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Plant Physiol. (1998) 116: 571-580
Relationship between CO2 Assimilation, Photosynthetic
Electron Transport, and Active O2 Metabolism in Leaves of
Maize in the Field during Periods of Low Temperature1
Michael J. Fryer,
James R. Andrews,
Kevin Oxborough,
David A. Blowers, and
Neil R. Baker*
Department of Biological Sciences, University of Essex, Colchester
CO4 3SQ, Essex, United Kingdom
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ABSTRACT |
Measurements of the quantum
efficiencies of photosynthetic electron transport through photosystem
II ( PSII) and CO2 assimilation (CO2) were made simultaneously on leaves of
maize (Zea mays) crops in the United Kingdom during the
early growing season, when chilling conditions were experienced. The
activities of a range of enzymes involved with scavenging active
O2 species and the levels of key antioxidants were also
measured. When leaves were exposed to low temperatures during
development, the ratio of
PSII/CO2 was elevated,
indicating the operation of an alternative sink to CO2 for
photosynthetic reducing equivalents. The activities of ascorbate
peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase,
glutathione reductase, and superoxide dismutase and the levels of
ascorbate and  -tocopherol were also elevated during chilling
periods. This supports the hypothesis that the relative flux of
photosynthetic reducing equivalents to O2 via the Mehler reaction is higher when leaves develop under chilling conditions. Lipoxygenase activity and lipid peroxidation were also increased during
low temperatures, suggesting that lipoxygenase-mediated peroxidation of
membrane lipids contributes to the oxidative damage occurring in
chill-stressed leaves.
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INTRODUCTION |
Exposure of maize (Zea mays) crops to low temperatures
during the early growing season in temperate regions results in
depressions in photosynthetic productivity and canopy development
(Miedema, 1982 ; Stirling et al., 1991; Baker and Nie, 1994).
Chill-induced decreases in CO2 assimilation in
maize leaves are associated with inhibition of photosynthesis involving
both increased dissipation of excitation energy in the PSII antennae
and photodamage to PSII reaction centers (Ortiz-Lopez et al., 1990;
Andrews et al., 1995; Fryer et al., 1995; Haldimann et al., 1996),
decreases in the activities of Benson-Calvin cycle enzymes
(Kingston-Smith et al., 1997), and poor development of the
photosynthetic apparatus (Nie and Baker, 1991; Nie et al., 1992, 1995).
Under such environmental stress conditions, which reduce the capacity
to assimilate C, it has been suggested that photosynthetic electron
flux to O2 will increase, resulting in the
increased production of superoxide,
H2O2, and hydroxyl radicals
(Asada, 1996). These active O2 species are
extremely damaging to lipids, proteins, and pigments unless they are
rapidly scavenged within the chloroplasts by a group of enzymes
consisting of SOD, GTR, DHAR, MDHAR, and APX (Asada, 1996). There is
some evidence, although not extensive, that increased levels of these
scavenging enzymes may play a role in limiting the degree of
photodamage experienced by maize at chilling temperatures (Jahnke et
al., 1991; Massacci et al., 1995; Hodges et al.,
1997).
In maize leaves at normal growth temperatures, the relationship between
photosynthetic electron transport and CO2
assimilation is highly conserved over a wide range of light intensities
and CO2 concentrations and also during the
induction of photosynthesis in dark-adapted leaves (Genty et al.,
1989). Examination of the quantitative relationship between electron
transport and CO2 assimilation in maize leaves in
air indicated that the majority of the reductants generated by electron
transport are consumed by CO2 assimilation and
that other sinks, such as N metabolism, O2
reduction via photorespiration, and the Mehler reaction, are minimal
(Edwards and Baker, 1993). However, when maize leaves are grown at low
temperatures the ratio of electron transport to
CO2 assimilation increases (Fryer et al., 1995;
Massacci et al., 1995). This indicates that there is an increased
allocation of reductants to sinks other than CO2 assimilation.
A similar increase in the ratio of electron transport to
CO2 assimilation was also observed when mangrove
(Cheeseman, 1994) and sweet sorghum (Massacci et al., 1996) leaves were
drought stressed. Therefore, it can be speculated that additional
electron sinks to CO2 assimilation, possibly
O2, develop during the imposition of
environmental stresses that impose restrictions on photosynthetic C
metabolism. Such a metabolic change could be a mechanism for preventing
photodamage to the photosynthetic apparatus that operates in
conjunction with increased dissipation of excitation in the PSII
antennae by nonradiative decay processes.
