JXB Advance Access published online on April 23, 2008
Journal of Experimental Botany, doi:10.1093/jxb/ern006
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SPECIAL ISSUE REVIEW PAPER |
Clusia: Holy Grail and enigma
Institut für Botanik, Technische Universität Darmstadt, Schnittspahnstrasse 3–5, D-64287 Darmstadt, Germany
* E-mail: luettge{at}bio.tu-darmstadt.de
Received 30 August 2007; Revised 11 December 2007 Accepted 7 January 2008
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Abstract |
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Clusia is the only genus with bona fide dicotyledonous trees performing Crassulacean acid metabolism (CAM). Clusia minor L. is extraordinarily flexible, being C3/CAM intermediate and expressing the photosynthetic modes C3, CAM, CAM-cycling, and CAM-idling. C3 photosynthesis and CAM can be observed simultaneously in two opposite leaves on a node and possibly even within the same leaf in the interveinal lamina and the major vein tissue, respectively. The relative activity of photosystem II (
PSII) indicating photosynthetic energy use, is larger under photorespiratory than under non-photorespiratory conditions due to the particular energy demand of photorespiration. The heterogeneity of PSII over the leaves as visualized by chlorophyll fluorescence imaging in the C3 mode is larger under non-photorespiratory conditions than under photorespiratory conditions. These observations indicate that photorespiration, presumably by its particular energy demand, synchronizes photosynthetic activity over the leaves. In the CAM mode, the heterogeneity of PSII is more dependent on the transitions between CAM phases. Free-running circadian oscillations of photosynthesis are strongly dampened in both the C3 and the CAM mode. Photorespiration is under circadian clock control in both the C3 and the CAM mode. PSII and the heterogeneity of PSII oscillate in phase with CO2 uptake and photorespiration only under non-photorespiratory conditions. Under photorespiratory conditions, PSII does not oscillate and there is no heterogeneity, again indicating the stabilizing function of photorespiration. Plants acclimatized to perform CAM switch to C3 photosynthesis during free-running oscillations while subjected to constant illumination. Key words: Circadian rhythmicity, photorespiration, photosynthetic physiotypes, photosystem II activity, physiotypic plasticity
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Introduction: the Clusia grail and its enigmas |
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 TopAbstract Introduction: the Clusia grail... Physiotypic plasticitySimultaneous C3 photosynthesis...PhotorespirationFree-running circadian...Circadian regulation of...CAM to C3 shift...ConclusionsReferences |
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According to medieval legends which climaxed in the great poem of Richard Wagner, Perceval arrived in the forest where the Holy Grail was hidden in the castle and, under the guidance of Gurnemanz, he saw the grail. However, he did not achieve a full understanding of its enigma (Wagner, 2005). The author of this review in 1983 arrived at the island of Trinidad and, under the guidance of Griffiths (Griffiths et al., 1986), on 26 March climbed Cerro del Aripo and saw Clusia. However, he did not achieve any understanding if its enigma. Perceval was released from the grail in disgrace. He roamed around learning about the miracle and proving his love and innocence; after a long time he returned and became master of the Holy Grail. The author left Trinidad without access to the wonders of Clusia. He moved around, first learning about the miracle from Tinoco Ojanguren and Vazquez-Yanez (1983) and Ting et al. (1985), and by proving his love and interest he was striving to become a master of the Clusia grail (Lüttge, 2007a).
Why should Clusia be considered a grail? It is extraordinarily unique as it is the only bona fide dicotyledonous tree genus performing Crassulacean acid metabolism (CAM) (Lüttge, 2007b). Its enigma remains after more than two decades of Clusia research; the enigma being why it is the only genus of trees performing CAM and yet, nevertheless, is ecologically highly successful. The Clusia grail embraces a huge ecological amplitude with extraordinary morphotypic and physiotypic plasticity and diversity (Lüttge, 2006, 2007c, d; Gustafsson et al., 2007; Lüttge and Duarte, 2007). A large variety of habitats are occupied by this genus, with extremes ranging from coastal sites to moist tropical forests and from highly exposed rock outcrops to shaded epiphytic canopy niches. There is a suite of morphological characteristics, with life forms such as terrestrial, epiphytic, hemi-epiphytic, and strangler, as well as physiological and biochemical attributes.
