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Sedoheptulose accumulation under CO2 enrichment in leaves of Kalanchoë pinnata: a novel mechanism to enhance C and P homeostasis?

In contrast to the well-documented roles of its mono- and bisphosphate esters, the occurrence of free sedoheptulose in plant tissues remains a matter of conjecture. The present work sought to determine the origin of sedoheptulose formation in planta, as well as its physiological importance. Elevated CO2 and sucrose induction experiments were used to study sedoheptulose metabolism in the Crassulacean acid metabolism (CAM) plants Kalanchoë pinnata and Sedum spectabile. Experimental evidence suggested that sedoheptulose is produced from the oxidative pentose phosphate pathway intermediate sedoheptulose-7-phosphate, by a sedoheptulose-7-phosphate phosphatase. Carbon flux through this pathway was stimulated by increased triose-phosphate levels (elevated CO2, compromised sink availability, and sucrose incubation of source leaves) and attenuated by ADP and inorganic phosphate (Pi). The accumulation of free sedoheptulose is proposed to act as a mechanism contributing to both C and P homeostasis by serving as an alternative carbon store under elevated CO2 or a compromised sink capacity to avoid sucrose accumulation, depletion of inorganic phosphate, and suppression of photosynthesis. It remains to be established whether this acclimation-avoiding mechanism is confined to CAM plants, which might be especially vulnerable to Pi imbalances, or whether some C3 and C4 plants also dispose of the genetic capacity to induce and accelerate sedoheptulose synthesis upon CO2 elevation.


Introduction
During the last 100 years, human activities have strongly contributed to the rising CO 2 levels in the atmosphere. The impact of this change and its putative correlation with global warming has been extensively debated (Rogelj et al., 2010). It is clear that the increasing CO 2 concentrations will also affect the production of future crops and algae, but the effect might differ greatly depending on the species, nutritional status, and other environmental factors (Xu et al., 2010). In general, elevation of atmospheric CO 2 concentration enhances photosynthesis and stimulates growth and productivity in most plant species covering different photosynthetic pathways, i.e. C 3 , C 4 , and Crassulacean acid metabolism (CAM) (Poorter and Navas, 2002;Ainsworth and Long, 2005;Ceusters and Borland, 2011). However, the initial enhancement is often found to diminish during long-term exposure to elevated CO 2 by excessive starch and sugar accumulation causing feedback inhibition of photosynthesis (Sage et al., 1989;Stitt, 1991). As this acclimation process is especially noted in annual C 3 plants whilst some C 3 trees and perennial CAM plants do not suffer from acclimation, the availability of sink capacity has been attributed as an important determinant for the longterm responses of plants to elevated CO 2 (Nobel and Israel, 1994;Ceulemans et al., 1995).
In contrast to the universal presence of its mono-and bisphosphate esters in the plant kingdom, free sedoheptulose only accumulates to a high extent in some members of the Crassulaceae family such as Sedum spectabile (La Forge and Hudson, 1917;Hegnauer, 1964). However, its presence, albeit at lower levels, in a range of other families including Apiaceae, Aquifoliaceae, Euphorbiaceae, Lamiaceae, Primulaceae, and Saxifragaceae suggests a broader range of occurrence (Tolbert et al., 1957: Okuda andMori, 1974;Häflinger et al., 1999;Soria et al., 2009). The only other free C7 sugar described in plants is mannoheptulose (Tesfay et al., 2012). Nordall et al. (1956) postulated that the presence of free sedoheptulose in amounts exceeding the steadystate concentration of its phosphate might be attributed to weak activity of sedoheptulokinase (EC 2.7.1.14), specifically producing sedoheptulose-7-phosphate from sedoheptulose. However, except for some studies involving sedoheptulokinase deficiency in humans, this enzymatic activity has been poorly studied (Kardon et al., 2008), and sedoheptulokinase activity has never been reported in plants. Alternatively, starting from sedoheptulose-7-phosphate, two routes might be considered in plant tissues that potentially yield the free heptose sugar, as both the Calvin-Benson cycle and the oxidative pentose phosphate pathway (oxPPP) contain sedoheptulose-7-phosphate as an intermediary reagent. Although the basic features of the oxPPP are well established (Kruger and von Schaewen, 2003), details on how exactly the pathway influences other processes are subject to further investigation. In addition to its putative origin, the role of free sedoheptulose in plants remains a matter of debate, except for serving as a precursor for the polyol volemitol in the horticultural hybrid polyanthus (Häflinger et al., 1999). A possible function as a carbohydrate reserve in CAM (such as starch or soluble sugars), essential to catalyse nocturnal CO 2 uptake, was ruled out earlier by Kull (1965Kull ( , 1967 based on the absence of clear changing diel patterns of sedoheptulose. Therefore, only speculation exists about its physiological roles in plant tissues (Kardon et al., 2008).
To gain more insight into the occurrence of free sedoheptulose and its physiological function in plant tissues, elevated CO 2 and sucrose induction experiments were used to study sedoheptulose metabolism in K. pinnata and S. spectabile, two closely related members of the Crassulaceae family performing CAM. The results indicated that sedoheptulose formation in planta might occur by a sedoheptulose-7-phosphate phosphatase acting on the oxPPP intermediate sedoheptulose-7-phosphate. Furthermore, experimental evidence suggested that sedoheptulose accumulation might confer a novel mechanism to contribute to C and P homeostasis in CAM plants. Other possible complimentary functions are also discussed.

