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  • Research article
  • Open Access

Effects of vitro sucrose on quality components of tea plants (Camellia sinensis) based on transcriptomic and metabolic analysis

  • †1, 2,
  • †3,
  • †1,
  • 3,
  • 1,
  • 1,
  • 1,
  • 1,
  • 3,
  • 1,
  • 3Email author and
  • 1Email author
†Contributed equally
BMC Plant Biology201818:121

https://doi.org/10.1186/s12870-018-1335-0

©  The Author(s). 2018

  • Received: 18 March 2018
  • Accepted: 31 May 2018
  • Published:

Abstract

Background

Tea plants [Camellia sinensis (L.) O. Kuntze] can produce one of the three most widely popular non-alcoholic beverages throughout the world. Polyphenols and volatiles are the main functional ingredients determining tea’s quality and flavor; however, the biotic or abiotic factors affecting tea polyphenol biosynthesis are unclear. This paper focuses on the molecular mechanisms of sucrose on polyphenol biosynthesis and volatile composition variation in tea plants.

Results

Metabolic analysis showed that the total content of anthocyanins, catechins, and proanthocyanidins(PAs) increased with sucrose, and they accumulated most significantly after 14 days of treatment. Transcriptomic analysis revealed 8384 and 5571 differentially expressed genes in 2-day and 14-day sucrose-treated tea plants compared with control-treated plants. Most of the structural genes and transcription factors (TFs) involved in polyphenol biosynthesis were significantly up-regulated after 2d. Among these transcripts, the predicted genes encoding glutathione S-transferase (GST), ATP-binding cassette transporters (ABC transporters), and multidrug and toxic compound extrusion transporters (MATE transporters) appeared up regulated. Correspondingly, ultra-performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-QQQ-MS/MS) analysis revealed that the content of non-galloylated catechins and oligomeric PAs decreased in the upper-stem and increased in the lower-stem significantly, especially catechin (C), epicatechin (EC), and their oligomeric PAs. This result suggests that the related flavonoids were transported downward in the stem by transporters. GC/MS data implied that four types of volatile compounds, namely terpene derivatives, aromatic derivatives, lipid derivatives, and others, were accumulated differently after in vitro sucrose treatment.

Conclusions

Our data demonstrated that sucrose regulates polyphenol biosynthesis in Camellia sinensis by altering the expression of transcription factor genes and pathway genes. Additionally, sucrose promotes the transport of polyphenols and changes the aroma composition in tea plant.

Keywords

  • Camellia sinensis, Polyphenol biosynthesis, Volatile
  • Sucrose induction
  • Transcriptomic and metabolic analysis

Background

The tea plant [Camellia sinensis (L.) O. Kuntze] is one of the most important economic crops cultivated in China, Japan, India, and other countries. Its leaves are used for making the tea beverage, one of three most widely consumed non-alcoholic beverages around the world because it contains abundant polyphenols, theanine, caffeine, and other secondary metabolites [1]. Among them, the polyphenol, also called tea polyphenol, is a collective term for phenolic acids and flavonoids including flavanols (catechins), anthocyanins, PAs (also named condensed tannins), and other special derivatives. Polyphenols account for 18–36% of the dry weight of tender leaves and are responsible for tea’s flavor [2–4]. Some studies have suggested that polyphenols play crucial roles in plant stress resistance. For example, they are crucial for protecting the tea plant against pathogens and insects [5, 6]. Additionally, polyphenols are the main functional ingredient in tea for preventing cancer, cardiovascular diseases, and obesity [7].

Studies have indicated that polyphenol biosynthesis in plants is influenced by chemical and physical factors, such as nutrients, hormones, and environmental conditions [8–13]. Among them, sucrose acts not only as carbon source for energy storage and sugar transportation, but also as a signal involved in metabolic processes such as anthocyanin synthesis in plants [14, 15]. Since the late twentieth century, the effects of sucrose on flavonoid and anthocyanin biosynthesis in grapes and radishes have been studied [16–18]. In Arabidopsis thaliana, sucrose induces anthocyanin biosynthesis through the upregulation of structural genes and positive transcription factors involved in the flavonoid biosynthesis pathway and potentially also through the concurrent down-regulation of the negative transcription factor, MYB-LIKE 2 (MYBL2) [19–21]. Previous studies also reported that sucrose could act as a signaling molecule, by first activating PRODUCTION OF ANTHOCYANIN PIGMENT 1 (PAP1) expression by a sucrose-specific signaling pathway and then triggering the expression of structural genes involved in anthocyanin and flavonoid biosynthesis [14, 19, 22, 23]. The sucrose-specific signaling pathway may be activated by different disaccharides, such as sucrose, maltose, and their breakdown products (glucose and fructose); however, sucrose is the most effective inducer of anthocyanin biosynthesis in Arabidopsis [23]. Liu et al. reported sucrose induction increases the content of non-galloylated catechins and up-regulates the expression of putative genes involved in their biosynthetic pathway in both tea callus and seedling [24]. Additionally, Wang et al. also reported sucrose up-regulates the expression of Camellia SINENSIS FLAVONOID 3′5′-HYDROXYLASE (CsF3′5′H), an important branch point gene involved in catechins biosynthesis [25]. In this study, test-tube tea plantlets were used to test for testing the effects of sucrose on polyphenol biosynthesis after 2, 7, 14, and 28d treatments. The results indicated that sucrose can increase the expression of structural genes involved in the biosynthesis of anthocyanins, catechins, and procyanidins. The sucrose specific induction machenism in tea plant is still unclear, one important reason is that we lack the information supported by accurate genome annotations.

Next-Generation Sequencing (NGS) based on the Illumina Hiseq 2000 platform provides a fast, cost-effective, and reliable approach to acquire abundant transcripts, especially for non-model organisms without reference genomic sequences [26]. In tea plants, the NGS technology has been used for analysis of putative genes associated with tea quality and stress response [27–29]. Here, it was performed to investigate the molecular mechanism of sucrose on polyphenol biosynthesis in tea plants and to provide a comprehensive analysis of the network of biochemical and cellular processes responding to sucrose.

In addition, we determined whether in vitro sucrose treatment affects the production of volatiles—the second group of compounds that affect tea taste and flavor in addition to polyphenols.

Results

Effects of sucrose on polyphenol accumulation

Similar sized test-tube tea plantlets were cultured on Murashige and Skoog standard medium (MS, Control) and MS supplemented with 90 mM sucrose (MS + 90 mM sucrose, Suc) for 28d (Fig. 1a). The stem of the plantlets grown on Suc for 9-14d began to turn red (Fig. 1b), while no red pigmentation was observed in the stem of the plantlets grown on MS or MS supplemented with 90 mM mannitol (data not shown). The anthocyanin levels were significantly different only in the lower part of the stem and were 7-fold higher than that in the control (Fig. 1c). Furthermore, the accumulation of total catechins and PAs in various organs of tea plants is affected by sucrose (Fig. 1d). The effects of sucrose treatment on polyphenol accumulation were observed after 7 and 14 days of treatment (Fig. 1d). However, the effects of sucrose on total catechins and PAs accumulation were not observed at 2d treatment (data not shown).
Fig. 1
Fig. 1

Effects of sucrose on polyphenol accumulation in test-tube tea plantlets. a. Test-tube tea plantlets; b. Red pigments accumulated in stems of plantlet after feeding sucrose; c. Anthocyanin levels are significantly different in the lower part of the stem; d. Accumulation of total catechins and PAs in various organs after 7, 14 and 28 d sucrose treatment. Note: * indicates significance at P < 0.05. The data represents the mean value of three biological replicates

