Sciknow Publications Ltd.

FBLS 2015, 3(2):39-43

Frontiers of Biological and Life Sciences DOI: 10.12966/fbls.06.03.2015
Attribution 3.0 Unported (CC BY 3.0)

Age Dependent Biochemical Responses in Camellia sinensis

Hrishikesh Upadhyaya*, Jayeeta Roy, and Sakhi Dutta
Department of Botany and Biotechnology, Karimganj College, Karimganj-788710, Assam, India
*Corresponding author (Email: hkupbl_au@rediffmail.com)

Abstract - Tea being perennial plant encounters different environmental stress. The present study aims at understanding various
biochemical responses during aging process in tea leaf. Total phenolic content of leaves increased with age of a plant, but it is not
sufficient to balance between ROS generation and antioxidant function of plant as evidenced by increased MDA content of the
leaf thus making the aged plant tissue less capable of coping with the prevailing environmental stress. Further, age dependent
changes in mineral nutrition and its effect on antioxidative defense function and detail analysis of regulation of nutrient trans-
porter during aging process can reveal better understanding of age dependent environmental stress adaptation mechanism in
Camellia sinensis.
Keywords - Aging, Biochemical Responses, Camellia sinensis

in plants (Tegischer et al., 2002), but there is scarcity of

1. Introduction
Tea is an important breverage which is prepared from the
leaves of Camellia sinensis (L.) O Kuntze, belonging to the
family Theaceae of angiosperm. It is not only a brevarage but
a good health drink. It is the source of vitamins, potent anti-
oxidants and other minerals including fluorine (Kabir, 2002;
Fujita & Yamagami, 2008). Tea plant being perennial crops,
is subjected to different environmental stresses. The annual
yield distribution and potential yield of tea is greatly influ-
enced by the seasonal fluctuations in weather variables such
as rainfall, temperature and drought etc. Various abiotic stress,
induces oxidative damage in tea plant and affects antioxidant
systems, altering different physiological and biochemical
processes (Upadhyaya & Panda, 2004a) and causes signifi-
cant crop losses. In the process of coping with changing
environment age of a plant is important as it may increase
generation of reactive oxygen species (ROS), such as supe-
roxide radicals (O2-), hydrogen peroxide (H2O2), hydroxyl
radical (.OH), alkoxyl radical (RO), singlet oxygen (.1O2)
within the cell of aging leaves. This leads to lipid peroxidation,
protein degradation, enzyme inactivation and affect nucleic
acid and almost every component of cell leading to cell death
which is similar to the effect of other abiotic stress (Panda,
2002;Upadhyaya & Panda, 2004b;Upadhyaya et al., 2008).
Although the biochemical damages caused by abiotic stresses
are retrievable (Upadhyaya & Panda, 2004b ; Upadhyaya et
al., 2008, 2012) depending upon the genotypes of plant and
duration and nature of stress exposure, but cellular changes
due to aging process are irretrievable in plants. However, the
intensity of damage due to aging varies depending on anti-
oxidant potential of the plants. There are few reports on
physiological and biochemical damages during aging process
information on age dependent biochemical variation in tea
plant. Antioxidant efficiency varies in different clonal varie-
ties of tea (Upadhyaya & Panda, 2013) and thus varied the
response of abiotic stress in different clones of tea (Hendique
& Manivel, 1990; Chakraborty et al., 2002; Upadhyaya &
Panda, 2013). The present investigation is an attempt to study
age dependent biochemical variation in tea plant.
Various hypotheses were proposed to account for the
change of leaf aspect in stressed plant. Previous study re-
vealed that stress induced oxidative damage of tea was cor-
related with dysfunction of antioxidant defense system
(Upadhyaya et al. 2008, 2011, 2013). However, plant species
may vary in their response under stress (Upadhyaya et al.,
2011, 2012) where the age of the plant might be an important
attributes. In order to better understand the age dependent
variation of plant response under natural condition, an expe-
riment was conducted with tea plant of different age group
collected from tea garden. In this study, we investigated the
biochemical responses in tea leaves of different age group
maintained at tea garden under natural conditions in order to
elucidate age dependent biochemical responses in tea leaves.
2. Materials and Methods
The experimental tea plant belongs to Genus Camellia of
order Ericales and family Theaceae of dicotyledonous group
of Angiosperms. Tea leaves of five different age group (1, 5,
10, 15 and 20 years old) were sampled from Durganagar Tea
Estate in a polybag and used for biochemical analysis. All the
experiments were repeated or replicated thrice depending on
the availability of the samples.
Concentration of chlorophyll a (Chl a), chlorophyll b (Chl
40 Frontiers of Biological and Life Sciences (2015) 39-43

