Annals of Botany 93: 629-652, 2004
© 2004 Annals of Botany Company
Ecophysiology of Crassulacean Acid Metabolism (CAM)
1 Institute of Botany, Technical University of Darmstadt, Schnittspahnstrasse 3–5, D-64287 Darmstadt, Germany
*For correspondence. E-mail: luettge{at}bio.tu-darmstadt.de
Received: 3 October 2003; Returned for revision: 17 December 2003; Accepted: 20 January 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONINPUTRECEIVERS: PLANT TYPESOUTPUTCONCLUSIONSLITERATURE CITED |
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• Background and Scope Crassulacean Acid Metabolism (CAM) as an ecophysiological modification of photosynthetic carbon acquisition has been reviewed extensively before. Cell biology, enzymology and the flow of carbon along various pathways and through various cellular compartments have been well documented and discussed. The present attempt at reviewing CAM once again tries to use a different approach, considering a wide range of inputs, receivers and outputs.
• Input Input is given by a network of environmental parameters. Six major ones, CO2, H2O, light, temperature, nutrients and salinity, are considered in detail, which allows discussion of the effects of these factors, and combinations thereof, at the individual plant level (‘physiological aut-ecology’).
• Receivers Receivers of the environmental cues are the plant types genotypes and phenotypes, the latter including morphotypes and physiotypes. CAM genotypes largely remain ‘black boxes’, and research endeavours of genomics, producing mutants and following molecular phylogeny, are just beginning. There is no special development of CAM morphotypes except for a strong tendency for leaf or stem succulence with large cells with big vacuoles and often, but not always, special water storage tissues. Various CAM physiotypes with differing degrees of CAM expression are well characterized.
• Output Output is the shaping of habitats, ecosystems and communities by CAM. A number of systems are briefly surveyed, namely aquatic systems, deserts, salinas, savannas, restingas, various types of forests, inselbergs and paramós.
• Conclusions While quantitative census data for CAM diversity and biomass are largely missing, intuition suggests that the larger CAM domains are those systems which are governed by a network of interacting stress factors requiring versatile responses and not systems where a single stress factor strongly prevails. CAM is noted to be a strategy for variable, flexible and plastic niche occupation rather than lush productivity. ‘Physiological syn-ecology’ reveals that phenotypic plasticity constitutes the ecophysiological advantage of CAM.
Key words: CAM, ecophysiology.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONINPUTRECEIVERS: PLANT TYPESOUTPUTCONCLUSIONSLITERATURE CITED |
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The first comprehensive review of Crassulacean Acid Metabolism (CAM) was published in 1960 (Wolf, 1960). Subsequently, CAM research has developed continuously and extensively, and has been reviewed widely in articles and books (Kluge and Ting, 1978; Osmond, 1978; Kluge, 1979; Queiroz, 1979; Cockburn, 1985; Ting, 1985; Winter, 1985; Lüttge, 1987, 1989a, 1993, 1998, 2002b, 2003a; Griffiths, 1988a; Winter and Smith, 1996a; Cushman and Bohnert, 1997, 1999; Cushman, 2001; Cushman and Borland, 2002; Dodd et al., 2002; Functional Plant Biology, 2002). This covers aspects of CAM ranging from ecosystems and ecology, physiology and metabolic pathways, cell biology, and transport and compartmentation to molecular biology.
The simplest definition of CAM, first described for species of the family Crassulaceae, is that there is (1) nocturnal uptake of CO2 via open stomata, fixation by phosphoenolpyruvate carboxylase (PEPC) and vacuolar storage of CO2 in the form of organic acids, mainly malic acid (phase I sensu Osmond, 1978), and (2) daytime remobilization of vacuolar organic acids, decarboxylation and refixation plus assimilation of CO2 behind closed stomata in the Calvin-cycle (phase III). Between these two phases there are transitions when stomata remain open for CO2 uptake for a short time during the very early light period (phase II) and reopen again during the late light period for CO2 uptake with direct assimilation to carbohydrate when vacuolar organic acid is exhausted (phase IV).
Phases II and IV respond very sensitively to environmental input parameters. However, versatility is greater than flexibility of expression of CAM phases. This characteristic has affected CAM terminology and definitions. Cockburn (1985) has made several fine distinctions, which are interesting as they distinguish between the performance of plants and plant organs with and without stomata, and also between terrestrial and aquatic plants. The following two distinctions are most important. First, CAM idling (Sipes and Ting, 1985), where stomata remain closed day and night and the day/night organic acid cycle is fed by internal recycling of nocturnally refixed respiratory CO2. Much has been written on this phenomenon of CAM in response to severe stress due to limitations of water availability (Griffiths, 1988b, 1989; Griffiths et al., 1989). Secondly, CAM cycling (Sipes and Ting, 1985), where stomata remain closed during the dark period but some nocturnal synthesis of organic acid fed by respiratory CO2 occurs, and where stomata are open during the light period with uptake of atmospheric CO2 and direct Calvin-cycle CO2 reduction (C3-photosynthesis) in addition to assimilation of CO2 remobilized from nocturnally stored organic acid.
CAM idling is considered as a form of very strong CAM, while CAM cycling is weak CAM. In the epiphytic Gesneriaceae Codonanthe crassifolia, Guralnick et al. (1986) observed CAM cycling in well-watered plants and CAM idling in drought-stressed plants. CAM cycling that scavenges respiratory CO2 appears to be a sort of prelude to ‘real’ CAM, and this work, as well as studies on the Portulacaceae (Guralnick and Jackson, 2001), suggests that it might have been a starting point for CAM evolution. Thus, the various forms of weak and strong CAM may be restricted to different individual species (Guralnick and Jackson, 2001), which then constitute different CAM physiotypes. However, they may also be expressed temporarily in one given species. For example, Sedum telephium has the potential to exhibit pure C3 characteristics when well-watered and a transition to CAM when droughted, including a continuum of different stages of CAM expression which are repeatedly reversible under changing drought and watering regimes (Lee and Griffiths, 1987).
Cockburn (1998) speculates that cycling of CO2 via malate into the Calvin cycle may also occur at higher than diurnal frequencies, and this ‘rapid-cycling CAM’ will be limited to daytime. However, this is difficult to distinguish from one-cell C4 photosynthesis that does not require the different cell types of mesophyll and bundle-sheath (Magnin et al., 1997; Freitag and Stichler, 2000, 2002; Akhani et al., 2003) and also from the function of stomatal guard cells that, in Cockburn’s view, are also close to performing CAM. Stomatal guard cells use malate synthesis by PEPC and vacuolar storage of potassium malate as osmoticum in turgor-driven opening movements and remobilize the malate during the closing movements. Photosynthetic carbon flow in some brown algae, where malate serves as an intermediate store of fixed CO2, has similarities to both CAM and C4 photosynthesis (Raven et al., 1985; Schmid and Dring, 1996; Keeley, 1996; Schmid et al., 1996). In higher plants, during nitrate reduction in the leaves, malic acid is synthesized by PEPC where the protons of the acid are used to neutralize the hydroxyl ions produced by nitrate reduction, and the malate anion is stored as its potassium salt in the vacuole during the day and remobilized again during the night (Winter et al., 1982a; Gerhardt and Heldt, 1984; see Lüttge and Clarkson, 1987). Thus, there are observed both acid fluctuations without malate fluctuations and malate fluctuations without acid fluctuations, neither of which could be called CAM. However, the case for CAM in some submerged freshwater plants is intriguing. In these plants, CO2 for dark fixation by PEPC is not taken up by the leaves but is supplied via the roots, protons of malic acid synthesized are exchanged for K+, and there are CAM-type malate oscillations without acid oscillations (Raven et al., 1988; Cockburn, 1998).