A primary aim of this study was to determine whether the
well-established chill-induced suppression of CO2
assimilation during chilling was associated with changes in the
relationship between electron transport and CO2
assimilation in maize leaves during the early growing season. If the
relationship between electron transport and CO2
assimilation changed, a secondary aim was to evaluate whether these
changes were associated with changes in the activity of active
O2-scavenging systems. Simultaneous measurements of the quantum efficiencies of linear electron transport through PSII
and of CO2 assimilation were made on leaves of
maize crops harvested from a field site in southeast UK during May and
June in 1994 and 1995, and the activities of a range of enzymes
involved with scavenging of active O2 species and
the levels of some key antioxidants in the leaves were determined.
Also, the extent of lipid peroxidation occurring in the leaves was
monitored, because this is another important source of active
O2 species that may be associated with the
environmental stress responses of leaves.
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MATERIALS AND METHODS |
Plant Material and Experimental Site
The experimental plot consisted of a 0.08-ha area of sandy, loamy
soil in northeast Essex, UK. Before the seeds were sown, the soil was
treated with N:P:K (2:1:1) fertilizer at a rate of 100 Kg
ha 1. Maize (Zea mays) cvs LG11 and
LG20.80 were sown on May 2, 1994, and May 1, 1995, respectively, in
randomized rows 0.33 m apart, at a depth of 6 cm, and at 0.2-m
intervals to give a population density of approximately 14 plants
m2, similar to that recommended for growers
(Tiley and Warbots, 1975). All measurements were made on the youngest
fully expanded leaves of the crop.
Climatologic Measurements
Environmental conditions were monitored by a weather station
(Delta-T Devices, Newmarket, UK) that was situated within 10 m of
the experimental plot. Measurements of PPFD and air temperature were
taken at 1-min intervals, and 30-min means were logged.
Measurements of Chlorophyll Fluorescence and CO2
Assimilation
Leaves were harvested from the field by cutting the base of the
leaf under water and were then transferred to an adjacent laboratory
and placed in a temperature-controlled leaf chamber, which was
described by Stirling et al. (1991). Measurements of chlorophyll
fluorescence and CO2 assimilation were made
simultaneously at 25°C over a PPFD range of 150 to 1500 µmol
m2 s1. At any given
PPFD the fluorescence yield at steady-state photosynthesis, Fs, and the maximum yield produced by a
0.5-s saturating flash (PPFD of 10,000 µmol
m2 s1),
Fm , were determined using a fluorimeter
(PAM-2000, Heinz Walz, Effeltrich, Germany). The
PSII was determined from the equation: PSII = (Fm  Fs)/Fm, as
originally described by Genty et al. (1989). Rates of
CO2 uptake by leaves in the light and respiration rates in the dark were measured at 25°C using an IR gas analyzer (model 225-Mk3, Analytical Development, Hoddeson, UK) as described by
Stirling et al. (1991).
The CO2 was determined by dividing the rate
of CO2 assimilation (corrected for respiratory
losses) by the rate at which quanta were absorbed, which was determined
using a Taylor integrating sphere. For analyses of the relationship
between PSII and
CO2, only data from leaves with rates of
CO2 assimilation greater than 2 µmol
m2 s1 were taken. The
dark respiration rates in leaves with lower CO2 assimilation rates were often similar to the photosynthetic rates and
could potentially give rise to large errors in the estimation of
CO2 due to the dark rate of respiration not
reflecting the respiration rate in the light. When photosynthetic rates
are considerably greater than respiration rates, such errors are
considerably reduced.
APX, GTR, MDHAR, and SOD Assays
Leaf tissue (approximately 85 mg; 2 × 103 m2) was harvested
from the field, and leaf area was immediately determined with a video-based leaf area meter (model AM, Delta-T Devices, Cambridge, UK).
Leaf tissue samples were then frozen in liquid N2
and stored at 80°C. Cell-free homogenates for antioxidant enzyme
assays were prepared essentially by the methods described by Jahnke et al. (1991). Leaf tissue samples were ground to a powder with liquid N2 and homogenized in 3 mL of ice-cold extraction
buffer (0.1 m
K2PO4, pH 7.0, 0.01 m sodium ascorbate, and 5 mm DTPA) and 30 mg of
insoluble polyvinylpolypyrrolidone. The homogenate was centrifuged at
3°C for 20 min at 16,000g. The cell-free supernatant
containing the antioxidant enzymes was then desalted by passing through
a disposable G-25 Sephadex PD-10 column of 9 mL total volume. The column was pre-equilibrated by running 25 mL of ice-cold
equilibration/elution buffer (0.1 m
K2PO4, pH 7.0, containing
200 µm DTPA) through it prior to sample application. The
cell-free extract was eluted with 3.5 mL of column
equilibration/elution buffer, the first 0.5 mL was discarded, and
the following 2.5 mL (the green chlorophyll-containing fraction) was
collected. Antioxidant enzymes were assayed in order of lability (Hull,
1990; Jahnke et al., 1991). APX was assayed immediately after
desalting, followed by MDHAR, GTR, and SOD.
