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Seasonal Variation of Phosphoenolpyruvate Carboxylase Specific Activity in Fifteen Species Exhibiting Facultative or Obligate Crassulacean Acid Metabolism
J. PlantPhysiol. Vol. 138. pp. 581-586{1991}
Seasonal Variation of Phosphoenolpyruvate Carboxylase Specific Activity in Fifteen Species Exhibiting Facultative or Obligate Crassulacean Acid Metabolism ELIZABETH
A.
H. PILON-SMITS, HENK 'T HART,
and JAN
VAN BREDERODE
Department of Botanical Ecology and Evolutionary Biology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands Received March 4,1991 . Accepted May 14, 1991
Summary
For 16 months the specific activity of the CAM key-enzyme PEP carboxylase (PEP-C) was determined monthly in twelve Sedum species previously shown to exhibit facultative or obligate CAM, as well as in Aeonium castello-paivae, Sempervivum nevadense and Kalanchoe daigremontiana. In all species examined the PEP-C activity showed a seasonal fluctuation with maxima during the summer from 8- to 30-fold higher than winter values. For most species the seasonal fluctuation correlated significantly with temperature and, to a lesser extent, with irradiation; no correlation was present with precipitation in garden-grown plants. Plants grown in greenhouse and garden showed corresponding seasonal fluctuations. Specific PEP-C activity, however, was restored to the summer level upon transfer to a growth chamber (21115 °C, LD) in October. To determine the controlling climatic factor for this growth room-induced increase in PEP-C activity, the influence of light intensity, photoperiod and temperature on PEP-C activity was investigated in Sedum rupestre. Daytime temperature appeared to be the most influential factor, whereas light intensity had a small but significant effect.
Key words: Sedum, Crassulacean acid metabolism, phosphoenolpyruvate carboxylase, seasonal fluctuation. Abbreviations: CAM = Crassulacean acid metabolism; PEP-C = phosphoenolpyruvate carboxylase.
Introduction
Crassulacean acid metabolism (CAM) is a physiological adaptation to arid environments, exhibited mostly by succulents and epiphytes (Kluge and Ting, 1978). The presence of CAM has been reported in at least twenty-five different families (Teeri, 1982; Winter, 1985) and is generally assumed to be of polyphyletic origin. CAM is a very variable characteristic: in addition to species showing obligate CAM, in many species the expression of CAM is highly dependent on environmental conditions such as water supply (Hoefner et al., 1987; Lee & Griffiths, 1987; Pilon-Smits et a!., 1990) and photoperiod (Brulfert et a!., 1982). A large variation in CAM has been reported for the genus Sedum (Knopf and Kluge, 1979; Muller and Kluge, 1983; © 1991 by Gustav Fischer Verlag, Stuttgart
Pilon-Smits et a!., 1991). As we reported earlier, obligate CAM is exhibited by Mexican species, whereas European species show more or less facultative CAM, which is waterstatus dependent (Pilon-Smits et a!., 1991). An additional factor, however, influences CAM-activity in Sedum. We observed earlier that the specific PEP-C activity is higher in summer than in winter. Moreover, the carbon isotope ratios shifted several promilles between May and July (Pilon-Smits et a!., 1991). A similar seasonal variation in PEP-C activity was reported for Portulaca ria afra (Guralnick and Ting, 1988). Also, Teeri et a!. (1986) reported a seasonal shift in oI3C value in well-watered plants of Sedum rubrotinctum. To study this season-dependent CAM-activity we followed the fluctuation in specific PEP-C activity for 16 months in twelve Sedum species (known to exhibit obligate or faculta-
582
ELIZABETH A. H. PILON-SMITS, HENK 'T HART, and JAN VAN BREDERODE
tive CAM), as well as in species from the genera Aeonium, Sempervivum and Kalanchoe (showing obligate CAM). The observed fluctuations in PEP-C activity were compared with those of several climatic factors. Growth room experiments were performed to investigate the influence of light intensity, photoperiod and temperature.
Materials and methods Plant material
A group of twelve Sedum species (exhibiting different CAM-activities) was selected, as well as representatives of three other genera. The species are listed in Table 1. Growth conditions
During 16 months, two plants of each species were grown in the greenhouse and, in the case of cold-resistant species, another pair in the garden under natural conditions. In the greenhouse the plants were well-watered and grown under natural light and temperature with a minimum temperature of 16°C. At the beginning of the last month (October) one of the two plants from each species was transferred from the greenhouse to a growth-chamber. Here plants were grown at 26klux, long days (16/8) and 21/15 dc. Branch samples were taken at the end of every month, in the afternoon. Light intensity
For light intensity experiments, 6-week old plants of Sedum ru· pestre were transferred from the greenhouse to the growth chamber. After an acclimatization period of 3 weeks at low LI (3 klux) they were subjected to four different LI-conditions: 3, 13.5, 24 and 33 klux. In each condition they were either well-watered or waterstressed. From each subgroup duplicate branch samples were taken in the afternoon, during 3 weeks. Photoperiod and temperature
For experiments concerning photoperiod and temperature three plants of S. rupestre were grown under long (16/8) or short days (8/16). Temperature was lowered, respectively, from 21/15°C to IS/10°C to 10/10 °C with 7-day intervals. The plants were wellwatered and grown at 3 klux. Biochemical methods
PEP-C extraction and assay were performed as described earlier (Pilon-Smits et al., 1990). Protein assay was carried out according to Bradford (1976).
