Transcriptomic analysis of postharvest toon buds and key enzymes involved in terpenoid biosynthesis during cold storage

Scientia Horticulturae 257 (2019) 108747

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Transcriptomic analysis of postharvest toon buds and key enzymes involved in terpenoid biosynthesis during cold storage

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Hu Zhaoa, , Shanshan Fenga, Wei Zhoub, Guoyin Kaib a Engineering Technology Research Center of Anti-aging Chinese Herb, Biology and Food Engineering College at Fuyang Normal University, Qinghe West Road 100, Fuyang 236037, China b Pharmacy College, Zhejiang Chinese medicine University, Gaoke Road, Fuchun Street, Hangzhou, 311402, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Transcriptome Volatile terpenoids Terpene synthase Cold storage Toona sinensis

Toon buds have been used in China as an important woody vegetable for its unique flavor. However, the changes of volatile terpenoids related to flavor and their biosynthesis in postharvest toon buds after cold storage remain largely unknown. Therefore, a transcriptomic database must be constructed to analyze the molecular mechanism of terpenoid biosynthesis in toon buds during cold storage. The chemical profiles of volatile terpenoids over postharvest storage periods were comparatively analyzed by gas chromatography–mass spectrometry. Results showed that the total content of volatile terpenoids and their oxidates, except for monoterpenes and a small portion of sesquiterpenes, increased significantly after cold storage. The transcriptome database derived from different cold storage times was established using BGIseq500 technology. Approximately 6.5 g of clean nucleotides were obtained and de novo assembled into 152 127 non-redundant unigenes, approximately 72.36% of which could be aligned to public databases. Many candidate genes involved in terpenoid biosynthesis were identified. Furthermore, 29 513 unigenes were demonstrated to be differentially expressed under different cold storage times. These differentially expressed genes were functionally annotated by gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes enrichment analyses. The expression of 20 putative unigenes involved in terpenoid pathways was confirmed by qRT-PCR, the results of which were consistent with the RNAseq data. These genes with different expression patterns correlated well with the changes in the volatile terpenoids in the toon buds during low-temperature storage. In summary, our results showed that the high expression levels of terpenoid backbone biosynthesis pathways contributed to sesquiterpene biosynthesis after cold storage, which led to high sesquiterpene accumulation in toon buds during low-temperature storage.

1. Introduction Toon bud, which is also called Chinese xiangchun shoot (Toona sinensis Roem), is a member of the Meliaceae family and is grown primarily for its edible bud. Chinese toon buds serve as an important dietary source because of their high nutrient content and therapeutic value (Xia et al., 2015; Shi et al., 2016). Every spring, numerous bioactive compounds, including polyphenols, flavonoids, and terpenes, begin to accumulate in toon buds and provide them with pharmacological activities associated with anti-inflammation, anticancer, antidiabetic, antioxidative, and antithrombotic properties (Hu et al., 2016; Shan et al., 2016; Wang et al., 2016). In addition to their influence on health, toon buds have a unique flavor that make them easily recognized and appreciated relative to other vegetables. As a seasonal vegetable, toon buds have only half a month of growth

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period and a considerably short shelf life at ambient temperature due to their high water content and vigorous respiratory rate after harvest. These properties lead to the high perishability of postharvest toon buds. Thus, low-temperature storage and modified atmosphere packaging are used to extend the shelf life of postharvest toon buds by reducing their respiration rate, surface damage, and browning (Zhu and Gao, 2017; Zhao et al., 2018). However, cold storage often leads to quality changes in postharvest fruits and vegetables. Significantly reduced titratable acidity, vitamin C content, and accelerated chlorophyll degradation have been observed in postharvest toon buds under cold storage (Chen et al., 2015; Li et al., 2017; Zhao et al., 2018). Volatile terpenoids, including mono- and sesquiterpenes and homoterpenes, are vital to the special flavor of Chinese toon buds. However, the effects of cold storage on the flavor of postharvest toon buds have been rarely investigated. The molecular mechanism of

Corresponding author. E-mail address: [email protected] (H. Zhao).

https://doi.org/10.1016/j.scienta.2019.108747 Received 9 July 2019; Received in revised form 3 August 2019; Accepted 5 August 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.

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2.3. RNA sample preparation, cDNA library construction, and sequencing

terpenoid biosynthesis in toon buds remains unclear (Hsu et al., 2012). Several key questions remain to be answered. (1) How can the content of flavor compounds in postharvest toon buds be changed under cold storage? (2) What is the molecular regulatory mechanism of terpenoid biosynthesis in postharvest toon buds? To answer these questions, we identified the key genes involved in terpenoid formation via RNA-seq sequencing. The changes in the volatile terpenoids were analyzed in toon buds during cold storage. This study revealed the dynamic changes in gene expression related to volatile terpenoid formation and provision of valuable information on the genetic regulation of terpenoid production in toon buds in response to cold storage.