MDHAR was assayed by a method modified from Hossain et al. (1984) and
Jahnke et al. (1991). The decrease in A340
due to the oxidation of NADH to NAD+ was
monitored over the linear 5-min period of the reaction by the
generation of monodehydroascorbate via the inclusion of ascorbate oxidase in the reaction mixture of 1 mL total volume. Extract (50 µL)
was mixed with 500 µm ascorbate, 150 µm
NADH, and 0.2 unit of ascorbate oxidase from Cucurbita sp.;
1 unit of ascorbate oxidase is defined by the manufacturer as the
amount that causes the oxidation of 1 µmol of ascorbate to
monodehydroascorbate per minute. The balance to 1 mL was made up by
monodehydroascorbate assay buffer (0.08 m
K2PO4, pH 7.8, containing
200 µm DTPA). The assay was repeated with twice the
volume of extract (100 µL) to check that there was a doubling of the
reaction rate. If this did not occur, then the ascorbate oxidase
solution had lost activity and had become limiting. The rate of
conversion of NADH to NAD+ was determined using
an extinction coefficient for NADH at 340 nm of 6.2 mm1
cm1.
GTR was assayed following a method modified from that of Schaedle and
Bassham (1977), Hossain et al. (1984), and Jahnke et al. (1991) based
on the decrease in A340 due to the
oxidation of NADPH to NADP+ over 5 min. The total
reaction mixture volume was 1 mL and contained 500 µm
oxidized glutathione, 100 µL of extract, 150 µm NADPH, and GTR assay medium (0.08 m
K2PO4, pH 7.8, and 200 µm DTPA). Correction was made for the non-GTR-dependent
oxidation of NADPH by excluding the oxidized glutathione from the
reaction mixture.
SOD was assayed by the NBT method modified from that described by Beyer
and Fridovich (1987). The assay is dependent on competition for the
photogenerated superoxide anion radical between the dye NBT (which is
oxidized to a fine purple formazan colloid that absorbs at 560 nm and
is stabilized in suspension by the presence of a detergent) and SOD in
the sample. The total reaction volume was 1 mL and contained from 30 to
800 µL of sample, to which was added 0.025% Triton X-100 (detergent)
and 57 µmol of NBT, the balance being made up of SOD assay buffer
(0.05 m K2PO4
containing 200 µm DTPA). The reaction was started by
adding 0.01 m Met and 1.13 µm riboflavin (the
superoxide anion radical photogeneration system) and placing the
reaction tube a preset distance from a 60-W fluorescent tube for 7 min.
The development of the purple coloration was then determined by
measurement of the A560 in a spectrophotometer blanked with SOD assay buffer. An inhibition curve
for A560 was constructed against an
increasing volume of sample. One unit of SOD was defined as that being
contained in the volume of extract that caused a 50% inhibition of the
SOD-inhibitable fraction of the NBT reduction (Beyer and Fridovich,
1987).
All enzyme assays were carried out at both 14 and 24°C using a
temperature-controlled cuvette. Temperature was monitored by a
calibrated thermocouple in the cuvette solutions. Each assay was the
mean from eight leaves. Leaf chlorophyll concentrations were determined
by the method of Hipkins and Baker (1986).
DHAR Assay
A separate extraction procedure, based on the method of Jahnke et
al. (1991), was used for the preparation of a cell-free extract for the
assay of DHAR due to the enzyme's lability. Leaf material
(approximately 0.20 g; 2 × 103 m2 area) was frozen in liquid
N2 and ground with a pestle to a powder in a
prechilled mortar. The powder was homogenized in PVP (30 mg) and 3 mL
of DHAR extraction medium (0.05 m
K2PO4 buffer, pH 6.5, containing 20% [v/v] glycerol, 2 × 104
m 4-chlororesorcinol, 2 mm DTPA, 1 mm PMSF, 1 mm benzamidine-HCl, 100 µm 2-mercaptobenzothiazole, 0.014 m
2-mercaptoethanol, 200 µm dehydroascorbate, and 5 mm  -amino-n-caproic acid). The homogenate was
immediately centrifuged at 3°C for 20 min. The supernatant was
used for the enzyme assay directly.
LOX Assay
Pro-oxidant enzymic lipid peroxidation (LOX activity) was assayed
polarographically by the uptake of O2 (Grossman
and Zakut, 1978). Extraction was by the method of Kar and Feierarbend
(1984). Leaf tissue (300 mg) was ground in liquid
N2 to a powder and then homogenized in 2 mL of
extraction buffer (50 mm
K2PO4 buffer, pH 7.5, containing 0.5% [v/v] Triton X-100). The extract was then centrifuged at 1000g at 4°C for 15 min. The supernatant
(0.5 mL) was added to 2.5 mL of buffered linoleate dispersion in a
Clark O2 electrode and O2
uptake was measured for 5 min at 24°C. Enzyme activity was expressed
in terms of O2 uptake per milligram of chlorophyll per unit of time. Buffered linoleate consisted of 230 mg of
linoleate dispersed with 274 mg of Tween 20 in 25 mL of distilled
water. The mixture was stirred and neutralized to pH 7.0 (using 1 m KOH) and then made up to 100 mL by adding 75 mL of
0.2 m
K2PO4 buffer, pH 6.5. All
experiments were replicated with a minimum of four leaves.
