Obligate CAM Research Articles

Regulation of the Degree of Manifestation of Obligate CAM Pathway in Sedum dendroideum by Salinity

Regulation of the Degree of Manifestation of Obligate CAM Pathway in Sedum dendroideum by Salinity

Effects of chilling on the photosynthetic performance of the CAM orchid Phalaenopsis.

Crassulacean acid metabolism (CAM) is one of the three main metabolic adaptations for CO2 fixation found in plants. A striking feature for these plants is nocturnal carbon fixation and diurnal decarboxylation of malic acid to feed Rubisco with CO2 behind closed stomata, thereby saving considerable amounts of water. Compared to the effects of high temperatures, drought, and light, much less information is available about the effects of chilling temperatures on CAM plants. In addition a lot of CAM ornamentals are grown in heated greenhouses, urging for a deeper understanding about the physiological responses to chilling in order to increase sustainability in the horticultural sector. The present study focuses on the impact of chilling temperatures (10°C) for 3 weeks on the photosynthetic performance of the obligate CAM orchid Phalaenopsis 'Edessa'. Detailed assessments of the light reactions were performed by analyzing chlorophyll a fluorescence induction (OJIP) parameters and the carbon fixation reactions by measuring diel leaf gas exchange and diel metabolite patterns. Results showed that chilling already affected the light reactions after 24h. Whilst the potential efficiency of photosystem II (PSII) (Fv/Fm) was not yet influenced, a massive decrease in the performance index (PIabs) was noticed. This decrease did not depict an overall downregulation of PSII related energy fluxes since energy absorption and dissipation remained uninfluenced whilst the trapped energy and reduction flux were upregulated. This might point to the presence of short-term adaptation mechanisms to chilling stress. However, in the longer term the electron transport chain from PSII to PSI was affected, impacting both ATP and NADPH provision. To avoid over-excitation and photodamage plants showed a massive increase in thermal dissipation. These considerations are also in line with carbon fixation data showing initial signs of cold adaptation by achieving comparable Rubisco activity compared to unstressed plants but increasing daytime stomatal opening in order to capture a higher proportion of CO2 during daytime. However, in accordance with the light reactions data, Rubisco activity declined and stomatal conductance and CO2 uptake diminished to near zero levels after 3 weeks, indicating that plants were not successful in cold acclimation on the longer term.

Light-responsive expression atlas reveals the effects of light quality and intensity in Kalanchoë fedtschenkoi, a plant with crassulacean acid metabolism.

BackgroundCrassulacean acid metabolism (CAM), a specialized mode of photosynthesis, enables plant adaptation to water-limited environments and improves photosynthetic efficiency via an inorganic carbon-concentrating mechanism. Kalanchoë fedtschenkoi is an obligate CAM model featuring a relatively small genome and easy stable transformation. However, the molecular responses to light quality and intensity in CAM plants remain understudied.ResultsHere we present a genome-wide expression atlas of K. fedtschenkoi plants grown under 12 h/12 h photoperiod with different light quality (blue, red, far-red, white light) and intensity (0, 150, 440, and 1,000 μmol m–2 s–1) based on RNA sequencing performed for mature leaf samples collected at dawn (2 h before the light period) and dusk (2 h before the dark period). An eFP web browser was created for easy access of the gene expression data. Based on the expression atlas, we constructed a light-responsive co-expression network to reveal the potential regulatory relationships in K. fedtschenkoi. Measurements of leaf titratable acidity, soluble sugar, and starch turnover provided metabolic indicators of the magnitude of CAM under the different light treatments and were used to provide biological context for the expression dataset. Furthermore, CAM-related subnetworks were highlighted to showcase genes relevant to CAM pathway, circadian clock, and stomatal movement. In comparison with white light, monochrome blue/red/far-red light treatments repressed the expression of several CAM-related genes at dusk, along with a major reduction in acid accumulation. Increasing light intensity from an intermediate level (440 μmol m−2 s−1) of white light to a high light treatment (1,000 μmol m–2 s–1) increased expression of several genes involved in dark CO2 fixation and malate transport at dawn, along with an increase in organic acid accumulation.ConclusionsThis study provides a useful genomics resource for investigating the molecular mechanism underlying the light regulation of physiology and metabolism in CAM plants. Our results support the hypothesis that both light intensity and light quality can modulate the CAM pathway through regulation of CAM-related genes in K. fedtschenkoi.

