Journal of Experimental Botany, Vol. 53, No. 378, pp. 2131±2142, November 2002 DOI: 10.1093/jxb/erf081

CO2-concentrating: consequences in crassulacean acid metabolism

Ulrich LuÈ ttge1 Institut fuÈr Botanik, Technische UniversitaÈt Darmstadt, Schnittspahnstrabe 3±5, D-64287 Darmstadt, Germany

Received 11 April 2002; Accepted 1 July 2002

Abstract crassulacean acid metabolism, oxidative stress, oxygen concentrating. The consequences of CO2-concentrating in leaf air- spaces of CAM plants during daytime organic acid decarboxylation in Phase III of CAM (crassulacean acid metabolism) are explored. There are mechanistic The overt phenomenon of internal consequences of internal CO2 partial pressures, CO2-concentrating: Phase III of CAM pCO2. These are (i) effects on stomata, i.e. high pCO2 i i Crassulacean acid metabolism (CAM) is a well-known eliciting stomatal closure in Phase III, (ii) regulation of modi®cation of photosynthesis that is covered in all the malic acid remobilization from the vacuole, malate basic textbooks of plant physiology and biochemistry and decarboxylation and re®xation of CO via Rubisco 2 which has been extensively reviewed (Osmond, 1978; (ribulose bisphosphate carboxylase/oxygenase), and Grif®ths, 1989; Winter and Smith, 1996a; Cushman and (iii) internal signalling functions during the transitions Bohnert, 1997; Dodd et al., 2002). The essential mechan- between Phases II and III and III and IV, respectively, ism of CAM is the acquisition of inorganic carbon (Ci) by in the natural day/night cycle and in synchronizing ± dark-®xation of bicarbonate (HCO3 ) via phosphoenolpyr- the circadian clocks of individual leaf cells or leaf uvate carboxylase (PEPC). This leads to an organic acid patches in the free-running endogenous rhythmicity (mainly malic acid)-concentrating effect in the dark period of CAM. There are ecophysiological consequences. when organic acid is stored in the central cell sap vacuole. Obvious bene®cial ecophysiological consequences In the subsequent light period organic acid is released from are (i) CO2-acquisition, (ii) increased water-use- the vacuole again, decarboxylated and the resulting CO2 ef®ciency, (iii) suppressed photorespiration, and (iv) assimilated in the Calvin cycle. These parts of the CAM reduced oxidative stress by over-energization of the cycle are called Phase I (nocturnal CO2-®xation via PEPC photosynthetic apparatus. However, the general and acid accumulation) and Phase III (daytime CO2- potency of these bene®cial effects may be questioned. recovery and assimilation), respectively. These phases are There are also adverse ecophysiological conse- separated by transition phases, Phase II in the morning and quences. These are (i) energetics, (ii) pH effects and Phase IV in the afternoon, respectively (Osmond, 1978; (iii) Phase III oxidative stress. A major consequence of see Fig. 1 in Dodd et al., 2002). CO2-concentrating in Phase III is O2-concentrating, Nocturnally accumulated vacuolar organic acid is a increased pCO2 is accompanied by increased pO2.Do i i CO2-store in the form of carboxyl groups and not in the reversible shifts of C /CAM-intermediate plants 3 form of free Ci. Raven and Spicer (1996) have explained in between the C ±CAM±C modes of photosynthesis 3 3 detail why the storage of Ci equivalent to the large amounts indicate that C3-photosynthesis provides better pro- of organic acid actually stored in CAM nocturnally tection from irradiance stress? There are many open (~500 mol m±3) would be a less operable and favourable questions and CAM remains a curiosity. option. Thus, strictly speaking, Phase I is not considered as CO2-concentrating, although it is an essential part of the Key words: Carbon dioxide concentrating, circadian clock, whole mechanism.

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ã Society for Experimental Biology 2002 2132 LuÈttge Conversely, the release and decarboxylation of organic leaves of C. rosea, now known as a CAM plant (Ball et al., acid during the major part of the light period (Phase III) 1991a, b), during the light period. He writes leads to a rapid build-up of high internal CO2-concentra- `In keiner P¯anze zirkuliert vielleicht eine so ungeheure tions effecting stomatal closure in the light. The internal Menge an Luft als in der Clusia rosea. Wenn man die CO2-concentrations behind closed stomata reported in the BlaÈtter dem Sonnenlicht aussetzt, so ergeben sie ... nicht literature are compiled in Table 1. They range from a eine einzige Luftblase.' 2-fold to an over 60-fold increase of internal related to `In no other plant perhaps as much air is circulating than external CO2-concentration. in the Clusia rosea. If one exposes the leaves to the sun Thus, when talking about CO2-concentrating in CAM light, they produce ... not a single air bubble.' this refers to the Phase III phenomenon. This will be the That means there is no obvious gas exchange in the light topic of this review when the mechanistic consequences of via the leaf surface. We now know, of course, that the CO2-concentrating for leaf functions and the eco- stomata are closed in Phase III of CAM, and hence there is physiological consequences for plant performance are no gas exchange. assessed. `Aus dem verwundeten Teile des Stengels faÈhrt aber mit ungeheurer Geschwindigkeit ein Strom von perlartigen LuftblaÈschen ... aus jedem dieser GefaÈûbuÈndel aus ± ein Historical recollections: CO2-concentrating and herrliches Schauspiel'. O2-concentrating `However, at the cut end of the petiole with an immense In the February of 1800 Alexander von Humboldt velocity a stream of pearl-like gas bubbles ... is released performed ecophysiological gas-exchange measurements from each of these vascular bundles, a magni®cent on Clusia rosea at Lake Valencia in Venezuela. It may be spectacle.' debated if he thus became the discoverer of CAM in We now know that behind the closed stomata there is a Clusia. He certainly made the correct observations. Once high internal gas pressure in Phase III. again, in 1937, W Hartenburg also made the right `Setzte ich den Apparat in den Schatten, so hoÈrte der observations, but like Alexander von Humboldt he did Luftstrom auf...' not present the appropriate explanation (Hartenburg, 1937; `Did I place the apparatus in the shade, the gas stream see LuÈttge, 1995). Thus, the discovery of CAM in Clusia ceased ...' appears to be rightly acknowledged to Tinoco Ojanguren That means that Alexander von Humboldt performed and Vazquez-Janez (1983). the appropriate control. What, however, Alexander von Humboldt was certainly `Luft aus dem Innern der Clusia rosea ... besteht aus 0,35 the ®rst to discover is the high internal O2-concentration in Oxygen und 0,65 Azote'.

