seasonal clonal variations and effects of stresses on quality chemicals and prephenate dehydratase...

ORIGINAL PAPER

Seasonal clonal variations and effects of stresses on qualitychemicals and prephenate dehydratase enzyme activity in tea(Camellia sinensis)

Vaishali Sharma • Robin Joshi • Ashu Gulati

Received: 21 June 2010 / Revised: 6 October 2010 / Accepted: 12 October 2010 / Published online: 16 November 2010

� Springer-Verlag 2010

Abstract Seasonal and clonal variations in catechins,

flavour component 2-phenylethanol and prephenate dehy-

dratase (PDT) enzyme were studied in tea clones repre-

senting both Assam and China varieties growing in Kangra

region of India. Catechins were analysed and quantified by

HPLC, and 2-Phenylethanol was quantified by GC. Assam

variety recorded higher amounts of catechins and PDT

activity than China variety in all the three growth flushes.

Activity of PDT and catechins content was high during

mains growth flush followed by early and backend flush. 2-

Phenylethanol content recorded higher levels in China

variety compared to Assam variety, and higher content was

observed in the early flush and decreased thereafter with

progress in season in both the varieties. Decrease in cate-

chins content, 2-phenylethanol and PDT activity was

observed in the tea shoots infested by Exobasidium vexans

over healthy shoots. Drought stress induced by withholding

water for a period of 8 days caused initial increase in the

contents of the catechins, 2-phenyethanol and PDT activity

and decreased with 3 day onwards with an increase in the

severity of water stress. Seasonal variations showed mod-

ulations in catechins and 2-phenylethanol in response

to changing environmental conditions, suggesting that

depending on the season there is higher flux of substrate

towards the required product.

Keywords Camellia sinensis � Catechins �2-phenylethanol � Prephenate dehydratase �Seasonal clonal variations � Growth flush

Abbreviations

PDT Prephenate dehydratase

DS Drought stress

VFC Volatile flavour compound

Introduction

Tea obtained from processed shoots of Camellia sinensis

is one of the most popular non-alcoholic beverages in

the world which is known for its flavour. The tea flavour

comprises both taste and aroma which is the most important

factor in determining the quality of tea. Agro-climatic

conditions, variety and geographical origin/locations

greatly influence the chemical composition of the tea shoot

[1, 2]. Catechins which contribute up to 30% dry weight [3]

are responsible for taste while volatile flavour compounds

constituting only 0.01–0.02% of the total dry weight are

responsible for aroma. Catechins also play a significant role

in plant defence and are reported to increase in stress. The

production of catechins in the tea plant increases on expo-

sure to light and decreases in shade [4]. There are several

reports of increase in flavonoid content in plants following

fungal infection and herbivore damage [5]. Catechins pos-

sess antioxidant activity and have been shown to exhibit

numerous medicinal properties including inhibition of car-

cinogenesis and mutagenesis [6, 7]. Flavour component 2-

phenylethanol has been known to have antimicrobial

properties, and its presence in plant reproductive structures

IHBT Publication No. 2128.

V. Sharma � R. Joshi � A. Gulati (&)

Institute of Himalayan Bioresource Technology,

Council of Scientific Industrial Research,

Palampur 176061, HP, India

e-mail: [email protected]

Present Address:V. Sharma

University of Vermont, Burlington, VT, USA

123

Eur Food Res Technol (2011) 232:307–317

DOI 10.1007/s00217-010-1379-3

suggests a protective role for flowers and fruits. 2-phenyl-

ethanol is potent insect attractants (http://www.pherobase.

com) and attracts different sets of pollinating and predatory

insects [8].

It has been established by isotope tracer experiments that

catechins and 2-phenylethanol are synthesized in higher

plants via shikimate pathway [9, 10]. The pathway leads to

the formation of aromatic amino acid phenylalanine which is

the primary precursor of catechins [11] and flavour com-

pound 2-phenylethanol [12] (Fig. 1). Prephenate dehydra-

tase (PDT; EC 4.2.1.51) is the enzyme responsible for the

formation of phenylalanine via phenylpyruvtae in microor-

ganisms [13]. In contrast to microorganisms, the metabolic

route from chorismate to phenylalanine in plants is still not

entirely known. Even though arogenate has been reported to

be a precursor for phenylalanine in plants, an enzyme con-

verting prephenate into arogenate has not yet been identified.

Moreover, six putative Arabidopsis isozymes were shown to

possess PDT activity when expressed in E. coli, and also

complemented a yeast PDT null mutant [14], implying that

these isozymes can also convert prephenate into phenyl-

pyruvate in vivo. Recent studies by Tzin et al. [15] showed

that Arabidopsis plants possess a functional metabolic route

from prephenate via phenylpyruvate into phenylalanine. A

number of plant species contain phenylpyruvate, which

serves as a precursor for a number of secondary metabolites

such as phenylacetaldehyde, 2-phenylethanol and 2-phen-

ylethyl-b-D-glucopyranoside [12, 16].

