Outline Tea (Camellia Sinensis) Origin in China

1/25/2019

PAG XXXVII San Diego 2019 Spreading of tea cultivation and consumption

Tea Tree Genome Sequencing Reveals Why Many Health-Beneficial Natural Products Are Concentrated in One Leaf

Outline Major specialized metabolites in one tea leaf

Water water others Components of tea leaves Dry weight (~25%) ~12-24% 1、Tea genome sequencing ~6% Tea Polyphenols (catechins, EGCG) ~5% 1-4% Saccharides (Sucrouse, etc) 2、 Production of major secondary 0.1 1-3% -1% Minerals 46-64% Amino acids (Theanine, Glutamic acid, etc) metabolites Caffeine Triterpenoids (saponins, sterols, …) Insolubles (Polysaccharides, Cellulose, Proteins, pigments, etc)

Theanine, 50-70 % of total amino acids in tea leaves Catechins, 12-24 % dry weight of leaf Caffeine, 0.3 -3 % of dry weight of leaf

Tea (Camellia sinensis) origin in China

 The rich tea tree germplasm, all Camellia species found in China. Camellia Questions unsolved: Section include 12 species and 6 varieties, originated from southwest tropic forests.

 About 2.58 million Acers of tea plantation 1.Why tea plants can synthesize galloylated monomeric catechins ? Accounting for more than 10 - 22 % on dry mass basis EGCG contents account for more than 70 % of total catechins.  China produces all six types of major teas, white, green, black, dark, and yellow. Only steaming green tea and CTC black teas are produced by other 2. How these highly bioactive compounds are stored in the leaves countries. without hurting themselves? Transport and storage

3. How theanine, the fresh and sweet taste-contributing free amino acids, is synthesized in tea leaves?

4. How caffeine biosynthesis is divergenet in tea tree from Cacao and Coffee and other plants ?

1 1/25/2019

Tea plant cultivars (Camellia sinensis (L.) O. Kuntze) ● Landscape of the tea plant genome

Small leaves, wider distribution areas, C. sinensis var. sinensis particularly adapted to north. Suitable (CSS) to make green tea, and other major types of teas. C. sinensis var. assamica (CSA) Large leaves, mainly distributed in tropical or subtropical worm areas. Higher levels of C. sinensis var. dehungensis catechins, suitable for make red or Puer teas， C. sinensis var. pubilimba

CSA genome is sequenced SNP markers in the genetic map (gray; a); SSR density (orange; b); TE density (c; Copia, orange; Gypsy, blue); Gene density (green; d); Transcription factors (black; e); Genes in syntenic blocks between tea and grape (each color for syntenic genes from each grape chromosome; f); and oriented paralogous genes (g). A 1-Mb sliding window was used to calculate the density of different elements.

(B) Estimation of divergence time between CSS and CSA using orthologous gene pairs within collinear blocks. Xia et al., MP, 2017 (C) DNA and protein sequence similarity of orthologous genes between CSS and CSA. The similarity of orthologous genes and coding proteins between CSS and CSA are 92.4% 和93.9%, respectively.

18 BAC libraries Two whole genome duplications in tea plant

● Expansion and contraction of gene families

Heterozygosity:~8 ‰, But Shucazao is low: 2.7‰

3 1 ←1← 2 ← ↑ 2 23 ← → ↓↓↓ 2 ↓ 31

30 Chromosomes in C. sinensis var. sinensis FISH (45srDNA probeset)： Tea ancestor diverged from its closet species kiwifruit lineage at around 80 mya. After that, root somatic cells (CSS) Diploid another WGD event occurred in tea lineage at ~30-40 mya. CSA and CSS diverged from their common ancestor 0.38-1.54 mya. Wei et al., PNAS, 2018

Genome parameters for Chinese and Assamica varieties Catechin biosynthesis:

● Estimated tea plant genome size: ~ 3.0 G ● Statistic comparisons on assembly and annotation of CSS and CAS

Coverage: 92% 85.4%

Wei et al., PNAS, 2018 Xia et al., MP, 2017 Wei et al., PNAS, 2018

2 1/25/2019

The major galloylated flavan-3-ol, EGCG in tea and other plants Enzyme families involved in acylation of plant metabolites

I: UDP-glucose:galloyl-1-O-β-D- Two enzyme families using phenolic compounds govern the acylation of secondary glucosyltransferase (UGGT) compounds:

II: Epicatechin:1-O-galloyl-β-D- I. BAHD-acyltransferases (BAHD-ATs) glucose O-galloyltransferase (ECGT) II. Serine CarboxyPeptidase-Like (SCPL)-acyltransferases (SCPL-ATs)

