Indian Journal of Exprimental Biology
Vol. 52, September 2014, pp. 898-904
Chloroplast ultra structure, photosynthesis and enzyme activities in regenerated
plants of Stevia rebaudiana (Bert.) Bertoni as influenced by copper sulphate
in the medium
Pourvi Jain, Sumita Kachhwaha & S L Kothari*
Department of Botany, University of Rajasthan, Jaipur 302 004, India
Received 2 April 2014; revised 5 June 2014
Stevia rebaudiana (Bert.) Bertoni is an important medicinal plant used as noncaloric commercial sweetener. Plants
regenerated with higher levels of copper sulphate in the medium exhibited enhanced activity of peroxidase and
polyphenoloxidase (PPO) enzymes. Transmission electron microscopy (TEM) revealed increase in size and number of
electron dense inclusions in the chloroplasts of plants regenerated at optimised level of copper sulphate (0.5µM) in the
medium. There was decrease in chlorogenic acid (CGA) content. Chl-a-fluorescence transient pattern (OJIP) showed that
the photosynthesis process was more efficient at 0.5µM CuSO4 in the medium.
Keywords: Chlorogenic acid, Chl-a-fluorescence transient pattern, Copper sulphate, Photosynthesis, Stevia rebaudiana
Stevia rebaudiana (Bert.) Bertoni is an economically
important source of non-caloric natural sweetener and
its extract is widely used in many countries as sugar
substitute, making it a major source of high potency
sweetener for the growing food market1,2. Stevia
rebaudiana, a Paraguayan perennial herb belongs to
family Asteraceae; its leaves accumulate a mixture of
eight different glycosides derived from the tetracylic
diterpene steviol3. These products taste intensely
sweet, e.g. Rebaudioside-A has been shown to be
320 times sweeter than sucrose on the weight basis4.
The accumulation of steviol glycosides in the cells of
S. rebaudiana largely depends on the development of
the chloroplast membrane system and the content of
the photosynthetic pigments5. The concentration of
glycosides in the leaves increases with delayed
flowering, long days and age of leaf tissue6. The
composition of tissue culture medium with different
concentrations of micronutrients to improves growth
and morphogenetic response, chlorophyll content
and biomass of the regenerated plants7-9. Copper is
known to be a vital component of electron transfer
reaction mediated by proteins such as superoxide
dismutase, cytochrome-c-oxidase and plastocyanin
but its concentration in the cells needs to be
——————
*Correspondent author
Telefax: + 91 141 2703439
E-mail: [email protected]
maintained at low levels10. However, the ability of
this essential metal ion to transfer electrons can be
hindered becoming toxic to cells when present in
excess as it is directly involved in the formation of
toxic reactive oxygen species11. Plants can regulate
their intracellular concentration of metal ions in
response to external stimuli, by modulating uptake,
transport, storage and secretion rates of metal ions12.
Excess of heavy metal stress in plants, can lead to
formation of reactive oxygen species (ROS) including
superoxide radicals, hydroxyl radicals and hydrogen
peroxide that damage the plant cells13. Antioxidant
enzymes such as peroxidases and polyphenoloxidases
can convert H2O2 to H2O in the plant cells and
neutralizes the toxic effects of H2O2.14 Polyphenol
oxidase (PPO) is a copper containing enzyme
localised on the thylakoids of chloroplasts and
involved in catalyzing the aerobic oxidation of
different phenolic compounds to quinines which are
autooxidised to dark brown pigments. Role of PPO as
an “oxygen buffer” has been postulated by Hulya
and Aylem15. The involvement and the role of
antioxidants in protection against oxidative stress has
also been demonstrated using transgenic plants16.
Plants are known to contain significant quantities
of various polyphenolic acids, such as coumaric acid
and caffeic acid, as well as their glycosides and esters.
Among these 5 -caffeylquinic or chlorogenic acid is
widely distributed and is present in high content in
JAIN et al.:STEVIA REBAUDIANA & CuSO4 AS MICRONUTRIENT
plants. Chlorogenic acid is of interest as a natural
antioxidant which had marked inhibitory activity
in relation to enzymes such as arginase17 and xanthine
oxidase18.
