Download Constraints on photosynthesis of C3 and C4 crop plants by

Copyright © NISC Pty Ltd
South African Journal of Botany 2004, 70(5): 751–759
Printed in South Africa — All rights reserved
SOUTH AFRICAN JOURNAL
OF BOTANY
ISSN 0254–6299
Constraints on photosynthesis of C3 and C4 crop plants by trichloroacetic
acid, an atmogenically generated pollutant
AJ Strauss1, GHJ Krüger1*, PDR van Heerden1, JJ Pienaar2 and L Weissflog3
Section Botany, School of Environmental Sciences and Development, North-West University, Potchefstroom Campus,
Hoffman Street, Potchefstroom 2520, South Africa
2
School of Chemistry and Biochemistry, North-West University, Potchefstroom Campus, Hoffman Street, Potchefstroom 2520,
South Africa
3
Department of Analytical Chemistry, Centre for Environmental Research, Leipzig-Halle, 15 Permoser Street, 04318 Leipzig,
Germany
* Corresponding author, e-mail: [email protected]
1
Received 26 January 2004, accepted in revised form 30 April 2004
This study examined the physiological and biochemical
bases of the inhibition of photosynthesis by
trichloroacetic acid (TCA) in Phaseolus vulgaris L. and
Zea mays L., representing dicotyledonous and monocotyledonous plants with C3 and C4 photosynthetic pathways respectively. TCA is a phytotoxic compound transformed in the atmosphere from chloro-organic substances (C2-CHCs) present in solvents and cleaning
agents used in the metal and textile industry, as well as in
combustion products released from coal-fired power stations and biomass burning. Photosynthetic gas
exchange and fast chlorophyll fluorescence kinetics
were measured in 3-week-old plants exposed for 3 days
to TCA concentrations ranging from 0.05–3.20g TCA-Na
m–2. Photosynthetic inhibition by TCA was observed in
both species, this being greater at the highest level of
TCA supplied in Z. mays (85% decrease) than in P. vulgaris (37% decrease), though at low levels of TCA supplied some photosynthetic stimulation was also
observed in P. vulgaris. Analysis of Rubisco activity
revealed that TCA decreased the activation state of the
enzyme in both species, especially in Z. mays, a possible
consequence also of a measured decrease in stomatal
conductance. However, the decline in net CO2 assimilation rate at saturating CO2 levels (Jmax) was also indicative
of a decrease in RuBP regeneration capacity. This osten-
sible mesophyll limitation was supported by chlorophyll
fluorescence measurements, which indicated a disruption of electron transport in photosystem II (PS II) reaction centres requisite for effective RuBP regeneration. It
is concluded that increased emission of C2-CHCs by
industry and veld-fire catastrophes pose a threat particular to grain production by monocotyledonous crops
especially in southern Africa which is experiencing
greater food demands from a rapidly expanding population.
Abbreviations: Γ = CO2 compensation concentration, A = net
CO2-assimilation rate at ambient atmospheric CO2-concentration (Ca), Jmax = net CO2-assimilation rate at saturating
intercellular CO2-concentration (Ci), C2-CHCs = C2-chlorohydrocarbons, CE = Carboxylation efficiency, CHCl3 = chloroform, DF = Total driving force for photosynthesis, E = transpiration, fw = fresh weight, GS = Stomatal conductance, kP
= Photochemical rate constant, kN = Non-photochemical rate
constant, l = Relative stomatal limitation of photosynthesis,
LSU = Rubisco large sub-unit, PIABS = performance index,
PSII = Photosystem II, ϕPo = Maximum yield of primary photochemistry, QA = Primary quinone acceptor, RuBP =
Ribulose-1,5-bisphosphate, TCA = trichloroacetic acid,
TECE = C2Cl4 = tetrachlorethene, tetrachloroethylene
Introduction
In addition to phytotoxic air pollutants such as NOx, ozone
and SO2, an increasingly important role is being played globally by highly volatile C2-chlorohydrocarbons (C2-CHCs) in
the degradation of natural vegetation and reduction in crop
yield (Weissflog et al. 2001).
Due to continued industrialisation, increasing use of the
C2-CHC tetrachloroethene (TECE) and trichloroethene
(TCE) as solvents and cleaning agents in the metal and textile industry, leads to the accumulation of these chloroorganic substances in the atmosphere (Midgeley and
McCulloch 1999). According to Fischer et al. (1982) and
Pearson (1982), 90–100% of the TECE produced escapes
into the environment. Alternative sources of these substances are combustion processes such as coal-fired power
752
stations emitting TECE (Garcia et al. 1992), and biomass
burning responsible for the emission of 4–30Gg yr–1 TCE
globally (Rudolph et al. 1995, 2000). Further sources of C2CHC pollutants are discussed by Weissflog et al. (2004).
When transported through the atmosphere, these compounds are exposed to oxidative, photolytic and hydrolytic
processes, which alter their original structure and thus their
chemical and physical properties, as well as their potential
phytotoxicity. Increased urban activities and veld-fire catastrophes increase the oxidation potential of the atmosphere
which leads to an increase in atmogenically produced TCA
in the regions on the lee side of the emitting source. Franklin
(1994) and Sidebottom and Franklin (1996) indicated that
one important tropospheric chemical breakdown pathway of
TECE in the planetary boundary layer, is oxidation by Cl–,
OH– and O2H–radicals respectively to form TCA. The worldwide occurrence of TCA, including the industrialised areas of
South Africa, is reported on by Weissflog et al. (2001, 2003).
