Transcriptional regulation of abscisic acid biosynthesis and signal transduction, and anthocyanin biosynthesis in ‘Bluecrop’ highbush blueberry fruit during ripening

Highbush blueberry (Vaccinium corymbosum) fruit accumulate high levels of anthocyanins during ripening, which might be controlled by abscisic acid (ABA), a signal molecule in non-climacteric fruits. For an integrated view of the ripening process from ABA to anthocyanin biosynthesis, we analyzed the transcriptomes of ‘Bluecrop’ highbush blueberry fruit using RNA-Seq at three ripening stages, categorized based on fruit skin coloration: pale green at ca. 30 days after full bloom (DAFB), reddish purple at ca. 40 DAFB, and dark purple at ca. 50 DAFB. Mapping the trimmed reads against the reference sequences yielded 25,766 transcripts. Of these, 143 transcripts were annotated to encode five ABA biosynthesis enzymes, four ABA signal transduction regulators, four ABA-responsive transcription factors, and 12 anthocyanin biosynthesis enzymes. The analysis of differentially expressed genes between the ripening stages revealed that 11 transcripts, including those encoding nine-cis-epoxycarotenoid dioxygenase, SQUAMOSA-class MADS box transcription factor, and flavonoid 3′,5′-hydroxylase, were significantly up-regulated throughout the entire ripening stages. In fruit treated with 1 g L−1 ABA, at least nine transcripts of these 11 transcripts as well as one transcript encoding flavonoid 3′-hydroxylase were up-regulated, presumably promoting anthocyanin accumulation and fruit skin coloration. These results will provide fundamental information demonstrating that ABA biosynthesis and signal transduction, and anthocyanin biosynthesis are closely associated with anthocyanin accumulation and skin coloration in highbush blueberry fruit during ripening.


Introduction
Climacteric fruits such as apple, banana, and tomato, generate a burst of ethylene at the onset of ripening [1][2][3]. The burst of ethylene accelerates ripening of climacteric fruits. These changes act as a signal of the initiation of ripening in all climacteric fruits. Ripening of climacteric fruits is also stimulated by exogenous ethylene. In contrast, non-climacteric fruits, a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 study [8], and the ABA solution was prepared with 5% ethanol containing 0.1% (v/v) Tween 80. All treatments were conducted after sunset to avoid the photodegradation of ABA [28]. Control fruit were treated with 5% ethanol containing 0.1% Tween 80 without ABA. These experiments were employed in a randomized complete block design with three replicates consisting of five shrubs each. Fifty fruit were randomly sampled from each replicate block at 5 days after treatment (DAT) when the fruit skin color began to change. All harvested fruit were immediately frozen in liquid nitrogen and stored at -80˚C until use for the quantification of individual anthocyanins and the expression profiling of their related transcripts.

Determination of fruit color
Fruit skin colors were measured using a spectrophotometer (CM-2500d; Minolta Co., Osaka, Japan) and described by the CIE L � , a � , and b � color space coordinates [29]. The L � value represents the lightness of colors, with a range of 0 to 100 (0, black; 100, white). The a � value is negative for green and positive for red. The b � value is negative for blue and positive for yellow. For each fruit, the values were measured at three different points along the fruit equator.

Determination of individual anthocyanin contents
Anthocyanins were extracted according to the method described by Gavrilova et al. [30], with some modifications. Approximately 5 g of ground fruit tissues was added to 10 mL of a solution containing acetone:acetic acid (99:1, v/v). The homogenates were sonicated for 15 min and centrifuged at 1,900 × g for 15 min. The supernatants were evaporated until dry using a rotary evaporator (EYELA N-1000S-W; Tokyo Rikakikai Co., Tokyo, Japan) at 37˚C, then completely redissolved in 10 mL of 20% methanol and filtered through a PTFE filter with a pore size of 0.45 μm (Whatman Inc., Florham Park, NJ, USA).