Contrarily the presence of copper directly
affects the photosynthetic process and its efficiency
in the chloroplast of regenerated leaves of
S rebaudiana. The fast Chlorophyll-a-fluorescence
transient pattern is a powerful tool for assessing
the photochemical electron transport as well as the
overall vitality and physiological performance of
oxygenic photosynthetic organisms and their
tissues19. It is a sensitive method for the detection
and quantification of changes induced in the
photosynthetic apparatus. Chl-a-fluorescence intensity
of dark-adapted photosynthetic organisms follow
a characteristic variation with time after the onset of
illumination. This effect is well known as “Kautsky
effect”20. In fluorescence induction curve, at the
minimal fluorescence (Fo) all the reaction centres
are open, and at maximal fluorescence (Fm) all
the reaction centres are closed. All oxygenic
photosynthetic material investigated so far showed
polyphasic fluorescence rise pattern consisting of
a sequence of phases denoted as O, J, I and P21. The
O-J-I-P transient pattern is defined by a certain time
during induction kinetics: O, first measurement at
onset of illumination, J at about 2 ms, I at about 30 ms
and P at about 500 ms22. Polyphasic chlorophyll
a fluorescence transient has been measured and
quantification was done by JIP test to study the
effect of copper sulphate on photosynthetic process
in S. rebaudiana. The aim of the study is to evaluate
the effect of optimization of micronutrient copper
on enzymatic activities, chloroplast ultrastructure
and photosynthesis in the regenerated plants of
S. rebaudiana.
Materials and Methods
Plant material—Stevia rebaudiana (SRB123
variety) plants were procured from Sun Fruit Pvt. Ltd.
Pune, India. Young primordial leaves surrounding
shoot tip and leaves of size < 1 cm from field grown
plants were used as explants.
Explant sterilization—The leaves were washed in
tap water and gently rinsed with 20% (v/v) extran
(Merck, India). The surface sterilization of explants
was carried out in 0.1% sodium hypochlorite solution
for 10 min. The explants were rinsed with five
changes of sterile distilled water. Murashige and
Skoog (MS) medium was prepared with 3% (w/v)
899
sucrose along with BAP (2.2 µM) and IAA (2.8 µM).
The medium was solidified with 0.8% Agar
(Qualigen, bacteriological grade). The pH of the
medium was adjusted to 5.8 before autoclaving at
121 °C and 1.2-1.3 kg/cm2 pressure for 20 min.
Culture establishment—Leaf explants were cut
from petiolar end and placed on sterile medium (with
dorsal side in contact with the medium) supplemented
with 6-benzylaminopurine BAP (2.2µM) and indole3-acetic acid (IAA; 2.8 µM) as per Jain et al8. All the
cultures were incubated in growth chamber at
26°C, 16 h photoperiod and 25 µ mol/ (m-2 s-1) light
intensity provided by white fluorescent tubes. After
4 weeks, the shoot buds induced directly on leaf
explants were excised from base along with some
portion of mother explant and placed on proliferation
medium supplemented with 6-benzylaminopurine
(BAP; 3.5 µM) and kinetin (Kn;1.8 µM). Cultures
were established in different vessels and were
observed regularly for 4 weeks. The leaves from
regenerated plants were used further as explants for
studying the effect of different concentration of
copper sulphate in the culture medium. The explants
were placed on culture medium supplemented with
BAP (2.2 µM) and IAA (2.8 µM) and sucrose
3% (w/v). This was considered as control induction
medium containing a usual CuSO4 level of 0.1 µM.
Different levels of CuSO4 (0.1, 0.5, 1, 2, 3, 5 µM)
were added in MS medium with BAP (3.5 µM) and
Kn (1.8 µM) at the same levels as the control
induction medium. The elongated shoots were sub
cultured in proliferation medium supplemented with
BAP (3.5 µM) and Kn (1.8 µM) along with different
levels of CuSO4 (0.1, 0.5, 1, 2, 3, 5 µM). The cultures
were kept under controlled conditions in culture
chamber and observed regularly for 4 weeks.