Table 1. Maximum internal CO2-concentrations and ci /ca-ratios (where ca was taken as 0.04%) measured in various CAM-plants in the light period, at midday, in Phase III of CAM by various methods

Note that the determination of pCO2 from measurements of gas exchange, i.e. leaf-conductance for water vapour, g , is only possible when i H2O stomata are open, at least to some extent, and that errors become large as stomata close in Phase III. Thus the values reported by Friemert et al. (1986) are approximations, showing the order of magnitude though.

CO2 Species Family pi (%) ci/ca Method Reference Opuntia ®cus- indica Cactaceae 1.30 32.5 Thermal conductivity Cockburn et al., 1979 Opuntia basilaris Cactaceae 2.50 62.5 Thermal conductivity Cockburn et al., 1979 Opuntia monacantha Cactaceae 0.12 3.0 Gas chromatography Spalding et al., 1979 Agave desertii Agavaceae 0.80 20.0 Thermal conductivity Cockburn et al., 1979 Yucca schidigera Agavaceae 0.40 10.0 Thermal conductivity Cockburn et al., 1979 AloeÈ vera Liliaceae 0.60 15.0 Thermal conductivity Cockburn et al., 1979 Ananas comosus Bromeliaceae 0.50 12.5 Thermal conductivity Cockburn et al., 1979 Ananas comosus Bromeliaceae 0.13 3.3 Gas chromatography Spalding et al., 1979 Cattleya sp. Orchidaceae 0.15 3.8 Thermal conductivity Cockburn et al., 1979 Phalaenopsis sp. Orchidaceae 0.23 5.8 Thermal conductivity Cockburn et al., 1979 Hoya carnosa Asclepiadaceae 0.08 2.0 Gas chromatography Spalding et al., 1979 Huernia sp. Asclepiadaceae 0.14 3.5 Gas chromatography Spalding et al., 1979 KalanchoeÈ gastonis-bonnieri Crassulaceae 0.27 6.8 Gas chromatography Spalding et al., 1979 KalanchoeÈ tomentosa Crassulaceae 0.35 8.8 Gas chromatography Spalding et al., 1979 KalanchoeÈ daigremontiana Crassulaceae 0.50 12.5 Gas chromatography Kluge et al., 1981 KalanchoeÈ tubi¯ora Crassulaceae 0.09 2.3 Gas exchange Friemert et al., 1986 Sedum praealtum Crassulaceae 0.29 7.3 Gas chromatography Spalding et al., 1979 Sedum morganianum Crassulaceae 0.26 6.5 Gas exchange Friemert et al., 1986 Sempervivum tectorum Crassulaceae 0.12 3.0 Gas exchange Friemert et al., 1986 CO2-concentrating in CAM 2133 `The air from the interior of Clusia rosea ... consists of With the new technique of chlorophyll ¯uorescence 0,35 oxygen and 0,65 "azote".' imaging (Daley et al., 1989; Siebke and Weis, 1995) it is That means that the gas in the leaf air spaces had 35% now possible (i) to obtain in addition a much better O2 and 65% N2. temporal resolution, (ii) to obtain spatial resolution over a As measurements in the ®eld are always dif®cult, later leaf, and (iii) to obtain some more insight into the the chemical O2-determinations were checked in the underlying processes, including the functions of CO2- laboratory of L-J Gay-Lussac in Paris and were found to evolution behind closed stomata, rather than just recording be too high by a systematic error of 5% (Quotations after the disappearance of malic acid. KraÈtz, 2001). Chlorophyll ¯uorescence imaging allows spatiotem- Using a gas chromatograph Spalding et al. (1979) poral monitoring of the relative quantum ef®ciency of rediscovered the high internal O2-concentrations during photosystem II, fPSII. When internal CO2-concentration, the light period, i.e. in Phase III of CAM sensu Osmond piCO2, is high it may saturate photosynthesis. This leads to (1978). They found values between 21.4% and 30.5% in high fPSII, and when fPSII is integrated over the whole leaf, various CAM species at midday and 41.5% in KalanchoeÈ òfPSII, high overall rates are obtained. The energy gastonis-bonnieri. In contrast to the high internal CO2- consumption of gluconeogenesis recovering the C3-carbon concentrations in Phase III, reported by Spalding et al. skeleton, pyruvate or PEP, originating from malate (1979) and elsewhere (see below), the high internal O2- decarboxylation, tends to lower fPSII. This effect is CO concentrations have largely been forgotten until very overridden by high pi 2 saturating photosynthesis, but it CO recently, when the ecophysiological implications of this may be discernible