RNA extraction
Total RNA was extracted from fruit at each stage as described by Jaakola et al. [31], with slight modifications. Extraction buffer (2% hexadecyltrimethylammonium bromide, 2% polyvinylpyrrolidone, 100 mM Tris-HCl [pH 8.0], 25 mM EDTA [pH 8.0], 2.0 M NaCl, and 2% β-mercaptoethanol) was heated to 65˚C and then 900 μL of the extraction buffer was transferred to a 2-mL microfuge tube containing 100 mg of powdered fruit tissues and incubated at 65˚C for 10 min. An equal volume of chloroform:isoamyl alcohol (24:1, v/v) was added, vortexed for 5 s, and centrifuged at 10,000 × g at 4˚C for 10 min. The supernatant of 750 μL was recovered and mixed with an equal volume of chloroform:isoamyl alcohol. Following the centrifugation as above, the supernatant of 600 μL was transferred to a new 2-mL tube, and an equal volume of 6 M LiCl solution was added. The mixture was incubated on ice for 30 min and centrifuged at 21,000 × g at 4˚C for 20 min to precipitate the RNA. The pellet was resuspended in 500 μL of preheated (65˚C) SSTE buffer (0.5% sodium lauryl sulfate, 1 M NaCl, 1 M Tris-HCl [pH 8.0], and 10 mM EDTA [pH 8.0]) while gentle shaking. An equal volume of chloroform:isoamyl alcohol was added, and the mixture was centrifuged at 21,000 × g at 4˚C for 10 min, then dried and resuspended in 20 μL diethyl pyrocarbonate-treated water. Finally, the solution was heated at 65˚C for 5 min to completely dissolve the RNA. The quality of the extracted RNA samples was assessed using a NanoDrop ND1000 (Thermo Fisher Scientific), following the confirmation of the RNA integrity using an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany).

RNA-Seq and sequence processing
Nine cDNA libraries were constructed for fruit at pale green, reddish purple, and dark purple stages from three replicates with 30 fruit each, using a TruSeq small RNA library preparation kit (Illumina, San Diego, CA, USA), and sequenced using an Illumina HiSeq 2000 system. The quality of the data produced was confirmed using the R package fastqcr (version 0.1.2). Adapters and low-quality reads, including short reads (< 36 bp) and reads with a Phred score Q � 20, were removed from the raw data using the R package QuasR (version 1.22.1). The trimmed reads were mapped to the reference highbush blueberry transcriptome (V. corymbosum RefTrans V1) from the Genome Database for Vaccinium using Cufflinks (version 2.2.1). The RNA-Seq data were deposited to the National Center for Biotechnology Information (NCBI) (accession No. PRJNA533973).

Gene ontology (GO) annotation and identification of differentially expressed genes (DEGs)
GO assignments were made to the mapped reads using InterProScan at the European Bioinformatics Institute through Blast2GO. The obtained GO terms were classified and plotted using WEGO (version 2.0).
To identify the DEGs, the mapped transcripts were functionally annotated using the KEGG database, and the expression levels of the transcripts were calculated as fragments per kilobase of transcript per million mapped reads (FPKM) using Cuffdiff (version 2.2.1). The FPKM values were normalized, and statistical analyses were performed on the fold change values using a Student's t-test at P < 0.05. The clustered DEGs based on their log 2 FPKM values were plotted using the R package pheatmap (version 1.0.12). The DEGs were identified from three replicates at each ripening stage.

Quantitative polymerase chain reaction (qPCR) analysis
Primer sets were designed using the NCBI PrimerBLAST. Sequences of the forward and reverse primers used for the qPCR are listed in S1 Table. The relative expression levels of the transcripts were determined using a Rotor-Gene Q (Qiagen, Valencia, CA, USA) and a Rotor-Gene SYBR Green PCR kit (Qiagen). The results were standardized to the expression level of the gene encoding glyceraldehyde 3-phosphate dehydrogenase, as described by Zifkin et al. [16]. The relative expression levels were plotted using the Prism program (version 8.0.2; GraphPad Software Inc., San Diego, CA, USA).

Statistical analysis
Statistically significant differences among means were determined by Student's t-test at P < 0.05 using the R 3.2.2 software package (http://www.r-project.org).

Fruit skin coloration and anthocyanin accumulation during ripening
The skin color of the 'Bluecrop' highbush blueberry fruit changed during ripening (Fig 1). With the calyx turning green to purple, the exocarp was mostly tinted red at reddish purple stage and then shifted bluer at dark purple stage (Fig 1). The reddish purple and dark purple stages indicated fruit at turning point and fully ripe stages, respectively [32]. Our previous study revealed that the skin coloration of 'Bluecrop' highbush blueberry fruit during ripening correlated with the accumulation of anthocyanins, especially of delphinidin and delphinidin derivatives [18].