Peroxidase and polyphenoloxidase assays—Fresh
leaves (1 g) were taken from regenerated shoots of
3-5 week old cultures and grounded in 0.05 M
potassium phosphate buffer (pH 7.0) with pre-chilled
mortar and pestle. The homogenate was centrifuged
for 20 min at 5000 rpm. After centrifugation, the
pellet was discarded and supernatant was mixed
with 70% cold acetone and centrifuged again at
5000 rpm for 10 min. The collected supernatant
was used for further enzyme assays. The reaction
mixture was prepared by mixing 0.1 mL enzyme
extract, 0.01 mL 20 mM Guaiacol and 0.1 mL
50 mM H2O2. Peroxidase activity was determined
spectrophotometrically by monitoring the formation
of tetraguaiacol at 470 nm after every 15 sec. One unit
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INDIAN J EXP BIOL, SEPTEMBER 2014
of peroxidase activity corresponds to the level of
enzyme activity which was expressed as moles
H2O2 destroyed/min/g fresh wt23. For PPO assay, the
reaction mixture was made by mixing 1 mL enzyme
extract, 3 mL catechol solution (0.01M catechol
freshly prepared in 0.1 M phosphate buffer, pH 6.0).
PPO activity was determined spectrophotometrically
by monitoring the formation of quinones at 495 nm
after every 15 seconds up to 5 min. One unit of
PPO activity corresponds to the level of enzyme
activity which was expressed as moles of quinones
formed/min/g fresh wt.
Estimation of chlorogenic acid—Standard curve
was constructed using dilution of a 10 mg %
commercial preparation of chlorogenic acid
(MP Biomedical, USA). Quantitative assay was
done spectrophotometrically at 315 nm. Fresh leaves
(100-150 mg) were crushed by pestle and mortar
in 100 volume of distilled water to prepare
homogenous green suspension. It was allowed to
stand for 1 min and was centrifuged at 9000 rpm
for 5-6 min. The supernatant was immediately
used for estimation of CGA content in leaves,
spectrophotometrically at 315 nm.
Measurement of fast Chl-a fluorescence induction
kinetics—The in vitro regenerated and potted plants
were kept in darkness for 15 min and examined for
Chl-a-fluorescence transient pattern (OJIP) using the
procedure described by Strasser et al24. Chlorophyll
fluorescence was measured using a plant efficiency
analyzer (Handy PEA, Hansa Tech Instruments Ltd.,
Norfolk, UK). The chlorophyll-a-fluorescence
transients were induced by the red light of
3000 µE m–2 s -1 and recorded from 10 µs up to 1 s.
All measurements were done on fully dark-adapted
attached leaves. From the first OJIP transients,
several bioenergetics parameters were derived
according to the equation of the JIP test
using Bioanalyzer program (R.M. Rodriguez,
Bioenergetics Laboratory, University of Geneva;
www.Unige.ch/sciences/biologie/bioen). Chlorophyll
‘a’ fluorescence at 20 µs, 2 ms, and 30 ms, and the
time required to achieve maximum fluorescence
are termed as the O, J, I, and P steps, respectively.
The J-I-P test refers to the analysis of structural
integrity and function of chloroplasts based on
fluorescence emission data25. Further details on OJIP
measurements and the use of different formulae are
described elsewhere26. The basic level of fluorescence
would vary with the amount of chlorophyll in the
same sample as well as the duration of light
and darkness. These differences in chlorophyll
fluorescence could be overcome by normalizations to
Fo, Fi, or Fm of the fast transients. This normalization
makes a comparison of the fluorescence transients
within the same figure easy27. Various photochemical
parameters like absorption, transmittance and electron
transport per cross section of leaf and per reaction
centre and photosynthetic yields are observed in
regenerated shoots at normal MS level and at higher
levels of copper sulphate in in vitro cultures and in
potted field transferred plants.