when pi 2 is lower. Thus, chlorophyll consequence of CO2-concentrating began to be discussed ¯uorescence images show some of the consequences of in more depth. internal CO2-generation from malate decarboxylation and CO2-concentrating. Questions I These events are not homogeneous in space over a CAM leaf of K. daigremontiana during the light period (Rascher, CO2-concentrating mechanisms and internal CO2-concen- 2001; Rascher et al., 2001). In the morning, some cells or trations in the leaf air spaces of CAM plants were reviewed patches of cells may start earlier than others to release by Grif®ths (1989) and in Winter and Smith (1996a;19 malic acid from the vacuoles, to decarboxylate malate and CO entries for `carbon dioxide±intracellular space' in the to raise pi 2. Since fPSII re¯ects the local availability of subject index). The major questions covered are the CO2 this can be seen as a patchiness of fPSII. This mechanism of CO2-concentrating itself, and among the inhomogeneity is particularly expressed during the transi- consequences the effects on photorespiration, light-stress tion between Phase II and Phase III. In phase II, stomata and photoinhibition, internal CO2-recycling, water-use- are still open and CO2 is both taken up from the ef®ciency, energy costs, and phylogenetic implications. atmosphere and produced in the leaf interior from malate These aspects are well, perhaps even overly, covered in the decarboxylation (Borland and Grif®ths, 1996). CO CAM literature. The present review will only allude to Increasing pi 2 is known to be the major internal control them insofar as it is necessary to fathom (i) some new parameter eliciting stomatal closure in Phase III. In K. mechanistic aspects of CAM-functions, i.e. non-linear daigremontiana stomata respond with a lag of about 15 min CO dynamics of CAM-leaves, including CO2-signalling versus with closing movements as pi 2 builds up (Bohn et al., CO the established functions of CAM-leaf metabolism, and (ii) 2001). Local gradients of pi 2 established due to the some ideas on adverse (eco-)physiological consequences desynchronization of leaf patches are maintained for a versus the established bene®cial (eco-)physiological con- while, because there is a severe constraint on CO2- sequences of CAM. diffusion inside the leaves of KalanchoeÈ daigremontiana. The uniformly spherical cells are densely packed, the Mechanistic consequences: spatiotemporal internal air space is only about 4±9% of the leaf volume non-linear dynamics of leaf functions and diffusion resistance is large (Maxwell et al., 1997; Rascher et al., 2001). Patchiness of fPSII is almost entirely CO -remobilization from malic acid/malate in Phase III CO 2 lost in Phase III when high internal pi 2 builds up Gas exchange measurements during most of Phase III (Table 1) overriding the limitations of CO2 diffusion. In when the stomata are ®rmly closed are not possible. The Phase III there is no CO2 limitation anywhere in the leaf. traditional method of recording malic acid remobilization However, inhomogeneity begins to peak again at the end of is to measure malate levels or titratable acidity in the cell Phase III, as some patches have ®nished malate decarbox- sap during the day. Rarely are samples taken at de®ned ylation earlier than others. At this time not only patches but locations over the leaf. Thus, this destructive and tedious also waves of fPSII moving over the leaf are seen. Such approach provides time series with a low temporal waves may be explained by a complex interaction of resolution and mostly no spatial resolution over the leaf. malate depletion, CO2 diffusion from neighbouring leaf 2134 LuÈttge zones into the depleted zones and the energy budget determined by decreasing needs of gluconeogenesis (Rascher, 2001). Hence, this non-linear spatiotemporal performance of the leaves during Phases II to IV from the beginning to the end of the day reveals several CO2-concentrating conse- quences. These observations elicit re¯ections on the regulation of CO2-remobilization from nocturnally stored malic acid/malate.