BAHD-ATs use acyl-CoA thioesters

Liu et al., JBC 2012 287: 44406. SCPL-ATs use 1-O-β-glucose esters Ikegami et al. (2007) as ‘energy-rich’ donor molecules Akagi et al. (2009) Terrier et al. (2009) In persimmon and grapevine Bontpart et al. New Phytologist 2015 208: 695

Biosynthesis of galloylated catechins Tea genome also contains a large family of BAHD family ATs highly expressed may involve SCPL1-A genes in apical buds and young leaves--- correlated with EGCG contents

Among 22 SCPL1A genes in tea genome, 4 SCPL1A generated during 30-40 mya WGD，15 SCPL1A generated by most recent tandem gene duplications.

14 SCPL1A genes are highly expression in young leaves and buds, where galloylated catechins are highly accumulated.

Biosynthesis of galloylated catechins Theanine biosynthesis: CsTS1 is a key enzyme may involve SCPL1-A genes

Among 22 SCPL1A genes in tea genome, 4 SCPL1A generated during 30-40 mya WGD，15 SCPL1A generated by most recent tandem gene duplications.

14 SCPL1A genes are highly expression in young leaves and buds, where galloylated catechins are highly accumulated.

Wei et al., PNAS, 2018

3 1/25/2019

茶氨酸 Proposed biosynthetic pathways for theanine in tea plants

Glutamine synthetase gene tissue expression patterns correlated with theanine

MALS

4 1/25/2019

CsGSs are bifunctional enzymes with both GS and TS activity

Standards Glu+EA+CsGSII-1b CsGSII-1b CsGSII-1b +

Glu+EA Glu+NH4 Abundance

Theanine St +

Glu+NH4 +CsGSII-1b

Abundance Abundance

Retention time (min) 8

Retention time (min) ) 1.5 6

4 1 CsACTIN

2 0.5

(mg/g FW) Abundance

Abundance 0 Theanine contents Expressionlevel 0

0 12 24 48 (Ratio to （） CsGSII-1a- CsGSII-1b- m/z m/z Incubation time h Control 320 45 asODN asODN 10 control 240 CsGSII-1a-asODN 1) 60 7.5 -

30 CsGSII-1b-asODN 1 g 160 5 - 40 15

80 2.5 20

(mg/g FW)

GS GS activity

(Peak (Peak area/hour)

(Peak area/ (Peak area/ hour)

Glutamine content Theanine content

0 Theanine content 0

0 0 (mmolmin 0 0.5 1 1.5 2 CsGSII-1a- CsGSII-1b- 0 0.5 1 1.5 2 0 14 24 Control Enzymes (μg) Enzymes (μg) Incubation time (h) asODN asODN

Convergent synthesis of caffeine in tea plants

Theophylline (1, 3- dimethylxanthine) 7-NMT 1-NMT Degradation 3-NMT

？

Caffeine Theobromine (1,3,7- (3,7- dimethylxanthine) trimethylxanthine) Kato et al., 2000, Nature 406:956 Ogawa et al., 2001, JBC 276:8213

Divergent and convergent evolution of NMTs for caffeine biosynthesis in tea plants

Camellia

Cacao Paralogue pairs

Xia et al. Mol Plant 2017 Wei et al., PNAS 2018 Coffea

5 1/25/2019

Functional redundancy and divergence of NMTs in tea genomes ? Camellia CsCTS1 and CaXMT1 has very different methyltransferase activity

7- 3- 1- X mX mX mX TB Pa TP CF 0.04 0.02

CSSNMT2 CSSNMT3 CSSNMT7 CSSNMT6 CSSNMT4 CSSNMT11 CSSNMT9 CSSNMT5 CSSNMT8 CSSNMT10 CSSNMT1 0.00 -0.02

0.04 0.02 XR

AU TCS1 0.00 -0.02 0.04 X 0.02 AU 7- 3- 1- CaXMT1 0.00 -0.02 X mX mX mX TB Pa TP CF 0.04 0.04 0.02 7-mX 3-NMT activity 0.02

AU 0.00 0.00 -0.02 -0.02

0.04 0.04 0.02 3-mX 1-NMT activity 0.02 XR

AU AU CaXMT1 0.00 0.00 -0.02 -0.02

0.04 0.04 0.02 1-mX 3-NMT activity 0.02 X

0.00 0.00

CSSNMT2 CSSNMT3 CSSNMT7 CSSNMT6 CSSNMT4 CSSNMT11 CSSNMT9 CSSNMT5 CSSNMT8 CSSNMT10 CSSNMT1 -0.02 -0.02 0.04 0.04 0.02 7-mX 0.02 TB 1-NMT activity AU