Kangra valley produces delicately flavoured orthodox tea

rich in catechins and volatile flavour components. The tea

crop yield is distributed over three growth flushes; the early

flush (March–May), mains flush (June–August) and backend

flush (September–November). The crop shows high sea-

sonal variations with quality of first flush declining reaching

a trough during the mains flush and recovering in the

backend flush. In this paper, we report the isolation and

activity patterns of PDT enzyme in relation to catechins and

2-phenylethanol in tea shoots plucked in different seasons

from different clones belonging to Assam and China varie-

ties growing in Kangra valley. The study would help in

better understanding the role of PDT enzyme involved in the

biosyntheses of catechins and 2-phenylethanol. Also, studies

on the impact of water stress and biotic stress caused by

Exobasidium vexans on the biochemical constituents that

determine quality attributes of tea were also presented.

Studies in this direction will be helpful in understanding the

extent of loss of quality attributes during various stresses.

Materials and methods

Chemicals

All catechin standards, ethylene diaminetetraacetic acid,

cysteine, sodium pyruvate, FAD and MgCl2, phenyl

pyruvate, Sephadex G-100, GC standards 2-phenylethanol,

ethyl caprate were purchased from Sigma Chemical Co.,

Sigma India. Methanol, acetonitrile and chloroform were

of HPLC grade and purchased from Merck, Mumbai. All

other chemicals used were obtained from Sigma Chemical

Co., India.

Plant material

Tea shoots (two and a bud) belonging to both Assam and

China tea varieties (40 no.) were collected in liquid nitro-

gen from bushes maintained under 7-day regular plucking

at the Institute’s Tea Experimental Farm [Palampur (HP,

India) at an elevation of 1,200 m] for consecutive two

years during three growth flushes. The shoots were

immediately dried to constant weight at power level 7 in aFig. 1 Biosynthetic pathway of flavan-3-ols, 2-phenylethanol, (1)

Prephenate dehydratase enzyme

308 Eur Food Res Technol (2011) 232:307–317

123

http://www.pherobase.com

http://www.pherobase.com

BPL-Sanyo Micro-Convection Domestic Oven model

BMC 900 T (34L) and kept in desiccators till further use.

The shoots were used for the estimations of total catechins

by spectrophotometry and catechins profile by HPLC.

Blister blight infected tea shoots, comprising the apical bud

and subtending three leaves, were graded into four cate-

gories, showing nil, ca 25%,ca 50% and C75% blistered

surface up to the third leaf [17]. The temperature and UV-B

data were collected with meteorological weather station for

the period under study. Categorization of clones is based on

the information provided in the literature and given by the

institutes that have released the clones [18]. Five cultivars

representing both Assam and China varieties were selected

based on their catechins profiles for further studies. Shoot

samples from these selected cultivars were divided into

three portions. One portion was dried to constant weight at

power level 7 in a BMC 900 T oven and kept in desiccators

till further use. The second portion was processed for

making acetone powder, while the third portion was ground

in liquid nitrogen and used for estimating catechins.

Acetone powder preparation

Fresh tea shoots were ground and repetitively washed with

chilled acetone (-20 �C) to prepare acetone powder.

Polyvinylpyrollidone (PVPP) was added while making

acetone powder. The acetone powder was dried under

vacuum and stored at -20 �C until use.

Extraction and estimations of total catechins

Total catechins were extracted from tea shoots by the

method of Singh et al. [19]. The method is based on the

formation of coloured complex of diazotized aryl-amine

with the A-ring of catechins. Tea shoots were ground in

liquid nitrogen, extracted with chilled acetone (initially

100% and later 60%), and the combined extract was par-

titioned between twice the volume of petroleum benzene

(v/v). The lower aqueous acetone layer containing cate-

chins and other polyphenolic compounds was removed,

dried, and the residue was dissolved in water to quantify

catechins using the freshly prepared diazotized sulpha-

nilamide (kmax of the coloured adduct at 425 nm).

Recovery experiment was always performed to estimate

the loss, and the data were accounted for while expressing

the amount of catechins. A standard curve was prepared

using d-(±)-catechin as standard.

Extraction and estimation of catechin profile

Apical bud and leaf samples were ground in liquid nitrogen

and extracted with aqueous methanol (70%) with inter-

mittent shaking followed by centrifugation at 1,400g for

10 min. The extraction steps were repeated resulting in a

final extract volume of 10 ml. The extracts were filtered

through a 0.5-lm Millipore filter before being injected on

an analytical semi-preparative Merck-Hitachi high-perfor-

mance liquid chromatography system fitted with a C-18

Lichrocart column (250 9 4.0 mm; 5 lm), along with suit-

able guard column. Samples were eluted with 0.1% ortho-

phosphoric acid in water (solvent A) and acetonitrile (solvent

B) as the mobile phase following Sharma et al. [20].