Results Seasonal fluctuation in specific PEP·C activity To study seasonal fluctuations in CAM, specific PEP-C activity was determined monthly from July 1989 to November 1990. The fifteen species used were previously shown to vary in CAM activity, as judged from oI3C values and water status dependent PEP-C activity (Pilon-Smits et al., 1991). The species used are listed in Table 1. The eight cold-resistant species were grown both in the greenhouse and in the
Table 1: List of species used in this study. CAM status based on carbon isotope ratio and water-status dependent PEP-C activity (PilonSmits et aI., 1991). Sedum Sect. Americana Frod. dendroideum Mo~ et Sesse nussbaumerianum Bitter
Origin
CAM status
Mexico Mexico
obligate obligate
Sect. Sedum acre L. album L. anglicum Huds. forsterianum Sm. fusiforme Lowe hirsutum All. nudum Aiton rupestre L. sediforme Oacq.) Pau tymphaeum Quezel and Contandr.
Hungary Greece France Spain Madeira Portugal Madeira Italy Portugal Greece
C3/facultative facultative C3lfacultative facultative facult/ obligate facultative N.D. facultative facultative facult/ obligate
Sect. Africana Frod. meyeri-johannis Engler
Kenya
facultative
Aeonium castello-paivae Bolle
Canary lsI.
obligate
Sempervivum nevadense Wale
Spain
obligate
Kalanchoe daigremontiana Hamet
cultivar
obligate
garden. At the beginning of the last month (October) one plant of each species was transferred from the greenhouse to the growth chamber. As shown in Fig. 1 all species show a similar seasonal fluctuation in specific PEP-C activity, due to changes in PEP-C activity (protein concentrations varied only slightly (factor < 2), tending to decrease with age). Plants of the same species grown in greenhouse and garden show remarkably similar results: even the monthly differences correspond. The figures for seasonal fluctuation of a species in greenhouse and garden show an average correlation of 0.85 ± 0.01 (n = 8). To determine the extent of external influences the specific PEP-C activity throughout the year was compared with different climatic factors. Specific PEP-C activity in all species (both in greenhouse and garden) is correlated at the 5 % level - or better - with temperature, as well as with irradiation in all but five cases. No correlation was found between specific PEP-C activity and precipitation in garden-grown plants. Obviously, the climatic factors temperature and irradiation are correlated with each other as well. By comparing partial correlations, however, the most influential factor can be appointed for most species. In seven species temperature clearly shows the highest partial correlation; these are Sedum acre, S. album, S. anglicum, S. forsterianum, S. nussbaumerianum, S. sediforme and Sempervivum nevadense. In four species both factors have equal partial correlations: S. dendroideum, S. meyeri-johannis, S. tymphaeum and Kalanchoe
Seasonal fluctuation in PEP-C activity
daigremontiana. In only three species specific PEP-C fluctuation correlates clearly better with irradiation. These are Sedum /usi/orme, S. nudum and Aeonium castello-paivae, all originating from the Macaronesian islands. Only the plants of Sedum hirsutum show different results in greenhouse and garden. Under greenhouse conditions irradiation appears to be more influential, whereas irradiation and temperature seem to be equally important in the garden. Influence 0/ light intensity, photoperiod and temperature When plants were transferred from the greenhouse to the growth chamber at the end of October, the initially low specific PEP-C activity was restored to its summer level within a month (Fig. 1). Apparently, the seasonal fluctuation is controlled by an external factor that varies in the same way in greenhouse and garden, and is optimal in these growth chamber conditions. Possible factors are photoperiod, light intensity and temperature. To investigate the influence of light intensity, Sedum rupestre plants were grown at four different light intensities, and under each condition either kept wet or completely dry for 3 weeks. Results are shown in Fig. 2. Under wet conditions the specific PEP-C activity remained at a low level. Plants grown at high light intensity (U) showed a small but significant (P
Variation
583
0/ PEP-C fluctuation between species
The amplitude of fluctuation in specific PEP-C activity varies between the species examined. Summer-winter differences (as obtained by the ratio of specific PEP-C activities in J~ly and December) range from 8-fold in species showing oblIgate CAM to 30-fold in species exhibiting facultative CAM. This seasonal variation appears to be correlated significantly (P < 0.1 %) with water-status dependent variation in specific PEP-C activity (as expressed by the ratio of PEP-C activities in drought stressed and well-watered plants, Pilon-Smits et al., 1991).