Hence, from all the above, it is apparent that a coherent and comprehensive definition of CAM is not straightforward (Holtum, 2002). The high versatility of CAM and CAM-like behaviour alluded to is certainly also related to the fact that there is nothing specific in the enzymatic complement of CAM. Although there are certainly CAM-specific isoforms of some of the enzymes involved, there are basically no CAM-typical enzymes (Lüttge, 1998, 2003a).
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INPUT |
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TOPABSTRACTINTRODUCTIONINPUTRECEIVERS: PLANT TYPESOUTPUTCONCLUSIONSLITERATURE CITED |
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Environmental parameter network
In ecophysiology principally we need to consider any possible environmental input to the organisms. Even if we only take the six environmental parameters which have been recognized as most important for CAM and which are studied most intensely, i.e. CO2, water, light, temperature, salinity and nutrients, and summarize their most prominent interactions we already arrive at a complex network. This is depicted in Figure 1, which represents a closed network where all factors are directly or indirectly connected and feeding back on each other. The major connections are listed in Table 1. This network structure should be kept in mind as I assess the ecophysiological impacts of the individual factors one by one and highlight some of their interactions.
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Carbon dioxide (CO2)
Generally, water is considered to be the most important factor and CAM to be an adaptation to water-shortage stress because transpirational water loss is minimized by CO2 acquisition via open stomata during the dark period and CO2 assimilation behind closed stomata during the light period. However, CAM is also observed in submerged freshwater plants (Keeley, 1996), where CO2 fixation via PEPC with its high affinity to its HCO3– substrate and in the absence of competition from C3-photosynthetic organisms during the dark period sustains an internal CO2-concentrating mechanism (Griffiths, 1989; Lüttge, 2002b). As well as some angiosperms, such submerged plants also include species of Isoëtes, a much more basic taxon of vascular plants’ phylogeny. CO2 may, therefore, be considered as the central factor and most important driving force for the earliest evolution of CAM (Griffiths, 1989). Thus, it might be assumed that early CAM evolution in Isoëtes occurred during geological times when atmospheric CO2 concentration, paCO2, was low. Indeed, the early evolution of the Isoëtales during the Permian, about 250 x 106 years ago, coincided well with a time of decreasing paCO2 during the Phanerozoic (see Berner, 1994; Laws et al., 2002). However, among more basic taxa, CAM is also frequently found in terrestrial, i.e. epiphytic and lithophytic, ferns (Ong et al., 1986; Winter et al., 1986; Holtum and Winter, 1999; Sayed, 2001). With its multifactor responses, and the fact that no special enzymes are required and a well-managed general enzyme complement suffices, CAM clearly evolved polyphyletically many times in the plant kingdom (see Lüttge, 2003b). Various facets of possible driving forces for the evolution of CAM have been considered by Raven and Spicer (1996).
When considering CO2, it is important to distinguish between atmospheric partial pressure, paCO2, the environmental factor sensu stricto, and internal partial pressure, piCO2, which are closely related to each other via stomatal opening and closing.
Above all else, CAM is a CO2-concentrating mechanism (for reviews see Griffiths, 1989; Lüttge, 2002b). This is due to the much higher substrate affinity of PEPC for HCO3– than of the C3-photosynthesis/Calvin cycle carboxylase Rubisco(ribulose-bis-phosphate carboxylase/oxgenase) for CO2. Thus, during the dark period a concentrated CO2 pool is built up in the form of vacuolar malic acid accumulation, and during phase III its remobilization in the light leads to internal CO2 concentrations that may be 2–60 times paCO2 (Table 2). For aquatic plants, this CO2-concentrating mechanism provides a benefit for CO2 acquisition. For terrestrial plants, the benefit of CO2-concentrating by CAM is considered to be related to water use, and will be discussed below.
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If internal CO2-concentrating of CAM is a benefit at low paCO2, it might be expected that the current man-made increase in paCO2 attenuates this advantage of CAM. However, most studies performed so far indicate a growth stimulation of CAM plants by elevated CO2 concentrations (Drennan and Nobel, 2000). Stimulation is related to phytohormone action (Li et al., 2002). In an orchid plantlet obtained from tissue culture, 1 % CO2 stimulated growth although PEPC and Rubisco levels were reduced (Gouk et al., 1997). Increased paCO2 compensated for the inhibitory effect of increased temperature on nocturnal CO2 fixation (Zhu et al., 1999). An induction of CAM in the C3/CAM-intermediate species Portulacaria afra was not mediated by alteration of CO2 availability (Huerta and Ting, 1988). A doubling of paCO2 from 370 to 750 p.p.m. had no significant effects in the CAM plant Agave vilmoriniana (Szarek et al., 1987). Doubling paCO2 increased the productivity of Opuntia ficus-indica by 35 % on average, mostly as a result of increases in both night-time and daytime CO2 uptake, while in Kalanchoë pinnata increased paCO2 mainly increased phase IV CO2 uptake (Winter et al., 1997). The responses of CAM plants to increased paCO2 are similar to those predicted for C3 plants, but much greater than those for C4 plants. There appears to be no expectation of downward acclimation (Wang and Nobel, 1996). Of course, the time scales of these observations are much too short to make any predictions on future changes. Overall, on a percentage basis effects are very small and in detail there are many uncertainties (Poorter and Navas, 2003). Currently, with the time-scales of ecosystem responses given, ecologically noticeable changes in habitat occupation by CAM plants may not be expected.
The consequences of elevated piCO2, i.e. CO2-concentrating, have been reviewed recently (Lüttge, 2002b). They mainly pertain to photorespiration, photoinhibition and oxidative stress. Earlier suggestions that these are minimized by CO2 concentrating in the internal air spaces of CAM organs in phase III to the extent shown in Table 2 are based on the expectation that Rubisco begins to become substrate saturated above 0·1 % CO2 (Berry and Downton, 1982). Thus, high piCO2 would suppress the oxygenase activity of Rubisco and hence photorespiration in the CO2/O2 substrate competition at Rubisco. The high photochemical work of saturated CO2 assimilation would prevent over-energization of the photosynthetic apparatus, and hence suppress photoinhibition and oxidative stress. However, observations contradict these expectations. Photorespiration, photoinhibition and oxidative stress in the light occur in CAM plants not only in phase IV, when they operate like C3 plants, but also in phase III with highly elevated piCO2. Substrate-saturated photosynthetic CO2 reduction behind closed stomata leads to elevated internal oxygen concentrations (Table 2), e.g. up to approx. 40 % in the CAM plant Kalanchoë gastonis-bonnieri (Spalding et al., 1979). This high piO2, as a consequence of high piCO2, supports the formation of reactive oxygen species. In fact, CAM plants have developed very effective antioxidative response systems (Castillo, 1996; Miszalski et al., 1998; Broetto et al., 2002).
Another function of piCO2 is signalling, and of particular ecophysiological relevance is regulation of stomatal opening. Increasing piCO2 affects stomatal closure. In general, the CO2 response sensitivity of guard cells is very variable between species and also for a given species due to acclimation (Frechilla et al., 2002). There appear to be no data on the critical level of piCO2 needed to close stomata in phase III of CAM. Apparently, stomata of CAM plants have a similar CO2 sensitivity to those of C3 plants, particularly in the range of 0–36 Pa, and a half-saturation constant of 19·6 Pa was observed for both C3 and CAM plants (Jewer et al., 1985). The internal CO2 concentrations in phase III are usually much higher and generally it is agreed that they are the most relevant cause of stomatal closure during daytime in this phase. Another interesting effect of piCO2-signalling is synchronization of the photosynthetic activities of individual cells or cell patches in leaves of CAM plants, e.g. during free-running circadian oscillations of CAM (Rascher et al., 2001; Rascher and Lüttge, 2002; Lüttge, 2002b).