Polyphenol, including phenolic acids, catechin monomers, oligomeric PAs, and flavonols, in different tissues of tea plantlets after 14d treatment was quantitatively measured using UPLC-QQQ-MS/MS (Table 1). Three types of phenolic acids were measured, including quinic acid, gallic acid derivatives (β-glucogallin, galloyl acid and galloylquinic acid), and hydroxycinnamic acid derivatives (caffeoylquinic acid and p-coumaroylquinic acid). The effect of sucrose on compound accumulation was different. For example, sucrose increased the content of galloylquinic acid, a special phenolic acid in the tea plant, increased in most parts of the plants, except for in the bud. However, the content of β-glucogallin, the precursor of galloylated catechins, significantly decreased by 84% in buds and by 71% in upper stems [30]. Monomers of flavanols (catechins) can be classified into non-galloylated and galloylated catechins and mainly exist in buds and upper stems. More non-galloylated catechins accumulated in buds and lower stems after sucrose treatment; however, their content in upper stems decreased significantly. Catechin (C) and epicatechin (EC) decreased by 69% in upper stems. The galloylated catechin content in buds and lower stems was not affected by sucrose, and its content in the 3rd leaf and upper stem decreased by 19%. Seven types of oligomeric PAs accumulated in the bud and 3rd leaf. Their content in lower stems increased 3-fold. However, their content in upper stems significantly decreased after sucrose treatment. For example, B2 (an oligomeric C or EC), decreased by 81%. The content of flavonols in the tea plant was also affected by sucrose. Among them, the flavonol with di-hydroxyl groups on the B-ring was significantly affected by sucrose, and its amount decreased by almost 40% in the third leaf and upper stems and by 14% in buds. However, its content increased by 1-fold in the lower stem.
Table 1

Effects of sucrose on polyphenol accumulation in different tissues of tea plantlets after 14d treatment using UPLC-QQQ-MS/MS

Compound

Control

Suc

ratio

Control

Suc

ratio

Control

Suc

ratio

Control

Suc

ratio

bud

bud

 

3rd leaf

3rd leaf

 

up-stem

up-stem

down-stem

down-stem

Phenolic acid (mg/g)

 Quinic acid

44.21 ± 2.01

86.06 ± 4.05

1.95

6.55 ± 0.23

7.45 ± 0.35

1.14

39.43 ± 1.89

40.19 ± 1.70

1.02

3.72 ± 0.15

6.19 ± 0.29

1.67

Gallic acid derivatives

  β-glucogallin

9.42 ± 0.41

1.47 ± 0.11

0.16

0.90 ± 0.05

0.97 ± 0.05

1.08

2.83 ± 0.12

0.81 ± 0.03

0.29

0.02 ± 0.00

0.01 ± 0.00

0.73

  galloyl acid

0.38 ± 0.01

0.36 ± 0.02

0.95

0.08 ± 0.00

0.10 ± 0.01

1.19

0.25 ± 0.01

0.16 ± 0.01

0.65

0.20 ± 0.01

0.03 ± 0.00

0.13

  galloylquinic acid

14.09 ± 0.9

13.29 ± 0.7

0.94

0.13 ± 0.01

0.47 ± 0.05

3.76

3.55 ± 0.16

6.71 ± 0. 32

1.89

0.09 ± 0.00

0.11 ± 0.01

1.15

  Summation

23.88 ± 1.32

15.12 ± 0.83

0.63

1.11 ± 0.06

1.54 ± 0.11

1.40

6.64 ± 0.29

7.68 ± 0.36

1.16

0.31 ± 0.01

0.14 ± 0.01

0.47

Hydroxycinnamic acids derivatives

  caffeoylquinic acid

0.16 ± 0.01

0.14 ± 0.01

0.90

0.14 ± 0.01

0.02 ± 0.00

0.17

0.12 ± 0.01

0.06 ± 0.00

0.52

ND

ND

 

  p-coumaroylquinic acid

2.29 ± 0.12

3.44 ± 0.15

1.51

ND

0.51 ± 0.02

 

0.45 ± 0.04

2.11 ± 0.13

4.65

ND

ND

 

  Summation

2.45 ± 0.13

3.59 ± 0.16

1.47

0.14 ± 0.01

0.53 ± 0.02

3.93

0.57 ± 0.05

2.17 ± 0.13

3.78

ND

ND

 

Flavanols (mg/g)

NongalloylatedCatechins

  catechin

2.79 ± 0.12

3.74 ± 0.16

1.34

0.86 ± 0.04

2.59 ± 0.13

3.02

5.51 ± 0.26

1.71 ± 0.08

0.31

0.99 ± 0.04

3.03 ± 0.13

3.06

  epicatechin

3.64 ± 0.21

6.26 ± 0.29

1.72

3.37 ± 0.15

3.81 ± 0.19

1.13

8.73 ± 0.31

2.75 ± 0.11

0.31

3.02 ± 0.13

4.47 ± 0.15

1.48

  gallocatechin

1.00 ± 0.06

2.66 ± 0.12

2.66

1.54 ± 0.08

2.30 ± 0.11

1.49

1.43 ± 0.06

1.34 ± 0.07

0.93

0.24 ± 0.01

1.05 ± 0.06

4.36

  epigallocatechin

13.91 ± 0.8

26.89 ± 1.20

1.93

10.47 ± 0.62

7.93 ± 0.38

0.76

13.99 ± 0.80

12.11 ± 0.71

0.87

4.24 ± 0.15

3.19 ± 0.14

0.75

  Summation

21.34 ± 1.19

39.55 ± 1.77

1.85

16.23 ± 0.89

16.63 ± 0.71

1.02

29.67 ± 1.36

17.90 ± 0.97

0.60

8.49 ± 0.33

11.74 ± 0.48

1.38

Galloylatedcatechins

  epicatechingallate

22.38 ± 1.09

20.75 ± 1.01

0.93

3.82 ± 0.15

3.58 ± 0.15

0.94

11.08 ± 0.84

9.29 ± 0.83

0.84

2.18 ± 0.13

2.05 ± 0.98

0.94

  epigallocatechingallate

89.03 ± 4.21

95.88 ± 4.67

1.08

18.29 ± 0.95

14.26 ± 0.68

0.78

52.19 ± 2.65

42.19 ± 2.05

0.81

6.30 ± 0.31

6.46 ± 0.31

1.03

  Summation

111.40 ± 5.30

116.63 ± 5.68

1.05

22.11 ± 1.10

17.84 ± 0.83

0.81

63.27 ± 3.49

51.48 ± 2.88

0.81

8.47 ± 0.44

8.51 ± 1.29

1.00

  total Catechins

132.74 ± 6.49

156.18 ± 7.45

1.18

38.34 ± 1.99

34.47 ± 1.85

0.90

92.94 ± 4.85

69.38 ± 3.85

0.75

16.97 ± 0.77

20.25 ± 1.77

1.19

Proanthocyanidins (area)

 m/z 865

ND

ND

 

ND

ND

 

ND

ND

 

490 ± 36

6057 ± 312

12.36

 m/z 577 PAs B2

33,626 ± 1670

52,158 ± 2600

1.55

17,040 ± 850

36,819 ± 1830

2.16

122,564 ± 6115

23,153 ± 1160

0.19

37,345 ± 1876

155,893 ± 7805

4.17

 m/z 729EC-ECG

17,582 ± 880

18,560 ± 930

1.06

2125 ± 105

3947 ± 185

1.86

15,214 ± 755

7597 ± 380

0.50

2089 ± 117

10,067 ± 515

4.82

 m/z 593EC-EGC or ECDG

2300 ± 110

6507 ± 320

2.83

5361 ± 260

17,280 ± 855

3.22

16,556 ± 815

5454 ± 267

0.33

3475 ± 184

20,418 ± 1015

5.88

 m/z 761EGC-EGCG

11,308 ± 565

21,097 ± 1050

1.87

3841 ± 180

4698 ± 225

1.22

6627 ± 325

9111 ± 438

1.37

909 ± 55

4932 ± 264

5.43

 m/z 745

3570 ± 178

5468 ± 270

1.53

2062 ± 105

3916 ± 185

1.90

3806 ± 185

3133 ± 148

0.82

ND

3992 ± 196

 

 m/z 609(EGC-EGC)

3809 ± 190

11,528 ± 570

3.03

11,219 ± 550

32,040 ± 1505

2.86

11,924 ± 585

5195 ± 246

0.44

2501 ± 129

17,566 ± 868

7.02

flavonols derivatives (area)

tri-hydroxyl in B-ring

  myricetin 3-O-galactoside

3929 ± 203

5100 ± 268

1.30

705 ± 42

ND

 