Discussion The results reported in this paper clearly show that in these temperate climate conditions specific PEP-C activity fluctuates seasonally in all species examined. The seasonal fluctuations in PEP-C activity are most probably due to differences in the production of PEP-C protein instead of activation of pre-existing PEP-C, as immunoblot experiments have shown previously in the case of drought-induced increase in PEP-C activity (Pilon-Smits et al., 1990). Specific PEP-C activity has been shown previously to be highly correlated with other CAM-criteria, namely oI3Cvalue and leaf thickness (Pilon-Smits et al., 1991). Consequently, we consider it to be a reasonably reliable indication of CAM-activity. The species investigated are a representative cross-section of all possible CAM variation. Therefore, it could well be possible that all CAM-species exhibit seasonal fluctuations in PEP-C activity - and possibly CAM activity - in temperate climates. The amplitude of seasonal variation in specific PEP-C activity of a species is highly correlated with its water-status depe.ndent variation. Both characteristics are probably reflectlons of the photosynthetic plasticity of the species. Apparently, even species known to exhibit obligate CAM are susceptible to environmental stimuli, since they show a seasonal fluctuation in specific PEP-C activity under these circumstances. Their yearly fluctuations are not as large as in
Spec PEP-C act. (mU/mg prot.) 2000 Spec . PEp·C act (mU/mg prot) _
Green house
~Iongday _
(21 C)
o~----L---~----~----~----~----~
o
6
10
13
17
21
time (d)
Fig. 2: Effect of light intensity and drought on specific PEP-C activity in Sedum rupestre. 0- - - - -0: 3 klux; • - - - --. : 13.5 klux; 6- - - - -6: 24 klux; .- - - - -.: 33 klux. Solid lines: dry; dotted lines: wet.
(15 C)
shortday
(10C)
Fig. 3: Influence of photoperiod and temperature on specific PEP-C activity in Sedum rupestre. Triplicate plants were grown at either short or long days, at day/night temperatures of, respectively, 21 / 15, 15/10 and 10/10°C (temperatures were lowered with 7-day intervals). black bar: short day (8/ 16); light-grey bar: long day (16/ 8); dark-grey bar: greenhouse (± 8/16, 16°C).
584
EUZABETH
A.
H . PILON-SMITS, HENK 'T HART,
and JAN
VAN B REDERODE
Spec. PEp·C activity (mUlmg prot.)
Spec. PEP-C a ctivity (mU/mg prot.) 2500
SOlA 700
B
*
2000
600 500
1500
400 1000
300 200
500
100
July'S9
Oct.
Jan '90
Apr.
July
Oct
July 'S9
5pec. PEP-C activit y (mU/mg prot.) 2000
Oct.
Ja n 9' 0
Apr .
July
Oct
Spec. PEP-C act ivity (m U/mg prot.)
C
D
3000 2500
*
1500 2000 1000
1500 1000
500 500 ..... 0 - -
0 July 'S9
Oct.
Jan. '90
Apr.
July
Oct.
Ju ly' S9
Oct.
Jan . 9' 0
Apr.
Ju ly
Oct.
Oct.
Jan. '90
Apr.
July
Oct.
Spec. PEP-Cact ivity (mU/mg prot.) 1000
E
3000 2500
SOO
2000 600 1500 400
1000
200
500
July'S9
Oct,
Jan. '90
Apr.
Ju ly
Oct.
July '8 9
Spec. PE P-C activity (mU/mg prot.)
Spec. PEP-C act ivity (mU/m g prot.)
G
2500
1000
*
2000
800
1500
600
1000
400
500
200
H ,,
,,
,,
,
'<,
,
*
*
'.,
,
q,
,, ,0 _____
0 Ju ly '89
Oct.
Jan. '90
Apr .
July
Oct,
July '89
Oct.
Jan. ' 90
Apr.
Ju ly
Oct.
Spec. PEP-C act iv ity (m U/mg prot .)
Spec. PEP-C activity (mU/ mg prot.) 1500
2000
1200
1500
900 1000 600 500
300
0 July '89
Oct.
Jan. '90
Apr.
Jul y
Oct.
July '89
Oct.
Jan . '90
Apr.
Ju ly
Oct.
Fig. 1: Seasonal course of specific PEP-carboxylase activity (A-O) and weather conditions (P-R). Solid lines: greenhouse; dotted lines: garden; stars: growth chamber (after 1 month). A: S.acre, B: S. sediforme, C: Sedum meyerijohannis, D: S. album, E: S. anglicum, F: S.dendroi· deum, G: S. nussbaumerianum, H: S. tymphaeum, I: S. nudum, J: S. Jusiforme, K: S. Jorsterianum, L: S. hirsutum, M: Sempervivum nevadense, N: Aeonium castello·paivae, 0: Kalanchoe daigremontiana, P: precipitation, Q: temperature, R: irradiance. Garden-grown plants of E, H, K, and L died in June, 1990.
species showing facultative CAM though. This phenomenon was briefly mentioned earlier (Muller and Kluge, 1983). The difference between obligate and facultative CAM becomes less clear when obligate CAM behaves like facultative in
temperate regions. Conversely, facultative CAM may behave like obligate in extreme climates, since latent CAM can readily be enhanced by drought or growth room conditions in facultative species in wintertime.
