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Utilization of fast chlorophyll A fluorescence

technique in assessing the salt/ion sensitivity
of mung bean and Brassica seedlings

Article in Journal of Plant Physiology · September 2001

Impact Factor: 2.56 · DOI: 10.1078/S0176-1617(04)70144-3

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J. Plant Physiol. 158. 1173 – 1181 (2001)
 Urban & Fischer Verlag
http://www.urbanfischer.de/journals/jpp

Utilization of fast chlorophyll a fluorescence technique in assessing the

salt/ion sensitivity of mung bean and Brassica seedlings

Amarendra N. Misra1 *, Alaka Srivastava2, 3, Reto J. Strasser2

1
Department of Botany, Utkal University, Bhubaneswar-751004, India
2
Laboratory of Bioenergetics, University of Geneva, CH-1254, Jussy-Geneva, Switzerland
3
Present address: Division of Biology and Medicine, Brown University, Providence, RI 02912, USA

Received January 17, 2001 · Accepted April 17, 2001

Summary
Effects of NaCl and KCl alone or in combination with CaCl2, Na2SO4 or K2SO4 on fast chlorophyll a
(Chl a) fluorescence kinetics of mung bean (Vigna radiata L.) and Brassica (Brassica juncea Coss.)
were studied. The concentrations of NaCl and KCl used were 100 mmol/L to 300 mmol/L and the con-
centration of CaCl2 was 5 mmol/L. Na2SO4 and K2SO4 used were in the range of 50 to 200 mmol/L.
Salt treatments were given to 6 d old seedlings and Chl a measurements were taken subsequently for
3 d after the salt treatment.
The fast Chl a fluorescence kinetics of mung bean and Brassica seedlings showed no significant
change within 3 d of experiments without the addition of salts/ions. The salt/ion treatments signifi-
cantly affected the Chl a fluorescence kinetics. The initial Chl a fluorescence level (F0) increased after
3 d with NaCl, which was enhanced by the addition of CaCl2. KCl and Na2SO4 or K2SO4 induced an
increase in F0 after 1d of salt treatments. CaCl2 alone and with NaCl or KCl enhanced FM after 1d of
treatment. KCl alone increased FM values after 1 d. However, the maximum Chl a fluorescence level
(FM), quantum yield of the primary photochemistry of PS II (φPo), quantum yield of electron transport
beyond QA – (φEo), efficiency that a trapped electron can move further ahead of QA – (ψo) and flux ratio
of electron transport (ETo/RC) decreased significantly with NaCl treatments after 3 d of treatment.
CaCl2 ameliorated these effects. ABS/RC and DI0/RC was enhanced several folds by NaCl treat-
ments and CaCl2 augmented these effects. Other salts and their combinations also showed en-
hancement in DI0/RC. However, Na salts showed amplified effects at equimolar concentrations com-
pared to K salts. The most prominent difference in the susceptibility of a system or genotype to differ-
ent salts or ions is due to the changes of the abundance of active reaction centers per leaf cross-sec-
tion (RC0/CS0).

Key words: Brassica – Brassica juncea – CaCl2 – Chl a fluorescence – ions – mung bean – salinity –
Vigna radiata

Abbreviations: ABS absorbance. – CS0 cross section. – d days. – DI0 dissipation of absorbed
quanta. – ET electron transport from QA – onwards. – ET0/RC, ABS/RC and DI0/RC are flux ratio of
electron transport, absorbance and dissipation per reaction center, respectively. – F0 initial Chl a fluo-

* E-mail corresponding author: [email protected]

0176-1617/01/158/09–1173 $ 15.00/0
1174 Amarendra N. Misra, Alaka Srivastava, Reto J. Strasser

rescence level. – FM maximum Chl a fluorescence level. – FV variable Chl a fluorescence measured as
FM – F0. – J and I intermediate steps of Chl a fluorescence rise between F0 = O level and FM = P levels
of Chl a fluorescence induction curve. – PS photosystem. – RC reaction center. – TR0 excitation
energy trapping. – φEo Quantum yield of electron transport beyond QA – . – φPo quantum yield of the
primary photochemistry of PS II. – ψo Efficiency that a trapped electron can move further ahead of
QA –

