Plant Physiol. (1996) 112: 1253-1 260
Effect of High Temperature on Photosynthesis in Beansl
II. CO, Assimilation and Metabolite Contents
Claudio Pastenes2* and Peter Horton
Robert Hill Institute, Department of Molecular Biology, University of Sheffield, Sheffield SI O 2TN,
United Kingdom
The effect of high temperatures on CO, assimilation, metabolite
content, and capacity for reducing power production in nonphotorespiratory conditions has been assessed in t w o different bean
(Phaseolus vulgarus 1.) varieties, Blue Lake (commercially available
i n the United Kingdom) and Barbucho (a noncommercially bred
Chilean variety), which are known t o differ in their resistance to
extreme high temperatures. Barbucho maintains i t s photosynthetic
functions for a longer period of time under extreme heat compared
with Blue Lake. The CO, assimilation rate was increased by increases in temperature, with a decrease in ratio of rates at temperatures differing by 10°C. It i s suggested that limitations to CO,
assimilation are caused by metabolic restrictions that can be differentiated between those occurring in the range of 20 t o 30°C and 30
to 35°C. It i s likely that changes i n the capacity for Calvin cycle
regeneration and starch synthesis affect photosynthesis in the range
of 20 to 30°C. But following an increase in temperature from 30 to
35"C, the supply of reducing power becomes limiting. From analysis
of adenylate concentration, transthylakoid energization, and, indirectly, NADPH/NADP+ ratio, it was concluded that the limitation
in the assimilatory power was due t o an oxidation of the NADPH/
NADP+ pool. In the range of 30 to 35"C, the photosystem I quantum yield increased and photosystem I I maintained i t s value. We
conclude that the reorganization of thylakoids observed at 30 t o
35°C increased the excitation of photosystem I, inducing an increase in cyclic electron transport and a decrease in the supply of
NADPH, limiting carbon assimilation.
High temperatures affect photosynthesis by altering
the excitation energy distribution by changing the structure of thylakoids (Berry and Bjorkman, 1980; Weis and
Berry, 1988) and by changing the activity of the Calvin
cycle and other metabolic processes such as photorespiration and product synthesis. Much of the attention has
been focused on the former aspect, since thylakoids are
highly sensitive to heat, whereas the restrictions imposed by high temperatures on carbon metabolism have
been nearly exclusively interpreted as the effect on CO,
'
Supported by a scholarship to C.P. from the Ministry of Planning of the Chilean Government.
Present address: Facultad de Ciencias Agrarias y Forestales,
Departamento de Producción Agrícola, Universidad de Chile,
Casilla 1004, Santiago, Chile.
* Corresponding author; e-mail cpasteneQabello.dic.uchi1e.cl;
fax 562-678-5700.
availability. The diffusion of CO, and O, and the affinity
for carboxylation of the Rubisco enzyme have been
proven to be affected by increasing temperatures (Hall
and Keys, 1983; Jordan and Ogren, 1984; Brooks and
Farquhar, 1985), but it has been argued that the stimulation of photorespiration cannot fully explain the effect
of high temperature. It has been suggested that a restricted electron transport may limit RuBP supply (Berry
and Bjorkman, 1980).
It also has to be considered that changes in temperature
induce variations in the activity of enzymes, and that because of their different Qlo values (Pollock and Rees, 1975)
such variations are not the same for a11 of the enzymes
acting in a particular pathway. In photosynthesis, particularly in the reactions directly involved in CO, assimilation
and SUCand starch synthesis, such a phenomenon is expected and there is evidence that changes in the properties
of enzymes such as cytosolic FBPase and Suc phosphate
synthase (Stitt and Grosse, 1988) caused by changes in
temperature contribute to the regulation of metabolism.
We studied the effect of temperature increases on photosynthesis in two bean varieties: BL, which is commercially available in the United Kingdom, and BA, a Chilean,
noncommercially bred variety. The two varieties are
known to differ in their resistance to extreme high temperatures; BA maintains its photosynthetic functions for a
longer period of time than BL in conditions of extreme heat
(C. Pastenes and P. Horton, unpublished data). In the
previous paper it was shown that increases in temperature
expose a restriction in photosynthesis above 30°C in the
two bean varieties, which is accompanied by changes in
thylakoid properties. In this paper, the alterations in carbon metabolism are explored and it is concluded that metabolic changes are at least in part a consequence of altered
thylakoid function.