586
ELIZABETH A. H. PILON-SMITS, HENK 'T HART, and JAN VAN BREDERODE
Multiple correlation of seasonal variation in PEP-C activity with different climatic factors indicated that in most species temperature is the most important factor. An exception, however, is formed by the species from Madeira and the Canary islands. In their case irradiation is the most influential factor. Perhaps the constant temperature in their natural habitat makes it an unsuitable factor to adapt seasonal processes to. From the growth room experiments it appears that in Sedum rupestre the most important external stimulus for the seasonal fluctuation in PEP-C activity is daytime temperature. High light intensity also results in a small but significant (P < 0.1 %) increase in specific PEP-C activity. Since the effect of light intensity is less than 2-fold in 3 weeks, whereas the growth-room induced rise of PEP-C activity in October involves a 5-fold average increase in specific PEP-C activity within a month, it seems unlikely that light intensity is a major factor, although it may have an additional effect. Whereas in S. rupestre drought alone is sufficient to enhance PEP-C activity, Borland and Griffiths (1990) reported enhancement of CAM in Sedum telephium only by a combination of drought and high PAR (photosynthetically active radiation). Photoperiod does not appear to influence the induction process in Sedum rupestre. In earlier experiments we observed no difference in specific PEP-C activity at photoperiods of either 12/12 or (LD)16/8 (Pilon-Smits et aI., 1990). A similar independence of photoperiod was reported for Sedum telephium (Lee and Griffiths, 1987) and Sedum spectabile (Brulfert et aI., 1988). In the latter the amplitude of CAM was somewhat enhanced by long days. Milller and Kluge (1983), on the other hand, reported photoperiod(LD)-dependency of CAM in Sedum sieboldii, S. spectabile, S. acre, S. telephium and S. coeruleum. A temperature shift from 21/15 °C to IS/10°C resulted in a rapid decrease in specific PEP-C activity, comparable with transfer to the greenhouse (16 0e). The critical daytime temperature, therefore, appears to lie between 16 and 21°C for Sedum rupestre. The mechanism of this temperature effect is not clear. Low night temperatures ( < 10 °C) are known to inhibit malate transport and other enzyme activities (Kluge and Ting, 1978). Perhaps additional temperature experiments will reveal more about the conditions needed for CAM-enhancement. Acknowledgements The investigations were supponed by the Netherlands Foundation for Biological Research (B.I.O.N.), with financial aid from the Netherlands Organization for the Advancement of Research (N.W.O.).
References BORLANDS, A. M. and H. GRIFFITHS: The regulation of CAM and respiratory recycling by water supply and light regime in the C3CAM intermediate Sedum telephium. Funct. Ecol. 4, 33 - 39 (1990). BRADFORD, M. M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72,248-254 (1976). BRULFERT, J., M. KLUGE, S. GU<;:LU, and o. QUEIROZ: Photoperiodism and CAM III: Different characteristics of the photoperiod-sensitive and non-sensitive isoforms of phosphoenolpyruvate carboxylase and CAM operation. Planta 154, 339-343 (1982). - - - - Combined effects of drought, daylength and photoperiod on rapid shifts in the photosynthetic pathways of Sedum spectabile, a CAM species. Plant Physiol. Biochem. 26, 7 -16 (1988). GURALNICK, L. J. and 1. P. TING: Seasonal patterns of water relations and enzyme activity of the facultative CAM plant Portulacaria afra (L.)Jacq. Plant Cell Environ. 11, 811-818 (1988). HOEFNER, R., L. VAZQUEZ-MoRENO, K. WINTER, H. J. BOHNERT, and J. M. SCHMITT: Induction of Crassulacean Acid Metabolism in Mesembryanthemum crystallinum by high salinity: mass increase and de novo synthesis of PEP-carboxylase. Plant Physiol. 83, 915-919 (1987). KLUGE, M. and 1. P. TING: Crassulacean Acid Metabolism. Springer Verlag, Berlin (1978). KNOPF, O. and M. KLUGE: Propenies of phosphoenolpyruvate carboxylase in Sedum species in relation to Crassulacean Acid Metabolism. Plant Cell Environ. 2, 73 -78 (1979). LEE, H. S. J. and H. GRIFFITHS: Induction and repression of CAM in Sedum telephium L. in response to photoperiod and water stress. J. Exp. Bot. 38,834-841 (1987). MULLER, D. and M. KLUGE: Immunological evidence for a crassulacean acid metabolism specific phosphoenolpyruvate carboxylase in Sedum and Kalanchoe species. Physiol. Veg. 21, 919-926 (1983). PILON-SMITS, E. A. H., H. 'T fuRT, andJ. VAN BREDERODE: Phosphoenolpyruvate carboxylase in Sedum rupestre (Crassulaceae): drought enhanced expression and purification. J. Plant Physiol. 136, 155-160 (1990). PILON-SMITS, E. A. H., H. 'T HART, J. A. N. MEESTERBURRIE, P . . NABER, R. KREULER, and J. VAN BREDERODE: Variation in Crassulacean acid metabolism within the genus Sedum: Carbon isotope composition and water status dependent phosphoenolpyruvate carboxylase activity. J. Plant Physiol. 137, 342 -347 (1991). TEERI, J. A.: Carbon isotopes and the evolution of C4 photosynthesis and crassulacean acid metabolism. In: NITACKI, M. H. (ed.): Biochemical Aspects of Evolutionary Biology, pp. 93 -130. Chicago (1982). TEERI, J. A., M. TURNER, andJ. GUREVITCH: The response of leaf water potential and Crassulacean Acid Metabolism to prolonged drought in Sedum rubrotinctum. Plant Physiol. 81, 678-680 (1986). WINTER, K.: Crassulacean Acid Metabolism. In: BARBER,J. and N. R. BAKER (eds.): Photosynthetic mechanisms and the environment, pp. 329-387. Elsevier (1985).