Strauss (2002) demonstrated that an inverse correlation
existed between the TCA content and the vitality of pine needles sampled over an air-pollution gradient which included a
part of the Vaal Triangle, a so-called ‘hot spot’ of anthropogenic air pollution in South Africa.
In the 1950s TCA was used in agriculture at an application
level of 15–30kg TCA ha–1 as herbicide against monocotyledonous grasses (Brian 1976). Barrens and Hummer (1951),
Zöttl (1953), Foy (1969) and Matolcsy et al. (1988) published
some information on the phytotoxic properties of TCA. They
inter alia pointed out that low concentrations of TCA caused
accelerated growth. Frank et al. (1992) reported that the
EC50 and EC10 values for bean-cell suspensions are 600 and
200µmol TCA l–1 respectively. Govindjee et al. (1997) conducted an in vitro investigation using spinach thylakoids to
characterise the effect of chloroacetates on photosystem II
(PSII) function. They found that these compounds, especially TCA, inhibit electron flow between QA and QB(QB–).
Photosynthesis is the principal process exploiting solar
energy for biosynthesis and primary production, thus avoiding damage to the photosynthetic apparatus must be one of
the main goals in environmental sciences (Brack and Franck
1998). The aim of the present investigation therefore was to
study the physiological and biochemical basis of the inhibition of photosynthesis by TCA on the whole-plant level in
Phaseolus vulgaris L. and Zea mays L., representing a C3
dicotyledonous and a C4 monocotyledonous plant, respectively. To achieve this aim TCA-induced constraints on photosynthesis of intact test plants were investigated under
strictly controlled conditions employing (1) infra-red gas
analysis to be able to discriminate between stomatal and
mesophyll limitation of photosynthesis, and (2) chlorophyll
induction kinetics (JIP test) which is a powerful non-invasive
tool to shed light on the mode of action of xenobiotics on the
primary processes of photosynthesis. In addition, in vitro
analyses of Rubisco activity, the primary enzyme in CO2assimilation, were carried out.
Materials and Methods
Experimental plants and treatment
Phaseolus vulgaris L. and Zea mays L. were used as exper-
Strauss, Krüger, Van Heerden, Pienaar and Weissflog
imental plants, representing C3- and C4-crop plants, respectively, to study the biochemical effect of TCA pollution.
Seeds of both species were obtained from the Agricultural
Research Council, Potchefstroom.
The experiments were conducted under controlled conditions in growth chambers (Conviron PGW 36, Controlled
Environment Ltd, Winnipeg, MB, Canada R3H 0R9) with sufficient incandescent light bulbs (General Electric,
Neodynium R80, 100W) and fluorescent lamps (General
Electric, Cool White, 150W) to maintain light intensities as
high as 1 300µmol photons m–2 s–1 at 1m distance. Seeds of
each species were planted in pots containing sterile sand
and watered daily with 250ml of distilled water. The plants
were subjected to a 15h/9h day/night period at 23°C/20°C.
The CO2-level was kept constant at the normal atmospheric
value of 350µmol mol–1.
After a period of two weeks the number of plants in each
pot was reduced to three. Complete Hoagland’s nutrient
solution (250ml) was administered to each pot every third
day onwards. The different treatments were initialised at this
point in time.
TCA was administered as sodium salt solution at different
concentrations to the growth medium of the test plants. The
concentrations applied were 0.05, 0.20, 0.80 and 3.20g
TCA-Na m–2. Investigations conducted by Weissflog et al.
(2000) employed a concentration of 6.0g TCA-Na m–2 in the
soil of large perennial trees like pines and birch, with clear
visual effects within four months of treatment. Thus lower
concentrations were used in these experiments, since the
treatments were administered to smaller, annual crop plants.
A higher level of 3.20g TCA-Na per m2 was also used to confirm that very high concentrations have a definite and drastic effect on the plants. The control pots received no TCANa.
The surface area of the soil in the pots was determined
and used to calculate the TCA-Na dosage for each treatment. The TCA-salt was dissolved in 100ml distilled water
before it was applied to the soil. The TCA-Na was administered only once.
Photosynthetic gas exchange
CO2 gas exchange was measured three days after TCAtreatment with an open circuit infra-red gas analysis system
(CIRAS-1, PP-systems, Hertz, UK) on four plants of each
TCA-Na-treatment. All measurements were conducted at a
leaf temperature of 23°C. A 2.5cm2 section of leaflet was
clamped in a software-controlled broad leaf cuvette with
built-in light and temperature control. Humidity in the
cuvette was maintained close to ambient conditions.
Irradiance during measurements was 1 200µmol m–2 s–1 to
ensure full activation of Rubisco (Taylor and Terry 1984).
For each external CO2 concentration (Ca) the ratio of CO2
assimilation rate (A) to intercellular CO2 concentration (Ci)
was automatically recorded. By increasing Ca at 5min intervals from 0–2 000µmol mol–1, A:Ci-response curves were
generated. The initial slope of the demand function (δA/δCi)
was computed by linear regression analysis. All other calculations were done according to Farquhar and Sharkey
(1982) and Chaves (1991). Relative stomatal limitation (l )
was calculated as the difference between the photosynthet-
South African Journal of Botany 2004, 70: 751–759
ic rate when Ci = 350µmol mol–1 (A0), and the actual rate at
Ca = 350µmol mol–1 (A), expressed as a percentage of A0.
Rubisco activity and protein content
Three days after the TCA-Na-treatment, 3.14cm2 leaf discs
were collected with a freeze clamp (cooled in liquid nitrogen)
from the youngest fully expanded leaf of five control- and
five TCA-Na-treated plants of both species. Sampling
occurred 5h after the onset of the light period at a light intensity of ±1 000µmol m–2 s–1. All leaf discs were stored at
–80°C for a maximum of two weeks.