This is widely covered in the reviews quoted and does not need to be reiterated here. In one of these reviews I addressed questions and lessons regarding Clusia (Lüttge, 2006). New questions and enigmas arise such as possible simultaneous performance of C3 photosynthesis and CAM within the same leaf, high dampening of clock-controlled free-running oscillations in both the C3 mode and the CAM mode of the C3/CAM intermediate Clusia minor L., and CAM to C3 shifts during free-running oscillations; more recent advances pertain to the role of photorespiration in the physiological performance and circadian rhythmicity of Clusia (Duarte, 2006; Duarte and Lüttge, 2007a, b), which will be addressed in this contribution.
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Physiotypic plasticity |
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The species C. minor is one of several C3/CAM intermediate species that have been described among Clusias and other genera. The most intensively studied C3/CAM species, including its molecular biology, is Mesembryanthemum crystallinum L. which has attained the status of a ‘model plant’. This is an annual species where a switch from C3 photosynthesis to CAM is elicited by various forms of environmental stress, particularly drought and salinity stress. It is still debated whether independent of and/or in addition to that there is an intrinsic developmental programme for a C3 to CAM shift as plants age, However, recent evidence clearly shows that M. crystallinum can complete its entire life cycle without ever showing CAM-type nocturnal CO2 uptake (Winter and Holtum, 2005, 2007). Be this as it may, with the short life time of this annual species, reversibility from CAM back to C3 photosynthesis is limited (Ratajczak et al., 1994). The situation for the perennial tree C. minor is completely different. The longevity of Clusia leaves in the field is at least 2 years (Olivares, 1997). Therefore, a one-off change between modes of photosynthesis in response to environmental factors would be of little benefit. Indeed, in Clusia, reversible switches back and forth between C3 photosynthesis and CAM are frequent and independent of any developmental programme (de Mattos and Lüttge, 2001).
However, the plasticity is even more extensive than that. Clusia minor is capable of expressing four different photosynthetic physiotypes: (i) pure C3 photosynthesis; (ii) pure CAM; (iii) CAM-cycling where stomata may remain closed during the night and respiratory CO2 is recycled in the dark period via phosphoenolpyruvate carboxylase (PEPC) and storage of organic acids for subsequent CO2 reduction in the light period via the Calvin cycle after decarboxylation of the organic acids, in addition to CO2 uptake via open stomata and fixation via ribulose-bisphosphate carboxylase/oxygenase (RubisCO); and (iv) CAM-idling where stomata remain closed night and day and respiratory CO2 is recycled (Lüttge, 2006, 2007c).
Clusia minor was shown to be so flexible that even two opposite leaves on one node were simultaneously performing C3 photosynthesis and CAM, depending on the respective conditions of irradiance and leaf to air water vapour pressure deficits they were subjected to (Lüttge, 2007c). Now there are indications that even different parts of a given leaf at the same time may perform C3 photosynthesis and CAM, and this is related to leaf anatomy.
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Simultaneous C3 photosynthesis and CAM in different parts within the same leaf |
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TopAbstractIntroduction: the Clusia grail...Physiotypic plasticitySimultaneous C3 photosynthesis...PhotorespirationFree-running circadian...Circadian regulation of...CAM to C3 shift...ConclusionsReferences |
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Chlorophyll fluorescence imaging of leaves allows a spatiotemporal resolution of the relative activity of photosytem II (
PSII) over individual leaves as a measure of photosynthetic energy use. This was used for separate recording of PSII over the lamina and the major vein of leaves of C. minor acclimatized to perform C3 photosynthesis and CAM, respectively (Fig. 1). The plants were kept at 25 °C and 120 µmol m–2 s–1 photosynthetically active radiation (PAR). These conditions and the relatively low irradiance in many previous trials have been found to be most reliable for keeping well-watered plants in the C3 mode of photosynthesis and to induce CAM by drought stress.