Water (H2O)
For terrestrial plants, the greatest benefit of CAM is considered to be increased water use efficiency (WUE) because stomatal opening during the dark period causes much less transpirational loss of water than opening during the light period. Indeed, the WUE of mol CO2 fixed : mol H2O transpired during night-time CO2 acquisition in CAM is estimated as about 6–30 x 10–3. Conversely, C3 plants have only 0·6–1·3 x 10–3 and C4 plants 1·7–2·4 x 10–3; but for phase IV of CAM, WUEs are similarly low, i.e. 1–4 x 10–3 (Black, 1973). Thus, WUE is related to CAM phases. That this is ecologically important is also seen in the fact that daytime phases II and IV are suppressed under the influence of drought stress, both in the laboratory (Smith and Lüttge, 1985: Lüttge, 1987) and in the field (Lee et al., 1989).
With this high WUE CAM plants might typically be expected to inhabit arid habitats. However, although CAM plants, such as cacti, agaves and euphorbs, often determine the physiognomy of deserts, CAM species inhabiting tropical rainforests far outnumber typical desert species (Table 3). Many of these species are epiphytic and subject to the particular problems of water supply in this habitat (Zotz and Hietz, 2001). It is estimated that approx. 57 % of all epiphytes are CAM plants. Therefore, high WUE might be the major ecophysiological trait of CAM and the foremost driving force of CAM evolution (Gil, 1986). However, Eller and Ferrari (1997) found that the C3 plant Othonna opima (Asteraceae) and the CAM plant Cotyledon orbiculata (Crassulaceae) with morphologically very similar succulent life-forms showed similar WUE under the same environmental conditions. Moreover, Pierce et al (2002) have even unravelled competitive abilities of CAM bromeliads of the genus Aechmea in an extremely wet tropical cloud forest. During the wet season, when rainfall, mist and wetting of leaf surfaces inhibit gas exchange, total CO2 uptake is superior in the CAM bromeliads compared with that of C3 bromeliads, where gas exchange is limited to the light period. During the dry season, the water-saving properties are to the advantage of CAM bromeliads. It is in particular the flexibility of CAM in contrast to C3 photosynthesis that supports the observed recent radiation of this photosynthetic pathway into cloud forests (Pierce et al., 2002). Based on comparative morphology of roots, of tank formation and of solute absorbing epidermal scales, Smith (1989) concluded that the common ancestor of extant epiphytic bromeliads must have been a pre-adapted, drought-tolerant terrestrial C3 type and that epiphytism and CAM evolved several times and independent of each other in the Bromeliaceae.
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In addition to CAM phase-dependent stomatal responses affecting WUE, CAM plants have other structural and functional ways of dealing with the water factor that are involved in short-, medium- and long-term storage of water.
Short-term effects are due to diurnal cycles of osmotic relations. The large vacuolar concentrations of nocturnally accumulated organic acids are osmotically active. The increased osmotic pressure (
) drives water uptake into the cells, which is associated with increased turgor pressure (P). This allows CAM plants extra acquisition of water, particularly towards the end of the dark period when vacuolar organic acid levels become rather high. It may be a particular advantage in moist, tropical forests with dew formation occurring mainly during the late dark period. During acid remobilization in phase III, P and decline again but the water gained is available to the plants (Lüttge, 1986; Eller and Ruess, 1986; Ruess et al., 1988; Eller et al., 1992; Murphy and Smith, 1998).
Special features of water transport across membranes are also involved in the cellular water storage of CAM plant cells. Ohshima et al. (2001) reported that the content of aquaporins, and hence water permeability, in the plasmalemma and tonoplast of leaf cells in some CAM plants is very low. Aquaporins have also been studied in the annual C3/CAM intermediate Mesembryanthemum crystallinum (Yamada et al., 1995; Kirch et al., 2000; Yamada and Bohnert, 2000), where a decrease in hydraulic conductivity of cell membranes has been shown upon CAM induction (Rygol et al., 1989; Trofimova et al., 2003).
Medium-term water storage occurs where CAM plants form external water reservoirs (phytotelmata), especially the tanks typical of many bromeliads. This allows, for example, water obtained from small occasional rainfalls, which can occur even during dry periods in seasonal precipitation regimes, to be stored for several days (Lee et al., 1989).
More long-term water storage is possible due to special non-green and non-photosynthetical water storage tissues, i.e. hydrenchymas. These may be peripheral tissues such as the large epidermal bladders of the annual C3/CAM intermediate species M. crystallinum (Haberlandt, 1904) and the epidermal and subepidermal layers of hydrenchyma cells of leaves of Peperomia and bromeliads (Gibeaut and Thomson, 1989; Lee et al., 1989; Horres and Zizka, 1995). Central water storage tissues are typical of leaf-succulent agaves and many stem-succulent CAM taxa.
The biophysical basis of such water storage and water remobilization as required under stress, i.e. cellular water transport, membrane hydraulic conductivities (Lp) and osmotic relations (, P and the cell wall elastic modulus, 
), have been studied extensively in M. crystallinum (Steudle et al., 1975, 1977) and in Agave deserti (Smith and Nobel, 1986, Smith et al., 1987). Under extended rainless periods, water can be remobilized from the water storage tissues to protect metabolically active tissue for many weeks (Schulte and Nobel, 1989). Water may also be transported from older to younger leaves (Donatz and Eller, 1993; Tüffers et al., 1995). In desert cacti and agaves, the reliance on internal water reserves may be coupled to CAM idling, so that there is no water loss by stomatal transpiration and only water loss by cuticular transpiration needs to be compensated for (Szarek and Ting, 1975; Holthe and Szarek, 1985; Lüttge et al., 1989). However, when the plants lose more than 50 % of their total water they die. Thus, regular seasonal rains are required to refill the reserves (Holthe and Szarek, 1985). For Peperomia magnoliaefolia this has been specified for different tissues. Entire leaves can lose up to 50 % of their total water, the water storing hydrenchyma can lose 75–85 %, but the photosynthetically active chlorenchyma suffers when it loses just 15–25 % (Schmidt and Kaiser, 1987).
The performance of the peripheral water storage tissue of epidermal bladders of M. crystallinum in relation with the diurnal osmotic oscillations given by malate accumulation and remobilization in the mesophyll has been analysed by Rygol et al. (1986, 1987, 1989) following diurnal courses of and P and hence water potential (
= P – ) in bladder and mesophyll cells. There was a clear water potential gradient between these two tissues at midday, constituting a driving force for transport of water from the bladders to the mesophyll cells, demonstrating the protective function of the water storage tissue at critical times of stress (see Lüttge, 2002a). Water movement and diel cycles of internal water distribution between water-storage hydrenchyma and photosynthetic chlorenchyma has also been demonstrated for A. deserti, Ferocactus acanthodes (Tissue et al., 1991) and Opuntia ficus-indica (Goldstein et al., 1991).
Water storing hydrenchymas are not green, and generally are considered not to participate in metabolic functions of CAM. Bladder cells of M. crystallinum have no function in this respect (Winter and Lüttge, 1976; Winter et al., 1981). Hydrenchyma of Crassula falcata might contribute to nocturnal CO2 fixation (Springer and Outlaw, 1988).
CAM also occurs in some resurrection plants that are desiccation-tolerant and can shift between biosis and anabiosis as they dry out and are rewatered, respectively. The resurrection plants Haberla rhodopensis and Ramonda serbica (Gesneriaceae) perform various transitions between C3 photosynthesis, CAM cycling and CAM idling as they dry out. CAM idling appears to be important during the first days after rewatering (Markovska et al., 1997; Markovska 1999).