Abbreviations: BA, variety Barbucho; BL, variety Blue
Lake; Chl, chlorophyll; FBP, Fru-1,6-bisP; FBPase, Fru-1,6bisphosphatase; F6P, Fru-6-P; G6P, Glc-6-P; MDH, malate dehydrogenase; PGA, glycerate-3-phosphate; QFsI, quantum yield
of PSI; QFSII, quantum yield of PSII; P,oo, PSI reaction center; qE,
energy-dependent nonphotochemical quenching; qN, nonphotochemical quenching; Qlo, ratio of rates at temperatures differTP, triose phosing by 10°C; RuBP, ribulose-1,5-bisphosphate;
phate.
1253
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Pastenes and Horton
1254
MATERIALS A N D METHODS
Bean plants (Phaseolus vulgaris L.) var BL and BA were
grown as described in the preceding paper (Pastenes and
Horton, 1996).
Gas Exchange and Freezing of Leaf Discs
CO, assimilation and transpiration were measured with
CO, and water vapor gas analyzers, respectively (both
model225 mark 3, ADC, Hertshire, UK), mounted in series
in an open system as described by Harris et al. (1983). Leaf
discs were obtained from heated attached leaves as described before (Pastenes and Horton, 1996), cut just before
the measurements, and placed in a square aluminum
chamber with temperature control. The leaf discs were
placed in the chamber with their borders immersed in a
water-filled depression to avoid dehydration. They were
illuminated with a cold lamp (modell500, Schott KL, Walz,
Effeltrich, Germany) through two fiberoptics at 45" opposite each other. The light intensity at the leaf surface was set
to 850 pmol m-'s-'. Air jets of 1000 pmol mol-I CO, and
2% O, in N, were directed to both leaf surfaces and after 40
min, when steady-state photosynthesis was reached, the
leaf discs were freeze-clamped, which cooled the leaf below 0°C in less than 0.1 s, as described by Doncaster et al.
(1989).
Metabolite and Enzyme Determination
Frozen leaf discs were divided into halves for the measurement of metabolites and NADPH-MDH and kept in
aluminum foi1 envelopes at -70°C until processed. Separate discs were taken for adenylate determination. Each
leaf disc was taken from a different plant.
Extraction was carried out in HCIO,, which has been
shown to result in high recovery of metabolites (Leegood
and Furbank, 1984). Metabolite determination was carried
out as described by Lowry and Passonneau (1972) and
Leegood (1993), measuring absorbance changes at 340 nm
minus 400 nm using a dual-wavelength spectrophotometer
(model DW-2000, SLM, Rochester, NY). Chl was estimated
by measuring the pheophytin content of the initial pellet
reserved from the leaf extraction (Vernon, 1960). For adenylate determination, no charcoal was added to the samples. NADPH-MDH activity was spectrophotometrically
assayed as described by Ashton et al. (1990). NADP-MDH
was fully activated by incubating the samples with 35
mmol of DTT under an atmosphere of N, at room temperature for 45 min.
535-nm Absorbance Changes
Measurements of 535-nm absorbance change and Chl
fluorescence were carried out simultaneously in a dualwavelength spectrophotometer (model DW-2000, SLM)
and a fluorometer (Walz) on leaf pieces inserted at 45°C to
the spectrophotometer light path, positioned in a glass
cuvette (Ruban et al., 1993). The cuvette was filled with
sufficient NaHCO,/Na,CO, buffer, pH 9, to cover the base
Plant Physiol. Vol. 1 1 2, 1996
of the leaf, and partially sealed with Parafilm (American
Can, Greenwich, CT) to maintain high CO, and humidity.
Quantum Efficiency of PSll and PSI
The simultaneous measurement of PSII and PSI quantum
yield was carried out according to the method described by
Schreiber et al. (1988) using two fluorometers (both PAM
101, Walz) to record fluorescence emission and
absorbance (830 nm). Measurements were made in leaves enclosed in a chamber with temperature regulation. Air containing 1000 pmol mol-l CO, and 2% O, in N, was passed
through the chamber over both leaf surfaces. The PSII and
PSI quantum yield were calculated after 20 to 25 min of
illumination (steady-state photosynthesis) and recorded
with a two-channel recorder. Actinic light was 800 pmol
m-2
s -1 and high-intensity pulse light was 1300 pmol m-*
sP1. The maximum fluorescence and P,,, oxidation levels
were recorded during the high-light pulse. One second
after the pulse, a 3-s dark interval was given to allow the
signal corresponding to the fully reduced leve1 of
to be
recorded. One leaf was used for each individual measurement and at least four replications were carried out for
each temperature.