Determination of Lipid Peroxidation
MDA content and other thiobarbituric acid-reacting substances were
assayed as indicators of the extent of lipid peroxidation in leaf
tissue by the method of Hodgson and Raison (1991). Leaf tissue (300 mg)
was ground in 4 mL of N2-degassed 10 mm Na2PO4 buffer, pH 7.4, and centrifuged at 1000g for 5 min.
Two-hundred microliters of the supernatant was added to a reaction
mixture containing 100 µL of 8.1% (w/v) SDS, 750 µL of 20% (w/v)
acetic acid, pH 3.5 (NaOH), 750 µL of 0.8% (w/v) aqueous
thiobarbituric acid, and 200 µL of distilled water. An identical
reaction mixture in which the 200 µL of supernatant was substituted
with an equal volume of buffer was simultaneously set up as an
absorbance blank. Both reaction mixtures were then incubated at 98°C
for 60 min. After cooling to room temperature, the mixtures were
centrifuged for 5 min. MDA concentration was calculated by subtracting
the A535 from the
A600 using a molar extinction
coefficient of 1.56 × 105
m1
cm1.
Ascorbate Content
Ascorbate was measured by a modification of the method of Omaye et
al. (1979), based on the coupling of dehydroascorbate to dinitrophenylhydrazine in the presence of thiourea as a mild reducing reagent, and conversion of the resulting dinitrophenylhydrazone to a
red compound by sulfuric acid. Each sample was ground in liquid
N2 with a little sand. The resulting powder
was then transferred into 1.5 mL of 6% (w/v) trichloracetic acid
solution and then centrifuged for approximately 7 min at
10,000g. The supernatant (0.5 mL) was then transferred
to two 2-mL tubes and diluted with 1.5 mL of trichloracetic acid. To
one tube was added 100 mg of HCl-washed activated charcoal to oxidize
any ascorbate to dehydroascorbate. Both tubes were sealed, shaken for 1 min, and centrifuged for 3 min at 10,000g. From each of the
two tubes, 0.5 mL of supernatant was pipetted into two additional 2-mL
tubes. Dinitrophenylhydrazine reagent (0.25 mL; 2 g of
2,4-dinitrophenylhydrazine and 4 g of thiourea in 100 mL of
25% [v/v] H2SO4)
was added to one tube of each pair. All four tubes were then
incubated for 3 h in a water bath at 37°C to allow the reaction
to run to completion. To all tubes 0.63 mL of
H2SO4 was then added
dropwise on ice. Reagent (0.25 mL) was added to the blank tubes. The
A540 of the sample relative to the blank
was measured. Standard curves were constructed using
l-ascorbic acid in the range of 0 to 4 mm.
-Tocopherol Content
Leaves (85 mg fresh weight per leaf) were ground to a fine powder
in liquid N2, which was transferred to an
ice-cold, 9-mL glass tissue homogenizer containing 2 mL of extraction
buffer (2 mm sodium isoascorbate, 5 mm
MgCl2, and 0.08 m
H2SO4, pH 6.8). The
macerate was then homogenized and -tocopherol was extracted by
vigorous shaking for 10 min with 1 mL of hexane. The upper organic
layer was separated by centrifugation for 10 min at 16,000g. The pellet was collected and twice reextracted before pooling the upper
organic layers and evaporating to dryness under
N2. Samples were stored in the dark at 80°C
under N2. Immediately prior to
chromatography, samples were dissolved in 100 µL of methanol. Separation was by reverse-phase chromatography (System Gold HPLC, Beckman) with a C18 ODS-1 column (length 25 cm,
i.d. 4 mm). The mobile phase was 3% dichloromethane in 97% methanol
(v/v) delivered at a flow rate of 1 mL/min with a 20-µL injection
loop. Detection was at 292 nm. Peak areas were integrated and the
column was calibrated by using known concentrations of purified
-tocopherol.
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RESULTS |
Environmental Conditions
The daily maximum and minimum temperatures and the daily
integrated photon flux experienced by the maize crops during May and
June in 1994 and 1995 are shown in Figure
1. In 1994 both minimum and maximum
temperatures increased during this period. In 1995, although there was
an increase in minimum temperature, maximum temperature did not show a
consistent increase during May. Light levels fluctuated markedly
throughout the experimental period in both years and demonstrate the
variable climatic conditions experienced by the crops.
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| Figure 1.
Daily integrated PPFD incident on the canopy and
the maximum and minimum air temperatures at the field site during May
and June 1994 (broken line) and 1995 (solid line).
