JXB Advance Access originally published online on April 11, 2008
Journal of Experimental Botany 2008 59(7):1851-1861; doi:10.1093/jxb/ern085
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RESEARCH PAPER |
Leaf succulence determines the interplay between carboxylase systems and light use during Crassulacean acid metabolism in Kalanchoë species*
Physiological Ecology Group, Department of Plant Sciences, Downing Street, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK
To whom correspondence should be addressed. E-mail: hg230{at}cam.ac.uk
Received 11 February 2008; Revised 26 February 2008 Accepted 28 February 2008
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Abstract |
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Top
Abstract
IntroductionThe photosynthetic physiology of Crassulacean acid metabolism was investigated in two Kalanchoë species with differing leaf succulence. The magnitude of CAM was higher for the more succulent leaves of K. daigremontiana, compared to the less succulent leaves of K. pinnata. High succulence was related to low mesophyll conductance: K. pinnata was able to maximize diurnal carbon gain by the C3 pathway, whereas increased succulence is associated with a higher commitment to the CAM cycle in K. daigremontiana. The Rubisco specificity factor,
, determining selectivity for CO2 over O2, was similar for both species (
88), and lower than that of Spinacea (95), but in contrast to C4 plants, the Rubisco KmCO2 (determined independently) was also lower in Kalanchoë spp. than in spinach. Differences in light use were related to the nature of the sink strength in each Phase of CAM, with PEPC activity resulting in low electron transport rates. Decarboxylation was marked by high, non-saturated rates of electron transport, with Rubisco activity and activation state increasing in both species during the course of the light period. The degree of succulence, and extent of CAM activity, was associated with a progressive inhibition of PSII photochemistry and potential Rubisco activity during the night in both species. Rubisco could be ‘woken up’ more rapidly in K. pinnata, whereas 45 min light acclimation was required for full recovery of electron transport and Rubisco activity in K. daigremontiana. Leaf morphology therefore seems to alter the expression of and dependence on CAM, but also the extent of co-regulation of carboxylase networks and light use capacity. Key words: Chlorophyll fluorescence, mesophyll conductance, PEPC, Rubisco activity and specificity
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Introduction |
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Crassulacean acid metabolism (CAM) is a modified form of C3 photosynthesis adopted by approximately 6% of vascular plant species (Winter and Smith, 1996a) as an adaptation to water deficit in terrestrial and epiphytic plants. CAM is a carbon-concentrating mechanism requiring a temporal separation of the C3 (ribulose 1,5-bisphosphate carboxylase oxygenase, Rubisco) and C4 (phosphoenol pyruvate carboxylase, PEPC) components, compartmentalized within a common cellular environment. By convention, CAM is divided into four Phases over the diel course (Osmond, 1978), namely Phase I, nocturnal CO2 fixation (atmospheric ±respiratory sources) mediated by PEPC and accumulation of malic acid within the vacuole; Phase II, atmospheric CO2 fixation at dawn which marks the transition between C4 and C3 activity; Phase III, decarboxylation of malic acid and fixation of the regenerated CO2 by Rubisco; Phase IV, a period of atmospheric CO2 fixation from the end of Phase III to dusk which latterly incorporates the shift from Rubisco to PEPC activity. The CAM pathway is known to be under circadian control and is subject to regulation by multiple oscillators which modulate elements of the pathway in line with environmental conditions (Borland et al., 1999; Lüttge, 2000; Dodd et al., 2003; Wyka et al., 2005).
A key morphological correlate of the capacity for CAM is leaf succulence (Winter et al., 1983; Borland et al., 1998). In a survey of the genus Kalanchoë (Crassulaceae) the extent of succulence has been positively correlated to both colonization of increasingly arid habitat and an increased contribution of CAM activity to total carbon gain (Kluge et al., 1993, 2001). By contrast, thinner-leaved, less-succulent species were more plastic in the expression of CAM and showed an increased proportion of net carbon gain via the C3 pathway (Kluge et al., 1993, 2001; Winter and Holtum, 2002; Winter et al., 2005). Leaf morphology has provided the basis for this study to compare the photosynthetic physiology of CAM plants, relating Rubisco kinetic properties and activation state, PEPC activity, and efficiency of light use.