Introduction was affected when superimposed by high irradiance (Sharma

and Hall 1992) and temperature stress (Larcher et al. 1990).
The problem of soil salinity is a major limiting factor for crop As the fluorescence parameters are suggested to be less af-
productivity in the arable land all over the world (Misra et al. fected by salt stress alone, it is imperative that the studies of
1997). Soil salinity is due to the accumulation of excess ion effects would have practical limitations using fluores-
amounts of Cl – , SO4 – 2, CO3 – 2 and HCO3 – salts of Na + , K + , cence measurements. However, in the present study we sug-
Mg + 2 and Ca + 2 (Greenway and Munns 1990). These salts af- gest emphatically that this technique can be used as a highly
fect plant growth and development. The most widely occur- sensitive method for salt and ion effects and their interactions
ring problem of soil salinity is the abundance of NaCl in soils in plants. Also, the genotype differences in salt or ion sensitiv-
(Misra et al. 1995). Salt affected areas get low precipitation, ity can be evaluated using fast fluorescence kinetic measure-
which facilitate the accumulation of soluble salts in the root ments.
growth zone of crops (Misra et al. 1997). Even the irrigation In the present study, we verify whether Chl a fluorescence
practices in salt-affected areas increase the agricultural prob- parameters can be used for screening salinity stress. We re-
lems by remobilizing salts from deeper soil layers. port the changes in Chl a fluorescence parameters of mung
Salinity affects plant growth, metabolism and photosyn- bean and Brassica leaves affected by NaCl and KCl alone or
thetic efficiency of crop plants (Misra et al. 1996, 1997, 1999). in combination with CaCl2. The effect of different SO4 – 2 salts
Mung bean is a salt-sensitive and Brassica is a salt-tolerant is compared with those of Cl – salts.
crop (Misra et al. 1995, 1996, 1999). Therefore, these crop
plants are most suitable for study of the impact of salinity or
ion effects on crops to assess the genetic basis of salinity re- Materials and Methods
sistance in plants. Misra et al. (1999) have reported that NaCl
salinity affects PS II photochemical efficiency, charge separa- Plant material
tion of primary charge pairs in PS II and pigment-protein com-
Mung bean (Vigna radiata L. cv. Sujata) and Brassica (Brassica jun-
plexes of thylakoid membranes. Besides NaCl, salts like KCl, cea Coss. cv. Pusa bold) were grown on nylon mess floated over dis-
Na2SO4 and K2SO4 also occur in salt-affected soils. Salinity tilled water at 25 ± 1 ˚C under continuous illumination of 125 µmol
stress effects are reported to be ameliorated by CaCl2 (Jime- m – 2 s –1. Six-day-old seedlings were transferred to distilled water con-
nez et al. 1997). taining NaCl or KCl (0mmol/L, 100mmol/L, 200mmol/L and 300mmol/L)
Chlorophyll a fluorescence is widely used as a tool to alone, or in combination with 5 mmol/L CaCl2. The Na2SO4 and K2SO4
screen the alternations in photosynthetic apparatus and its salt treatments were given at 0 mmol/L, 50 mmol/L, 100 mmol/L and
functional efficiency (Havoux et al. 1988, Belkhodja et al. 200 mmol/L concentrations. Each treatment had minimum 3 replicates
with at least 5 plants in each replicate.
1994, Jimenez et al. 1997). This could be used as a tool for
detecting the salt effects on plants (Smillie and Nott 1982,
Bongi and Loreto 1989). However, Jimenez et al. (1997) sug-
gested that this tool can only be used as an indicator of salt
Fluorescence measurements
stress in rose plants when additional stress of high light is Plant Efficiency Analyzer (PEA, Hansatech Ltd., UK) measures fast
added. These authors have suggested that the evaluation of fluorescence kinetics of leaves at room temperature. The leaf samples
fluorescence parameters are not clear-cut for salt stress, as it were dark-adapted for 5 min before the fluorescence measurements.
is for other stress factors like water stress, chilling, freezing Dark-adapted leaves were illuminated homogeneously over an area of
4 mm dia with an array of 6 LED (λ max. 650 nm, 600 W m – 2) and fluo-
and air pollution. However, changes in fluorescence param-
rescence signals were detected using a PIN photodiode after passing
eters have been used as an early warning system for salinity
through a long-pass filter (50 % transmission at 720 nm). Fluorescence
stresses (Smillie and Nott 1982, Bongi and Loreto 1989), al-
transients were recorded from 10 µsec to 1 sec with the data acquisi-
though these changes were less specific and not as dra- tion rate of 10 µsec for first 2 msec and then at the rate of 1msec with a
matic. Even quantum yield of the primary photochemistry of resolution of 12 bits. The fluorescence signal at 40 µsec after the onset
PS II (Fv/FM ratio) was reported to be unaffected by salinity of illumination was considered as F0 (Srivastava et al. 1995, Strasser
(Brugnoli and Lauteri 1991, Brugnoli and Bjorkman 1992) or and Strasser 1995, Strasser et al. 1999, 2000).
 Assessment of salt/ion sensitivity by Chl a fluorescence 1175