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Relationship between Electron Transport and CO2
Assimilation
Consistent with the findings of Stirling et al. (1994), the
ability of leaves to assimilate CO2 was depressed
in May, when periods of chilling were experienced, and increased
markedly as temperature increased through June, as illustrated by the
changes in the representative photosynthetic light-response curve of
leaves harvested in May and June 1995 (Fig.
2).
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| Figure 2.
Response of CO2 assimilation,
corrected for dark respiratory losses, to incident PPFD for the
youngest mature leaf of a maize crop sampled on May 16 ( ), May 23 ( ), and June 26 ( ) of 1994. ses of three leaves are
shown.
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An effective way to examine the relationship between linear electron
transport and CO2 assimilation in maize leaves is
to determine the CO2 and
PSII over the range of PPFDs used to determine a photosynthetic light-response curve (Genty et al., 1989; Edwards and
Baker, 1993). Previously, plots of PSII
against CO2 for mature maize leaves over a
wide range of PPFDs and environmental conditions showed a remarkable
correlation between the two parameters, with the ratio of
PSII/CO2 remaining
constant within a range of approximately 11 to 13 (Edwards and Baker,
1993). A value of 12 for
PSII/CO2 implies
that six electrons must be transported through PSII for each molecule
of CO2 assimilated (Edwards and Baker, 1993).
Measurements of PSII and
CO2 at a range of PPFDs between 150 and 1500 µmol m2 s1 were made
on maize leaves throughout May and June in 1994 and 1995. Plots of
PSII against CO2
were then constructed from data collected from all of the leaves
monitored on any given sampling day. Representative examples of such
plots for days in 1994 and 1995 are shown in Figure
3. The predicted relationship between PSII and CO2
(PSII/CO2 = 12) for
mature maize leaves is shown by the dashed lines in Figure 3. The
majority of leaves harvested during May and June exhibit a relationship
between PSII and
CO2 that deviates from that predicted for
mature maize leaves. It is only toward the end of June in 1995 that the
data points on the PSII against
CO2 plot fall close to the predicted line
(Fig. 3).
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| Figure 3.
Relationship between PSII and
CO2 for leaves harvested on selected days
during May and June of 1994 (A) and 1995 (B). In 1995 some leaves were
measured in an atmosphere containing 2% O2 (); all
other data ( ) were obtained from leaves in an atmosphere containing
21% O2. The dashed lines indicate the expected
relationships for mature, nonstressed maize leaves (see text).
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In some cases, toward the end of June, a number of points determined at
the lower PPFDs (high CO2 values) fell below
the line (Fig. 3). Such deviations at low PPFDs from the predicted linear relationship have been reported in a range of species and may be
associated with the errors involved with correcting
CO2 assimilation rates for respiratory losses
using dark respiration rates (Edwards and Baker, 1993). During May and
early to mid-June, the majority of data points fell well above the
predicted line, thus demonstrating an unusually high
PSII/CO2. The
magnitude of the deviations of
PSII/CO2 from the
predicted value is perhaps most clearly demonstrated by plotting
PSII against CO2
for all leaves monitored in May and June of 1994 and 1995 (Fig.
4). It can be seen clearly from these
plots that the majority of the data points fall above the line for the
predicted relationship of PSII and
CO2 in mature maize leaves and that many
data points fall considerably above the predicted line. This would
suggest that frequently during May and June the rate of electron
transport through PSII is in considerable excess of that required to
sustain the observed rate of CO2 assimilation.
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| Figure 4.
Relationship between PSII and
CO2 for all leaves analyzed during May and
June of 1994 (A) and 1995 (B). In 1995 some leaves were measured in an
atmosphere containing 2% O2 (); all other data ()
were obtained from leaves in 21% O2. The dashed lines indicate the expected relationships for mature, nonstressed maize leaves (see text).
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If during the period of early crop development the proportion of
electron equivalents resulting from PSII photochemical activity that
are used for CO2 assimilation in leaves is
considerably lower than that found in nonstressed leaves, then,
clearly, sinks other than CO2 assimilation for
the products of electron transport must be operating. A prime candidate
for an alternative electron acceptor is O2,
either via a Mehler reaction or photorespiration. Although it is
commonly assumed that nonstressed, mature maize leaves do not exhibit
any significant level of photorespiration due to the high
CO2 concentration in the bundle-sheath cells, it
is possible that under chilling stress conditions the
CO2-concentrating mechanism may not operate
efficiently, and therefore the CO2 concentration would be considerably lower and may permit oxygenation of ribulose 1,5-bisphosphate by Rubisco. This possibility was examined by reducing
the O2 concentration from 21 to 2% in the
atmosphere of leaves when PSII
andCO2 were being measured in 1995. This reduction in O2 concentration had no major effect
on the relationship between PSII and
CO2 (Figs. 3 and 4) and is an indication
that the onset of photorespiration during periods of low temperature could not account for the additional sink for electron equivalents.