Regulation of CO2-remobilization from malic acid/ malate in Phase III

Is anything known about how and where CO2-remobiliza- tion from malic acid/malate is regulated in Phase III (Fig. 1)? Are there (and if so where are they) control points of metabolite ¯uxes in membrane-transport and enzyme- reaction steps? Is there any mass-action type feedback? (i) CO2-consumption by Rubisco: Is it the rate of CO2- consumption by Rubisco and assimilation in the Calvin cycle which regulates via a CO2-demand? In favour of this assumption is the observation that malate consumption in Phase III is accelerated by increased light intensity enhancing photosynthesis (Kluge, 1968; Thomas et al., 1987). Against this is the sheer CO2-concentration build- ing up when it oversaturates C3-photosynthesis (Rubisco and Calvin cycle). Substrate saturation of C3-photosyn- thesis is between 0.1% and 0.4% (Berry and Downton, 1982). CO2-assimilation during Phase III of CAM in KalanchoeÈ daigremontiana saturates at 0.2% (Maxwell et al., 1998). Fig. 1. Malic acid/malate remobilization and decarboxylation and Thus, according to the data of Table 1, there was strong build-up of high internal CO2-concentrations in Phase III of CAM and substrate oversaturation of Rubisco in two Opuntia control points discussed in the text, i.e. (i) CO2-consumption by Rubisco, (ii) consumption of cytoplasmic malate by decarboxylation, species, but not in a third species studied, and some and (iii) malic acid ef¯ux from the vacuole. CA=carbonic anhydrase. oversaturation was observed in Agave desertii, AloeÈ vera, and perhaps Ananas comosus (although only in one of the two measurements) and K. daigremontiana. Such over- control eliminated, they found the cross-over point where saturation would rule out a regulation of remobilization via net CO2-exchange became zero, i.e. uptake balanced CO release, at 0.06%. This is much lower than most p 2 the CO2 demand of Rubisco. On the other hand, this seems i to be the exception rather than the rule. In the majority of values in Table 1. In addition, patchiness of fPSII cases in Table 1 Rubisco would operate at or below independent of stomatal reactions (Rascher, 2001) also substrate saturation, and this allows the assumption that the suggests that CO2-mediated regulation may be an internal regulation of remobilization is by Rubisco and that carbon effect related to Rubisco activity. dioxide is functioning as a controlling factor in decarboxy- (ii) Consumption of cytoplasmic malate by decarboxy- lation (Cockburn and Patel, 2002). lation: Is the regulation of remobilization at the point of CO There appear to be no data on the critical level of pi 2 malate decarboxylation, where the demand for cytoplas- needed to close stomata in Phase III. The CO2-response mic malate would determine cytoplasmic malate supply sensitivity of guard cells, in general, is very variable from the vacuole? A mass action effect at this point is quite between species and also for a given species due to possible because all of the three malate decarboxylating acclimation (Frechilla et al., 2002). Hence, it is hard to say enzymes occurring in different CAM plants, namely NAD- CO if this effect is just saturated at the pi 2-levels given and NADP-dependent malic enzymes and phosphoenol- (Table 1), and thus, a critical control point, or if it is pyruvate-carboxy-kinase, catalyse readily reversible reac- oversaturated. The latter is assumed. Cockburn and Patel tions. Driving the process towards decarboxylation (2002) have performed a set of interesting experiments. requires reasonably high cytoplasmic malate levels. In When they exposed leaves of various CAM plants with the fact, measurements of cytoplasmic pH during the diurnal epidermis removed, leaf discs or leaf strips, with stomatal CAM cycle in K. daigremontiana have shown a drop by CO2-concentrating in CAM 2135 ± 0.3 pH units at midday (Hafke et al., 2001). This may be CO2-®xation (Phase I), where HCO3 is the substrate of due to both high levels of malic acid and CO2 in the PEPC. By contrast, obviously nothing is known about the cytoplasm (Fig. 1). With a buffer capacity of about 65 mM role of CA in Phase III (Raven and Spicer, 1996). + CO H per pH unit for a pHcyt of about 7.5, this corresponds to High pi 2 may be a downstream consequence of the a cytoplasmic acid load equivalent to 10 mM malic acid. operation of the regulation network (Fig. 1) including CA- CO (iii) Malic acid ef¯ux from the vacuole: A third functions. pi 2-levels saturating or oversaturating photo- possibility is that it is malic acid ef¯ux from the vacuole synthesis in Phase III do not appear to be an upstream which determines the whole process. The mechanism by requirement of CAM-function inasmuch as often levels which this occurs is still uncon®rmed, i.e. diffusion of saturating Rubisco are not attained (see above and Table 1). malic acid (LuÈttge and Smith, 1984) or a carrier- or In conclusion then, while the precise position and function CO channel-mediated process (see LuÈttge et al., 2000). What is of control points is not certain, clearly pi 2 may exert a known is that it is a passive process (LuÈttge and Smith, signalling function between the rate of CO2-consumption 1984). It can be regulated, however, by changing tonoplast by Rubisco and malic acid remobilization from the properties. During nocturnal malic acid accumulation, vacuole. which has osmotic consequences, turgor