Photosynthetic adaptation and survival strategy of Duvalia velutina in an extremely arid environment

Leafless Duvalia velutina Lavranos (Apocynaceae) is an arido-active stem succulent common in the arid region southwest of the Arabian Peninsula. This region is characterized by a short wet season with erratic rainfall and a long dry season with high temperature and high irradiance. We investigated the survival strategy of D. velutina by studying nurse association, gas exchange, and chlorophyll fluorescence. Results showed that D. velutina exhibited the strict nurse association with shade for protection against heat and high irradiance. Results also showed that D. velutina is an obligate CAM plant with ample physiotypic plasticity involving a shift to CAM-idling under prolonged drought. Chlorophyll fluorescence measurements revealed water stress-induced reduction of PSII activity occurring in concomitance with a marked rise of nonphotochemical quenching and chlorenchyma anthocyanin content. These results reflected photoprotective capacity involving nonradiative excess energy dissipation and antioxidative attributes. We concluded that the complex survival strategy of D. velutina in its natural arid habitat includes a multifaceted interplay of nurse association, physiotypic plasticity, and photoprotective mechanisms.

Identification of obligate C<sub>3</sub> photosynthesis in Dendrobium

Dendrobium is one of the three largest genera in the Orchidaceae and is distributed throughout various habitats. We investigated photosynthesis in seven Dendrobium species and cultivars by comparing their leaf δ13C values, titratable acidity, and CO2 exchange in well-watered and drought-stressed conditions. In addition, the leaf thickness and mesophyll succulence index (Sm) were measured in well-watered conditions. Our results indicate that Dendrobium loddigesii is a typical obligate (or constitutive) CAM plant because the leaf δ13C values were -14.47 and -14.66‰ in both conditions, respectively. Others showed the leaf thickness of 0.31-0.89 mm and their δ13C values ranged from -25.68 to -28.37‰. These are not the CAM plants but they could not be classified as obligate C3 or C3/CAM intermediate plants. Dendrobium crepidatum and Dendrobium fimbriatum were further identified as the obligate C3 plants because the net CO2 uptake was positive during daytime and negative during nighttime in both conditions. In contrast, Dendrobium chrysotoxum, Dendrobium nobile, and D. nobile 'V1' and 'V4', showed no positive net CO2 uptake and low ΔH+ values during nighttime under well-watered conditions, indicating C3 photosynthesis. However, they showed the positive net CO2 uptake and large ΔH+ values during nighttime after drought-stress (21 or 28 days without H2O), indicating CAM photosynthesis. Therefore, these four species and cultivars were identified as C3/CAM intermediate (inducible or facultative) plants. In brief, obligate CAM, C3/CAM intermediate, and obligate C3 plant types all exist in the section of Dendrobium. To the best of our knowledge, this is the first report of the obligate C3 plants in Dendrobium, and these diverse photosynthetic pathways may explain their varied environmental adaptations.

Photosynthetic activity of stems in two Clusia species

In stems of Clusia, CO 2 concentrated in the xylem sap in CAM trees can be fixed by PEPC and Rubisco, while in C 3 trees only Rubisco is engaged. The photosynthetic characteristics of 7–8-year-old stems of two tropical trees representing the Clusiaceae family were compared: Clusia multiflora Kunth. described as an obligate C3 and Clusia rosea Jacq. as an obligate CAM plant. Photosynthetic gas exchange, xylem CO2 concentration, chlorophyll distribution, 13C discrimination, daily malate and citrate fluctuations and the abundance of Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) and PEPC (phosphoenolpyruvate carboxylase) proteins were measured in leaves and stems. In stems of both species a low CO2 efflux (in the range of 0.05–0.1 µmol m−2 s−1) was observed as a result of extremely low cork conductance for water vapor (0.15–0.2 mmol m−2 s−1). This led to the CO2 concentration in xylem sap reaching 5.2 (CO2*) mmol l−1. The substantial amount of chlorophyll in the outer part of the bark and light-induced decrease of CO2 concentration within the xylem can be explained by photosynthetic activity in this tissue. Moreover, Western blotting analyses proved the presence of Rubisco in the stems of both Clusia species; however, PEPC was only found in C. rosea. Additionally, daily fluctuations in the concentration of citrate and malate (higher than in leaves) and significant enrichment in 13C in C. rosea stems were observed. These facts allow us to conclude that the examined stems of C. rosea and C. multiflora represent specific types of photosynthetic metabolism.