Initial Rubisco activity was measured according to the
method of Keys and Parry (1990). Initial activity is the activity of the enzyme under the growth conditions at the time of
sampling without prior in vitro activation with bicarbonate.
Each leaf disc was ground to a fine powder with liquid nitrogen in a pre-cooled mortar and rapidly extracted with 0.8ml
ice-cold extraction buffer containing 100mM Bicine-NaOH
(pH 8.0), 20mM MgCl2, 50mM β-mercaptoethanol, 5mM
phenylmethylsulfonyl fluoride (PMSF) and 30mg insoluble
polyvinylpolypyrrolidone (PVPP). The crude extract was
centrifuged at 10 000 x g at 4°C for 1min. Clarified supernatant (25µl) was immediately used for the measurement of
initial Rubisco activity in reaction mixture (0.5ml) containing
100mM Bicine-NaOH (pH 8.2), 20mM MgCl 2, 10mM
NaH14CO3 (0.5µCi µmol–1) and 400µM ribulose-1,5-bisphosphate (RuBP). After 1min the reaction was terminated by
addition of 0.2ml 10M formic acid. Acidified samples were
evaporated to dryness in an oven at 60°C. After addition of
3.5ml scintillation cocktail to each vial, the 14C incorporated
into 3-phosphoglycerate were determined by liquid scintillation spectrometry. Total chlorophyll content of leaf samples
were determined according to the method of Wintermans
and De Mots (1965).
For the quantification of Rubisco protein content leaf
discs were extracted with 3ml extraction buffer containing
50mM Tris-HCl (pH 6.8), 1mM EDTA, 3mM DTT, 6mM
PMSF and 30mg insoluble PVPP. The crude extract was
centrifuged at 10 000 x g at 4°C for 15min. The soluble-protein content of the supernatant was determined according
to the method of Bradford (1976). Proteins (4µg) were separated by SDS-PAGE at 200V at 4°C for 75min. After separation, proteins were transferred to a PVDF-membrane. The
blotted membrane was blocked and probed with primary
antibody specific to the Rubisco large subunit (LSU) followed by horseradish peroxidase-conjugated anti-rabbit
secondary antibody. The signal was developed by chemiluminescence (BM Chemiluminescence Western Blotting Kit,
Roche Molecular Biochemicals, Mannheim, Germany).
Data analysis of Western blots was done with a Syngene
Genius Bio Imaging System (Syngene, Cambridge CB4
1TF, UK).
Fast chlorophyll fluorescence kinetics
Three days after the TCA-Na-treatment, fluorescence induction curves of 4 control- and TCA-treated plants were recorded with a fluorimeter (PEA, Hansatech Instruments,
Kingslyn, UK). Measurements were conducted predawn 1h
753
prior to the start of the following light period. Each fluorescence induction curve was analysed according to the JIPtest (Strasser and Strasser 1995). The following data from
the original measurements were used: the maximal fluorescence intensity, FP, equal to FM since the excitation intensity
was high enough to ensure the closure of all reaction centres (RCs) of PSII; the fluorescence intensity at 50µs considered as the intensity F0 when all RCs are open; the fluorescence intensity at 300µs (F300µs) required for the calculation of the initial slope M0 = (dV/dt)0 ≅ (δV/δt)0 of the relative
variable fluorescence (V) kinetics and the fluorescence
intensity at 2ms (J step) denoted as FJ.
The JIP-test represents a translation of the original data to
biophysical parameters (for the conceptual approach and
analytical derivation see Krüger et al. 1997 and Van
Heerden et al. 2003). The average value of each of these
parameters for all pre-dawn measurements was calculated.
The parameters which all refer to time zero (start of fluorescence induction) are: (a) the specific energy fluxes (per
reaction centre, RC) for absorption (ABS/RC), trapping
(TR0/RC), electron transport (ET0/RC) and dissipation at the
level of the antenna chlorophylls (DI0/RC); (b) the flux ratios
or yields, i.e. the maximum quantum yield of primary photochemistry (ϕPo = TR0/ABS = FV/FM), the efficiency with which
a trapped exciton, having triggered the reduction of QA to
QA–, can move an electron further than QA– into the electron
transport chain (ψ0 = ET0/TR0), the quantum yield of electron
transport (ϕEo = ET0/ABS = ϕPo • ψ0) and the quantum yield
of dissipation (ϕDo = DI0/ABS = 1 – ϕPo); (c) the phenomenological energy fluxes (per excited cross section, CS) for
absorption (ABS/CS), trapping (TR0/CS), electron transport
(ET0/CS) and dissipation (DI0/CS); (d) the fraction of active
PS II-reaction centres per total absorption (RC/ABS) and per
excited cross section (RC/CS).
The initial stage of photosynthetic activity of a RC-complex
is regulated by three functional steps namely absorption
events of light energy (ABS), trapping of excitation energy
(TR) and conversion of excitation energy to electron transport (ET). Recently a multi-parametric expression of these
independent steps contributing to photosynthesis, the performance index (PIABS), was introduced (Strasser et al. 1999,
Tsimilli-Michael et al. 2000):
PIABS =
γ
1–γ
•
ϕPo
1–ϕPo
•
ψo
1–ψo
where γ is the fraction of reaction centre chlorophyll (ChlRC)
per total chlorophyll (ChlRC + Antenna). Therefore γ/(1 – γ) =
ChlRC/ChlAntenna = RC/ABS. This expression can be deconvoluted into two JIP-test parameters and estimated from the
original fluorescence signals as RC/ABS = RC/TR0 •
TR0/ABS = [(F2ms – F50µs)/4(F300µs – F50µs)] • FV/FM. The factor
4 is used to express the initial fluorescence rise per 1ms.