Extraction of 2-phenylethanol

Two grams of the leaf sample dried as described above was

ground in a pestle and mortar with a pinch of sucrose

(*0.08 gm) and 0.6 mL distilled water. The ground sample

was extracted with 5–6 mL chloroform and filtered through

cotton. The residue was washed with more chloroform to

make up the volume to 10 mL. Chloroform extract was dried

over anhydrous sodium sulphate and concentrated under an

atmosphere of nitrogen to 0.2 mL. A volume of 0.5 ll of the

concentrate was injected into gas chromatograph.

Estimation of 2-phenylethanol

The concentrated volatile flavour extracts were analysed on

Agilent 7890 GC equipped with FID and GC ChemStation

and EZChrom Elite chromatography data systems.

GC conditions: Fused silica HP-1 column (10 m 9 0.53

mm id, stationary phase SE-30, film thickness 2.65 lm),

nitrogen as carrier gas with flow rate of 30 mL/min. The

injector temperature was kept at 220 �C with split ratio of

1:3. FID temperature was kept at 230 �C. GC column

temperature was programmed isotherm at 40 �C for 2 min

and increased to 180 �C at the rate of 10 �C/min and final

hold at 180 �C for 5 min.

Identification of 2-phenylethanol

Identification was done by comparing retention indices

with the reference standards run under similar conditions,

and quantification of liberated flavour components was

done by peak area calculation using ethyl caprate as

internal standard.

Quantification of 2-phenylethanol

Concentration (mg/g dried shoot)

¼ ½Area of sample peak� � ½Conc:ethyl caprate�½Area of ethyl caprate peak]

Quantification of 2-phenyl ethanol was done by peak

area calculation using the known concentration of ethyl

Eur Food Res Technol (2011) 232:307–317 309

123

caprate (density: 0.862) used as internal standard. Factors

like dilution factor and injection volume were taken care of

in calculation.

Prephenate dehydratase extraction and partial

purification

The enzyme PDT was extracted from acetone powder in a

solution containing 0.1 M phosphate buffer (pH 7.5), 1 mM

EDTA, 5% cysteine, 5 mM sodium pyruvate, 0.2 mM FAD

and 1 mM MgCl2 according to Goers and Jensen [21]. All

enzyme procedures were carried out at 4 �C. The extract was

centrifuged at 3,000g for 30 min to remove the cell debris

from the extract. Solid ammonium sulphate was added to 55%

of saturation by stirring for 30 min. The sample was centri-

fuged at 7,000g for 30 min, and the pellet was dissolved in a

small volume of 0.1 M phosphate buffer (pH 7.5). The sample

was loaded on a Sephadex G-100 column equilibrated with the

same buffer, and desalted sample was collected and used for

estimation of PDT activity.

Prephenate dehydratase assay

PDT was assayed by measuring the rate of conversion of

prephenate to phenylpyruvate according to Gething et al.

[22]. The activity was assayed in 0.5 mL reaction mixture

containing 0.5 mM barium prephenate, 20 mM Tris HCl

(pH 8.2), 1.0 mM EDTA, 0.01% bovine serum albumin

and 20 mM 2-mercaptoethanol. After a pre-incubation of

5 min at 37 �C, the enzyme extract was added to the

reaction mixture. The reaction was terminated after 5 min

by the addition of 0.8 mL of 1.5 M NaOH. The absorbance

was measured at 320 nm to determine the concentration of

phenylpyruvate formed. The enzyme activity was expres-

sed in terms of total absorbance units per gram fresh weight

of tea shoot under the assay conditions. The amount of

phenylpyruvate released was quantified by calibration

curve prepared using pure phenylpyruvate.

Drought stress treatment

Two-year-old tea plants were raised in plastic pots in an

environment-controlled room (temperature, 25 ± 1 �C;

RH, 70–80%). Drought stress (DS) was imposed by with-

holding water during the entire 8-day treatment period.

After the 8-day treatment period, the plants were watered.

Control samples were harvested at the corresponding times

from untreated control plants that were kept well watered.

Statistical analysis

All determinations were run in triplicate, and the results

were reported as the mean and standard deviation.

Statistical variance analysis of independent data with three

replicates was performed using ANOVA.

Results and discussion

The catechins and volatile flavour compound 2-phenyl-

ethanol present in the tea shoots give an indication of the

potential of a clone or a cultivar to make good quality tea.

The tea flavour shows high seasonal variations in quality.

In higher plants, secondary metabolites catechins and

volatile flavour compound 2-phenylethanol are biosynthe-

sized via shikimate pathway with phenylalanine and tyro-

sine as the primary precursors. The present studies were

conducted with the objective of elucidating the role of PDT

enzyme in the biosynthesis of catechins and flavour com-

pound 2-phenyl ethanol in tea. It is reported that the tea

clones within the same cultivar or variety exhibit different

catechin contents and aroma profiles [1, 23, 24]. The tea

clones growing at the IHBT Tea Experimental Farm were

evaluated for seasonal variations.