Light quality modulates metabolic synchronization over the diel phases of crassulacean acid metabolism.

Temporal compartmentation of carboxylation processes is a defining feature of crassulacean acid metabolism and involves circadian control of key metabolic and transport steps that regulate the supply and demand for carbon over a 24h cycle. Recent insights on the molecular workings of the circadian clock and its connection with environmental inputs raise new questions on the importance of light quality and, by analogy, certain photoreceptors for synchronizing the metabolic components of CAM. The present work tested the hypothesis that optimal coupling of stomatal conductance, net CO2 uptake, and the reciprocal turnover of carbohydrates and organic acids over the diel CAM cycle requires both blue and red light input signals. Contrasting monochromatic wavelengths of blue, green, and red light (i.e. 475, 530, 630nm) with low fluence rates (10 μmol m(-2) s(-1)) were administered for 16 hours each diel cycle for a total treatment time of 48 hours to the obligate CAM bromeliad, Aechmea 'Maya'. Of the light treatments imposed, low-fluence blue light was a key determinant in regulating stomatal responses, organic acid mobilization from the vacuole, and daytime decarboxylation. However, the reciprocal relationship between starch and organic acid turnover that is typical for CAM was uncoupled under low-fluence blue light. Under low-fluence red or green light, the diel turnover of storage carbohydrates was orchestrated in line with the requirements of CAM, but a consistent delay in acid consumption at dawn compared with plants under white or low-fluence blue light was noted. Consistent with the acknowledged influences of both red and blue light as input signals for the circadian clock, the data stress the importance of both red and blue-light signalling pathways for synchronizing the metabolic and physiological components of CAM over the day/night cycle.

Acclimation to high salinity in the invasive CAM plant Aptenia cordifolia

Background: Invasive species, such as Aptenia cordifolia, grow rapidly in different habitats, colonising large areas of soil surface. However, mechanisms of acclimation to salt stress have not been explored thus far in this obligate CAM plant. Aims: We evaluated the capacity of this species to withstand salt stress in terms of growth, photoprotection and changes in phytohormones. Results: This species was not only very resistant to extreme salinity (up to 650 mM NaCl) in the short term (14 days) but also to high salt concentrations (150 mM NaCl) in the longer term (42 days); improvements in leaf water status and fresh biomass production were also observed. This occurred concurrently with an increase in zeatin levels, while concentrations of other phytohormones were unaffected. Furthermore, plants were able to maintain the F v/F m ratio above 0.80 – indicating little damage to PSII – for 42 days at 150 mM NaCl, or above 0.50 for 14 days at extreme salinity (up to 650 mM NaCl). Conclusions: We conclude that A. cordifolia has not only evolved mechanisms to adapt to high salinity, but it is able to take advantage of such conditions by increasing leaf water content and fresh biomass production, which underpin its ecological competence in saline environments.

The photosynthetic pathway of the roots of twelve epiphytic orchids with CAM leaves

The photosynthetic pathway of the roots (both the white velamentous main portions and the green, nonvelamentous tips) was investigated in twelve taxa (natural species and intergeneric hybrid cultivars) of epiphytic orchids having CAM leaves. All organs contained chlorophyll, and the a/b ratios indicate that the organs, especially the roots, are likely shade-adapted. Stable carbon isotope ratios of the tissues were near −15‰ for all organs, a value typical of obligate (constitutive) CAM plants. Values for root tissues were slightly lower (more negative) than those of the leaves. The presence of CAM in the leaves of these orchids did not ensure that their roots performed CAM photosynthesis. Further work is needed to address the questions raised in this study and to determine if the photosynthetic roots of these taxa are capable of assimilating atmospheric CO2.

