Indian Journal of Experimental Biology
Vol. 42, December 2004, pp. 1208- 1211
Modulation of osmotic stress effects on photosynthesis and respiration by
temperature in mesophyll protoplast of pea
Padmanabh Dwivedi 1* & AS Raghavendra
Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India
Received 24 April 2004; revised 24 August 2004
Exposure of mesophyll protoplast of pea to osmotic stress decreases the rate of photosynthesis while stimulating
marginally the respiratory rate of mesophyll protoplasts. The interaction of osmotic and temperature stress during the
modulation of photosynthetic and respiratory rates of pea (Pisum sativum var Azad P1) mesophyll protoplasts was
investigated. The protoplasts were exposed to either iso-osmotic (0.4 M) or hyper-osmotic (1.0 M) concentration of sorbitol
at 15° and 25°C. The rates of photosynthesis and respiration were studied. At optimum temperature of 25°C, there was a
decrease in photosynthesis (<10%) at hyper-osmoticum (osmotic effect), whereas respiration increased marginally (by about
15%). Low temperature (15 °C} aggravated the sensitivity of both respiration and photosynthesis to osmotic stress. At l5°C,
the decrease in photosynthesis due to osmotic stress was >35%, while the respiratory rate was stimulated by 30%. The
relative proportion of cytochrome pathway decreased by about 50% at both 15°C and 25°C while that of alternative pathway
increased, more so, at 15°C, when the mesophyll protoplasts were subjected to hyper-osmoticum stress. The titration
experiments showed that extent of engagement of alternative pathway was higher, the slope value was slightly higher for
15°C compared to 25°C. Low temperature modulates the effect of hyper-osmoticum stress on photosynthesis and
respiration, and results in increased participation of alternative pathway.
Keywords: Alternative pathway, Cytochrome pathway, Osmotic stress, Protoplast, Respiration, Temperature stress
Respiratory electron transport pathways of plant
mitochondria comprise the cytochrome (cyt) pathway
and the alternative pathway 1• The relative contribution
of these two pathways to total respiration depends on
environmental conditions. The response of plant
respiration to abiotic stress varies with the stress
factor and also with duration of the treatment or
exposure to such stress factors 2. Nutrient deficiency,
anoxia and low light induce the increased
participation of alternative pathway in plant tissues 3A.
Osmotic stress is known to prolong the induction
phase, inhibit photosynthetic carbon metabolism and
stimulate respiration in protoplasts at 25 oc as well as
induce increased capacity of alternative pathway 5 .
Low temperature stress induces alternative pathway
respiration in potato tubers\ maize 6 and mung bean
plants 7 . However, there is no clear information on the
extent and engagement of these two respiratory
pathways under the combined effect (interaction) of
osmotic and low temperature stress. The present
article attempts to study the pattern of cytochrome
and alternative pathways in pea mesophyll protoplasts
1
Present address: Department of Botany, Arunachal University,
Itanagar 791 111, India
*Correspondent author: [email protected]
exposed to osmotic stress, by using 1.0 M sorbitol
(hyper-osmoticum) in comparison to 0.4 M sorbitol
(iso-osmoticum), at two temperatures (15 °C and
25°C). This study will indicate if the effect of osmotic
stress on photosynthesis and respiration is modulated
by temperature. Sorbitol is known to be a non permeating osmoticum and has been used by other
workers to impose osmotic stress on leaf slices,
protoplasts or chloroplasts 8. Since protoplasts do not
have barriers like the cell wall and intercellular gases,
they take up externally added compounds, like
inhibitors, quickly and the effect of these compounds
can be observed in a few minutes.
Materials and Methods
Plants of pea (Pisum sativum L. cv Azad) were
raised from seeds (Pocha Seeds Company, Pune,
India) in plastic trays filled with soil and farmyard
manure. The plants were grown outdoor under a
natural photoperiod of approximately 12 hr and
average daily temperature of 30°C day/20°C night.
The first and second fully unfolded leaves were
picked from 8-10 day old plants and used for
protoplast isolation.
Isolation of mesophyll protoplasts-Mesophyl!
protoplasts were isolated using digestive enzymes of
DWIVEDI & RAGHAVENDRA: MODULATION OF OSMOTIC STRESS EFFECTS
cellulase and macerozyme, as already described 9 . The
preparation had about 90-95% intact protoplasts. The
metabolic vigour of the protoplasts was checked
routinely by measuring photosynthetic 0 2 evolution.
Average ranged from 102 to 110 ).!mole 0 2 h( 1 mg- 1 chi.