pressure in- creases. This may have effects on the tonoplast which Circadian rhythmicity of CAM a complication: are important for the switching between nocturnal net oscillatory CO2-concentrating? malic acid accumulation and daytime net malic remobil- The circadian rhythmicity of CAM is a complication. In ization (LuÈttge et al., 1975, 1977; LuÈttge and Ball, 1977; the normal day/night cycle, switching between nocturnal Steudle et al., 1980; Smith et al., 1984). In fact, this is also net-accumulation and daytime net-remobilization and back part of a model suggesting the function of the tonoplast as again to net-accumulation of malic acid can be readily the hysteresis switch in the oscillator of circadian explained by the changing external conditions shifting the rhythmicity of CAM (see below). As in option (ii), this equilibria. Equilibrium thermodynamics, therefore, pro- would lead to rather high cytoplasmic malate concentra- vide suf®cient explanation for the ®lling and emptying, tions in Phase III. respectively, of the vacuole. However, with the endogen- A cytoplasmic malate concentration of 10 mM corres- ous circadian rhythmicity under constant environmental ±3 ponds to 10 mol m CO2 if considered decarboxylated to conditions this does not apply. pyruvate. A 2.5% CO2 partial pressure in the aerenchyma The study of circadian rhythmicity needs continuous (Opuntia basilaris) would correspond to only about one- recording of long time-series. Therefore, for technical tenth, i.e. 1.1 mol m±3 CO . In leaves of K. daigremontiana reasons, it is mainly gas exchange, CO (J ) and water 2 2 CO2 the volumes of cytoplasm and air space are about 0.5±1% vapour (J ), that is monitored. Occasionally malate H2O and about 3.6±8.8%, respectively (Rona et al., 1980; Smith analyses are available and enzyme activities, PEPC and and Heuer, 1981; and Maxwell et al., 1997; Rascher, 2001, Rubisco were followed both directly by enzyme analyses respectively). Thus, the air-space volume is about 10 times and indirectly by the online collection of samples from the the cytoplasmic volume, and the above rough estimates air ¯ow through gas-exchange systems to measure the CO would not reject the possibility that even the highest pi 2 different carbon-isotope discrimination by the two en- measured (Table 1) is a consequence of equilibria estab- zymes (Grams et al., 1997). However, these techniques are lished via strong vacuolar malic acid ef¯ux and build-up of tedious, destructive (malate analyses, enzyme extracts) or cytoplasmic malate levels. However, it is still necessary to expensive (carbon-isotope analyses) and long, densely know much more about the rates of the enzyme reactions sampled, time-series are not available. Little is known CO involved in establishing these equilibria. about the internal partial pressures of CO2 and O2, pi 2 and O A very important enzyme in these equilibria is carbonic pi 2, during the circadian oscillations. From the point of ± anhydrase (CA). CO2 and HCO3 may diffuse in parallel in view of this review, however, the question must be asked the cytosol and in the chloroplast stroma. In terrestrial C3 whether there is a circadian rhythmicity of CO2-concen- plants CA enhances the diffusion of Ci from the site at trating. which atmospheric CO2 dissolves in cell wall or apoplast Bohn et al. (2001) have calculated the ratio of internal to CO water to the site of CO2 consumption by Rubisco (Raven external p 2 (ci/ca)inKalanchoeÈ daigremontiana from and Spicer, 1996). In the same way it could mediate CO measurements of J and the derived leaf-conductance 2 H2O release into the leaf air spaces of CAM plants in Phase III for water vapour, g . They have studied entrainment of H2O (Fig. 1). In C3-plants CA is mainly expressed in the the CAM-cycle in K. daigremontiana under imposed chloroplast stroma, although there is also activity in the external rhythms of light intensity of 140 mmol m±2 s±1 cytosol (Reed, 1979). Tsuzuki et al. (1982) did not ®nd (lower) and 250 mmol m±2 s±1 (higher) with a period of 24 h cytosolic CA in some of the CAM plants they tested, while and 16 h, respectively. They found oscillations of ci/ca Holtum et al. (1984) found in all CAM plants tested that between a minimum of 0.64 and a maximum of 1.03 at the cytosolic CA activity exceeded the requirement of dark low and high light intensities, respectively. ci/ca-oscilla- 2136 LuÈttge tions of similar magnitude are obtained during endogenous the gene for PEPC-kinase, the enzyme which regulates rhythmicity under constant external conditions (Tomasz PEPC-activity, is under circadian control (Nimmo et al., Wyka, personal communication). Thus, there was some 1987, 2001; Carter et al., 1991, 1996; Kusumi et al., 1994; feeble CO2-concentrating keeping ci/ca not too much Hartwell et al., 1996, 1999), and in fact many other genes, CO below unity or even raising pi 2 slightly above the especially of enzymes involved in CAM are clock- atmospheric partial pressure during the peaks of photo- controlled genes (Boxall et al., 2001), it appears that synthesis. This is an order of magnitude less than the CO2- circadian CAM performance is under the control of a post- concentrating in Phase III of the normal CAM cycle, where translational machinery or oscillator. The PEPC-kinase ci/ca for K. daigremontiana was 12.5 (Table 1). gene is under the control of metabolism and cytoplasmic/ Does chlorophyll-¯uorescence