Total RNA was extracted from the toon buds by using TRIzol Reagent (Invitrogen, Life Technologies, USA) following the manufacturer’s protocol. DNA contamination was then removed with DNaseI (Invitrogen, Life Technologies, USA). Total RNA sample quality control, including concentration and purity, was identified using a NanoDrop™ UV–vis Spectrophotometer (Thermo Fisher Scientific, USA) and an Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, USA). Messenger RNA (mRNA) was isolated from total RNA by using oligo-dTcoated magnetic beads. Then, mRNA was fragmented using divalent cations in combination with heat in an appropriate fragmentation buffer. First-strand cDNA was synthesized using an N6 random primer and M-MuLV Reverse Transcriptase. Subsequently, second-strand cDNA synthesis was performed using DNA polymerase I and ribonuclease H. The synthesized cDNA was subjected to end repair and was then 3′ adenylated. Adaptors were ligated to the ends of these 3′ adenylated cDNA fragments. The ligation products were purified, and many rounds of PCR amplification were performed to enrich the purified cDNA template by using a PCR primer. The PCR products were denatured by heat, and the single-strand DNA was cyclized by splint oligo and DNA ligase. Library quality was assessed on the Agilent Bioanalyzer 2100 System (Agilent Technologies, Santa Clara, USA). The libraries were sequenced on the BGIseq500 platform (BGI, Wuhan, China, http:// www.seq500.com/en/).

2. Materials and methods 2.1. Plant material collection The excellent cultivar of T. sinensis, which is called “heiyouchun” in our study, was obtained from Taihe County, Anhui Province, China, in April 2018. The toon buds picked were pooled in insulation containers with ice bags and rapidly delivered to the laboratory of Fuyang Normal University, Fuyang City. Robust toon buds, which were free from visual blemishes and mechanical damage, were selected by uniform size (length of 10–15 cm). The toon bud samples were immediately stored at 4 °C with 85%–95% relative humidity. After 0, 3, and 5 days of cold storage, the samples were taken out randomly and named as BYC0, BYC3, and BYC5, respectively. Fifteen toon buds were selected in each sampling time as three biological replicates. The toon buds selected were snap frozen in liquid nitrogen and stored at –80 °C.

2.4. Sequencing read filtering and de novo assembly Raw reads obtained by the BGIseq500 platform were filtered to remove low-quality reads (reads with > 10% Q-value < 20 bases), adaptor-polluted reads, and unknown base “N” reads by using internal soft filtering. After filtering, the total clean reads from three libraries were assembled into unigenes by using the Trinity method (Grabherr et al., 2011). In brief, Trinity contains three independent software modules: Inchworm, Chrysalis, and Butterfly. Trinity partitions sequence data into many individual de Bruijn graphs, each representing transcriptional complexity at a given gene or locus. Each graph is then independently processed to extract full-length splicing isoforms and to tease apart transcripts derived from paralogous genes. Trinity assembly results are called transcripts. With the use of Tgicl, we performed gene family clustering to obtain the final unigenes for downstream analysis. The unigenes were divided to two types. One type is a cluster, in which the prefix CL refers to the cluster ID behind it. One cluster comprises several unigenes with > 70% similarity. The other type is singleton, the prefix for which is a unigene.

2.2. Volatile terpenoid analysis To analyze the volatile terpenoids of postharvest toon buds from different cold storage times, we collected samples of frozen toon buds and rapidly ground them in liquid nitrogen. Frozen toon bud powder (0.25 g) were packed in 10 ml glass vials added with 10 μl of 1.0 μg μl−1 capric acid lactone (Aladdin Chemistry Co., Ltd., Shanghai, China) as the internal standard. The vials were tightly sealed with septa (PTFEbutyl synthetic rubber). The toon buds’ headspace volatiles were extracted from the samples and concentrated using a headspace solidphase microextraction manual headspace sampler with 65 μm polydimethylsiloxane (PDMS) fiber (Supelco, USA). The extraction conditions were as follows. The sample vials were heated in a water bath at 80 °C for 1 h. Splitless injection mode was performed to thermally desorb the volatile terpenoids for 5 min at 250 °C. The analysis was carried out with HS-GC–MS (Agilent HS-7697A/GC-7890A/MS-5975C, DB-5MS gas chromatographic column, Agilent, USA) using helium as the carrier gas with a flow rate of 1.0 ml min−1. The control procedure of column temperature was as follows: 50 °C for 1 min, followed by 10 °C min−1 up to 100 °C, held 100 °C for 1 min, followed by 4 °C min−1 up to 200 °C, held at 200 °C for 1 min, continued to be held up to 280 °C at 3 °C min−1, and held at 280 °C for 7 min. The volatile terpenoids were identified by comparing the retention times of individual peaks with those in the chemical database NIST. Retention index (RI) was calculated via the injection of a series of C10 to C40 straight-chain n-alkanes (50 mg l−1 in n-hexane) purchased from Fluka (Madrid, Spain). The volatile compound standards used for the identification of target compounds were purchased from Sigma–Aldrich (Madrid, Spain), Merck (Darmstadt, Germany), and Fluka (Madrid, Spain). The standards were of the highest quality available. Sodium chloride was obtained from Merck (Darmstadt, Germany). Volatile terpenoid quantity was calculated by peak area normalization with capric acid lactone as the internal standard; the units were expressed as μg kg−1 on a fresh weight basis. All GC–MS analyses were designed to three biological replicates, and data are presented as mean ± standard deviation (SD).

