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Does Enzymatic Hydrolysis of Glycosidically Bound Volatile Compounds Really Contribute to the Formation of Volatile Compounds During the Oolong Tea Manufacturing Process?

Authors:
  • South China Botanical Garden, Chinese Academy of Sciences

Abstract and Figures

It was generally thought that aroma of oolong tea resulted from hydrolysis of glycosidically bound volatiles (GBVs). In this study, most GBVs showed no reduction during oolong tea manufacturing process. beta-Glycosidases either at protein or gene level were not activated during the manufacturing process. Subcellular localization of beta-primeverosidase provided evidence that beta-primeverosidase was located in the leaf cell wall. The cell wall remained intact during the enzyme-active manufacturing process. After the leaf cell disruption, GBVs content reduced. These reveal that during the enzyme-active process of oolong tea, non-disruption of the leaf cell walls resulted in impossibility of interaction of GBVs and -glycosidases. Indole, jasmine lactone, and trans-nerolidol were characteristic volatiles produced from the manufacturing process. Interestingly, the three volatiles contents reduced after the leaf cell disruption, suggesting that mechanical damage with the cell disruption, which is similar to black tea manufacturing, did not induce accumulation of the three volatiles. In addition, 11 volatiles with flavor dilution factor ≥44 were identified as relative potent odorants in the oolong tea. These results propose that enzymatic hydrolysis of GBVs was not involved in the formation of volatiles of oolong tea, and some characteristic volatiles with potent odorants were produced from the manufacturing process.
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Does Enzymatic Hydrolysis of Glycosidically Bound Volatile
Compounds Really Contribute to the Formation of Volatile
Compounds During the Oolong Tea Manufacturing Process?
Jiadong Gui,
,,§
Xiumin Fu,
,,§
Ying Zhou,
,
Tsuyoshi Katsuno,
Xin Mei,
,
Rufang Deng,
Xinlan Xu,
Linyun Zhang,
#
Fang Dong,
Naoharu Watanabe,
and Ziyin Yang*
,,,
Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement, South China Botanical Garden,
Chinese Academy of Sciences, Xingke Road 723, Tianhe District, Guangzhou 510650, China
University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China
Provincial Key Laboratory of Applied Botany South China Botanical Garden, Chinese Academy of Sciences, Xingke Road 723,
Tianhe District, Guangzhou 510650, China
Tea Research Center, Shizuoka Prefectural Research Institute of Agriculture and Forestry 1706-11 Kurasawa, Kikugawa 439-0002,
Japan
#
College of Horticultural Science, South China Agricultural University, Wushan Road, Tianhe District, Guangzhou 510642, China
Guangdong Food and Drug Vocational College, Longdongbei Road 321, Tianhe District, Guangzhou 510520, China
Graduate School of Science and Technology, Shizuoka University, 3-5-1 Johoku, Naka-ku, Hamamatsu 432-8561, Japan
*
SSupporting Information
ABSTRACT: It was generally thought that aroma of oolong tea resulted from hydrolysis of glycosidically bound volatiles
(GBVs). In this study, most GBVs showed no reduction during the oolong tea manufacturing process. β-Glycosidases either at
protein or gene level were not activated during the manufacturing process. Subcellular localization of β-primeverosidase provided
evidence that β-primeverosidase was located in the leaf cell wall. The cell wall remained intact during the enzyme-active
manufacturing process. After the leaf cell disruption, GBV content was reduced. These ndings reveal that, during the enzyme-
active process of oolong tea, nondisruption of the leaf cell walls resulted in impossibility of interaction of GBVs and
β-glycosidases. Indole, jasmine lactone, and trans-nerolidol were characteristic volatiles produced from the manufacturing
process. Interestingly, the contents of the three volatiles was reduced after the leaf cell disruption, suggesting that mechanical
damage with the cell disruption, which is similar to black tea manufacturing, did not induce accumulation of the three volatiles. In
addition, 11 volatiles with avor dilution factor 44were identied as relatively potent odorants in the oolong tea. These results
suggest that enzymatic hydrolysis of GBVs was not involved in the formation of volatiles of oolong tea, and some characteristic
volatiles with potent odorants were produced from the manufacturing process.
KEYWORDS: aroma, glycosidically bound volatiles, indole, β-primeverosidase, tea, volatile
INTRODUCTION
Tea (Camellia sinensis) aroma (volatility and odor-activity) is
one of the main sensory properties which are decisive for the
quality of the nal product. The tea volatile compounds can be
divided into four major classes according to their metabolic
origin: terpenoids, phenylpropanoids/benzenoids, fatty acid de-
rivatives, and carotenoids.
1
Monoterpenoids are enzymatically
synthesized de novo from acetyl CoA and pyruvate provided by
the carbohydrate pools mainly in plastids and the cytoplasm,
2
such as linalool (fresh oral odor), linalool oxides (oral), and
geraniol (oral, rose-like).
3
Most volatile benzenoids and
phenylpropanoids are primarily derived from phenylalanine,
4
such as 2-phenylethanol (oral, rose-like), benzyl alcohol (weak
oral), and phenylacetaldehyde (oral, rose-like).
3
Fatty acid-
derived straight-chain alcohols, aldehydes, ketones, acids, esters,
and lactones are basically formed by three processes, α-oxidation,
β-oxidation, and the lipoxygenase pathway,
4
such as hexanol
(green note), (Z)-3-hexen-1-ol (green leaf-like), and methyl
jasmonate (oral, jasmine-like).
3
The oxidative cleavage of
carotenoids leads to the production of apocarotenoids through
catalysis of a family of carotenoid cleavage dioxygenases,
5
such as
β-ionone (oral).
3
Moreover, volatiles occur in tea leaves not
only as free forms but also as glycosidically bound forms, which
are more water-soluble and less reactive than their free aglycon
counterparts.
6
Many glycosidically bound volatiles (GBVs)
have been isolated and identied in tea leaves, such as the
β-primeverosides and β-glucopyranosides of alcoholic volatiles
including benzyl alcohol, 2-phenylethanol, 1- phenylethanol, methyl
salicylate, (Z)-3-hexenol, linalool, linalool oxides, and geraniol, and
some GBVs that are hydrolyzed to form nonalcoholic volatiles
including benzaldehyde, coumarin, and damascenone.
715
Received: March 17, 2015
Revised: July 24, 2015
Accepted: July 26, 2015
Published: July 26, 2015
Article
pubs.acs.org/JAFC
© 2015 American Chemical Society 6905 DOI: 10.1021/acs.jafc.5b02741
J. Agric. Food Chem. 2015, 63, 69056914
Many attempts have been done to improve or modify the
volatiles of tea leaves either by treating leaves during growth
of the plants (raw materials) or by postharvest treatment of
leaves during the tea manufacturing process.
1
Biotic stress
(insect attack) and abiotic stress (light) have been utilized for
the improvement of volatiles in raw tea leaves. As a typical
example of biotic factors, the famous Taiwan oolong tea
(Oriental Beauty) has a unique aroma reminiscent of ripe fruit
and honey that is induced by the attack of tea green leaf-
hoppers.
16
As an example of abiotic factors, Gyokuro and
Tencha, known as the nest teas in Japan, are produced from
tea leaves under shading treatment. It was found that tea leaves
kept in darkness signicantly increased levels of volatiles,
especially volatile phenylpropanoids/benzenoids.