Seasonal and clonal variations in quality chemicals

and PDT activity

Tea shoots belonging to different clones of Assam and

China varieties showed qualitative and quantitative varia-

tions in total catechins and 2-phenylethanol contents and

PDT activity over various growth flushes. The clones

recorded significant seasonal variations (p \ 0.1) in their

catechins content. The clones belonging to Assam variety

recorded higher amounts of total catechins, epigallocate-

chin gallate (EGCG), epicatechin gallate (ECG), epigallo-

catechin (EGC), catechin than the clones of China variety

(Table 1) under Kangra valley conditions. EGCG recorded

the highest content in all the clones irrespective of the

variety followed by EGC. Similar trends were shown by

ECG, catechin and EC which showed least variation among

the different varieties. The average total catechins content

of Assam variety (20%) was higher than China variety

(15%). Similar trends in variations in catechins contents

were observed by Saijo et al. [25]. Air temperature, sun-

shine hours and distribution of rainfall influence the quality

of tea. Catechins mainly EGC recorded higher content

during mains flush when the Sun rays are the strongest

followed by early and backend flush in both Assam and

China varieties (Tables 2, 3). Similar observations were

also recorded by Sanderson [4]. The higher catechins

content could be attributed to a rise in temperature as the

biosynthesis of catechins increases by an increase in tem-

perature. The temperature and UV-B data collected from

meteorological weather station during the study were cor-

related with total catechin contents during the period.


123

Catechins content was highest during mains growth flush

(June–August) when the average day temperatures were

higher followed by early (March–May) and backend

flush (September–November) (Table 4). A high positive

correlation (r2 = 0.80–0.98 for different clones under

study) was observed between an increase in cate-

chins content and temperature for both varieties. An

increase in catechins content was also observed with the

increase in UV-B (Table 5). A positive correlation

(r2 = 0.83–0.97 for different clones under study) was

observed between the catechins content and UV-B.

Similar trends were also recorded by Hahlbrock [26],

who observed increase in anthocyanins and flavanol

contents in response to UV irradiation in Arabidopsis.

These UV-absorbing compounds are thought to provide a

means of protection against UV-B damage and sub-

sequent cell death.

2-phenylethanol recorded higher content in China vari-

ety compared to Assam variety (Table 6). Both the varie-

ties recorded higher content during the early flush and

decreased with progress in season (Table 6). The variations

were more pronounced in China variety compared to

Assam variety. The results elucidated preponderance of

flavour imparting biochemicals in China variety over

Assam variety in Kangra valley. Slight changes in the

climate factors result in noticeable changes in the catechins

and aroma complex of teas [24, 27]. In Kangra valley,

early growth flush coincides with dry weather with cooler

nights and desiccating winds that favour the biogenesis of

aroma.

PDT activity was monitored in tea clones during the

three growth flushes (Fig. 2). Significant variations

(p \ 0.05) were observed in PDT activity extracted from

freshly plucked shoots of two varieties for the three growth

flushes. Clones of Assam variety showed high PDT activity

in all the three flushes compared to China variety. Activity

of PDT was high during mains growth flush followed by

early and backend flush. A strong positive correlation was

observed between total catechins and PDT activity for both

Assam and China variety in three growth flushes

(r2 = 0.92 for Assam variety and r2 = 0.93 for China

variety). PDT enzyme can be linked to catechins biosyn-

thesis in the tea varieties. An increase in PDT activity was

also observed with the increase in temperature and UV-B

(Table 7). PDT recorded positive correlation with tem-

perature (r2 = 0.67–0.81 for different clones under study)

and UV-B (r2 = 0.77–0.91 for different clones under

study). Similar effect of UV-B on shikimate reductase and

catechins content has also been reported from tea grown in

Assam [28].

Table 1 Catechins content of tea clones growing at IHBT Tea

Experimental Farm

Accession no. % Catechins (dw)

