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The effect of leaf age and salt stress on malate accumulation and phosphoenolpyruvate carboxylase activity in Mesembryanthemum crystallinum
Plant Science Letters, 7 (1976) 341--346 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
341
THE EFFECT OF LEAF AGE AND SALT STRESS ON MALATE ACCUMULATION AND PHOSPHOENOLPYRUVATE CARBOXYLASE ACTIVITY IN MESEMBR YANTHEMUM CR YSTALLINUM
D.J. VON WILLERT, G.O. KIRST, S. TREICHEL and K. VON WILLERT lnstitut fiir Botanik, Fachbereich Biologie der Technischen Hochschule, Schnittspahnstr. 10, D-6100 Darmstadt (W. Germany) (Received March 22nd, 1976) (Revision received April 26th, 1976) (Accepted May 28th, 1976)
SUMMARY
Prior to treatment with 300 mM NaCI Mesembryanthemum crystaUinum leaves of different ages have only small differences in phosphoenolpyruvate (PEP) carboxylase activity and do not accumulate malate at night, indicating that they behave like C3-plants. After 12 days of NaC1 treatment the mature leaves exhibit a 20-fold increase in PEP carboxylase activity and a significant accumulation of malate during the night. These features are characteristics of crassulacean acid metabolism (CAM) plants. The PEP carboxylase activity in the youngest leaf did not increase until the leaf reached a certain stage of development. Since the youngest leaves always showed the lowest PEP carboxylase activity and never accumulated malate at night they do not contribute to a CAM. The results provide evidence for the existence of two regulatory processes in Mesembryanthemum crystallinum both causing a change from C3 to CAM. One process is under environmental control (NaCl concentration) and occurs only in mature leaves. The second process is an ontogenetic response and is associated with maturation.
INTRODUCTION
Mesembryanthemum crystallinum responds to salination with the development of a CAM [1]. The main characteristic of a CAM is a diurnal fluctuation in malate production. At the end of the night leaves have a high malate content which is largely depleted during the subsequent day. It has been shown Abbreviations: CAM, Crassulacean acid metabolism; PEP, phosphoenolpyruvate.
342
that in Mesembryanthemum crystallinum the accumulation of malate at night is associated with the ability to fix carbon dioxide non-autotrophically [2]. The enzyme involved in CO2 dark fixation is PEP carboxylase which in the case of M. crystallinum and other species of the Mesembryanthemaceae has been reported to undergo significant activation during salt treatment [3,4]. Recently Jones [5] has shown that leaves of different ages from the CAM plant Bryophyllum fedtschenkoi differ in their ability to accumulate malate during the night. Mature leaves exhibit a CAM, young leaves do not, and intermediate leaves are intermediate in their behaviour. Thus not all leaves of a CAM plant contribute to malate accumulation. A similar ontogenetic response in the leaves of another CAM plant has been reported by Lerman et al. [6]. Their investigations suggest that PEP carboxylase is more active in mature than in young leaves. This paper describes the changes in the activity of PEP carboxylase and the ability to accumulate malate in leaves of M. crystallinum as a function of NaC1 treatment and leaf age. MATERIALS AND METHODS
M. crystallinum plants were grown in a controlled environment chamber with a photoperiod of 12 h. Temperature in the day was 25°C and at night 18°C. The plants were irradiated with incandescent light at an intensity of 10 klux. 10 weeks after germination salt treatment was started by watering the plants with 300 mM NaC1. Leaves were numbered so that the first formed leaf pair was given number 1. At the start of the experiment, day 0, leaves 3, 4, 5 and 6 were analysed, leaves 1 and 2 at this stage were dead. By the end of the experiment, day 37, the 7th leaf pair had developed and was included in the analysis. At the end of the light period PEP carboxylase activity was assayed. Leaves were cut off and homogenized in a mortar with twice their weight of buffer containing Tris--bicine (200 mM, pH 8.5) and MgC12 (5 raM). The homogenate was filtered and centrifuged at 45 000 g for 10 min. The resulting crude supernatant was immediately used. PEP carboxylase activity (EC 4.1.1.31) was assayed at 30°(3 by coupling the reaction with malate dehydrogenase (EC 1.1.1.37). The oxidation of NADH was measured at 340 nm in a Beckman 24 Kinetik spectrophotometer. The standard assay system contained Tris--bicine (100 mM, pH 7.5), PEP (1.5 mM), NADH (0.15 mM), NaHCOs (5 mM), MgC12 (2 mM), and crude extract in a total volume of 2 ml. The content of malate in the leaves was determined enzymatically [7]. Leaves were harvested and f r e e z e . t i e d at the beginning and end of the dark period. RESULTS Prior to salt treatment the activity of the PEP carboxylase in the different leaves of M. crystallinum was at a low level. At that time maximum activity