2.5. Sequence alignment and functional annotation To annotate the function of toon bud transcriptome, we searched the unigenes against seven public databases with certain alignment programs. The unigenes were annotated into the National Center for Biotechnology Information (NCBI) non-redundant nucleotide (Nt) database by using the Blastn program. They were also annotated into NCBI non-redundant protein (Nr), EuKaryotic Orthologous Groups (KOG), Kyoto Encyclopedia of Genes and Genomes (KEGG), and SwissProt using the Blastx or Diamond program with a threshold E value of 10−5. The products annotated on Nr were functionally classified into the Gene Ontology (GO) database by using the BLAST2GO program (Conesa et al., 2005). GO terms were divided into three modules, namely, molecular function (MF), biological process (BP), and cellular component (CC) ontologies. InterProScan 5 software was used to annotate the unigenes into the InterPro database. 2.6. Detection of differentially expressed genes (DEGs) The number of clean reads for each unigene was calculated and 2

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and γ-muurolene (113%). The extension of storage of up to 5 days caused a further increment in the contents of volatile terpenoids, except for monoterpenoid and part of sesquiterpenoids, such as α-pinene, Dlimonene, α-cadinene, ylangene, α-copaene, α-bourbonene, and β-elemene, which had significantly lower contents in BYC5 than in BYC0.

normalized to fragments per kb per million reads (FPKM) (Mortazavi et al., 2008). The Bowtie2 and RSEM methods (RNA-Seq expression estimation by expectation maximization) were used to estimate each unigene and its isoform expression levels from RNA-Seq data with a statistical model that considers reads that map to multiple positions (Li and Dewey, 2011). To identify the DEGs that were regulated at the transcript level, we used DESeq in comparing the unigene expressions at different storage times (BYC0, BYC3, and BYC5). A false discovery rate of ≤ 0.001 and absolute value of log2Ratio ≥ 2 were set as the threshold for significant difference in unigene expression (Reiner et al., 2003). The DEGs were then subjected to GO classification and KEGG pathway enrichment analysis.

3.2. Transcriptome sequencing, de novo assembly, and annotation Three toon bud samples of BYC0, BYC3, and BYC5 obtained from three cold storage time points were processed as described in Materials and Methods. The cDNA libraries were used for high-throughput sequencing using the BGIseq500 platform. The sequencing and assembly statistics are shown in Table 2. Approximately 221.37 Mb raw reads were generated from BYC0, BYC3, and BYC5, > 88% of which reached the quality standards and satisfied the requirements for assembly after adaptor and low-quality sequences were discarded. By a three-step approach with the Trinity software, the filtered high-quality reads were subjected and assembled into 279 074 contigs. The combined contigs in the BYC0, BYC3, and BYC5 libraries were further assembled into 152 127 non-redundant unigenes, which could represent the global transcriptomes of postharvest toon buds for three cold storage time points. The length of the transcripts ranged from 200 bp to 17 285 bp, with an average length of 973 bp and N50 transcript length of 1 690. The NCBI’s nucleotide and non-redundant SwissProt, KEGG, KOG, Interpro, GO, and Intersection databases were chosen to annotate the transcripts. The percentage of transcripts annotated was 72.36% on the basis of BLASTx with an E-value threshold of 10−5. The species distribution revealed that approximately 74 405 unigenes (48.91%) highly matched with sequences from Citrus sinensis.

2.7. Quantitative real-time RT-PCR verification A series of enzyme genes related to terpenoid backbone, monoterpenoid, sesquiterpenoid, and other terpenoid biosynthesis was chosen to verify their expression patterns in various storage times of toon buds by using quantitative RT-PCR. As mentioned previously, the total RNA of nine samples (each cold storage time point included three biologically repeated samples) was independently extracted using TRIzol Reagent (Invitrogen, Life Technologies, USA). In accordance with the manufacturer’s protocol, TransScript All-in-One cDNA supermix (TransGene Biotech, Beijing) was used in reverse transcription for the synthesis of first-strand cDNA, which was then stored at −20 °C for subsequent qRT-PCR analysis. The gene-specific primers (Supplementary file 1) for qRT-PCR were designed using Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and synthesized by Generay Biotechnology Company (Shanghai). The actin gene of T. sinensis was used as the internal housekeeping gene control (Zhao et al., 2017). The qRT-PCR reaction was carried out in an Applied Biosystem 7300 Real-time PCR System (ABI, USA). The 20 μl volume of reaction mixture was composed of 2 μl of dilute template cDNA, 2 μl of primer pairs, 10 μl of GoTaq® qPCR Master Mix (Promega, USA), and 6 μl of deionized water. The setting program of qPCR reaction was as follows: denaturation at 95 °C for 20 s, followed by 40 cycles of 95 °C for 15 s, annealing at 60 °C for 30 s, and then extension at 72 °C for 30 s. After the reactions, primer specificity was evaluated by melting curve analysis. Each gene in qPCR quantity was analyzed in three biological replicates and three technical replicates. The relative expression levels of the selected genes were normalized to T. sinensis actin gene by using the 2−ΔΔCt method.