Under field conditions, photosynthetic responses to light are a key determinant of plant fitness (Külheim et al., 2002). Photosynthetic control of electron transport ensures that the supply of ATP and NADPH is balanced with consumption (Foyer et al., 1990). The latter is determined by photosynthetic sink strength and requires that light use efficiency is high under light-limiting conditions and that an increasing proportion of light energy is dissipated non-photochemically at intensities above light saturation. In C3 plants the major photosynthetic sinks are the Rubisco carboxylation and oxygenation reactions, whereas in CAM plants in each of the three light period Phases, the sink strength and light dependency depends on the extent of PEPC activity early and late in the photoperiod (de Mattos et al., 1997; Maxwell et al., 1999; de Mattos and Lüttge, 2001; Griffiths et al., 2002a, b).
Early in Phase II, PEPC-driven CO2 assimilation is light independent and a low Rubisco sink demand is indicated by a minimal efficiency of PSII photochemistry (Maxwell et al., 1999; Rascher et al., 2001). Decarboxylation (Phase III) is the most energetically demanding process of the CAM pathway, constituting both the Calvin Cycle and the gluconeogenic recovery of pyruvate to the level of storage carbohydrate (Winter and Smith, 1996b; Schöttler et al., 2002). Rubisco regulation is also distinct for CAM plants (Maxwell et al., 1999; Griffiths et al., 2002a). In this paper, the underlying variations in Rubisco specificity factor, kinetics, and activation during CAM are explored. These are compared to more general trade-offs in Rubisco kinetic properties (Tcherkez et al., 2006), as compared with plants adapted to high internal CO2 concentrations (C4: Kubiens et al., 2003; Kubiens and Sage, 2004) or drought stress (Delgado et al., 1995; Galmés et al., 2005).
During CAM, Phase IV photosynthesis is most similar to conventional C3 photosynthesis, allowing the low mesophyll conductance associated with succulent tissues to be determined (when CO2 supply is limited and photorespiration promoted: Maxwell et al., 1997, 1998; Griffiths et al., 2000). Secondly, towards dusk Rubisco activity declines as PEPC activity increases, which reduces the sink demand (Maxwell et al., 1999). Differences in the activity of Rubisco and expression of the CAM phases have implications for diurnal energy demand and light use efficiency, and at night, there maybe a decrease in potential PSII photochemical efficiency in CAM-induced leaves of Mesembryanthemum crystallinum (Schöttler et al., 2002).
Two contrasting species were investigated: Kalanchoë daigremontiana (Hamet et Perr.) a thick-leaved, succulent species and Kalanchoë pinnata (Lam.) Pers., a thinner-leaved less-succulent relation, which differ in their commitment to CAM (Winter et al., 2005). It was hypothesized that: (i) Rubisco kinetic properties (specificity factor and Km for CO2) would differ from C3 species; (ii) high succulence would engender both an increased capacity for CAM and a higher commitment to C4 processes; (iii) low succulence would allow increased direct C3 activity during extended daytime phases; (iv) electron transport would diagnose carboxylase activity and sink demand; and (v) the extent of inhibition, and recovery, of electron transport and Rubisco activity in the dark period would be related to leaf succulence.
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Materials and methods |
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Plant material
Mature plants of K. daigremontiana and K. pinnata were grown and maintained under greenhouse conditions at the Botanic Garden, University of Cambridge, UK. The experimental plants were watered daily and received an all-purpose commercially available liquid fertilizer (Baby-Bio, Pbi Home and Garden Ltd, Enfield, UK) twice weekly. Initial gas exchange experiments were undertaken in the greenhouse under natural light using the third fully expanded leaf of both species in July 2001. Leaf thickness was determined for five replicates using a micrometer and leaf succulence derived from the weight per unit area of freshly harvested leaf disc samples (n=5). Additional measurements of Rubisco activity and PEPC Ki for malate, over light and dark periods were made on these plant stocks maintained in a Sanyo Controlled Environment Chamber in October 2002, with a 14 h photoperiod, PAR at leaf height of 175 µmol m–2 s–1, and day–night temperature of 25/18 °C set to mimic summer conditions in the greenhouse.
Assay for soluble protein
To 1–10 µl of extract, distilled water was added to bring the combined volume to 500 µl. To this, 500 µl of Coomassie reagent was added. After 5–10 min, the extinction at 595 nm was measured. A standard calibration curve was constructed using 1.5–12.5 µl of 1 mg ml–1 BSA.
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Specificity factor of Rubisco |
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Purification of Rubisco
All extraction and purification steps were carried out at <4 °C. Approximately 200 g of frozen tissue was added to 1000 ml of extraction buffer (200 mM HEPES (pH8.0), 20 mM MgCl2, 10 mM NaHCO3, 5 mM R-mercaptoethanol, 10 mM EDTA, 1% (w/v) PVPP, and for Kalanchoë species, 2% (w/v) PEG 6000) in a precooled Waring blender.