Light (h
)
Light has two important functions in CAM. First, it acts as the energy source of photosynthesis and second, it affects expression and performance of CAM via signalling systems.
Light and photosynthesis
Intensity of photosynthetically active radiation (PAR, or photosynthetic photon flux density, PPFD), during the day (phase III) determines the rate of organic acid mobilization from the vacuole (Kluge, 1968; Barrow and Cockburn, 1982; Thomas et al., 1987). The rate-limiting step in this process may be the directly light-dependent assimilation of CO2 via Rubisco in the chloroplasts, malate decarboxylation in the cytosol, or malate efflux from the vacuole (Lüttge, 2002b). The high piCO2 built up in phase III is evidentally important in the regulation of the process, where a central role is played by carbonic anhydrase mediating the pH-dependent HCO3–/CO2 equilibria in the cytosol and chloroplast stroma, and the supply of the proper substrate CO2 to Rubisco. To date, this enzyme has been unduly neglected in CAM research (Tsuzuki et al., 1982; Holtum et al., 1984; Raven and Spicer, 1996; Lüttge, 2002b). PPFD during the light period determines the degree of nocturnal organic acid accumulation during the subsequent dark period, i.e. the amplitude of the day/night oscillations of CAM, because light-driven photosynthesis and gluconeogenesis fill the carbohydrate stores required for PEP synthesis via glycolysis during the dark period as a precursor for phase I CO2 fixation by PEPC (Nobel and Hartsock, 1983).
The energy requirement for carbon flow of the CAM cycle is higher than in C3 photosynthesis. Estimates of stoichiometries of ATP : NADPH : CO2 are 3 : 2 : 1 for C3 photosynthesis and 4·8 : 3·2 : 1 for CAM with malate oscillations, and up to 5·9 : 3·9 : 1 for CAM with malate plus citrate oscillations (Winter and Smith, 1996b). It may be asked then, if CAM can get under energy limitation. This may be the case for epiphytic CAM plants in very moist cloud forests during the rainy season (Pierce et al., 2002). However, this may be the exception, as generally PPFD in CAM plant habitats is not limiting (Lüttge, 2002b).
Conversely, there may be surplus irradiance and over-energization of the photosynthetic apparatus in CAM. This is normally observed in C3 plants at sun-exposed sites and elicits the various mechanisms of non-photochemical energy dissipation and photo-protective, as well as photo-destructive, photoinhibition. With the strong interactions of some input factors (Fig. 1) these processes are not governed by irradiance alone. Although in Kalanchoë daigremontiana water stress per se did not affect primary photochemical activity, e.g. potential quantum yield of photosystem II when leaves were darkened overnight, it increased susceptibility to photoinhibitory light stress during the day (Lu et al., 2003). It has often been argued that the high piCO2 built up in phase III of CAM protects plants from over-energization during the time of the day when the highest irradiance prevails due to substrate-saturated photochemical work using most of the excitation energy of photosynthesis (Gil, 1986). However, it has already been noted, whilst discussing the factor CO2 above, that this is not the case. For protection, CAM plants possess the entire complement of energy dissipation methods known also from C3 plants, namely photorespiration, radiative energy dissipation via zeaxanthin and the futile xanthophyll cycle of epoxidation and deepoxidation, D1-protein turnover, etc. (reviewed by Lüttge, 2000, 2002b; and more recently by Lu et al., 2003).
Light and signalling
High light intensities may elicit CAM expression in C3/CAM intermediate species, such as Guzmania monostachia (Maxwell et al., 1994, 1995, 1999; Maxwell, 2002) and Clusia minor (where it is under the control of an UV-A/blue light receptor) (Grams and Thiel, 2002).
A signalling function of light related to CAM is obvious in the photoperiod, i.e. long-day dependent induction of CAM in the facultative CAM plant Kalanchoë blossfeldiana ‘Tom Thumb’. Phytochrome, the red-light receptor involved in photoperiodisms, elicits CAM expression (Brulfert et al., 1973, 1975). Long-days also enhance CAM expression in M. crystallinum (Cheng and Edwards, 1991; Guralnick et al., 2001a), where CAM induction is known to be mostly related to salinity and drought stress, and phytochrome is involved (Cockburn et al., 1996). In K. blossfeldiana ‘Tom Thumb’ long-day regimes induce both flowering and a shift from C3 photosynthesis to CAM (Brulfert et al., 1973). Both flowering and CAM in plants are controlled by the ‘biological clock’, where blue light and red light are basically important environmental input factors, and cryptochromes (blue light receptors) and phytochromes are essential elements of input pathways (Lüttge, 2003a, b). To date, it is not clear where the output pathways that produce the overt phenomena of flowering and CAM branch. Other species of Kalanchoë, such as K. daigremontiana and K. tubiflora, although also long-day plants for the induction of flowering, are obligate CAM plants.
In C3/CAM intermediate species, light responses of stomata also change dramatically when CAM is induced. In Portulacaria afra, blue-light and red-light responses of stomata in the C3-state are lost in the CAM-state. Signals such as piCO2, high water-vapour pressure differences between leaves and the atmosphere, high temperature and low water potential are excluded as being inhibitory signals for stomatal opening in response to blue and red light during CAM. The inhibition is also observed in isolated epidermal peels and different signalling must be involved (Lee and Assmann, 1992). The xanthophyll zeaxanthin is probably involved in the signal transduction chain from light to stomatal opening (Zhu et al., 1998). In M. crystallinum after the C3–CAM transition, the opening response of guard cells to blue and white light is lost, together with light-dependent xanthophyll formation (Tallman et al., 1997).
Temperature (T)
The major interactions of the temperature factor determining CAM performance are with individual enzymes, membranes, respiratory activity and stomatal movement. Temperature often may not be decisive per se but acts by modulating the impact of other factors (Kluge and Ting, 1978). Nobel (1996) notes that under conditions of cultivation, temperature is not a major factor affecting the productivity of CAM plants.
The contention that for optimal performance of CAM plants need relatively low night temperatures and high day temperatures goes back to in vitro studies by Brandon (1967) of temperature optima of key enzymes in the metabolic pathway, where the enzymes of nocturnal malate synthesis, PEPC and malate dehydrogenase reach their optimum at 35 °C and the decarboxylating enzymes, e.g. malic enzyme, at above 53 °C. In vitro PEPC enzymology shows that the active phosphorylated form of the enzyme is stabilized at low temperatures (3 °C or less) while higher temperature promotes dephosphorylation (Carter et al., 1995), so that inhibition of the enzyme by allosteric effectors is also lower at low and higher at high temperatures (Buchanan-Bollig and Kluge, 1981; Carter et al., 1995). Thus, from overall performance of the counteracting enzymes of carboxylation and decarboxylation, where lower temperatures favour the former and higher temperatures the latter (Buchanan-Bollig et al., 1984), it was concluded that rather cool night temperatures somewhat below 20 °C would be most favourable for dark fixation in CAM. However, due to the complexity of temperature interactions and the frequent temperature acclimation, considering temperature optima of enzymes in vitro is a simplification. Comparing three growth temperature regimes, Israel and Nobel (1995) found that PEPC and Rubisco had maximal activities at 45/35 °C day/night while total daily CO2 uptake was greater at 30/20 °C and 15/5 °C.