343
0,2
j"
days
0
12day~
I/
~Tdays
> 0,1
\
o
0_ bJ o_
i 3
4
5
6
i
4 leaf pair 3
O~ 0
•
i
i
i
i
i
i
i
5
6
3
4
5
6
7
number
Fig. 1. Activity o f the PEP carboxylase (tamales fixed CO~/h/g fr.wt.) in the leaf pairs o f Mesembryanthemum crystaUinum at 3 intervals following treatment with 300 mM NaCI. At 37 days, open circles represent the activity o f the PEP carboxylase in the 3 leaves which had developed during salt treatment on a branch originating in the axil o f the 3rd leaf o f the main shoot. Filled circles represent the activity in the leaves of the main shoot. Leaves were numbered so that the first formed leaf pair was given number 1.
..~ E
so01
o
300
2
ol
0 days
12days
5
11o
E
10C
8 K E
3
4
6
3
4
5
N 6
37 days
3
4
5
6
7
{eaf pair number
o~ Fig. 2. A m o u n t o f overnight malate consumption or accumulation in the different leaves of Mesembryanthemum crystallinum during a dark period o f 12 h at 18°C at 3 different intervals following treatment with 300 mM NaCI. Leaves were numbered so that the first formed leaf pair was given number 1.
344 was found in middle leaf pairs; activity was lower in both younger and more mature leaves (Fig. 1). With the exception of the youngest leaf pair PEP carboxylase activity increased significantly during a 12~tay treatment with 300 mM NaC1. No significant activation was observed in the youngest leaf pair; the subapical leaf pair exhibited intermediate activation. The differences in PEP carboxylase activity between different leaves of a Mesembryanthemum plant at the beginning of the salt treatment became more and more accentuated with proceeding treatment (Fig.l). After 37 days on salt a further leaf pair had developed; this exhibited the lowest PEP carboxylase activity. The 6th leaf pair had matured. This process was associated with an increase in the activity of the PEP carboxylase. A similar response was shown by the 5th leaf pair where activity also increased with maturation. During the 37 days of salt treatment, branches grew from the axils of the 3rd and 4th leaf pairs. These branches carried small leaves which were more succulent than the leaves of the main shoot. Even in the leaves of these branches, which developed under salt treatment, the activity of PEP carboxylase increased with increasing leaf age (Fig. 1). After a salt treatment of 37 days the activity in the youngest leaves of both branch and main shoot were identical, and not significantly higher than in the youngest leaf prior to salination. Since the branch grew faster than the main shoot the oldest leaf of the branch must be compared with the 6th leaf of the main shoot; they exhibited comparable activities. At the beginning of the salt treatment none of the leaves was able to form and store malate during the night, but instead showed a slight consumption. During salt treatment the ability to accumulate malate at night developed only in the mature leaves or during maturation. The top leaf pair did not show any accumulation of malate even after 37 days of salt treatment (Fig. 2). DISCUSSION There are two significant aspects of the data reported here: (1) Jones [5] has provided good evidence that different leaves of a CAM plant can have different daily cycles of leaf resistances. For mature leaves lower resistances were recorded at night and higher resistances at the day. The opposite was found with young leaves. Since leaf resistance reflects stomatal opening this means that mature leaves open their stomata at night and young leaves at the day. The CO2 concentration in the substomatal spaces was assumed to control nocturnal opening of stomata in CAM plants [8]. Different internal CO2 concentrations should be the result of different capacities for dark CO2 fixation. Since the level of PEP carboxyiase is responsible for the ability to fix CO2 during the night, our findings, that the activity of the PEP carboxylase increases with increasing leaf age provides enzymatic support for the hypothesis. This means that NaCl-treated, CAM~xhibiting Mesembryanthemum plants behave like other well known CAM plants which do not need a special treatment for the development of a CAM [5,6].