ND

2367 ± 123

 

ND

269 ± 12

 

  myricetin 3-O- glucoside

6797 ± 346

6940 ± 359

1.02

1220 ± 58

1301 ± 72

1.07

3577 ± 185

3404 ± 164

0.95

ND

260 ± 10

 

  Summation

10,726 ± 549

12,040 ± 627

1.12

1925 ± 100

1301 ± 72

0.68

3577 ± 185

5771 ± 287

1.61

ND

529 ± 30

 

di-hydroxyl in B-ring

  quercetin 3-O-galactosylrutinoside

2539 ± 136

2235 ± 126

0.88

780 ± 48

489 ± 34

0.63

2465 ± 131

1025 ± 55

0.42

806 ± 45

684 ± 28

0.85

  quercetin3-O-glucosylrutinoside

9680 ± 496

8675 ± 456

0.90

3933 ± 208

2379 ± 126

0.60

5641 ± 291

3847 ± 184

0.68

793 ± 45

2704 ± 136

3.41

  quercetin 3-galactoside

1404 ± 87

1367 ± 78

0.97

428 ± 30

ND

 

1376 ± 62

674 ± 31

0.49

290 ± 18

208 ± 12

0.72

  quercetin 3-O-glucoside

2465 ± 138

1630 ± 89

0.66

911 ± 42

850 ± 45

0.93

1284 ± 58

783 ± 45

0.61

168 ± 7

526 ± 32

3.14

  Summation

16,088 ± 857

13,907 ± 749

0.86

6052 ± 328

3717 ± 201

0.61

10,766 ± 542

6330 ± 315

0.59

2056 ± 115

4122 ± 208

2.00

mono -hydroxyl in B-ring

  kaempferol-3-O-galactosylrutinoside

338,752 ± 16,950

290,468 ± 14,530

0.86

61,932 ± 3085

39,007 ± 1968

0.63

137,928 ± 6870

130,099 ± 6485

0.94

23,498 ± 1164

18,979 ± 1001

0.81

  kaempferol3-O-glucosylrutinoside

853,325 ± 42,664

753,945 ± 37,665

0.88

206,694 ± 10,345

120,862 ± 6055

0.58

316,408 ± 15,808

334,177 ± 16,675

1.06

23,691 ± 1135

37,778 ± 1982

1.59

  kaempferol-3-O-galactoside

ND

933 ± 59

 

154 ± 10

287 ± 28

1.86

447 ± 31

484 ± 30

1.08

ND

255 ± 10

 

  kaempferol-3-O-glucoside

ND

20,072 ± 1008

 

1491 ± 85

ND

 

6994 ± 350

9054 ± 446

1.29

496 ± 71

199 ± 9

0.40

  Kaempferol-3-O-rhamnosylgalactoside

25,559 ± 1289

26,315 ± 1315

1.03

11,173 ± 560

5296 ± 276

0.47

9333 ± 456

11,450 ± 564

1.23

1567 ± 76

2223 ± 124

1.42

  Summation

1,217,636 ± 60,903

1,091,733 ± 54,577

0.90

281,445 ± 14,085

165,452 ± 8327

0.59

471,109 ± 23,515

485,263 ± 24,200

1.03

49,252 ± 2396

59,434 ± 3126

1.21

 total flavonols

1,244,449 ± 62,309

1,117,680 ± 55,953

0.90

289,422 ± 14,513

170,470 ± 8600

0.59

485,453 ± 24,242

497,364 ± 24,802

1.02

51,309 ± 2511

63,556 ± 3334

1.24

Note: ND indicates that the polyphenol was not detected; the data represents the mean value of three biological replicates

Digit indicates the ratio of Suc / Control

Effects of sucrose on volatile compounds

Four types of volatile compounds were measured using GC/ MS, including terpene derivatives, aromatic derivatives, lipid derivative and other compounds, the effect of sucrose on their accumulation was different (see Additional file 1: Table S1). For example, the content of α-farnesene belonging to sesquiterpenoid diterpenoid increased 5.77-fold; the expression of one transcript (Unigene 46,443), which was predicted as the key biosynthetic gene encoding farnesene synthase, was significantly upregulated 3-fold after 2 and 14 days of sucrose treatment (see Additional file 2: Table S2). Here, 33 terpene derivatives were detected and classified into monoterpenoid sesquiterpenoid diterpenoid; these compounds were biosynthesized via methylerythritol phosphate (MEP) and mevalonate (MVA) pathways (see Additional file 3: Figure S1). The expression of HMGR (CL12062.Contig1) and DXS (Unigene57617) and DXR (Unigene46601) as the key genes involving in terpenoid backbone pathway were up-regulated by sucrose. The expression of one transcript (CL1850.Contig3 encoding linalool synthase) was not significantly affected by sucrose; and the content of linalool and geraniol in tea leaf only decreased by 4%. Additionally, the expression of 1 transcript (Unigene9305 encoding (E)-nerolidol synthase) was up-regulated by sucrose after 2d; however, its expression was down- regulated by sucrose after 14d; and the content of the (E)-nerolidol only decreased by 5%.

Effects of sucrose on the expression of key structural genes related to polyphenol biosynthesis using qRT-PCR

For further analysis of the effects of sucrose on polyphenol biosynthesis at the transcriptional level, Quantitative real-time-PCR (qRT-PCR) was used to test the expression of 11 key structural genes involved in the polyphenol biosynthetic pathway (Fig. 2). Their expression significantly increased 3-fold after 2d treatment. After 7d, the expression of Chalcone synthase (CHS), Flavanone 3-hydroxylase (F3H), Flavonoid 3′-hydroxylase (F3′H), Leucoanthocyanidin reductase (LAR), and Anthocyanidin reductase (ANR) increased 1-fold. After 14d, the effect of sucrose on the above genes was less noticeable.
Fig. 2
Fig. 2

Effects of sucrose on expression of key structural genes involved in polyphenol biosynthesis using qRT-PCR. Note: * indicates significance with |log2 Ratio| ≥ 1. The data represents the mean value of three biological and three technical replicates

Sequencing, de novo assembly, and functional annotation

To obtain the overall transcriptional levels of genes in the tea plant treated by sucrose after 2 and 14d, four normalized cDNA libraries (2d: 2nd D Control and Suc; 14d: 14th D Control and Suc) were constructed for transcriptome sequencing. Based on the Illumina Hiseq 2000 platform, 21,381,193,620 nucleotide (nt) bases were generated from all libraries in total and about 237.6 million clean reads (94.94% of the raw reads) were achieved for de novo assembly (see Additional file 4: Table S3). Finally, a total of 118,843 transcripts were obtained with an average length of 1212 nt and a N50 of 1999 nt (see Additional file 5: Table S4).

To predict the functions of the assembly transcripts, a total of 82,459 transcripts (69.38% of all assembled Unigenes) were annotated using the NR (Non-redundant protein database), NT (Non-redundant nucleotide database), Swiss-Prot (Annotated protein sequence database), KEGG (Kyoto encyclopedia of genes and genomes), COG (Clusters of orthologous groups of protein), and GO (Gene ontology) databases based on two levels of sequence similarity, sequence-based and domain-based alignments, with an e-value<1e-5 (see Additional file 6: Table S5).

Analysis of DEGs responding to sucrose

Using the fragments per kb per million reads (FPKM) method, the DEGs between two samples were identified with a significant threshold of |log2 Ratio (FPKM Control-vs-Suc) | ≥ 1 and the false discovery rate (FDR) of ≤0.001 based on the P-value threshold set as ≤1e-5. A total of 8384 DEGs were detected in 2nd D Control-vs-Suc. Among them, 6187 DEGs (73.80% of the total DEGs) were up-regulated. A total of 5571 DEGs were detected in 14th D Control-vs-Suc, and only 2146 DEGs (38.52% of the total DEGs) were up-regulated (see Fig. 3).
Fig. 3
Fig. 3

Statistics of DEGs from tea plants responding to sucrose. Note: DEGs were classified into two classes; the red bar indicates up-regulated and the green bar indicates down-regulated, the digit indicates the number of DEGs

GO function and KEGG pathways analysis of DEGs responding to sucrose

To better understand the biological functions of DEGs responding to sucrose, GO and KEGG analyses were performed for comparisons of 2nd D Control-vs-Suc and 14th D Control-vs-Suc. GO functional enrichment analysis indicated that 49 and 48 GO terms were classified into three ontologies which changed significantly between 2nd D and 14th D Control-vs-Suc (see Additional file 7: Figure S2).