The expression RC/ABS shows the contribution to the PIABS
due to the RC-density on a chlorophyll basis. The contribution of the light reactions for primary photochemistry are
estimated according to the JIP-test as (ϕPo/(1 – ϕPo)) =
TR0/DI0 = kP/kN = FV/FM. The contribution of the dark reactions are derived as (ψ0/1 – ψ0)) = ET0/(TR0 – ET0) = (FM –
F2ms)/(F2ms – F50µs).
According to the definition, the performance index is a
754
Strauss, Krüger, Van Heerden, Pienaar and Weissflog
DFABS = log(PIABS) = log
( ) ( ) ( )
RC
ϕPο
+ log
ABS
1–ϕPο
+ log
ψο
1–ψο
Statistical analyses
Significant differences between the means of different treatments were determined by analysis of variance, using
Student’s t-test of honest significant difference.
25
Phaseolus vulgaris (C3)
15
g TCA m–2
0.00
0.05
0.20
0.80
3.20
10
5
0
-5
25
(b)
Zea mays (C4)
20
15
10
Results and Discussion
5
Photosynthetic gas exchange
0
The A:Ci-response curves obtained for the different TCAtreatments not only show the effect of TCA concentration on
photosynthetic gas exchange, but also illustrate the difference in gas-exchange kinetics between C3 (P. vulgaris) and
C4 (Z. mays) plants. The CO2-assimilation capacity (A) of the
C3-plant has a higher dependency on CO2-supply (Ci) and
has a lower carboxylation efficiency (CE) (shallower initial
slope of the A:Ci-response curve) with a concomitant higher
CO2-compensation concentration of c. 60µmol mol–1 (Figure
1a). In contrast to the C3-plant, the C4-plant is less dependent on CO2-supply, has a much higher CE (steeper initial
slope) with a concomitant lower CO2-compensation concentration of less than 10µmol mol–1 (Figure 1b). Even superficially viewed Figure 1 indicates that TCA-Na, at all levels
used, inhibited CO2-assimilation to a larger extent in Z. mays
than in P. vulgaris. Figure 2 compares the effect of TCA-Na
at different concentrations on various A:Ci-response curvederived photosynthetic gas exchange parameters of P. vulgaris and Z. mays. At all TCA-Na-concentrations used the
CO2-assimilation capacity (A) of Z. mays was impaired to a
higher degree than in P. vulgaris. At the highest TCA-Nalevel used, the reduction in A was 85% in Z. mays, compared
to 39% in P. vulgaris (Figure 2a). The concomitant decrease
in CE at this TCA-Na-level was 83% and 37%, which corresponded to an increase in CO2-compensation concentration
(Γ) of 43% and 36% for Z. mays and P. vulgaris respectively. Note that already at a TCA-Na-level of 0.8g m–2 marked
changes occurred in most gas-exchange parameters of both
species. In both species and especially in the case of Z.
mays, Ci increased with increase in TCA-Na-concentration
(data not shown) although stomatal conductance (Gs)
decreased and stomatal limitation of photosynthesis (l )
increased (data not shown). This fact as well as the data discussed above, point at TCA-Na-induced mesophyll limitation
(a)
20
ASSIMILATION RATE (µmol CO2 m–2 s–1)
product of expressions of the form [pi/(1 – pi)], where the pi
(i = 1, 2, …, n) stand for probabilities or fractions. Such
expressions are well known in chemistry, with pi representing e.g. the fraction of the reduced and (1 – pi) the fraction
of the oxidised form of a compound, in which case log(pi/(1
– pi)) expresses the potential or driving force for the corresponding oxido-reduction reaction (Nernst equation).
Extrapolating this inference from chemistry, the log (PIABS)
can be defined as the total driving force (DFABS) for photosynthesis of the observed system, created by summing the
partial driving forces for each of the several energy bifurcations (at the onset of the fluorescence rise O-J-I-P):
-5
200 400 600 800 1 000 1 200 1 400
INTERCELLULAR CO2 CONCENTRATION
(µmol mol–1)
Figure 1: A:Ci response curves of P. vulgaris (a) and Z. mays (b)
plants treated with different concentrations of TCA. The plotted values represent the mean of four replicates. The dotted lines indicate
the ambient CO2 concentration of 350µmol mol–1
(inhibition of the photochemical and/or enzymatic processes) of photosynthesis in both species, possibly due to a
decrease in supply of reducing power (Von Caemmerer and
Farquhar 1981, Farquhar and Sharkey 1982). This deduction is further supported by the fact that Jmax were reduced
by TCA-Na at all levels in Z. mays and above 0.2g m–2 in P.
vulgaris, indicating that the regeneration capacity of RuBP
was inhibited. On the other hand, as mentioned briefly
above, in both species increasing TCA-Na-concentrations
lead to a decrease in Gs (at the highest TCA-Na concentration: 81% and 61% for Z. mays and P. vulgaris, respectively). Calculation of the percentage stomatal limitation (l ) from
the A:Ci curves according to Farquar and Sharkey (1982) for
the different TCA-Na-treatments, revealed an increase in l of
1 900% (Z. mays) and 41% (P. vulgaris) at the 3.2g TCA-Na
m–2-treatment. At the 0.8g m–2-treatment the corresponding
values already increased by 570% and 29% respectively.