Total EGC EC EGCG ECG Catechin

IHBT 11A 16.70 2.90 0.07 12.90 0.60 0.27

IHBT 12A 16.30 3.74 0.89 10.03 0.91 0.41

IHBT 13A 22.00 5.60 1.45 13.50 0.90 0.25

IHBT 14A 18.63 4.40 0.84 12.41 0.71 0.27

IHBT 16A 16.96 3.44 0.36 12.19 0.67 0.28

IHBT 17A 24.90 7.00 1.35 15.20 0.92 0.39

IHBT 18A 16.34 2.45 0.41 11.45 0.89 0.53

IHBT 20A 20.30 6.59 0.96 11.80 0.63 0.28

IHBT 24A 21.03 4.30 0.47 15.19 0.70 0.28

IHBT 30A 18.53 3.06 1.14 12.40 0.71 0.49

IHBT 32A 23.30 6.52 3.50 12.30 0.42 0.31

IHBT 33A 15.70 1.63 0.04 12.40 0.90 0.33

IHBT 41A 20.60 2.44 0.85 14.40 1.12 0.53

IHBT 52A 24.80 5.38 0.84 16.94 0.95 0.44

IHBT 1C 16.80 3.70 0.41 11.86 0.56 0.25

IHBT 2C 18.70 4.68 0.04 12.90 0.64 0.27

IHBT 9C 10.2 2.30 0.20 7.91 0.50 0.25

IHBT 37C 17.44 4.45 0.9 10.71 0.79 0.31

IHBT 39C 9.43 0.59 ND 6.95 0.76 0.44

IHBT 40C 20.70 5.10 0.53 14.01 0.68 0.26

IHBT 44C 14.80 2.60 1.00 10.10 0.67 0.27

IHBT 45C 19.00 5.60 0.25 11.80 0.70 0.26

IHBT 46C 13.90 1.58 0.03 11.31 0.66 0.25

IHBT 47C 13.50 1.73 0.01 10.74 0.60 0.27

IHBT 49C 17.00 4.60 0.53 10.71 0.70 0.27

IHBT 50C 10.8 0.64 ND 9.01 0.59 0.25

IHBT 55C 19.20 4.10 0.82 12.92 0.70 0.27

IHBT 70C 16.00 4.60 0.36 10.12 0.54 0.25

IHBT 73C 12.50 0.60 ND 10.72 0.50 0.25

Values are the mean of three replicates. A Assam variety, C China

variety

Table 2 Variations in catechins during three growth flushes in

Assam variety

Growth flush Catechins (mg g-1 dw)

Total EGC EGCG ECG EC Catechin

Early flush 105b 12b 72a 10b 7.5a 5.3a

Mains flush 151a 31a 79a 28a 7.7a 5.4a

Backend flush 81c 10b 54b 9b 5.7b 4.8b

Values are the mean of three different determinations as analysed on

HPLC

Values within a column having common letters do not differ signif-

icantly at 0.1


123

Concentration of catechins, 2-phenylethanol and PDT

activity in response to leaf age

Because the most actively growing tissue of the tea plant,

i.e. the apical bud and the associated leaves up to the

second node, commonly referred to as two and a bud, are

used to manufacture high-quality tea [29], we examined the

effects of leaf age on the concentrations of catechins, 2

phenylethanol and PDT activity. The composition and

content of catechins present in tea shoot showed higher

values for total catechins in young tea leaf components

(first leaf and apical bud) over the mature components

Table 3 Variations in catechins during the three growth flushes in

China variety

Growth flush Catechins (mg g-1 dw)

Total EGC EGCG ECG EC Catechin

Early flush 90b 10b 62a 10b 7.0a 1.5a

Mains flush 104a 17a 65a 14a 7.1a 1.6a

Backend flush 65b 10b 31b 9b 5.8b 1.1b

Values are the mean of three different determinations as analysed on

HPLC

Values within a column having common letters do not differ signif-

icantly at 0.1

Table 4 Effect of temperature on catechins content in tea clones

Month Mean

temperature ( �C)

Tea clones (mg g-1 dw)

Assam China

IHBT 16 IHBT30 IHBT41 IHBT2 IHBT50

March 15.0 97.2e 101d 102.06e 80.0f 77.5c

April 20.0 103.8d 108.2c 109.73d 87.7e 87.6b

May 22.1 107.1d 112.5c 113.0d 93.2d 91.66b

June 24.0 129.5c 130.7b 131.9c 100.8c 94.46b

July 25.5 151.3a 156.9a 161.3a 127.8a 111.6a

August 22.0 134.3b 131.4b 139.9b 111.0b 96.4b

September 20.1 97.8e 96.9d 95.26f 82.0f 70.5d

October 17.0 86.0f 87.4e 85.5 g 70.9 g 55.0e

November 13.6 78.4g 79.3f 79.1 h 62.8 h 51.3e

CV% 1.129 1.119 1.067 1.393 1.626

SEM± 0.408 0.414 0.353 0.413 0.457

Values are the mean of three different determinations. Values for the same column sharing the same letters did not differ significantly at 0.05

Table 5 Effect of UV-B on catechins content in tea clones

Month Mean UV-B

(Watts M-2 9 102)

Tea clones (mg g-1 dw)

Assam China

IHBT 30 IHBT 41 IHBT 16 IHBT 2 IHBT 50

March 23.0 101d 102.06e 97.2e 80.0f 77.5c

April 25.0 108.2c 109.73d 103.8 d 87.7e 87.6b

May 29.0 112.5c 113.0d 107.1d 93.2d 91.66b

June 36.0 130.7b 131.9c 129.5 c 100.8c 94.46b

July 37.0 156.9 a 161.3a 151.3 a 127.8a 111.6a

August 26.4 131.4b 139.9b 134.3 b 111.03b 96.4b

September 22.4 96.9d 95.26f 97.8e 82.0f 70.5d

October 21.6 87.4e 85.5 g 86.0f 70.9 g 55.0e

November 20.0 79.3f 79.1 h 78.4 g 62.8 h 51.3e

CV% 0.859 0.862 0.953 1.199 1.818

SEM± 1.799 2.589 2.880 2.307 1.571



123

(second, third and fourth leaves) (Table 8). EGCG

recorded higher values in the bud and first leaf and

decreased with the maturity of the leaves. EGC also

recorded higher contents in first leaf and decreased with

maturity of the leaves. Like Japanese teas [26], Kangra

tea also recorded higher catechins levels in young leaves

in comparison with older leaves. Studies of Barman et al.