RESULTS
Gas Exchange
The CO, assimilation rate measured in BA and BL (Fig.
1)increased when the temperature was raised from 20 to
35°C. The largest increase was observed from 20 to 25°C
(Qlo = 1.96), with a smaller increase from 25 to 30°C (Qlo
1.22), and an even smaller increase from 30 to 35°C (QIo
1.08); these results are the same as those recorded for O,
evolution (Pastenes and Horton, 1996).
The transpiration rate was increased four times, from
1.8 to about 8 mmol H,O m-' s-l (Fig. I), nearly proportional to the increase in temperature from 20 to 35°C.
Based on the results of calculations done according to the
method of von Caemmerer and Farquhar (1981), the
internal concentration of CO, was maintained nearly
constant at about 950 pmol mol-l, with an increasing
conductance for CO, (data not shown). These parameters
suggest that no limitation to photosynthesis occurs at
high temperatures due to changes in resistance to CO,;
on the contrary, the internal concentration of CO, is
maintained nearly constant throughout the thermal regime imposed on the leaves. This relationship between
temperature and CO, conductance has not always been
reported as being positively correlated (Mukohata et al.,
1971), mainly because the thermal effect on stomatal
conductance depends on the water status of the leaf
compared with the air. Therefore, if the vapor pressure
difference is allowed to increase with temperature, the
stomatal conductance will become limiting to photosynthesis, mostly in C, plants in normal air (Monson et al.,
1982). Obviously, only a significant negative effect of
temperature on stomatal conductance would have been
limiting to photosynthesis under the experimental conditions used, since the atmosphere was enriched in CO,
--
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1255
High Temperature and Photosynthesis in Beans
but in BL its concentration changed less markedly, resembling the changes observed in TP (Fig. 2E).
The relative changes in level of each metabolite depended on the temperature range examined and in some
cases were insensitive to changes in temperature, and CO,
assimilation increased. Such changes in metabolite pools
upon increases in temperature have been interpreted as
resulting from specific temperature-dependent regulation
of the enzymes of the Calvin cycle and of starch or SUC
synthesis (Leegood, 1993). This regulation can be investigated by examining ratios between certain metabolites.
The G6P/F6P ratio has been reported to be three times
higher in the cytosol than in the stroma (Dietz and Heber,
1984; Gerhardt et al., 1987). G6P/F6P calculated from the
metabolite data (Fig. 2F) was markedly decreased in BL;
25 -
-
15
-
10
l
4
I
N
20
m
I
0
n
a -
E
ON
2
2E
6 4 -
2 -
n l
15
I
I
I
I
20
25
30
35
40
300
-
G
PGA
RUBP/PGA
- 0.4
T e m p e r a t u r e ("C)
200 -
Figure 1 . CO, assimilation (A) and transpiration (B) rates upon increases in temperature from 20 to 35°C in BL (O) and BA (O). Each
point represents the average and SE of at least eight samples.
0.2
100 -
TA c i
to a level saturating for photosynthesis, accompanied by
a low O, concentration.
"1
TP/RUBP
0.0
3.0
3
(D
-t
0
U
o
-.
-+
80
(D
Metabolite Content
60
The concentration of the major hexose phosphate, G6P,
decreased with the increase in temperature from 20 to 35°C
in both varieties, from 200 to 110 nmol mg-I Chl in BL, and
from 170 to 110 nmol mg-I Chl in BA (Fig. 2A). It is
important to note that even though there appears to be a
difference in shape of the temperature response between
the varieties, the important feature is the consistent decrease in the hexose phosphate concentration with temperature increase. Similarly, a decrease in the F6P content (Fig.
2A) from 120 to 80 nmol mg-' Chl in BL and from 95 to 55
nmol mg-' Chl in BA was observed between 20 and 35°C.
PGA, the first product after CO, incorporation into the
Calvin cycle, appears to maintain its concentration with
increasing leaf temperature in both varieties (Fig. 2B). In
the TPs (i.e. glyceraldehyde phosphate and dihydroxyacetone phosphate) the changes are less clear between 20 and
30"C, but as temperature was increased to 35"C, a sharp
decrease in the concentration was observed (Fig. 2C). FBP,
on the other hand, showed a strong and continuous decline
in both varieties (Fig. 2D), from about 31 nmol mg-' Chl at
20°C to about 10 nmol mg-' Chl at 35°C. RuBP decreased
when temperature was increased from 30 to 35°C in BA,
7
1
40
2o
t
1
1
1
I
1
1
1
0.5
n
-.
