S17-008

Marked modulation by temperature of phosphoenolpyruvate carboxylase

from leaves of Amaranthus hypochondriacus, a C4 plant, compared to that in
Pisum sativum, a C3 species
Bhaskarrao Chinthapalli, AS Raghavendra

Department of Plant Sciences, School of Life Sciences, University of Hyderabad,

Hyderabad 500 046, India. Fax: +91-40-3010145, email: asrslrs@uohyd.ernet.in

Keywords: Arrhenius plots, Cold sensitivity, C4 enzymes, PEPC, Temperature

Introduction

The temperature optima for photosynthesis and growth in C4 plants are higher than those
for C3 plants. The cold sensitivity C4 pathway has been suggested to be related to the cold
sensivitity of key C4 enzymes, such as pyruvate phosphate dikinase or PEP carboxylase,
PEPC (Kingston-Smith et al. 1997; Du et al. 1999). However, McWilliam and Ferrar
(1974) have suggested that the high temperature tolerance of C4 plants is due to not the
greater thermostability of their PEP carboxylase, but heat-resistant protein synthesizing
machinery, which replaces the PEP carboxylase denatured by high temperature. Selinioti
et al. (1986) observed that light and temperature interact during the activation of PEPC
and this might be important for C4 plants, such as A. paniculatus.

On illumination, the activity of PEPC is enhanced by 2-3 fold along with a marked
decrease in the malate sensitivity of the enzyme. These changes during the light
activation are due mainly to the phosphorylation of the enzyme (Chollet et al. 1996,
Vidal and Chollet 1997, Parvathi et al. 2000a). Compared to the extensive literature on
the properties and mechanism of light activation of PEPC, in C4 plants, the literature on
the regulation by temperature of PEPC is quite limited (Rajagopalan et al. 1994). The
present study is an attempt to characterize the temperature responses of PEPC from a
typical C4 plant, Amaranthus hypochondriacus and compared with those of a C3 plant
Pisum sativum. Experiments were conducted on leaf discs so as to simulate physiological
situation in vivo and to also assess the reversibility of temperature effects.

Materials and Methods

Plants of Amaranthus hypochondriacus L. (cv. AG-67) and Pisum sativum L. (cv. Arkel)
were raised from seeds. The plants were grown outdoors in the field under a natural
photoperiod of approximately 12 h and temperature of 30 – 40 oC day/25 – 30 oC night.
The upper fully expanded leaves were harvested, about 2 – 3 h after sunrise. Leaf discs
(each of ca. 0.2 mm2) were prepared from 4- to 6- week-old plants of Amaranthus
hypochondriacus and 8- to 10-day-old plants of Pisum sativum.

The preparation of leaf extracts and the assay of PEPC are all described in detail
elsewhere (Parvathi et al. 2000b). The sensitivity of PEPC to malate was checked using
either 0.5 mM malate (in case of C4 species) or 2 mM malate (in C3 species).Chlorophyll
was estimated by extraction with 80% acetone (Arnon 1949).

Thirty leaf discs were floated on distilled water in a 5 cm diameter Petri dishes and were
left in darkness for 2 h. After predarkening, the leaf discs were incubated 30 min at
required temperature in the range of 15oC to 55oC in a themostatically temperature
controlled water bath at each temperature. The temperature range tested in A. hypochon-
driacus (and other C4 plant) was 15oC to 55oC and for P. sativum (and other C3 species)
was 15oC – 50oC. At the end of 30 min at each temperature, the leaf discs were extracted
(as described above) for determining PEPC activity.

Results

The optimal temperature for PEPC activity in leaf discs of A. hypochondriacus (C4) was
45 oC compared to 30 oC in P. sativum (C3 species) (Fig. 1A). The response of enzyme
to temperature was quite dramatic when plotted as the % of maximum activity (Fig. 1B).

A 90
100
Activity (% of maximum)

80
Pisum sativum
80

70 Amaranthus hypochondriacus
60

Inhibition % of control
40 60
20
Amaranthus hypochondriacus A
50
1400 B 40
Activity (µmol mg Chl h )
-1

1200 Amaranthus hypochondriacus

Pisum sativum
1000 30
-1

800
20
600
400 10
200
Pisum sativum
0
10 15 20 25 30 35 40 45 50
10 15 20 25 30 35 40 45 50
o
o
Temperature C
Temperature C

Fig. 1. The activity of PEPC in extracts from leaf Fig. 2. Effect of temperature on inhibition by
discs of A. hypochondriacus (C4 plant) and malate of PEPC activity in extracts from leaf
P. sativum (C3 species) incubated at varying discs exposed to varying temperature in
temperatures. The activity of PEPC is either as % A. hypochondriacus (C4 plant) and P. sativum (C3
of maximum (A) or units of activity (B). species).