[30] on allocation of 14C assimilates in tea showed

that high-yielding clones retained lower amount of

photosynthates (saccharides) in the maintenance leaves

(source) and allocated higher percentage towards the

pluckable shoots (sink), indicating that more precursors

are available for catechin biosynthesis in young leaves

when compared to older ones. Similarly, PDT activity

was high in younger leaves (apical bud and up to third

leaf) but low levels were observed in mature leaves

(Table 9). These results are indicative of more precursor

availability for catechin and VFCs biosynthesis in

younger leaves. 2-phenylethanol recorded higher levels in

3rd leaf compared to the young components of the China

tea shoot viz. bud, first leaf and second leaf, while there

was not much difference in the content from 2nd to 3rd

leaf in Assam variety (data not shown). Similar results

have been obtained for black teas made from leaves with

different levels of maturity [31]. The levels of floral

aroma compounds recorded higher values in teas made

from three and a bud than in teas made from two and a

bud [27].

Effect of blister blight on catechins, 2-phenylethanol

and PDT activity

Studies on the effect of foliar disease blister blight caused

by Exobasidium vexans on catechins, 2-phenylethanol

contents and PDT activity showed a decrease in quality

chemicals viz. catechins and 2-phenylethanol content in the

tea shoots infested by E. vexans over healthy shoots.

Decrease was more pronounced in tea shoots that were

50% or more infested by blister blight over healthy shoots

(Table 10). PDT activity also recorded decrease in tea

shoots infested by E. vexans over healthy shoots

(Table 10). Similar results were also observed by Gulati

et al. [17]. The loss in the activity of PDT and quality

chemicals could be due to the fact that this fungus causes

extensive damage to the young succulent tissue mainly the

palisade and the epidermal layers containing the stomata

resulting in lower photosynthetic rates and thereby indi-

rectly reducing the carbon flow through the shikimate

pathway.

Table 6 Seasonal clonal variations in volatile flavour component

2-phenylethanol in Assam and China tea variety

Cultivars 2-Phenylethanol content*

Early flush Mains flush Backend flush

IHBT 30 (Assam) 0.204c 0.149b 0.108b

IHBT 41 (Assam) 0.174d 0.138b 0.100b

IHBT 02 (China) 0.271a 0.228a 0.171a

IHBT 50 (China) 0.229b 0.197a 0.142a

* Values are the mean of three different determinations as analysed

on GC. mg/g ethyl caprate taken as standard. Values for the same

column sharing the same letters did not differ significantly at 0.1

100

120

140

160

180

200

220

240

260

280

IHBT 41 IHBT 16 IHBT 30 IHBT 2 IHBT-50

China VarietyAssam Variety

Tea Clones

Pre

phen

ate

dehy

drat

ase

activ

ity (

AU

g-1

FW

)

EarlyBackendMains

Fig. 2 Seasonal and clonal

variations in prephenate

dehydratase activity in Assam

and China tea variety. Activity

is expressed in terms of total

absorbance units (AU) at

320 nm per g fresh weight of tea

shoot. Values are the

mean ± SD of three different

determinations (p \ 0.05)


123

Effect of drought stress on catechins, 2-phenylethanol

and PDT activity

We found that the drought stress decreased catechins, 2-

phenylethanol starting from 3 day and onwards. Similar

trend was also seen in the PDT activity. On 8 day of

the drought stress, the concentrations of ECs had

decreased by 23, 21 and 15%, respectively, compared

with values at 0 day. The decrease in 2-phenylethanol

was 16% compared with the enzyme activity which was

39% in Assam variety. The decrease was less pro-

nounced in catechins and its components in China

variety, while the decrease was more pronounced in the

2-phenylethanol content and PDT activity in China

variety compared to the values on 0 day in the Assam

variety. Drought is known to down-regulate the

expression of phenylalanine ammonia-lyase (PAL) in

tea [32]. The decrease in PDT activity could be due to

feedback inhibition. Since the activity of PAL is down

regulated in drought stress, the phenylalanine already

present does not deaminate to yield trans-cinnamic acid.

PDT activity was found to be inhibited by phenylala-

nine (Vaishali Sharma and Ashu Gulati unpublished

Table 7 Effect of temperature and UV-B on prephenate dehydratase activity in tea clones

Month/mean

temperature ( �C)

Mean UV-B

(watts M-2 9 10-2)

Prephenate dehydratase activity (AU g-1 FW)*

IHBT 16 IHBT30 IHBT41 IHBT2 IHBT50

March/15.0 23 181.6d 199.4d 207.5e 174.567c 174.66c

April/20.0 25 190.53c 214.8e 227.066d 178.73c 176.93c

May/22.1 29 193.13c 200.63d 203.33f 169.43c 157.5d

June/24.0 33 219.23b 240.76c 251.86c 190.167b 179.2c

July/25.5 37 258.0a 265.0 a 275.83a 202.033a 199.3a

August/22.0 27.0 221.26b 250.4b 266.13b 193.46b 185.36b

September/20.1 22.4 181.7d 193.16f 196.76f 160.8d 133.33e

October/17.0 21.6 182.0d 193.0f 197.0f 161.0d 133.0e

November/13.6 20 178d 182.43g 185.36g 156.433d 121.8f

CV% 0.658 0.597 0.603 0.837 0.918

SEM± 0.658 0.448 0.429 0.471 0.567


* Enzyme Activity is expressed in terms of total absorbance units at 320 nm per g fresh weight of tea shoot