The tissue was homogenized seven times for 15 s with 15 s intervals in between. The clarified extracts were fractionated with ammonium sulphate and material precipitating between 31% and 42% (spinach), or 50% and 90% (Kalanchoë) was collected by centrifugation (20 min, 4 °C, 20 000 g). Precipitates were resuspended in anion exchange buffer (25 mM HEPES, 10 mM MgCl2, 10 mM NaHCO3, 2.5 mM R-mercaptoethanol, and 5 mM EDTA) and loaded onto a 250x20 mm column packed with an anion exchange resin (DEAE Fractogel Tentacle, Merck, Merck Biosciences Ltd, Nottingham, UK). Bound proteins were then eluted with a linear NaCl gradient (0–750 mM NaCl) in anion exchange buffer created by a peristaltic gradient pump (Model EP-1 Econo Pump, Bio-Rad Laboratories Ltd, Hemel Hempsted, UK) and fractions were collected (Model 2110 Fraction collector, Bio-Rad Laboratories Ltd, Hemel Hempsted, UK), and monitored at 280 nm. Fractions with the highest Rubisco activity (>33% Rubisco activity of peak fraction) were combined, desalted, and separated from other proteins using size exclusion chromatography (waterjacketed Superdex 200 Highload column, 26/60, Pharmacia, Amersham plc, Little Chalfont, UK).
Aliquots from each purification step were retained to determine Rubisco specific activity and recovery on the basis of total extractable protein. The purity of the Rubisco preparations was also assessed by SDS-PAGE.
Measurement of the specificity factor
Rubisco samples were placed in the main body of glass cuvette 50 mM Bicine pH 8.3, 20 mM MgCl2, 0.1 mM 6-PG, and 0.2 mg ml–1 carbonic anhydrase (bovine erythrocyte) and made up to a volume of 900 µl. 1.5 nmol of RuBP in 100 µl of the reaction buffer were placed in a sidearm. The reaction vessels were placed in a shaking waterbath at 25 °C and the buffers were purged at a rate of 0.3 l min–1 for 90 min with a humidified gas mixture of 0.04% CO2, 40% O2, and 59.6% N2. The gas mixture was produced by blending pure CO2, O2, and N2 via mass flow controllers connected to a 5.0 l mixing volume.
The concentration of CO2 was monitored with an infrared CO2 analyser (ADC 225 mk III, Hoddesdon, UK), and the concentration of O2 with an oxygen electrode (Hansatech LD2, Norwich, UK). After equilibration, the RuBP in the sidearm was mixed into the main chamber to allow complete catalysis (60 min). 20 mg Dowex resin, pretreated with 1 M KOH and 1 M HCI and washed with 18.2 M
water, was added to the reaction mixture to remove Mg2+ ions before analysis using an anion chromatography system as described by Uemura et al. (1996). Proteins and the resin were removed using spin columns with an exclusion size of 5 kDa. The filtrate was diluted with 18.2 water by 2.5 to ensure 3-PGA and 2-PG were separated for peak area detection, and aliquots of 25 µl were injected into an HPLC system (Dionex DX-AQ 1110, Leeds, UK). The reaction products were detected by chemically suppressed conductivity after separation on an AS11 column (Dionex, Leeds, UK) using 20 mM NaOH as eluent. The elution profile and peak areas of the compounds were recorded with the software package Peaknet (Dionex, Leeds, UK).
Highly reproducible results (SD 
5%) were obtained, provided that 2-PG and 3-PGA were completely separated, the AS11 column was regularly regenerated, and that the Rubisco preparations were free from phosphatase activity. The specificity factor, , was calculated using the equation:
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PGA* and PG* are the peak areas of 3-PGA and 2-PG, respectively, and the value of 0.0375 represents the ratio of the solubilities of CO2 and O2 in the aqueous Phase (Uemura et al., 1996).