O
"C
15 20 25 30 35 40 20 25 30 35 40
T e m p e r a t u r e ("C)
Figure 2. Metabolite concentration and metabolite relationship in
bean leaves of BL (O and) . and BA (O and O), subjected to different
temperatures from 20 to 35°C. Each point represents the average and
SE of at least five samples.
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Copyright © 1996 American Society of Plant Biologists. All rights reserved.
1256
Pastenes and Horton
the value was nearly 75% lower at 35°C than at 20°C.In BA,
however, this ratio appeared to be constant from 20 to
35"C,although there is an indication of a decrease at 30°C.
The decline in the G6P/F6P ratio in BL suggests that there
was a trend toward stromal compartmentation of the hexose phosphates induced by temperature (Gerhardt et al.,
1987), suggesting a possible limitation in the pathway to
starch synthesis. The difference in the extent to which the
ratio declined between the varieties could be due to the fact
that SUCand starch accumulate in different proportions,
depending on the time the plant has been exposed to light
(Gerhardt et al., 1987;Neuhaus and Stitt, 1990). However,
the leaves were always darkened for at least 1h and taken
randomly from the growth room during the day.
A well-known effect of temperature on photosynthesis is
the increase of photorespiration due to a change in gas
solubility, affecting the actual concentration of CO, reaching the acceptor compared with O, (Ku and Edwards, 1977
Hall and Keys, 1983; Jordan and Ogren, 1984), but also
because of a decline in the CO,/O, specificity of Rubisco,
as demonstrated by in vitro and in vivo experiments (Jordan and Ogren, 1984; Brooks and Farquhar, 1985). Even
though high CO, concentration greatly reduced the probability for photorespiration, changes in Rubisco activity
could still affect the rate of CO, assimilation at a given
temperature. However, the data shown in Figure 2G suggest that any change in Rubisco activity did not limit
photosynthesis in either variety; on the contrary, the observed decrease in the RuBP/PGA ratio is evidence of an
increase in the flux through Rubisco.
The regeneration sequence in the Calvin cycle comprises
the metabolic reactions in the stroma, from TP to RuBP.
Hence, the ratio TP/RuBP is an indicator of the capacity of
the cycle to regenerate the RuBP (Neuhaus and Stitt, 1989),
and as shown in Figure 2H, this ratio tended to increase in
both varieties when temperature was increased from 25 to
30°C.This suggests that a possible limitation in photosynthesis appears in the regeneration phase of the Calvin
cycle, simultaneously with the threshold at which the Qlo
for CO, assimilation rate declines. The TP/RuBP ratio
reached the highest level at 30"C,and declined markedly at
35"C,suggesting a new constraint in the assimilation rate
when the temperature was raised to 35°C.The ratio FBP/
F6P (Fig. 21) was maintained nearly constant in both bean
varieties when temperature was increased from 20 to 25"C,
and decreased from 25 to 35°C.These data suggest that the
FBPase-catalyzed reaction does not limit the response of
photosynthesis to temperature.
The two-step reaction for the reduction of CO, is driven
by the ATP and NADPH produced by the electron transport in thylakoids. Therefore, the PGA/TP ratio serves to
estimate the assimilatory power (Heber et al., 1986), the
product between the redox ratio (NADPH/NADP), and
the phosphorylation potential (ATP/ ADP). The stromal
pH must also be known, but it has been determined to
saturate at rather low light intensities, varying between 7.5
and 8.2 (Heldt et al., 1973).
The PGA/TP ratio in both bean varieties (Fig. 2J) was
approximately constant between 20 and 30"C, but in-
Plant Physiol. Vol. 1 1 2, 1996
creased dramatically when the leaf temperature was increased from 30 to 35"C,revealing a significant restriction
of ATP and/or NADPH supply. The possibility that a
restriction on ATP supply was responsible for the effect of
high temperature on photosynthesis was tested directly by
measuring the adenylate content of freeze-clamped leaf
discs. The ATP/ADP ratio decreased approximately 15% in
both varieties when the temperature increased from 20 to
30°C (Fig. 3), and was maintained as temperature was
further increased from 30 to 35°C. The ATP/ADP ratio
coincides with the lowest value reported in in vivo experiments (Dietz and Heber, 1984; Dietz and Heber, 1986;
Neuhaus and Stitt, 1989;Rao et al., 1990), and this ratio is
known to be low in conditions of increasing light and CO,,
as used here (Dietz and Heber, 1984;Diet and Heber, 1986).