The decrease at 15 oC was much higher in case of A. hypochondriacus (C4 plant) than
that of P. sativum (C3 species). Similarly the decrease in activity of PEPC at
temperatures above 40 oC was much greater in the case of P. sativum (C3 species) than
that in A. hypochondriacus (C4 plant). Thus, the C4 PEPC was more sensitive to sub-
optimal temperatures and less sensitive to supra-optimal temperatures than that of C3
species. As the temperature was raised from 15 oC to 50 oC, there was a marked decrease
in malate sensitivity of PEPC in C4 plants (up to 77 %) was more than that in C3 species
(about 30%), when the enzyme was assayed at 0.05 mM bicarbonate (Fig. 2). There is a
decrease in malate sensitivity as the temperature was raised from 15 oC to 50 oC. The
extent of malate inhibition was always higher in case of P. sativum (C3 species) than that
of A. hypochondriacus (C4 plant).

Arrhenius plots were made by using the PEPC activity determined at a range 10 oC to 45
o
C in case of A. hypochondriacus (C4 plant) and 10 oC to 35 oC in case of P. sativum (C3
plant). The activation energy of PEPC was less at higher temperatures than that at cold
temperature (Table 1). The activation energy of PEPC from A. hypochondriacus was less
than that from P. sativum in the temperature range of 10 to 27 oC. However, the
activation energy of PEPC from A. hypochondriacus was less than that of P. sativum
above the temperature of 27 oC. The activation energy increased by almost 2 fold over
the absence of malate at temperatures around 10 oC to 17 oC in both A. hypochondriacus
(C4 plant) and P. sativum (C3 species) (Table 1). Arrhenius plots exhibited abrupt
changes or “break points” at only one point of 17 oC in A. hypochondriacus (C4 plant),
while at two points corresponding 17 oC and 27 oC in P. sativum (C3 species) (data not
shown). The patterns of Arrhenius curves in presence of malate were quite similar to
those in the absence.

Table 1. Activation energy (Kcal mol-1) of PEPC in extracts from leaf discs exposed to
different temperatures in A. hypochondriacus (C4 plant) and P. sativum (C3 species).
Amaranthus Pisum
Temperature range (oC) hypochondriacus sativum
Activation energy (kcal mol-1)
Enzyme activity
(absence of malate)
10-17 13.8 18.3
17-27 3.8 9.1
27-35 or 27-45* 2.9 1.23
Enzyme activity
(presence of malate)
10-17 23.0 31.0
17-27 6.1 12.5
27-35 or 27-45* 4.5 1.03
*The range was 27-35 oC for P. sativum and 27-45 oC for A. hypochondriacus.

Discussion

The results from this study demonstrate that temperature can cause quite dramatic
changes in not only the activity but also the regulatory properties of PEPC in both C3 and
C4 plants. The optimal temperature for PEPC in A. hypochondriacus (C4) was 45 oC
compared to 30 oC in P. sativum (C3 species) is not surprising. The activities of PEPC
and NADP-ME in desalted extracts from different species of sugarcane showed no large
changes after incubating the enzyme at various temperatures from 30 oC to 0 oC (Du et al.
1999). In contrast, PEPC in extracts of P. maximum lost up to 50% of its activity after
incubation for 60 min at 0 oC while the enzyme from P. miliaceum was stable (Krall and
Edwards 1993). Our results confirm that the C4 PEPC is quite sensitive to sub-optimal
temperatures compared to the PEPC of C3 species.

The steep increase in the activity of PEPC with temperature, particularly above 15 oC,
could be physiologically significant, as during the day the temperatures are expected to
rise on typically clear and sunny day. A combination of light and warm temperature
could amplify the photoactivation of the PEPC, as observed in case of Egeria densa
(Casati et al. 2000).

Our results demonstrate for the first time the dramatic changes by temperature in the
sensitivity of PEPC to malate. As the temperature was raised from 15 oC to 50 oC,
decrease in malate sensitivity of PEPC in C4 plants (85 to 45%) was more than that in C3
species (65 to 39%) (Fig. 2). The changes induced in malate sensitivity were much more
strongly manifested in C4 plants than those of C3 species. The extent of malate inhibition
is quite high in case of P. sativum (C3 species) than that of A. hypochondriacus (C4 plant).
Lowering the temperature from 25 to 3 oC not only decreased the catalytic capacity of
PEPC but also causes a considerable reduction (about 10 fold) in the sensitivity of malate
of both the phosphorylated and dephosphorylated forms of PEPC when assayed in vitro
(Carter et al.1995). In C4 species the effect of malate on PEPC with varying temperature
has occasionally been reported (Wu and Wedding 1987; Carter et al. 1995).

The increase in activation energy of PEPC in presence of malate, appears to be logical.

Malate increased the activation energy of PEPC in both C3 and C4 species (Table 1). For
Amaranthus cruentus the activation energy also rapidly increases below 20 oC, but it is
not clear whether it extrapolates to infinity at the same temperature as in Sorghum
bicolor, or at a slightly lower temperature (Laisk and Edwards 1997). Further, studies are
essential to characterize molecular basis of modulation by temperature of PEPC in C4
plants.

Acknowledgements

Our work is supported by a grant (to ASR) from University Grants Commission
(No. 3-174/2001/SR-II), New Delhi.

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