345
(2) Two modes of regulation of the CO2 fixation pathway must be distinguished. One process is the NaCl-promoted change from C3 to CAM. This change can be controlled by environmental conditions and might be an adaptive reaction. We have outlined that this change is not carried out by the entire plant but is restricted to mature leaves. Young leaves will not develop CAM features unless they have reached a certain stage of development. The second process concerns the change from C3-photosynthesis to CAM during ageing. This process is not limited to the adaptive phase in Mesembryanthemum; it continues even in totally adjusted plants, indicating that ageing phenomena are involved. Carbon pathway changes have been reported for the C4 plant Portulaca oleracea [9]. Here the change towards an increased C3-photosynthesis with increasing leaf age is more likely to be an ontogenetic response than an environmentally controlled process. Similar conclusions had been drawn for the shift from C3 to CAM in Bryophyllum fedtschenhoi and Bryophyllum daigremontianum [5,6]. Our results agree with this concept. Regardless of the duration of the salt treatment the youngest leaves possess the lowest activity of PEP carboxylase and show no accumulation of malate. PEP carboxylase activity and malate accumulation only increase during maturation. The view that the change from C3 to CAM in M. crystallinum involves a function of age and is not solely an environmentally controlled process is supported by the finding that the appearence of CAM in leaves of the branches which have developed during salt treatment depends on leaf age. On the other hand, mature leaves of very old non-saline Mesembryanthemum plants exhibit only a weak CAM. This might be interpreted as a NaCl-enhanced ageing [10,11]. The common suggestion that an increased succulence associated with NaC1 treatment or cell expansion is responsible for the increased CAM [12,13] does not seem to be correct, since it has been demonstrated that increased succulence is not a prerequisite for malate accumulation in M. crystallinum [14]. In earlier reports [2,13] it has been shown that within 12 to 14 days leaves take up all the NaC1 needed for osmotic and salt adjustment. After this period no significant increase in the ion content of the leaves took place. On the other hand, content of Na ÷ and C1- depends on leaf age increasing with increasing leaf age. Since NaCI is known to be a strong inhibitor of the PEP carboxylase [3,15] one would expect that if NaC1 interacts with PEP carboxylase in the intact leaf or in the crude extract that with increasing leaf age the activity of the PEP carboxylase should decrease. The opposite was found. Thus if there was an inhibition of the enzyme it must be reversed when the crude extract was diluted 100-fold in the enzyme assay. ACKNOWLEDGEMENT This research was supported by the Deutsche Forschungsgemeinschaft. We thank Dr. J.A. Raven and Dr. J. Sprent (University of Dundee) for critical advice in preparing this manuscript.
346 REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
K. Winter and D.J. yon Wfllert, Z. Pflanzenphysiol., 67 (1972) 166. D.J. yon Willert, Z. Pflanzenphysiol., 72 (1974) 64. S.P. Treichel, G.O. Kirst and D.J. yon Willert, Z. Pflanzenphysiol., 71 (1974) 437. S. Treichel, Plant Sci. Lett., 4 (1975) 141. M.B. Jones, Plants (Berl.), 123 (1975) 91. J.C. Lerman, E. Deleens, A. Nato and A. Moyse, Plant Physiol., 53 (1974) 581. M. Kluge, Plants (Berl.), 80 (1968) 859. T.F. Neal~, Aust. J. Biol. Sci., 26 (1973) 705. R.A. Kennedy and W.M. Laetach, Plants (Berl.), 115 (1973) 113. D.J. yon Willert and D. Kramer, Planta (Berl.), 107 (1972) 227. J.T. Prkco and J.W. O'Leary, Physiol. Plant., 27 (1972) 95. M. Kluge, O.L. Lange, M.V. Eichman and R. Schmidt, Plants (Berl.), 112 (1973) 357. K. Winter, Plants, 109 (19"/2) 135. D.J. yon Willert, Plant Sci. Lett., 4 (1975) 225. C.B. Osmond and H. Greenway, Plant Physiol., 49 (1972) 260.