Physiological evolution and association between physiology and growth form inAeonium(Crassulaceae)

Aeonium(Crassulaceae) comprises species that display a remarkable degree of morphological diversity, including rosette trees, candelabrum shrubs, highly branched shrubs, and woody rosettes. Greenhouse studies have documented that the genus is also diverse in basic photosynthetic metabolism, with C3, CAM, and C3‐CAM intermediates present. However, the degree of photosynthetic diversity present in natural populations and between species has never been surveyed. We analyzed stable isotopes of carbon from field‐collected species ofAeoniumand confirmed that these species are diverse physiologically. Using these data, each species sampled was coded as CAM, C3, or intermediate. This trait was optimized using parsimony onto one arbitrarily selected cladogram for Macaronesian Crassulaceae that had been previously published. These analyses indicated that the ancestral physiological condition for the entire Macaronesian clade of Crassulaceae is C3photosynthesis. WithinAeonium, one subclade consists largely of species with obligate CAM, one is exclusively intermediate, and two subclades are more variable in photosynthetic metabolism. The ancestral character state for theAeoniumclade is equivocal when the intermediate carbon isotope values were coded as a third character state; however, when these intermediates are considered polymorphic (i.e., facultative CAM), C3photosynthesis also was ancestral inAeonium. Concentrated changes tests were conducted to explore associations between CAM and three growth‐form attributes, including the candelabrum shrub and rosette tree growth‐forms and monocarpy. These tests revealed no significant association between CAM and monocarpy. The candelabrum shrub growth‐form is also not linked with CAM, but CAM has evolved twice within taxa displaying this growth‐form. In contrast, the hypothesized correlation between CAM and the rosette tree growth‐form was neither rejected nor supported.

Molecular Evidence for the Occurrence of H<sup>+</sup>-Transporting V-ATPase Subunit D and Two Different Forms of Subunit E in Leaves of the Obligate CAM Species Kalanchoë daigremontiana

Membrane proteins of purified tonoplast vesicles from leaves of KalanchoĂŤ daigremontiana Hamet et Perrier were solubilized by the non-ionic detergent Triton X-114 and subsequently separated by MonoQ® anion-exchange chromatography. Special attention was given to the range of molecular masses around 30 kDa comprising the central stalk subunit peptides of the H+-transporting V-ATPase. Three polypeptides of apparent molecular masses of 32, 33 and 34 kDa were separated. Proteolytic fragments were obtained by trypsin digestion. Analysis by matrix-assisted laser desorption ionization (MALDI) mass spectrometry of tryptic fragments of the 32 and 33 kDa peptides and protein data- bank comparisons showed that they are two different forms of subunit E. N-terminal amino acid sequencing of tryptic fragments of the 34 kDa peptide showed that it is subunit D. This work provides for the first time unequivocal molecular evidence that the central stalk of the V-ATPase of the obligate CAM plant K. daigremontiana includes subunit D and different forms of subunit E.

An isozyme of the NADP-malic enzyme of a CAM plant, Aloe arborescens, with variation on conservative amino acid residues.

In Aloe arborescens, an obligate CAM plant, Western analysis detected three major isoforms of NADP-malic enzyme (NADP-ME), 72kDa with a pI of 6.0, 65kDa with a pI of 5.6 and 65kDa with a pI of 5.5. Among them, the 65kDa protein with a pI of 5.5 was leaf-specific, and the 65kDa protein with a pI of 5.6 was found only in roots, whereas the 72kDa protein was uniformly detected in both organs. Activity staining indicated enzyme activity of both 65kDa NADP-MEs but little activity of the 72kDa protein. A cDNA clone encoding a leaf-abundant NADP-ME, AME1, was isolated. Deduced amino acid sequence of AME1 showed a high degree of homology to known NADP-MEs, but it was also found that AME1 contained substitutions on five conservative amino acid residues, some of which have been predicted to be important for their enzyme activity. Transgenic rice carrying the aloe AME1 gene efficiently produced an additional 65kDa protein with a pI of 5.5 as an active NADP-ME. These results indicate that AME1 corresponds to the leaf-specific 65kDa NADP-ME, which may be involved in CAM photosynthesis. It was also shown that substitutions of these conservative amino acid residues identified in AME1 still allowed it to give enzyme activity.