Active O2 Species Metabolism
If increased Mehler-APX cycle activity accounts for the additional
sink for electrons during May and early June, then increased activities
of active O2 and radical-scavenging enzymes and
levels of antioxidants might be expected. The maximum extractable
activities of APX, DHAR, GTR, MDHAR, and SOD were determined at 25°C
for leaves harvested on May 16, May 23, and June 26, 1995 (Table
I). When the enzyme activities were
expressed on the basis of leaf area APX and DHAR decrease from May to
June, SOD increased and GTR and MDHAR showed no significant changes.
However, if the enzyme activities are expressed on the basis of
chlorophyll content a very different picture emerges. The activities of
all of the enzymes decreased significantly from mid May to the end of
June: APX and DHAR by more than 70%, GTR and MDHAR by approximately
60%, and SOD by 50% (Table I).
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Table I.
Antioxidant enzyme activities assayed at 25°C and
associated antioxidant contents extracted from maize leaves harvested
from the field on May 16, May 23, and June 26, 1995
Data are the means ± se of four independent
replicates.
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It has been reported that the activities of enzymes involved in
scavenging active O2 species and radicals in
maize leaves can be extremely temperature sensitive (Jahnke et al.,
1991). In the context of the protection from photoxidation in the field it was important to determine whether the enzymes assayed maintained a
high activity at a representative field temperature. Consequently, the
enzyme extracts were all assayed at 14°C (Table
II). Unlike the situation when the
enzymes were assayed at 25°C (Table I), APX, DHAR, GTR, and MDHAR
activities on a unit leaf area basis all decreased from May to late
June. The activity of SOD showed no significant change.
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Table II.
Antioxidant enzyme activities assayed at 14°C
after extraction from maize leaves harvested from the field on May 16, May 23, and June 26, 1995.
Data are the means ± se of four independent
replicates.
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When the activities at 14°C (a temperature better reflecting the day
temperature that the leaves in the field might experience in mid-May)
were expressed on a unit chlorophyll basis, all enzymes exhibited very
large decreases in activity from May to late June. By comparing the
activities of each enzyme per unit chlorophyll in extracts made from
leaves harvested on May 16 and assayed at 14°C with those from leaves
harvested on June 26 and assayed at 25°C (Fig.
5), we could evaluate the magnitude of
the change in potential activity of the enzymes operating in leaves in
the field from mid-May, when temperatures are low, to the end of June,
when temperatures are higher. The activity of DHAR in mid-May was
almost 6-fold that in late June, and the activities of APX, MDHAR, and SOD were elevated approximately 2- to 3-fold, whereas there was a much
smaller enhancement of GTR activity.
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| Figure 5.
Ratio of activities of enzymes assayed at 14°C
for leaves harvested on May 16 to the activities assayed at 25°C for
leaves harvested on May 26, 1995. Data are given for APX, DHAR, GTR, MDHAR, and SOD. ses of three replicates are shown.
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Two key antioxidants in chloroplasts involved in active
O2 and radical scavenging are ascorbate and
-tocopherol. The level of ascorbate on a unit leaf area basis did
not change significantly throughout May and June; however, on a unit
chlorophyll basis it decreased by more than 50% (Table I). The level
of -tocopherol actually increased per unit area from May to late
June; however, a 35% decrease occurred during this period when
expressed per unit chlorophyll.
Lipid peroxidation by LOX in leaves generates both singlet oxygen and
superoxide anion radicals (Lynch and Thompson, 1984). LOX is an enzyme
normally involved in wound responses in tissues and catalyzes the
reaction of O2 with free, polyunsaturated fatty acids to form conjugated lipid hydroperoxides. It is quite possible that LOX could be an important initiator of oxidative damage under chilling-stress conditions. Extractable LOX activity was greater for
leaves harvested on May 16 compared with June 26 (Table
III). The elevated LOX activity in
mid-May is consistent with the higher MDA content in the leaves on May
16 compared with June 26 (Table III); MDA is a product of lipid
peroxidation.
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Table III.
Relative LOX activity and extent of lipid
peroxidation, as monitored by MDA levels, from maize leaves harvested
from the field on May 16, May 23, and June 26, 1995
Data are the means ± se of four independent
replicates.
