Whole-transcriptome Analysis Reveals the Promising Bioresource of Total Flavonoid from Bitter-Pit Apples, Especially the Pitted Parts

Background: Apple fruits are rich in avonoids, and play important roles in human-health protection against chronic diseases. However, pitter pit in apple has affected apple fruit production worldwide. There must be some application values could be exploited from the bitter-pit apples so as to reduce the loss caused by bitter pit. Results: In the present study, the inuence of bitter pit on the total avonoid content and avonoid biosynthesis in apples was investigated using the aluminum chloride colorimetric method, whole-transcriptome sequencing and qRT-PCR analysis. The results showed that the total avonoid content in bitter-pit apples (BG), pitted parts (BGBB) and non-pitted parts (JKBF) was 4.28-fold, 4.68-fold and 0.57-fold respectively as that in healthy apples (JKG). By RNA-Seq analysis, 26, 23 and 3 DEGs involved in avonoid biosynthesis were enriched in JKG vs. BG, JKG vs. BGBB and JKG vs. JKBF comparisons, respectively. Eight DEGs [CYP98A3(1), CYP98A3(2), BADH, DAT, HCT(1), HCT(2), CHI(1) and CHI(2)], were selected to be validated by qRT-PCR analysis, and the consistent expression patterns with RNA-Seq analysis were obtained, the results showed that the 8 DEGs were upregulated in BG and BGBB but downregulated in JKBF when compared with JKG. Conclusions: The avonoid accumulation and biosynthesis in apples, especially the pitted parts, were stimulated greatly by bitter pit, while depressed slightly in non-pitted parts. The results indicated that the bitter-pit apples, especially the pitted parts, could be used as the promising bioresource of total avonoid for the therapeutic utilization in human chronic diseases. The expression patterns of the 8 candidate genes were determined by RNA-Seq and analyzed to determine the FPKM values. The results showed that these genes were upregulated in JKG vs. BG and JKG vs. BGBB, with FPKM ratios of 1.43–29.42 and 1.83–52.08, respectively, while they were downregulated in JKG vs. JKBF, with FPKM ratios of 0.34–0.82. The expression levels of the 8 candidate genes were validated by qRT-PCR analysis, and the results showed that the relative expression levels of these genes were increased in JKG vs. BG and JKG vs. BGBB comparisons, with ratios of 1.05–7.17 and 1.07–8.03, respectively, while the relative expression levels were decreased in JKG vs. JKBF comparison, with expression ratios of 0.12–0.96.

Whole-transcriptome sequencing, which employs next-generation sequencing (NGS) technologies to sequence complementary DNAs (cDNAs), is an advanced and widely-used method for the massively parallel sequencing of total RNA in plants with a higher resolution than Sanger sequencing and microarray-based methods [32][33][34][35]. In the present study, the total avonoid content was determined using the aluminum chloride colorimetric method, then whole-transcriptome sequencing was used to evaluate the differences in total RNA, including messenger RNAs (mRNAs), long noncoding RNAs (lncRNAs), circular RNAs (circRNAs) and microRNAs (miRNAs) [36][37][38][39], especially genes related to avonoid biosynthesis, in healthy apple fruit, bitter-pit fruit and different parts of bitter-pit fruit.
Finally, the expression of the candidate differentially expressed genes (DEGs) involved in avonoid biosynthesis was validated by qRT-PCR analysis. The results of this study will provide new insights into the in uence of bitter pit on the avonoid biosynthesis and accumulation in apples by investigating the in uence of bitter pit on avonoid biosynthesis and accumulation in apple fruit and evaluating the potential bioresources of avonoids for therapeutic utilization from bitter-pit apple fruit.

