Volatile Glycosylation in Tea Plants

Presentation by:Mangesh Shivaji Khadase

Roll No. 15AG64R18Department of Agricultural and Food

Engineering Indian Institute of Technology, Kharagpur, WB.

Introduction Tea is considered to be one of the big three non-alcoholic beverages. In 2013, 5.07 million tons of tea was produced worldwide. A key element that determines a tea’s quality is its aroma. Tea plants store aroma compounds stably in the water-soluble

diglycoside form called β-primeverosides. While β-primeveroside of aromas does not produce aroma in and of

itself, a diglycoside-specific glycosidase called β-primeverosidase, present in the tea plants, cleaves this diglycoside into aroma compounds and sugar moiety, and liberates the aroma compounds.

However, until now it was totally unknown how β-primeverosides of aromas are produced in tea plants. This seminar focuses on the process by which β-primeverosides are produced in an attempt to elucidate the mechanism of aroma storage.

Camellia sinensis

Family - Theaceae Genus - Camellia Species - sinensis

Evergreen shrub or small tree.

Native to East Asia, the Indian Subcontinent and Southeast Asia.

Two major varieties:  Camellia sinensis var. sinensis Camellia sinensis var. assamica Mainly cultivated in tropical

and subtropical climates, in areas with at least 127 cm. of rainfall a year

Types of TeaGreen Tea• Not Fermented• Mainly China and Japan

Oolong Tea• Semi Fermented• Produced in Taiwan

Black tea• Fermented• Highest Production

Herbivore Attack

Mechanical Wounding

Endogenous Developments

C10

C15

C9

C16

C6

phenyl- propanoids

sesquiterpenoids

norisoprenoids

green leaf alcohols

monoterpenoids

Different Aroma Volatiles

• attractants for both parasitic and predatory insects for herbivores

benzyl alcohol

• it also act as a attractant for herbivory2-PE• induce defense responses in neighbouring

plants(Z)-3-hexenollinaloolgeraniol

• potential activity toward microorganisms and fungi

• potent apoptosis-inducing activity in plant cells

Biosynthesis pathway of geranyl β-primeveroside from geraniol.

CsGT1 (UGT85K11) and CsGT2 (UGT94P1) are the two glycosyltransferases that catalyze the sequential glucosylation and xylosylation of geraniol, resp.

Results

Organ Specific Composition• Aroma monoglycosides and diglycosides

extracted from fresh leaves and stems.• Various β-primeverosides, as well as

monoglycosides of aroma compounds in tea leaves, quantified by LC-MS

• The results show that geranylpri and linalyl-pri were detected mainly.

• These suggest that the metabolic activity of the glycosylation machinery is higher in growing young tissues.

• The total amounts of 2PE-pri, benzyl-pri, and hexenyl-pri increased in the mature leaves, whereas those of geranyl-pri and linalyl-pri decreased.

Biochemical characterization of CsGT1.• CsGT1, catalyzes the glucosylation of

Geraniol• Peak was observed at retention time of

10.2 min • CsGT1 was assigned as Cs_UGT85K11

Sugar Acceptor Specificity:geraniol 100%eugenol 84%

(Z)-3-hexenol 62%Benzyl alcohol 48%

2-PE 9.2%linalool 1.4%

Biochemical characterization of CsGT2.

Geranyl-glc 100%2PE-glc 16%

Linalyl-glc 12%Eugenyl-glc 2%

UDP-Xyl 100%UDP-Glc 30%UDP-Gal 3%

• Peak was observed at 4.9 min• CsGT2 was assigned as

UGT94P1

• Sugar Acceptor Specificity:

• Sugar Donor Specificity:

Homology Modelling and Mutagenesis Analysis of CsGT2

Gene Expression and Phylogenetic Analysis of CsGT1 and CsGT2

CsGT1 and CsGT2 highly expressed in young leaves.

Expression of CsGT1 and CsGT2 decreased with leaf maturation.

• Sequence Analysis:CsGT1 and CsGT2 only share 27% amino acid identity.

• Phylogenetic analysis:CsGT1 and CsGT2 belong to differentclades, OG2 and OG8, respectively.