Theoretical considerations for the OJIP transients

The fast fluorescence kinetics of all oxygenic photosynthetic materials
show a transient fluorescence rise known as O, J, I and P (Strasser
and Strasser 1995). The maximal fluorescence intensity FP denotes
the FM values where all the reaction centers (RCs) are physiologically
closed. The F0 state indicates the physiological state when all the RCs
are open.
Energy fluxes per RC and per CS, and flux ratios or yield ratios
have been derived using the theory of energy fluxes in biomembranes
(Sironval et al. 1981) and the experimental values of the OJIP-fluores-
cence transient values. The constellation of their values at any instant
is considered as an expression of the function of the system (Kruger
et al. 1997).
Absorbance (ABS) refers to the photon absorbed by the antenna
pigment molecules. A part of the absorbed energy is trapped (TR) by
the RC, and the other portions dissipated as heat and fluorescence.
The trapped energy is converted to redox energy by electron trans-
port (ET) to QA and QB. The different yields of absorbed energy and
the specific fluxes at time zero can be derived according to the JIP-
test (Strasser et al. 2000) as follows:
TR0/ABS = φPo = (1 – F0)/FM = FV/FM TRo/RC = M0 · (1/VJ)
ET0/TR0 = ψo = 1 – VJ ABS/RC = M0 · (1/VJ) · (1/φPo)
ET0/RC = M0 · (1/VJ) · ψo ET0/ABS = φEo = φPo · ψo
Where:
VJ = (F2ms – F0)/(FM – F0) M0 = (dV/dt)0 = 4.(F300 µs – F0)/(FM – 0)
The total absorption and active RC per CS has been calculated as fol-
lows:
RC/CS0 = φPo · (VJ/M0) · F0 ABS/CS0 = F0.

Performance index
The product of the specific fluxes, the performance indices (PI) can
be calculated using the JIP test as suggested by Srivastava et al.
(1999) and Strasser et al. (2000).
PIABS = (RC/CS) [φPo / (1– φPo)] [ψo/(1– ψo)]
By using the Nernst’s equation the log PI can be defined as a driving
force for photosynthesis.
Driving force (DFABS) = log (PIABS)

Results and Discussion

The fluorescence characteristics of mung bean and Brassica
seedlings changed with the concentration and nature of salt/
ion treatments (Fig. 1). There was no significant change in
the fluorescence kinetics within 3 d of experiments without the
addition of salts/ions in mung bean seedlings (Fig. 1A). Since
the salt/ion-induced changes in fluorescence characteristics
were not significantly different from each other after 1 d of
Figure 1. Effect of salts on the fluorescence induction kinetics of mung
treatment, only the data after 3 d are shown and the differ-
bean [A – D] and Brassica [E, F] seedlings. Six day old seedlings were
ences are interpreted in terms of salt or ion basis. NaCl salt
treated with different salts and measurements were done after 1, 2 and
induced an increase in F0 and a decrease in FM of mung bean 3 d. Flourescence kinetics shown in A and E are control seedlings after
seedlings (Fig. 1 D). The F0 value of 200 mmol/L NaCl-treated 1, 2 and 3 d of treatment. Transients shown in B – D and F are obtained
seedlings were enhanced by 16 % of that of the control seed- after 3d of NaCl [D, F], NaCl + CaCl2 [C] and KCl [B] treatments.
lings. The decrease in FM values was much more drastic than
the changes in F0 values. Compared to the effects of NaCl,
KCl treatment showed a decrease in the FM values only at
1176 Amarendra N. Misra, Alaka Srivastava, Reto J. Strasser