Chlorophyll content-Chlorophyll content of the
protoplast suspension was determined, after extraction in
80% (v/v) acetone 10 .
Photosynthetic oxygen evolution and respiratory
oxygen uptake--Photosynthesis and respiration were
measured by monitoting 0 2 evolved and 0 2 uptake,
respectively at 25°C using Clark type oxygen electrode
(Hansatech , U.K.). Calibration of oxygen content in
electrode chamber was done with air saturated water.
The reaction medium of 1 ml in the oxygen electrode
chamber contained 0.4M, Sorbitol; 1 mM, CaClz; 1 rnM,
MgClz; l rnM, bicarbonate; 10 rnM, HEPES-KOH (pH
7.5) and protoplasts containing 10 )J.g ml- 1 chlorophyll.
To impose osmotic stress, the concentration of sorbitol
was altered from 0.4 to 1.0 M. Other components of
reaction medium were unaltered. The protoplast reaction
mi xture was maintained in dark and the rate of
respiration was monitored for 3-5 min. The respiratory
rates of protoplasts in the reaction mixture were linear
up to at least 20 min. The mitochond1ial inhibitors were
added to the reaction mixture to make up the required
final concentrations.
Extent
and
engagement
of
alternative
parhway-Engagement of alternate pathway was
estimated according to the method of Yani and
Raghavendra 11 • Respiration was measured in presence of
increasing concentration of salicylhydroxarnic acid
(SHAM) 0 - 4 mM, with or without 200 ~ of
potassium cyanide (KCN). The first set of values was
plotted against the second. The slope of this plot gives
the fraction of the capacity of alternative pathway
engaged. Per cent (extent) of cytochrome and alternative
pathways out of total respiration was calculated
following the established formulae 11 • Concentration of
SHAM and KCN at which 0 2 uptake was minimum was
chosen for this calculation.
Chemicals-Cellulase and macerozyme were from
Yakult Honsha Co. Ltd., Japan. The laboratory
chemicals used were procured from either E. Merck
(India) Ltd., Loba Chernie or Himedia Laboratories, all
from India and fine biochemicals from Sigma Chemical
Company, USA.
Results and Discussion
The respiratory rates of protoplasts subjected to
1.0 M of sorbitol (osmotic stress) were higher th an
those in 0.4 M of sorbitol (iso-osmoticum) . The 0 2
uptake by pea mesophyll protoplasts was sensitive to
the mitochondrial inhibitors. The respiratory rate of
protoplasts decreased as the concentration of KCN
(Fig 1a) or SHAM (Fig. 1b) was increased.
The photosynthetic rate was minimally reduced (<
10%) at 25 °C, whereas it was lowered by >35 % at
(b )
(a)
20
1209
"';'
.s=
u
18
Cl
E
";'
.s=
1.0 M sorbitol
16
N
0
0
E
.2;
14
Q)
.;,:
ro
......
12
0.4 M so rbito l
a.
:::1
N
0
10
0.0
0.1
0.2
0.3
KC N (m M)
0.4
0.5
0
1
2
3
4
SHAM (m M)
Fig_. 1- Sensitivity of respirati o n in pea meso ph yll protoplasts to-( a) KCN ; (b) SHAM. [Respiratory rates of 0 2 uptake (ftm ole 0 2 hr' 1mg
chi 1) at 0.4 and 1.0 M of sorbitol].
INDIAN J EXP BIOL, DECEMBER 2004
1210
the plot was higher (P> 1.0) under 1.0 M sorbitol
stress compared to the value of 0.83-0.84, under
normal 0.4 M sorbitol, at both l5 °C and 25 °C (Fig.
2a, b). The titration experiments indicated the
increased engagement of alternative pathway, when
protoplasts were subjected to osmotic stress.
Rate of photosynthesis of the mesophyll protoplast
declined considerably, whereas respiratory rate was
maximally high at 15°C compared to 25 °C, when
mesophyll protoplast was subjected to higher
osmotiCUQ1 concentration. Temperature modulated the
effect of osmotic stress on both photosynthesis and
respiration. The respiratory rate of protoplast under 1.0
M of sorbitol were higher at 15° and 25°C. The P value
at 1.0 M of sorbitol was above 1.0, whereas under
0.4 M of sorbitol (normal), it was below 1.0 at both the
temperature. Thus, titration experiments revealed an
increase in the engagement of alternative pathway at
15° and 25°C under hyper-osmoticum stress. The
proportion of cyt pathway under these conditions
lowered down. The findings in the present study
complement with others where it has been suggested
that the increased rate of respiration at low temperature
involves a greater participation by the alternative
pathwa/·12 .