AU 0.00 0.00 -0.02 -0.02 0.04 0.04 Pa 3 -NMT activity 0.02 3-mX 0.02 AU 0.00

AU 0.00 -0.02 -0.02 0.04 0.04 0.02 1-mX

AU CsXMT2 0.02 TP 7-NMT activity 0.00

AU 0.00 -0.02 CsTCS1 -0.02 0.04 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 0.02 TB 分钟

AU 0.00

-0.02

CSSNMT1 CSSNMT2 CSSNMT3 CSSNMT7 CSSNMT6 CSSNMT4 CSSNMT11 CSSNMT9 CSSNMT5 CSSNMT8 CSSNMT10 0.04 Pa 0.02

AU 0.00 -0.02 0.04 TP 0.02

AU CsXMT1 0.00 -0.02 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 Methyltransferase 进化树分钟

Camellia CsXMT2-6 has all 1,3,7-positional methyltransferase activity Evolution of secondary metabolism genes among various plant species

7- 3- 1- X mX mX mX TB Pa TP CF 0.04

0.02

0.00

-0.02 0.04 SAM XR 0.02

AU CsXMT2-6

0.00

-0.02 0.04 XR 0.02

AU 0.00 7- 3- 1- -0.02 X mX mX mX TB Pa TP CF 0.04 7-mX 3-NMT activity to TB 0.04 0.02

AU 0.02

AU 0.00 0.00 -0.02 -0.02 0.04 3-mX 1-NMT activity to TP 0.04 0.02 X

AU 0.02 SAM CsXMT2-6

AU 0.00 R 0.00 -0.02 -0.02 0.04 3-NMT activity to TP 1-mX 0.04 0.02 1-NMT activity to CF

AU 0.02

AU TB 0.00 0.00 -0.02 LAR 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50-0.02 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 WGD 分钟 ANS 0.04

0.02 7-NMT activity to CF SCPL AU TP 0.00 GTD -0.02 CHSs 0.04 3-NMT activity to CF DFRs 0.02

AU Pa SCPLs 0.00

-0.02

0.04

0.02

AU X 0.00

-0.02 In tea linearage, GTD seems contribute more to specialized metabolite generation 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 分钟

： Camellia CsXMT1-4 and CsXMT1-4 have special methyltransferase activity Conclusions  Tea plants are diploid tree with a large size and high heterozygosity of 7- 3- 1- X mX mX mX TB Pa TP CF genome 0.04

0.02

AU 0.00  Two WGD may contribute to tea lineage speciation, but recent TGD of key -0.02 0.04 genes mostly contribute to generation of caffeine, galloyated catechins 0.02 CsXMT1-4 AU 7- 3- 1- 0.00 mX mX mX TB Pa TP CF biosynthesis in tea plants. -0.02 X 0.04 0.04

0.02

0.02 X AU

AU 0.00 0.00 R -0.02 -0.02 0.04 SCPLs or other ATs may be involved in generation and diversification of 0.04  0.02 0.02 AU CsXMT2-4 AU 7-mX 0.00 galloylated catechins in tea plants. 0.00 -0.02

-0.02 0.04

0.04 0.02 TB

0.02 0.00 AU 3-mX 0.00 -0.02

0.04 Theanine is generated by bifunctional GSs, acting as a reservoir in tea -0.02  0.04 0.02 TP

0.02 1-NMT exchanger activity to 7-mX 0.00 plant. AU 1-mX 0.00 -0.02 7-mX 0.04 -0.02 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 Pa10.00 10.50 11.00 11.50 12.00 12.50 13.00 0.02 3-NMT activity 分钟 AU Diverse variations in XMT sequences and the convergent XMT activities may 0.00 

-0.02 0.04 explain the concentration of caffeine alkaloids in tea plant leaves.

0.02 X

0.00

-0.02 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 分钟

6 1/25/2019

Acknowledgements

Lab members: Anhui Agricultural University: Dr. Penghui Li Dr. Xiaochun Wan Dr. Linlin Liu Dr. Chaolin Wei Dr. Gaoyang Zhang Dr. Yang Hua Dr. Muhammand Zulfiqu Ali Dr. Liping Gao, …. Guangbiao She BGI Shuiwei Yu Dr. Shancen Zhao Yingying Li Dr. Qiang Gao Jihong Yang Songbo Wang, Chun Liu, Xuecheng Zhao SIBS, CAS, Yanrui Zhang Drs. Han Bin, and Ying Lu University of Georgia-Atlanta Dr. Jeffery Bennetzen Funding: NSFC, MST, Government of Anhui Province, …

Thanks for your attention