200 mmol/L and no significant effect on fluorescence param- (ψo) and the probability that an absorbed photon will move an
eters were observed at 100 mmol/L (Fig. 1 B). Na2SO4 treat- electron into the electron transport chain (φEo). The changes
ment was also less detrimental for mung bean seedlings at in fluorescence intensities by salinity is in sharp contrast to
equimolar concentrations of the ions (fluorescence curves not the earlier reports that Fv/FM ratio was unaffected by salinity
shown). Treatment of CaCl2 with NaCl salt treatments retarded (Brugnoli and Lauteri 1991, Brugnoli and Bjorkman 1992). In
the decrease in FM and enhancement in F0 up to 200 mmol/L the present study, there was no need to superimpose high ir-
NaCl (Fig. 1 C). The seedlings treated with NaCl along with radiance (Sharma and Hall 1992) or temperature stress (Larch-
CaCl2 showed a F0 rise of 28 % and FM decrease of 46 % over er et al. 1990) to bring about the changes in Chl fluores-
control values (Fig. 1C). cence parameters. The discrepancy by earlier authors could
As that of the mung bean seedlings there were no changes have been due to the use of resistant genotypes, where the
in the fluorescence kinetics of Brassica seedlings during the effect of salinity was not manifested unless the system was
growth period from 1d to 3 d (Fig. 1E). However, the F0 values severely stressed either by imposing another stress or by
increased and FM values decreased gradually with an in- high concentrations of salts. However, earlier workers used
creasing concentration of NaCl (Fig. 1 F). The F0 rise and FM the changes in fluorescence parameters as an early warning
decrease indicates a block in the electron transport to QA – system for salinity stresses (Smillie and Nott 1982, Bongi and
(Krause and Weis 1991). NaCl salinity is reported to affect wa- Loreto 1989).
ter oxidation (PS II photochemical efficiency) and charge sepa- Na2SO4 treatment affected φPo in mung bean seedling only
ration of primary charge pairs in PS II of mung bean and Bras- by 20 % of control value compared to 90 % in NaCl treatments
sica seedlings (Misra et al. 1999). Both QA – and QB – charge re- at equimolar concentrations of Na + ion (Fig. 2). CaCl2 treat-
combination with S states were affected by NaCl salinity. ment augmented the quantum yield to the control value only
Figure 2 shows the effect of different salts on the quantum up to 200 mmol/L NaCl treatment. NaCl treatment (at
yield of the primary photochemistry of PS II (φPo), the effi- 300 mmol/L) with CaCl2 caused a decrease of φPo to 42 % of
ciency that a trapped electron can move further ahead of QA – the control value (Fig. 2). This suggests that CaCl2 could only
retard the detrimental effects of NaCl by counter-ion effects
and defer the toxic effects of NaCl to a higher concentration.
Compared to that of mung bean seedlings, the changes in
φPo in NaCl salt-treated seedlings of Brassica showed a slow
and gradual decrease with increasing salt concentrations
(Fig. 2). The decrease at 300 mmol/L NaCl was 26 % of con-
trol values. Salts of K + alone or in combination with CaCl2
showed no significant change in φPo of mung bean (Fig. 2)
and Brassica (data not shown). These results corroborate the
report on amelioration of NaCl salinity stress effects by CaCl2
(Jimenez et al. 1997).
The electron transport per trapped photon molecule (ψo)
was enhanced at 100 mmol/L NaCl but decreased to 42 %
and 49 % of control values, respectively at 200 mmol/L and
300 mmol/L concentrations (Fig. 2). Addition of CaCl2 en-
hanced the ψo of control seedlings and retarded the NaCl-
induced decrease in ψo of mung bean seedlings. However,
the ψo value of seedlings treated with 300 mmol/L NaCl and
CaCl2 decreased relatively to same value as that without
CaCl2 (Fig. 2). Na2SO4 treatments decreased ψo value only
up to 16 % at 300 mmol/L NaCl.
The quantum yield for electron transport beyond QA – (φEo =
ET0/ABS) which is a product of φPo and ψo is also depicted in
Figure 2. φEo was retarded to less than 8 % at 200 mmol/L and
300 mmol/L NaCl and CaCl2 ameliorated the effect up to
200 mmol/L NaCl. Na2SO4 treatments also affected φEo. How-
ever, the decrease in φEo values of Na2SO4-treated seedlings
were only 22 % of control value (Fig. 2).
Figure 2. The effect of different concentrations of Na- (A, C, D) and The performance index (PI) of the salt-stressed seedlings
K-salts (B) on the quantum efficiency φPo (A, B) or flux ratios ψo (C) showed an exponential relationship with φEo (Fig. 3). Brassica
and φEo (D) of mung bean seedlings after 3 d of treatment. Note the seedlings treated with Na + salts and mung bean seedlings
changes in φPo of Brassica seedlings treated with NaCl (A). with K + salts were grouped in clusters. However, mung bean
 Assessment of salt/ion sensitivity by Chl a fluorescence 1177