3.3. Functional classification The KOG database is a eukaryote-specific version of the Cluster of Orthologous Groups tool for identifying ortholog and paralog proteins (Tatusov et al., 2003). All proteins in the KOG database are assumed to have been evolved from an ancestor protein and are thus either orthologs or paralogs. The KOG annotation showed that 77 422 (50.89%) unigenes were assigned to KOG categories. Among the 25 KOG categories, the cluster for “General function prediction only” represented the largest group, followed by “signal transduction mechanisms” and “posttranslational modification, protein turnover, chaperones” (Fig. 1). In addition, 2 596 unigenes (3.35% of total KOG hits) were categorized into “secondary metabolites biosynthesis, transport and catabolism.” GO, an international general normalization of gene functional annotation system (GO Annotation), was used to categorize the functions of the predicted unigenes. A total of 68 669 unigenes (45.14%) were assigned to at least one GO term. On the basis of sequence homology, the unigene sequences from postharvest toon buds were assigned to 53 functional groups under three main sections: 135 942, 127 886, and 74 366 were assigned to the BP, CC, and MF sections, respectively (Fig. 2). As a result, metabolic process (35 089) and cellular process (31 833) were prominently represented in the BP section. In the CC section, the cell (24 399) and cell part (24 288) represented the majority. For the MF section, binding (32 890) and catalytic activity (32 038) constituted the majority. These results indicated that most of the unigenes in GO annotation were involved in metabolism and fundamental biological regulation. The KEGG pathway database lays particular emphasis on the functional annotations of enzymes and the biochemistry pathway of genes related to their metabolic networks. To identify important metabolic pathways in the postharvest toon buds, we assigned 73 739 (48.47% of the total unigenes) unigene sequences to 135 KEGG pathways. All transcripts were grouped into six categories, namely, cellular processes, environmental information processing, genetic information processing, human diseases, metabolism, and organismal systems. Most of the unigenes from the postharvest toon buds were mapped to the

3. Results 3.1. Determination of volatile terpenoids The results of the SPME GC–MS analysis of volatile terpenoids in postharvest toon buds subjected to different cold storage times were shown in Table 1. The most important and abundant terpenoid compound was β-caryophyllene (3396.88 ± 235.22 μg kg−1), followed by isolongifolene, 9,10-dehydro (2503.93 ± 44.81 μg kg−1), D-cadinene (1680.22 ± 47.32 μg kg−1), isoaromadendrene epoxide (1343.32 ± 100.30 μg kg−1), calamenene (1158.16 ± 15.31 μg kg−1), and α-copaene (1116.28 ± 71.89 μg kg−1), As shown in Table 1, postharvest toon buds were affected over cold storage times. A significant increase in the total contents of volatile terpenoids was observed with the extension of storage time. Compared with those at harvest (BYC0), significantly high contents of volatile terpenoids and their oxide were noted in BYC3. Arranged in order of content abundance after 3 days of cold storage, alloaromadendrene oxide showed the highest increment (910%), followed by isoaromadendrene epoxide (621%), α-bulnesene (407%), caryophyllene oxide (344%), isolongifolene, 9,10-dehydro (283%), isocalamendioldehydroxy (216%), trans-β-farnesene (206%), ledene oxide (152%), αcurcumene (146%), cis-α-farnesene (145%), caryophyllene (131%), 3

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Table 1 Volatile compounds in postharvest toon buds at three different storage times at low temperature. Peak 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

Rt (min)

Volatile compounds

5.48 10.87 15.35 16.03 16.23 16.47 16.61 17.12 17.54 17.67 17.96 18.14 18.50 18.60 18.73 19.13 19.21 19.30 19.78 20.09 20.41 20.51 20.66 20.93 21.07 21.38 22.35 22.62 23.38 24.45 31.12 34.41

α-Pinene D-Limonene α-Cadinene Ylangene α-Copaene α-Bourbonene β-Elemene β-Caryophyllene Caryophyllene trans-β-Farnesene α-Guaiene α-Bisabolene cis-α-Farnesene α-Humulene Alloaromadendrene γ-Muurolene trans-β-Ionone α-Curcumene δ-Humulene β-Bisabolene d–Cadinene Calamenene Dihydroactinidiolide α-calacorene Ledene oxide Isocalamendiol-dehydroxy Aromadendrene-dehydro α-Bulnesene Caryophyllene oxide Alloaromadendrene oxideIsolongifolene,9,10-dehydro Isoaromadendrene epoxide

0 day of cold storage c

24.13 ± 1.22 198.36 ± 22.72c 154.51 ± 9.51b 159.12 ± 14.86c 1116.28 ± 71.89c 113.08 ± 7.98b 193.79 ± 8.35b 3396.88 ± 235.22a 247.83 ± 30.66a 896.35 ± 24.09a 605.26 ± 37.75a 153.23 ± 12.44a 667.20 ± 28.87a 785.16 ± 47.53b 200.97 ± 9.21b 650.17 ± 14.94b 666.03 ± 62.08b 404.15 ± 7.58b 428.40 ± 29.98a 384.79 ± 16.78a 1680.22 ± 47.32b 1158.16 ± 15.31b 286.94 ± 10.69a 120.14 ± 14.07a 113.14 ± 12.23a 112.13 ± 26.26a 332.30 ± 46.41b 85.98 ± 8.54a 69.78 ± 10.75a 163.13 ± 4.73a 2503.93 ± 44.81a 1343.32 ± 100.30a

3 day of cold storage b

16.99 ± 1.39 56.34 ± 1.65b 124.81 ± 5.82a 91.17 ± 12.08b 780.01 ± 36.42b 88.19 ± 5.37a 261.81 ± 29.57c 2473.28 ± 184.37c 573.61 ± 38.52c 2739.19 ± 310.57b 1067.73 ± 24.37b 171.31 ± 7.48b 1637.80 ± 103.16b 655.04 ± 5.01a 349.05 ± 16.95c 1381.99 ± 77.71c 615.32 ± 54.63b 992.58 ± 89.82c 517.38 ± 6.70b 752.18 ± 26.74c 1034.60 ± 25.28a 1055.04 ± 59.02a 269.23 ± 32.02a 172.71 ± 25.21b 285.31 ± 13.38c 353.90 ± 23.38c 428.11 ± 20.48c 436.21 ± 32.94c 309.49 ± 6.70c 1648.27 ± 91.72c 9580.56 ± 88.72c 9684.88 ± 364.90c