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Rubisco initial and total activity assay and determination of the KmCO2 values |
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Frozen tissue samples (0.25 g) were homogenized in 1 ml extraction buffer (450 mM HEPES (pH 8.0), 40 mM MgCl2, 5 mM R-mercaptoethanol, 20 mM EDTA, 1% (w/v) PVPP, and for Kalanchoë species, 1% (w/v) PEG 6000) and centrifuged for 30 s at 4 °C, 10 000 g, and the supernatant decanted and used for further determinations of Rubisco initial and total (as well as maximal) activity. Aliquots were retained for the determination of total protein. The radiometric assays were conducted at 21 °C, with measurements of initial activity started immediately by the addition of 100 µl of extract to an assay mixture of 250 mM Bicine (pH 8.2), 12.5 mM NaH14CO3 (0.05 Ci ml–1), 25 mM MgCl2, and 0.625 mM RuBP, and stopped after 1 min by the addition of 200 µl of 10 M formic acid. Measurements of total activity were made following 10 min of preincubation to obtain full carbamylation of Rubisco, with 100 µl of extract for 10 min at 21 °C in 375.5 µl of a mixture of 250 mM Bicine (pH 8.2), 12.5 mM NaH14CO3 (0.05 Ci ml–1), and 25 mM MgCl2. The reaction was started by the addition of 12.5 µl of stock RuBP to give a final concentration of 0.5 mM in the assay. The reaction was allowed to proceed for 1 min and terminated by the addition of 200 µl 10 M formic acid. Samples were dried at 125 °C and the acid stable product counted by liquid scintillation spectrometry.
The KmCO2 assays were conducted at 25 °C in airtight 5 ml reaction vials, flushed beforehand with N2 for 4 min at 0.3 l min–1 to remove all O2 and CO2. After the addition of 0.25 µmol RuBP and varying amounts (0.4 mM–14.86 mM) of [14C]bicarbonate, the reactions were started by the addition of pre-activated Rubisco (10 min at 25 °C in 20 mM [14C] bicarbonate). After 1 min the reactions were stopped by the addition of 200 µl 10 M formic acid. The assay mixtures were oven-dried at 125 °C and the radioactivity of the acid-stable product was counted by liquid scintillation spectrometry. The Km values for CO2 were determined using half reciprocal plots; CO2 concentrations in the assay were calculated using the Henderson–Hasselbalch equation and the pK value of 6.37 at 25 °C. The Km values obtained for the CAM plant Rubiscos were compared and standardized against the KmCO2 value for Spinacea oleracea determined simultaneously (Parry et al., 1989; Delgado et al., 1995).
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PEPC Ki for malate |
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Using the method of Borland and Griffiths (1997), 250 mg of frozen leaf tissue was homogenized at 4 °C in 1 ml of extraction buffer, (as described for Rubisco, see above), and after centrifugation (2 min at 10 000 g), the supernatant was desalted into 100 mM MES (pH 7.2), 10 mM MgSO4 using NAP 5 columns (Amersham plc, UK). The Ki for malate was then determined by the addition of varying amounts of malate to 900 µl of an assay mix of 100 mM MES (pH 7.2), 5 mM MgCl2, 2 mM PEP, 0.25 mM NADH, 10 mM sodium bicarbonate, and 10 U malate dehydrogenase. The assay was conducted at 21 °C and initiated by the addition of 100 µl of extract.
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Rubisco and PEPC data presentation |
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Data for Rubisco and PEPC assays are presented as total activity in crude extracts, together with the activation state (%) determined as the difference between initial and total activities. Samples were collected at approximately hourly intervals during a day–night cycle for the plants in the controlled environment chamber. For ease of presentation, Rubisco data are presented from three of the houly samples (each comprising three replicate leaf samples), from 3–6 h (early) and 12–13.5 h (late) into the 14 h photoperiod; for PEPC, the mean of three hourly samples are presented for samples 2–5 h (early) and 6-9 h (late) into the 10 h dark period. For Rubisco analyses during the light response curves conducted late in the dark period, samples were taken at intervals from duplicate leaves.
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Leaf-sap titratable acidity |
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For the greenhouse-grown plants, six replicate samples were taken from both species at approximately houly intervals over the diel course. Leaf discs of known area were cut and immediately weighed (fresh weight, FW) and then frozen at –20 °C. Individual leaf discs were thawed and boiled for 20 min in 4 ml distilled water. The extracts were allowed to cool and titrated to neutrality against 10 mM NaOH. Leaf-sap acidity was expressed on an area basis. The magnitude of CAM was quantified as the mean maximum–minimum leaf sap acidity (
H+). Decarboxylation rate was calculated from the slope of the linear Phase of the reduction in titratable acidity during Phase III. |
Gas exchange |
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Net atmospheric CO2 exchange over the diel course was measured for three replicate leaves using a portable IRGA (LI-6400, Li-Cor, Nebraska, USA) fitted with the ‘Sun and Sky’ chamber. Incident light was recorded as the intensity within the chamber at leaf level, accounting for any attenuation through the chamber head and variations due to leaf orientation. Leaf temperature was maximally 1 °C above ambient and did not exceed 30 °C. Atmospheric CO2 was supplied from outside the greenhouse via a 20.0 l buffering volume at a flow rate of 300 ml min–1. Mesophyll conductance (gm) was assessed for four replicates using combined gas exchange and chlorophyll fluorescence following the procedure described by Maxwell et al. (1997). Measurements were restricted to the period when only Rubisco-mediated atmospheric CO2 uptake occurs during Phase IV.