A total of 3553 DEGs (7.46% of all the transcripts aligned to the KEGG database) were annotated and 29 KEGG pathways were enriched significantly in the 2nd D Control-vs-Suc comparison based on a Q-value of ≤0.05. Among them, the most enriched pathway was “flavonoid biosynthesis” (Table 2). In 14th D Control-vs-Suc comparison, 2009 DEGs (4.22% of all the transcripts aligned to KEGG databases) were annotated and 20 KEGG pathways were significantly enriched with the same threshold. The most enriched pathway was that for “plant-pathogen interaction” (Table 3). A total of 17 KEGG-enriched pathways were common between second and fourteenth D Control-vs-Suc. Of the 12 KEGG pathways specific to the second D Control-vs-Suc comparison, one was the KEGG-enriched pathway for anthocyanin biosynthesis (Fig. 4).
Table 2

Gene ontology analysis of DEGs obtained from tea plants treated by sucrose after 2d

 

Pathway

DEGs genes

All genes

Q-value

(3553)

(47655)

1

Flavonoid biosynthesis

87 (2.45%)

314 (0.66%)

2.35E-25

2

Biosynthesis of secondary metabolites

530 (14.92%)

4746 (9.96%)

1.33E-20

3

Phenylpropanoid biosynthesis

124 (3.49%)

653 (1.37%)

1.76E-20

4

Stilbenoid, diarylheptanoid and gingerol biosynthesis

63 (1.77%)

233 (0.49%)

3.38E-18

5

Flavone and flavonol biosynthesis

44 (1.24%)

165 (0.35%)

1.41E-12

6

Phenylalanine metabolism

52 (1.46%)

234 (0.49%)

1.76E-11

7

Plant hormone signal transduction

291 (8.19%)

2615 (5.49%)

4.76E-11

8

Zeatin biosynthesis

63 (1.77%)

365 (0.77%)

5.88E-09

9

Cutin, suberine and wax biosynthesis

30 (0.84%)

116 (0.24%)

1.65E-08

10

Pentose and glucuronateinterconversions

70 (1.97%)

452 (0.95%)

6.26E-08

11

DNA replication

44 (1.24%)

244 (0.51%)

4.79E-07

12

Carotenoid biosynthesis

40 (1.13%)

212 (0.44%)

4.95E-07

13

Limonene and pinene degradation

34 (0.96%)

170 (0.36%)

1.05E-06

14

Metabolic pathways

902 (25.39%)

10,454 (21.94%)

1.79E-06

15

Ether lipid metabolism

130 (3.66%)

1142 (2.4%)

8.47E-06

16

Starch and sucrose metabolism

129 (3.63%)

1141 (2.39%)

1.24E-05

17

Diterpenoid biosynthesis

22 (0.62%)

105 (0.22%)

6.04E-05

18

Tryptophan metabolism

22 (0.62%)

107 (0.22%)

7.84E-05

19

Other glycan degradation

47 (1.32%)

328 (0.69%)

8.46E-05

20

Endocytosis

156 (4.39%)

1526 (3.2%)

2.40E-04

21

Glycerophospholipid metabolism

160 (4.5%)

1577 (3.31%)

2.69E-04

22

Glucosinolate biosynthesis

15 (0.42%)

64 (0.13%)

3.18E-04

23

Isoflavonoid biosynthesis

15 (0.42%)

72 (0.15%)

1.25E-03

24

Plant-pathogen interaction

309 (8.7%)

3440 (7.22%)

1.60E-03

25

Monoterpenoid biosynthesis

10 (0.28%)

41 (0.09%)

3.38E-03

26

Anthocyanin biosynthesis

6 (0.17%)

20 (0.04%)

1.26E-02

27

Cysteine and methionine metabolism

40 (1.13%)

339 (0.71%)

1.27E-02

28

Base excision repair

29 (0.82%)

228 (0.48%)

1.51E-02

29

Homologous recombination

36 (1.01%)

323 (0.68%)

4.46E-02

Table 3

Gene Ontology analysis of DEGs obtained from tea plants treated by sucrose after 14d

 

Pathway

DEGs genes

All genes

Q-value

(2009)

(47655)

1

Plant-pathogen interaction

275 (13.69%)

3440 (7.22%)

3.78E-23

2

Phenylpropanoid biosynthesis

64 (3.19%)

653 (1.37%)

3.04E-08

3

Zeatin biosynthesis

41 (2.04%)

365 (0.77%)

6.03E-07

4

Flavonoid biosynthesis

37 (1.84%)

314 (0.66%)

6.41E-07

5

Plant hormone signal transduction

159 (7.91%)

2615 (5.49%)

5.74E-05

6

Stilbenoid, diarylheptanoid and gingerol biosynthesis

26 (1.29%)

233 (0.49%)

1.37E-04

7

Biosynthesis of secondary metabolites

256 (12.74%)

4746 (9.96%)

3.87E-04

8

Diterpenoid biosynthesis

15 (0.75%)

105 (0.22%)

5.25E-04

9

Glycerophospholipid metabolism

96 (4.78%)

1577 (3.31%)

3.06E-03

10

DNA replication

23 (1.14%)

244 (0.51%)

3.55E-03

11

Phenylalanine metabolism

22 (1.1%)

234 (0.49%)

4.47E-03

12

alpha-Linolenic acid metabolism

17 (0.85%)

164 (0.34%)

5.98E-03

13

Starch and sucrose metabolism

71 (3.53%)

1141 (2.39%)

7.18E-03

14

Isoflavonoid biosynthesis

10 (0.5%)

72 (0.15%)

7.18E-03

15

Limonene and pinene degradation

17 (0.85%)

170 (0.36%)

7.18E-03

16

Monoterpenoid biosynthesis

7 (0.35%)

41 (0.09%)

1.12E-02

17

Ether lipid metabolism

69 (3.43%)

1142 (2.4%)

1.39E-02

18

Nitrogen metabolism

18 (0.9%)

203 (0.43%)

1.68E-02

19

Phosphatidylinositol signaling system

33 (1.64%)

465 (0.98%)

1.74E-02

20

Flavone and flavonol biosynthesis

15 (0.75%)

165 (0.35%)

2.62E-02

Fig. 4
Fig. 4

The pathways significantly enriched by DEGs after 2d and 14d sucrose treatment. Note: the horizontal coordinates indicate percent of DEGs, the vertical coordinates indicate significantly enriched pathways of differentially expressed genes

Effects of sucrose on polyphenol biosynthesis based on transcriptome sequencing

Based on the ratio of FPKM Control-vs-Suc, most of the transcripts involved in the phenylpropanoid and flavonoid pathways were up-regulated 2-fold or more after 2d of treatment. Additionally, the expression of transcripts annotated as Phenylalanine ammonialyase (PAL), Dihydroflavonol 4-reductase (DFR), LAR, and Anthocyanidin synthase (ANS) was notably up-regulated. After 14 days of treatment, the expression of only PALB increased 1-fold, whereas others were not affected by sucrose (Fig. 5). These results indicate that tea polyphenol biosynthesis is comprehensively affected by sucrose.
Fig. 5
Fig. 5

Effects of sucrose on the expression of structural genes related to polyphenol biosynthesis in tea plants after 2d and 14d. Note: Red indicates significant up-regulation, blue indicates no difference, green indicates significant down-regulation. Digit indicates the number of Unigenes

Effects of sucrose on the expression of transcription factors involved in polyphenol biosynthesis based on transcriptome sequencing

Polyphenol biosynthesis in plants is regulated by transcription factors (TFs) including R2R3-MYB, bHLH, and WD40 [31, 32]. In this study, 37 DEGs were predicted to be MYB members and were classified into three types: R1 (4 DEGs), R2R3 (29 DEGs), and R1R2R3 (4 DEGs). Most DEGs (23/37) were up-regulated after sucrose treatment for 2 days, and only five DEGs were up-regulated after sucrose treatment for 14 days (Table 4). Additionally, the phylogenetic tree, including 29 R2R3-MYBs and 126 Arabidopsis R2R3-MYBs, were classified into 13 subgroups (see Additional file 8: Figure S3). Phylogenetic analysis indicated that 33 bHLHs were dispersed into 15 subfamilies (see Additional file 9: Figure S4), and 21 of them were up-regulated after sucrose treatment for 2d (Table 5).
Table 4

Analysis of DEGS-predicted as R2R3-MYB obtained from tea plants treated by sucrose

Gene ID

Gene

2ndD

14thD

Type

Subgroups

Putative function clade and gene function

length

fold

fold

No.