Plant material and sampling
The closely related Kalanchoë pinnata and Sedum spectabile species belong both to the Crassulaceae and perform CAM (Brulfert et al., 1988;Lüttge et al., 1991). Plants were originated from leaf cuttings and grown under controlled greenhouse conditions (Leuven, Belgium). The elevated CO 2 experiment was performed with K. pinnata plants that were divided equally between two greenhouse compartments (Leuven, Belgium). Control plants were grown under ambient atmospheric CO 2 concentrations (~380 μmol mol -1 ). The CO 2 -treated plants were exposed to ~700 μmol mol -1 , as described previously (Ceusters et al., 2008). All other environmental conditions in both compartments were identical. During the day, a minimum temperature of 21 °C was maintained, whilst at night a minimum of 19.5 °C was achieved. Between 6:00 and 22:00 h, additional artificial lighting was provided [photosynthetic photon flux density (PPFD)=30 μmol m -2 s -1 ] and the average integrated daily PPFD was ~7 mol photons m -2 d -1 . Plants were watered twice weekly with a conventional nutrient solution, as described by Londers et al. (2009).
After 12 weeks, young fully developed leaves were cut in the afternoon (15:00 h) from plants (n=5) under both CO 2 regimens and immediately frozen in liquid nitrogen to arrest any enzyme activity.
Purification of sedoheptulose and confirmation by nuclear magnetic resonance (NMR) About 50 g of leaf material (cut into pieces of 1 cm 2 ) was combined with 100 ml of Milli-Q water, boiled for 15 min, and homogenized with a Waring blender. The homogenate was centrifuged at 20 000g for 20 min. The supernatant was put on a mixed-bed Dowex column [50 ml resin of acetate -(Ac -) and 50 ml of H + Dowex; Acros Organics, Morris Plains, NJ, USA] and further concentrated with a Rotavap to 15 ml, which was loaded in three 5 ml aliquots onto a Ca-Dowex column (50 × 300 mm) and fractionated. Sedoheptulose-containing fractions were checked with analytical high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and combined. Excess glucose was removed by the addition of glucose oxidase (500 U). The resulting glucuronic acid was removed by loading the mixture onto 50 ml Ac -Dowex. The column flowthrough was concentrated to 4 ml with a Rotovap and subjected in ten 400 μl aliquots to preparative HPAEC-PAD as described previously (Vanhaecke et al., 2006). Manually collected fractions were neutralized, combined, and loaded onto an active charcoal column (25 × 150 mm, 12-20 mesh; Sigma). The column was subsequently washed with 500 ml Milli-Q water. Sedoheptulose was eluted with 20 % ethanol, which was subsequently removed with a Rotovap. Finally, the remaining volume was lyophilized (LSL Secfroid) and pure sedoheptulose (10 mg) was dissolved in 0.6 ml D 2 O for NMR analysis. Spectra were recorded at 22 °C on a Bruker Avance II 600 equipped with a 5 mm TCI HCN Z gradient cryoprobe. Bruker Topspin 2.1 software was used to process all spectra. The two-dimensional pulse programs of 1 H-1 H correlation spectroscopy (COSY), heteronuclear single quantum correlation (HSQC), heteronuclear multiple bond correlation (HMBC), and nuclear Overhauser effect spectroscopy experiments were carried out as described previously (Vanhaecke et al., 2008).
Induction with sucrose K. pinnata leaves were cut in pieces of 0.5 cm 3 and for the (3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) experiment they were first incubated overnight in 100 ml of 20 μM DCMU (or water as a control) in Petri dishes (17 cm diameter), before transferring them for 2 d in 100 ml of 200 mM sucrose (or water) with or without DCMU in the dark or in continuous light (PPFD=100 μmol m -2 s -1 ) at 22 °C. Sucrose induction was also performed by placing flower-decapitated S. spectabile stem parts (with leaves included) in a beaker with 200 mM sucrose for 2 d under continuous light. Subsequently, sugar concentrations and sedoheptulose-7-phosphate phosphatase activities were determined in the leaves.