5 day of cold storage 0a 24.57 ± 3.33a 368.66 ± 18.27c 35.36 ± 3.32a 644.34 ± 6.04a 85.09 ± 3.56a 86.75 ± 7.41a 2923.56 ± 38.66b 312.26 ± 4.28b 2795.20 ± 121.96b 2461.16 ± 98.03b 151.54 ± 4.28a 3588.98 ± 37.11c 981.66 ± 13.87c 178.10 ± 7.78a 567.02 ± 62.56a 422.51 ± 71.01a 344.45 ± 16.30a 1648.88 ± 58.21c 410.07 ± 45.99b 2088.27 ± 156.02c 1318.96 ± 168.79c 280.68 ± 40.56a 216.78 ± 12.95c 154.06 ± 9.18b 163.13 ± 98.00b 179.70 ± 6.52a 360.92 ± 20.89b 176.67 ± 14.80b 448.59 ± 27.44b 5665.60 ± 159.97b 1866.55 ± 80.56b

Note: Data presented are the means ± standard error of pooled data (n=3). The letters a, b, and c in superscript indicate the significant differences between the cold storage times of postharvest toon buds according to Duncan’s multiple range test at P < 0.05.

to the biosynthesis of various secondary metabolite pathways (some unigenes were reused in several KEGG pathways), which were sorted into 23 subcategories, with phenylpropanoid biosynthesis (ko00940), terpenoid backbone biosynthesis (ko00900), and carotenoid biosynthesis (ko00906) representing the largest subcategories (Supplementary file 2). These data provided a rich source of information for further mining particular genes associated with the quality and flavor of postharvest toon buds.

Table 2 Summary of sequencing, assembly, and annotation of postharvest toon bud transcriptome. Sequencing statistics

BYC 0

BYC 3

BYC 5

Total raw reads (Mb) Total clean reads (Mb) Total clean bases (Gb) % GC content Assembly statistics Number of contigs Unigenes

74.60 65.52 6.55 41.52

77.12 65.68 6.57 41.06

69.65 65.21 6.52 41.13

81 614 50 665 All 152 127 973 1 690

100 185 64 355

97 275 62 738

Total number of transcripts Average transcript length N50 transcript length Range of transcript lengths Number of transcripts < 500pb Number of transcripts ≥ 500pb Annotation statistics Annotated transcripts by Nt Annotated transcripts by Nr Annotated transcripts by SwissProt Annotated transcripts by KEGG Annotated transcripts by KOG Annotated transcripts byInterpro Annotated transcripts by GO Annotated transcripts byIntersection Total annotated transcripts

3.4. DEGs of postharvest toon buds during cold storage Among the annotated unigenes, 96 379 genes were expressed in three samples at different cold storage times; and 5 316, 5 485, and 5 464 genes were found to be expressed specifically at BYC0, BYC3, and BYC5, respectively (Fig. 4A). Digital gene expression profile analysis was performed to identify the DEGs in BYC0, BYC3, and BYC5. The DEGs between two samples were identified by comparing those in BYC3 and BYC0, in BYC5 and BYC0, and in BYC5 and BYC3 (Fig. 4B). Specifically, 29 513 DEGs, including 15 542 upregulated and 13 971 downregulated genes, were identified in BYC3 relative to BYC0. The functional annotations and statistics for all DEGs between BYC3 and BYC0 are in Supplementary file 3. The functions of the 29 513 DEGs were classified by Blast2GO (Fig. S1). Among the BPs, the dominant subcategories included metabolic processes, cellular processes, and single-organism processes. In CC, a large number of subcategories included membrane, cell, cell part, and membrane part. Catalytic activity and binding were dominant in the MF section. GO enrichment analysis suggested that the upregulated DEGs related to biological regulation, regulation of BP, signaling in BP section, DNA binding, and nucleic acid binding transcription factor activity were more than the downregulated ones in the MF section. However, the downregulated genes were greater in number than the updownregulated ones in the CC section. As for

72 295 79 832 89 800 (59.03 %) 104 027 (68.38 %) 66 666 (43.82 %) 73 739 (48.47 %) 77 422 (50.89 %) 84 693 (55.67 %) 68 669 (45.14 %) 37 034 (24.34 %) 110 077 (72.36 %)

metabolism category (42 054), followed by genetic information processing (15 180), organismal systems (3 721), cellular processes (3 712), and environmental information processing (3 406), whereas those transcripts related to the category of human diseases (318) represented a minority (Fig. 3). Notably, 3 398 transcripts of toon buds were related 4

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Fig. 1. KOG functional classification of unigenes in toon buds.

signal transduction in KEGG pathways (Fig. S6) were more than the downregulated ones.