CL5525.Contig4

955

476.9a

–

R2R3

other

Trichome development-regulated: AtMYB82 [69]

Unigene18972

1084

17.02a

0.41b

R1R2R3

Unknown

 

Unigene35962

3506

13.97a

0.49b

R1R2R3

Unknown

Unigene12085

975

13.54a

0.32b

R2R3

6

Anthocyanin biosynthes-related: AtMYB75and AtMYB90 [54, 70, 71]

Unigene41846

938

4.98a

–

R2R3

6

Secondary cell wall formation-related: AtMYB75 [72]

Unigene35958

3304

6.28a

–

R1R2R3

Unknown

 

CL8695.Contig1

1179

5.47a

–

R2R3

5

Seed pigmentation biosynthesis -controlled: AtMYB123 [48, 73]

Unigene11002

1229

2.93a

–

R2R3

5

 

Unigene7972

1143

5.41a

–

R2R3

9

Seed germination and reproductive development-related AtMYB17 [74, 75]

CL1441.Contig4

2364

2.85a

–

R2R3

9

Petal development: AtMYB16 [76]

 

Repressor of cell outgrowth: AtMYB106 [77]

Unigene24177

714

4.91a

–

R2R3

other

 

Unigene20350

1829

2.20a

–

R2R3

other

CL12359.Contig1

3219

2.56a

–

R2R3

other

CL5017.Contig2

1322

4.04a

0.34b

R2R3

1

Hypersensitive response: AtMYB30Cooperates with BES1 to regulate

CL8708.Contig1

1933

2.91a

–

R2R3

1

brassinosteroid-induced gene Expression; abiotic stress response, SA–mediated pathway AtMYB30 [77]

Unigene13855

767

3.84a

–

R2R3

15

Epidermal cell fate specification: AtMYB23 [78]

Trichome development: AtMYB0 and AtMYB23,

CL7877.Contig1

887

3.25a

–

R2R3

15

Root hair patterning-controlled AtMYB66 [79]

Unigene1868

527

2.68a

–

R1

Unknown

 

Unigene16731

1118

2.41a

–

R2R3

14

Axillary meristem initiation in roots-related: AtMYB36 [80]

CL3134.Contig13

4926

2.40a

–

R1R2R3

Unknown

 

CL13057.Contig1

995

2.31a

–

R2R3

4

The battle against UV by repressing C4H: AtMYB4 [81]

CL13057.Contig2

827

–

2.64a

R2R3

4

 

CL2339.Contig1

1129

2.24a

–

R2R3

21

Lignin, xylan and cellulose biosynthesis-regulated: AtMYB52, AtMYB54 and AtMYB69 [82]

 

Ovule and fruit development: AtMYB117 [83]

ABA hypersensitivity and drought tolerance: AtMYB52 [84]

CL8255.Contig3

1314

–

2.02a

R2R3

7

Flavonol glycosides-related: AtMYB11, AtMYB12 and AtMYB111 [34]

CL6408.Contig3

1494

2.01a

–

R2R3

2

Shoot apex morphogenesis: AtMYB13 [85]

CL9344.Contig1

1068

–

0.25b

R2R3

2

Cold stress tolerance: AtMYB14 and AtMYB15 [86, 87]

CL6408.Contig1

1557

–

0.45b

R2R3

2

 

CL5350.Contig2

1322

–

0.16b

R2R3

2

Unigene48919

574

0.41b

–

R2R3

2

CL1581.Contig2

1552

–

0.18b

R1

Unknown

CL7764.Contig2

980

–

0.15b

R1

Unknown

Unigene6794

537

–

2.47a

R2R3

other

Unigene36358

1700

–

2.01a

R2R3

other

AS1 leaf morphogenesis (polarity specificity) and plant immune response: AtMYB91 [88];

 

Rough-sheath development: AtMYB91 [89]

Unigene11308

1618

–

2.10a

R2R3

13

Stomatal closure: AtMYB61 [90];

 

Multiple aspects of plant resource allocation-controled: AtMYB61 [91]

Unigene38120

1427

–

0.47b

R2R3

22

Stomatal closure-regulated: AtMYB44,AtMYB70, AtMYB73 and AtMYB77 [92, 93]

 

Auxin signaling pathway- modulated: AtMYB77 [94];

Unigene39226

735

0.49b

–

R2R3

20

GA metabolism and signaling involved in regulation starvation responses:AtMYB62 [95];

 

Cell separation processes-related: AtMYB116 [96]

Unigene2945

935

0.44b

–

R1

Unknown

 

Note: “a”indicates significant up-regulation; “–”indicates no difference; “b”indicates significant down-regulation. Unknown and other indicate Unigene is not grouped

Table 5

Analysis of DEGS-predicted as bHLH obtained from tea plants treated by sucrose

GeneID

Gene

2ndD

14thD

Subfamily

Gene name

Putative function clade and gene function

length

fold

fold

No.

in Arabidopsis

Unigene60798

496

1967.8a

–

3

AtbHLH18

 

Unigene26720

1512

15.20a

–

 

AtbHLH25

CL2783.Contig8

2320

280.50a

–

25

AtbHLH74

Regulation root growth: AtbHLH74 [97]

CL4342.Contig3

2304

2.02a

–

  

CL9935.Contig2

1894

7.50a

0.42b

25

AtbHLH137

Unigene21382

845

4.85a

–

25

AtbHLH63

Unigene29122

545

8.35a

2.14a

1

AtbHLH33

Cold tolerance: AtbHLH33,AtbHLH116(ICE1),AtbHLH61and AtbHLH93 [98]

 

AtbHLH116

Stomatal differentiation: AtbHLH33(ICE2)and AtbHLH116 [99];

AtbHLH61

Drought stress:AtbHLH116(ICE1) [100]

,AtbHLH93

 

CL1034.Contig1

3358

–

0.30b

1

AtbHLH35

CL1034.Contig2

889

–

0.27b

 

AtbHLH27

Drought stress:bHLH27 [100]

CL1034.Contig5

942

–

0.27b

AtbHLH29

Iron Uptake-regulated:AtBHLH29 [101]

CL1768.Contig1

648

4.33a

–

10

AtbHLH57,

 
 

AtbHLH67,

AtbHLH70

CL12543.Contig1

1074

3.58a

–

10

AtbHLH71

CL9545.Contig2

1190

2.38a

–

10

AtbHLH94

CL9545.Contig1

813

2.31a

–

 

AtbHLH96

Unigene17438

326

2.29a

–

 

CL13089.Contig1

2067

0.37b

–

10

AtbHLH57

Unigene32633

1085

3.54b

–

9

AtbHLH91

 

AtbHLH10

AtbHLH89

Unigene10835

1585

0.34b

–

26

AtbHLH69

Female gametophyte development;

 

AtbHLH66

Response to phosphate deficiency stress:AbHLH69, AbHLH66 [53]

Unigene2520

732

2.89a

–

16

AtbHLH135

 

Unigene5385

844

2.74a

–

5

AtbHLH42

Anthocyanin biosynthesis (GL3, EGL3, TT8) [53]

Unigene21617

2490

2.35a

–

 

Regulate proanthocyanidin biosynthesis [49, 51]