ABA as a positive regulator of anthocyanin accumulation during ripening
In the ABA-treated fruit, the calyx turned dark purple and the exocarp changed to red or purple at 5 DAT, but untreated fruit remained green (Fig 2). Although the L � value of the ABAtreated fruit was not significantly different from that of untreated fruit, the a � and b � values of the ABA-treated fruit were significantly higher and lower, respectively, than those of untreated fruit (Table 1). These results indicated that the ABA-treated fruit were redder and bluer than untreated fruit. Accelerated skin coloration by ABA application has also been reported in other non-climacteric fruits, including strawberry [12,33], grape [10,17,27], and sweet cherry [14].
The ABA application also accelerated the accumulation of individual anthocyanins in 'Bluecrop' highbush blueberry fruit (Table 2). At 5 DAT, no anthocyanins were detected in untreated fruit, while the ABA-treated fruit accumulated four anthocyanins: cyanidin, malvidin, delphinidin, and petunidin 3-O-glucosides (Table 2). However, neither pelargonidin nor peonidin 3-O-glucosides were found in the ABA-treated fruit ( Table 2). According to our previous study in 'Bluecrop' highbush blueberry fruit [18], cyanidins, malvidins, and delphinidins began to accumulate from reddish purple stage and petunidins were accumulated at dark purple stage, but no pelargonidins were accumulated throughout the entire ripening stages. No pelargonidin accumulation was also observed in 'Jersey' highbush blueberry fruit regardless of ABA application [8]. The absence of pelargonidin is a common characteristic of the fruits of the Ericaceae [34,35]. The ABA application promoted the accumulation of anthocyanins, especially of delphinidin derivatives, with a temporary increase in ABA content and thus accelerated fruit skin coloration [8].

Transcriptome and GO analyses
As the results of RNA-Seq in 'Bluecrop' highbush blueberry fruit during ripening, the trimmed reads ranged from 25,552,078 to 28,546,644 with Q30 of 94.9 to 98.2% and GC content of 48.0 to 48.9% (Table 3). The total bases of average 2.58 to 2.88 × 10 9 were obtained (Table 3).
Of the 25,766 assembled transcripts, 10,998 transcripts were assigned and classified into 44 groups within the three GO categories: cellular component, molecular function, and biological process (Fig 3). The majority of the GO terms (51.7%) were assigned to molecular function,  Table 1. Chromaticity values of 'Bluecrop' highbush blueberry fruit at 5 days after the treatment with or without 1 g L -1 (±) ABA at pale green stage (ca. 30 days after full bloom). 0% were assigned to biological process and cellular component, respectively (Fig 3). Transcripts associated with binding and catalytic activity were typical in molecular function, while those associated with cell and cell part were highly represented in cellular component. For biological process, metabolic process and cellular process were the most highly represented groups. These two dominant groups in each GO category for 'Bluecrop' highbush blueberry fruit (Fig 3) have also been observed in the same cultivar [36]
The BCH was significantly up-regulated from pale green to reddish purple stages (S2 Table), as observed in kiwifruit [15]. However, ABA accumulation was significantly reduced in two allelic dsm2 mutants of rice, which lacked a functional BCH protein [38]. NCEDs and their isoforms have been identified in many plant species, including bilberry [17,39,40], tomato [41,42], and grape [43,44,45]. These enzymes were differentially expressed depending on tissues, developmental stages, and environmental conditions [46]. NCED expression was reported to temporarily increase with the increased ABA contents during ripening of grape [9] and 'Rubel' highbush blueberry fruits [16]. Of the six NCEDs, two NCEDs (XLOC_002223 and XLOC_021727) were also significantly up-regulated from pale green to reddish purple stages (S2 Table). From reddish purple to dark purple stages, one NCED (XLOC_024952) was upregulated, while the remaining three NCEDs were down-regulated (S2 Table). The AAO3 was significantly down-regulated from reddish purple to dark purple stages, but its FPKM values remained low throughout the entire ripening stages (Fig 4B).
In Arabidopsis, ABA application induced the transcriptional expression of the PYR/PYL/ RCAR family [49]. In strawberry fruit, similarly, ABA application enhanced the up-regulation of a PYR among the family during ripening, while no coloration occurred in the PYR-silenced fruit regardless of ABA application [5]. However, up-regulation of the four PP2Cs and downregulation of the one SnRK2 and one ABF in the present study (S2 Table) were contradictory to the results in Arabidopsis [49]. The question of if there is ABA signal transduction process specific for highbush blueberry fruit remains to be answered unequivocally.
In the present study, 21 transcripts were annotated to encode four ABA-responsive transcription factors (ten TDRs, seven MYBs, two bHLHs, and two WDRs) (Fig 5). Of the ABA-  [48]). (B) Heatmap of the log 2 FPKM of candidate transcripts involved in ABA signal transduction responsive transcription factors, TDR (XLOC_020802) was most highly expressed throughout the entire ripening stages (Fig 5B). However, two TDRs (XLOC_008525 and XLOC_012094) were significantly down-regulated from pale green to reddish purple stages and their expressions remained low thereafter (S2 Table). Four MYBs (XLOC_002719, XLOC_004086, XLOC_014506, and XLOC_024956) and one bHLH (XLOC_007502) were significantly upregulated from reddish purple to dark purple stages, but the others were not significantly regulated throughout the entire ripening stages (S2 Table).
In bilberry fruit, TDR and MYB were found to be sequentially up-regulated during ripening and the up-regulations were enhanced by ABA application, increasing the anthocyanin accumulation [39]. ABA application also enhanced the expressions of bHLH as well as MYB in grape fruit [51]. Silencing TDR and MYB led to a decrease in anthocyanin accumulation and its associated gene expressions in sweet cherry [14] and bilberry fruits [39].  (Fig 6A) [16,52]. These enzymes and their related genes in anthocyanin biosynthesis have been discovered in several plant species, including Arabidopsis, maize, grape, and petunia [53], and the anthocyanin biosynthesis pathway was reported to be highly conserved in angiosperms [54].
Following the hydroxylation, individual anthocyanins are biosynthesized at different levels depending on the reactions of DFR and ANS with their respective substrates [52,53]. In strawberry fruit, one DFR isoform specifically reacted with dihydrokaempferol to biosynthesize pelargonidin derivatives, the most abundant anthocyanins in these fruit [59].
Glucosylation, galactosylation, and arabinosylation were the major glycosylation processes, which were catalyzed by the actions of various sugar transferases, in highbush blueberry fruit [8,18]. In our previous study, we determined 22 anthocyanins resulted from these three glycosylation processes in 'Bluecrop' highbush blueberry fruit [18]. In the present study, however, three out of five transcripts encoding UFGTs for glucosylation were up-regulated throughout  Table), but the transcripts associated with galactosylation and arabinosylation were not found (Fig 6B). Similarly to our report, no transcripts associated with galactosylation or arabinosylation have been found in the other transcriptome analyses of highbush blueberry fruit [23][24][25][26][27].
Leucoanthocyanidins and anthocyanidins can be diverted to flavan 3-ols by the actions of LAR and ANR, respectively [16]. Since LAR and ANR compete with ANS and UFGT for their respective common substrates, the up-regulations of ANSs and UFGTs and the down-regulations of LARs and ANR (S2 Table) might also contribute to anthocyanin accumulation in highbush blueberry fruits during ripening. Following the glycosylation, OMT methoxylates the Bring of anthocyanidin skeletons at the 3 0 -, and 3 0 -and 5 0 -positions [51,60]. The transcript encoding OMT was found to be down-regulated in 'Bluecrop' highbush blueberry fruit throughout the entire ripening stages (S2 Table) as observed in grape fruit, despite of the accumulation of methoxylated anthocyanins [57].