This means that in addition to mesophyll limitation, TCAinduced stomatal limitation also plays a role in the inhibitory
effect of TCA on photosynthesis of both species, particularly
in Z. mays.
South African Journal of Botany 2004, 70: 751–759
ASSIMILATION RATE
(µmol CO2 m–2 s–1)
(a)
25
Phaseolus vulgaris (C3)
Zea mays (C4)
20
15
10
300
(b)
250
200
150
100
In vitro Rubisco activity
The effect of TCA-treatment on initial Rubisco activity
(expressed on a total chlorophyll basis) in P. vulgaris and Z.
mays is illustrated in Figure 3. In both the control and TCAtreated plants, initial Rubisco activity was consistently higher in P. vulgaris than in Z. mays. This finding was corroborated by Western blot analysis, which revealed a higher
steady-state Rubisco LSU-protein content in this species
(results not shown). Both species showed a marked sensitivity towards TCA with respect to Rubisco activity. Even at
the lowest TCA-Na-concentration of 0.05g m –2, initial
Rubisco activity was decreased (P < 0.05) by between 18%
to 21% in both species. Up to a concentration of 0.8g TCANa m–2 the inhibitory response in the two species was fairly
similar. At the highest concentration of 3.2g TCA-Na m–2,
however, initial Rubisco activity was decreased to a much
larger extent in Z. mays (73%, P < 0.01) than in P. vulgaris
(24%, P < 0.01). Western blot analysis indicated that TCANa-treatment did not induce any significant reduction in
steady-state Rubisco LSU-protein content in the two species
(results not shown). The decrease in initial Rubisco activity,
50
0.5
(c)
Phaseolus vulgaris (C3)
Zea mays (C4)
5.0
0.4
0.3
RUBISCO ACTIVITY (µmol min–1 mg–1 chlorophyll)
CO2 COMPENSATION
CARBOXYALTION EFFICIENCY STOMATAL CONDUCTANCE
CONCENTRATION (µmol mol–1)
(mol CO2 m–2 s–1)
(µmol m–2 s–1)
5
755
0.2
0.1
0.0
(d)
80
60
20
10
0.0
0.5 1.0 1.5 2.0 2.5 3.0
TCA CONCENTRATION (g m–2)
Figure 2: Effect of different concentrations of TCA applied to the
soil of potted P. vulgaris (C3) (closed circles) and Z. mays (C4) (open
circles) on various parameters of photosynthetic gas exchange
namely (a) CO2 assimilation rate at Ca = 350µmol mol–1, (b) stomatal conductance at Ca = 350µmol mol–1, (c) carboxylation efficiency and (d) CO2 compensation concentration. Each datapoint represents the mean of four replicates ± SE
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
TCA CONCENTRATION (g m-2)
Figure 3: Effect of different TCA concentrations applied to the soil
on the in vitro Rubisco activity in leaves of P. vulgaris (C3) and Z.
mays (C4) plants. Each data-point represents the mean of six replicates ± SE
756
Strauss, Krüger, Van Heerden, Pienaar and Weissflog
without any significant decrease in steady state Rubisco protein content, suggests a TCA-Na-mediated decrease in the
activation state of the enzyme, particularly in Z. mays. The
inhibition in initial Rubisco activity might largely explain the
TCA-Na-induced decrease in CE that was measured in both
species.
A decrease in Rubisco activation state could be an indirect
consequence of a TCA-mediated decrease in stomatal conductance (lower availability of CO2 at the site of carboxylation) or because of direct effects of TCA on enzyme regulation itself. It is important to note that the decrease in initial
Rubisco activity and stomatal conductance did not show the
same TCA-Na-dose dependency. Whereas initial Rubisco
activity already decreased at the lowest TCA-Na-concentration in both species, stomatal conductance only decreased
significantly at a TCA-Na-concentration of 0.8g m–2 or higher. This finding suggests that the decrease in Rubisco activation state is a direct consequence of TCA on enzyme regulation and not mediated through a decrease in stomatal
conductance. Inhibition of Rubisco activity by TCA will result
in a slower rate of CO2 draw-down into the chloroplast, with
a concomitant increase in Ci, which will ultimately act as a
trigger for stomatal closure. Taken together, our results
regarding Rubisco activity provides further support for the
presence of severe mesophyll limitation of photosynthesis.
Chlorophyll a fluorescence kinetics
The behaviour of the photosynthetic apparatus of P. vulgaris
and Z. mays upon TCA-Na-treatment was followed by the
analysis of the recorded fluorescence transients according
to the JIP-test. The average fast fluorescence transients presented on a logarithmic scale and normalised on 50µs (F0)
and 2ms (FJ) of P. vulgaris (Figure 4a) and Z. mays (Figure
4b) treated with different TCA-concentrations, indicate that
the major effect of TCA occurred in the multiple turn-over
events of PSII function, i.e. in the transition J (2ms) to P
(~700ms). The transition from O to J reflects mainly photochemical reactions leading to the reduction of the electron
acceptor QA, while the further transient J to P is strongly
affected by the subsequent dark reactions in the electron
transport chain (Strasser et al. 1999). Note that the TCAinduced deviations are larger in the case of Z. mays. The
transients also indicate stimulation of PSII-activity at the lowest and up to the second lowest TCA-levels in Z. mays and
P. vulgaris respectively. Figure 5 depicts the fractional reduction of the active RCs per excited cross section, along with
the fractional changes in the specific (per RC) and phenomenological (per CS) energy fluxes through PSII, namely
ABS/RC, TR0/RC, ET0/RC, ABS/CS, TR0/CS, ET0/CS as well
as the quantum efficiency of primary photochemistry (ϕp0 =
FV/FM), as influenced by different TCA-Na-concentrations.