341
THE EFFECT OF LEAF AGE AND SALT STRESS ON MALATE ACCUMULATION AND PHOSPHOENOLPYRUVATE CARBOXYLASE ACTIVITY IN MESEMBR YANTHEMUM CR YSTALLINUM
D.J. VON WILLERT, G.O. KIRST, S. TREICHEL and K. VON WILLERT lnstitut fiir Botanik, Fachbereich Biologie der Technischen Hochschule, Schnittspahnstr. 10, D-6100 Darmstadt (W. Germany) (Received March 22nd, 1976) (Revision received April 26th, 1976) (Accepted May 28th, 1976)
SUMMARY
Prior to treatment with 300 mM NaCI Mesembryanthemum crystaUinum leaves of different ages have only small differences in phosphoenolpyruvate (PEP) carboxylase activity and do not accumulate malate at night, indicating that they behave like C3-plants. After 12 days of NaC1 treatment the mature leaves exhibit a 20-fold increase in PEP carboxylase activity and a significant accumulation of malate during the night. These features are characteristics of crassulacean acid metabolism (CAM) plants. The PEP carboxylase activity in the youngest leaf did not increase until the leaf reached a certain stage of development. Since the youngest leaves always showed the lowest PEP carboxylase activity and never accumulated malate at night they do not contribute to a CAM. The results provide evidence for the existence of two regulatory processes in Mesembryanthemum crystallinum both causing a change from C3 to CAM. One process is under environmental control (NaCl concentration) and occurs only in mature leaves. The second process is an ontogenetic response and is associated with maturation.
INTRODUCTION
Mesembryanthemum crystallinum responds to salination with the development of a CAM [1]. The main characteristic of a CAM is a diurnal fluctuation in malate production. At the end of the night leaves have a high malate content which is largely depleted during the subsequent day. It has been shown Abbreviations: CAM, Crassulacean acid metabolism; PEP, phosphoenolpyruvate.
342
that in Mesembryanthemum crystallinum the accumulation of malate at night is associated with the ability to fix carbon dioxide non-autotrophically [2]. The enzyme involved in CO2 dark fixation is PEP carboxylase which in the case of M. crystallinum and other species of the Mesembryanthemaceae has been reported to undergo significant activation during salt treatment [3,4]. Recently Jones [5] has shown that leaves of different ages from the CAM plant Bryophyllum fedtschenkoi differ in their ability to accumulate malate during the night. Mature leaves exhibit a CAM, young leaves do not, and intermediate leaves are intermediate in their behaviour. Thus not all leaves of a CAM plant contribute to malate accumulation. A similar ontogenetic response in the leaves of another CAM plant has been reported by Lerman et al. [6]. Their investigations suggest that PEP carboxylase is more active in mature than in young leaves. This paper describes the changes in the activity of PEP carboxylase and the ability to accumulate malate in leaves of M. crystallinum as a function of NaC1 treatment and leaf age. MATERIALS AND METHODS
M. crystallinum plants were grown in a controlled environment chamber with a photoperiod of 12 h. Temperature in the day was 25°C and at night 18°C. The plants were irradiated with incandescent light at an intensity of 10 klux. 10 weeks after germination salt treatment was started by watering the plants with 300 mM NaC1. Leaves were numbered so that the first formed leaf pair was given number 1. At the start of the experiment, day 0, leaves 3, 4, 5 and 6 were analysed, leaves 1 and 2 at this stage were dead. By the end of the experiment, day 37, the 7th leaf pair had developed and was included in the analysis. At the end of the light period PEP carboxylase activity was assayed. Leaves were cut off and homogenized in a mortar with twice their weight of buffer containing Tris--bicine (200 mM, pH 8.5) and MgC12 (5 raM). The homogenate was filtered and centrifuged at 45 000 g for 10 min. The resulting crude supernatant was immediately used. PEP carboxylase activity (EC 4.1.1.31) was assayed at 30°(3 by coupling the reaction with malate dehydrogenase (EC 1.1.1.37). The oxidation of NADH was measured at 340 nm in a Beckman 24 Kinetik spectrophotometer. The standard assay system contained Tris--bicine (100 mM, pH 7.5), PEP (1.5 mM), NADH (0.15 mM), NaHCOs (5 mM), MgC12 (2 mM), and crude extract in a total volume of 2 ml. The content of malate in the leaves was determined enzymatically [7]. Leaves were harvested and f r e e z e . t i e d at the beginning and end of the dark period. RESULTS Prior to salt treatment the activity of the PEP carboxylase in the different leaves of M. crystallinum was at a low level. At that time maximum activity