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It can be seen in Fig. 1 that, in the lamina, in the C3 mode
PSII increases rapidly in the early morning while in the CAM mode the increase is more gradual because in the morning both PEPC and RubisCO contribute to fixation of CO2 (phase II of CAM), but only RubisCO involves photochemical energy use. Over the major vein, the pattern is CAM like in both cases, i.e. in the C3-acclimatized leaves the green vein tissue may have retained a CAM activity. The enigma posed is whether this is just a side-effect of incomplete regulation during acclimation or a selected useful trait in the physiology of the Clusia leaves. Succulent leaves of obligate CAM plants, for example in the genus Kalanchoë, normally have a simple anatomy with only densely packed isodiametric cells. In contrast, the lamina of leaves of C. minor has a bifacial structure with an adaxial multilayered palisade parenchyma (Fig. 2A) and an abaxial spongy parenchyma with large intercellular air spaces (Fig. 2B). However, the green tissue above the major veins of the leaves is more ‘Kalanchoë- or CAM-like’ with densely packed spherical cells. Lateral intercellular gas diffusion of CO2 has a signalling function in synchronizing photosynthetic activity in leaves of K. daigremontiana Hamet et Perrier but it is hindered by the large resistance provided by the dense packing of the leaf cells (Duarte et al., 2005). The different behaviour of lamina and vein in C. minor can be explained by only weak coupling by lateral gas diffusion between the lamina and the green vein tissue (Duarte and Lüttge, 2007b; Lüttge, 2007e). Performance of CAM by the vein tissue even under C3 conditions in the lamina might be useful for retrieval of respiratory CO2 in the area of the bundles. However, this still remains an intriguing question.
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Photorespiration |
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There is ample evidence that like all other photosynthesizing organisms CAM plants are also subject to light stress and oxidative stress, are performing photorespiration, and are furnished with mechanisms of non-photochemical quenching of photosynthetic excitation, i.e. thermal energy dissipation via zeaxanthin and the xanthophyll cycle and related mechanisms. In an earlier CAM workshop, Heber et al. (2002) underlined this point forcefully. It was often argued, however, that in CAM these phenomena were largely restricted to transitions in the morning and afternoon, when stomata were open and atmospheric CO2 was assimilated (phases II and IV). It was thought that the effect of CO2 concentrating behind closed stomata saturating the carboxylation activity of RubisCO when nocturnally stored organic acid was remobilized (phase III) would suppress oxidative stress and photorespiration. The observation that concomitantly with CO2, O2 is also concentrated within the leaves during phase III greatly modified this view (Lüttge, 2002a).
Duarte (2006; Duarte and Lüttge, 2007b) was the first to study photorespiration online together with gas exchange and PSII (Fig. 3) in plants of C. minor acclimatized to perform C3 photosynthesis and CAM, respectively, by using drought stress as a differentiating parameter. He used an array of instruments co-ordinated for simultaneous measurements of the relevant photosynthetic parameters net CO2 exchange, leaf conductivity for water vapour, effective quantum yield of photosystem II (PSII), and its spatiotemporal dynamics and heterogeneity visualized by chlorophyll fluorescence imaging. The heterogeneity of PSII was calculated from chlorophyll fluorescence images by using the nearest neighbour principle based on cellular automaton algorithms (Hütt and Neff, 2001; Duarte and Lüttge, 2007b). Photorespiration was recorded using a device automatically applying air with only 1% O2 which allowed establishment of non-photorespiratory conditions for 20 min at intervals during the online measurements. Photorespiration (
) was obtained by the difference between net CO2 uptake under non-photorespiratory conditions (1% O2 in the air: giving total carboxylation acitivity of RubisCO, 
-max) minus net CO2 uptake under photorespiratory conditions (21% O2 in the air: giving total carboxylation minus oxygenation activity of RubisCO, = 
-max– ):
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) were measured by infrared gas analysis. A synopsis of the pattern obtained is shown in Fig. 3. Photorespiration was obvious in the C3 mode and in phases II and IV of CAM. It could not be measured in phase III of CAM because the stomata were closed so that the air with only 1% O2 did not have access to the leaf interior. That photorespiration was greater in early phase IV than later on may be an after-effect of O2 concentrating during phase III inside the leaves. In the C3 mode,
PSII, indicating photosynthetic energy use, was much larger under photorespiratory conditions than under non-photorespiratory condtions. This agrees with the fact that photorespiration consumes more energy than CO2 assimilation (Osmond and Grace, 1995; Heber et al., 2001; Heber, 2002). A similar pattern is seen in phases II and IV of CAM.