Seasonal Variation of Phosphoenolpyruvate Carboxylase Specific Activity in Fifteen Species Exhibiting Facultative or Obligate Crassulacean Acid Metabolism ELIZABETH
A.
H. PILON-SMITS, HENK 'T HART,
and JAN
VAN BREDERODE
Department of Botanical Ecology and Evolutionary Biology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands Received March 4,1991 . Accepted May 14, 1991
Summary
For 16 months the specific activity of the CAM key-enzyme PEP carboxylase (PEP-C) was determined monthly in twelve Sedum species previously shown to exhibit facultative or obligate CAM, as well as in Aeonium castello-paivae, Sempervivum nevadense and Kalanchoe daigremontiana. In all species examined the PEP-C activity showed a seasonal fluctuation with maxima during the summer from 8- to 30-fold higher than winter values. For most species the seasonal fluctuation correlated significantly with temperature and, to a lesser extent, with irradiation; no correlation was present with precipitation in garden-grown plants. Plants grown in greenhouse and garden showed corresponding seasonal fluctuations. Specific PEP-C activity, however, was restored to the summer level upon transfer to a growth chamber (21115 °C, LD) in October. To determine the controlling climatic factor for this growth room-induced increase in PEP-C activity, the influence of light intensity, photoperiod and temperature on PEP-C activity was investigated in Sedum rupestre. Daytime temperature appeared to be the most influential factor, whereas light intensity had a small but significant effect.
Key words: Sedum, Crassulacean acid metabolism, phosphoenolpyruvate carboxylase, seasonal fluctuation. Abbreviations: CAM = Crassulacean acid metabolism; PEP-C = phosphoenolpyruvate carboxylase.
Introduction
Crassulacean acid metabolism (CAM) is a physiological adaptation to arid environments, exhibited mostly by succulents and epiphytes (Kluge and Ting, 1978). The presence of CAM has been reported in at least twenty-five different families (Teeri, 1982; Winter, 1985) and is generally assumed to be of polyphyletic origin. CAM is a very variable characteristic: in addition to species showing obligate CAM, in many species the expression of CAM is highly dependent on environmental conditions such as water supply (Hoefner et al., 1987; Lee & Griffiths, 1987; Pilon-Smits et a!., 1990) and photoperiod (Brulfert et a!., 1982). A large variation in CAM has been reported for the genus Sedum (Knopf and Kluge, 1979; Muller and Kluge, 1983; © 1991 by Gustav Fischer Verlag, Stuttgart
Pilon-Smits et a!., 1991). As we reported earlier, obligate CAM is exhibited by Mexican species, whereas European species show more or less facultative CAM, which is waterstatus dependent (Pilon-Smits et a!., 1991). An additional factor, however, influences CAM-activity in Sedum. We observed earlier that the specific PEP-C activity is higher in summer than in winter. Moreover, the carbon isotope ratios shifted several promilles between May and July (Pilon-Smits et a!., 1991). A similar seasonal variation in PEP-C activity was reported for Portulaca ria afra (Guralnick and Ting, 1988). Also, Teeri et a!. (1986) reported a seasonal shift in oI3C value in well-watered plants of Sedum rubrotinctum. To study this season-dependent CAM-activity we followed the fluctuation in specific PEP-C activity for 16 months in twelve Sedum species (known to exhibit obligate or faculta-
582
ELIZABETH A. H. PILON-SMITS, HENK 'T HART, and JAN VAN BREDERODE
tive CAM), as well as in species from the genera Aeonium, Sempervivum and Kalanchoe (showing obligate CAM). The observed fluctuations in PEP-C activity were compared with those of several climatic factors. Growth room experiments were performed to investigate the influence of light intensity, photoperiod and temperature.