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DISCUSSION |
The data presented in Figures 3 and 4 demonstrate that during the
early growth season maize leaves exhibit values of
PSII/CO2 that are
considerably greater than would be expected for mature, nonstressed
leaves. In extreme cases this ratio was increased by a factor of 3.5, implying that 21 electrons are transported through PSII for each
molecule of CO2 assimilated, compared with 6 electrons in nonstressed leaves. It would appear that electron sinks
other than CO2 must be operating to sustain such
high PSII/CO2 values. Initially it was thought that photorespiration may be a
significant sink for electrons in leaves experiencing low temperatures because chilling may inhibit the
CO2-concentrating mechanism in the bundle-sheath
cells. This would result in a decreased CO2 concentration and allow oxygenation of ribulose 1,5-bisphosphate by
Rubisco. However, when leaves with high
PSII/CO2 values
were exposed to an atmosphere containing 2% O2
to inhibit photorespiration, no significant decreases in
PSII were observed (Figs. 3 and 4). Consequently, photorespiration can be ruled out as a major sink for
electrons in these leaves.
Direct reduction of O2 via a Mehler reaction is
an obvious candidate for dealing with the increase in electron flux
relative to CO2 assimilation in chill-stressed
leaves. The increased activities per unit chlorophyll of APX, DHAR,
GTR, MDHAR, and SOD, coupled with the increased levels of the
antioxidants ascorbate and -tocopherol, in leaves in mid-May
compared with late June (Tables I and II) would be consistent with this
hypothesis. However, when the enzyme activities and antioxidant
contents are expressed on the basis of unit leaf area, only small
differences are found between mid-May and late June (Tables I and II).
Because the rate of generation of active O2
species via the Mehler reaction in chilled leaves will be related to
the rate of light capture by the antennae pigments, the enzyme
activities and antioxidant contents expressed on a chlorophyll basis,
rather than on an area basis, may be more relevant to the issue of
changing electron sinks. However, comparison of the rates of the enzyme
activities on a chlorophyll basis from leaves harvested in mid-May and
late June, assayed at temperatures similar to those experienced in the
field during the day (Fig. 5), does not give overwhelming support for
the Mehler reaction acting as the major sink for electrons. APX, DHAR,
MDHAR, and SOD all exhibit 2-fold increases in activity in mid-May
compared with late June, but GTR activity is increased by only
approximately 15%.
It would appear from the elevated LOX activity and MDA content in
leaves in mid-May compared with late June (Table III) that photosynthetic-reducing equivalents are not the only source of oxidative stress during periods of low temperatures. LOX activity is
normally associated with peroxidation of lipids during leaf senescence
and in response to plant tissue wounding (Kar and Feierabend, 1984;
Lynch and Thompson, 1984; Thompson et al., 1987; Croft et al., 1993;
Saravitz and Siedow, 1996). The higher LOX activity in maize leaves
during periods of low temperatures might suggest that increased LOX
synthesis is a leaf response to chilling stress. Alternatively,
increased LOX activity may be a response to increased lipid
peroxidation produced as a result of chill-induced photo-oxidative events. In either case, LOX-mediated peroxidation of membrane lipids is
likely to make a significant contribution to the oxidative damage
occurring in chill-stressed leaves.
Aside from the Mehler reaction, there are two other possible metabolic
explanations for why the ratio of
PSII/CO2 is
increased when leaves are exposed to periods of low temperatures. It is well established that for C4 plants to maintain a
high CO2 concentration in the bundle sheath
leakage of CO2 from the bundle sheath to the
mesophyll must be compensated for by overcycling of the
C4-acid cycle relative to the net rate of C
assimilation (Hatch, 1987; Furbank et al., 1990). An additional energy
requirement is associated with this overcycling, because ATP is
required for PEP synthesis (Furbank et al., 1990). It has been
estimated that in mature, nonstressed C4 leaves
the C4-acid cycle runs 25% faster than the net
rate of photosynthesis (Farquhar, 1983; Evans et al., 1986; Henderson
et al., 1992). If the rate of overcycling relative to net
photosynthetic C assimilation were to increase at chilling temperatures, then this would result in an increase in
PSII/CO2.
Another factor that could modify
PSII/CO2 is the
rate of operation of a Q cycle around the Cyt
b/f complex relative to linear photosynthetic
electron transport. Operation of a Q cycle can potentially
increase the ratio of ATP to NADPH produced by linear electron
transport (Ort, 1986), which could modify the quantum yield of
CO2 assimilation. Furbank et al. (1990) estimated
that in the absence of a Q cycle increasing the
C4-acid overcycling from 0 to 100% of total C
assimilation would increase the quantum requirement of
C4 photosynthesis from approximately 18 to 24. When a Q cycle is operating, this change from no
C4-acid overcycling to 100%
C4-acid overcycling would increase the quantum
requirement from 12 to 15. Operation of a Q cycle was
estimated to decrease the quantum requirement from 18 to 12 if no
C4-acid overcycling occurred and from 24 to 15 at
100% overcycling.