17
Besides the
modication of raw materials, the manufacturing processes have
signicant inuences on the compositions of volatiles in nal
tea products. In general, fermented teas including oolong tea
and black tea have more volatiles and aroma properties than
nonfermented green tea. This was generally thought to be due
to the involvement of hydrolysis of GBVs in the manufac-
turing process.
18
The GBVs are present within vacuoles, while
β-primeverosidase, one major glycosidase involved in the hy-
drolysis of GBVs, was presumed to be localized in cell walls.
18
This compartmentation of substrates and enzymes in plant cells
implies that interactions between the enzyme and the substrate
do not happen in intact tea leaves.
19
However, during the
manufacturing process, tea leaf tissues and cells are disrupted,
which results in the possibility of interactions between the
glycosidase and the GBVs and liberation of the free volatiles.
During the manufacturing process of black tea, the levels of
primeverosides of the volatiles decreased greatly, and the
glycosidase activities showed a peak during withering but were
drastically reduced after rolling, suggesting that enzymatic hy-
drolysis of GBVs mainly occurred during the stage of rolling.
20
Therefore, enzymatic hydrolysis of GBVs is proposed as an
important process during the manufacturing of black tea. In
contrast, most GBVs showed no reductions in their contents
during the manufacturing process of oolong tea. The contents
of GBVs increased after the solar-withering stage, and reached
their highest levels in the nal stages of oolong tea produc-
tion.
21
This result led us ask whether enzymatic hydrolysis of
GBVs really contributes to the formation of volatiles during the
oolong tea manufacturing process. To answer this question, we
monitored changes in contents of free volatiles and GBVs,
glycosidase enzyme activities, and β-glycosidase gene expression
level during the manufacturing process of oolong tea. We
attempted to give direct evidence of subcellular localization of
β-primeverosidase, and also investigated the eect of the manu-
facturing process on the leaf cell structure. Finally relatively
potent odorants occurring in oolong tea product were evaluated
by gas chromatographymass spectrometer/olfactometry
(GCMS/O). The results help us nd out the truth of forma-
tion of volatiles in oolong tea, and advance our understanding
of dierences of formation of volatiles between oolong tea and
black tea.
MATERIALS AND METHODS
Chemicals. Benzaldehyde, benzyl alcohol, β-damascenone,
δ-decalactone, ethyl decanoate, Furaneol, 2-hexen-1-ol, 3-hexenyl
acetate, indole, β-ionone, jasmine lactone, 3-methylnonane-2,4-dione,
methyl salicylate, 2-phenylethanol, and vanillin were purchased from
Wako Pure Chemical Industries Ltd., Japan. α-Farnesene, (Z)-3-
hexen-1-ol, linalool, linalool oxides, methyl jasmonate, trans-nerolidol,
geraniol, polyvinylpolypyrrolidone (PVPP), XAD-2, and β-glucosidase
were purchased from Sigma-Aldrich Company Ltd., USA. β-Prime-
verosidase and p-nitrophenyl-β-primeveroside were purchased from
Amano Enzyme Inc., Japan. p-Nitrophenyl-β-D-glucopyranoside
was purchased from Aladdin Industrial Co. Shanghai, China.
[15N]Anthranilic acid (15N% = 98%) was purchased from Cambridge
Isotope Laboratories Inc., Cambridge, MA. Porapak Q cartridge was
purchased from Waters Corporation, USA. Coomassie Blue R250,
30% acrylamide/Bis solution, N,N,N,N-tetramethylethylenediamine
(TMEMD), sodium dodecyl sulfate (SDS), and 2×SYBR Green
Universal PCR Mastermix were purchased from Bio-Rad Laboratories,
USA. Quick RNA isolation Kit was purchased from Huayueyang
Biotechnology Co., Ltd., China.
Plant Materials and Manufacturing Process of Oolong Tea.
One bud and two or three leaves of Camellia sinensis var. Jinxuan were
plucked at the Tea Experiment Station in the South China Agricultural
University (Guangzhou, China) in October, 2014. The manufacture of
oolong tea was carried out according to the general method,
21
as
shown in Figure S1 (Supporting Information). The freshly plucked tea
leaves (P) were exposed to sunlight (43 °C and 93,500 Lux) for
70 min as solar withering (SW). Afterward, the tea leaves were indoor-
withered (IW) for 2 h at temperature of 27 °C and a relative humidity
of 70%, and subsequently turned over by 5 times (T1T5) every
1.5 h. Then the tea leaves were parched in a tea-ring roller machine at
250 °C for 23 min to inactivate the enzymatic activity and x the
sample (F). Finally, the tea leaves were rolled at room temperature for
15 min and dried at 105 °C for 1.5 h. The samples from P, SW, IW,
and T1T5 were immediately frozen by liquid nitrogen for enzyme
activity and gene expression proling. Three replicates were processed
according to the oolong tea manufacturing.
Extraction and Analysis of Volatiles in Tea Samples. One
gram of samples (nely powdered) was extracted by 2.5 mL of dichlo-
romethane containing 5 nmol of ethyl decanoate as an internal
standard in a shaker at room temperature overnight. The extraction
solution was dried over anhydrous sodium sulfate and concentrated to
400 μL using nitrogen stream (MIULAB NDK200-1, MIU Instru-
ments CO., Ltd., Hangzhou, China). One microliter of the concentrate
was subjected to GCMS QP2010 SE (Shimadzu Corporation, Japan)
equipped with a SUPELCOWAXTM 10 column (30 m ×0.25 mm ×
0.25 μm, Supelco Inc., Bellefonte, PA, USA). The injector temperature
was 240 °C. The splitless mode was used with a splitless time of 1 min,
and helium was the carrier gas with a velocity 1.0 mL/min. The GC
oven temperature was 60 °C for 3 min, ramp of 4 °C/min to 240 °C,
and then 240 °C for 20 min. The mass spectrometry was operated
with full scan mode (mass range m/z40200).
Extraction and Analysis of GBVs in Tea Samples. Analysis of
GBVs used the enzymatic hydrolysis combined with the GCMS
analysis method, which was the same as described previously.
15,22
Five
hundred milligrams (fresh weight) of sample (nely powdered) was
extracted with 2 mL of cold methanol by vortexing for 2 min followed
by ultrasonic extraction in ice-cold water for 10 min. The extracts were
mixed with 2 mL of cold chloroform and 0.8 mL of cold water for
phase separation. The resulting upper layer was dried and dissolved in
1 mL of water. The resulting solution was mixed with 30 mg of PVPP,
stood for 90 min, and was centrifuged (16400g,4°C, 10 min), and
repeated with the supernatant. The nal supernatant was loaded to an
Amberlite XAD-2 column (1 mL) and eluted with 5 mL of water,
5 mL of pentane:dichloromethane (2:1), and 5 mL of methanol.
The methanol eluent was dried using nitrogen stream, and redissolved
in 400 μL of 50 mM citric acid buer (pH 6.0) containing
β-primeverosidase and β-glucosidase, and reacted at 37 °C for 14 h.