Total avonoid content
The total avonoid content in apple fruit was determined using the aluminum chloride colorimetric method, and the results showed that the total avonoid content in BG and BGBB was signi cantly higher than that in JKG, with ratios of 4.28 (BG/JKG) and 4.68 (BGBB/JKG), respectively, while the total avonoid content in JKBF was lower than that in JKG, with a ratio of 0.57 (JKBF/JKG) ( Fig. 1). At the same time, we found the total avonoid content in BGBB was a little higher than that in BG, with ratio of 1.09 (BGBB/BG), and the total avonoid content in JKBF was signi cantly lower than that in BG and BGBB, with ratios of 7.55 (BG/JKBF) and 8.26 (BGBB/JKBF), respectively.

RNA-Seq and quality ltering
Healthy and bitter-pit apple fruit of uniform ripeness and size were chosen to study the in uence of bitter pit on different expression of avonoid biosynthesis-related genes. Twelve cDNA libraries of the whole transcriptome were generated for RNA-SEq. We obtained 963.948 M raw reads containing 144.592 G nucleotides by RNA-SEq. After clean-up and quality ltering, 862.772 M clean reads containing 124.435 G nucleotides were obtained (Table 1). The clean Q30 percentages were 94.1%, indicating that the RNA-seq results were of high quality and suitable for use in further analyses. By miRNA sequencing, 4331 reads associated with 3753 target genes were obtained, among which 2571 genes were upregulated and 1182 genes were downregulated. Of the 41894 total RNA sequences subjected to sequence analysis and annotation, 15852 were known, and 26042 were novel (Table 2).  The RNA-Seq data were submitted to SRA database in NCBI with the accession number of PRJNA640254.

Analysis of sequence length distribution
The sequence length distribution of the transcripts is depicted by FPKM values in  Similar results were obtained for the sequence-length distribution of genes (