are more detrimental to photosynthetic systems than Na2SO4.

Also equimolar concentrations of Cl – is more toxic than SO4 – 2
ions (Fig. 4). The scatter plots for individual data points (open
circles) also suggest that the variations in NaCl-treated seed-
lings were bigger compared to that of Na2SO4 or in combina-
tion with CaCl2. Such an analysis suggests that stress effects
for ions or salts and their concentrations can be analyzed
through such double logarithmic plots.
A comparison of the slope for log PIABS versus log φEo = log
ET0/ABS shows a distinct pattern of salt effects for NaCl and
KCl (Fig. 5). The slope (m) for KCl-treated seedlings was 4.05
and that for NaCl salt was 2.58. Thisanalysis depicts that as
log (PIABS) is positively correlated to log (φEo = ET0/ABS), the
Figure 3. The relationship in the probability that an absorbed photon
lower ‹m› values would represent a smaller decrease in the
will move an electron into the electron transport chain (φEo = (ET0/ABS)
photosynthetic performance, when the yield for electron trans-
rel.)) and the performance index (PIABS (rel.)) of Na + or K + -treated
mung bean and Brassica seedlings. port decreases.
The slope analysis for different salts and their ions were
analyzed by combining the salts of the similar ions (Table 1).
This analysis confirms our hypothesis that the ‹m› value
(where m = ∆ log PI/∆ log φEo) derived from the fast Chl a fluo-
rescence kinetics can be taken as an indicator for salt or ion
effects on photosynthetic efficiency analysis during salt
stress. Also the tolerance and susceptibility of the genetic
systems can be determined through this analysis. So, we pro-
pose that this analysis can be used for a wide range of appli-
cations from agronomy to biotechnological analysis of sus-
ceptibility of genotypes or photosynthetic systems to salts or
ions. The differences in ‹m› values for mung bean seedlings
under salt stress are clearly in an increasing order starting
with NaCl, NaCl + CaCl2, Na2SO4, KCl, KCl + CaCl2, K2SO4 (Ta-
ble 1). The ionic effects compared by this analysis depicts

Figure 4. The logarithmic relationship between φEo (= (ET0/ABS)rel.),

and the performance index (PIABS)rel. of mung bean seedlings treated
with different concentrations of Na + salts with or without the addition of
CaCl2. The bold closed circles denote the average values of each
treatment (n = 5–7) and the small open circles represent the individual
values for the treatments. Note the linear relationship between the two
log functions and the salt concentration dependence of these func-
tions. The four clusters show the different salts or their combined ef-
fects to the similar extent.

seedlings treated with NaCl salts showed minimum values for

PI and φEo. The log (ET0/ABS) rel. as a function of log (PIABS)
rel. for different Na-salt treatments are shown as a straight line
Figure 5. Analysis of the slope angles (m) for the linear realtionship
in a double logarithmic plot in Figure 4. The two log functions
between log φEo (= log (ET0/ABS)rel.) and log performance index (=
have a linear relationship suggesting that the effects of differ-
log (PIABS)rel.) of mung bean seedlings treated with different concen-
ent salts are through their ionic interactions with the photosyn-
trations of K + (closed circles) and Na + (open circles) salts. The relative
thetic systems. The relative effects of salts or ions can be de- values of ‹m› can be taken as an index for effectiveness of the salts
tected from such double log plots. The dose responses and on the photosynthetic systems. The lower the value of ‹m› the lesser
gradual time course of action of salts can also be determined the inhibitory effect of the salts on the quantum efficiency of photosyn-
from the linearity of this curve. It is very clear that NaCl salts thetic electron transport (φEo) and the performance index (PIABS)rel.
1178 Amarendra N. Misra, Alaka Srivastava, Reto J. Strasser