343
0,2
j"
days
0
12day~
I/
~Tdays
> 0,1
\
o
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i 3
4
5
6
i
4 leaf pair 3
O~ 0
•
i
i
i
i
i
i
i
5
6
3
4
5
6
7
number
Fig. 1. Activity o f the PEP carboxylase (tamales fixed CO~/h/g fr.wt.) in the leaf pairs o f Mesembryanthemum crystaUinum at 3 intervals following treatment with 300 mM NaCI. At 37 days, open circles represent the activity o f the PEP carboxylase in the 3 leaves which had developed during salt treatment on a branch originating in the axil o f the 3rd leaf o f the main shoot. Filled circles represent the activity in the leaves of the main shoot. Leaves were numbered so that the first formed leaf pair was given number 1.
..~ E
so01
o
300
2
ol
0 days
12days
5
11o
E
10C
8 K E
3
4
6
3
4
5
N 6
37 days
3
4
5
6
7
{eaf pair number
o~ Fig. 2. A m o u n t o f overnight malate consumption or accumulation in the different leaves of Mesembryanthemum crystallinum during a dark period o f 12 h at 18°C at 3 different intervals following treatment with 300 mM NaCI. Leaves were numbered so that the first formed leaf pair was given number 1.
344 was found in middle leaf pairs; activity was lower in both younger and more mature leaves (Fig. 1). With the exception of the youngest leaf pair PEP carboxylase activity increased significantly during a 12~tay treatment with 300 mM NaC1. No significant activation was observed in the youngest leaf pair; the subapical leaf pair exhibited intermediate activation. The differences in PEP carboxylase activity between different leaves of a Mesembryanthemum plant at the beginning of the salt treatment became more and more accentuated with proceeding treatment (Fig.l). After 37 days on salt a further leaf pair had developed; this exhibited the lowest PEP carboxylase activity. The 6th leaf pair had matured. This process was associated with an increase in the activity of the PEP carboxylase. A similar response was shown by the 5th leaf pair where activity also increased with maturation. During the 37 days of salt treatment, branches grew from the axils of the 3rd and 4th leaf pairs. These branches carried small leaves which were more succulent than the leaves of the main shoot. Even in the leaves of these branches, which developed under salt treatment, the activity of PEP carboxylase increased with increasing leaf age (Fig. 1). After a salt treatment of 37 days the activity in the youngest leaves of both branch and main shoot were identical, and not significantly higher than in the youngest leaf prior to salination. Since the branch grew faster than the main shoot the oldest leaf of the branch must be compared with the 6th leaf of the main shoot; they exhibited comparable activities. At the beginning of the salt treatment none of the leaves was able to form and store malate during the night, but instead showed a slight consumption. During salt treatment the ability to accumulate malate at night developed only in the mature leaves or during maturation. The top leaf pair did not show any accumulation of malate even after 37 days of salt treatment (Fig. 2). DISCUSSION There are two significant aspects of the data reported here: (1) Jones [5] has provided good evidence that different leaves of a CAM plant can have different daily cycles of leaf resistances. For mature leaves lower resistances were recorded at night and higher resistances at the day. The opposite was found with young leaves. Since leaf resistance reflects stomatal opening this means that mature leaves open their stomata at night and young leaves at the day. The CO2 concentration in the substomatal spaces was assumed to control nocturnal opening of stomata in CAM plants [8]. Different internal CO2 concentrations should be the result of different capacities for dark CO2 fixation. Since the level of PEP carboxyiase is responsible for the ability to fix CO2 during the night, our findings, that the activity of the PEP carboxylase increases with increasing leaf age provides enzymatic support for the hypothesis. This means that NaCl-treated, CAM~xhibiting Mesembryanthemum plants behave like other well known CAM plants which do not need a special treatment for the development of a CAM [5,6].