Materials and methods Plant material
A group of twelve Sedum species (exhibiting different CAM-activities) was selected, as well as representatives of three other genera. The species are listed in Table 1. Growth conditions
During 16 months, two plants of each species were grown in the greenhouse and, in the case of cold-resistant species, another pair in the garden under natural conditions. In the greenhouse the plants were well-watered and grown under natural light and temperature with a minimum temperature of 16°C. At the beginning of the last month (October) one of the two plants from each species was transferred from the greenhouse to a growth-chamber. Here plants were grown at 26klux, long days (16/8) and 21/15 dc. Branch samples were taken at the end of every month, in the afternoon. Light intensity
For light intensity experiments, 6-week old plants of Sedum ru· pestre were transferred from the greenhouse to the growth chamber. After an acclimatization period of 3 weeks at low LI (3 klux) they were subjected to four different LI-conditions: 3, 13.5, 24 and 33 klux. In each condition they were either well-watered or waterstressed. From each subgroup duplicate branch samples were taken in the afternoon, during 3 weeks. Photoperiod and temperature
For experiments concerning photoperiod and temperature three plants of S. rupestre were grown under long (16/8) or short days (8/16). Temperature was lowered, respectively, from 21/15°C to IS/10°C to 10/10 °C with 7-day intervals. The plants were wellwatered and grown at 3 klux. Biochemical methods
PEP-C extraction and assay were performed as described earlier (Pilon-Smits et al., 1990). Protein assay was carried out according to Bradford (1976).
Results Seasonal fluctuation in specific PEP·C activity To study seasonal fluctuations in CAM, specific PEP-C activity was determined monthly from July 1989 to November 1990. The fifteen species used were previously shown to vary in CAM activity, as judged from oI3C values and water status dependent PEP-C activity (Pilon-Smits et al., 1991). The species used are listed in Table 1. The eight cold-resistant species were grown both in the greenhouse and in the
Table 1: List of species used in this study. CAM status based on carbon isotope ratio and water-status dependent PEP-C activity (PilonSmits et aI., 1991). Sedum Sect. Americana Frod. dendroideum Mo~ et Sesse nussbaumerianum Bitter
Origin
CAM status
Mexico Mexico
obligate obligate
Sect. Sedum acre L. album L. anglicum Huds. forsterianum Sm. fusiforme Lowe hirsutum All. nudum Aiton rupestre L. sediforme Oacq.) Pau tymphaeum Quezel and Contandr.
Hungary Greece France Spain Madeira Portugal Madeira Italy Portugal Greece
C3/facultative facultative C3lfacultative facultative facult/ obligate facultative N.D. facultative facultative facult/ obligate
Sect. Africana Frod. meyeri-johannis Engler
Kenya
facultative
Aeonium castello-paivae Bolle
Canary lsI.
obligate
Sempervivum nevadense Wale
Spain
obligate
Kalanchoe daigremontiana Hamet
cultivar
obligate
garden. At the beginning of the last month (October) one plant of each species was transferred from the greenhouse to the growth chamber. As shown in Fig. 1 all species show a similar seasonal fluctuation in specific PEP-C activity, due to changes in PEP-C activity (protein concentrations varied only slightly (factor < 2), tending to decrease with age). Plants of the same species grown in greenhouse and garden show remarkably similar results: even the monthly differences correspond. The figures for seasonal fluctuation of a species in greenhouse and garden show an average correlation of 0.85 ± 0.01 (n = 8). To determine the extent of external influences the specific PEP-C activity throughout the year was compared with different climatic factors. Specific PEP-C activity in all species (both in greenhouse and garden) is correlated at the 5 % level - or better - with temperature, as well as with irradiation in all but five cases. No correlation was found between specific PEP-C activity and precipitation in garden-grown plants. Obviously, the climatic factors temperature and irradiation are correlated with each other as well. By comparing partial correlations, however, the most influential factor can be appointed for most species. In seven species temperature clearly shows the highest partial correlation; these are Sedum acre, S. album, S. anglicum, S. forsterianum, S. nussbaumerianum, S. sediforme and Sempervivum nevadense. In four species both factors have equal partial correlations: S. dendroideum, S. meyeri-johannis, S. tymphaeum and Kalanchoe
Seasonal fluctuation in PEP-C activity
daigremontiana. In only three species specific PEP-C fluctuation correlates clearly better with irradiation. These are Sedum /usi/orme, S. nudum and Aeonium castello-paivae, all originating from the Macaronesian islands. Only the plants of Sedum hirsutum show different results in greenhouse and garden. Under greenhouse conditions irradiation appears to be more influential, whereas irradiation and temperature seem to be equally important in the garden. Influence 0/ light intensity, photoperiod and temperature When plants were transferred from the greenhouse to the growth chamber at the end of October, the initially low specific PEP-C activity was restored to its summer level within a month (Fig. 1). Apparently, the seasonal fluctuation is controlled by an external factor that varies in the same way in greenhouse and garden, and is optimal in these growth chamber conditions. Possible factors are photoperiod, light intensity and temperature. To investigate the influence of light intensity, Sedum rupestre plants were grown at four different light intensities, and under each condition either kept wet or completely dry for 3 weeks. Results are shown in Fig. 2. Under wet conditions the specific PEP-C activity remained at a low level. Plants grown at high light intensity (U) showed a small but significant (P
Variation
583
0/ PEP-C fluctuation between species
The amplitude of fluctuation in specific PEP-C activity varies between the species examined. Summer-winter differences (as obtained by the ratio of specific PEP-C activities in J~ly and December) range from 8-fold in species showing oblIgate CAM to 30-fold in species exhibiting facultative CAM. This seasonal variation appears to be correlated significantly (P < 0.1 %) with water-status dependent variation in specific PEP-C activity (as expressed by the ratio of PEP-C activities in drought stressed and well-watered plants, Pilon-Smits et al., 1991).