The first important point to make is, as was revealed by
the gas exchange and Rubisco activity data, that photosynthesis in Z. mays was inhibited more severely than in P. vulgaris. In both species, ET0/CS (Figure 5a and 5b) was much
more affected than TR0/CS and thus also ϕPo, due to the
large changes in the efficiency with which trapped excitons
can move further than QA– in the electron transport chain
(ψ0) (Figure 5c) [Note that ET0/CS = TR0/CS • ψ0]. At the 3.2g
Figure 4: Average polyphasic fast fluorescent transients on a logarithmic time scale, normalised on F0 and FJ (2ms) of the plants treated with different TCA-concentrations, illustrating that the major
effect of TCA occurred in the multiple turnover events of PSII function, i.e. in the transition J (2ms) to P (~700ms). The transition from
O to J reflects mainly photochemical reactions leading to the reduction of the electron acceptor QA–, while the further transient J to P
(the fluorescence maximum) is strongly affected by the subsequent
dark reactions in the electron transport chain (Strasser et al. 1999).
The transients represent the average of 30 measurements of each
treatment
TCA-Na m–2-treatment the decrease (P < 0.01) in ψ0 was
26% and 19% for Z. mays and P. vulgaris, respectively. It is
important to note that ET0/CS (Figure 5b) and also ψ0
(Figure 5c), increased relative to the control at the lowest
(0.05g TCA-Na m–2) TCA-Na-treatment. This observation
corresponded to the CO2-assimilation data for P. vulgaris
(see Figure 1) and the visual observation that both P. vulgaris and Z. mays treated with this concentration of TCA-Na,
grew slightly more vigorously than the control plants. Our
data thus corroborates the early finding of Barrens and
Hummer (1951) and Zöttl (1953) that very low concentrations of TCA caused accelerated growth, which suggested
that TCA has plant hormonal (auxin) properties at very low
concentrations. With increasing TCA-Na-concentrations,
ET0/CS was increasingly inhibited, especially in Z. mays
(Figure 5b). With increasing TCA-Na-concentrations, both
species showed an increase in apparent antenna size
(increase in ABS/RC) (Figure 5a and 5b), which indicates
that a fraction of the RCs was inactivated (Krüger et al.
1997). Also illustrated by the latter figure is the fact that for
South African Journal of Botany 2004, 70: 751–759
TRo/CS
(a)
ABS/CS
1.3
1.2
1.1
1
757
Phaseolus vulgaris
ETo/CS
0.8
0.7
Phi(Po)
RC/CS
0.5
ETo/RC
ABS/RC
TRo/RC
Zea mays
TRo/CS
(b)
ABS/CS
1.3
1.2
1.1
1
ETo/CS
0.8
0.7
Phi(Po)
ABS/RC
RC/CS
0.5
ETo/RC
TRo/RC
(c)
0.60
g TCA m–2
0.00
0.05
0.20
0.80
3.20
Phaseolus vulgaris
Zea mays
0.55
yo
0.50
0.45
0.40
0.35
0.00
0.05
0.20
0.80
3.20
TCA CONCENTRATION (g m–2)
Figure 5: Spider plots visualising the fractional reduction of the
active RCs per excited cross section, along with the fractional
changes in the specific (per RC) and phenomenological (per CS)
energy fluxes through PSII (a and b), as well as the efficiency with
which a trapped exciton can move electrons further than QA– in the
electron transport chain (ψo) (c), as influenced by different TCA-concentrations
both species, and especially in Z. mays, increasing TCAconcentrations lead to a decrease in the density of active
PSII reaction centres (RC/CS). This ‘switching off’ of a fraction of RCs was compensated for by an increase in the specific trapping flux (TR0/RC) as well as apparent antenna size
(ABS/RC), leading to only a very moderate decrease in ϕPo
(FV/FM). This phenomenon illustrates the power of the JIPtest, which deconvolutes the behaviour of PSII into several
functional and structural parameters, to quantify the behaviour of plants upon environmental stress. The results presented in Figure 5 suggest that the primary processes of
photosynthesis are regulated at the level of the reaction centre (RC/CS; inactivation or ‘switching-off’), the level of excitation energy trapping (TR0/RC) and the level of transformation of trapped excitation energy to energy of reducing
equivalents (ET0/TR = ψ0) (Krüger et al. 1997, Van Heerden
et al. 2003). The change in the performance index (PIABS),
which is a multi-parametric expression taking into consideration the independent parameters of absorption (chlorophyll
concentration), the quantum efficiency of trapping and the
probability of the conversion of trapped excitation energy to
electron transport, illustrates the differential effect of TCA on
P. vulgaris and Z. mays (Figure 6), showing that the latter is
more severely affected. At the highest TCA-Na-level administered, the decrease in PIABS was 51% (P < 0.01) for P. vulgaris opposed to 69% (P < 0.01) for Z. mays. The contribution of the regulatory steps is visualised by Figure 7 showing
the TCA-induced changes in the total driving force (DF), as
well as in the constituent partial driving forces namely, log
RC/ABS, log ϕPo/(1 – ϕPo) and log ψ0/(1 – ψ0). Figure 7 is
based on the following equation (for the conceptual
approach and derivation of the equation see Strasser et al.