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Chlorophyll fluorescence |
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Chlorophyll fluorescence was measured in situ using a MiniPam fluorometer (Heinz Walz GmbH, Effeltrich, Germany), powered from a mains electricity supply, with integral leaf clip, PFD sensor, and thermocouple. Rapid light response curves (LRC) were used to analyse the dynamic response of linear photosynthetic electron transport (ETR) and thermal dissipation of light energy (non-photochemical quenching, NPQ) to a range of light intensities. Leaves were marked around the leaf clip and measurements were always made on the same area of leaf for five replicate plants. The plants were dark-acclimated for a period of 10 min and then exposed to light of known intensity for a period of 20 s (range 0–
1600 µmol photon m–2 s–1). At each intensity, the operating efficiency (
PSII), ETR, and NPQ were calculated.
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, where 
is the maximal nocturnal dark-adapted value of maximal fluorescence predetermined for each replicate leaf area. The potential operating efficiency was quantified as Fv/Fm where 
and Fo is the steady-state fluorescence level in the dark. Diurnal LRCs were performed on the same day/night as gas exchange at specific times relative to the timing of the Phases of CAM: 05.00 h (early Phase II); 07.00 h (late Phase II); 12.00 h (mid-Phase III), and 16.00 h (mid-Phase IV). An identical protocol was undertaken for nocturnal LRCs with measurements made at night. The induction light was provided by a cold light source (FL-440; Heinz Walz GmbH), with leaves exposed to 300 µmol photon m–2 s–1 for a period of 45 min with LRCs and Rubisco activity measured firstly in the dark, and then at 5, 15, and 45 min over the acclimation period.
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Results |
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Leaves of K. daigremontiana were more succulent (1.98 kg m–2) and thicker (1.93 mm) as compared to K. pinnata (0.93 kg m–2 and 0.95 mm, respectively; Table 1). K. daigremontiana leaves had lower values of mesophyll conductance (gm=0.058 mol CO2 m–2 s–1 bar–1) as compared to K. pinnata (gm=0.100 mol CO2 m–2 s–1 bar–1; Table 1). On an area basis, overall levels of acidity were considerably higher for K. daigremontiana compared to leaves of K. pinnata, confirmed by a
H+ of 300±8 mmol H+ m–2 for the former and 162±7 mmol H+ m–2 for the latter (Table 1). Net acidification continued for 2 h into the day for K. daigremontiana and for 1 h in leaves of K. pinnata (Phase II; Fig. 1), whilst decarboxylation was observed between approximately 08.00–18.00 h for the former and from 07.00–12.00 h for the latter. Decarboxylation rate during Phase III was 5.6 µmol CO2 m–2 s–1 (r2=0.991) and 4.9 µmol CO2 m–2 s–1 (r2=0.962) for K. daigremontiana and K. pinnata, respectively (Table 1).
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Large interspecific differences in the duration and activity of the daytime Phases of CAM were apparent from measurements of gas exchange (Fig. 2). Maximum daytime CO2 uptake was observed at 07.00 h during Phase II in leaves of K. daigremontiana (3.97 µmol CO2 m–2 s–1) at a PFD of 199 µmol photon m–2 s–1 (Fig. 2A). Phase III occurred from 09.00 h to 15.00 h, followed by a second period of daytime atmospheric CO2 fixation of approximately 5–6 h duration (Fig. 2A). By contrast, higher rates of light-dependent atmospheric CO2 fixation occurred for much of the day in K. pinnata, interrupted by a much shorter period of stomatal closure from 11.30–13.30 h (Fig. 2B). Variations were in part due to changes in the natural light environment in the greenhouse (Fig. 2A, B) as well as an inherent capacity for CAM. Differences were also apparent in patterns of nocturnal assimilation during Phase I. In leaves of K. daigremontiana, CO2 assimilation rate increased to a maximum and was maintained throughout the night (Fig. 2A). By contrast, a gradual increase in CO2 uptake occurred overnight to a similar maximum rate of 3.66 µmol CO2 m–2 s–1 at around 01.30 h and then decreased to a low level for the rest of the night in K. pinnata (Fig. 2B).