Another target of temperature is the cell membranes, where temperature directly affects fluidity, and hence permeability, which is very important for organic acid compartmentation (Friemert et al., 1988). An inverse relation of diurnal heat tolerance of CAM plants to tissue acid levels is probably related to this, when higher temperatures increase tonoplast permeability and acid efflux from the vacuole and the acid load may exert detrimental effects in the cytosol (Kappen and Lösch, 1984; Lehrum et al., 1987). The tonoplast of CAM plants also shows very pronounced acclimation to growth temperatures. In a process of homeoviscous adaptation in K. daigremontiana, the membrane order or rigidity is much enhanced at growth temperatures of 34/25 °C day/night compared with that of 25/17 °C. This reduces malic acid permeability of the tonoplast and allows controlled malate accumulation/remobilization during the CAM cycle at elevated temperatures. Homeoviscous adaptation is due both to changed lipid composition and to lipid/protein interactions (Kluge et al., 1991b; Kliemchen et al., 1993; Behzadipour et al., 1998).
Compartmentation of malic acid modulated by temperature effects on the tonoplast is also relevant for interactions with respiration and associated acclimation (Medina and Osmond, 1981). Labelling studies with stable carbon isotopes have shown that the flux of malate through mitochondria was approx. 100 % at the beginning, 60 % in the middle and 70–100 % at the end of the dark period. This is related to the CO2 fixation rate, which is highest in the middle of the night when a higher proportion of the malate formed may first pass the mitochondria, and to the electrochemical energy gradient at the tonoplast against which malic acid is accumulated, which is highest at the end of the night (Kalt et al., 1990). In these phytotron experiments, temperature was kept constant throughout the whole night. However, in the field, where temperature gradually decreases during the night towards the early morning, such interactions will be more important.
Temperature effects on air humidity may largely determine relationships between temperature and stomatal opening. Stomata of the CAM plant K. pinnata are highly sensitive to air humidity (Medina, 1982). Maximum rates of dark CO2 fixation were similar at all temperatures between 12 and 25 °C within a given range of leaf/air water vapour pressure differences. However, the onset of nocturnal net dark CO2 fixation and the time to reach a peak rate were delayed as temperature increased, and hence total CO2 uptake and malate accumulation were reduced with increasing temperature during the dark period.
In summary, temperature relations suggest that diurnal temperature changes with lower night-time and higher daytime temperatures are favourable for CAM. In the C3/CAM intermediate Clusia minor, C3 to CAM shifts are strongly enhanced when day/night temperature differences are increased and are less sensitive to absolute temperatures (Haag-Kerwer et al., 1992). Conversely, there is also a large amount of evidence that CAM can be well expressed under constant temperatures (e.g. Lüttge and Beck, 1992). Frequently in the tropics, day and night temperatures are not very different and CAM plants grow well and show lush occupation of such habitats (Kluge and Ting, 1978; Plant, Cell and Environment, 1986).
Nutrients
There is a lot of information on nutrient relationships of epiphytic CAM plants, because mineral nutrition in the epiphytic habitat has been studied extensively. However, problems of mineral nutrient acquisition and particular solutions such as the formation of tanks and other phytotelmata, epiphytic root systems and mycorrhiza and myrmecophytism pertain to epiphytes in general and are not CAM-specific, although CAM plants obviously also make use of these adaptations (Benzing, 1983, 1990; Lüttge, 1989a).
Nutrient levels and element responses have also been studied in CAM desert succulents (Nobel, 1983; Nobel and Berry, 1985). In the chlorenchyma of cultivated cacti and agaves, levels of Ca, Mg and Mn tended to be higher than in most other agronomic plants. The rather high levels of Ca in these plants are noteworthy, and are also observed in many other CAM taxa such as Aloë and Crassulaceae (Crassula, Kalanchoë; Karmarkar and Joshi, 1969; Phillips and Jennings, 1976; Rössner and Popp, 1986; Meyer and Popp, 1997) as well as Clusia (Ball et al., 1991a; Oliavares and Aguiar, 2002), and thus these CAM plants are calcitrophic species. Together with K+, Na+ and Mg2+, Ca2+ serves as a counter-ion for a background pool of carboxylates (of several tens of millimolar in some cases), which does not oscillate diurnally and is thought to help in osmotic stabilization (Phillips, 1980; Smith et al., 1996). With a plethora of ionic dissociation equilibria involved, it is not easy to measure and calculate to what extent calcium is free or bound and sometimes contrasting conclusions are reached (Schomburg, 1994; Meyer and Popp, 1997; Behzadipour, 1999). This is important, because Ca2+ can bind to negatively charged groups of proteins and lipids, and hence decrease membrane fluidity/permeability (Schomburg, 1994; Behzadipour, 1999). Although this might change diurnally during the CAM cycle in relation to changing levels of Ca2+ binding organic acids, and might be involved in the regulation of switches between net acid accumulation and remobilization (Kluge and Schomburg, 1996), so far, there is no unequivocal evidence for such a mechanism (Schomburg, 1994; Meyer and Popp, 1997; Behzadipour, 1999).
The importance of N has been evaluated on theoretical grounds because it may be predicted that CAM plants might need less N than C3 plants, and thus have a higher nitrogen use efficiency (NUE) (Griffiths, 1989; Raven and Spicer, 1996). In C3 plants, Rubisco may account for 50 % or more of the total soluble leaf protein (Björkman et al., 1976; Ku et al., 1979), but CAM plants would need less Rubisco due to their CO2-concentrating mechanism, and hence bind less N in the Rubisco protein. Enzyme analyses in K. pinnata by Winter et al. (1982b) support this expectation. Since it is known that in Kalanchoë species CAM expression is related to leaf age and increases as leaves mature, they analysed activities and amounts of PEPC and Rubisco in leaves of increasing age and CAM expression. Activity and amount of Rubisco decreased while PEPC increased. In leaves of plants that were supplied with nitrate, the amount of enzyme protein related to total soluble protein in young and mature leaves was, respectively, 30 and 17 % for Rubisco and 1 and 10 % for PEPC. Nitrate and phosphate deficiency have positive effects on CAM expression in M. crystallinum (Paul and Cockburn, 1990) and N deficiency has positive effects on CAM performance in K. blossfeldiana (Ota, 1988a). Santos and Salema (1991, 1992) studied mineral nutrition of the facultative CAM species K. lateritia. CAM was best expressed at intermediate N supply.
Conversely, it could be argued that the high levels of piCO2 oversaturating Rubisco in phase III of CAM could possibly make it beneficial for CAM plants to have more of the enzyme. Widmann et al. (1990) found that in K. daigremontiana and K. tubiflora NUE was less than expected for CAM plants. The ability of succulent C3 and CAM species to use N was highly species-specific and varied with age and environmental conditions. CAM plants were no better adapted to N-deficient habitats than C3 plants, which could use limiting N more efficiently by increasing transpiration and exploiting the soil more effectively (Widmann et al., 1993). In the aquatic CAM plant Littorella uniflora, CAM did not increase NUE (Baattrup-Pedersen and Madsen, 1999).
Contrasting observations are also related to NH4+ vs. NO3– preference of CAM plants. Ota (1988b) and Ota et al. (1988) found that in K. blossfeldiana CAM was more pronounced under NO3– than under NH4+ nutrition. In both cases, drought stress increased CAM performance but the discrepancy between NO3– and NH4+ treatments increased. It was suggested that NH4+ depresses PEPC activity and CAM (Ota and Yamamoto, 1991). Conversely, Fernandes et al. (2002) found that when tanks of the terrestrial CAM bromeliad Neoregelia cruenta were supplied with 5 mM NH4NO3 there was a preference for NH4+ uptake over NO3– uptake of more than 11-fold within 24 h. In two other bromeliads, NO3– was found to be a poor N source but was effective together with NH4+ (Nievola et al., 2001). It could be argued that the use of reduced N, including organic N, is a specific adaptation of tank-forming bromeliads where putrefying litter and dead animals produce reduced N and low molecular organic N compounds which may be particularly important in the epiphytic habitat (Endres and Mercier, 2001, 2003). On the other hand, Clusia species fed via their roots also use reduced N (Wanek et al., 2002a) and Arndt et al. (2002) observed that although potentially all N sources supplied to the roots of C. minor could be used, there was a strong preference for NH4+ over NO3– and glycine.