From the calculations of Furbank et al. (1990) it can be seen that a
C4 leaf at optimal growth temperature, operating
a Q cycle but having no C4-acid overcycling,
will have a quantum requirement for CO2
assimilation of approximately 12, which would increase to 24 if the
Q cycle ceased to operate and 100%
C4-acid overcycling occurred. If we assume the
most extreme, but highly unlikely, scenario that maize leaves that have
developed at optimal growth temperatures operate a Q cycle but have no
C4-acid cycling and that in leaves that have
developed at chilling temperatures the Q cycle ceases to
operate and 100% C4-acid cycling occurs, then a
doubling of the quantum requirement for C4
photosynthesis from 12 to 24 would occur at chilling temperatures.
Clearly, this doubling of the quantum requirement would not be
sufficient to account for the observed 3.5-fold increase in
PSII/CO2 (and the
quantum requirement for CO2 assimilation) when
maize leaves experience low temperatures in the field. Although
Q-cycle operation and C4-acid cycling
may be factors that could be modified by chilling, even the most
extreme changes in these activities could not account for the observed
increases in
PSII/CO2.
It is possible that the high
PSII/CO2 values for
leaves experiencing low temperatures may be due to inaccuracies in the measurement of PSII and
CO2. To determine
CO2, the rate of respiratory
CO2 evolution in the light is estimated from the
dark respiration rate. If the rate of respiration in the light relative to that in the dark increases at low temperatures, then this would result in an underestimation of CO2 assimilation
in chilled leaves and an elevated
PSII/CO2. However,
it seems unlikely that the magnitude of any such chill-induced changes
in respiratory activity in the light would be sufficiently large to
account for the large increases in
PSII/CO2.
A potential source of error in the estimation of
PSII is the overestimation of
Fm, which would lead to an overestimation of PSII. Kramer et al. (1995) showed that the
saturating light pulse (0.5 s at a PPFD of approximately 10,000 µmol
m2 s1) used to reduce
QA during the measurement of
PSII will also reduce the plastoquinone pool,
which will result in a loss of the quenching due to oxidized
plastoquinone prior to applying the saturating light pulse. However,
this error will be significant only when leaves have highly oxidized
plastoquinone pools at steady-state photosynthesis, as would be found
at low PPFDs, and would then only result in overestimations of
PSII of less than 10%. All of the
PSII measurements in this study were made on
leaves exposed to PPFDs between 150 and 2000 µmol
m2 s1. Over this PPFD
range qP is low (data not shown), indicating that
QA is not highly oxidized. As there is a close
relationship between the redox state of QA, as
estimated from qP, and the plastoquinone pool,
errors due to quenching by oxidized plastoquinone did not affect
significantly the
PSII/CO2 values
obtained in this study.
It is possible that differences in the optical properties of the maize
leaves at different times could result in differences in
PSII/CO2. In maize
leaves at high PPFDs, measurement of PSII using fluorescence excitation of 560 and 660 nm produces different values, but this is not the case at low PPFDs (Kingston-Smith et al.,
1997). This has been attributed to the differential penetration of the
560 and 660 nm radiation into the leaf. Because measurements of
PSII at high PPFDs using 560 and 660 nm of
fluorescence excitation for maize leaves harvested from a field plot
throughout May and June in 1997 produced similar results (within 10%,
data not shown), it is unlikely that such errors are important in the
context of the high
PSII/CO2 values
observed during periods of chilling.
This study demonstrates that
PSII/CO2 is
considerably elevated when maize leaves are exposed to low temperatures
in the field. Although chill-induced inhibition of a Q
cycle, an increase in the overcycling of C4
acids, and possible errors in measurement of
PSII and CO2 would
produce increases in this ratio, these factors cannot account for the
magnitude of the increases observed. The chill-induced increases in
PSII/CO2 imply that
the rate of linear electron transport relative to
CO2 assimilation increases and alternative
electron acceptors to CO2 must become available. Increased levels of active O2- and
radical-scavenging enzymes and levels of antioxidants in the chilled
leaves suggest that O2, via a Mehler reaction, is
a candidate for such an alternative electron acceptor.
| |
FOOTNOTES |
1
This work was supported by grants from the
Biotechnology and Biological Sciences Research Council of the United
Kingdom and from the Commission of the European Communities (grant no.
AIRI-CT92-0205 to N.R.B.).
*
Corresponding author; e-mail baken{at}essex.ac.uk; fax
44-1206-873416.
Received August 19, 1997;
accepted October 23, 1997.
| |
ABBREVIATIONS |
Abbreviations:
APX, ascorbate peroxidase.
DHAR, dehydroascorbate reductase.
DTPA, diethylenetriaminepentaacetic acid.
GTR, glutathione reductase.
LOX, lipoxygenase.
MDA, malondialdehyde.
MDHAR, monodehydroascorbate reductase.
NBT, nitroblue tetrazolium.
CO2, quantum efficiency of CO2
assimilation.
PSII, relative quantum efficiency of PSII
electron transport.
qP, photochemical quenching
coefficient.
SOD, superoxide dismutase.
| |
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Abstract
Introduction