Afterward, 144 mg of sodium chloride was added to the reaction
solution, and stood for 15 min followed by addition of 5 nmol of ethyl
decanoate as an internal standard. The solution was extracted with
400 μL of dichloromethane and centrifuged, and the dichloro-
methane fraction was dried over anhydrous sodium sulfate, followed
by GCMS analysis. The GCMS conditions were the same as
described above.
Determination of Enzyme Activities of β-Glucosidase and
β-Primeverosidase. The enzyme assay method referred to the previous
Journal of Agricultural and Food Chemistry Article
DOI: 10.1021/acs.jafc.5b02741
J. Agric. Food Chem. 2015, 63, 69056914
6906
reports.
20,23
One gram of nely powdered tea leaves was ground in ice-
cold acetone and ltered using a suction pump (T-50.2L, Tianjin
Jinteng Experiment Equipment Co., Ltd., Tianjin, China). The
acetone-insoluble powder on the lter paper was washed with ice-
cold acetone several times until clean and colorless acetone ltrate was
obtained. The acetone-insoluble powder was dried by nitrogen stream
to remove residual acetone and stored at 80 °C. For each 0.25 g of
acetone-insoluble powder, 6.25 mL of ice-cold 50 mM sodium citrate
buer (pH 6.0) was added to dissolve the powder, accompanied by
0.125 g of PVPP. After vortexing twice for 30 s at 0 °C and
centrifuging at 10000g(4 °C, 20 min), the supernatant was used as
the crude enzyme solution. The substrate, either p-nitrophenyl-β-D-
glucopyranoside or p-nitrophenyl-β- primeveroside, was dissolved in
90 μL of 50 mM sodium citrate buer (pH 6.0) and incubated at
37 °C for 5 min. Ten microliters of crude enzyme solution was
added, and then the whole reaction mixture was incubated at 37 °C
for 60 min. The reaction was stopped by adding 140 μL of 0.2 M
sodium carbonate, resulting in yellow color of the mixture, which was
Figure 1. Changes in free volatiles during the manufacturing process of oolong tea. P, freshly plucked tea leaves; SW, solar withering; IW, indoor
withering; T1T5, turn over by 5 times; F, xing. Data represent the mean value ±standard deviation of three in independent experiments
performed in triplicate. The y-axis unit is peak area ratio of analyte to internal standard per g dry weight tea leaves. The volatiles in the pane showed
signicant increase during the manufacturing process of oolong tea.
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DOI: 10.1021/acs.jafc.5b02741
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measured at 400 nm by a spectrophotometer (Innite M200 PRO,
TECAN, Switzerland). The amount of p-nitrophenol was determined
according to a calibration curve.
Transcript Expression Analysis of β-Primeverosidase,9/13-
Lipoxygenase (LOX), and Terpene Synthases (TPS). Total RNA
was isolated immediately after dissection. The reactions were perfor-
med using Power SYBR Green PCR Master Mix in a 20 μL volume
containing 10 μL of Power SYBR Green PCR Master Mix (2×),
0.2 μM each specic forward and reverse primer. Two microliters of
10-fold diluted template was used for a 20 μL PCR reaction. The
encoding elongation factors (EFs) were used as internal reference
gene.
24
The EFs,β-primeverosidase,
15
LOX,
25
and TPS
26
specic primers
of qRT-PCR are shown in Table S1 (Supporting Information). The
qRT-PCR was carried out on Roche LightCycle 480 (Roche Applied
Science, Mannheim, Germany) under the condition of one cycle of
95 °C for 10 min, 40 cycles of 95 °C for 15 s, and 60 °C for 1 min.
A melt curve was performed at the end of each reaction to verify PCR
product specicity. The 2ΔΔct method was used to calculate the
relative expression level.
27
Changes in mRNA levels of the target genes
were normalized to that of EFs.
Observation of Tea Leaf Cell Structure. The tea leaf samples of
P, T1, T2, and T5 stages were cut into approximately 1 mm ×2mm
pieces and xed in 0.1 M phosphate buer (pH 7.2) containing 2%
glutaraldehyde and 2.5% paraformaldehyde. After 6 times washing with
0.1 M phosphate buer, the leaf samples were postxed in 1% osmium
tetroxide for 4 h and washed with 0.1 M phosphate buer. Then the
xed leaf samples were dehydrated and embedded in at molds using
EPON812 resin. Ultrathin sections (80 nm) were cut by ultramicro-
tome (Leica UC7, Leica Microsysteme GmbH, Wetzlar, Germany),
which then were stained by 4% uranyl acetate and 2% lead citrate.
Ultrathin sections were observed by a transmission electron micro-
scope (JEOL JEM-1010, Tokyo, Japan) operating at 100 kV.
Subcellular Localization of β-Primeverosidase in Tobacco.
To construct vectors for expression of GFP-fusion protein, the stop
codon of β-primeverosidase coding sequence was replaced by XmaI
restriction site, with the following primers: 5-GTCGACATGATGG-
CAGCGAAAGGG-3(forward), 5-CCCGGGGCTTGAGGAGGA-
ATTTCTT-3(reverse). The PCR product of β-primeverosidase
cDNA fragment, in which the stop codon was eliminated, was cut with
SalI/XmaI and then cloned into the SalI/XmaIsitesofthe
pCAMBIA3300-GFP vector. The reorganization vector was trans-
formed into Agrobacterium GV3101 by freezethaw. The overnight
Agrobacterium cultures were sedimented at 5000gfor 1 min. The pellet
was resuspended in a solution containing 10 mM MgCl2,10mM
morpholineethanesulfonic acid (pH 5.6), and 100 μM acetosyringone
to OD600 = 0.4.
28,29
Leaves of Nicotiana benthamiana were inltrated
by using a syringe without a needle, and then the tobacco was grown
on a 16 h light/8 h dark under 25 °C regime for 45 days. Protoplasts
were prepared as follows: tobacco leaves were cut into 23cm
2pieces
Figure 2. Eect of disruption of tea leaf cell on amounts of free
volatiles and total GBVs. CK-1, The 4th turn over, at which stage tea
leaf cell is not disrupted; CK-2, the 5th turn over, at which stage tea
leaf cell is not disrupted; cell disruption-1, after turn over 1 (T1), parts
of tea leaves were completely crushed to cause disruption of leaf cell,
and indoor-withered at room temperature for 4.5 h; cell disruption-2,
after turn over 1 (T1), parts of tea leaves were completely crushed to
cause disruption of leaf cell, and indoor-withered at room temperature
for 6.0 h. Data represent the mean value ±standard deviation of three
independent experiments performed in triplicate. The y-axis unit is
peak area ratio of analyte to internal standard per g dry weight tea
leaves.
Figure 3. Eect of disruption of tea leaf cell on the transformation
from [15N]anthranilic acid to [15N]indole. (A) GCMS chromatog-
raphy (m/z118) of [15N] indole from CK and leaf cell disruption. The
[15N]anthranilic acid feeding experiment started from the turn over 1
(T1). CK, the 5th turn over, at which stage tea leaf cell is not
disrupted; leaf cell disruption, after T1, parts of tea leaves were
completely crushed to cause disruption of leaf cell, and indoor-
withered at room temperature for 6.0 h. (B) Data represent the mean
value ±standard deviation of three independent experiments
performed in triplicate. The relative content was calculated based on
peak area of m/z118 of [15N] indole.