Ecophysiologically it is important to consider the N factor in relation to the action of other factors, especially light and water. Generally, chlorophyll contents, rates of photosynthesis and quantum yield decline under N limitation and this also holds true for some factor combinations in CAM plants, e.g. the bromeliads Bromelia humilis (Fetene et al., 1990) and N. cruenta (Fernandes et al., 2002), and K. pinnata (Lüttge et al., 1991a). With the latter, a multifactor study was performed where plants were grown at high and low irradiance, with and without N- and H2O-deficiency and analysed with and without transfer from low to high and from high to low irradiance (Lüttge et al. 1991a, b). The plants showed highly varying NUE under these different conditions. Sufficient N supply overall had positive effects. It also allowed flexible responses to irradiance. For example, when well-watered and N-supplied shade-grown plants were transferred to high light conditions, total day/night net CO2 uptake was much increased, particularly due to phase IV photosynthesis. Shade plants when well supplied with N were not photoinhibited in high light. Similar observations were made by Fernandes et al. (2002) with N. cruenta where high light and high N leaves behaved similarly to shade plants. In B. humulis, NUE (expressed as mol CO2 taken up per mole leaf N) was reduced by more than 50 % by N deficiency in both high light and low light grown plants (Fetene et al., 1990). Nitrate nutrition at high light also promoted CAM performance in C. minor (Franco et al., 1991).
Overall, there is no clear evidence supporting particularly high NUE in CAM plants. Multifactorial effects and responses in relation to N nutrition of CAM plants obviously show a larger variability than any differences between C3 and CAM plants.
Salinity
One of the major effects of salinity is osmotic stress, and hence there are intimate relationships to drought stress or ‘water stress’. Therefore, considering CAM as a major photosynthetic accommodation to water stress, CAM might be expected to be a prominent trait among halophytes. Moreover, halophytes are often succulent as they sequester NaCl in large central vacuoles, which is called ‘salt succulence’ (Ellenberg, 1981). It might be speculated that this is a good preadaptation for CAM, inasmuch as it is argued that the development of succulent leaf anatomy with large uniform spherical cells, typical of many CAM plants (e.g. Kalanchoës), is a first step towards the evolution of CAM (Guralnick and Jackson, 2001; Guralnick et al., 2001b).
However, observations do not support this expectation as, in general, halophytes are not CAM plants and CAM plants are not halophytes. There are some isolated reports that CAM plants may cope well with mild NaCl stress, e.g. individuals of coastal populations of the cactus Opuntia humifusa (Silverman et al., 1988) and some Macronesian Sempervivioideae (Lösch and Kappen, 1981; Lösch 1990), but generally CAM plants, including desert succulents, are highly salt sensitive (Nobel, 1983; Nobel and Berry, 1985). CAM plants inhabiting highly saline ecosystems, such as coastal and inland salinas, are effectively functional salt excluders at the root level, such as some cacti (Nobel et al., 1984) and, mainly, stress avoiders displaying either morphological root system dynamics in response to periodic stress or complete escape from the saline substratum by retreat to epiphytic niches.
The single conspicuous exception to the above is the annual facultative halophyte and facultative CAM species Mesembryanthemum crystallinum (Lüttge, 1993, 2002a; Adams et al., 1998; Cushman and Bohnert, 2002). This plant can grow well in the absence of NaCl but has its growth optimum at several hundred mM NaCl in the medium and can complete its life cycle at 500 mM NaCl (Winter, 1973; Lüttge, 2002a). It is a species with inducible CAM. It exclusively performs C3 photosynthesis during the early stages of its growth when kept at low salinity. Although it has an inherent developmental programme that gradually shifts its metabolism to CAM, even if continuously kept at low salinity, high salinity rapidly induces CAM in young plants and strongly enhances the age-dependent CAM (Adams et al., 1998; Lüttge, 2002a). Many responses to salinity and CAM induction have been described. At the membrane level, the proton pumping V-ATPase of the tonoplast is enhanced while the proton pumping V-pyrophosphatase is down-regulated. Changes to membrane fluidity suggest molecular modifications of the tonoplast. At the cellular level, there is a change of photosynthetic metabolism based on inductive changes of gene expression at the molecular level with a regulatory network involving phytohormones and secondary messengers and where transcriptional and post-translational regulation also interact. This has been covered frequently in other reviews (Lüttge, 1993; Cushman and Bohnert, 1997, 1999, 2002; Cushman, 2001) so the keynotes given above will suffice here. The inductive responses of gene expression in M. crystallinum also offered the opportunity to identify CAM-specific enzymes (Meyer et al., 1990). Although the enzymatic machinery required for CAM is not basically different from the complement of housekeeping enzymes, the identification of new isoforms of enzymes after the induction of CAM in M. crystallinum helps in the identification of particularly important key enzymes of CAM.
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TOPABSTRACTINTRODUCTIONINPUTRECEIVERS: PLANT TYPESOUTPUTCONCLUSIONSLITERATURE CITED |
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The receivers of environmental inputs are the various types of plants. Basically, we distinguish genotypes and phenotypes. The genotype comprises the complete genetic information of the plant, and thus represents a given constitution. As such it is not affected by environmental input. Due to a genetic developmental programme ontogenetically it generates a certain phenotype, which is always the direct receiver of environmental input. Responses of gene expression to environmental input are due to feedback from the phenotype, e.g. in M. crystallinum where there is a developmental programme with expression of CAM as the plants age but where environmental stresses, such as salinity and drought, accelerate CAM expression in time and enhance the degree of CAM expression.
Genotypes
To date, there is insufficient access to CAM genotypes. With the planned sequencing of M. crystallinum’s genome (Meyer et al., 1990; Adams et al., 1998), it may be possible, at some time, to handle complete genomic information of a C3/CAM intermediate species. Moreover, there are mutants of M. crystallinum, one of which is CAM deficient (J. Cushman, pers. comm.), and this may advance our understanding of the extent to which there is genomic anchorage of the CAM syndrome.
Hence, at the genetic level, we remain restricted to indirect information, as given by molecular analyses of isoenzymes essential for CAM. Genes may differ for isoenzymes within a given plant species, for example PEPC expressed in various plant organs and serving housekeeping functions and CAM-type dark fixation of CO2, respectively (Gehrig et al., 1998). Different genes may encode isoenzymes of C3 and CAM plants, or different isoenzymes before and after induction of CAM in C3/CAM intermediate species (Brulfert et al., 1982, 1985; Brulfert and Queiroz, 1982; Groenhof et al., 1990; Slocombe et al., 1993; Gehrig et al., 1995). Following the evolutionary genetic trend in conservation and diversification, respectively, of key enzymes, e.g. PEPC, is another important approach.
Some information is also being gradually gathered by molecular studies of phylogenetic relationships among CAM taxa. Because of the relationships between genotypes and phenotypes, it is important in ecology to obtain comparative genetic information so that molecular analyses of populations become increasingly essential in ecophysiology. For CAM, research, to date, has been mostly restricted to the family Bromeliaceae and the genera Clusia and Kalanchoë. The research is based on the detection of DNA polymorphisms by DNA amplification from arbitrary short primers (RAPD–PCR fingerprinting) and, more recently, on the study of internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA.