Sugar extraction and quantification
Leaves were boiled in 2 volumes of water for 15 min and further homogenized with a mortar and pestle. The extract was centrifuged at 13 200 rpm for 5 min. Subsequently, 200 μl of the supernatant was added to a mixed-bed Dowex column (300 μl Dowex H + and 300 μl Dowex Ac -; both 100-200 mesh; Acros Organics). The column was eluted six times with 200 μl ddH 2 O (Vergauwen et al., 2000). From the supernatant, an aliquot was analysed using HPAEC-PAD, as described previously (Vergauwen et al., 2000). Peak quantification and identification was performed using the external standards method (Vergauwen et al., 2000). No losses in sedoheptulose occurred during the applied extraction procedures, as verified by adding a known amount of sedoheptulose to an extract from a non-sedoheptulose producing species followed by quantification of the amount found after the purification procedure (not shown).

Enzyme extraction and enzyme activity measurements
Standardized procedures were followed (Eisenthal and Danson, 1992). Leaves were extracted in 2 volumes of extraction buffer (20 mM KMES, pH 6.0, containing 2 mM mercaptoethanol, 2 mM MgCl 2 , 10 mM NaHSO 3 , 0.1 % Polyclar and 1 mM PMSF). The extract was dialysed overnight against 500 volumes of 20 mM KMES buffer. After dialysis, the enzyme was centrifuged for 5 min at 16 000g. For the sedoheptulose-7-phosphate phosphatase assay, an enzyme aliquot was combined with 2 mM sedoheptulose-7-phosphate, 4 mM MgCl 2 , 50 mM KMES (pH 6.0) (final concentrations), with (or without) the extra addition of 4 mM ADP or 4 mM potassium phosphate (pH 6.0) and incubated at 30 °C. After 0, 3, and 20 h of incubation, an aliquot of 10 μl was removed and added to 50 μl of 20 μM mannitol (internal standard) with 0.04% sodium azide and heated to 90 °C for 5 min. Identical concentrations and treatments were used in the sedoheptulose kinase assay, except that sedoheptulose and ATP (4 mM) were used as substrates. All reaction products were analysed by HPAEC-PAD, as described above.
Chloroplast isolation S. spectabile chloroplasts were isolated using the method of Kley et al. (2010) with minor modifications. A layer of 3 ml of 50 % Percoll (GE Healthcare) was covered with 7 ml of chloroplasts and centrifuged in a Sigma 203 centrifuge (13020 swing-out rotor) at 2800 rpm for 10 min. After washing in 10 ml Clp buffer (Kley et al., 2010), the chloroplasts were spun down at 2500 rpm for 5 min.

Data analysis
Data were analysed using the statistical software package SAS Enterprise Guide 4.0. Before carrying out statistical tests, the normality of the datasets was checked by means of the Kolmogorov-Smirnoff statistic (P>0.05). Means were compared using a two-sample t-test (α=0.05).