that the order of ion toxicity on photosynthetic efficiency of

Table 2. Ion sensitivity of performance index and activities of mung
mung bean seedlings are Cl – > Na + > SO4 – 2 > K + (Table 1).
beean and Brassica seedlings after 3 d of treatment with different
The ‹m› values for NaCl salt-stressed mung bean and Bras-
salts and their combinations. The relative values are calculated from
sica seedlings were 2.58 and 2.2, respectively (Table 1). The
the ratio between the treated seedlings and the untreated (control)
‹m› values for interactions of ions and plants are also shown seedlings.
in Table 1 that can be used in the crop efficiency analysis. pPIABS (rel.) = – log [PIABS (Treatment) / PIABS (Control)].
We have analyzed the relative performance index of the pPICS (rel.) = – log [PICSo (Treatment) / PICSo (Control)].
salt-stressed plants (Table 2). The -log values (pPI) or the where PICso = F0 * PIABS.
driving force for electron transport, in terms of the absorbed
Treatments pPIABS (rel.) ET0/ABS (rel.) pPICSo (rel.) ET0/CS0 (rel.)
quanta and the cross section of the leaf, showed that the rela-
tive performance index [pPIABS(rel.) and pPICSo(rel.)] was Mung bean
maximum for NaCl treatment in mung bean. CaCl2 amelio- NaCl 3.352 0.051 3.289 0.058
rated the effect of NaCl in such a way that pPIABS(rel.) and NaCl + CaCl2 0.105 0.919 0.092 0.946
pPICSo(rel.) values approached 0.1. Similar effects were seen Na2SO4 0.590 0.655 0.516 0.782
for Brassica seedlings with NaCl treatment, although the Mung bean
pPIABS(rel.) and pPICSo(rel.) values were less than a seventh KCl 0.556 0.689 0.569 0.669
of that of mung bean seedlings (Table 2). The pPIABS(rel.) val- KCl + CaCl2 0.106 0.945 0.121 0.913
ues for Na2SO4, K2SO4 and KCl treatments were nearly 0.5 to K2SO4 0.666 0.706 0.653 0.727
0.6. CaCl2 salt also showed countering action to KCl. The Brassica
pPIABS(rel.) and pPICSo(rel.) values were similar to those of NaCl 0.485 0.650 0.523 0.734
NaCl. These observations along with our earlier analysis of NaCl + CaCl2 0.056 0.992 0.110 0.835
the slope values, clearly suggests that in terms of counter-ion
effects CaCl2 acts with Cl – ions (comparing the values of
NaCl and KCl in Table 2). These effects were shown to be due salt-stressed seedlings. This analysis also showed that the
to an enhancement in the electron transport efficiency in the relative susceptibility of mung bean seedlings to NaCl salinity,
compared to that of Brassica seedlings or other salts, was
very high. This can be depicted by the analysis of pPI(rel.)
values and/or relative electron transport efficiency of salt-
Table 1. Regression analysis for log [(PI) rel.] and log [(ETo/ABS) rel.]
stressed seedlings (Table 2). Differential effects of ions can
for ion effects on mung bean and Brassica seedlings. The slope is
given as «m». The regression equation is log [PI (rel.)] = m * log [ETo/ also be assessed from this analysis.
ABS (rel.)]. The performance index in the leaves is the cumulative ef-
R2 and n denote the Correlation coefficient and number of samples, fects of the functions of ψo, φPo, CS0/ABS and RC0/CS0 (Sri-
respectively. vastava et al. 1999). Figure 6 shows the component analysis
for the changes in performance index (PI) of the salt-stressed
Plant Salt or iron m R2 n
mung bean and Brassica seedlings. The components which
Mung bean NaCl 2.582 0.998 68 were most affected by salt stress in mung bean are φPo, RC0/
NaCl + CaCl2 2.960 0.999 75 CS0 and the electron transport (ψo) per trapped electrons.
Na2SO4 3.116 0.988 80 The significant difference in Brassica and mung bean seed-
lings was the increase in RC0/CS0 of Brassica seedlings
KCl 3.477 0.986 65
KCl + CaCl2 3.869 0.998 60 treated with Na + salts (Fig. 6). The susceptibility of photosyn-
K2SO4 4.375 0.999 50 thetic systems at the φPo level to different stress factors are
well documented (Kruger et al. 1997, Srivastava and Strasser
Na + ion 2.612 0.996 223
1997, Ouzounidou et al. 1997). In this study we suggest that
K + ion 3.894 0.978 175
there are differences in the changes in φPo in the susceptible
Cl – 2.589 0.997 143
Ca + + + Cl – 3.000 0.994 267 and tolerant systems, as well with different salt or ion treat-
SO4 – 3.392 0.952 130 ments. However, the most prominent differences in the sus-
Combined salts 2.633 0.991 398 ceptibility of a system or genotype to different salts or ions are
due to damage of active RC, which results in decreased RC0/
Brassica NaCl 2.208 0.992 70
CS0 and the photochemical efficiency of PS II. Although the
NaCl + CaCl2 2.676 0.993 70
Na + ion 2.283 0.985 140 electron transport efficiency beyond QA – decreased along
with the number of reaction centers and electron donation to
Mung bean and NaCl 2.5487 0.996 138
QA, the severity of the decrease in φPo suggests that the do-
Brassica combined NaCl + CaCl2 2.878 0.995 145
nor side is relatively more affected than the acceptor side.
Na2SO4 3.093 0.985 64
Salt-induced acceptor side inhibition of charge transfer was
Plant x Na + 2.800 0.994 363
reported earlier (Misra et al. 1999, Sahu et al. 1998). The salt-
 Assessment of salt/ion sensitivity by Chl a fluorescence 1179