Clusia is a large genus of neotropical species of woody plants and trees. The number of species is estimated to be approx. 300 (Pipoly et al., 1998). The family Clusiaceae and the subfamily Clusioideae with the genus Clusia have a minimum phylogenetic age of 90 x 106 years (Gustafsson et al., 2002). ITS sequencing of nuclear ribosomal DNA has been used by two independent groups to determine the molecular phylogeny of Clusia. Vaasen et al. (2002) studied 17 species and Gehrig et al. (2003) studied 31 species, with 12 species being common to both studies. Clusia is considered as being a monophyletic genus. The two studies both arrived at the conclusion that in the genus CAM developed independently more than once, i.e. the idea that CAM may have evolved only once from a C3 ancestor of Clusia is ruled out. It is now considered incorrect that all Clusia species might have at least a small and weak capacity for CAM, as proposed by Grams et al. (1998). Clusia multiflora, which is currently taken as the ‘model C3 species’ of the genus, is not at the bottom of the molecular phylogenetic trees. In other respects, observations and conclusions of the two research groups are variable. Vaasen et al. (2002) did not find agreement between their molecular data and phylogenetic trees based on morphological studies of flowers, fruits and vegetative parts. However, Gehrig et al. (2003) found close correlations between their cladograms of Panamanian Clusia species and three morphologically defined groups of Central American Clusias. Considering the large variety of CAM physiotypes (with very weak to strong CAM) which Clusia genotypes can generate and the scarcity of bona fide obligate C3 species, one group thinks that CAM is dominant in the genus (Lüttge, 1996, 2000; Vaasen et al., 2002) while the other group appears to consider that ‘some species of Clusia exhibit CAM’ (Gehrig et al., 2003).
The Bromeliaceae comprise approx. 2800 species (Luther and Sieff, 1998). Martin (1994) has identified the photosynthetic pathway of 249 species, of which 69 % show CAM capacity with varying degrees of CAM expression. The family itself is considered monophyletic. Among the three subfamilies, the Bromelioideae are probably monophyletic and the Tillandsioideae possibly so, while the Pitcarnioideae are definitely polyphyletic (Crayn et al., 2000). CAM has evolved polyphyletically several times within each subfamily (Smith, 1989; Crayn et al., 2000). Thus, again it is difficult, if not impossible, to trace a specific CAM genome.
The only obvious case where CAM is related to phylogenetic sections is in the genus Kalanchoë. According to the taxonomy of Boiteau and Allorge-Boiteau (1995) there are three sections, marking an evolutionary trend from Kitchingia to Bryophyllum to Eukalanchoe. Comparative DNA polymorphism and ITS sequencing of nuclear ribosomal DNA confirm these relationships based on morphological characteristics, and the degree of CAM expression follows the same evolutionary trend. First, facultative, drought-inducible but, once induced, extremely flexible CAM is found in all groups of thin-leaved, not very succulent species in Boiteau’s section Kitchingia and in the first two groups of the section Bryophyllum. Second, obligate, but flexible, CAM (especially with respect to making use of daytime phase IV CO2 uptake) is found in the remaining groups of the section Bryophyllum. Third, obligate, uniform CAM is found in the section Eukalanchoe (Gehrig et al., 1997, 2000). Thus, the coincidence between the degree of CAM expression and the infrageneric phylogenetic position of the species suggests that the diversity of CAM pattern in the genus is not due solely to phenotypic flexibility but is largely determined genotypically. Hence, in this genus CAM may indeed have evolved monophyletically from an ancestor with dominating CO2 acquisition by the C3 pathway and only weak nocturnal CO2 fixation gradually advancing to the development of full CAM. The genus is also a good example of how separation of populations and segregation may have driven speciation and evolution. The diversification centre of Kalanchoë is the large island of Madagascar. The centre of adaptive radiation is thought to be located in the humid regions on the eastern side (Boiteau and Allorge-Boiteau, 1995) where CAM does not appear to be of selectional advantage and from where plants have spread into the arid regions in the dry south and from there to arid sites in eastern continental Africa (Gehrig et al., 2001). Currently, this is still reflected by the distribution of species of the three sections with weak, medium and strong CAM at the community level in the different ecosystems of the island.
Phenotypes
For phenotypes, morphotypes and physiotypes can be distinguished. An invaluable checklist of CAM plants documented over the past 25 years with information on morphotypes and physiotypes has been produced by Sayed (2001; see also Smith and Winter, 1996). The morphotypes are structural life forms, as delineated by comparative morphology and anatomy. The physiotypes are physiological life forms as delineated by comparative physiology, biochemistry, biophysics and molecular biology.
Morphotypes.
In physical appearance, there is no typical morphotype of CAM plants. CAM is expressed in a large range of morphologically very different life forms. There is only one morphological/anatomical feature that is common to all CAM plants, namely more or less strongly pronounced succulence. This is often, but not only, due to the formation of large water storage tissues, because nocturnal organic acid accumulation and the associated osmotic adjustments in the photosynthetically active tissue always require large cells with the central vacuole occupying a high proportion (up to 98 %) of the total cell volume (Steudle et al., 1980; Lüttge and Smith, 1984). Such succulence has even been considered as a prerequisite for the evolution of the biochemical CAM cycle (Guralnick and Jackson, 2001; Guralnick et al., 2001b). CAM expression and leaf succulence are often closely correlated (Teeri et al., 1981; Winter et al., 1983; Kluge et al., 2001).
Table 4 gives an overview of CAM plant morphotypes. The data in the table do not aim to be systematic or complete; several of the patterns distinguished overlap, e.g. rosettes are also leaf-succulents, etc. However, it may help to give a general overview (see also table in Sayed, 2001). Simple CAM leaf-succulents, such as Kalanchoë species, are uniformly composed of large spherical cells. Other CAM leaf-succulents have special non-green water storage tissues that can be peripheral, hypodermal or central in the mesophyll. Stem-succulents are characteristic life forms of arid sites and deserts and have central water storage tissues. Many epiphytes are CAM plants, most being either bromeliads or orchids although some belong to other taxa. Epiphytic cacti are normally spineless. Some cacti are liana-like climbers. All Cactaceae perform CAM, but among the three subfamilies, Pereskioideae, Opuntioideae and Ceroideae, the Pereskioideae grow leaves seasonally and leaves may also occur in the Opuntioideae, and those leaves perform C3 photosynthesis while the succulent green cladodes and stems perform CAM (Nobel and Hartsock, 1987). Many CAM plants are rosettes, some with special features like the water-storing phytotelmata of tank-forming bromeliads. Leaves of submerged, freshwater rosette plants perform CAM, but in amphibious plants CAM does not occur when the leaves are exposed to the air (Keeley, 1996; Robe and Griffiths, 2000). Thus, CAM is distributed among all kinds of morphotypes, providing various potential adaptive advantages, but there is no single one that appears particularly well suited for CAM. An enigma is posed by the last line of Table 4. Why are there almost no CAM trees? Among stem-succulent dicotyledons there are species that reach a spectacular size, for example columnar cacti, candelabrum euphorbias and some Didieraceae, which have been called ‘tree-succulents’ (Ellenberg, 1981). Moreover, Vareschi (1980) has referred to cactus forests. However, these are not real trees with secondary thickening growth, and the sensation of being in a forest with a closed canopy when walking in such habitats does not so much originate from the columnar cacti but rather from the acacias and other woody plants scattered among them. Moreover, there is the CAM-performing Yoshua tree of the genus Yucca with a special monocotyledonous type of secondary thickening. However, the only true dicotyledonous trees performing CAM are species of Clusia. The question why there are not more dicotyledonous trees with CAM cannot, at present, be answered. The great success of CAM Clusias, with an extraordinarily large ecological amplitude covering coastal sites of rocks and dunes, savannas, rock outcrops, gallery forests, rainforests and cloud forests (Lüttge, 1996), seems to rule out that tree structure and CAM function are not compatible. Is it the high plasticity, flexibility and speciation rate within the genus Clusia that led to a first evolution of CAM among trees?