CsGT1 ≈ cassava UGT85K4 and UGT85K5

CsGT2 – GGT which includes morning glory DUSKY (UGT79G16) and tomato NSGT1

Developmental Stages of Tea Leaves

Young leaves

Mature Leaves

Relative Transcript Abundance of CsGT1, CsGT2 and β-PD

CsGT1 CsGT2 B- PD0

2

4

6

8

10

12

14

16

18

11.7

15.6

0.09

5.7

0.09 0.0021 1 1

4.6

0.90.005

Young Leaves Matured Leaves Stem Root

Discussion

CsGT1 and CsGT2 Catalyze the Two Glycosylation Steps of Volatile Monoterpenes and Alcohols

CsGT1 belongs to the UGT85 family. It shows similarities in structure and function to kiwi AdGT4

and grape VvGT14, VvGT16, VvGT17, and VvGT19 that were recently shown to catalyze the glucosylation of small terpenes and primary alcohols that are accumulated as glycosides in ripe kiwi and grape.

This suggests that the machinery behind the glucosylation of monoterpenes and primary alcohols is fairly conserved among phylogenetically discrete various plant species.

CsGT2 was identified as a novel UGT that specifically catalyzes the 6-O-xylosylation of the sugar moiety of aroma monoglucosides, the second step of glycosylation in the biosynthesis of β -primeverosides.

Sugar Donor Specificity of CsGT2 for UDP-Xyl• Mutagenesis experiments (Ile-141→Ser-141) revealed that the

unique Ile-141 of CsGT2 is a residue determining the specificity toward UDP-Xyl.

• CsGT2-I141S mutant’s considerably higher specificity for UDP-Glc suggests that the xylosyltransferase evolved from a glucosyltransferase by the acquisition of the crucial Ile-141.

• The unique sugar donor specificity of CsGT2 for UDP-Xyl is due to hydrophobic bulky side chain of the Ile residue which hinders the access of sugar donors with a functional group at the C6 position to the substrate pocket of GGT xylosyltransferases.

• In contrast, the CsGT2-I141S mutant showed a preference for UDP-Glc instead of UDP-Xyl as a sugar donor.

Putative Physiological Roles of CsGT1 and CsGT2 inVolatile Metabolism

Schematic illustration of a mode of action of CsGT1, CsGT2, and β -PD in the volatile metabolism in C. sinensis.

CsGTs - storage of VOCsβ –PD - release of VOCs

Conclusion• β-Primeverosides are the most abundant form of aroma

diglycosides in C. sinensis, and they are commercially and physiologically important for tea aroma quality in dietary beverages and for chemical defense against herbivores in the tea plant.

• CsGT1 and CsGT2 catalyze the sequential glucosylation and xylosylation of aromas, respectively, leading to the production of aroma β -primeverosides.

• The transcripts of the two CsGTs and β -PD in young tea leaves, together with the localization of geranyl-pri in epidermal cells of young tea leaves, strongly support the potent physiological role of VOCs in chemical defense.

Future Prospects:• The elucidation of the aroma storage mechanism in tea plants

offers the following prospects: Identifying the varieties, cultivars or parts of tea plants based

on the activity of CsGT1 and CsGT2, will facilitate development of new tea products by choosing specific varieties or parts.

Analyzing temporal regulation of CsGT1 and CsGT2 and environmental factors affecting their activities will help ascertain the best time and suitable parts of the plant to harvest. This knowledge will in turn lead to the new cultivating and processing applications that bring out the aroma of tea.

CsGT1 and CsGT2 will serve as useful genetic markers for breeding new tea cultivars with focus on aroma.

References• Ohgami, S.; Ono, E.; Horikawa, M.; Murata, J.; Totsuka, K.; Toyonaga,

H.; Ohba, Y.; Dohra, H.; Asai, T.; Matsui, K.; Mizutani, M.; Watanabe, N.; Ohnishi, T. Volatile glycosylation in tea plants: Sequential glycosylations for the biosynthesis of aroma β-primeverosides are catalyzed by two Camellia sinensis glycosyltransferases. Plant Physiol. 2015, 168, 464.

• Gui, J.D.; Fu, X.M.; Zhou, Y.; Katsuno, T.; Mei, X.; Deng, R.F.; Xu, X.L.; Zhang, L.Y.; Dong, F.; Watanabe, N.; et al. Does enzymatic hydrolysis of glycosidically bound volatile compounds really contribute to the formation of volatile compounds during the oolong tea manufacuring process? J. Agric. Food Chem. 2015, 63, 6905–6914.