In the C3 mode, the heterogeneity of PSII was much larger under non-photorespiratory conditions than under photorespiratory conditions. This shows that the higher energy demand of photorespiration homogenizes energy use over the leaves while with carboxylase activity of RubisCO alone, patchiness or heterogeneity is building up. It is assumed that lateral CO2 gas diffusion within leaves is responsible for spatiotemporally synchronizing the activities of cells over the leaf lamina or suppressing patchiness and heterogeneity (Lüttge, 2007e). Evidently with the CO2 substrate of RubisCO alone in the C3 mode, heterogeneity is maintained. However, with the second substrate, O2, also involved in both lateral diffusion and particular energy demand as indicated by PSII energy use, is synchronized over the leaves. Again this poses an enigma and an intriguing question. Why might there be a requirement for such energy compensation over the leaves and why should photorespiration be exploited for energy disposal rather than thermal dissipation. Or are both just different faces of the same coin?
In the CAM mode, the situation is much more complex due to the greater number of interacting and counteracting factors that are involved. Heterogeneity depends not only on the relative energy demand of CO2 fixation and photorespiration but also on the expression of CAM modes, and, as previous studies (Rascher et al., 2001; Rascher and Lüttge, 2002) showed, particularly on the latter. Thus, a direct comparison with the C3 mode is not easy. For example, in phase II of CAM, both carboxylating enzymes, PEPC and RubisCO, are active. Only RubisCO is consuming photosynthetic energy and under non-photorespiratory conditions it is somewhat favoured in the competition with PEPC. Therefore, while PEPC is down-regulated during phase II, energy demand should increase. Simultaneously in the dynamics of phase II, however, malate remobilization and internal CO2 concentrating set in, with both the synchronization signalling by CO2 and the concentrating of CO2 favouring the carboxylase function of RubisCO with its lower energy demand than the oxygenase function and photorespiration.
Under photorespiratory conditions, the heterogeneity of PSII was high in phase II when both PEPC (not using photosynthetic excitation energy) and RubisCO were active, and much higher than in the C3 mode. It was very low in phase III, when both CO2 and O2 were concentrated inside the leaves, high concentrations supporting lateral diffusion within the leaves. It was still low in early phase IV when particularly high oxygen concentrations may still have been effective inside the leaves, and it was increased in the later phase IV to levels comparable with those of the C3 mode. Under non-photorespiratory conditions, the heterogeneity was similar in phases II and IV but much lower than in the C3 mode. Therefore, it appears that in the CAM mode, spatiotemporal dynamics of heterogeneity are a particular consequence of changing CO2 and O2 regimes inside the leaves during the transitions of CAM phases.
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Free-running circadian oscillations of photosynthesis in the C3 and CAM modes: rapid dampening |
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The circadian rhythmicity of photosynthesis was originally studied in depth in unicellular algae (for a review, see Lüttge, 2002b). Circadian rhythmicity of photosynthesis in vascular C3 plants (Hennessey and Field, 1991; Li et al., 1992; Hennessey et al., 1993) and C4 plants (Britz et al., 1987) has also been described. Circadian rhythmicity in CAM is particularly well studied physiologically and biochemically, especially in obligate CAM plants of the genus Kalanchoë (Wilkins, 1992; Borland et al., 1999; Lüttge, 2002a, b). Many genes in green plants are under clock control, including genes relevant for photosynthesis (Harmer et al., 2000; Dodd et al., 2005). In relation to CAM, some studies at the molecular level were also performed, especially with respect to circadian oscillations of the activity of the key enzyme of dark fixation of CO2, PEPC, and its phosphorylation by PEPC kinase (Carter et al., 1991; Hartwell et al., 1996, 1999, 2002). More detailed molecular studies on the circadian expression of various clock genes were performed with M. crystallinum (Boxall et al., 2005). The overt outputs of CAM oscillators mainly studied so far were gas exchange, i.e. of CO2 and water vapour, and night/day changes of organic acid levels. Occasionally carbon isotope ratios,
13C, were also used to characterize oscillations of the activities of the carboxylating enzymes, PEPC and RubisCO (Grams et al., 1996). More recently oscillations of PSII and its spatiotemporal dynamics during CAM were also studied using chlorophyll fluorescence imaging (Rascher et al., 2001; Rascher and Lüttge, 2002).