b), total chlorophyll and total carotenoid (Cx+c) concentra-
tions were analyzed following Lichtenthaler (1987) and
Shabala et al. (1998) methods, respectively. 100mg of leaf
samples were placed in a 25-ml glass vial, added with 10 ml of
95.5 % acetone, and the glass vials were sealed with parafilm
to prevent evaporation and then stored at 4 oC for 48h. The
concentrations of Chl a, Chl b and Cx+c were measured using
an UV-visible spectrophotometer at 662, 644 and 470 nm
respectively. A solution of 95.5 % acetone was used as a blank.
The Chl a, Chl b, total chlorophyll and carotenoid concentra-
tions in the leaf tissues were calculated according to the
following equations:
Chl a = 9.784D662 0.99D644
Chl b = 21.42D644 -4.65D662
Total chlorophyll = Chl a + Chl b
1000D470 1.90Chl a 63.14 Chl b
Cx+c =
214
where Di is an optical density at the wavelength i.
The total phenolic content was extracted from the tea
leaves in 2N HCl. Tea leaves (0.25 g) were boiled for 30 min
in 5 ml of 2 N HCl. After cooling, the homogenate was fil-
tered through a Whatman paper and the filtrate used to de-
termine total phenolic compounds. Samples (5 ml) were
mixed with 0.75 ml of 1.9 M Na2CO3 plus 0.25 ml of Fo-
linCiocalteau phenol reagent. This mixture was kept in dark,
at room temperature (25oC), for one hour, before its absorp-
tion was read at 750 nm (Herrig et al., 2002).
The total sugar content was extracted from the tea leaves in
80% ethanol. Tea leaves (0.1g) were grind in 5ml of 80%
ethanol. Then it was boiled for 20 min. After cooling, cen-
trifugation was carried out at 2000 g for 5 min. Supernatants
(1ml) were mixed with 4ml anthrone. Then boiled it for 8 min,
the absorbance of the supernatants was measured at 630 nm
(Hedge & Hofreiter, 1962).
The level of lipid peroxidation, expressed as MDA content,
was determined as 2-thiobarbituric acid (TBA) reactive me-
tabolites. Plant fresh tissues (0.2 g) were homogenized and
extracted in 10 ml of 0.25% TBA made in 10 ml trichloroa-
cetic acid (TCA). Then extract was heated at 95oC for 30 min
and then rapidly cooled in ice. After centrifugation at 10000 g
for 10 min, the absorbance of the supernatant was measured at
532 nm. Non-specific turbidity was corrected by subtracting
the absorbance value taken at 600 nm. The concentration of
MDA was calculated using extinction coefficient of 155m
M-1cm-1 (Zhang et al., 1992).
Each experiment was repeated thrice and data presented
are with mean standard error ( SE ). The data analysis was
carried out using MS Excel and SPSS 10. The results were
subjected to ANOVA and Tukeys post-hoc test was used for
comparison between pairs of treatments.

3. Results and Discussion

Table 1. Changes in Chla, Chlb, Chla: Chlb, total Chlorophyll and total Carotenoid contents in leaves of tea [Camellia sinensis L.
(O) Kuntze] of different age group. Data presented are mean SE (n=3). Values with the same letter within column are not
significantly different by Tukeys post-hoc test at P0.5

Age of plant Chl a Chl b Chla/Chlb Total Chlorophyll Total Carotenoid

(mg g-1FW) (mg g-1FW) Ratio (mg g-1FW) (mg g-1FW)

1yr 6.340.15a 5.080.44 a 1.260.10 a 11.420.50 a 5.950.20 a

5yr 5.120.28 a 4.070.14 ab 1.260.03 a 9.190.42 b 5.700.14 a
10 yr 3.860.64 b 2.990.42 b 1.280.05 a 6.851.06 bc 4.140.63 ab
15 yr 3.000.12 b 3.980.19 ab 0.750.04 b 6.980.31bc 4.770.04 ab
20 Yr 2.020.45 c 3.950.32 ab 0.5120.03 c 5.970.75 bc 4.960.09 ab

In the present investigation, an attempt has been made to
study the biochemical responses of tea leaf during aging
process by analyzing the biochemical changes in leaf of tea
plant of different age group collected from garden. The
marked reduction of total Chl with the progressive age of the
plant due to the decrease of both Chl a and Chl b contents. Chl
b was degraded more than Chl a due to age induced senes-
cence process in tea leaf. Such reduction in Chl content has
been ascribed to loss of chloroplast membranes, excessive
swelling, distortion of the lamellae vesiculation, and the
appearance of lipid droplets (Kaiser et al., 1981) during abi-
otic stress in tea (Mukhopadhyay and Mondal, 2014; Upad-
hyaya and Panda, 2004b; Upadhyaya et al., 2008, 2011, 2013).
Age induced degradation of both total chlorophyll and caro-
tenoid in tea plant (Table.1). Decrease in chlorophyll and
carotenoid due to oxidative stress as a result of age induced
ROS generation as reported earlier in case of abiotic stress
(Upadhyaya et al., 2008, 2013). Such degradation of chloro-
phyll pigments may eventually decrease photosynthetic effi-
ciency in plants which might be one of the potent causes of
reduction in growth of plant. However, earlier study also
provides evidence for a significant positive relationship be-
tween transpiration efficiency and leaf chlorophyll concen-
tration in plant (Sheshshayee et al., 2006). As depicted in
 Frontiers of Biological and Life Sciences (2015) 39-43 41