Detection of sedoheptulose in K. pinnata by NMR
The studied sugar adopted three conformations in a D 2 O solution. The 13 C signals of the three anomeric carbon atoms could easily be recognized by their typical chemical shift close to 100 ppm (Fig. 1). Starting from these quaternary carbons, assignment in the individual spin systems was possible using a combination of two-dimensional HMBC, HSQC-total correlation spectroscopy, HSQC, and COSY spectra. The observed ratio of three conformations and the obtained 13 C data corresponded to those published for d-sedoheptulose-7-phosphate (Charmantray et al., 2009), despite minor differences that are expected from its phosphorylation state. Chemical shift assignments and 3 J HH couplings within each ring are listed in Table 1. The obtained NMR spectra could be differentiated clearly from those obtained for mannoheptulose (Waschke et al., 2011).

Sedoheptulose accumulation under elevated CO 2 in K. pinnata in vivo
In the leaves of K. pinnata, CO 2 elevation caused the levels of glucose and fructose to increase (P<0.05), whilst sucrose was maintained at a similar level compared with control plants (P>0.05; Table 2). Initially, free sedoheptulose was present only at values representing 20-30 % in comparison with hexoses and sucrose, but after 12 weeks of CO 2 enhancement, sedoheptulose had increased by 500 % (P<0.05) and became the dominant sugar in the leaves of K. pinnata. No other major carbohydrate accumulations were detected by HPAEC-PAD.

Artificial induction of sedoheptulose accumulation in isolated leaves of K. pinnata
To gain more insight into the strong accumulation of sedoheptulose noted under CO 2 enrichment, additional experiments were set up under ambient CO 2 involving the elimination of sinks by isolating source leaf pieces and incubating them in water or 200 mM sucrose under continuous illumination for 4 d.
Incubation of source leaves in a water solution showed a steady small but significant increase (P<0.05) in hexose sugars over a time course of 4 d ( Fig. 2A, B). After 1 d of incubation, sucrose levels decreased threefold (P<0.05) and remained invariable thereafter for the considered time period (Fig. 2C). Sedoheptulose showed a tenfold increase (P<0.05) after 3 d of incubation (Fig. 2D). Feeding the leaves with 200 mM sucrose solution resulted in a massive accumulation of 20 and 40 times the original concentration for glucose and fructose, respectively ( Fig. 2A, B). Glucose build-up only occurred for 1 d, whilst fructose enhancement took about 2 d before reaching a maximum steady-state level (P>0.05). The accumulation of sucrose was much more limited compared with the hexoses, and showed only a threefold increase over 3 d (Fig. 2C), suggesting that the sucrose concentration is strictly controlled in K. pinnata. After the first day of incubation in a 200 mM sucrose solution, sedoheptulose showed a small increase (P<0.05), similar to that in leaves incubated in a water solution (P>0.05) (Fig. 2D). However, over the next 2 d, sedoheptulose increased at a much higher rate, resulting in a 30-fold accumulation after 3 d.

Inhibition of photosynthesis reduces sedoheptulose formation
Leaf pieces of K. pinnata were incubated overnight in the presence and absence of DCMU, an inhibitor of photosynthesis. Thereafter, they were kept under continuous light and in the dark for 2 d, respectively (±DCMU), both in the presence or absence of 200 mM sucrose (Fig. 3). Without additional sucrose, only the treatment with light and without DCMU yielded a significant amount of sedoheptulose, whilst only negligible amounts were detected in the light plus DCMU condition and in the dark. All sucrose-treated leaf pieces accumulated high levels of glucose and fructose, and showed an increased level of sucrose and sedoheptulose. However, in the light without DCMU, the sedoheptulose:sucrose ratio was substantially higher than in the three other conditions (Fig. 3). These results suggested that photosynthesis stimulates sedoheptulose formation. Nevertheless, synthesis also appeared to be possible in the dark, although to a lower extent. This was further corroborated using etiolated  K. pinnata material (not shown). When green stem parts (with leaves) of S. spectabile were induced with 200 mM sucrose, sedoheptulose concentrations in the leaves increased from 6.7 mM (control) to 15.5 mM.