most of the DEGs in KEGG enrichment classification, the metabolic pathways and biosynthesis of secondary metabolites constituted more downregulated DEGs than upregulated ones, whereas the number of upregulated DEGs in plant-pathogen interaction and plant hormone signal transduction was more than that of downregulated ones (Fig. S2). Through a comparison of two libraries between BYC5 and BYC0, 16 091 upregulated genes and 18 519 downregulated genes were identified between the two samples. A total of 34 610 DEGs were identified between BYC5 and BYC0, and this number exceeded that between BYC3 and BYC0. The statistical information for these DEGs is summarized in Supplementary file 4. The results of the GO functional classification between BYC5 and BYC0 were similar to those for the comparison of BYC3 and BYC0. GO enrichment analysis indicated that approximately half of the DEGs were upregulated and that half were downregulated in the 5 day library (Fig. S3). In KEGG classification, the number of downregulated genes was significantly higher than that of upregulated ones in most of the groups, except for the plant hormone signal transduction group (Fig. S4). A total of 9 770 DEGs were counted between BYC5 and BYC3. The functional annotations and statistical information of all 9 770 DEGs were summarized in Supplementary file 5. GO functional classification indicated that the most of the 9 770 DEGs were involved in metabolic processes, cellular processes, and single organism processes in the BP section; in cell and cell parts in the CC section; and in catalytic activity and binding in the MF section. GO enrichment analysis and KEGG classification showed that most of these genes were downregulated at BYC5, whereas those upregulated genes related to nucleic acid binding transcription factor activity in GO analysis (Fig. S5) and plant hormone

3.5. Identification of putative structural genes with potential relevance to terpenoid backbone biosynthesis pathway Despite the availability of qualitative and quantitative research on terpenoids in toon buds, structural genes with potential relevance to biosynthetic pathways remain largely unknown (Hsu et al., 2012; Wang et al., 2014). The analysis of the transcriptome data showed that 84 unigenes coded for six enzymes in the mevalonate (MVA) pathway were located in the cytoplasm and that 76 unigenes coded for seven enzymes in the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway were located in plastid, which synthesized the common precursor isopentenyl diphosphate (IPP) for downstream terpenoid pathways (Fig. 5). Similar to those in other organisms, the structural genes for upstream terpenoid backbone biosynthesis pathway were presented as multi-members in toon buds (Supplementary file 6). For example, 18 unigenes were predicted to code for hydroxymethylglutaryl-CoA reductase (HMGR), which is the first key regulatory enzyme in the MVA pathway to form MVA. Twenty unigenes were predicted to code for 1-deoxy-D-xylulose5-phosphate synthase (DXS), which catalyzes the conversion of pyruvate and D-glyceraldehyde 3-phosphate into 1-deoxy-D-xylulose-5phosphate in the first step in the MEP pathway. Four unigenes were predicted to code for isopentenyl-diphosphate delta-isomerase (IPPI), which catalyzes the isomerization of IPP to dimethylallyl diphosphate. Forty-nine unigenes were annotated to encode prenyltransferases in terpenoid pathways: fifteen unigenes were for geranyl diphosphate 5

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Fig. 2. GO classification of unigenes in toon buds.

indicated that they were arranged into four main clades (Fig. 6). Among the eight putative TPSs, four TPSs (CL15101.Contig2_All annotated to TPS11, CL24441.Contig3_All annotated to germacrene D synthase, CL364.Contig2_All annotated to α- farnesene synthase, and CL16320.Contig1_All annotated to β-caryophyllene synthase) were clustered with an available Humulus lupulus sesquiterpene synthase (HlSTS1, B6SCF5.1). CL10756.Contig1_All was annotated in NCBI as a homologous sequence of limonene synthase, which was gathered with H. lupulus monoterpene synthase (HlMTS1, B6SCF3.1) in the same group. Two unigenes (CL3842.Contig4_All, Unigene1152_All) and one unigene (CL2079 Contig10_All) were found to be probably involved in diterpenoid and triterpenoid biosynthesis, respectively. CL3842.Contig4_All and Unigene1152_All were clustered with Citrus clementina entkaur-16-ene synthase, whereas CL2079 Contig10_All exhibited a close relationship with C. sinensis and Glycine max squalene synthesis.

synthase (GPPS), seven unigenes were for farnesyl pyrophosphate synthase (FPPS), and twenty-seven unigenes were for geranylgeranyl pyrophosphate synthase.

3.6. Identification of putative terpene synthase (TPSs) in toon bud transcriptome TPS is a multigene family of enzymes responsible for the biosynthesis of monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), or triterpenes (C30) with prenyl diphosphates (GPP, GGPP or FPP) as substrates. The specific gene families of TPS enrich the diversity of terpene carbon skeletons. All members of TPS were searched against toon bud unigenes by using PFAM motif PF01397 (N-terminal TPS domain) (Finn et al., 2008). As a result, 106 unigenes were blasted to putative TPS, and 27 of these unigenes were found to contain fulllength coding sequences. To understand their possible functions and evolutionary relationship with other species, we performed phylogenetic analysis and then aligned eight typical putative protein sequences to their homologs in other species with Mega 6.0 software. The results 6

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Fig. 3. Categories of KEGG metabolism pathways of unigenes in toon buds.

synthase (HMGS), CL21706.Contig1_All 3-hydroxy-3-methylglutarylCoA reductase (HMGR), CL19752.Contig1_All mevalonate kinase (MK), CL1120.Contig8_All phosphomevalonate kinase (PMK), CL6705.Contig5_All diphosphomevalonate decarboxylase (PMDC), CL2905.Contig2_All DXS, CL7187.Contig1_All 1-deoxy-D-xylulose 5phosphate reductoisomerase (DXR), CL16321.Contig2_All 2-C-methylD-erythritol 4-phosphate cytidylyltransferase (MCT), CL5593.Contig9_All 4-(Cytidine 5′-diphospho)-2-C-methyl-D-erythritol

3.7. Quantitative PCR validation and expression analysis of candidate genes involved in terpenoid pathway during cold storage To confirm the low temperature response of candidate genes related to terpenoid biosynthesis and examine their expression patterns during cold storage, we selected 20 unigenes for qRT-PCR analysis. The unigenes included CL2590.Contig5_All acetyl-CoA C-acetyltransferase (AACT), CL24228.Contig2_All 3-hydroxy-3-methylglutaryl-coenzyme A

Fig. 4. Distribution of genes of postharvest toon buds at different cold storage times. A: Venn diagram illustrating the expression patterns of genes for different cold storage days (left). B: number of DEGs in each cold storage day (right). 7

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Fig. 5. Biosynthetic pathway of monoterpenes and sesquiterpenes.