imaging allow the black vacuolar malate compartmentation (Borland et al., 1999; box to be denuded? In Rascher's experiments (Rascher, Nimmo, 2000). The tonoplast was shown to have oscillator CO 2001; Rascher et al., 2001) imaging only provided relative function. pi 2 being directly related to malate compart- data on fPSII for comparisons within a given time series mentation and metabolism, namely, malate decarboxyl- and no absolute measurements of fPSII which would allow ation, may indeed be an important internal signal. It may the values obtained during circadian rhythmicity to be act directly or in a signal transduction chain, where pH compared with those of the diurnal dark/light cycle. effects and membrane potential transients may also be During the circadian time series òfPSII (integrated over the involved (Stahlberg et al., 2001). Whether this signalling CO whole leaf) was low when J was zero and stomata were function of p 2 is necessary for the normal day/night CO2 i obviously closed. This is in contrast to Phase III of the cycle of CAM is not known. Perhaps not in Phase III, when CO CO normal day/night cycle of CAM when there is CO2- pi 2 is homogenously high. However, pi 2-signalling concentrating and when òf was high as J was zero. may be important in the regulation of changes between PSII CO2 Does this mean that there is no CO2-concentrating during Phases II and III and III and IV, respectively, as alluded to the circadian rhythm? Or, perhaps, CO2-concentrating may by the transients of inhomogeneity at these times as only be weak, as suggested by the experiments of Bohn discussed above. et al. (2001; and Tomasz Wyka; see above), and could be overridden by the energy requirements of gluconeogenesis Established bene®cial ecophysiological which tend to lower fPSII. This then might support the idea that the strong CO - consequences: but are there not open 2 questions? concentrating observed in Phase III of the normal day/ night CAM cycle is a consequence of equilibria established There are four ecophysiological consequences of CO2- as discussed above, but does not occur during the non- concentrating in CAM, which are well established in the linear dynamics of circadian oscillations. On the other literature and widely accepted. hand, pronounced patchiness does occur during the circadian rhythm, and it also oscillates. Its level even CO2-acquisition increases considerably during the ongoing free running Nocturnal CO2-®xation and daytime interior CO2-concen- oscillations. Adjacent leaf patches of 5±10 mm diameter trating by submerged plants in fresh water is a mechanism (approximately 30±60 cells) and 15±30 mm apart may get of CO2-acquisition avoiding daytime competition with desynchronized, particularly during phases of fPSII non-CAM photosynthesizers (Grif®ths, 1989; Keeley, decline. They synchronize again as fPSII increases. Most 1996). CO2-concentrating itself is the ecophysiological likely, as in the entrainment experiments of Bohn et al. bene®t here. This may have been the original driving force (2001), the sequence of events is a reduction of net CO2- of CAM-evolution as it is expressed in plants as CO uptake, J ,asp 2 increases, followed by a decrease of phylogenetically early as the lycopodiopsid IsoeÈtes CO2 i gH2O. (Grif®ths, 1989; Keeley, 1996). However, is this bene®cial Thus, there must be at least some internal CO2- consequence not restricted to fresh-water plants? concentrating. Internal CO2 is thought to be the synchro- nizing signal. However, as seen above, due to the diffusion Water-use-ef®ciency (WUE) restriction inside the leaf of K. daigremontiana this In terrestrial CAM plants Phase III CO2-concentrating requires high internal CO2-concentrations. They are high allows CO2-assimilation behind closed stomata. This enough in Phase III of normal CAM where patchiness is minimizes transpirational water loss during the hottest low and much lower, even at their highest levels, in part of the day with the largest atmospheric water vapour circadian oscillations where patchiness remains high pressure de®cit, while stomata are open during the night and synchronization/desynchronization shows strong for CO2 acquisition. CAM plants have the highest water- dynamics. use-ef®ciency (WUE). What does this say about CAM and its Phase III CO2- This has led to a widely held view that CAM plants concentrating phenomenon? Although it was found that typically are inhabitants of the driest arid sites. True, there CO2-concentrating in CAM 2137 c. 1800 species of stem succulent cacti and leaf succulent Table 2. Ratios of internal concentrations of O2:CO2 at agaves, the CAM plants of deserts. However, among the midday in various CAM plants (Spalding et al., 1979) orchids and bromeliads there is an estimated 10 000 CAM O /CO species which are typical elements of the ¯ora of the 2 2 moister shaded, semi-shaded and semi-exposed rainforests Ambient air 633 Opuntia monacantha 205 (LuÈttge, 2000). Does this mean that WUE needs to be Ananas comosus 161 challenged as the major ecophysiological trait of CAM and Hoya carnosa 285 foremost driving force of CAM-evolution? Or is this Huernia sp 174 KalanchoeÈ gastonis-bonnieri 155 explained by the fact that most of the CAM-species of KalanchoeÈ tomentosa 88 tropical forests are epiphytes with their own particular Sedum praealtum 81 problems of water supply as they do not root in a soil substrate (Zotz and Hietz, 2001)?