Discussion The results reported in this paper clearly show that in these temperate climate conditions specific PEP-C activity fluctuates seasonally in all species examined. The seasonal fluctuations in PEP-C activity are most probably due to differences in the production of PEP-C protein instead of activation of pre-existing PEP-C, as immunoblot experiments have shown previously in the case of drought-induced increase in PEP-C activity (Pilon-Smits et al., 1990). Specific PEP-C activity has been shown previously to be highly correlated with other CAM-criteria, namely oI3Cvalue and leaf thickness (Pilon-Smits et al., 1991). Consequently, we consider it to be a reasonably reliable indication of CAM-activity. The species investigated are a representative cross-section of all possible CAM variation. Therefore, it could well be possible that all CAM-species exhibit seasonal fluctuations in PEP-C activity - and possibly CAM activity - in temperate climates. The amplitude of seasonal variation in specific PEP-C activity of a species is highly correlated with its water-status depe.ndent variation. Both characteristics are probably reflectlons of the photosynthetic plasticity of the species. Apparently, even species known to exhibit obligate CAM are susceptible to environmental stimuli, since they show a seasonal fluctuation in specific PEP-C activity under these circumstances. Their yearly fluctuations are not as large as in
Spec PEP-C act. (mU/mg prot.) 2000 Spec . PEp·C act (mU/mg prot) _
Green house
~Iongday _
(21 C)
o~----L---~----~----~----~----~
o
6
10
13
17
21
time (d)
Fig. 2: Effect of light intensity and drought on specific PEP-C activity in Sedum rupestre. 0- - - - -0: 3 klux; • - - - --. : 13.5 klux; 6- - - - -6: 24 klux; .- - - - -.: 33 klux. Solid lines: dry; dotted lines: wet.
(15 C)
shortday
(10C)
Fig. 3: Influence of photoperiod and temperature on specific PEP-C activity in Sedum rupestre. Triplicate plants were grown at either short or long days, at day/night temperatures of, respectively, 21 / 15, 15/10 and 10/10°C (temperatures were lowered with 7-day intervals). black bar: short day (8/ 16); light-grey bar: long day (16/ 8); dark-grey bar: greenhouse (± 8/16, 16°C).
584
EUZABETH
A.
H . PILON-SMITS, HENK 'T HART,
and JAN
VAN B REDERODE
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Fig. 1: Seasonal course of specific PEP-carboxylase activity (A-O) and weather conditions (P-R). Solid lines: greenhouse; dotted lines: garden; stars: growth chamber (after 1 month). A: S.acre, B: S. sediforme, C: Sedum meyerijohannis, D: S. album, E: S. anglicum, F: S.dendroi· deum, G: S. nussbaumerianum, H: S. tymphaeum, I: S. nudum, J: S. Jusiforme, K: S. Jorsterianum, L: S. hirsutum, M: Sempervivum nevadense, N: Aeonium castello·paivae, 0: Kalanchoe daigremontiana, P: precipitation, Q: temperature, R: irradiance. Garden-grown plants of E, H, K, and L died in June, 1990.
species showing facultative CAM though. This phenomenon was briefly mentioned earlier (Muller and Kluge, 1983). The difference between obligate and facultative CAM becomes less clear when obligate CAM behaves like facultative in
temperate regions. Conversely, facultative CAM may behave like obligate in extreme climates, since latent CAM can readily be enhanced by drought or growth room conditions in facultative species in wintertime.