1999 and Van Heerden et al. 2003):
DF = log(PIABS) = log RC/ABS + log ϕPo/(1 – ϕPo) + log
ψ0/(1 – ψ0)
Both plant species showed similar patterns with regard to
the effect of the different TCA-concentrations. The partial
driving force affected most was the conversion of trapped
excitation energy to electron transport beyond QA, namely
log ψ0/(1 – ψ0). The decrease in this parameter was 29% for
P. vulgaris and 44% for Z. mays at the highest TCA-Na-level
used. This finding is in accordance with the TCA-induced
changes in the fluorescence transients depicted by Figure 4.
Note that an increase of c. 10% (P. vulgaris) and c. 30% (Z.
mays) in this parameter occurred at the lowest TCA-Na-level
used, indicating that the stimulatory effect of TCA-Na at low
concentrations, similar to the inhibitory effect at higher concentrations, is partly connected to conversion of trapped
excitation energy to electron transport by PSII. Our data thus
corroborates the results of the in vitro experiment of
Govindjee et al. (1997) using thylakoid membranes indicating that TCA inhibits electron flow between QA and QB(QB–).
Conclusion
Although both of the species investigated were affected by
TCA-Na-treatment, Zea mays (C4) was more severely affected than Phaseolus vulgaris (C3). This was evident from the
photosynthetic gas-exchange data as well as Rubisco activity, pointing to mesophyll as well as stomatal limitation of
photosynthesis, and a decrease in the activation state of relevant enzymes. Even though Z. mays was affected more
than P. vulgaris, higher levels of TCA caused severe inhibition in both species. Visual observations and chlorophyll a
fluorescence data showed an initial stimulation in growth
and PSII activity in both species, and P. vulgaris in particular, but not at higher TCA-Na-concentrations. Again, as corroborated by the previous data, higher levels of TCA-Na had
758
Strauss, Krüger, Van Heerden, Pienaar and Weissflog
6
PlABS
12
10
8
6
4
Phaseolus vulgaris (C3)
Zea mays (C4)
0.00
0.05
0.20
0.80
3.20
TCA CONCENTRATION (g m–2)
Figure 6: Effect of different TCA concentrations applied to the soil
on the vitality (PIABS) of potted P. vulgaris and Z. mays plants. Each
datapoint represents the mean of thirty measurements ± SE
a more severe effect on Z. mays, influencing electron transport in particular. The difference in response between P. vulgaris and Z. mays is most probably due to the large morphological and biochemical differences between C3 and C4
plants.
The stimulatory effect of TCA could have a negative influence on ecosystems, if certain species growth is enhanced
as a result of exposure to low levels of pollution by TCA.
The data presented proves that TCA has serious implications for crop production and particularly the monocotyledonous crops. An increase in the emission of C2-CHCs by
industry and veld-fire catastrophes pose a threat to agricultural production and its ability to keep pace with the growing
food demand for the rapidly expanding populations of southern Africa.
The results of this investigation not only provide data
about the physiological and biochemical basis of the
inhibitory effect of TCA on crop plants, but also provide information to indicate an area of risk, which may stimulate
efforts to develop pollution abatement strategies in respect
to C2-CHC-air pollution.
References
Barrens KC, Hummer RW (1951) Basic herbicidal studies with
derivatives of TCA. Agricultural Chemistry 6: 48–121
Brack W, Franck H (1998) Chlorophyll a fluorescence: A tool for the
investigation of toxic effects in the photosynthetic apparatus.
Ecotoxicological and Environmental Safety 40: 34–41
Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilising the principle of
protein-dye binding. Analytical Biochemistry 72: 248–254
Brian RC (1976) The history and classification of herbicides. In:
Audus LJ (ed) Herbicides: Physiology, Biochemistry, Ecology.
Academic Press, London
Chaves MM (1991) Effects of water deficits on carbon assimilation.
Journal of Experimental Botany 42: 1–16
Farquhar GD, Sharkey TD (1982) Stomatal conductance and pho-
DRIVING FORCE = log (PlABS)
14
5
log (RC/ABS)
log jPo/(1–jPo)
log Yo/(1–Yo)
log (RC/ABS)
log jPo/(1–jPo)
log Yo/(1–Yo)
4
3
2
1
0.00
0.05
0.20
0.80
3.20
TCA CONCENTRATION (g m–2)
Figure 7: Effect of different TCA-Na concentrations applied to the
soil of potted P. vulgaris (hatched bars) and Z. mays plants (solid
bars) on the partial driving forces of primary photosynthesis, namely absorption of light energy (log RC/ABS), quantum efficiency of
primary photochemistry (log ϕPo/[1 – ϕPo]), and probability of the
conversion of trapped excitons to electron transport beyond QA (log
ψo/[1 – ψo])
tosynthesis. Annual Review of Plant Physiology 33: 317–345
Fischer M, Friesel P, Koch G, Roβkamp E (1982) Evaluation of
Water Pollution Caused by Chlorinated Ethylenes. Report of the
Institut für Wasser-, Boden und Lufthygiene, Berlin. Final draft to
the European Community (EC)
Foy C (1969) The chlorinated aliphatic acids. In: Kearney PC,
Kauffmann DD (eds) Degradation of Herbicides. Marcel Dekker,
New York, pp 207–254
Frank H, Scholl H, Sutinen S, Norokorpi Y (1992) Trichloroacetic
acid in conifer needles in Finland. Annual Botanical Fennici 29:
263–267
Franklin J (1994) The atmospheric degradation and impact of perchloroethylene. Toxicological Environmental Chemistry 46:
169–182
Garcia JP, Beyne-Masclet S, Mouvier G, Masclet P (1992)
Emissions of volatile compounds by coal-fired power stations.