Sedoheptulose is produced from sedoheptulose-7phosphate in vitro
Because it proved practically impossible to obtain substantial amounts of proteins from K. pinnata, the close relative species S. spectabile, known to accumulate high levels of sedoheptulose under standard conditions in its organs (La Forge and Hudson, 1917; Fig. S1 at JXB online), was used for protein extraction. Both species belong to the Crassulaceae and are situated in the same subfamily, Sedoideae (Mort et al., 2001). Desalted protein extracts of sucrose-induced leaves were incubated with 2 mM sedoheptulose-7-phosphate. Fig. 4A shows the time-dependent formation of sedoheptulose from sedoheptulose-7-phosphate. No sedoheptulose kinase activity (the reverse reaction) could be detected by combining sedoheptulose and ATP (not shown), strongly suggesting that the sedoheptulose concentration in planta might be controlled by the sedoheptulose-7-phosphate supply and the sedoheptulose-7-phosphate phosphatase activity. Strikingly, the addition of both ADP and inorganic phosphate (P i ) at a concentration of 4 mM attenuated the sedoheptulose-7-phosphate phosphatase activity (Fig. 4A). This was not observed for the non-specific phosphatases present in enzyme extracts of the C 3 plant wheat (Fig. 4B), suggesting the presence of a rather specific sedoheptulose-7-phosphate phosphatase in S. spectabile that can be regulated by ADP and P i . Moreover, sedoheptulose-7-phosphate phosphatase activities increased to 490 nmol mg -1 of protein min -1 in the leaves of sucroseinduced stems, compared with 25 nmol mg -1 of protein min -1 in control leaves (Table S1 at JXB online). Unfortunately, our continued efforts to purify this enzyme remained unsuccessful due to enzyme instability or removal of an essential co-factor.

Sedoheptulose formation probably originates in the cytosol via the oxPPP and is phloem transportable
To determine the putative subcellular location of sedoheptulose synthesis in S. spectabile, chloroplasts were isolated and lysed and the supernatant was analysed. The amount of sedoheptulose was negligible (Fig. S1), arguing for the actual synthesis of sedoheptulose in the cytosol via the oxPPP, as the Calvin-Benson cycle solely acts inside the chloroplasts. However, the absence of sedoheptulose in freshly prepared chloroplasts should be confirmed by non-aqueous fractionation methods. Compared with leaves under full light (maximal activities up to 1100 nmol mg -1 of protein min -1 were recorded), much lower sedoheptulose-7-phosphate phosphatase activities (up to 33 nmol mg -1 of protein min -1 ) could be detected in S. spectabile roots, despite the fact that roots also accumulate sedoheptulose, although to a lower extent than leaves (Nordall et al., 1956;Fig. S1). This implied that sedoheptulose is produced mainly in the leaves and transported to the roots through the phloem, as suggested previously by Liu et al. (2002). Typically, the honeydew of aphids still partly contains phloem-derived sugars, although substantial sugar pattern modulation occurs by the action of glycosyl transferases in the aphid gut (e.g. producing melezitose from sucrose; Vantaux et al., 2011). Intriguingly, next to melezitose as a major sugar, honeydew from S. spectabile-resident aphids contained substantial amounts of sedoheptulose ( Fig.  S2 at JXB online), providing further evidence for the phloem transport of sedoheptulose in S. spectabile.