586
ELIZABETH A. H. PILON-SMITS, HENK 'T HART, and JAN VAN BREDERODE
Multiple correlation of seasonal variation in PEP-C activity with different climatic factors indicated that in most species temperature is the most important factor. An exception, however, is formed by the species from Madeira and the Canary islands. In their case irradiation is the most influential factor. Perhaps the constant temperature in their natural habitat makes it an unsuitable factor to adapt seasonal processes to. From the growth room experiments it appears that in Sedum rupestre the most important external stimulus for the seasonal fluctuation in PEP-C activity is daytime temperature. High light intensity also results in a small but significant (P < 0.1 %) increase in specific PEP-C activity. Since the effect of light intensity is less than 2-fold in 3 weeks, whereas the growth-room induced rise of PEP-C activity in October involves a 5-fold average increase in specific PEP-C activity within a month, it seems unlikely that light intensity is a major factor, although it may have an additional effect. Whereas in S. rupestre drought alone is sufficient to enhance PEP-C activity, Borland and Griffiths (1990) reported enhancement of CAM in Sedum telephium only by a combination of drought and high PAR (photosynthetically active radiation). Photoperiod does not appear to influence the induction process in Sedum rupestre. In earlier experiments we observed no difference in specific PEP-C activity at photoperiods of either 12/12 or (LD)16/8 (Pilon-Smits et aI., 1990). A similar independence of photoperiod was reported for Sedum telephium (Lee and Griffiths, 1987) and Sedum spectabile (Brulfert et aI., 1988). In the latter the amplitude of CAM was somewhat enhanced by long days. Milller and Kluge (1983), on the other hand, reported photoperiod(LD)-dependency of CAM in Sedum sieboldii, S. spectabile, S. acre, S. telephium and S. coeruleum. A temperature shift from 21/15 °C to IS/10°C resulted in a rapid decrease in specific PEP-C activity, comparable with transfer to the greenhouse (16 0e). The critical daytime temperature, therefore, appears to lie between 16 and 21°C for Sedum rupestre. The mechanism of this temperature effect is not clear. Low night temperatures ( < 10 °C) are known to inhibit malate transport and other enzyme activities (Kluge and Ting, 1978). Perhaps additional temperature experiments will reveal more about the conditions needed for CAM-enhancement. Acknowledgements The investigations were supponed by the Netherlands Foundation for Biological Research (B.I.O.N.), with financial aid from the Netherlands Organization for the Advancement of Research (N.W.O.).
References BORLANDS, A. M. and H. GRIFFITHS: The regulation of CAM and respiratory recycling by water supply and light regime in the C3CAM intermediate Sedum telephium. Funct. Ecol. 4, 33 - 39 (1990). BRADFORD, M. M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72,248-254 (1976). BRULFERT, J., M. KLUGE, S. GU<;:LU, and o. QUEIROZ: Photoperiodism and CAM III: Different characteristics of the photoperiod-sensitive and non-sensitive isoforms of phosphoenolpyruvate carboxylase and CAM operation. Planta 154, 339-343 (1982). - - - - Combined effects of drought, daylength and photoperiod on rapid shifts in the photosynthetic pathways of Sedum spectabile, a CAM species. Plant Physiol. Biochem. 26, 7 -16 (1988). GURALNICK, L. J. and 1. P. TING: Seasonal patterns of water relations and enzyme activity of the facultative CAM plant Portulacaria afra (L.)Jacq. Plant Cell Environ. 11, 811-818 (1988). HOEFNER, R., L. VAZQUEZ-MoRENO, K. WINTER, H. J. BOHNERT, and J. M. SCHMITT: Induction of Crassulacean Acid Metabolism in Mesembryanthemum crystallinum by high salinity: mass increase and de novo synthesis of PEP-carboxylase. Plant Physiol. 83, 915-919 (1987). KLUGE, M. and 1. P. TING: Crassulacean Acid Metabolism. Springer Verlag, Berlin (1978). KNOPF, O. and M. KLUGE: Propenies of phosphoenolpyruvate carboxylase in Sedum species in relation to Crassulacean Acid Metabolism. Plant Cell Environ. 2, 73 -78 (1979). LEE, H. S. J. and H. GRIFFITHS: Induction and repression of CAM in Sedum telephium L. in response to photoperiod and water stress. J. Exp. Bot. 38,834-841 (1987). MULLER, D. and M. KLUGE: Immunological evidence for a crassulacean acid metabolism specific phosphoenolpyruvate carboxylase in Sedum and Kalanchoe species. Physiol. Veg. 21, 919-926 (1983). PILON-SMITS, E. A. H., H. 'T fuRT, andJ. VAN BREDERODE: Phosphoenolpyruvate carboxylase in Sedum rupestre (Crassulaceae): drought enhanced expression and purification. J. Plant Physiol. 136, 155-160 (1990). PILON-SMITS, E. A. H., H. 'T HART, J. A. N. MEESTERBURRIE, P . . NABER, R. KREULER, and J. VAN BREDERODE: Variation in Crassulacean acid metabolism within the genus Sedum: Carbon isotope composition and water status dependent phosphoenolpyruvate carboxylase activity. J. Plant Physiol. 137, 342 -347 (1991). TEERI, J. A.: Carbon isotopes and the evolution of C4 photosynthesis and crassulacean acid metabolism. In: NITACKI, M. H. (ed.): Biochemical Aspects of Evolutionary Biology, pp. 93 -130. Chicago (1982). TEERI, J. A., M. TURNER, andJ. GUREVITCH: The response of leaf water potential and Crassulacean Acid Metabolism to prolonged drought in Sedum rubrotinctum. Plant Physiol. 81, 678-680 (1986). WINTER, K.: Crassulacean Acid Metabolism. In: BARBER,J. and N. R. BAKER (eds.): Photosynthetic mechanisms and the environment, pp. 329-387. Elsevier (1985).