Figure 6. Component analysis for the decrease in the log perform-

ance index (driving force) of salt-affected seedlings. The variations in
salt treatments and genotypes are characterized by the differences
between their photosynthetic components. A several-fold difference
between the functions of φPo and RC0/CS0 can be taken into account
as the major contributory factor for the decreased photosynthetic per-
formance of plant systems to salt treatments.

induced damage to the reaction center could be due to ion

induced rapid turn-over. Further studies are needed to prove
this hypothesis by evaluating the genetic variations in sus-
ceptibility of plant systems to various stress factors.
The salt/ion-induced changes in the phenomenological pa-
rameters of fluorescence kinetics are shown in a pipeline
Model (Fig. 7 A, B). In this model the OJIP values were taken
to express PS II activities in terms of the cross section unit
(Leaf model, right panel) and per unit reaction center (mem-
brane model, left panel). NaCl salt treatment in mung bean
seedlings inhibited the electron transport beyond QA – per

Figure 7. The pipeline model for the salt/ion-induced changes in the

phenomenological parameters of fluorescence kinetics of mung bean
(A) and Brassica (B). The leaf model (right panel) shows phenomeno-
logical fluxes or apparent activities per cross section (CS0) and the
membrane model (left panel) shows the specific activities per unit
reaction center (RC). The density of active photosynthetic units in-
volved in the reduction of QA, per cross section, is shown as small
open circles in the leaf model. The small closed circles (A) demon-
strate inactive photosynthetic units and the small hatched circles (B)
represent newly synthesized units. The arrows indicate fluxes for light
absorbance (ABS), excitation energy trapping (TR0), energy dissipa-
tion (DI0) and electron transport (ET0) beyond QA – . The width of each
arrow denotes the relative size of the fluxes or the antenna. Note that
the dimentions of the figure in the membrane model (left panel, A) for
NaCl 2000 mmol/L is reduced 10 × to fit into the plane of the paper.
1180 Amarendra N. Misra, Alaka Srivastava, Reto J. Strasser