345
(2) Two modes of regulation of the CO2 fixation pathway must be distinguished. One process is the NaCl-promoted change from C3 to CAM. This change can be controlled by environmental conditions and might be an adaptive reaction. We have outlined that this change is not carried out by the entire plant but is restricted to mature leaves. Young leaves will not develop CAM features unless they have reached a certain stage of development. The second process concerns the change from C3-photosynthesis to CAM during ageing. This process is not limited to the adaptive phase in Mesembryanthemum; it continues even in totally adjusted plants, indicating that ageing phenomena are involved. Carbon pathway changes have been reported for the C4 plant Portulaca oleracea [9]. Here the change towards an increased C3-photosynthesis with increasing leaf age is more likely to be an ontogenetic response than an environmentally controlled process. Similar conclusions had been drawn for the shift from C3 to CAM in Bryophyllum fedtschenhoi and Bryophyllum daigremontianum [5,6]. Our results agree with this concept. Regardless of the duration of the salt treatment the youngest leaves possess the lowest activity of PEP carboxylase and show no accumulation of malate. PEP carboxylase activity and malate accumulation only increase during maturation. The view that the change from C3 to CAM in M. crystallinum involves a function of age and is not solely an environmentally controlled process is supported by the finding that the appearence of CAM in leaves of the branches which have developed during salt treatment depends on leaf age. On the other hand, mature leaves of very old non-saline Mesembryanthemum plants exhibit only a weak CAM. This might be interpreted as a NaCl-enhanced ageing [10,11]. The common suggestion that an increased succulence associated with NaC1 treatment or cell expansion is responsible for the increased CAM [12,13] does not seem to be correct, since it has been demonstrated that increased succulence is not a prerequisite for malate accumulation in M. crystallinum [14]. In earlier reports [2,13] it has been shown that within 12 to 14 days leaves take up all the NaC1 needed for osmotic and salt adjustment. After this period no significant increase in the ion content of the leaves took place. On the other hand, content of Na ÷ and C1- depends on leaf age increasing with increasing leaf age. Since NaCI is known to be a strong inhibitor of the PEP carboxylase [3,15] one would expect that if NaC1 interacts with PEP carboxylase in the intact leaf or in the crude extract that with increasing leaf age the activity of the PEP carboxylase should decrease. The opposite was found. Thus if there was an inhibition of the enzyme it must be reversed when the crude extract was diluted 100-fold in the enzyme assay. ACKNOWLEDGEMENT This research was supported by the Deutsche Forschungsgemeinschaft. We thank Dr. J.A. Raven and Dr. J. Sprent (University of Dundee) for critical advice in preparing this manuscript.
346 REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
K. Winter and D.J. yon Wfllert, Z. Pflanzenphysiol., 67 (1972) 166. D.J. yon Willert, Z. Pflanzenphysiol., 72 (1974) 64. S.P. Treichel, G.O. Kirst and D.J. yon Willert, Z. Pflanzenphysiol., 71 (1974) 437. S. Treichel, Plant Sci. Lett., 4 (1975) 141. M.B. Jones, Plants (Berl.), 123 (1975) 91. J.C. Lerman, E. Deleens, A. Nato and A. Moyse, Plant Physiol., 53 (1974) 581. M. Kluge, Plants (Berl.), 80 (1968) 859. T.F. Neal~, Aust. J. Biol. Sci., 26 (1973) 705. R.A. Kennedy and W.M. Laetach, Plants (Berl.), 115 (1973) 113. D.J. yon Willert and D. Kramer, Planta (Berl.), 107 (1972) 227. J.T. Prkco and J.W. O'Leary, Physiol. Plant., 27 (1972) 95. M. Kluge, O.L. Lange, M.V. Eichman and R. Schmidt, Plants (Berl.), 112 (1973) 357. K. Winter, Plants, 109 (19"/2) 135. D.J. yon Willert, Plant Sci. Lett., 4 (1975) 225. C.B. Osmond and H. Greenway, Plant Physiol., 49 (1972) 260.