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Different life stages of CAM plants may involve morphotypic differences. Often it is simply plant size that affects ecophysiological performance, particularly with respect to water relations (Schmidt et al., 2001; Zotz et al., 2001; Hietz and Wanek, 2003). In stem-succulent CAM plants, the larger surface/volume ratio of small plants compared with that of larger ones leads to a higher relative water loss in the former compared with that of the latter and makes survival under extended dry conditions more difficult (Jordan and Nobel, 1979, 1982; Lerdan et al., 1992).
However, different life stages may produce more clearly different morphotypes. Clusias are hemiepiphytic stranglers. Seedlings may start growth terrestrially and develop as independent trees. Alternatively, they may start growth epiphytically and grow aerial roots, some of which gain soil contact while others act as holdfasts and eventually strangle the host, resulting in Clusia eventually becoming an independent tree as the host dies and rots away (Ball et al., 1991a). However, the expression of CAM is strongly under environmental control and is not correlated with the life form of the seedling, epiphyte, strangler or tree. Thus, there is no ontogenetic programme for CAM expression (Ball et al., 1991b; Wanek et al., 2002b).
Among tank-forming, epiphytic CAM bromeliads, many species have a different morphotype in the juvenile stage. Juvenile plants form small rosettes of narrow leaves not overlapping at their bases and thus, do not form phytotelmata (Schmidt and Zotz, 2001). They therefore have particular problems of water and nutrient acquisition similar to the so-called atmospheric bromeliads which can attach themselves to any support, including fence wires and telephone cables, and are restricted to the absorption of water and nutrients, via epidermal scales or trichomes, from rain, aerosols and dust. Juvenile stem-succulents, e.g. cacti, are handicapped in not being able to store sufficient water compared with that of larger adult individuals, because the juvenile plants have larger surface area : volume or chlorenchyma : hydrenchyma ratios (Jordan and Nobel, 1979, 1982).
With respect to life cycles, most CAM plants are perennial. Many CAM plants are monocarpic or hapaxanthic, i.e. they die after flowering either in their second year as biennial plants, e.g. Kalanchoës, or after many years in the vegetative state once flowering has occurred, e.g. Agaves. Apart from M. crystallinum and M. nodiflorum (Winter and Troughton, 1978) and Crassula siberiana, an annual leaf-succulent growing in granitic outcrops in New South Wales, Australia (Brulfert et al., 1991), there are no other reports of annual CAM plants.
Physiotypes.
Photosynthetic physiotypes are the major physiotypes that can be distinguished for the metabolic cycle of CAM. ‘CAM cycling’, ‘full CAM’ and ‘CAM idling’ have already been mentioned above. Full CAM may show considerable plasticity due to the flexible expression of the various CAM phases, and this comprises CAM idling. It is also noteworthy that bona fide obligatory CAM plants may turn totally to C3 photosynthesis under certain conditions, e.g. K. daigremontiana at 7 °C (Kluge, 1969). Even the extremely atmospheric CAM bromeliad Tillandsia usneoides, with its apparent strong constitution for endurance of stress, shows high plasticity and may perform C3 photosynthesis to a considerable extent as conditions allow (Kluge et al., 1973; Haslam et al., 2002, 2003). However, all these flexible reactions of a given plant or species can still be regarded as comprising the ‘full CAM’ physiotype. Conversely, CAM cycling may be a separate physiotype, as it appears that some species only express CAM cycling and never advance to stronger CAM. This is also sometimes considered as a first step in the evolution of CAM.
In addition, there are the true intermediates (see also Winter and Smith, 1996b; Sayed, 2001). There are a fair number of C3 photosynthesis/CAM intermediates that can switch between full C3 photosynthesis and full CAM. Some of them have already been mentioned above where the impact of environmental factors eliciting such switches have been discussed, e.g. photoperiod in K. blossfeldiana ‘Tom Thumb’, salinity in M. crystallinum. Guzmania monostachia appears to be the only real C3/CAM intermediate species in the large CAM plant family Bromeliaceae (Maxwell et al., 1994, 1995, 1999; Maxwell, 2002), although there is some evidence that Nidularium innocentii may also be C3/CAM (see Griffiths, 1989). The large genus Clusia, that has only one morphotype of entire and somewhat leathery succulent leaves, comprises three photosynthetic physiotypes, i.e. C3, C3/CAM and CAM (Lüttge, 1999). The list of species compiled by Sayed (2001) includes highlighting of CAM-inducible, i.e. C3/CAM, intermediate plants.
Sage (2002) has questioned whether CAM and C4 photosynthesis are compatible. It appears, however, that there genuinely are some C4/CAM intermediate species, e.g. Peperomia camptotricha (Nishio and Ting, 1993), Portulaca oleracea (Koch and Kennedy, 1980, 1982; Mazen, 1996) and Portulaca grandiflora (Koch and Kennedy, 1980, 1982; Ku et al., 1981; Kraybill and Martin, 1996; Guralnick and Jackson, 2001; Guralnick et al., 2002). Only succulent C4 dicotyledons are capable of diurnal fluctuations of organic acids, where dark-respiratory CO2 is trapped in bundle sheaths by PEPC and the water storage tissue in the succulent leaves may also participate in the fixation of internally released CO2 (Ku et al., 1981). In Portulaca, this may be a form of CAM cycling in leaves with C4 photosynthesis, while stems perform CAM idling (Guralnick et al., 2002).
However, although C4 photosynthesis and weak CAM occur in the same leaves, they are separated in space and do not occur in the same cells. This incompatibility of C4 photosynthesis and CAM may be due to anatomical, biochemical and evolutionary incompatibilities. The separation of malate synthesis and decarboxylation in space in C4 photosynthesis and in time in CAM, respectively, and the primary evolution of C4 photosynthesis for scavenging photorespiratory CO2 and of CAM for scavenging respiratory CO2 (CAM cycling) may be the most important backgrounds of these incompatibilties. Although single cells may perform C4 photosynthesis, there is intracellular compartmentation of carboxylation and decarboxylation, and these cells never perform CAM. Unlike C3–CAM coupling, there is never C4–CAM coupling and both pathways only occur side by side in C4/CAM intermediate species (Sage, 2002).
In summary, the CAM physiotypes CAM, CAM idling, C3/CAM and C4/CAM can be distinguished.
Roots have been largely neglected in CAM research. Of course, as CAM is a mode of photosynthesis, the focus has naturally been on the green tissues of CAM plants, but roots provide the essential support by the pedosphere and cannot be disregarded in the ecophysiology of CAM. Some information is available on green aerial roots of epiphytic orchids that may perform CAM (Winter et al., 1985; Goh and Kluge, 1989; Kluge et al., 2001). A lot of information is available on epiphyte roots and their role in anchorage and nutrition including morphotypic distinctions of root systems of bromeliads, namely soil roots, tank roots and anchorage roots (Smith et al., 1986; Smith, 1989). Cacti may sacrifice their fine roots during periods of severe drought stress, which they may overcome by many weeks of CAM idling with continuously closed stomata, and then rapidly regrow new roots when precipitation occurs and soil water becomes available again (Kausch, 1965; Lüttge et al., 1989).
A systematic functional study of CAM root systems was performed on CAM desert succulents, i.e. cacti (mainly Ferocactus acanthodes and Opuntia ficus-indica) and agaves (mainly Agave deserti). The performance of these roots is conspicuous and may be considered as a pronounced CAM- or at least desert-succulent physiotype. The roots have rectifier properties and respond opportunistically to changing water potential () gradients between soil and root tissue. When soil > root they maintain a high hydraulic conductivity, Lp, for water uptake. When the soil dries out and