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with a sterile scalpel blade. Two grams of leaf pieces was transferred to
50 mL tubes containing 10 mL of buer solution (50 mM 2-(N-
morpholino)ethanesulfonic acid, 0.6 M mannitol, and 10 mM calcium
chloride), containing 1% cellulose and 0.05% macerozyme. The tubes
were incubated at 25 °C on a reciprocating shaker (50 rpm) for 4 h.
Protoplasts were washed and held in buer solution. Confocal
Figure 4. Changes in GBVs during the manufacturing process of oolong tea. P, freshly plucked tea leaves; SW, solar withering; IW, indoor withering;
T1T5, turn over by 5 times; F, xing. Data represent the mean value ±standard deviation of three independent experiments performed in
triplicate. The y-axis unit is peak area ratio of analyte (free volatiles from enzymatic hydrolysis of GBVs) to internal standard per g dry weight tea
leaves.
Figure 5. Changes in β-glycosidase enzyme activity, β-Primeverosidase,LOX, and TPS expression levels during the manufacturing process of oolong
tea. P, freshly plucked tea leaves; SW, solar withering; IW, indoor withering; T1T5, turn over by 5 times. Data represent the mean value ±standard
deviation of three independent experiments performed in triplicate. (A, B) One unit was dened as the formed amount of p-nitrophenyl by the
action of per mg protein per minute. (C, D, E, F, G) Transcript abundance was calculated based on the dierence in cycle threshold (Ct) values
between target gene and internal reference gene transcripts by the normalized relative quantication 2ΔΔCt method. The expression level of gene
from P sample was dened as 1.
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DOI: 10.1021/acs.jafc.5b02741
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microscope images were taken using a Zeiss LSM 510 META confocal
laser microscope (Cal Zeiss, Jena, Germany) with a 100×oil objective
under 488 nm excitation wavelength for GFP detection.
Eect of Disruption of Tea Leaf Cell on Amounts of Free
Volatiles and GBVs. After turn over 1 (T1), parts of tea leaves were
completely crushed to cause disruption of leaf cell, and indoor-
withered at room temperature for 4.5 and 6.0 h, respectively. After-
ward, 1 g of treated sample was used for analyses of free volatiles and
GBVs, which methods are described as above. The amounts of free
volatiles and GBVs of the treated samples were compared with those
of T4 and T5, respectively.
Administration of [15N]Anthranilic Acid into Tea Leaves and
Analysis of [15N]Indole. Fifty microliters of solution of [15N]-
anthranilic acid (20 mM) was injected into each tea leaf at the T1
stage. One part of the tea leaves with [15N]anthranilic acid was com-
pletely crushed to cause disruption of leaf cell and indoor-withered at
room temperature for 6.0 h, while the other part of the tea leaves with
[15N]anthranilic acid followed the oolong tea manufacturing and was
processed to the T5 stage. The amount of [15N]indole was analyzed by
GCMS as described above. The characteristic ion of [15N]indole is
m/z118.
Identication of Relatively Potent Odorants of Oolong Tea.
The relatively potent odorants were evaluated using GCMS/O and
aroma extraction and dilution analysis (AEDA), as described
previously.
3033
The oolong tea product powders (2 g) were incubated
in hot distilled water (40 mL, 8090 °C) for 5 min and centrifuged for
10 min at 3000g. The resultant supernatant (30 mL) was loaded on a
Porapak Q cartridge (2 mL volume, 5080 mesh), eluted with 3 mL
of water, and 3 mL of pentane:diethyl ether (1:1, v/v) as aroma
extract. Afterward, the aroma extract was added with ethyl decanoate
(20 μg/20 μL) as an internal standard, dried over anhydrous sodium
sulfate, and concentrated to 100 μL in a stream of nitrogen to give an
original odor concentrate sample.
The original odor concentrate of the sample infusion was stepwise
diluted with pentane:diethyl ether (1:1, v/v) 1:4 = 41, 1:16 = 42,
1:64 = 43, 1:256 = 44, 1:1024 = 45, 1:4096 = 46, and 1:16384 = 47. The
aliquots (2 μL) of each sample were analyzed by GCMS/O. The
avor dilution (FD) factors of the odorants were determined by
AEDA.
The GC/MSD (5975C, Agilent Technologies Inc., USA) equipped
with a DB-WAX capillary column (60 m ×0.25 mm i.d., 0.25 μmlm
thickness, Agilent Technologies Inc., USA) was used for mass spectro-
metric identication. The splitless mode was used with a splitless time
of 1 min. The split ratio at the time of the split vent liberation was
20:1. Helium was used as a carrier gas with a ow rate of 2 mL/min.
The injector temperature was 240 °C. The GC oven was maintained at
40 °C for 2 min, increased by 5 °C/min to 240 °C, and was kept at
this temperature for 25 min. The mass spectrometer was operated in
full scan mode with a mass range of m/z20280. For GCMS/O, the
euent was split into the olfactory detection port (Gerstel K.K.,
Japan) and MS using two deactivated, uncoated fused silica capillaries
(106 cm ×0.15 mm i.d., 139 cm ×0.1 mm i.d.) at the end of the
capillary. Volatile compounds were identied by direct comparison
with the Kovats GC retention indices (RI), mass spectra, and those of
the authentic specimens.
RESULTS AND DISCUSSION
Indole, Jasmine Lactone, and trans-Nerolidol Were
Signicantly Increased at the Turn Over Stage. To nd
out which volatiles, especially those also occurring in glyco-
sidically bound form, have big changes during the manufactur-
ing process, we monitored changes of free volatiles from the
plucking process to xing process (enzymatic reaction termi-
nation) of oolong tea. Among the manufacturing processes, the
turn over process (T1T5) had a signicant inuence on the
contents of volatiles (Figure 1). In the present study, not all the
free volatiles had big changes during the manufacturing process
of oolong tea. The three volatiles including indole, jasmine
lactone, and trans-nerolidol had signicantly big increase at the
turn over process (Figure 1). In addition, most fatty acid-
derived volatiles such as 1-hexanol, (E)-2-hexen-1-ol, (Z)-
hexen-1-ol, etc., linalool oxides, and α-farnesene increased at
T1T5, whereas some free volatiles such as linalool, geraniol,
diendiol I, benzyl alcohol, 2-phenylethanol, and methyl
salicylate, which also occur in glycosidically bound form, did
not show signicant changes during the process (Figure 1).
Wang et al. rst reported that the contents of free alcoholic
aroma compounds remained almost unchanged or slightly
decreased, but jasmine lactone and indole signicantly
increased in the nal oolong tea products compared to dried
fresh tea leaves.
21
In addition, increase of indole during the
oolong manufacturing process was also demonstrated by a
direct analysis in real time mass spectrometry.
34
Our previous
study on comparison of volatiles of green teas, oolong teas, and
black teas from dierent cultivars and regions also proposed
that jasmine lactone and indole were characteristic volatiles in
oolong tea products.
35
Furthermore, the present study indi-
cated that these volatiles mostly accumulated at the turn over
Figure 6. Subcellular localization analysis of GFP-β-primeverosidase. Upper photos: GFP-β-primeverosidase is visible in cell wall structures at the
tobacco leaf cell. Lower photos: Green uorescence was not shown in the protoplast cell prepared from the tobacco leaves (the upper photos), which
proved that the GFP-β-primeverosidase protein is localized in the cell wall.