Transmission electron microscopic studies—For
Electron Microscopy, fixation of leaves of
regenerated plants was done in 2.5% glutaraldehyde
and 23% paraformaldehyde, made in 0.1 M Sodium
phosphate buffer (pH 7.4). The fixed tissues were
transferred in phosphate buffer to SAIF (Sophistical
Analytical Instrumentation Facility (DST) AIIMS.
Sample processing, microtomy and electron
microscopy was done in AIIMS, New Delhi.
Statistical analysis and experimental design—Each
treatment consisted of 3 replicates. One way analysis
of Variance (ANOVA) was applied in order to
evaluate the effect of different concentrations of
micronutrient copper on Peroxidase and PPO enzyme
activity and on CGA content. The statistical analysis
was conducted by Fisher’s least significance
difference (P + 0.05)28.
Results
Effect of CuSO4 on enzyme activities and CGA
content—Activities of peroxidase and PPO enzymes
increased in plants regenerated at higher levels of
copper sulphate in the medium. With increase in
copper levels in culture medium there was
considerable decrease in antioxidant (CGA) content in
the leaves of regenerated plants (Fig.1).
Effect of CuSO4 on photosynthetic activity—
Photosynthetic performance of leaf samples taken
from potted plant (20 days old field transferred plant)
as autotrophic control, in vitro regenerated plant
at 0.1µM CuSO4 as heterotrophic control, in vitro
regenerated plant at 0.5 µM CuSO4 and in vitro
regenerated plant at 1 µM CuSO4 was measured using
the Handy plant efficiency analyzer. Effect of copper
sulphate was observed on different photochemical and
biochemical parameters during photosynthesis in
radar plot which revealed that there was no variation
at different parameters in leaves of field transferred
JAIN et al.:STEVIA REBAUDIANA & CuSO4 AS MICRONUTRIENT
plant. At MS level of copper sulphate a slight
variation in different parameters was observed while
plants regenerated at higher levels of copper showed
maximum variations in different parameters (Fig. 2).
Effect of CuSO4 on chloroplast ultrastructure—
TEM studies were carried out to observe the effect of
copper sulphate on ultrastructure of the chloroplast in
Stevia rebaudiana. Plants regenerated at the MS level
of CuSO4 had symmetric and asymmetric oblong
shape chloroplasts situated along thick cell wall.
Magnified view of single chloroplast showed the
presence of single electron dense inclusion body
present in stroma (Fig. 3a). Plants regenerated at
higher levels of copper in the medium had higher
number of electron dense inclusion bodies in their
chloroplasts and the size of the inclusion bodies were
also larger along with large vacuoles present in
mesophyll cells (Fig. 3 b-d).
Fig.1—Effect of CuSO4 on enzyme activity and antioxidant
content of regenerated plants of Stevia rebaudiana (a) Peroxidase
activity in leaf, (b) Polyphenol oxidase activity in leaf, (c) CGA
in leaves
901
Discussion
The appropriate level of micro nutrients is essential
for the optimal functioning and growth of plants.
Copper has been considered as one of the essential
micronutrients29. It has also been reported that copper
plays an important role in several metabolic activities
including protein and carbohydrate metabolism31.
Numerous copper containing enzymes (cytochrome
oxidase, ascorbate oxidase, phenolase, laccase,
diamine oxidase, super oxide dismutase, and quinol
oxidase) have been identified in plants31. Copper is
directly involved in the photosynthetic electron
transport chain as a constituent of plastocyanin, a
copper containing protein32. Plastocyanin operates as
an electron carrier between the two photo systems.