Table 8 Concentration of catechins in response to leaf age (4 and a bud)

Shoot component Catechin (mg g-1 dw)

Total EGC EGCG ECG Catechin EC

Bud 149 ± 0.11 55 ± 0.07 64 ± 0.13 16 ± 0.11 5.0 ± 0.11 9.0 ± 0.06

First leaf 151 ± 0.07 57 ± 0.12 64 ± 0.11 16 ± 0.06 5.0 ± 0.20 9.0 ± 0.11

Second leaf 140 ± 0.12 54 ± 0.11 59 ± 0.21 17 ± 0.12 4.5 ± 0.11 6.0 ± 0.12

Third leaf 133 ± 0.11 50 ± 0.10 56 ± 0.16 15 ± 0.11 4.4 ± 0.10 5.7 ± 0.11

Fourth leaf 100 ± 0.08 30 ± 0.11 50 ± 0.14 9.0 ± 0.15 4.0 ± 0.14 4.8 ± 0.12

CV% 1.006 0.607 0.665 0.335 0.192 0.222

SEM± 0.454 0.998 0.551 0.708 0.723 0.825

Values are the mean of three different determinations (p \ 0.05)

Table 9 Concentration of prephenate dehydratase in response to leaf

age

Tea shoot component Prephenate dehydratase (AU g-1 FW)*

Bud 201.0a

First leaf 184.6b

Second leaf 159.8c, d

Third leaf 168.3c

Fourth leaf 147.4d

* Activity is expressed in terms of total absorbance units (AU) at

320 nm per g fresh weight of tea shoot. Values for the same column

sharing the same letters did not differ significantly at 0.1


123

work). This is in accordance with the studies conducted

on the enzyme with phenylalanine in Alcaligenes eu-

trophus by Friedrich et al. [33]. Recovery in catechins

and 2-phenylethanol along with the enzyme after

rehydration is higher in China variety than in Assam

variety grown in Kangra valley (Table 11, 12).

In conclusion, we isolated PDT enzyme from fresh tea

shoots and demonstrated a strong correlation between the

catechins concentrations and PDT activity indicating a role

of the enzyme in the biosynthesis of catechins. The two tea

varieties showed seasonal and clonal variations in quality

chemicals and PDT enzyme activity indicating the depen-

dence of metabolic carbon flux through shikimate pathway

towards catechins or 2-phenylethanol on the season and

agro-climatic conditions. This is supported by our results

that catechin contents and PDT activities are the highest

during mains flush indicating more precursor availability

for catechin biosynthesis during mains flush. Further in

depth studies on regulatory role of PDT and other branch

point enzymes like prephenate dehydrogenase involved in

tyrosine biosynthesis may help us to better understand the

kinetics of the synthesis of catechins and 2-phenylethanol

in fresh tea shoots. Further, this study gives a better

understanding of the effects of factors such as temperature,

Table 10 Changes in catechin, 2-phenylethanol and prephenate

dehydratase activity due to infection by Exobasidium vexans

Infection

level

Catechins

(mg g-1 dw)

2-Phenylethanol

(GC peak

area 9 107)

Prephenate

dehydratase Activity

(AU g-1 FW)*

Nil 126 13.2 300

Ca 25% 104 3.8 193

Ca 50% 84 1.96 151

C75% 70 0.55 120

CV% 0.757 0.651 0.236

SEM± 0.874 0.145 0.114

* Enzyme activity is expressed in absorbance units at 320 nm per g

fresh weight of tea shoot. Values are mean of three readings and

significant at 0.05 probability level

Table 11 Variations in catechins, 2-phenylethanol contents and prephenate dehydratase activity in tea shoots of Assam variety under drought

stress

Treatment Catechin (mg g-1 dw) Prephenate dehydratase

Activity* (AU g-1 FW)

2-Phenylethanol

(GC peak area 9107)Total EGC EGCG ECG EC

Control 14.85 ± 0.35 4.45 ± 0.28 7.43 ± 0.13 2.11 ± 0.09 0.55 ± 0.03 241 ± 0.35 0.221 ± 0.23

Control-1 15.09 ± 0.11 4.57 ± 0.11 7.64 ± 0.15 2.19 ± 0.15 0.55 ± 0.03 258 ± 0.33 0.231 ± 0.18

Control-2 14.88 ± 0.24 4.38 ± 0.23 7.56 ± 0.23 2.13 ± 0.21 0.52 ± 0.11 249 ± 0.28 0.234 ± 0.24

Control-3 14.58 ± 0.18 4.16 ± 0.38 7.39 ± 0.21 2.09 ± 0.14 0.51 ± 0.08 231 ± 0.18 0.228 ± 0.24