Atmospheric Environment 26A: 1589–1592
Govindjee, Xu C, Schansker G, Van Rensen JJS (1997)
Chloroacetates as inhibitors of photosystem II: Effects on electron
acceptor side. Journal of Photochemistry and Photobiology B:
Biology 37: 107–117
Keys AJ, Parry MAJ (1990) Ribulose bisphosphate
carboxylase/oxygenase and carbonic anhydrase. Methods in
Plant Biochemistry 3: 1–15
Krüger GHJ, Tsimilli-Michael M, Strasser RJ (1997) Light stress provokes plastic and elastic modifications in structure and function of
photosystem II in camellia leaves. Physiologia Plantarum 101:
265–277
Matolcsy M, Nädasy M, Andriska V (1988) Pesticide Chemistry.
Elsevier, Amsterdam, pp 577–580
Midgeley PM, McCulloch A (1999) Production sales and emissions
South African Journal of Botany 2004, 70: 751–759
of halocarbons from industrial sources. In: Hutzinger O (ed) The
Handbook of Environmental Chemistry. Vol. 4, Part E, Reactive
Halogen Compounds in the Atmosphere. Springer, Berlin, pp
155–190
Pearson CR (1982) C1- and C2-halocarbons. In: Hutzinger O (ed)
The Handbook of Environmental Chemistry. Vol. 3, Part B,
Anthropgenic Compounds. Springer, Berlin, pp 69–88
Rudolph J, Khedim A, Koppmann R, Bonsang B (1995) Field study
of the emissions of methyl chloride and other halocarbons from
biomass burning in Western Africa. Journal of Atmospheric
Chemistry 22: 67–80
Rudolph J, Von Czapiewski K, Koppmann R (2000) Emissions of
methyl chloroform (CH3CCl3) from biomass burning and the tropospheric methyl chloroform budget. Geophysics Research
Letter 27: 1887–1890
Sidebottom H, Franklin J (1996) The atmospheric fate and impact of
hydrochlorofluorocarbons and chlorinated solvents. Pure Applied
Chemistry 68: 1757–1769
Strasser BJ, Strasser RJ (1995) Measuring fluorescence transients
to address environmental questions: The JIP-test. In: Garab G
(ed) Photosynthesis: Mechanisms and Effects. Vol. V. Kluwer
Academic Publishers, The Netherlands, pp 4325–4328
Strasser RJ, Srivastava A, Tsimilli-Michael M (1999) Screening the
vitality and photosynthetic activity of plants by fluorescence transient. In: Behl RK, Punia MS, Lather BPS (eds) Crop
Improvement for Food Security. SSARM, Hisar, India, pp 72–115
Strauss AJ (2002) The Effect of C2-Chlorohydrocarbons and their
Phytotoxic Metabolites on Photosynthesis of Crop Plants and
Natural Vegetation. MSc Thesis, Potchefstroom University for
Christian Higher Education, Potchefstroom, South Africa
Taylor SE, Terry N (1984) Limiting factors in photosynthesis. V.
Photochemical energy supply co-limits photosynthesis at low
values intercellular CO2 concentration. Plant Physiology 75:
82–86
Tsimilli-Michael M, Eggenberg P, Biro B, Köves-Pechy K, Vörös I,
Strasser RJ (2000) Synergistic and antagonistic effects of arbuscular mycorrhizal fungi and Azospirillum and Rhizobium nitrogenfixers on the photosynthetic activity of alfalfa, probed by the
chlorophyll a polyphasic fluorescence transient OJIP. Applied Soil
Ecology 462: 1–14
Edited by CF Musil
759
Van Heerden PDR, Tsimilli-Michael M, Krüger GHJ, Strasser RJ
(2003) Dark chilling effects on soybean genotypes during vegetative development: parallel studies of CO2 assimilation, chlorophyll
a fluorescence kinetics O-J-I-P and nitrogen fixation. Physiologia
Plantarum 117: 476–491
Von Caemmerer S, Farquhar GD (1981) Some relationships
between the biochemistry of photosynthesis and the gas
exchange of leaves. Planta 153: 376–387
Weissflog L, Elansky N, Putz E (2000) Ecotoxicological risk in the
Caspian Catchment Area. ECCA, Final report EU-project: IC15
CT96–0106
Weissflog L, Krüger G, Elansky N, Putz E, Pfennigsdorf A, Seyfarth
K, Nüchter M, Lange C, Kotte K (2003) Input of trichloroacetic
acid into the vegetation of various climatic zones — measurements on several continents. Chemosphere 52: 443–449
Weissflog L, Krüger G, Kellner K, Pienaar J, Lange C, Strauss R,
Elansky N, Pfennigsdorff A, Ondruschka B (2004) Air pollutionderived trichloroacetic acid contributing to degradation of vegetation in Southern Africa. South African Journal of Science 100:
289–293
Weissflog L, Pfennigsdorff A, Martinez-Pastur G, Puliafito E,
Figueroa D, Elansky N, Nikonov V, Putz E, Krüger G, Kellner K
(2001) Trichloroacetic acid in the vegetation of polluted and
remote areas of both hemispheres — Part I. Its formation, uptake
and geographical distribution. Atmospheric Environment 35:
4511–4521
Wintermans JFGM, De Mots A (1965) Spectrophotometric characteristics of chlorophylls a and b and their pheophytins in ethanol.
Biochimica et Biophysica Acta 109: 448–453
Zöttl H (1953) Untersuchungen über die Wirkung von
Trichloroacetat und anderen Halogenacetaten auf planzliches
Gewebe. Zeitschrift fur Naturforschung 8b: 317–323