Transcriptional expression in ABA-treated fruit
The effects of ABA treatment on the transcript expression were confirmed by qPCR analysis against the annotated transcripts, which were found to be highly up-regulated during ripening. All transcripts examined except for CHI (XLOC_013012) were more highly expressed in the ABA-treated fruit at 5 DAT than in untreated fruit (Fig 7). These included NCED The ABA-mediated up-regulation of NCED in 'Bluecrop' highbush blueberry fruit (Fig 7) was also previously observed in other non-climacteric fruits, including grape [43], strawberry [12], and bilberry [17], implying that the endogenous ABA contents increased during ripening. Exogenous ABA as well as the increased endogenous ABA might accelerate the ripening process, including fruit skin coloration and cell softening.
The up-regulations of TDR and eight genes (CHS, F3H, F3 0 H, F3 0 5 0 H, DFR, ANS, UFGT, and OMT) (Fig 7) might contribute to the anthocyanin accumulation (Table 2) and the associated fruit skin coloration (Fig 2 and Table 1). In ABA-treated grape [61], strawberry [12], and blueberry fruits [8], however, the levels of anthocyanin accumulation and its associated gene expression were dependent on the concentration, timing, and duration of the ABA application. Despite these differences, the transcription factors and genes were similarly expressed in those fruits during ripening [8,12,61,62]. More detailed information on ABA effects under various environmental and experimental conditions is needed to understand the ripening process of naturally grown fruits and to explore the possible implementation of ABA application in agricultural fields.
Supporting information S1 Table. Sequences of forward and reverse primers used for quantitative polymerase chain reaction.