Suppressed photorespiration mitochondrial enzyme fumarase as an indicator of the Rubisco has two competing substrates, CO and O . Phase function of the tricarboxylic acid cycle. Its activity was 2 2 much reduced in Mesembryanthemum crystallinum in the III CO2-concentrating elevates substrate levels for the carboxylating activity of Rubisco so that it can operate at CAM-state as compared to the C3-state. High mitochon- drial activity during the light period decreases photo- or close to substrate saturation beginning above 0.1% CO2 (Berry and Downton, 1982; Table 1). This suppresses the respiration because it consumes O2 and generates CO2 and oxygenase activity of Rubisco, i.e. photorespiration hence tends to increase the internal CO2:O2 ratio (KroÈmer, (Osmond et al., 1982; Cushman and Bohnert, 1997), and 1995). Thus, low mitochrondrial activity in the CAM-state may indicate increased photorespiration as a protection hence, the loss of CO2 related to photorespiration. However, CO -concentrating itself may lead to some loss against stress by high O2-levels (Miszalski et al., 2001). 2 Therefore, can suppressed photorespiration really be of CO2, namely by diffusion out of the leaf at the very high considered as a bene®cial consequence of CO2-concen- CO2 gradient between the leaf air spaces and the atmos- phere (Friemert et al., 1986). trating? The photorespiratory machinery remains active in CAM plants (Osmond et al., 1982; Whitehouse et al., 1991; Prevented overenergization of the photosynthetic Edwards et al., 1996; Heber et al., 1996). It is needed in energy transduction pathway Phase IV when plants perform C3-photosynthesis with Another long-accepted bene®cial consequence of Phase III open stomata and atmospheric CO2. But what about Phase CO2-concentrating and operation of Rubisco near substrate III? To what extent is O2-concentrating as a consequence saturation was the reduction or even prevention of stress of CO2-concentrating counteracting the effect of the latter? due to over-energization of the photosynthetic apparatus In most cases O2/CO2 ratios in the leaves in Phase III and production of reactive oxygen species (ROS). This has remain well below those in the ambient atmosphere by a even been considered to have been a major driving force factor of 0.1±0.5 (Spalding et al., 1979; Table 2). for the evolution of CAM (Gil, 1986). Conversely, it is Nonetheless, photorespiration may make important con- now known that CAM plants are subject to photoinhibi- tributions to oxygen metabolism in Phase III of CAM tion, not only in Phase IV when stomata are open and CO2 (Thomas et al., 1987). Catalase (CAT), the H2O2 scaveng- is taken up from the atmosphere, but also in Phase III with CO ing enzyme in photorespiration, remained unchanged the very high pi 2 (see LuÈttge, 2000, for a review). during C3 to CAM shifts in the C3/CAM intermediate This is not the occasion to get involved in the species Sedum album under mild drought stress increasingly sophisticated discussion of protective acute (Castillo, 1996). In the C3/CAM intermediate species photoinhibition or scaling down of photosystem II ef®- Mesembryanthemum crystallinum CAT activity was sig- ciency, with electrical or zeaxanthin cycle quenching of ni®cantly reduced in the CAM-state compared to the C3- potential quantum yield of PSII, destructive but protective state. However, during the diurnal cycle it increased in the D1-protein turnover and chronic destructive photoinhibi- light period and showed a peak in the late afternoon tion. It is just important to note that in the CO2- (Niewiadomska et al., 1999). Nothing is known about the concentrating Phase III, photoinhibition does occur; the O O dynamics of pi 2 in the light period and to what extent pi 2 xanthophyll cycle is involved (Adams et al., 1987; Adams, remains high at the end of Phase III when the malic acid 1988; Keiller et al., 1994; Herzog et al., 1999; see LuÈttge, CO store is exhausted, pi 2 declined and Phase IV stomatal 2000, for a review) and acclimation to high light stress may opening has not (yet) occurred. Under particular water occur in CAM plants (Adams et al., 1987; Winter and stress Phase IV stomatal opening may not be expressed at Awender, 1989; Fetene et al., 1990; LuÈttge et al., 1991; all (Smith and LuÈttge, 1985). Photorespiration may then Robinson and Osmond, 1994; Adams and Demmig- become very important. Miszalski et al. (2001) used the Adams, 1996). 2138 LuÈttge Therefore, is reduced oxidative stress really to be regulation in that it inhibits PEPC and the vacuolar H+- considered as a bene®cial consequence of CO2-concen- transporting V-ATPase, thus avoiding futile recycling of trating in Phase III of CAM? What are the bene®ts of C3- CO2 into malate and vacuolar malic acid during Phase III. photosynthesis contrasted with CAM? In the studies of C3/ However, the high apoplastic and leaf air-space CO2- CAM-intermediate plants the question mostly asked is concentrations must have effects on cell wall pH (Fig. 1). what drives the shift from C3-photosynthesis to CAM? These effects may be quite important when piCO2 attains When reversible shifts occur in both directions, which is several per cent, although these cases may be rare the case in the perennial C3/CAM-intermediate Clusia (Table 1). There may also be other consequences, for minor, what drives a shift from CAM to C3-photosynthesis example, on plasma membrane electrical potentials (de Mattos et al., 2001)? In fact, for C. minor it was shown (Hedrich et al., 2001). This has been completely over- that it was not so much CAM per se which offered looked so far. There is no work on apoplastic pH in CAM ecological advantages but the ¯exibility given by CAM cells in Phase III nor any discussion of it in the CAM and C3 options (Herzog et al., 1999). Moreover, in growth literature. chamber experiments when C. minor switched from CAM Phase III oxidative stress to C3-photosynthesis, CO2-uptake over 24 