The osmotic stress (hyper-osmoticum) at 1.0 M of
sorbitol might cause water loss and thereby resulting
in a change in biophysical characteristics of
membranes. Exposure of plants to temperature between
l5 °C under hyper-osmoticum. The respiratory rate was
increased by about 15% at 25°C, but it increased
maximally by 30% at 15 °C. (Table 1) under 1.0 M of
sorbitol. When protoplasts were subjected to osmotic
stress (1.0 M sorbitol), the relative proportion of
cytochrome pathway decreased at both 15°C (from 42
to 22%) and 25 °C (55 to 27%), while the proportion of
alternative pathway increased from 28 to 48%, and 26
to 38% at l5 °C and 25°C, respectively. The osmotic
effect has no effect on extent of residual respiration at
15 °C, but it increased from 19 to 35% at 25 °C.
The titration experiments done to assess the
capacity of alternative pathway showed that slope of
Table 1- lnteraction of osmotic and temperature stress during the
modulation of photosynthetic and respiratory rates of pea
mesophyll protoplasts. The data are shown as per cent change at
1.0 M of sorbitol (hyper-osmoticum) compared to 0.4 M of
sorbitol (iso-osmoticum) at varying temperature.
[Values are averages of two different experiments]
Temperature(°C)
Respiration
Photosynthesis
15
20
25
30
130
108
115
110
63
67
94
98
Whereas the average photosynthetic rate (~-tmole 0 2 evolved hr" 1
mg chr 1) at 15° and 25°C under 0.4 M of sorbitol was 56.0 and
103.0 respectively, the respiratory rate (~-tmole 0 2 consumed hr" 1
mg chr 1) under similar conditions of temperature and osmoticum
were 6.9 and 7.8 , respectively.
140
130
-...
(a) 15 °C
0.4 M sorbitol (p=0.83)
110
•
0.4 M sorbitol
u
70
•
•
0
M •o,bltol(p=l.13)
•
60
30
(~=0.84)
~0
.
· •~·
.
1.0 M sorbitol (p=1.07)
N
0
•
•
120
0
c0
(b) 25 °C
70
50
60
0 2 uptake (% of control)
40
(SHAM+ KCN]
80
70
80
90
100
110
120
0 2 uptake (% of control)
[SHAM+ KCN]
Fig. 2-Extent and engagement of alternative pathway in mesophyll protoplasts of pea subjected at either normal osmoticum (0.4 M
sorbitol) or hyper-osmoticum of 1.0 M sorbitol at-( a) I5 °C; and (b) 25 °C. Rate of 0 2 uptake as % of control at di ffe re nt concentrati on of
SHAM in the absence of KCN is plotted against a similar set of values obtained in the presence of KCN (0.2 mM). The data are the
averages of three separate experiments done on different days.
DWIVEDI & RAGHAVENDRA: MODULATION OF OSMOTIC STRESS EFFECTS
0°-l5°C (chilling stress) causes chilling injury which
leads to alterations in metabolic processes, decrease in
enzymic activities, changes in membrane fluidity and
inhibition of photosynthetic capacity 13 . The osmotic
stress leads to accumulation of superoxide radicals,
which may lead to lipid peroxidation 14 . Such
oxidative degradation of phospholipids and other
unsaturated lipids of cell membrane lead to leak of
membranes 15 • As a result of membrane damage under
osmotic and low temperature stress, the chances of
participation of alternative pathway increases and
there is reduction of cyt pathway, as evident in the
present study .
An inherent limitation of metabolic inhibitors is the
possibility of their unspecific and multiple effects on
different processes in cells 16 . In the present study,
however, inhibitors were used at low concentration
and for relatively short-term assays to minimize the
problems. The mesophyll protoplasts, during the
present study, did not rupture on exposure to 1.0 M of
sorbitol stress. They remained intact as observed
under the microscope. It was evident from the present
study that there was rise in engagement of alternative
pathway in pea mesophyll protoplasts in response to
hyper-osmoticum and low temperature stress. The
residual respiration varied between 19-35% and this
might be due to a complex of several oxygen
consurmng processes, in non-respiratory extra
mitochondrial reactions. In conclusion, the present
study showed that the low temperature treatment
modulates the effect of hyper-osmoticum stress on
photosynthesis and respiration, and resulted in
increased participation of alternative pathway. Plants
appear to adjust to abiotic stress by switching over to
alternative pathway under changing environmental
conditions.
The author (PO) acknowledges the support from
INSA, New Delhi, for awarding INSA Visiting
Fellowship. The work was supported by a grant from
DST (SP/SO/A-12/98), New Delhi, India.
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