Figure 1, total sugar content of leaf also changes with the sugar content of the mature plant could be attributed to the
progressive age of the plant. Total sugar content of leaf re- degradation of photosynthetic pigments resulting in reduction
mains higher at younger plant (1 and 5 year plant) and it of photosynthetic efficiency of plant.
declines with the progressive age of the plant. Such lower

Fig. 1. Changes in total sugar contents in leaves of tea [Camellia sinensis L. (O) Kuntze] of different age group. Data presented
are mean SE (n=3). Bars with the same letter are not significantly different by Tukeys post-hoc test at P0.5
The important biochemicals in determining tea quality thesis of phenolic might helped in maintaining the health
include the green leaf tea catechins and their oxidation quality of tea, as the most abundant phenolic present in tea are
products (theaflavins and thearubigins) which are responsible catechins which are potent contributors of many beneficial
for most of the plain black tea attributes. Catechins are the health properties of tea (Cabrera et al., 2006). However, plants
most abundant polyphenols present in tea plant which makes phenolic metabolites are also reported to function as free
it a potent health drink. Although total phenolic content in tea radical scavengers and mutagenesis inhibitors (Cabrera et al.,
leaves increases with progressive age of the plant as depicted 2006). In case of tea, different catechins may be the important
in Fig. 2 it might not be sufficient to cope with ROS generated phenolic compounds contributing to free radical protection
with progressive age of the plant as evidenced by increased and anti mutagenesis property of the plant.
MDA content of leaf (Fig. 3). However, increased biosyn-

Fig. 2. Changes in total phenolic contents in leaves of tea [Camellia sinensis L. (O) Kuntze] of different age group.
Data presented are mean SE (n=3). Bars with the same letter are not significantly different by Tukeys post-hoc test at P0.5
42 Frontiers of Biological and Life Sciences (2015) 39-43
Fig. 3. Changes in MDA content in leaves of tea [Camellia sinensis L. (O) Kuntze] of different age group. Data presented are
mean SE (n=3). Bars with the same letter are not significantly different by Tukeys post-hoc test at P0.5
Membrane integrity declines with advancing age of plant.
In aging plant tissues, lipid peroxidation plays a key role in
the loss of membrane integrity (Zhang, 1992). This study
presents evidence of extensive lipid peroxidation during
prolonged storage of plant. Selected features of the deterior-
ative processes occur during aging of plant tissues.
Lipid peroxidation refers to the oxidative degradation of
lipids. It is the process in which free radicals "steal" electrons
from the lipids in cell membranes, resulting in cell damage.
The quantification of the end-products of lipid peroxidation,
is to be specific, malondialdehyde (MDA). The most com-
monly used test is called a TBARS assay (Thiobarbituric acid
reactive substances assay). Thiobarbituric acid reacts with
malondialdehyde to yield a fluorescent product. The present
investigation also showed that lipid peroxidation increased
with progressive age of the plant suggesting loss of membrane
intrigrity.
4. Conclusion
In conclusion, it may be suggested that aging affect the plants
ability to cope with environmental stress. The present findings
reveals that various biochemical alterations occurs during
aging process in tea leaf which could be correlated with age
induced loss in growth and productivity of crop plant. Al-
though, total phenolic content of leaves increased with age of
a plant, it is not sufficient to balance between ROS generation
and antioxidant function of plant as evidenced by increased
MDA content of the leaf thus making the aged plant tissue less
capable of coping with the prevailing environmental stress.
Further, age dependent changes in mineral nutrition and its
effect on antioxidative defense function and detail analysis of
regulation of nutrient transporter during aging process can
reveal better understanding of age dependent environmental
stress adaptation mechanism in Camellia sinensis.
Acknowledgement
The plant material used in the project was procured from
Durganagar Tea Estate, Karimganj, Assam, India and we
expressed thanks to the manager of the Tea Estate for pro-
viding the same. We sincerely acknowledge the use of facility
in the IBH, and under Star College programme at Karimganj
College supported by DBT, Govt. of India during this work.
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