Journal of Agricultural and Food Chemistry Article
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J. Agric. Food Chem. 2015, 63, 69056914
6910
process.
36,37
During the oolong tea manufacturing process, tea
leaves are exposed to various stresses, such as plucking (wounding),
solar withering (drought, heat, and UV/light radiation), indoor
withering (drought), and turn over (wounding).
16
Therefore, the
aroma formation in tea leaves during the manufacturing process of
oolong tea is proposed to be the stress-responsive biochemical
reactions of juvenile leaves of tea plants. Indole is a very repre-
sentative stress-responsive volatile compound in the manufacturing
process of oolong tea. Indole occurs with very trace amount in
intact tea leaves. However, indole level increases rapidly after
plucking.
38
Also indole increased during the withering process
of green tea leaves even at a relatively low temperature (15 °C,
for 16 h).
33
In the present study, the turn over led to more
accumulations of indole (Figure 1). This evidence suggests that
wounding stress from the turn over process is a key factor
regulating formation of indole. Interestingly, black teas contain
much less indole,
35
although wound stress is also involved in
the manufacturing process of black tea. Tea leaves are still
relatively intact and alive from the plucking to the turn over
process of oolong tea, whereas the structure of the leaf cells is
disrupted during the rolling process and the contents of the
cells are completely mixed during the rolling process of black
tea.
20,21
Amounts of indole, jasmine lactone, and trans-nerolidol
reduced after tea leaf cell disruption (Figure 2), suggesting that
mechanical damage with tea leaf cell disruption, which is similar
to the manufacturing process of black tea, does not induce the
accumulation of indole, jasmine lactone, and trans-nerolidol. As
anthranilic acid is one of precursors of indole,
33
we investigated
the formation of indole by feeding of stable isotope-labeled
compound [15N]anthranilic acid to the tea leaves. It was found
that the amount of [15N] indole in tea leaf with cell dis-
ruption was much lower than that in tea leaf without disruption
(Figure 3). This suggests that indole may be oxidized by P450
enzymes after the leaf cell disruption.
39
Therefore, the pathway
leading from anthranilic acid to indole may be a key step of
formation of indole during the oolong tea process. As the genes
involved in the pathway leading from anthranilic acid to indole
are still unknown in plants, we are attempting to identify the
involved genes and will investigate their responses to the
stresses from the manufacturing process of oolong tea. In addi-
tion, it would be very interesting to investigate whether dis-
ruption of leaf cell and complete mixture of contents of cell
may lead to the pathway competition between indole, jasmine
lactone, and trans-nerolidol and other metabolites.
Most GBVs Did Not Show Reduction during the
Manufacturing Process. To conrm whether enzymatic
hydrolysis of GBVs occurs during the manufacturing process of
oolong tea, we monitored changes of GBVs from the plucking
process to xing process (enzymatic reaction termination) of
oolong tea. No GBVs were reduced in their contents during the
enzyme-active process (Figure 4), which is consistent with the
previous report.
21
This suggests that enzymatic hydrolysis of
GBVs may not happen during the manufacturing process of
oolong tea. The GBVs occur in vacuoles, whereas hydrolases
such as β-primeverosidase were presumed to be localized in cell
walls.
18
This compartmentation of substrates and enzymes in
plant cells leads to little possibility of hydrolysis of GBVs in
nondisrupted tea leaf cells. After additional treatment was done
to disrupt tea leaf cell, GBVs were reduced (Figure 2), further
demonstrating that lack of disruption of tea leaf cell during the
oolong tea process resulted in unavailability of interaction of
Figure 7. Transmission electron micrograph of the tea leaf cells during
the oolong tea manufacturing process. P, plucking; T1, turn over 1;
T2, turn over 2; T5, turn over 5; V, vacuole; CW, cell wall. (A, D, G, J)
Multicells on the edge of sample tea leaves in P, T1, T2, and T5
respectively. Bar, 5 μm. (B, E, H, K) Single cell on the edge of sample
tea leaves in P, T1, T2, and T5 respectively. Bar, 2 μm. (C, F, I, L) Cell
wall of the cell on the edge of sample tea leaves in P, T1, T2, and T5
respectively. Bar, 0.1 μm.
Table 1. Identication of Potent Odorants (FD Factor 44)
in Oolong Tea
RI identied compound odorant quality FD factor (4n)
1538 linalool fresh oral 5
1716 3-methylnonane-2,4-dione fresh oral 5
1821 β-damascenone honey 5
1840 unidentied
a
minty 5
1883 unidentied
a
fresh oral 4
1945 β-ionone oral 4
2017 Furaneol
a
sweet 7
2200 δ-decalactone oral, sweet 6
2266 jasmine lactone oral, sweet 6
2431 indole animal-like, oral 4
2546 vanillin sweet 5
a
The MS spectrum is provided as Figure S2.
Journal of Agricultural and Food Chemistry Article
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substrates and enzymes and no hydrolysis of GBVs. Inter-
estingly, the previous report
21
and the present study found that
the amounts of some GBVs increased (Figure 4). Currently,
most studies on GBVs have concentrated on glycosidases,
whereas little information on glycosyl transferases is available.
1
Recently glycosyl transferases in tea leaves have been func-
tionally characterized;
40
it will be helpful for our understanding
of the accumulation of GBVs during the oolong tea manu-
facturing process.
β-Glycosidases Were Not Activated during The
Manufacturing Process, and Interaction of β-Glycosi-
dases and GBVs Were Unavailable. To investigate whether
β-glycosidases were activated during the manufacturing process,
we monitored enzyme activities and gene expression levels of
β-glycosidases from the plucking process to the fth turn over
process of oolong tea. The enzyme activities of β-glucosidase
and β-primeverosidase did not show increase during the manu-
facturing process (Figures 5A and 5B). Moreover, expression
level of β-primeverosidase showed reduction trend at the turn
over process (Figure 5C). Although many stresses including
drought, heat, wounding, and UV/light radiation are involved
in the oolong tea manufacturing process, our study indicates
that β-glycosidases were not activated under these stresses.
This also reveals that contact of substrate (GBVs) and enzyme
(β-glycosidases) is a key point determining whether enzymatic
hydrolysis of GBVs happens in the tea manufacturing process.
Previously, Mizutani et al. found that β-primeverosidase
is N-glycosylated, and has an N-terminal signal sequence, thus
presumed that β-primeverosidase may locate in the cell
wall.
18
However, a direct evidence of subcellular localization of
β-primeverosidase in tea leaves is still unavailable. In the present
study, subcellular localization of β-primeverosidase provided
more evidence that β-primeverosidase is located in the leaf cell
wall (Figure 6). Moreover, we investigated the cell structures
of tea leaves from the manufacturing process of oolong tea.
From the P to T1 process, the leaf cells showed complete and
unwounded. The content was divided into several parts and
showed the clear boundary (Figure 7AF). From the T2 to T5
process, the cells shrank and distorted intensely, while the cell
walls remained well and unwounded (Figures 7GL). These
results strongly support that lack of disruption of tea leaf cell
during the oolong tea process resulted in unavailability of inter-
action of GBVs (substrates located in vacuole) and β-glycosidases
(enzymes).