Circadian oscillations of net CO2 exchange, , and leaf conductance for water vapour, 
, of plants of C. minor acclimatized to the C3 and CAM mode, respectively, were recorded by Duarte (2006) and Duarte and Lüttge (2007a, Fig. 11.1) under constant illumination with a PAR of 120 µmol m–2 s–1. The endogenous rhythms were highly dampened after a few periods. In the C3 mode there were four clear endogenous periods at 30 °C and five at 25 °C in both and . At 21 °C, about six periods in were clearly expressed but looked arrhythmic. In the CAM mode there were a few more endogenous periods at 30 °C, i.e. up to nine, which had highly dampened amplitudes, however. At 25 °C and 21 °C, there were 5–6 endogenous periods, which again were highly dampened. At 25 °C effective quantum yield of PSII, 
F/F'm, was also measured using a pulse amplitude modulated fluorometer (Fig. 4). The endogenous oscillations had a clearly larger amplitude in the CAM mode than in the C3 mode, and persisted for longer. However, in both modes, the oscillations of F/F'm dampened out more rapidly than those of and , and the dampening was associated with an overall decline of F/F'm. This was also correlated with a general reduction of in the arrhythmic stage attained after the loss of rhythmicity which must have been the reason for a similarly reduced 
, explaining the reduced F/F'm by substrate limitation of the carboxylase activity of RubisCO, and hence energy use of PSII.
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It is intriguing that in both modes of photosynthesis the endogenous rhythm of C. minor was rapidly dampened out. For the C3 mode, a comparison with the obligate C3 plant Phaseolus vulgaris L. is interesting, where rhythmicity of photosynthesis was highly dampened under certain conditions but under other conditions it has been shown to last for many endogenous periods (Hennessey and Field, 1991). For the CAM mode, comparison can be made with the obligate CAM plant K. daigremontiana where the endogenous gas exchange rhythm continues with unchanged amplitude for very many periods over several weeks (Lüttge and Beck, 1992). Is the strong dampening a special property of C. minor? Endogenous circadian rhythmicity may enhance productivity of plants (Dodd et al., 2005) and it is regularly argued in the literature that it is important for organisms to anticipate diurnally changing conditions in order to be fit by preparedness (Lüttge, 2002b). Looked at in this way, endogenous circadian rhythmicity could be a hindrance for plasticity needing flexibility. Thus, it would make sense if strongly dampened rhythmicity were an intrinsic property of a plant as ecophysiologically as flexible as C. minor. This is one more of its enigmas.
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Circadian regulation of photorespiration |
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The complement of technical approaches for simultaneous online measurements of CO2 and water vapour gas exchange, the separate activities of carboxylation and oxygenation of RubisCO, absolute values of effective quantum use efficiency of PSII, and relative quantum use efficiency of PSII with spatiotemporal resolution used in the work of Duarte (2006, see also above) allowed the demonstration for the first time of the existence of circadian control of photorespiration in plants. Using the C3/CAM intermediate C. minor, clock-controlled photorespiration could be revealed for both modes of photosynthesis (Duarte, 2006; Duarte and Lüttge, 2007a).
In both the C3- and the CAM-acclimatized plants of C. minor, and display circadian oscillations (Figs 5, 6). The oscillations of and are in phase, which shows that both substrate reactions of RubisCO oscillate in phase and are not under separate circadian clock control. PSII oscillates in phase with and , but only under non-photorespiratory conditions. Under photorespiratory conditions, it does not oscillate. As PSII is a measure of photosynthetic energy use, this suggests that under varying energy demand during the oscillations of carboxylation and oxygenation activities of RubisCO, photorespiration with its particularly high energy demand (Osmond and Grace, 1995; Heber et al., 2001; Heber, 2002) has a compensating effect. It confirms conclusions obtained from experiments under a normal dark/light regime that photorespiration stabilizes photosynthetic energy use (see above).