cross section (ET0/CS) due to the inactivation of the reaction effect of plants. Comparison of the salt/ion effects on the pho-
centers (closed black circles). The trapping efficiency per tosynthetic efficiency of mung bean seedlings also showed
cross section (TR0/CS) also decreased. However, NaCl treat- that KCl was less inhibitory to electron transport processes
ment increased the aborption (ABS/CS) and heat dissipation compared to equimolar concentrations of NaCl. Addition of
(DI0/CS). These effects were several times greater in the CaCl2 along with NaCl, which completely blocked the elec-
membrane model (left panel) where because of the inactiva- tron transport in mung bean, could completely ameliorate the
tion of the active reaction centers, the ABS, DI0 and TR0 val- inhibitory effect of NaCl. Also it is possible to differentiate be-
ues increased in terms of specific activity of active reaction tween the susceptible and tolerant genotypes for salt suscep-
centers (Fig. 7 A, left panel). Equimolar quantity of NaCl tibility.
showed less detrimental effect on Brassica seedlings (Fig. The most prominent differences in the susceptibility of a
7 B), compared to the mung bean seedlings (Fig. 7 A). The system or genotype to different salts or ions are due to dam-
reaction centers remained active and there was even synthe- age of active RC, which results in decreased RC0/CS0 and
sis of new reaction centers (hatched circles) in Brassica the photochemical efficiency of PS II. Although the electron
seedlings treated with 200 mmol/L NaCl, which was com- transport efficiency beyond QA – decreased along with the
pletely inhibitory to mung bean (compare Fig. 7 A with 7 B). number of reaction centers and electron donation to QA, the
Like mung beans, the electron transport (ET0) of Brassica severity of the decrease in φPo suggests that the donor side is
also decreased. However, absorbance (ABS) and dissipation relatively more affected than the acceptor side.
(DI0) per cross section or per reaction center increased with The effect of individual components of salt/ion(s) and their
NaCl treatment, although the quantum of change in Brassica combined effects can be deciphered using the analysis of
seedlings was less than 10 × than that of mung bean seed- performance index and the slope (m) analysis. This analysis
lings. This phenomenon is distinct depiction of genetic varia- confirms our hypothesis that the ‹m› value (where m = ∆ log
bility in the salt tolerance of these two crops (Misra et al. PI/∆ log φEo) derived from the fast Chl a fluorescence kinetics
1999). Addition of CaCl2 along with NaCl, which completely can be taken as an indicator for salt or ion effects on photo-
blocked the electron transport in mung bean, could com- synthetic efficiency analysis during salt stress. Also the toler-
pletely ameliorate the inhibitory effect of NaCl (Fig. 7A). Com- ance and susceptibility of the genetic systems can be deter-
parison of the salt/ion effects on the photosynthetic efficiency mined through this analysis. So, we propose that this analysis
of mung bean seedlings also showed that KCl was less inhib- has a wide range of applications from agronomy to biotech-
itory to electron transport processes compared to equimolar nological analysis of susceptibility of genotypes or photosyn-
concentrations of NaCl (Fig. 7 A). The ABS, TR0 and DI0 val- thetic systems to salts or ions. The differences in ‹m› values
ues of KCl-treated seedlings decreased compared to the con- for mung bean seedlings under salt stress showed that the
trol (no salt) seedlings. The pigment content of the leaf also detrimental effects of different salts are in the decreasing or-
decreased (data not shown). This resulted in lesser damage der starting with NaCl, NaCl + CaCl2, Na2SO4, KCl, KCl + CaCl2,
to the reaction center complexes (black circles). The degra- K2SO4. The ionic effects compared by this analysis depicts
dation of leaf pigments was induced by KCl and therefore, di- that the order of ion toxicity on photosynthetic efficiency of
luting the Chl per cross section leads to a decrease in the ab- mung bean seedlings are Cl – > Na + > SO4 – 2 > K + . Mung
sorbance and trapping of photons per cross section. The les- bean seedlings were found to be more susceptible to NaCl
ser damaging effect of KCl in mung bean, as compared to salt stress compared to the Brassica seedlings. Also, the ‹m›
NaCl, appears to be through its effect on pigment metabo- values for interactions of ions and plants can be used in the
lism. NaCl treatment might have a direct effect of Na + on the crop efficiency analysis.
reaction center complex itself leading to severe salt stress ef-
fects. The augmentation of photosynthetic efficiency by CaCl2 Acknowledgements. ANM is thankful to University of Geneva for sup-
in salt-stressed mung bean seedlings to the control values port during the Summer of 1998 and 2000.
could be due to repletion of Ca + in the NaCl-treated seedlings
which could have been replaced from the photosynthetic sys-
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