h (dark ®xation plus light ®xation of external CO2) more than doubled. The O2-concentrating consequence of CO2-concentrating This implies that the integrated daily photon utilization for in planta has been demonstrated above. Studies of O2- the photochemical work of CO2-assimilation increased. exchange by mass spectroscopy of stable oxygen isotopes The cost paid by the plant is water because, concomitantly, (16O, 18O) and the main electron ¯ow of chloroplasts have WUE was reduced to a third (de Mattos et al., 2001). revealed the strong O2 production in CAM plants in the However, this observation supports the idea that C3- light (Thomas et al., 1987). Experiments measuring O2 photosynthesis may provide superior protection from evolution in the leaf disc O2-electrode, where leaf discs of irradiance stress compared to CAM. This was already CAM plants have been arti®cially exposed to 5% ambient suggested some time ago by the ®nding that an increase of CO2, were performed (Adams et al., 1987; Adams and photosynthetically active radiation from 360 to 1200 mmol Osmond, 1988; Borland and Grif®ths, 1989; Maxwell ±2 ±1 m s led to a suppression of night-time CO2-®xation and et al., 1998), and Osmond et al. (1996) note the action of much increased daytime CO2-®xation in Clusia minor, but the `O2-pump' leading `to ever-increasing O2-concentra- only when plants were well watered (Schmitt et al., 1988; tion in the experimental chamber as malate decarboxyla- LuÈttge, 1996). tion proceeds'. Thus Phase III CO2-concentrating not only stays short of preventing photorespiration and photoinhibi- tion (see above), but the corresponding O -concentrating Adverse ecophysiological consequences 2 even may be one of the major adverse consequences. O If some of the established bene®cial consequences of CO2- High pi 2 supports formation of aggressive reactive concentrating are being questioned, is it also necessary to oxygen species (ROS) such as hydrogen peroxide (H2O2),

d ± d even pinpoint some adverse consequences? There are at superoxide (O2 ) and the hydroxyl radical (OH ). CAM least three, of which the third is a major one: plants are especially equipped to deal with this oxidative stress by the increased expression of an antioxidative Energetics response system (ARS). In the C3/CAM-intermediate CAM is more costly energetically than C3-photosynthesis. Sedum album this appeared to be especially important The stoichiometrics of paper-biochemistry have been during the C3 to CAM transition when the activities of worked out in every detail (Winter and Smith, 1996b). ascorbate peroxidase, superoxide dismutase (SOD), Energy demand was also measured under laboratory gluthatione reductase, and monodehydroascorbate reduc- conditions (Maxwell et al., 1998). Whether this is a tase as ROS scavenging enzymes were increased (Castillo, limiting factor under actual environmental conditions with 1996). In the C3/CAM-intermediate M. crystallinum, adverse ecophysiological consequences is much less clear. where CAM-induction is elicited by salt stress, it is often It might be in shaded habitats of CAM plants in tropical dif®cult to distinguish ARS expression in response to forests. However, as an example, among shaded, semi- salinity from requirements of CAM, for example, the up- shaded and exposed plants of the CAM-bromeliad regulation of cytosolic CuZn-dependent SOD (Hurst and Bromelia humilis it was the shaded individuals, which Ratajczak, 2002). It appears, however, that up-regulation showed the lushest growth (Lee et al., 1989). of mitochrondrial Mn-dependent SOD is a typical reaction to the induction of CAM (Miszalski et al., 1998; Broetto pH et al., 2002). Phase III CO2-concentrating has pH-effects. The acidi®- By cross-tolerance this may also explain the obser- cation of the cytosol by 0.3 pH units (Hafke et al., 2001; vation that CAM plants are less sensitive to the see above) may have useful consequences in cellular oxidative stress given by SO2 and O3 than C3 plants CO2-concentrating in CAM 2139

Table 3. O2-evolution and SO2-oxidation by acidi®ed (loaded Acknowledgements with malic acid) and deacidi®ed (malic acid broken down) I thank Richard Leegood and Uwe Rascher for reading this review protoplasts of KalanchoeÈ daigremontiana (Miszalski et al., and for valuable suggestions prior to publication. 1997) ±1 In O2-evolution measurements either no bicarbonate or 10 mmol l bicarbonate was added to the medium. This is not speci®ed by the References authors for the SO2-oxidation measurements. For direct comparison ±1 with rates of O2-evolution, rates of SO2-oxidation h were calculated Adams III WW. 1988. Photosynthetic acclimation and taking four times the SO2 oxidized in the ®rst 15 min of the photoinhibition of terrestrial and epiphytic CAM tissues experiment shown. growing in full sunlight and deep shade. Australian Journal of Plant Physiology 15, 123±134. Rate (mmol mg±1 chlorophyll h±1) Deacidi®ed Acidi®ed Adams III WW, Demmig-Adams B. 1996. Energy dissipation and ± O2-evolution without HCO3 8.6 15.7 the xanthophyll cycle in CAM plants. In: Winter K, Smith JAC, ± O2-evolution with 10 mM HCO3 13.2 32.3 eds. Crassulacean acid metabolism: biochemistry, ecophysiology SO2-oxidation 9.2 22.0 and evolution. Ecological Studies, Vol. 114. Berlin, Heidelberg, New York: Springer Verlag, 97±114. Adams III WW, Osmond CB. 1988. Internal CO2 supply during photosynthesis of sun and shade grown CAM plants in relation to photoinhibition. Plant Physiology 86, 117±123. (Olszyk et al., 1987; Miszalski et al., 1997). This is Adams III WW, Osmond CB, Sharkey TD. 1987. Responses of not an effect of day-time stomatal closure as shown by two CAM species to different irradiances during growth and using protoplasts or leaf discs of KalanchoeÈ daigre- susceptibility to photoinhibition by high light. Plant Physiology montiana with the epidermis stripped off. The larger 83, 213±218. 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