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The highest energy demand during endogenous periods was reached in the peaks of
. In the CAM mode when photorespiration is suppressed, the energy consumed due to the oxygenation activity of RubisCO may be diverted to other energy-consuming processes. In the normal external dark/light rhythm of CAM, organic acid synthesis and transport into the vacuoles mainly occur in the dark and use respiratory energy. In constant light, the energy for these processes can also be supplied by the light reactions of photosynthesis but only in direct competition with the Calvin cycle and photorespiration. This competition evidently must be much larger in the peaks than in the troughs of the rhythm. Such a competition of photochemical work of photosynthesis and photorespiration with non-photochemical work of organic acid synthesis via PEPC and its transport across the tonoplast is reflected in the observation that the linear correlation between PSII and the sum of the carboxylation and oxygenation rates of RubisCO (measured as maximum rates of CO2 uptake under 1% O2, see above) is less pronounced in the CAM-acclimatized (r2=0.75) than in the C3-acclimatiaed (r2=0.96) plants (Fig. 7).
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The curves of Figs 5 and 6 and the corresponding chlorophyll fluorescence images of Figs 8 and 9 show that under photorespiratory conditions in both modes there was no heterogeneity of
PSII over the leaves during the oscillations of photosynthesis and photorespiration. However, there was heterogeneity of PSII under non-photorespiratory conditions, and the spatial structure showed oscillations between homogeneous and heterogeneous states. Except for the first circadian phases, heterogeneity was low in the peaks and high in the troughs of , , and PSII, i.e. high photosynthetic activity synchronized events over the leaves.
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CAM to C3 shift during circadian oscillations |
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In the experiments of Duarte (2006; Duarte and Lüttge, 2007a) when the plants of C. minor were acclimatized to perform C3 photosynthesis, they showed the C3 pattern of gas exchange under an external dark/light (DL) regime with respiratory loss of CO2 in the dark and strong CO2 uptake in the light just before application of continuous light (LL) and again when they were returned to DL subsequent to LL. That means they remained in the C3 mode to which they had been acclimatized. The plants acclimatized to the CAM mode in DL prior to LL showed a CAM pattern with nocturnal CO2 uptake (phase I), an early morning peak of CO2 uptake (phase II), followed by stomatal closure (phase III), and again net CO2 uptake in the afternoon (phase IV). However, when they were returned to DL subsequent to LL, they showed a C3 pattern with daytime net CO2 uptake. This means that in contrast to the C3 mode plants, they did not remain in the mode to which they had been acclimatized but changed to the opposite mode, the C3 mode, which they did not perform before LL, i.e. they switched the mode either during LL or at the onset of DL after LL.
This observation, that during the endogenous oscillation the CAM-acclimatized plants of C. minor shift to C3 photosynthesis, has an interesting analogy in the obligate CAM plant K. daigremontiana (Fig. 10). In this plant it was seen that the circadian rhythm of malic acid levels is strongly dampened during 3–4 endogenous periods under LL without any reduction of the amplitudes of the gas exchange rhythm, which continues unchanged for many periods (Fig. 10; Wyka and Lüttge, 2003). It is important to underline this observation here because it does not fully rule out that vacuolar malic acid accumulation is a central pacemaker process in K. daigremontiana as is somewhat imprecisely quoted occasionally (Boxall et al., 2005). The situation is much more complex as also correctly evaluated by Borland and Taybi (2004). The observations imply that during the initial circadian periods CAM-type malic acid oscillations may well be the pacemaker with, however, an increasing contribution from C3-like processes during the rhythm, without that being noticed in the overt rhythmic output of gas exchange. This also becomes obvious when nocturnal malate accumulation is prevented artificially in experiments (Wyka et al., 2004). The findings with C. minor show that carboxylase and oxygenase functions of RubisCO are under similar circadian control (see above), i.e. they appear to be controlled together and not separately, and hence it is RubisCO itself which must be under clock control. This leads to the assumption that there are independent C3-like and CAM-like oscillators which are working in a trade-off-like fashion in the obligate CAM plant K. daigremontiana, where both oscillators together are important to maintain rhythmicity even when the contribution of CAM is reduced. In C. minor, both oscillators dampen out, with the loss of rhythmicity after a few periods in both modes of photosynthesis.