Unigene23312

1076

2.49a

–

13

AtbHLH106

Abiotic stress-involved in cold, salt, ABA and drought stress:

 

AtbHLH107

AtbHLH106 [102]

Unigene47124

874

2.47↑

0.43b

27

AtbHLH128,

 

Unigene39259

789

–

0.00b

 

AtbHLH129

Regulation root elongation and ABA response:AtbHLH129 [103]

 

AtbHLH80

 

AtbHLH81

AtbHLH122

Drought and osmotic stress tolerance, ABA catabolism repression: AtbHLH122 [100]

 

AtbHLH130

 

Unigene28617

886

2.23a

–

15

AtbHLH133

 

AtbHLH68

CL8951.Contig3

2042

–

0.30b

15

AtbHLH123

Unigene38437

809

2.20a

–

19

AtbHLH149

CL496.Contig1

889

2.19a

–

31

AtbHLH140

Unigene20853

1750

–

2.87a

31

AtbHLH87

Flower and fruit development, initiation/maintenanceofaxillary

meristems [53]

CL2917.Contig5

3168

–

0.28b

2

AtbHLH3

Male fertility-affected:AtbHLH3(JAM3) [104]

Unigene63328

1505

–

4.65a

2

AtbHLH14

 

CL10048.Contig2

1395

–

0.05b

7

AtbHLH92

Tolerance to NaCl and osmotic stresses: bHLH92 [105]

CL1061.Contig1

2440

–

0.10b

7

AtbHLH41

 

Note:“a”indicates significant up-regulation; “–”no difference; “b” indicates significant down-regulation

The R2R3-MYBs, bHLH, and WD40 TFs, could act as regulators of polyphenol biosynthesis individually or jointly. The R2R3-MYBs in Subgroup (Sg) 4 and Sg7 were predicted to be negative and positive regulators, respectively, for controlling the production of flavonols via regulating the up-stream genes involved in polyphenol biosynthetic pathway [33, 34]. However, the R2R3-MYBs in Sg5 and Sg6 require both bHLH (subfamily 2, 5, and 24) and WD40 for construction into a ternary complex MYB-bHLH-WD40 (MBW) for positively regulating down-stream genes involved in polyphenol biosynthetic pathway [31, 35, 36]. Here, 7 DEGs were classified into the above mentioned 4 subgroups of R2R3-MYBs. After 2d sucrose treatment, the expression of 3 DEGs (Unigene12085, Unigene 41,846 and CL8695 Contig1) in Sg6 and Sg5 were significantly up-regulated 6-fold; and the expression of CL13057.Contig2 in Sg4 was down-regulated significantly (Fig. 6a). Additionally, 2 DEGs (Unigene 21,617, Unigene 5385) in Subfamily 5 of bHLHs were up-regulated by sucrose (Fig. 6b). Based on the same method, only one transcript (Unigene25483) was predicted to be involved in the MBW complex, and its expression was not affected by sucrose (Fig. 6c).
Fig. 6
Fig. 6

Effects of sucrose on the expression of R2R3-MYB (a), bHLH (b) and WD40 (c) involved in polyphenol biosynthesis. Note: The phylogenetic tree was constructed based on amino acid sequences using MEGA5 according to the neighbor-joining method. GenBank accession numbers: MYB-Sg4: AtMYB4 (AEE86955), HlMYB1 (CAI46244), DvMYB2 (BAJ33514), GmMYBZ2 (ABI73970); MYB-Sg5: OsMYB3 (BAA23339), AtMYB123 (Q9FJA2), GhMYB38 (AAK19618); MYB-Sg6: AtMYB75 (AEE33419), AtMYB90 (AEE34503); MYB-Sg7: AtMYB11 (XP_002876680), AtMYB12 (O22264), AtMYB111 (XP_002865729), VvMYBF1 (ACV81697), MdMYB22 (AAZ20438), LjMYB12 (BAF74782). bHLH-Sg5: AtbHLH12 (Q8W2F1), AtbHLH42 (Q9FT81), AtbHLH1 (Q9FN69), AtbHLH2 (Q9CAD0), DrMYC1 (AEC03343), Vv_010152 (CAN62848.1); bHLH-Sg2: AtbHLH3 (O23487), AtbHLH14 (O23090), GmMYC2like (XP_003528771), VvMYC4 (XP_002279973), Pt_002299425 (XP_002299425). WD40: PFWD (BAB58883), InWDR (BAE94407), PhAN11 (AAC18914), VvWDR1 (NP_001268101), MdTTG1 (ADI58760), AtTTG1 (CAB45372), ZmPAC1 (AAM76742)

Effects of sucrose on the expression of genes involved in polyphenol transport

In plants, transporters (ABCs and MATEs), and GSTs are involved in polyphenol transporting. These transporters are found in many species including Arabidopsis TT19 and TT12 genes (AtTT19; AtTT12), the grape GST and ABCC1 genes (VvGST19; VvABCC1), the maize MRP3 gene (ZmMRP3), and the Medicago truncatula MATE (MtMATE) [37–42]. In the present study, 22, 15, and 21 DEGs were predicted to encode GST, ABC, and MATE-transporters, respectively. Phylogenetic analysis showed three transcripts closely corresponding to the above 3 transporters (Fig. 7). Among them, the expression of the ABC (CL11884.Contig7) and MATE (Unigene47970) decreases significantly by sucrose after 2d, and their expression increases after 14d (Additional file 10: Table S6). However, the expression of the GST (Unigene24131) responds to sucrose opposite of the above mentioned two transcripts (Additional file 10: Table S6). The above results indicate there could be different transporters and GSTS for transporting the polyphenol in tea plants.
Fig. 7
Fig. 7

Effects of sucrose on the expression of Unigenes encoding transporters related to flavonoid. a. Glutathione S-transferase;b. ABC transporters;c. mate transporters. Note: The phylogenetic tree was constructed based on amino acid sequences using MEGA5 according to the neighbor-joining method. All protein sequences used in this figure were provided in Additional file 13: Txt S1

Using qRT-PCR for transcriptome sequencing validation

To validate the results of transcriptome sequencing, 30 DEGS were randomly selected to be analyzed by qRT-PCR. We found that 83.33% of the total transcripts expression was consistent with the results from transcriptome sequencing, including 11 genes involved in polyphenol biosynthesis. Detailed information regarding the selected DEGs and 11 genes is presented in Additional file 11: Figure S5.

Discussion

The mechanisms of sucrose effects on tea polyphenol biosynthesis

In the past decades, exploration of tea polyphenol biosynthesis and their influencing factors have become a hotspot for research in plant secondary metabolism [30, 43]. Due to self-incompatibility, rich genetic diversity, and the large genome in tea plants, little genomic information is available and the molecular mechanisms of tea polyphenol biosynthesis are still unclear [44, 45]. Our previous research demonstrated tea polyphenol shared a similar biosynthetic pathway to other plants, such as shikimic acid, phenylpropanoid, and flavonoids synthetic pathways [2]. Its biosynthesis is also affected by sucrose, light, and other factors [24, 46].

Studies have demonstrated sucrose-specific transcriptional regulation of polyphenol biosynthesis in plants. For example, Boss et al. reported that the expression of DFR involved in anthocyanin and PAs biosynthesis in grape was induced by sucrose treatment, and they speculated that the accumulation of the two metabolites in grape berry skin could be attributed to sugar accumulation during grape berry development [47]. According to microarray data, it was revealed that anthocyanin biosynthesis in Arabidopsisis is stimulated by sucrose which acts as a signal to activate PAP1, a TF for activating the expression of structural genes involved in anthocyanin biosynthetic pathway, such as PAL, Cinnamate 4-hydroxylase (C4H), 4-coumaroyl-CoA ligase (4CL), and others [19, 23]. However, the structural gene F3′5′H and transcriptional factor PAP2 are not affected by sucrose [19]. In tea plants, Wang et al. found the expression of Cs F3′5′H increased 15-fold by feeding sucrose [25]. Liu et al. reported that sucrose induced the accumulation of catechins and upregulated the expression of putative genes involved in their biosynthetic pathway [24]. In this study, the total content of catechins and PAs significantly increases by sucrose induction for 7d and the accumulation of anthocyanin increases 7-fold in the stems of tea plantlets after 14d sucrose treatment. Only after 2d treatment, the expression of structural genes involved in their biosynthesis is up-regulated based on qRT-PCR and transcriptome sequencing. After 14d, the effects of sucrose were not detected.