Besides β-primeverosidase, we also investigated the expression
levels of LOX that is a key gene that involved in formation of
fatty acid-derived volatiles and jasmine lactone,
1,25
and TPS
that are possibly involved in formation of trans-nerolidol in
tea leaves.
26
LOX is proposed as a wounding stress-response
gene.
25
LOX gene expression level signicantly increased at
the turn over process (Figure 5D), which may be one of the
reasons that most free fatty acid-derived volatiles and jasmine
lactone increased at that process (Figure 1). Similarly, TPS1, 2,
and 3 gene expression levels also signicantly increased at the
turn over process (Figures 5E,F,G). The present study provides
important hints that the wounding stress at the turn over
process may activate some key genes involved in formation of
volatiles.
Furaneol, Jasmine Lactone, δ-Decalactone, Linalool,
Vanillin, β-Ionone, β-Damascenone, 3-Methylnonane-
2,4-dione, and Indole Were Identied as Relatively
Potent Odorants in Oolong Tea. The above evidence shows
that enzymatic hydrolysis of GBVs did not signicantly con-
tribute to the formation of volatiles of oolong tea. To further
nd out which volatiles contribute to avor of oolong tea,
GCMS/O and AEDA were employed to determine the
contribution of each volatile to the quality of the teas aroma
and avor. The results revealed 49 odor-active peaks with FD
factors between 41and 47in the nal oolong tea product.
Among them, the 11 compounds showed as relatively potent
odorants with the higher FD factor (44)(Table 1). AEDA is
most frequently used for identifying ordor-active compounds in
Figure 8. Hypothetical model of dierent formations of volatiles between the oolong tea process and the black tea process.
Journal of Agricultural and Food Chemistry Article
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J. Agric. Food Chem. 2015, 63, 69056914
6912
tea, and evaluating the avor changes that occur during the
manufacturing of tea beverages.
33,4145
Based on this technique,
many volatile compounds were identied as the most important
components in Japanese and Chinese green teas and black tea
beverages. However, little information on potent odorants in
oolong tea is available. In the present study, Furaneol, jasmine
lactone, δ-decalactone, linalool, vanillin, β-ionone, 3-methylno-
nane-2,4-dione, β-damascenone, indole, and two other com-
pounds were identied as relatively potent odorants in oolong
tea (Table 1). Although linalool was one of the potent odorants,
its formation was not from enzymatic hydrolysis of glycosides of
linalool (Figures 1 and 4). Jasmine lactone and indole were
identied as relatively potent odorants, further suggesting that
they were characteristic compounds derived from the
manufacturing process of oolong tea (Figures 1 and 2)and
partly contributed to the aroma of oolong tea. Other compounds
showed high FD factors, suggesting that some compounds with
low concentrations may have a comparatively low olfactory
threshold detected by human and contribute to the characteristic
aroma of oolong tea.
Formation of Volatiles Is Dierent between Oolong
Tea and Black Tea. Oolong tea and black tea are fermented
teas and have more aroma characters than green tea. Therefore,
analysis and identication of volatile compounds during the
manufacturing process of oolong tea and black tea have been
well done. Based on the previous reports and the present study,
we propose a hypothetical model of dierent formations of
volatiles between the oolong tea process and the black tea
process (Figure 8). (1) For oolong tea, turn over is a key pro-
cess to produce tea aroma and increase of wounding intensity
could lead to accumulations of indole, jasmine lactone, and
trans-nerolidol, which may be characteristic aromas of oolong
tea. However, such wounding is not sucient for disruption of
tea leaf cell, which is essential for interaction of substrates and
enzymes. (2) For black tea, rolling is a key process to produce
tea aroma. At this process, complete disruption of tea leaf
cell leads to interaction of substrates and enzymes and many
GBVs are hydrolyzed to release free alcoholic aroma com-
pounds. The results help us nd out the direct evidence of
formation of volatiles in oolong tea, and advance our un-
derstanding of dierences of formation of volatiles between
oolong tea and black tea.
ASSOCIATED CONTENT
*
SSupporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jafc.5b02741.
Table of studied primers of qRT-PCR, ow chart of
oolong tea manufacturing process, and mass spectra
(PDF)
AUTHOR INFORMATION
Corresponding Author
*Tel: +86-20-38072989. E-mail: zyyang@scbg.ac.cn.
Author Contributions
§
J.G. and X.F. equally contributed to this work.
Funding
This study was supported by 100 Talents Programme of the
Chinese Academy of Sciences(Y321011001 and 201209), and
the Foundation of Science and Technology Program of
Guangzhou (2014J4100219).
Notes
The authors declare no competing nancial interest.
ABBREVIATIONS USED
AEDA, aroma extraction and dilution analysis; EFs, elongation
factors; FD, avor dilution; GBVs, glycosidically bound
volatiles; GCMS/O, gas chromatographymass spectrome-
ter/olfactometry; IW, indoor-withered; LOX, 9/13-lipoxyge-
nase; P, freshly plucking tea leaves; PVPP, polyvinylpolypyrro-
lidone; RI, retention indices; SW, solar withering; T1T5,
turned over by 5 times; TMEMD, N,N,N,N-tetramethyle-
thylenediamine; TPS, terpene synthases
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Geraniol is a potent tea odorant and exists mainly as geranyl glycoside in Camellia sinensis. Understanding the mechanisms of geraniol biosynthesis at molecular levels in tea plants is of great importance for practical improvement of tea aroma. In this study, geraniol and its glycosides from tea plants were examined using liquid chromatography coupled with mass spectrometry. Two candidate geraniol synthase (GES) genes (CsTPS) and two Nudix hydrolase genes (CsNUDX1-cyto and CsNUDX1-chlo) from the tea genome were functionally investigated through gene transcription manipulation and gene chemical product analyses. Our data showed that in tea leaves, levels of geranyl β-primeveroside were dramatically higher than those of geranyl β-glucoside, while free geraniol was undetectable in this study. A tempo-spatial variation of geranyl β-primeveroside abundance in tea plants existed, with high levels in young and green tissues and low levels in mature or non-green tissues. Cytosolic CsNUDX1-cyto showed higher hydrolysis activity of geranyl-pyrophosphate to geranyl-monophosphate (GP) in vitro than did chloroplastidial CsNUDX1-chlo. A transgenic study revealed that expression of CsNUDX1-cyto resulted in significantly more geranyl β-primeveroside in transgenic Nicotiana benthamiana compared with non-transgenic wild-type, whereas expression of CsNUDX1-chlo had no effect. An antisense oligo-deoxynucleotide study confirmed that suppression of CsNUDX1-cyto transcription in tea shoots led to a significant decrease in geranyl β-primeveroside abundance. Additionally, CsNUDX1-cyto transcript levels and geranyl β-primeveroside abundances shared the same tempo-spatial patterns in different organs in the tea cultivar "Shucha Zao," indicating that CsNUDX1-cyto is important for geranyl β-primeveroside formation in tea plants. Results also suggested that neither of the two candidate GES genes in tea plants did not function as GES in transgenic N. benthamiana. All our data indicated that CsNUDX1-cyto is involved in geranyl β-primeveroside production in tea plants. Our speculation about possible conversion from the chemical product of CsNUDX1-cyto to geranyl β-primeveroside in plants was also discussed.