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Contrasting patterns of ETR and NPQ were observed during the phases of CAM for the more succulent leaves of K. daigremontiana and the less succulent leaves of K. pinnata (Fig. 3). In both species, the lowest Jmax and levels of ETR at all light intensities was observed at dawn (05.00 h; Fig. 3A, B) concomitant with the highest levels of NPQ (Fig. 3C, D). ETR saturated at a rate of 19 µeq m–2 s–1 (Fig. 3A; K. daigremontiana) and 34 µeq m–2 s–1 (Fig. 3B; K. pinnata at 05.00 h). Equally, light response curves were very similar for all leaves during Phase III (Fig. 3). During decarboxylation, the highest and unsaturated rates of electron transport were observed for both species (Fig. 3A, B) linked to the lowest levels of NPQ (Fig. 3C, D). At 07.00 h (late Phase II) overall ETR had increased with a Jmax of 57.9 µeq m–2 s–1, saturated at 600 µmol photon m–2 s–1 (Fig. 3A) in leaves of K. daigremontiana and was associated with the second highest values of NPQ observed through the day (Fig. 3C). The LRC of ETR for K. daigremontiana at 16.00 h was saturated at 400 µmol photon m–2 s–1 with a Jmax of 93.9 µeq m–2 s–1 (Fig. 3A) and moderate NPQ (Fig. 3C). In marked contrast, a low photochemical efficiency was not observed during late Phase II in the less succulent leaves of K. pinnata and the response of ETR and NPQ measured at 07.00 h and 16.00 h were very similar (Fig. 3B, D).
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A comparison of Rubisco kinetic properties was made on Rubisco purified to homogeneity to derive Rubisco specificity factor (Srel,
), whilst KmCO2 and Rubisco activity were from crude Rubisco extracts, with activities compared early and late in the light period (Table 2). For the Rubisco specificity factors, absolute values for CAM plants are compared with those realized for spinach using the same analytical system, to allow for systematic shifts between laboratories using contrasting methodologies. Specificity factors were significantly lower for both CAM species relative to spinach (87 and 88, respectively, P 0.05; Table 2), with KmCO2 also significantly lower for K. daigremontiana relative to spinach (P 0.05; Table 2). Diurnal time-courses of Rubisco activity and activation state, and night-time PEPC activity (as PEPC Ki for malate), were compared for the two species across the diel cycle. For ease of presentation, data from three sampling points early and late in the photopheriod or dark period are compared for each enzyme.
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Total extractable Rubisco activity was lower in K. daigremontiana than K. pinnata, both early and late in the photoperiod (Table 2). Rubisco total activity and activation state increased late in the afternoon in both species, consistent with previous studies (Maxwell et al., 1999; Griffiths et al., 2002a). For PEPC, activation state, as indicated by the Ki for malate, increased dramatically towards the end of the dark period, with carboxylation strength (as Ki) higher in the more succulent K. daigremontiana (Table 2). The limited predawn recovery of ETR, and potential link to Rubisco activity, was monitored during a 45 min period of light acclimation imposed 3 h before dawn (Fig. 4). Initial dark-acclimated measurements showed the clear inhibition of electron transport rate (consistent with Fig. 3), and over the following 45 min a gradual increase in ETR was observed for K. daigremontiana and Rubisco activity occurred (Fig. 4A, B). By contrast, there was a more rapid recovery of both ETR and Rubisco activity in K. pinnata, also consistent with the predicted response from comparative rates of ETR and NPQ during light acclimation (Fig. 3). However, the potential operating efficiency of PSII (Fv/Fm) remained close to optimal values throughout the night and was not significantly different between species (data not shown).