Discussion
In contrast to the well-documented roles of the phosphate esters of sedoheptulose, either in the regenerative part of the Calvin cycle or in the oxPPP, the occurrence of substantial amounts and the physiological role of free sedoheptulose in a range of CAM plants has remained enigmatic, despite the fact that sedoheptulose was discovered almost a century ago in S. spectabile (La Forge and Hudson, 1917). Here, we have shed light on the putative importance of sedoheptulose anabolism in the CAM plants K. pinnata and S. spectabile.
We showed that sedoheptulose, whose identity was confirmed by NMR (Fig. 1, Table 1), was produced from sedoheptulose-7-phosphate in vitro, probably by the activity of a specific sedoheptulose-7-phosphate phosphatase (Fig. 4) operating in the leaves. Increased sedoheptulose-7-phosphate phosphatase activities were noticed concomitantly with elevated sedoheptulose concentrations in the leaves of sucroseinduced S. spectabile stems, suggesting that this enzyme is involved in the biosynthesis of sedoheptulose in planta. Sedoheptulose-7-phosphate constitutes an intermediary product in two pathways in plants: the Calvin-Benson cycle and the oxPPP. In contrast to the first, which is restricted to a plastid origin, the oxPPP can be completed in the cytosol alone or involve compartmentalization between cytosol and chloroplast, depending on species, tissue, developmental stage, and environmental conditions (Kruger and von Schaewen, 2003). The absence of sedoheptulose in chloroplasts suggested an oxPPP-mediated synthesis of sedoheptulose in the cytosol. However, by converting sedoheptulose-7-phosphate to sedoheptulose, significant amounts of carbohydrate may be withdrawn from the actual oxPPP. This observation prompted further investigation into its physiological significance.
Under control conditions, free sedoheptulose concentrations were only of minor importance in K. pinnata, but doubling of the atmospheric CO 2 concentration caused sedoheptulose to increase by 500 % whilst sucrose was not influenced in source leaves (Table 2). Strict metabolic control of the amount of sucrose under CO 2 elevation has been noted previously in other CAM plants, which show no downward acclimation of photosynthesis in response to elevated CO 2 , but the precise underlying mechanisms have not been elucidated (Wang and Nobel, 1996;Ceusters and Borland, 2011). It is well known that, as well as reducing expression of genes implicated in photosynthesis, accumulated sucrose might reduce the rate of its own synthesis. This might cause sugar phosphates to accumulate and inorganic phosphate (P i ) pools to be depleted in the cytosol and in chloroplasts, ultimately inhibiting photophosphorylation and leading to long-term acclimation of photosynthesis under elevated CO 2 in a range of plants (Stitt, 1991;Sheen, 1994). In different CAM plants, a lack of long-term acclimation has been attributed mainly to increased rates of phloem sucrose transport to supply growing sinks and avoiding sucrose accumulation in the source leaves (Wang and Nobel, 1996). In addition to this mechanism, we propose in our model (Fig. 5) that oxPPP-mediated sedoheptulose formation in the cytosol in K. pinnata might function as an excess carbon escape valve to deal with a surplus of triose phosphates originating from the chloroplasts (Calvin-Benson cycle), and as such avoiding sucrose accumulation under CO 2 enrichment when sink capacities might become saturated. To test this hypothesis, source-sink transport was compromised by  (Fig. 2). The sucrose concentration inside the leaves rose slightly from 1.5 to about 5 mM, representing only a threefold increase, indicating that there is a strict metabolic control over the sucrose concentration inside the leaves but without the possibility of diverting excess sucrose to sinks. Initially, significant amounts were converted to glucose and fructose (Fig. 2), but after 2 d, the levels of these sugars were also saturated and a massive 30-fold increase in sedoheptulose level occurred (Fig. 2). These results demonstrated that sedoheptulose synthesis can act as an alternative or additional mechanism to deal with excess carbohydrate in the leaf or even at the cell level, whilst elevated increased phloem transport of sucrose requires coordination between different plant organs. In this context, sedoheptulose formation offers a higher degree of flexibility in comparison with sucrose. Both sugars are phloem transportable, but when this possibility is restricted, significant accumulation of sedoheptulose can take place inside source leaves without suppressing photosynthesis. Moreover, CAM plants are particularly well established to store large amounts of sugars in the typically large central vacuoles, which dominate about 95% of the cell's volume (Steudle et al., 1980;Kenyon et al., 1985). Of course, the drawback of carbon through sedoheptulose formation in vivo should only be active when triose phosphates are accumulating excessively, i.e. when sink capacity is limited and triose phosphates are synthesized at high rates under elevated CO 2 .