OJIP transient pattern was used to monitors
function of chloroplasts in mesophyll cells of Stevia
rebaudiana under higher levels of copper sulphate in
in vitro regenerated plants. Similarly OJIP transient
pattern were used for revealing the effects of high salt
stress on PSI in wheat leaves26. Functional integrity of
pea mesophyll protoplasts during isolation process
was monitored by OJIP transient studies. Their results
demonstrated that the OJIP transients could be
successfully used to study the quality of mesophyll
protoplasts at different isolation steps. The protoplast
maintained their integrity and photosynthesis status
well and their performance was similar to intact
leaves33. In the current study the effect of
micronutrient copper was analysed on photosynthesis
process. The plant efficiency analyser was used and
OJIP transient pattern was generated for assessing the
photosynthesis process in live Stevia plants.
Shikanai et al.34 also reported copper as an essential
micronutrient required for photosynthesis process.
The author reported that the leaves of copper deficient
plants and Arabidopsis thaliana mutants, exhibited
reduced chlorophyll content and photosynthetic
electron transport into the chloroplast.
The ultra structure study of chloroplast showed
increase in inclusion bodies in chloroplasts at
higher level of copper sulphate. Earlier electron dense
inclusion bodies inside thylakoids were reported
in chloroplasts of some other plant species
The experiments conducted on Perilla ocymoides
showed that such a modified type of thylakoids were
dominant when short day plants were grown under
continuous illumination. It is of interest to note that
both Stevia and Perilla are the short-day plant that
display increased vegetative growth without transition
902
INDIAN J EXP BIOL, SEPTEMBER 2014
Fig.2—Effect of CuSO4 on different phytochemical and biochemical parameters during photosynthesis in regenerated leaves of
Stevia rebaudiana as depicted by Radar plot between Fo and Fm. [Control plant in pot: Green in vitro regenerated plant at MS levels
of CuSO4 (0.1 µM): Red, in vitro regenerated plant at (0.5 µM) levels of CuSO4: Blue, in vitro regenerated plant at (1µM) levels
of CuSO4: Brown]
to flowering stage when they are grown under long
day conditions35. Moreover, the content of the steviol
glycosides in Stevia leaves grown under long days
was found to be higher than that in the leaves of
the same plants grown under short day. However
the chemical nature of these inclusions remains so
far poorly understood and most likely may be
different for different plant species. According to
Ladygin et al.5 such inclusion bodies observed in
thylakoids of chloroplasts of Stevia cells are not
steviol glycosides, but might be their precursor.
However, in the thylakoids of Nymphoides indica
chloroplasts, the electron dense inclusion bodies
represented phenols35. It was suggested that in
the chloroplasts of leaves of juvenile plants,
these inclusion bodies might serve as an inhibitors
of flowering and they could converse into
inactive forms, for example glycosides as may be
required. It is most likely that prolonged period
of intensive production of chloroplast isoprenoids
(phytols, carotenoids and plastoquinone-9) in
chloroplasts, the process essential for photosynthesis
as a whole precedes an active formation of the
steviol glycosides in these organelles during
ontogenesis. These metabolites are shown to be
capable of being synthesized in chloroplasts following
an alternative pathway36. However, the observed
positive correlation between the development
and activity of the photosynthetic apparatus and
production of the steviol glycosides suggests that
synthesis of isoprenoids in Stevia chloroplasts is
somewhat related to secondary metabolite production
in chloroplast of Stevia rebaudiana. Therefore, the
production of both isoprenoids and steviol glycosides
in chloroplasts could be stimulated upon increasing
the concentrations of precursors of these compounds.
The initial step of synthesis of steviol glycosides occurs
in chloroplasts using alternative glyceraldehydes-3-
JAIN et al.:STEVIA REBAUDIANA & CuSO4 AS MICRONUTRIENT
903
and Bioenergetics Laboratory, University of Geneva,
Switzerland for plant efficiency analysis of Stevia
rebaudiana
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Fig 3.—Transmission electron micrograph showing chloroplast
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Conclusion
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Acknowledgement
Pourvi Jain gratefully acknowledges Council of
Scientific and Industrial Research (CSIR), New Delhi
for the award of SRF and RA (Research Associateship)
and Professor Reto J. Strasser, Director, Microbiology
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