In Arabidopsis, the correct expression of BANYULS (BAN) as a key gene of PAs biosynthesis is necessary for activation of TT2 (AtMYB123, an R2R3-MYB TF encoded by the TRANSPARENT TESTA2 gene) and TT8 (AtbHLH42, a bHLH TF encoded by the TRANSPARENT TESTA8 gene) together with TTG1 (AtTTG1, a WD-repeat protein encoded by the TRANSPARENTTESTA GLABRA1gene) [48–50]. TT2 cannot be replaced by any other AtMYB [51]. Additionally, the genes of Sg4, 5, 6, and 7 R2R3-MYB and the Subfamily2, 5, and 24 bHLH are all involved in flavonoid biosynthesis [35, 52]. Based on their amino acid sequence alignment, it was found that 7 R2R3-MYB and 4 bHLH are predicted to participate in flavonoid biosynthesis in tea plants [53]. In the present study, seven DEGs were classified into the aforementioned four subgroups of the R2R3-MYBs and four DEGs into bHLH subfamilies 5 and 2. Among them, the expression of 3 transcripts (Unigene12085, Unigene41846, and CL8695.Contig1) in R2R3-MYB Sg6 and Sg5 were up-regulated 6-fold; this finding is consistent with those of studies indicating that sucrose can induce the expression of PAP1/MYB75, which is essential for sucrose-induced anthocyanin biosynthesis [19, 23, 48, 54]. In addition, Unigene5385 corresponded to TT8 and its expression was significantly increased by sucrose treatment for 2d, indicating that it might be involved with others in regulating the accumulation of anthocyanins and PAs [55, 56]. Notably, only one transcript (Unigene25483) corresponds closely to AtTTG1, consistent with the results reported in C. sinensis [53]. However, it was not affected by sucrose, possibly because WD40 proteins have no catalytic activity and act as docking platforms for MYB and bHLH proteins in regulating flavonoid biosynthesis [48, 51, 53, 57].

As described above, it is inferred the accumulation of tea polyphenol might be directly due to high expression of their structural genes which could be synergistically regulated by TFs.

The mechanisms of sucrose effects on tea polyphenol transport

Based on analysis of UPLC-QQQ-MS/MS, the non-galloylated catechins and oligomeric PAs were significantly induced by sucrose in bud, 3rd leaf, and lower stems after 14d treatment; however, their content in upper stems decreased significantly, especially C, EC, and their oligomeric PAs. This suggests there was flavonoid transport in tea plants. Extensive research shows GST, ABC, and MATE transporters could be involved in flavonoid transport and there are at least three mechanisms, GST-linked, Vesicle trafficking (VT), and MATE transporters [38, 39, 42, 58–61]. In the present study, only three transcripts annotated as GST, ABC, and MATE were involved in flavonoid transport, and their expression was differently affected by sucrose. As described above, it is inferred that there are varieties of proteins for synergistically transporting tea polyphenol in tea plants. However, the molecular mechanisms remain unclear.

Impact of sucrose on the volatile

It is known that the flavor of tea is basically determined by taste (non-volatile compounds) and aroma (volatile compounds) [62]. The tea polyphenol is crucial for tea taste, and the terpene derivatives including monoterpenoid and sesquiterpenoid are important aroma ingredient due to their delectable fruit fragrance and low detection threshold [63]; for example, linalool and geraniol have fruity and sweet floral scents [62]. Previous research indicated that linalool, geraniol, nerolidol, ionone, and jasmone were identified as odour-active in many types of green teas [64, 65]. In the present study, (Z)-jasmone and β-ionone content increased by 2.63 and 0.57-fold, respectively; however, linalool, geraniol and nerolidol were not significantly affected by sucrose. As the biosynthetic pathway volatile compounds is complicated, and the molecular mechanisms involving in volatile compounds affected by sucrose need to be further studied.

Conclusions

In this paper, the test-tube tea plantlets were used for investigating the effects of sucrose on polyphenol biosynthesis. Metabolomics and transcriptomics analyses indicated that sucrose up-regulation of anthocyanins, catechins, and PAs biosynthesis. Sucrose controls the expression of structural and regulating genes. Additionally, sucrose promotes the transport of polyphenol in Camellia sinensis by the predicted transporters GST, ABC, and MATE involved in polyphenol transport. In summary, these results and analyses present valuable resources for better understanding the biosynthesis molecular mechanisms underlying the main characteristics of secondary metabolites in the tea plant and help improve the nutritional quality of tea.

Methods

Plant materials and cultivation conditions

The test-tube tea plantlets [Camellia sinensis (L.) O. Kuntzevar. cultivar Nongkangzao] were initially grown in vitro on classical solid MS medium and then transferred to solid MS supplemented with 90 mM sucrose for sucrose feeding studies with 10 h of light (42 μmol/m2 s) at 24 ± 1 °C. Correspondingly, similar sized test-tube tea plantlets were transferred to classical solid MS medium for the control under the same conditions. In the above experiments, the tea plantlets were incubated on MS supplemented with 90 mM mannitol for the osmotic control.

For metabolic analysis of polyphenol, the samples of different organs (the buds, third leaves, and the upper and lower stems) were collected from the tea plantlets cultivated after 2, 7, 14, and 28d. Meanwhile, samples of leaves were also collected from the tea plantlets cultivated after 2, 7, 14 and 28d for analysis of polyphenol biosynthesis at the transcriptional level. All the collected samples were immediately frozen in liquid nitrogen and stored at − 80°Cuntil use. In this study, approximately 10 independent tea plants were collected for one biological replicate; and three biological replicates were used for analysis.

Chemicals and reagents

The compounds viz., quinic acid, β-glucogallin, galloyl acid, galloylquinic acid, caffeoylquinic acid, p-coumaroylquinic acid, catechin, epicatechin, gallocatechin, epigallocatechin, epicatechingallate, epigallocatechingallate, procyanidin B2, myricetrin, quercitrin, and kaempferitrinwere obtained from Sigma (St Louis, MO, USA) and Axxora Co. and Ltd. (Lausanne,Switzerland). Cyanidin chloride was procured from Axxora Co. and Ltd. (Lausanne, Switzerland). HPLC grade acetic acid, methanol, and acetonitrile were bought from Tedia Co., Ltd. (Fairfield, OH, USA). Concentrated hydrochloric acid, vanillin, and other solvents used for extraction were acquired from Sinopharm Chemical Reagent Co., Ltd. (Shang-hai, China).

Extraction and quantitative analysis of the polyphenol

Extraction and quantitative analysis of the polyphenol was performed with UPLC-QQQ-MS/MS as suggested by Jiang et al. [2]. The total catechins were extracted and quantitatively analyzed using 1% vanillin–HCl (w/v) according to the methods described by Wang et al. [66].

Spectrophotometry analysis of anthocyanins was carried out as described by Pang et al. and the molar absorbance of cyanidin-3-O-glucoside was used for calculating the total anthocyanin concentration [67].

The total PAs were extracted and quantitatively analyzed using spectrophotometry by the methods reported by Jang et al. and their concentration was converted by using a standard curve of procyanidin B2 [2].

Extraction and analysis of the volatile compounds

Extraction and analysis of the volatile compounds collected from the samples of the leaves of tea plantlets cultivated after14 d were performed with a headspace-solid phase microextraction (HS-SPME) fiber, coupled with gas chromatography (Agilent 7697A) and mass spectrometry (Agilent 7890A) (GC/MS). In brief, 0.3 g of leaves samples were cut up and put in the 20 ml headspace bottle 4 mL by adding boiling double distilled water dissolved 0.8 g KCl. After incubation for 1.5 min, the volatile compounds were collected using a 50/30 μm DVB/CAR/PDMS SPME fiber (Supelco, PA, USA) for 50 min at 70 °C and then desorbed into the GC injection port at 250 °C for 5 min. Subsequently, the volatile compounds were resolved by BD-5 capillary column (30 m × 0.25 mm × 0.25 μm, Agilent) for GC/MS analysis according to Han et al. [64].