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Discussion |
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As predicted, the thicker, more succulent leaves of K. daigremontiana showed an increased commitment to CAM, manifested as a higher
H+ and nocturnal CO2 uptake, together with prolonged PEPC activity during Phase II (indicated by continued TA accumulation; Fig. 1) and an extended period of decarboxylation. By contrast, the leaves of K. pinnata showed a more plastic response to prevailing light conditions and were able to supplement nocturnal carbon fixation with extensive daytime atmospheric CO2 fixation directly by Rubisco (see also Winter, 1980; Winter and Tenhunen, 1982; Winter et al., 2005). Although it is clear that succulence is a prerequisite of CAM (Teeri et al., 1981; Kluge et al., 2001), surprisingly few studies have considered the implications of this type of cellular morphology on the photosynthetic physiology of CAM, which contrasts to similar models available for C3 plants (Evans and von Caemmerer, 1996; Roderick et al., 1999a, b). As shown in this study and elsewhere (Kluge et al., 1993, 2001; Kluge and Brulfert, 1996), high leaf succulence is associated with an increased magnitude of CAM. Although it is not clear what determines the capacity for CAM many factors could be involved including vacuolar capacity (Borland and Griffiths, 1997; Lüttge, 2002) or relative investment in C4 enzymes (Winter et al., 1982; Borland et al., 1998), or reassimilation capacity in the light (Griffiths et al., 2002a).
Dominance of C4 carboxylation offers a selective advantage in terms of maintaining carbon fluxes in semi-arid habitats with predictable rainfall, but the associated increase in succulence is deleterious for CO2 fixation via the C3 pathway and ultimately productivity (Borland et al., 1994; Borland and Dodd, 2002). Succulent leaves have a high degree of cell to cell contact (Balsamo and Uribe, 1988), low volumes of intercellular airspace (Smith and Heuer, 1981), and a very low volume of mesophyll cells appressed to the intercellular air spaces (Slaton and Smith, 2002). This results in extremely low values of mesophyll conductance of CO2 and Rubisco carboxylation/oxygenation rates for CAM plants (Table 1; Maxwell et al., 1997, 1998). The subsequent gm also applies to C4 carboxylation processes (Evans and von Caemmerer, 1996) since the diffusive supply of CO2 (and conversion to 
) occurs in the cytosol where on-line discrimination methods have shown the limitations imposed by mesophyll at night, as well as by day (Griffiths et al., 2007).
Because of these internal limitations, and the daily feast and famine of internal CO2 supply experienced within leaves of CAM plants, it is hypothesized that the Rubisco specificity factor () would differ for CAM, as compared to C3 species. The HPLC methodology produced data similar to Uemura et al. (1996) for spinach (94.7 versus 93.7), which is at the upper end of the range found using alternative techniques (Jorden and Ogren, 1981; Kane et al., 1994). Relative to the measurements made on spinach, for both Kalanchoë species was 7% lower than spinach, which is similar to shifts seen in Rubisco from C4 plants (Jorden and Ogren, 1981; Kane et al., 1994), although Uemura et al. (1996) found a value of 96.4 for Zea mays. The independently-determined values for KmCO2 were lower than spinach (Table 2), with this shift consistent when measured at 60% O2 (data not shown), whereas previous studies on C4 plants have shown values 2–3 times higher than spinach (Yeoh et al., 1980; Jorden and Ogren, 1981; Bird et al., 1992). The higher KmCO2 in C4 plants, also found by Kubiens and Sage (2004), is consistent with a higher rate of enzyme turnover (kcat) (Tcherkez et al., 2006) and could reduce the investment in catalytic protein (Kubiens et al., 2003; Kubiens and Sage, 2004). In contrast, the low KmCO2 and specificity factor in the CAM Kalanchoë species perhaps support the high catalytic and carboxylation potential during Phase III (analogous to C4: Griffiths et al., 2002a), as well as the low internal CO2 supply during Phase IV (potentially 110 ppm or less, Maxwell et al., 1997; Griffiths et al., 2000, 2002a). Since most CAM plants only develop relatively modest water deficits (Dodd et al., 2002), the systematic changes in seen in drought-tolerant C3 plants (Delgado et al., 1995; Galmés et al., 2005) are perhaps less important than a variable CO2 regime as a driver for CAM Rubisco catalytic properties.
In terms of the co-regulation of carboxylases, there were changes in affinity and activity as either light period (Rubisco) or dark period (PEPC) progressed, consistent with earlier studies (Maxwell et al., 1999; Griffiths et al., 2002; Dodd et al., 2003). Meanwhile, PEPC activity in the light period was indicated by the low rates of linear electron transport and high non-photochemical quenching. In contrast to K. pinnata, Phase II C4 activity was more protracted in the K. daigremontiana leaves showing a continued inhibition of electron transport at 07.00 h. Extended diurnal PEPC activity is commonly observed (particularly in the genus Clusia; Roberts et al., 1997) and is advantageous since CO2 fixation can proceed in the face of light and CO2-limitation of Rubisco. However, PEPC is not a strong sink for electron transport. In both species an increase in NPQ occurred during Phase II to prevent the capacity for light use and dissipation being exceeded. The small increase in electron transport at 07.00 h in K. daigremontiana suggests the onset of an additional sink and indicates increasing Rubisco activity (Maxwell et al., 1999). With the exception of the measurements (close to dawn) ETR and NPQ were similar during Phases II and IV for K. pinnata, suggesting that Rubisco is the dominant sink for much of the day in these plants.