... Studies have shown that benzeneacetaldehyde, dimethyl sulfide significantly increased during the shaking process of oolong tea [24,45]. Moreover, it has been reported that the accumulation of (Z)-3-hexenol during the shaking process was a stable phenomenon, due to the de novo synthesis pathway, rather than enzymatic hydrolysis [46,47]. ...
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... To learn more about the optical process that causes alterations in flavonoid glycosides throughout withering, a TMT-labeling proteomics investigation was used to examine the variations in the after-harvest leaf proteome. Because of this, major alterations in metabolites and previous studies on glycosidic compounds metabolites have tended to use comparatively short withering or spreading times (<12 h) [24,25]. As a result, the proteomes of leaves that had been withering for 0 h, 12 h, and 30 h were studied. ...
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... Notably, indole, farnesene, E-nerolidol, and ocimene, the most important aroma components of oolong tea (Lin et al., 2013;Yang et al., 2013;Baldermann et al., 2014), were clustered in Cluster 2. In contrast to the accumulation pattern of cluster 1 and cluster 2, the volatile metabolites in cluster 3 including hexanal, (Z)-3-hexenal, (Z)-3-hexen-1-ol, cis-3-hexenyl isovalerate, (Z)-2-penten-1-ol, (E)-2-non-enal, and 1-heptanol were abundant in fresh leaves (YL stage) and began to decrease approximately 40 min after the wounding treatment ( Supplementary Figure 4 and Supplementary Dataset 2). Our results suggest that most of the substances with a grass odor such as (Z)-3-hexen-1-ol (Wang et al., 2017), 1-heptanol (Wang et al., 2017), and hexanal (Wen et al., 2014) gradually disappear within 40 min after wounding treatment, while some substances with flower and fruit flavors gradually accumulate after 40 min of wounding treatment, which may be the potential reason why it is necessary to keep tea leaves motionless after wounding treatment for a while during oolong tea possessing (Gui et al., 2015;Zeng et al., 2019a;Chen et al., 2020). ...
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Understanding extensive transcriptional reprogramming events mediated by wounding during the oolong tea manufacturing process is essential for improving oolong tea quality. To improve our comprehension of the architecture of the wounding-induced gene regulatory network, we systematically analyzed the high-resolution transcriptomic and metabolomic data from wounding-treated (after turnover stage) tea leaves at 11 time points over a 220-min period. The results indicated that wounding activates a burst of transcriptional activity within 10 min and that the temporal expression patterns over time could be partitioned into 18 specific clusters with distinct biological processes. The transcription factor (TF) activity linked to the TF binding motif participated in specific biological processes within different clusters. A chronological model of the wounding-induced gene regulatory network provides insight into the dynamic transcriptional regulation event after wounding treatment (the turnover stage). Time series data of wounding-induced volatiles reveal the scientific significance of resting for a while after wounding treatment during the actual manufacturing process of oolong tea. Integrating information-rich expression data with information on volatiles allowed us to identify many high-confidence TFs participating in aroma formation regulation after wounding treatment by using weighted gene co-expression network analysis (WGCNA). Collectively, our research revealed the complexity of the wounding-induced gene regulatory network and described wounding-mediated dynamic transcriptional reprogramming events, serving as a valuable theoretical basis for the quality formation of oolong tea during the post-harvest manufacturing process.
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The delicate aroma of Bao-chung tea comes from oxidation, followed by fixation in the pan-firing step. Traditionally, the timing of pan-firing has been based on odor perception by tea masters and lacks relevant scientific research. Pan-firing at three different green-note intensities and three stirring sequences was used to explore the relationship between the compositions of volatile organic compounds (VOCs) before pan-firing and in the finished tea. Pan-firing decreased green leaf volatiles and increased the ratio of terpenoid volatiles. The characteristic VOCs of the finished tea were highly related to VOCs before pan-firing (R² = 0.97). Principal component analysis revealed that the traditional judgment of the pan-firing step is based on nonanal, β-linalool, and cis- and trans-linalool oxides. The timing of pan-firing is crucial for VOCs, and VOC composition before pan-firing can be used to predict desired tea aroma.
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Dianhong tea (DHT) is popular for its pleasant caramel-like aroma. In this study, the aroma profile of high-grade DHT have been studied using gas chromatography-mass spectrometry (GC-MS) and gas chromatography-olfactometry (GC-O) combined with headspace solid phase microextraction (HS-SPME). A total of 52 aroma-active compounds were identified by GC-O coupled with aroma extract dilution analysis (AEDA) and odor specific magnitude estimation (Osme). Among them, quantification of 21 aroma-active compounds indicated that the content of linalool (5928 µg/kg) was the highest in high-grade DHT, followed by phenylethanol (3923 µg/kg) and phenylacetaldehyde (1801 µg/kg). Sensory-directed aroma recombination and omission tests further verified that phenylacetaldehyde, linalool, geraniol and 3-ethyl-2,5-dimethylpyrazine were important contributors to the overall sensory characteristics of high-grade DHT which dominated mainly by floral, sweet and caramel-like odors. This work will provide a theoretical reference for comprehensively understanding the aroma characteristic of DHT.
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Reliable reference selection for the accurate quantification of gene expression under various experimental conditions is a crucial step in qRT-PCR normalization. To date, only a few housekeeping genes have been identified and used as reference genes in tea plant. The validity of those reference genes are not clear since their expression stabilities have not been rigorously examined. To identify more appropriate reference genes for qRT-PCR studies on tea plant, we examined the expression stability of 11 candidate reference genes from three different sources: the orthologs of Arabidopsis traditional reference genes and stably expressed genes identified from whole-genome GeneChip studies, together with three housekeeping gene commonly used in tea plant research. We evaluated the transcript levels of these genes in 94 experimental samples. The expression stabilities of these 11 genes were ranked using four different computation programs including geNorm, Normfinder, BestKeeper, and the comparative ∆CT method. Results showed that the three commonly used housekeeping genes of CsTUBULIN1, CsACINT1 and Cs18S rRNA1 together with CsUBQ1 were the most unstable genes in all sample ranking order. However, CsPTB1, CsEF1, CsSAND1, CsCLATHRIN1 and CsUBC1 were the top five appropriate reference genes for qRT-PCR analysis in complex experimental conditions.
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Camellia sinensis synthesizes and emits a large variety of volatile phenylpropanoids and benzenoids (VPB). To investigate the enzymes involved in the formation of these VPB compounds, a new C. sinensis short-chain dehydrogenase/reductase (CsSDR) was isolated, cloned, sequenced, and functionally characterized. The complete open reading frame of CsSDR contains 996 nucleotides with a calculated protein molecular mass of 34.5 kDa. The CsSDR recombinant protein produced in Escherichia coli exhibited dehydrogenase-reductase activity towards several major VPB compounds in C. sinensis flowers with a strong preference for NADP/NADPH co-factors, and showed affinity for (R)/(S)-1-phenylethanol (1PE), phenylacetaldehyde, benzaldehyde, and benzyl alcohol, and no affinity for acetophenone (AP) and 2-phenylethanol. CsSDR showed the highest catalytic efficiency towards (R)/(S)-1PE. Furthermore, the transient expression analysis in Nicotiana benthamiana plants validated that CsSDR could convert 1PE to AP in plants. CsSDR transcript level was not significantly affected by floral development and some jasmonic acid-related environmental stress, and CsSDR transcript accumulation was detected in most floral tissues such as receptacle and anther, which were main storage locations of VPB compounds. Our results indicate that CsSDR is expressed in C. sinensis flowers and is likely to contribute to a number of floral VPB compounds including the 1PE derivative AP.