RNA extraction and qRT-PCR analysis

Total RNA was extracted as described by Zhao et al. [53]. The RNA concentration, quality, and integrity were measured by using spectrophotometry (Agilent2100) and gel electrophoresis. The single-stranded complementary deoxyribonucleic acid (cDNA) was synthesized using Prime-Script™ (Takara, Dalian, Code: DRR037A) for qRT-PCR analysis. All the primer sequences were designed using Primer Premier 6.0 and the selected Unigene IDs are detailed in the additional file (see Additional file 12: Table S7). The qRT-PCR assays were performed by using a CFX96™ optical reaction module (Bio-RAD, USA) and the detailed detection system was the same as previously described by Zhao et al. [53]. The resultant relative expression values were normalized against the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and evaluated from the mean value of three biological and three technical replicates by the 2-ΔΔCT method [68].

Library construction, RNA-seq and de novo assembly

Library Construction and de novo assembly were performed by Beijing Genome Institute (BGI; Shenzhen, China). Briefly, the specific operations are summarized as follows: the mRNA isolated from the total RNA was fragmented into smaller pieces to create templates for synthesizing the first-strand cDNA. Using the first-strand cDNA as templates, the double-stranded cDNA was produced with random primers (Japan, Takara). Subsequently, these cDNA fragments were processed by end repair using DNA polymerase and polynucleotide kinase and ligation of adapters to produce approximately 200 bp fragments. Finally, these fragments were purified by using Qiaquick Gel Extraction Kit (Qiagen) and enriched with PCR to construct cDNA libraries.

In this study, four cDNA libraries (2d: 2nd D Control and Suc; 14d: 14th D Control and Suc) were examined by using the Agilent 2100 Bioanalyzer and were sequenced using Illumina HiSeq™ 2000. The clean reads were obtained from the raw reads by removing the low-quality reads and the reads with adaptors or unknown nucleotides larger than 5%. Based on assembly of clean reads separately, Unigenes were the resulting sequences after removing redundancy and short contigs separately using the short reads assembling program–Trinity.

Bioinformatics analysis of the assembled Unigenes

By using BLASTx (E-value 10− 5) against the database of NR, NT, GO, Swiss-Prot, COG, and KEGG, the assembled Unigenes were annotated for functional analysis and their expression levels were calculated by the fragments per kb per million reads (FPKM). Differentially expressed genes (DEGs) were identified with a significant threshold of|log2 Ratio of FPKM (Control-vs-Suc)| ≥ 1 and FDR ≤ 0.001 based on the P-value threshold set as ≤1e− 5. Based on FDR ≤ 0.05, KEGG Pathway analysis was performed to ascertain the main biochemical and signal transduction pathways of DEGs.

Phylogenetic analysis of transcription factors and transport proteins involved in polyphenols

The phylogenetic trees for transcription factors and transport proteins were constructed according to the method as described by Zhao et al. [53]. Briefly, the MEGA 5.0 software was used for the phylogenetic analysis and the neighbor-joining statistical method was carried out based on amino acid sequences. The Bootstrap method with 1000 replicates was performed for evaluating the tree nodes. By using the p-distance method, evolutionary distances were computed. All the sequences used for the alignment were retrieved from The Arabidopsis Information Resource (TAIR, Carnegie Institution for Science Department of Plant Biology, USA), the UniProt Database (UniProt, Switzerland), and the National Center for Biotechnology Information (NCBI, USA).

Availability of supporting data

The transcriptome sequencing data based on the Illumina Hiseq 2000 platform obtained from leaves of Camellia sinensisare available in NCBI SRA (https://www.ncbi.nlm.nih.gov/sra/ with accessions SRR5427581,SRR5427580,SRR5427578 and SRR5427577.

Notes

Abbreviations

4CL: 

4-coumaroyl-CoA ligase

ABCtransporter: 

ATP-binding cassette transporter

ANR: 

Anthocyanidin reductase

ANS: 

Anthocyanidinsynthase

At: 

Arabidopsis thaliana

C: 

Catechin

C4H: 

Cinnamate 4-hydroxylase

cDNA: 

Single-stranded complementary deoxyribonucleic acid

CHI: 

Chalconeisomerase

CHS: 

Chalcone synthase

Cs: 

Camellia sinensis

DEGs: 

Differentially expressed genes

DFR: 

Dihydroflavonol 4-reductase

EC: 

Epicatechin

ECGT: 

Epicatechin:1-O-galloyl-β-D-glucose-O-galloyltransferase

F3′5′H: 

Flavonoid3′,5′-hydroxylase

F3′H: 

Flavonoid 3′-hydroxylase

F3H: 

Flavanone 3-hydroxylase

FDR: 

False discovery rate

FLS: 

Flavonol synthase

FPKM: 

Fragments per kb per million reads

GAPDH: 

Glyceraldehyde-3-phosphate dehydrogenase

GST: 

Glutathione S-transferase

LAR: 

Leucoanthocyanidinreductase

MATE transporter: 

Multidrug and toxic compound extrusion transporter

MBW: 

MYB-bHLH-WD40

MS: 

Murashige and Skoog standard medium

NGS: 

The next-generation sequencing

PAL: 

Phenylalanine ammonialyase

PAP1: 

Production of anthocyanin pigment 1

PAP2: 

Production of anthocyanin pigment 2

PAs: 

Proanthocyanidins

qRT-PCR: 

Quantitative real-time-PCR

Sg: 

Subgroup

TF: 

Transcription factor

TT12: 

Transparent testa 12

TT19: 

Transparent testa19

TT2: 

Transparent testa 2

TTG1: 

Transparent testa glabra1

UGT: 

UDPG-glucosyltransferase

UPLC-QQQ-MS/MS: 

Ultra-performance liquid chromatography-triple quadrupole mass spectrometry

Declarations

Acknowledgements

We would like to thank professor Frank Obrock and Katie Fonseca for professional writing services.

Funding

This work was funded in the framework of the Natural Science Foundation of China (31570694; 31470689; 31300577). LG, the funder of NSF (31570694) and TX, the funder of NSF (31470689) and Specialized Research Fund for the Doctoral Program of Higher Education (20133418130001) conceived and supervised this study. YQ, the funder of the Natural Science Foundation for Higher Education of Anhui Province (KJ2017A441) and the Natural Science Foundation of Suzhou University (2016jb02) performed, designed the experiments and wrote the manuscript. YJL, the funder of NSF(31270730) and Natural Science Foundation of Anhui Province, China (1408085QC51) revised the manuscript. The funders of the Special Foundation for Independent Innovation of Anhui Province, China (13Z03012), the Biology Key Subject Construction of Anhui and ‘Hundred Talents Program’ of the Chinese Academy of Sciences(39391503–7), Anhui Major Demonstration Project for the Leading Talent Team on Tea Chemistry and Health and the Innovative Research Team in University (IRT1101) had no role in the experiment design, data analysis, decision to publish or preparation of the manuscript but supported this study.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors’ contributions

TX and LG conceived and supervised this study. YQ and SZ performed the experiments and designed the experiments. SY designed the GC/MS method and analysed the date. JX and YZL analyzed the data. YQ and YJL wrote and edited this manuscript. XD participated in sample collection. XJ designed the UPLC-QQQ-MS/MS method. WW and ML performed RNA preparation. All authors read and approved the final manuscript.

Competing interest

The authors declare that they have no competing interests.

Ethics approval and consent to participate

Not applicable.

Publisher’s Note

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Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, 130 West Changjiang Rd, Hefei, 230036, Anhui, China
(2)
School of Biological and Food Engineering, Suzhou University, 49 Middle Bianhe Rd, Suzhou, 234000, Anhui, China
(3)
School of Life Science, Anhui Agricultural University, 130 West Changjiang Rd, Hefei, 230036, Anhui, China

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