Our findings that incubating leaf pieces in a water solution without extra sucrose in the dark or under photosynthesisinhibiting conditions (DCMU) resulted in only marginal sedoheptulose formation (Fig. 3) confirm this view, and significant sedoheptulose accumulation was restored when leaf pieces were incubated in a water solution in the light.
Diverting sedoheptulose from triose phosphates via glucose-6-phosphate in the oxPPP also involves the liberation of P i and reducing power (NADPH) (Fig. 5). NADPH might be utilized in biosynthetic processes, for the production of antioxidants (e.g. ascorbate, glutathione), or, after conversion to NADH, oxidized in the mitochondrial electron transport chain. An elevated amount of reducing power could also favour CAM operation under elevated CO 2 because it energizes the conversion of oxaloacetic acid to malate during nocturnal CO 2 uptake. However, previous measurements from Winter et al. (1997) showed that K. pinnata only showed an elevated daytime uptake of CO 2 , whilst nocturnal sequestration and consequently malate formation remained unaffected upon CO 2 treatment. As already mentioned above, the availability of P i is crucial to drive photosynthesis, and in this respect, CAM plants can be particularly vulnerable to P i imbalances as they generally utilize more ATP and as such more P i to convert carbon, originating from CO 2 , into sugars during photosynthesis (Iglesias et al., 1993;Winter and Smith, 1996). Moreover, pyruvate phosphatase dikinase also requires both ATP and P i to convert pyruvate to phosphoenolpyruvate (PEP) during the daytime (Fig. 5), and as such these reactions might compete  Silvera et al., 2010;Holtum et al., 2005). Dotted arrows represent multiple steps. Free sedoheptulose formation under elevated CO 2 in K. pinnata is indicated and originates from the action of a sedoheptulose-7-phosphate phosphatase acting on the oxPPP intermediate sedoheptulose-7-phosphate. A negative feedback control by ADP and P i is proposed, contributing to both C and P homeostasis. Glc6P, glucose-6-phosphate; PGA, 3-phosphoglycerate; PEP, phosphoenolpyruvate; Pyr, pyruvate; Sedo, sedoheptulose; Sedo-7-P, sedoheptulose-7-phosphate; Suc, sucrose; TP, triose phosphate. (This figure is available in colour at JXB online.) with direct Rubisco assimilation for P i during the transition phases II and IV, leading to P i limitation in the chloroplast. It is clear that the production and transport of PEP are crucial for the functioning of CAM plants to provide consistent carbohydrate storage each day to ensure the continuation of nocturnal carboxylation via PEP carboxylase (Ceusters et al., 2010;Borland et al., 2011). As such, the formation of sedoheptulose is not only beneficial with regard to carbon allocation under elevated CO 2 but also contributes to P i homeostasis. Of course, this operation is only useful when chloroplast and cytosolic P i and ADP are low, and this is completely in line with our finding that P i and ADP exerted a negative feedback control on sedoheptulose-7-phosphate phosphatase activity (Figs 4 and 5). Besides the proposed functions of sedoheptulose in CO 2 -enriched K. pinnata, the free heptose sugar might also serve a role in cellular antioxidant mechanisms. CAM habitats are generally characterized by high solar radiations, e.g. exposed terrestrial or epiphytic habitats where photosynthetic active radiation is not limiting photosynthesis but overenergization of the photosynthetic apparatus in CAM plants might constitute a real threat (Lüttge 2002). Moreover, it has been proposed that sugar(like) compounds, when accumulating at higher concentrations, might act as reactive oxygen species scavengers (Van den Ende and Valluru, 2009;. Typically, resurrection species maintain higher soluble sugar levels compared with closely related nonresurrection species (Djilianov et al., 2011).
In conclusion, we propose that sedoheptulose formation in planta occurs via the oxPPP intermediary sedoheptulose-7-phosphate, probably by a dedicated cytosolic sedoheptulose-7-phosphate phosphatase, although the exact subcellular localization is a subject for further investigation. Furthermore, it is proposed that sedoheptulose formation contributes to both C and P homeostasis in K. pinnata by acting as an alternative carbon store under elevated CO 2 to avoid sucrose accumulation and depletion of P i . Our experimental evidence (elevated CO 2 , external sugar, and light) strongly suggest a stimulated flux of C through this pathway, mediated by increased triose-phosphate levels but attenuated by ADP and P i . It remains to be established whether this mechanism is confined to CAM plants, which might be especially vulnerable to P i imbalances, or whether some C 3 and C 4 plants also dispose of the genetic capacity to induce and accelerate sedoheptulose synthesis upon CO 2 elevation.

Supplementary data
Supplementary data are available at JXB online. Fig. S1. Typical chromatograms showing the sugar composition of different organs and isolated chloroplasts of S. spectabile. Fig. S2. Typical chromatogram showing the sugar composition of honeydew from S. spectabile-resident aphids. Table S1. Sedoheptulose-7-phosphate (S7P) phosphatase activities and sedoheptulose concentrations in leaves derived from sucrose-induced (200 mM, 2d) stem fragments of S. spectabile compared with an untreated control.