GO enrichment analysis of DEGs
For the JKG vs. BG comparison, a total of 4643 genes (3922 upregulated and 721 downregulated) were highly enriched in 3 classes and 34 sub-classes, of which 1065, 2632 and 946 genes were annotated with GO terms related to "molecular function", "biological process" and "cellular component", respectively (Fig. 2a). For the JKG vs. BGBB comparison, a total of 4760 genes (3121 upregulated and 1639 downregulated) were highly enriched in 3 classes and 32 sub-classes, of which 1357, 2605 and 798 genes were annotated with GO terms related to "molecular function", "biological process" and "cellular component", respectively (Fig. 2b). For the JKG vs. BG comparison, a total of 2156 genes (501 upregulated and 1655 downregulated) were highly enriched in 3 classes and 34 sub-classes, of which 457, 1107 and 592 genes were annotated with GO terms related to "molecular function", "biological process" and "cellular component", respectively (Fig. 2c).
The avonoid biosynthesis pathway in KEGG database from JKG vs. BG (Fig. 4a), JKG vs. BGBB (Fig. 4b) and JKG vs. JKBF (Fig. 4c) indicated that the DEGs involved in avonoid biosynthesis were upregulated in JKG vs. BG and JKG vs. BGBB while downregulated in JKG vs. JKBF, which was consistent with the KEGG pathway enrichment analysis.
Apples have been reported to be rich in avonoids, and the biosynthesis and accumulation of the total avonoid content in apple fruit are in uenced by many biotic and abiotic factors [16, 19, 25-31].Abid et al. [26] observed a signi cant increase in the total amount of phenolic compounds and avonoids of apple juice after high-pressure processing (HPP) at 450 MPa/25 °C/10 min. Fernández-Jalao et al. [31] observed that high-pressure processing (400 MPa/35 °C/5 min) increased 30% of total avonols and maintained total phenolic compounds in S-apples, and increase 54% of total phenolic compounds in I-apple treated at 600 MPa/35 °C/5 min. Lower temperatures and increased exposure to light during maturity and harvest might improve the total phenolic and total avonoid content in the apple peel [25,28].Liu et al. [19] found that the total avonoid content in the peels and pulps of three Xinjiang red-esh apple lines were 1.23-1.61 times and 1.43-3.49 times higher than those of the control. Sha q and Singh [29] found that a single preharvest spray application of L-phenylalanine (100 mg L − 1 ), when applied 3-4 weeks prior to the anticipated commercial fruit maturity, increased the accumulation of avonoids in the fruit skin without adversely affecting the fruit quality. Zhang et al. [30] found that, during the whole growth period of fruit development, the avonoid content in 'Fuji' and 'Starkrimson' apples treated by differently pollinated trees was 4.24%-19.63% higher than that in control apples, with signi cant differences among different cultivars.
In the present study, the total avonoid content was determined using the aluminum chloride colorimetric method, with 4.28-fold, 4.68-fold and 0.57-fold of total avonoid content in BG, BGBB and JKBF, respectively, than in JKG, indicating that bitter pit signi cantly stimulates avonoid biosynthesis and accumulation in bitter-pit apples, especially the pitted parts. Liu et al. [19] found that the total avonoid content in apple peels was 2.14-4.64-fold as that in apple pulp. In this present study, the total avonoid content in BGBB was a little higher than that in BG, and the total avonoid content in JKBF was signi cantly lower than that in BG and BGBB, the results were consistent with that of Liu et al. [19], which may due to the location of the pitted part (BGBB) just beneath the peels and the location of the non-pitted part (JKBF) mostly far from the peels.
Many genes are involved in the avonoid biosynthesis pathway and result in differences in avonoid content in fruit. Xu et al. [27] found signi cant differences in the avonoid components and contents among 3 strains, i.e., Hongcui NO.1, Hongcui NO.2 and Hongcui NO.4, which might result from variations in the expression of transcription factors and the structure of genes related to avonoid biosynthesis. Cao et al. [40] found that PpMYB15 and PpMYBF1 were involved in regulating avonol biosynthesis in peach fruit. In the present study, based on the results of whole-transcriptome sequencing, GO enrichment analysis and KEGG pathway enrichment analysis, 8 DEGs related to avonoid biosynthesis, namely, CYP98A2 (1), CYP98A2 (2), BADH, DAT, MdHCT (1), MdHCT (2), CHI (1) and CHI (2), were selected for validation of their differential expression in JKG vs. BG, JKG vs BGBB and JKG vs JKBF by qRT-PCR analysis.
In terms of biotechnology, cytochrome P450 (CYP) enzymes show promise in the synthesis of high-value chemicals and natural products [41]. The major function of CYPs is to catalyze the biosynthesis of endogenous compounds such as hormones and avonoids, and participate in the oxidative metabolism of many exogenous compounds [42]. Plant BAHD acyltransferases constitute a large family of acyl CoA-utilizing enzymes whose products include small volatile esters, modi ed anthocyanins, constitutive defense compounds and phytoalexins [43]. Deacetylvindoline 4-O-acetyltransferase (DAT) is the terminal enzyme in the synthesis of the alkaloid vindoline [44]. Hydroxycinnamoyl-coenzyme A (CoA) hydroxycinnamoyl transferases (HCTs) belong to the BAHD acyltransferase family and play important roles in the metabolism of biosynthetic intermediates and some specialized metabolites [45]. Chalcone isomerase (CHI) is an important enzyme in the plant avonoid biosynthetic pathway that catalyzes bicyclic chalcone into tricyclic (S)-avanones and ensures adequate substrates for the pathway [46].
In the present study, RNA-Seq analysis and qRT-PCR validation showed that the expression of the 8 selected DEGs in JKG vs. BG, JKG vs. BGBB and JKG vs. JKBF had consistent variation trends, that is, the 8 DGEs involved in avonoid biosynthesis were upregulated in BG and BGBB but downregulated in JKBF compared with JKG, which were consistent with the result of total avonoid content determined using the aluminum chloride colorimetric method, indicating that the biosynthesis and accumulation of avonoids in apple fruit, especially the pitted parts (BGBB), was stimulated by bitter pit disorder.