The slight decrease in the ATP / ADP ratio is accompanied
by a stimulation in CO, assimilation and is likely to be the
effect of more efficient catalytic reactions in the Calvin
cycle (Heber et al., 1986). Rather than a failure in ATP
synthesis, there is an increase in the rate of ATP consumption with the concomitant relative increase in ADP.
535-nm Absorbance
Light scattering at 535 nm has been chosen as an indirect
indication of the energization capacity of thylakoids, since
such a signal has been related to the formation of qE by
increasing the pH in the chloroplasts (Krause, 1973;Ruban
et al., 1992, 1993).The extent of the difference between the
light / dark steady-state absorbance change upon illumination decreased with the increase in temperature from 20 to
30°C (data not shown). This is consistent with the decrease
in qN over this temperature range (Pastenes and Horton,
1996).As temperature was increased from 30 to 35"C, the
extent of the difference between steady-state light / dark
levels was maintained or slightly increased, suggesting
that at 35°C thylakoids are able to accumulate protons in
the lumen at least up to a level sufficient to cause A,,,
comparable to that observed at 30°C (data not shown).
05l
15
I
I
I
I
20
25
30
35
40
Temperature ("C)
Figure 3. Effect of increasing leaf temperature o n ATP/ADP ratio in
BL (O)and BA (O). Each point represents the average and SE of at least
four samples.
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High Temperature and Photosynthesis in Beans
1257
NADP-MDH Activity
The reversible light-dark activation-inactivation of the
enzyme NADP-MDH is strongly influenced by the
NADPH/NADP ratio; a high ratio favors a more active
enzyme (Scheibe and Jacquot, 1983; Ashton et al., 1990).
Figure 4 shows that the in vivo activity of NADP-MDH
expressed as a percentage of the total activity measured is
temperature-insensitive in the range of 20 to 30"C, with an
average value above 30%. This is similar to results previously reported by Holaday et al., (1992) in bean leaves at
27°C. However, at 35°C the activation state decreased to
lower than 20% in both varieties, indicating a decrease in
the NADPH / NADP ratio.
0 3
-
02 -
n
". i
I
02
I
I
0.3
0.4
Quantum Efficiencies of PSll and PSI
I
I
O5
0.6
0.7
@PSI
As expected, @psII increased with temperature in both
varieties, reaching a maximal value at 30°C and maintained
at 35°C (Fig. 5). Similar data were obtained when, , ,CD,
was
measured simultaneously with O, evolution (Pastenes and
Horton, 1996).,@
,,
follows a similar pattern, but there is a
continued increase in this parameter from 30 to 35"C, translating into a decrease in the ratio @psII/@FsI over this
thermal range. A nonlinear relationship between CDpsII and
aFSIhas been reported when an imbalance is created between the capacity of electron transport and the sink for
reducing equivalents in the stroma (Harbinson et al., 1990a,
1990b).Under such circumstances, this is interpreted as the
occurrence of photosynthetic control over electron transport involving cyclic electron flux through PSI (Harbinson
et al., 1990b). These observations, however, were later suggested to be an artifact, due to the incapacity of the method
to detect those PSI centers that are reduced and incapable
of being oxidized due to acceptor-side limitations. This
deviation is likely to occur in conditions of decreasing CO,
under constant light intensity and during photosynthetic
induction (Klughammer and Schreiber, 1994). A similar
deviation from linearity is observed in Figure 5 when tem-
i
T
1
I
I
I
I
I
20
25
30
35
40
Temperature ("C)
Figure 4. In vivo activity of NADP-MDH expressed as the percentage
from maximal activity on temperature. O, BL; O, BA. Each point
represents the average and SE of at least five samples.
