyanin synthesis gene expression, respectively (An et al. 2018). In this study, the
expression of ARF5, Aux/IAA4 and Aux/IAA16
was inhibited in ABA8, Suc8 and AS8 treatments, especially in AS8 treatment,
suggesting that ABA and sucrose partially suppressed auxin signaling pathway to
weaken the inhibitory effect of auxin on anthocyanin
biosynthesis.
Conclusion
ABA,
sucrose and ABA + sucrose treatments promoted anthocyanins accumulation and ABA +
sucrose treatment had the greatest
effect, which supplied the details of the effects of ABA and/or sucrose on the global transcriptome modification
during strawberry coloration. Identification and expression analysis of MBW
complex, WRKY, Aux/IAA and ARF showed that most of them may act as negative regulatory
factors for anthocyanin synthesis. Strong transcript signals of DFR, ANS, ANR
and UFGT in structural genes contributed to the accumulation
of anthocyanins in sucrose and ABA + Sucrose treatment. Meanwhile, GST protein
is also a positive regulator for anthocyanin accumulation.
Acknowledgements
The authors acknowledge the
support from National Natural Science Foundation of China (3180817), Key
projects of Sichuan Provincial Science and Technology Department (2018NZ0126) and the State Education
Ministry, Key projects of Sichuan Provincial Education Department (172A0319)
and Service Station Projects of New Rural Development Research Institute,
Sichuan Agricultural University (2018, 2020).
Author Contributions
YL and XZ designed the experiments and wrote the
manuscript. CG performed most of the experiments and analyzed results. All
authors discussed the results and commented on the manuscript. All authors have
read and agreed to the published version of the manuscript.
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