As predicted, decarboxylation during Phase III constituted the largest sink for electron transport in both species. Assuming an absence of pyruvate oxidation during Phase III, Winter and Smith (1996b) have calculated a requirement of 4.3 ATP and 2.9 NADPH per CO2 assimilated as compared to 3 ATP and 2 NADPH per CO2 fixed by the Calvin cycle. This increase in energetic demand is clearly mirrored by an increase in Jmax and the lack of light saturation observed for light response curves made at this time. As advocated by Schöttler and co-workers (2002), future work is now required to determine whether alternative processes such as cyclic flux also feature during this period to meet the additional ATP requirement.
The light response curves measured during the dark period and the subsequent responses to a short period of light acclimation reinforce the differences between the two species in terms of commitment to the CAM pathway. The lack of recovery of electron transport after 5 min in the light for leaves of K. daigremontiana was also found in the succulent leaves of two other species (K. fedtschenkoi and K. marneriana, data not shown) but not in any of the thinner, less succulent leaves of K. pinnata (also K. blossfeldiana and young leaves of K. daigremontiana, data not shown) and may be interpreted as a diagnostic of the extent of CAM activity. The potential efficiency of PSII photochemistry (Fv/Fm) decreases at dawn in leaves of CAM-induced Clusia minor and Mesembryanthemum crystallinum (de Mattos et al., 1999; Schöttler et al., 2002). Griffiths et al. (2002b) also showed that the quantum yield for O2 evolution decreased at dawn in Guzmania monostachia, but a reduction in Fv/Fm was not observed in this study (data not shown).
The low linear electron transport rates in association with PEPC may reflect a light-dependent process, such as state transitions, or a low activation state of Rubisco. The slow increase in Rubisco activity, following light induction in the dark in leaves of K. daigremontiana, in turn, may be a function of the very low levels of Rubisco activase protein found in Phase I CAM leaves (Griffiths et al., 2002a). A more rapid recovery of whole chain electron transport may result when higher light intensities are applied for longer at night (Osmond et al., 1996). It has now been shown that the rate of ETR was directly associated with induction of Rubisco, when CAM leaves were illuminated in the dark period, confirming the relationship between Rubisco activation and electron transport sink strength (Fig. 4).
In summary, this work has confirmed that under well-watered conditions the expression of CAM is dependent on leaf succulence. Overall, Rubisco kinetic properties (specificity factor and Km for CO2) did differ from C3 species, but perhaps reflect the daily feast and famine in CO2 supply, rather than drought or elevated CO2 alone. Highly succulent leaves (K. daigremontiana) are more committed to the CAM pathway, with a lower mesophyll conductance, and suppressed Rubisco activity and electron transport capacity in the dark period. Less succulent leaves (K. pinnata) show increased plasticity, with Rubisco activity more easily activated in the dark period, and atmospheric CO2 uptake maximized by day by direct C3 processes. The degree of succulence and expression of CAM is manifested as differences in the timing and duration of the CAM phases. The regulation and sink strength of each carboxylase affects light use efficiency, and fluorescence-derived ETR is directly related to the extent of Rubisco activity and activation. In conclusion, more succulent CAM plants tend to express a higher degree of nocturnal CO2 fixation and daytime uptake, strongly inhibit or down-regulate Rubisco activity at night, and are probably more committed to CAM because of low mesophyll conductances (Griffiths et al., 2007).
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Acknowledgements |
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This research was supported by The Royal Society UK, NERC, and The Isaac Newton Trust. We are grateful to Alex Goodall and Pete Michna for plant maintenance. Professor Martin Parry, Dr John Androlojc, and Dr Alfred Keys at Rothamsted Research, Harpenden, UK, were generous in their enthusiastic advice in developing the Rubisco purification and assay procedures. Wanne Kromdijk and Aline Horwath kindly provided data analysis.
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* This paper is dedicated to Barry Osmond, who encouraged us to investigate the dark side of CAM.
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