Control-4 14.23 ± 0.12 4.01 ± 0.11 7.27 ± 0.38 2.038 ± 0.11 0.51 ± 0.05 214 ± 0.24 0.224 ± 0.28

Control-5 13.85 ± 0.12 3.88 ± 0.17 7.09 ± 0.45 1.94 ± 0.17 0.51 ± 0.08 191 ± 0.15 0.219 ± 0.35

Control-6 13.72 ± 0.27 3.78 ± 0.15 6.95 ± 0.11 1.91 ± 0.11 0.48 ± 0.10 182 ± 0.38 0.204 ± 0.19

Control-8 13.36 ± 0.22 3.67 ± 0.21 6.79 ± 0.22 1.77 ± 0.12 0.46 ± 0.08 169 ± 0.36 0.195 ± 0.29

Control-9 13.28 ± 0.15 3.59 ± 0.18 6.58 ± 0.21 1.65 ± 0.16 0.45 ± 0.05 148 ± 0.28 0.179 ± 0.13

Control-10 12.95 ± 0.17 3.55 ± 0.25 6.45 ± 0.14 1.54 ± 0.18 0.44 ± 0.08 128 ± 0.35 0.165 ± 0.19

DS-Day 1 15.45 ± 0.31 4.61 ± 0.34 7.70 ± 0.25 2.21 ± 0.18 0.58 ± 0.07 284 ± 0.24 0.243 ± 0.33

DS-Day 2 14.91 ± 0.15 4.35 ± 0.42 7.46 ± 0.25 2.21 ± 0.12 0.58 ± 0.07 272 ± 0.33 0.238 ± 0.25

DS-Day 3 14.26 ± 0.24 4.16 ± 0.11 7.26 ± 0.84 2.01 ± 0.15 0.53 ± 0.03 251 ± 0.44 0.229 ± 0.37

DS-Day 4 13.22 ± 0.35 3.77 ± 0.14 6.89 ± 0.57 1.82 ± 0.28 0.51 ± 0.01 229 ± 0.38 0.225 ± 0.39

DS-Day 5 12.50 ± 0.39 3.61 ± 0.21 6.63 ± 0.54 1.64 ± 0.34 0.41 ± 0.01 189 ± 0.24 0.211 ± 0.38

DS-Day 6 11.87 ± 0.42 3.41 ± 0.19 6.36 ± 0.52 1.52 ± 0.65 0.40 ± 0.02 165 ± 0.34 0.199 ± 0.39

DS-Day 8 11.28 ± 0.19 3.27 ± 0.29 6.14 ± 0.45 1.46 ± 0.85 0.37 ± 0.05 149 ± 0.23 0.186 ± 0.47

Day 9 11.42 ± 0.14 3.31 ± 0.12 6.31 ± 0.42 1.49 ± 0.11 0.38 ± 0.05 161 ± 0.15 0.201 ± 0.38

Day 10 11.68 ± 0.21 3.38 ± 0.11 6.44 ± 0.25 1.53 ± 0.19 0.4 ± 0.05 184 ± 0.22 0.209 ± 0.33

CV% 0.789 0.621 0.758 0.863 0.356 0.856 0.226

SEM 0.541 0.145 0.314 0.259 0.189 1.356 0.183

* Activity is expressed in terms of total absorbance units at 320 nm per g fresh weight of tea shoot. Values are the mean ± SD of three different



123

UV-B, fungal and drought stress on the biosynthesis of

quality chemicals from tea.

Acknowledgments Authors are grateful to the Director, Institute of

Himalayan Bioresource Technology, Palampur, India for support of

this research. They thank Dr. R.K. Sud for providing plant material and

Mr. R.K. Tandon for technical support. VS acknowledges Council of

Scientific and Industrial Research (CSIR), India for financial assistance

as Senior Research Fellow. Authors acknowledge financial assistance

received from CSIR under the projects ‘‘Niche pathway engineering in

tea’’ and ‘‘High value products from agro-forestry resources from

Himalayan region and quality product development including facility for

evaluation of nutraceutical/value added products’’.

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DS-Day 8 9.32 ± 0.19 3.41 ± 0.12 4.38 ± 0.14 1.34 ± 0.14 0.19 ± 0.08 132 ± 0.31 0.245 ± 0.33

Day 9 9.38 ± 0.22 3.48 ± 0.12 4.38 ± 0.22 1.32 ± 0.11 0.21 ± 0.03 149 ± 0.23 0.258 ± 0.24

Day 10 9.55 ± 0.24 3.53 ± 0.14 4.48 ± 0.12 1.34 ± 0.14 0.21 ± 0.02 157 ± 0.42 0.270 ± 0.18

CV% 0.895 0.865 0.889 0.588 0.258 0.559 0.289

SEM 0.507 0.122 0.452 0.359 0.145 0.985 0.158

* Activity is expressed in terms of total absorbance units at 320 nm per g fresh weight of tea shoot. Values are the mean ± SD of three different



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