Fig. 6. Phylogenetic tree of terpene synthases. Phylogenetic analysis of five putative T. sinensis TPS protein sequences with their homologs from other plants indicates that they are clustered into three main clades, namely, monoterpenoid synthase, sesquiterpenoid synthase, and diterpenoid synthase.

these candidates detected by qPCR were consistent with the results of the RNA-Seq analysis (Supplementary file 7), suggesting that the RNASeq results were accurate and reliable.

kinase (CMK), CL20567.Contig1_All 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MDS), CL2842.Contig2_All 4-hydroxy-3-methylbut-2-enyl-diphosphate synthase (HDS), CL7336.Contig1_All 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR), CL12867.Contig1_All IPPI, CL2082.Contig4_All geranyl diphosphate synthase (GPPS), CL5749.Contig3_All farnesyl diphosphate synthase (FPPS), CL10756.Contig1_All limonene synthase (LS), CL16320.Contig1_All (E)-β-farnesene synthase (BFS), CL24441.Contig1_All germacrene D synthase (GDS), and CL2079.Contig1_All squalene synthase (SQS). The results showed that those unigenes encoding AACT, HMGS, HMGR, MK, PMK, PMDC, DXS, DXR, MCT, CMK, MDS, HDS, and IPPI were significantly upregulated with increasing cold storage time (Fig. 7). Especially at BYC3, the expression levels of AACT, HMGS, PMK, PMDC, DXR, MCT, and IPPI were remarkably higher than those at BYC0. At BYC5, the expression levels of HMGR, MK, DXS, and CMK were further enhanced. However, those unigenes encoding GPPS, FPPS, LS, GDS, and SS showed different change trends, and their relative expression levels apparently declined during cold storage stages. Interestingly, some unigenes related to monoterpenoid biosynthesis, such as GPPS and LS, were expressed at much lower levels at BYC3 and BYC5 compared with those encoding sesquiterpenoid biosynthesis (FPPS, BFS, and GDS). The mRNA levels of

4. Discussion Toon bud is an important woody vegetable that is highly valuable commercially for its unique aromatic flavor and nutrition. However, postharvest toon buds deteriorate quickly due to their high water content and antioxidant component under room temperature storage, leading to rapid bud browning, wilting within a day, and seriously reduced market value (Zhu and Gao 2018). Cold storage effectively slows down toon bud browning and reduces nutrition loss. However, low temperatures induce chilling injury and ultimate quality deterioration (Zhao et al., 2018). Toon bud flavor is an important quality attribute that affects consumer acceptability. Toon bud flavor constitutes numerous volatile compounds, approximately 20 of which actually contribute to flavor. Most volatile compounds include monoterpenes and sesquiterpenes, which determine flavor quality. In general, volatile compounds in toon buds are directly related to consumer acceptability. Thus, the flavor of toon buds must be preserved during storage. However, to the best of our knowledge, the effects of cold storage time on 8

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Fig. 7. Expression profiles of putative candidate genes related to terpenoid biosynthesis of toon buds during low-temperature storage. Error bars represent standard errors of 3 biological replicates of 15 toon buds. The bars with different letters are significantly different at P < 0.05. The gene names, gene ID in transcriptome data, and primer sequences used for qRT-PCR analysis are shown in Supplementary file 1.

the flavor compound composition of toon buds have not been widely studied. In our present work, volatile terpenoid compounds, except for monoterpene and some sesquiterpenes, were strengthened in postharvest toon buds after cold storage. Brizzolara et al. (2018) reported that volatile organic compounds generally accumulate under low-temperature conditions. Morales (2014) also observed that the raspberry “Maravilla” cultivar undergoes increased total volatile compound content during low-temperature storage. Our results were consistent with their studies. However, contrasting findings show that aromas in “Nanguo” pears and raspberry “Sevillana” cultivar show decreased response to low-temperature storage (Shi et al., 2018; Morales et al., 2014). These results showed that different behaviors for diverse cultivars were subjected to low-temperature storage. Notably, terpenoid oxides in toon buds significantly increased under low-temperature storage possibly due to the following. First, volatile terpenoids in toon buds can be spontaneously oxidated when exposed to air. Lan’s et al. (2016) showed terpene oxides of grapes increase during over-ripening due to spontaneous oxidation, particularly at sub-zero temperatures. Second, some unigenes encoding CYP450, which efficiently convert terpenoid into its oxide, were highly expressed following cold storage (data not shown). To explore the molecular mechanism of flavor compound accumulation in toon buds during low-temperature storage, we performed a global survey of transcriptome profiles during cold storage periods via RNA-Seq. The results showed that 221.37 Mb raw reads were obtained and subsequently assembled into 152 127 fin. l unigenes with an average length of 973 bp. The unigenes generated were more than those