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A crude enzyme was extracted from fresh tea leaves and it showed β-glucosidase activity, using p-nitrophenyl-β-D-glucoside as a substrate. The non-volatile fraction of a hot-water extract from green tea was incubated with the crude enzyme extract at 37°C for 30 min. After this enzymatic hydrolysis, the formation of (Z)-3-hexenol, linalool oxide I, linalool, methyl salicylate, geraniol, benzyl alcohol and 2-phenylethanol was observed. Geranyl-β-D-glucoside was synthesized by the Koenigs-Knorr reaction. When it was reacted with the crude enzyme extract of tea leaves, geraniol was identified on GC as an enzymatic hydrolysis product. Thus, geranyl-β-D-glucoside seems to be the precursor of geraniol in tea leaves. © 1990, Japan Society for Bioscience, Biotechnology, and Agrochemistry. All rights reserved.
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A crude enzyme was extracted from fresh tea leaves and it showed β-glucosidase activity, using p-nitrophenyl-β-d-glucoside as a substrate. The non-volatile fraction of a hot-water extract from green tea was incubated with the crude enzyme extract at 37°C for 30 min. After this enzymatic hydrolysis, the formation of (Z)-3-hexenol, linalool oxide I, linalool, methyl salicylate, geraniol, benzyl alcohol and 2-phenylethanol was observed. Geranyl-β-d-glucoside was synthesized by the Koenigs–Knorr reaction. When it was reacted with the crude enzyme extract of tea leaves, geraniol was identified on GC as an enzymatic hydrolysis product. Thus, geranyl-β-d-glucoside seems to be the precursor of geraniol in tea leaves.
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Tea plants (Camellia sinensis) store volatile organic compounds (VOCs; monoterpene, aromatic, and aliphatic alcohols) in the leaves in the form of water-soluble diglycosides, primarily as β-primeverosides (6-O-β-D-xylopyranosyl-β-D-glucopyranosides). These VOCs play a critical role in plant defenses and tea aroma quality, yet little is known about their biosynthesis and physiological roles in planta. Here we identified two UDP-glycosyltransferases (UGTs) from C. sinensis: UGT85K11 (CsGT1) and UGT94P1 (CsGT2), converting VOCs into β-primeverosides by sequential glucosylation and xylosylation, respectively. CsGT1 exhibits a broad substrate specificity toward monoterpene, aromatic and aliphatic alcohols to produce the respective glucosides. On the other hand, CsGT2 specifically catalyzes the xylosylation of the 6'-hydroxy group of the sugar moiety of geranyl β-D-glucopyranoside, producing geranyl β-primeveroside. Homology modeling, followed by site-directed mutagenesis of CsGT2, identified a unique isoleucine 141 residue playing a crucial role in sugar donor specificity toward UDP-xylose. The transcripts of both CsGTs were mainly expressed in young leaves, along with β-primeverosidase (β-PD) encoding a diglycoside-specific glycosidase. In conclusion, our findings reveal the mechanism of aroma β-primeverosides biosynthesis in C. sinensis. This information can be used to better preserve tea aroma during the manufacturing process, and to investigate the mechanism of plant chemical defenses. Copyright © 2015, Plant Physiology.
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Tea aroma is one of the most important factors affecting the character and quality of tea. Recent advances in methods and instruments for separating and identifying volatile compounds have led to intensive investigations of volatile compounds in tea. These studies have resulted in a number of insightful and useful discoveries. Here we summarize the recent investigations into tea volatile compounds: the volatile compounds in tea products; the metabolic pathways of volatile formation in tea plants and the glycosidically-bound volatile compounds in tea; and the techniques used for studying such compounds. Finally, we discuss practical applications for the improvement of aroma and flavor quality in teas.
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The volatile fractions of three famous Chinese green tea cultivar (Longjing, Maofeng, Biluochun) infusions were prepared by a combination of the adsorptive column method and the SAFE techniques. The aroma extract dilution analysis (AEDA) applied to the volatile fractions revealed 58 odor-active peaks with FD factors between 41 and 47. Forty-six of the odorants, which included six odorants which have not been reported in the literature in the Chinese green tea (2-isopropyl-3-methoxypyrazine, 2-ethenyl-3,5-dimethylpyrazine, cis-4,5-epoxy-(E)-2-decenal, 4-ethylguaiacol, (E)-isoeugenol, and 3-phenylpropionic acid), were identified or tentatively identified by GC-MS and GC-O. Among the perceived odorants, 4-hydroxy-2,5-dimethyl-3(2H)-furanone, 3-hydroxy-4,5-dimethyl-2(5H)-furanone, coumarin, vanillin, geraniol, (E)-isoeugenol, and 2-methoxyphenol showed high FD factors in all the cultivars, irrespective of the cultivar or harvesting season, suggesting that these seven odorants are essential for the aroma of the Chinese green tea. On the other hand, the contents of the odorants, FD factors of which were uneven between the cultivars, were suggested to influence the characteristic aroma of each cultivar. In addition, the formation mechanism of (E)-isoeugenol, one of the odorants which have not been reported in the literature with a high FD factor common to all the cultivars, was investigated, and it was suggested that the (E)-isoeugenol content of the tea products has a close correlation with the manufacturing process of the tea leaves.
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Our previous study found that 1-phenylethanol (1PE) was a major endogenous volatile compound in tea (Camellia sinensis) flowers and can be transformed to glycosically-conjugated 1PE (1PE-Gly). However, occurrences of 1PE-Gly in plants remain unknown. In this study, four 1PE-Gly have been isolated from tea flowers. Three of them were determined as (R)-1PE -D-glucopyranoside ((R)-1PE-Glu), (S)-1PE-Glu, and (S)-1PE -primeveroside ((S)-1PE-Pri), respectively, based on evidences of NMR, MS, LC-MS, and GC-MS. The other one was identified as (R)-1PE-Pri based on the data of LC-MS and GC-MS. Moreover, these 1PE-Gly were chemically synthesized as the authentic standards to further confirm their occurrences in the tea flowers. 1PE-Glu had higher molar concentration than 1PE-Pri in each floral stage and organ. The ratio of (R)- to (S)- differed between the 1PE-Glu and the 1PE-Pri. In addition, a 1PE-Gly hydrolase -primeverosidase recombinant protein produced in Escherichia coli exhibited high hydrolysis activity towards (R)-1PE-Pri. However, -primeverosidase transcript level was not highly expressed in the anther part, which accumulated the highest contents of 1PE-Gly and 1PE. This suggests that 1PE-Gly may not be easily hydrolyzed to liberate 1PE in the tea flowers. This study provides evidences of occurrences of 1PE-Gly in plants for the first time.