Figure 5. Correspondence of quantum yields of PSll and PSI in BL
(O) and BA (O) upon increasing temperature from 20 to 35OC. Each
point represents the average and SE of at least four samples.
perature was increased from 30 to 35°C in both bean varieties. aPsI
tends to continue increasing, compared with
which seems to maintain a constant value. To make
sure that such a response was not due to an incapacity of
the light pulse for oxidizing P,,, centers the maximal signal
amplitude corresponding to the absorbance difference between the dark-adapted state (a11 P,,, in a reduced state)
and the high-intensity light pulse after far-red light (a11
in an oxidized state) was measured according to Klughammer and Schreiber (1994) at 30 and 35°C. This amplitude
was not different from that obtained in steady-state photosynthesis, according to the procedure described in "Materials and Methods" (data not shown). More specifically,
no further increase in the signal was recorded when applying the light pulse after far-red light, illumination, or at
steady state, suggesting that no acceptor-side limitation is
significant at high temperatures. Such a limitation is in fact
not expected from the NADP-MDH data that indicated
decreased NADPH/ NADP with temperature increases.
DISCUSSION
The rate of increase in CO, assimilation with increasing
temperatures progressively decreases until reaching a leve1
beyond which only a small increase in gas exchange is
observed. The maximum change in rate is observed in the
range of 20 to 25"C, reaching nearly a maximum assimilation rate at 30°C. A further temperature increase to 35°C
results in only a minor change in the gas-exchange rate.
It has been reported previously that temperature limits
CO, availability, either because of physiological responses
of leaves, which results in increases in resistance to the gas
diffusion (Mukohata et al., 1971; Monson et al., 1982), or
because of the relative solubility of CO, and O,, which
compete for the same acceptor (Ku and Edwards, 1977;
Hall and Keys, 1983; Jordan and Ogren, 1984). It has also
been reported that heat alters the functioning of Rubisco by
varying the substrate specificity of the enzyme (Jordan and
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Copyright © 1996 American Society of Plant Biologists. All rights reserved.
1258
Pastenes and Horton
Ogren, 1984; Brooks and Farquhar, 1985) and its activity
(Weis, 1981; Santarius et al., 1991).It is clear from the data
presented here, however, that the restrictions in CO, assimilation are not linked to the availability of CO, (Fig. 1)
and, very importantly, are not related to the activity of
Rubisco (Fig. 2). That Rubisco activity did not limit photosynthesis, however, does not mean that its activity was not
affected. For example, Kobza and Edwards (1987) found
that mild increases in temperature resulted in reduced
Rubisco activity without limiting the rate of photosynthesis, and maintained a low RuBP/PGA ratio.
The observed response of CO, assimilation to temperature seems to be related to the capacity for final product
synthesis and to the capacity of the Calvin cycle to regenerate RuBP, as well as to the failure of the system to
maintain a necessary assimilatory power. These restrictions
became evident at different temperatures, as shown by the
different temperature responses of various metabolites. In
general, the changes occurring from 20 to 30°C can be
distinguished from those observed from 30 to 35"C, although some changes were continuous throughout the
temperature range.
The activity of SUCphosphate synthase and cytosolic
FBPase do not appear to restrict Suc synthesis, since there
was no accumulation of hexose phosphates (Fig. 2A). In
fact, studies with partially purified SUCphosphate synthase
have shown that this enzyme has a relatively high Q1, and
that the cytosolic FBPase becomes less sensitive to inhibition by Fru-2,6-bisphosphate and AMP (Stitt and Grosse,
1988).The decrease in the hexose phosphate leve1 indicates
that Suc synthesis is favor'ed by high temperatures (Stitt
and Grosse, 1988).
G6P and F6P are present in the chloroplast and cytosol
within the pathways for starch and Suc formation, respectively, and are interconverted by the enzyme phosphoglucose isomerase. This reaction is in equilibrium in the cytosol, but displaced from equilibrium in the stroma toward
G6P, where it has been proposed to have a rate-limiting
function in starch synthesis (Dietz, 1985; Gerhardt et al.,
1987). The stromal compartmentation of G6P and F6P in
BL, demonstrated by the decrease in G6P/F6P ratio (Fig.
2F), suggests that starch synthesis does not match the
increase in the capacity for SUCsynthesis as the temperature increases from 20 to 30°C.
In the regeneration phase of the Calvin cycle, the carbon
flux is mainly controlled by stromal FBPase, sedoheptulose-1,7-bisphosphatase, and ribulose-5-phosphate kinase
(Buchanan, 1980). The increase in the TP/RuBP ratio (Fig.