Conclusions
In summary, the accumulation of the total avonoid content and the expression of genes involved in avonoid biosynthesis in bitter-pit apples (BG), especially the pitted parts (BGBB), were signi cantly stimulated but that in non-pitted parts (JKBF) were slightly decreased by bitter pit when compared to the healthy apples (JKG), indicating that bitter-pit apples, especially the pitted parts, could be used as an alternative promising bioresource of total avonoids, which are widely used for therapeutic applications in human chronic diseases.

Apple fruit samples
Apple fruits (Malus domestica Borkh. cv. Yanfu2, a bud mutation of 'Fuji' apple by Yantai Fruit Tree Institute of Shandong) were harvested at the commercial maturity stage from an orchard located in Yedian, Mengyin, Shandong Province, China (), and healthy fruit and bitter-pit fruit of uniform ripeness and size were selected for experiments. Twenty grams of apple longitudinal slices except the core were sampled from healthy apples (JKG) and bitter-pit apples (BG) respectively, and 20 g of the pitted parts (BGBB, just include the pitted spot, as little as possible the part without pitted symptom) and non-pitted parts (JKBF, the part at least 1.5 cm away from the pitted spot) were collected from the bitter-pit apples respectively, each sample was repeated for three times. The samples were packed with tinfoil and preserved in liquid nitrogen as soon as possible after collection.

Detection of total avonoid content
Apple fruit samples weighing 0.5 g were used to detect the total avonoid content using the aluminum chloride colorimetric method [47] at 415 nm (λ max of quercetin) with a U-3900 UV/VIS spectrophotometer (Hitachi High-Tech Science, Japan). The total avonoid content from apple fruit samples (fresh weight, FW) was expressed as mg kg − 1 .

Transcriptomic pro ling
The total RNA extraction, preparation of whole-transcriptome libraries and Illumina deep sequencing using the Illumina HiSeq™ 2500 system were performed by Beijing Ori-Gene Science and Technology Corp., LTD (Beijing, PR China).
Total RNA was extracted separately from the 12 samples using TRIzol Reagent (Tiangen Biotech CO., LTD, Beijing, China). RNA purity were assessed by A260/A280 (> 1.8) and A260/A230 (> 1.6) using Nanodrop, and the yield and quality were determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and an RNA 6000 Nano LabChip Kit (Agilent, CA, USA), RIN > 7.0. Strand-speci c adapters were added to the fragmented RNA (approximately 200 nt in length) before reverse transcription followed the manufacturer's instructions. pathway enrichment analysis using the GOseq R package and KOBAS software, respectively [49,50]. All DEGs were characterized by GO classi cation and KEGG pathway enrichment analysis using a hypergeometric test and Bonferroni's correction, with corrected P-values < 0.05 [51]. The GO terms and KEGG pathways of differentially expressed miRNA targets were annotated using the Annot8r annotation tool against the UniProt database- [52].
qRT-PCR-based validation of avonoid biosynthesis-related DEGs qRT-PCR was conducted to verify the expression of 8 DEGs involved in the avonoid biosynthesis pathway and to assay the same samples to validate the bioinformatic and RNA-seq results. PCR primers were designed with Primer Premier 6.0 (PREMIER Biosoft International, Palo Alto, CA, USA) based on the nucleotide sequences obtained by transcriptional sequencing. The 8 selected genes and their speci c primers are listed in Table 5. qRT-PCR using the previously obtained total RNAs as the templates was performed using the FastQuant RT Kit (with gDNAase) (TIANGEN Biotech CO., LTD, Beijing, China) and 2 × RealStar Green Mixture (GenStar Technologies Company Inc., Chino, CA, USA) in an ABI Prism 7500 Sequence Detector (Applied Biosystems, Waltham, USA). MdActin was used as an endogenous control. The 2 −ΔΔCt comparative threshold cycle (Ct) method was used to evaluate the relative expression levels of target genes [53]. The values reported represent the averages of 3 biological replicates.

Declarations
Ethics approval and consent to participate Not applicable.

Consent to publication
Not applicable.