previously obtained for two cultivars T. sinensis (66 331) (Zhao et al., 2017), suggesting that numerous novel unigenes were generated in toon buds under low-temperature storage. For gene annotation, 152 127 unigenes were blasted and annotated to existing open databases, including Nt, Nr, SwissProt, KEGG, KOG, Interpro, GO, and Intersection. Furthermore, 110 077 unigenes were identified by BLASTx, and 48.91% of the unigenes showed high homology to those of C. sinensis, reflecting a close evolutionary relationship between the two species. Through comparison of gene expression among three toon bud samples, 29 513 DEGs were revealed. The DEGs were enriched in 20 pathways involved in photosynthesis, biosynthesis of secondary metabolites, and plant hormone signal transduction. According to the flavor compound analysis, terpenoid biosynthesis was explored to reveal the molecular mechanism of volatile terpenoid accumulation. Terpenoid metabolism in plants has been widely studied, and many genes involved in the pathway have been identified (Niu et al., 2015; Vranová et al., 2013). In the present study, 159 unigenes were involved in terpenoid backbone biosynthesis, which included two metabolic pathways, that is, the pathways of MVA in the cytoplasm and those of MEP in the plasmid. For downstream terpenoid pathways, that is, monoterpene, sesquiterpene, diterpenoid, and triterpenoid biosynthesis, 106 unigenes encoding putative TPSs were found in the toon bud transcriptome. The number of the TPSs identified seemed to account for the diversity of terpenoid components in T. sinensis. Phylogenic tree analysis of the putative TPSs showed that most of them were clustered with sesquiterpene synthase, which was consistent with the fact that sesquiterpenes are the most abundant terpenoids related to flavor in T. 9

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Construction Universities in Zhejiang Province (No. ZYAOX2018027), and the Innovation Program for College Students (No. 201810371031). We would like to thank Professor Wei Shu and Doctor Liu Jinjin from the State Key Laboratory of Tea Plant Biology and Utilization for providing the support needed to detect volatile terpenoids in toon buds by GC–MS.

sinensis. Twenty unigenes involved in terpenoid biosynthesis were selected for time-dependent expression profiling by qPCR under low-temperature storage conditions. Accordingly, gene-specific primers were used, as designed in Table S1. Seven genes from the MVA pathway, seven genes from the MEP pathway, one FPPS gene, one GPPS gene, one MTS gene, two STS gene, and one SS gene were selected for validation. A distinct differential expression pattern of these genes was observed. Compared with the control (0 day of cold storage), these genes involved in the MVA and MEP pathways showed higher expression levels after 3 or 5 days of cold storage. IPP is a major precursor of terpenoid biosynthesis in toon buds. The upregulated expression of genes related to terpenoid backbone biosynthesis promoted the accumulation of the IPP precursor of terpenoids biosynthesis, which contributed to the increase of volatile terpenoid compounds. However, the downstream genes of terpenoid backbone biosynthesis, namely, GPPS, FPPS, MTS, STS, and SS, which mediated the biosynthesis of monoterpene, sesquiterpene, and other terpenoids in the branched metabolic pathway, presented lower gene expression levels in toon buds at 3 or 5 days of cold storage in comparison with the control. As shown in Fig. 7, the relative expression levels of GPPS and MTS related to monoterpene biosynthesis declined more obviously than those of FPPS, STS, and SS related to sesquiterpene and other terpenoids biosynthesis. The result indicated that after 3 or 5 days of cold storage, the genes of the key enzymes FPPS, STS, and SQS in the sesquiterpene pathway maintained higher expression levels than those in the monoterpene pathway, resulting in the biosynthesis of additional sesquiterpenes in postharvest toon buds at 3 or 5 days of cold storage. Moreover, the high activation level of terpenoid backbone biosynthesis, together with the decrease in monoterpene pathway, might have contributed to the high biosynthesis level of sesquiterpene and other terpenoids. Our study showed that the differential expression of genes involved in the terpenoids biosynthesis pathway led to different accumulation patterns of volatile terpenoid components during postharvest cold storage. However, why terpenoid backbone biosynthesis had a high activation level in postharvest toon buds remains unknown and must be further investigated in future studies.

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5. Conclusion We employed high-throughput next-generation sequencing technology to obtain the transcriptome data of postharvest toon buds in response to low-temperature storage. The results will benefit the identification of genes involved in volatile terpenoid compound accumulation in postharvest toon buds. In addition, the present work constitutes the largest unigene dataset of toon buds during low-temperature storage and will thus help researchers in exploring the genes involved in toon buds and their response to low-temperature storage. Furthermore, some unigenes associated with terpenoid biosynthesis showed significant differential expression, implying that these potential unigenes may contribute to volatile terpenoid compound accumulation in postharvest toon buds during low-temperature storage. Thus, the transcriptome sequence generated in this study represents a valuable resource for further research, such as functional genomics, evolutionary analyses, and breeding of T. sinensis that are rich in flavor components. Declaration of Competing Interest The authors have no conflicts of interest to declare. Acknowledgments This work was supported by grants from the Natural Science Key Foundations of the Anhui Bureau of Education (No. KJ2019A0515), the Open Fund of Advantaged and Characteristic Disciplines (Traditional Chinese Medicine of Zhejiang Chinese Medical University) for Key 10