2H) suggests that a possible limitation is induced in the
regeneration pathway by temperature when it is increased
from 25 to 30°C. If a limitation does exist in this pathway,
it is unlikely that it is at the stromal FBPase, since the ratio
FBP/ F6P (Fig. 21) indicates that the FBPase-catalyzed reaction is favored by increases in temperature, particularly in
the range of 25 to 30°C. In addition, there is no accumulation of FBP.
Beyond 30°C the assimilation of CO, does not respond to
temperature and a new limitation to photosynthesis appears, this time related to the capacity to reduce PGA to
Plant Physiol. Vol. 112, 1996
TPs (Fig. 2J). The increase in the ratio of PGA/TP means
that high temperature affects the capacity to generate the
assimilatory power, i.e. the synthesis of ATP and
NADPH needed to sustain further increases in CO, assimilation. It is unlikely that such a restriction originated
from a decrease in the Pi pool in the chloroplast, since no
hexose accumulation was observed in the current study.
On the contrary, hexose tended to decrease (Fig. 2A).
Therefore, the restriction in the photosynthetic process at
30 to 35°C resulted from a more subtle change in thylakoid function.
The capacity for phosphorylation in chloroplasts is not
negatively affected by heat and, more specifically, the transition from 30 to 35°C does not show any substantial decrease in either the 535-nm absorbance change (data not
shown) or in the ATP/ADP ratio (Fig. 3). This suggests that
protons accumulate in the chloroplast lumen and also that
membrane-bound ATPases respond to that energization
according to substrate availability. On the contrary, the
NADPH / NADP ratio, indirectly measured through the
activation state of NADP-MDH, decreases during the transition from 30 to 35°C. This effect is accompanied by an
imbalance between the quantum yields of PSI and PSII
(Figs. 4 and 5), suggesting that it is the supply of NADPH
that limits carbon assimilation.
The limitation of NADPH supply could arise from inactivation of electron transport, perhaps at PSII. However,
this would lead also to an unaltered ratio of PSII/PSI
efficiency. Alternatively, the restriction in supply of
NADPH could arise from the changes in thylakoid function
and organization that occur at this temperature range
(Pastenes and Horton, 1996). The transition to state 2 and
the increase in PSII, may limit the supply of electrons from
PSII while stimulating PSI excitation. Although this situaoxidation, an increase in
tion tends to increased
reduction without a concomitant increase in PSII efficiency
could be explained by an increase in the rate of cyclic
electron flow around PSI. This cyclic flow could be stimulated by the lateral redistribution of Cyt b6/f (Vallon et al.,
1991).
The putative increase in PSI cycle electron flow could
have a physiological role. Even though the ATP/ ADP does
not change, an increase in permeability of the thylakoids
due to high temperatures (Emmett and Walker, 1973; Santarius et al., 1991) could still occur if cyclic electron transport is simultaneously enhanced, hence counteracting proton leakage. This would be consistent with the limiting
factor in PGA reduction being NADPH. If this is the case,
the structural changes leading to a favoring reduction of
the Cyt bf complex, like those associated with state transitions, would help to prevent a decrease in ATP, but at the
expense of NADPH. Therefore, such structural changes to
thylakoids caused by increases in temperature, namely
state transitions and PSII, centers, would be significant in
enabling plants to carry out photosynthesis in conditions of
proton leakage. This is perhaps in addition to a role in the
protection of PSII from overexcitation in conditions of high
light intensities associated with high temperatures in the
field (Havaux et al., 1991).
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High Temperature and Photosynthesis in Beans
Differences between t h e varieties w e r e observed i n the
various metabolite ratios ( e g . G6P/F6P) a n d suggest some
variation i n the temperature response of carbohydrate metabolism. Similarly, differences i n thylakoid function were
a p p a r e n t f r o m t h e difference i n qN profiles (Pastenes a n d
Horton, 1996). However, neither of these a r e strongly reflected i n the steady-state rate of photosynthesis, which
responded similarly to temperature i n BL a n d BA.
ACKNOWLEDGMENTS
We thank Richard Leegood and Paul Quick for the advice on
metabolite determination methods and discussion and Pam
Scholes for technical assistance in different experiments. C.P.
thanks the Overseas Research Students award scheme (UK) for
partly supporting the postgraduate studies in 1994, and the Universidad de Chile.
Received March 6, 1996; accepted August 3, 1996.
Copyright Clearance Center: 0032-0889/96/112/ 1253/08
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