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It can be envisaged that the PEPC kinase gene (Carter et al., 1991; Hartwell et al., 1996, 1999, 2002; Borland et al., 1999) is at a lower hierarchical level than the central clock genes and regulated downstream of them. It also appears to be under direct metabolic control as its regulation depends on cytoplasmic malate levels (Borland et al.. 1999). These, in turn, are determined by the malic acid oscillations of vacuolar influx and efflux. Thus, within the CAM-like oscillator we have the two interconnected PEPC kinase and vacuolar malate oscillators. As regards the C3-like oscillator the decisive C3 elements which make an increasing contribution during circadian rhythmicity in the CAM mode in ongoing LL are not known. They could be involved in RubisCO activation or regulation of stomatal guard cell movements, or both. Possibly upstream clock components play a role, i.e. central clock genes. The central clock genes have been studied in the C3/CAM intermediate M. crystallinum in great detail (Boxall et al., 2005). In M. crystallinum they are the same as in the C3 annual Arabidopsis thaliana (L.) Heynh. There are genes whose expression only oscillates in either the C3 mode or the CAM mode, and other genes oscillate in both modes of photosynthesis. The most important ones are CCA1 (circadian clock associated), LHY (late elongated epicotyl), TOC1 (timing of CAB expression), ELF 3 and ELF 4 (early flowering 3 and 4), ZTL (zeitlupe), and FKF1 (flavin-binding kelch repeat F-box 1). Among them, CCA1/LHY, TOC1, and ELF 4 oscillate in both the C3 and the CAM mode. Under LL, the phases of the oscillations of the TOC1 transcript in the C3 state and in the CAM state in M. crystallinum are strongly offset against each other, and the phases of CCA1 and LHY are slightly offset in both modes. Unfortunately, the study of Boxall et al. (2005) does not cover the physiological and biochemical background of the molecular performance of the plants, and their molecular analyses only cover the first two endogenous circadian periods. What is therefore not known is if in M. crystallinum the endogenous rhythm was only connected to malic acid oscillations in the early periods which Boxall et al. (2005) measured and if later the CAM rhythm may have shifted to a more C3-like behaviour as in K. daigremontiana and C. minor. Evidently the message of Fig. 10 needs to be borne in mind in future molecular studies.
Taken together, these observations reveal a network with heterarchical regulatory levels from putatively upstream central clock gene elements to downstream biochemical and biophysical pacemakers of overt rhythmic output. The nodes, i.e. the various functional elements, in the network linked via edges are intricately interconnected by feedback loops.
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Conclusions |
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The uniqueness of the Clusia grail remains with the major question: why is Clusia the only bona fide dicotyledonous tree genus having CAM but which nevertheless is ecologically so successful?
The other intriguing questions and enigmas to be elucidated by future work are:
- Are C3 photosynthesis and CAM in parallel in different cells of the same leaf accidental or controlled?
- Would a robust circadian clock impair ecophysiological flexibility?
- Is there circadian control of photorespiration-related genes? So far this does not appear to be known. Harmer et al. (2000) do not particularly mention photorespiration and gene products involved in it among the ‘key pathways’ they discuss in orchestrated transcription in Arabidopsis.
- In the shift back to C3 photosynthesis during free-running oscillations in the CAM mode, which and where is the organizer?
Beyond the medieval legends, the quest for the Holy Grail has roots in very ancient pagan myths with implications of fitness and fertility, plenty, perfection, and uniqueness. Perhaps more detailed specific studies may unravel similar plasticity in the expression of modes of photosynthesis and fitness in niche occupation in C3/CAM intermediate species of other genera, but as far as we can see the uniqueness of Clusia of being the only dicotyledonous trees with such capacities remains.
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Acknowledgements |
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I thank Professor Howard Griffiths for his kindness in inviting me to the C4–CAM workshop in Cambridge, notwithstanding my retirement, and for his suggestion of the title for this review. My article dwells strongly on the work in the thesis of my last PhD student Dr Heitor M Duarte whose contributions to the Holy Grail of Clusia are most gratefully acknowledged.
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