Comparative Study of the C3/CAM Intermediate Species Clusia parviflora Saldanha et Engi. and the Obligate CAM Species Clusia hilariana Schlecht. Growing Sympatrically Exposed and Shaded in the Coastal Restinga of Brazil

Abstract: The C3/CAM intermediate species, C/usia parviflora Saldanha et EngI., and the obligate CAM species Clusia hilariana Schlecht., occur sympatrically in the coastal sand dune vegetation of the Restinga of Brazil. Their photosynthetic activity at an exposed and at a shaded site was compared by measuring gas exchange (porometry), chlorophyll a fluorescence parameters, organic acid levels (malic and citric) and carbon isotope ratios. At the shaded site, low photosynthetic photon flux densities (PPFD) strongly restricted photosynthetic activity. However, C parviflora could readily make use of light flecks. At the exposed site, C. parviflora was much less affected by photoinhibition than C. hilariana. The CAM species showed higher apparent rates of linear photosynthetic electron transport (ETR) and higher effective quantum yield of PSII (ΔF/F'm) than did C. parviflora during high insolation in the middle of the day, i.e., the time of Phase Ill of CAM. Nevertheless, it suffered much more severe acute photoinhibition that was not reversible after 20 min of darkening during this time, and even some chronic photoinhibition not reversible overnight. Comparative studies of sympatric physiotypes with different modes of photosynthesis of a given leaf morphotype, as available in the genus Cksia, challenge some CAM dogmas, e.g., CAM may not always be superior at exposed sites and may not always provide better photoprotection at high PPFD. However, the idea that C3/CAM plasticity allows occupation of a wider range of habitats is supported.

One Morphotype, Three Physiotypes: Sympatric Species of Clusia with Obligate C3 Photosynthesis, Obligate CAM and C3‐CAM Intermediate Behaviour

Abstract: Among about 150 species of the genus of Clusia, morphologically very similar neotropical shrubs and trees, 20 have been studied ecophysiologically and 39 original publications were screened here for key data on the potential performance of the various species. I have collected data on carbon isotope discrimination (873C), day‐night oscilliations of malate (Amal), citrate (Acitr) and titratable protons (AH), maximum rates of net CO2 exchange in the dark (Dark Jco2) and in the light (Light Jco2) apparent rate of photosynthetic electron transport at PS II (ETR), effective quantum yield of non‐cyclic electron transport at PS II (AF/F'm) at 1000 umol m‐2 s‐1 PPFD, and potential quantum yield of PS II of leaves adapted to short periods of darkness at midday (Fv/Fm midday) and after relaxation overnight (Fv/Fm predawn). The 20 species can be arranged from predominant C3 photosynthesis to principally CAM. There may be only a few bona fide obligatory CAM species in the genus, but it is doubtful if there are obligatory C3 species. The data were then used to provide comparisons of intrinsic capabilities for Clusia species occurring sympatrically at two sites each, in Brazil and Venezuela. No clear advantage of CAM emerges from these comparisons under the stress of high insolation and low water availability at these sites. Different Clusia species are successful in different ways and with different intrinsic ecophysiological capacities. The conventional expectations in CAM as a drought and light stress adaptation are confounded in Clusia to the extent that on some occasions C3 photosynthesis seems to be the superior strategy. However, it appears, that C3/CAM plasticity which is so widespread in the genus, with many species and potentially rapid speciation, allows a particularly wide ecological amplitude.

One Morphotype, Three Physiotypes: Sympatric Species ofClusiawith Obligate C3Photosynthesis, Obligate CAM and C3-CAM Intermediate Behaviour

One Morphotype, Three Physiotypes: Sympatric Species of<i>Clusia</i>with Obligate C₃Photosynthesis, Obligate CAM and C₃-CAM Intermediate Behaviour

Ecophysiological investigations on plants of the genus Plectranthus(Lamiaceae). Influence of environment and leaf age on CAM, gasexchange and leaf water relations in Plectranthus marrubioides Benth.

Plectranthus marrubioides , a leaf succulent native to the Higher Escarpment of Yemen, shows obligate CAM. Even in the youngest leaves nocturnal accumulation of malic and, to a lower extent, of citric acid (≈ 10% that of malic acid) was measurable. Patterns of gas exchange were typically that of CAM plants in mature leaves while expanding leaves exhibited CAM-cycling. Gas exchange patterns were very flexible. In response to changes in the environmental conditions they could reversibly be switched between all variations of CAM. Decreasing daytime temperature from 35 to 20°C or varying light intensities from 150 to 600 μmol m −2 S −1 did not influence nocturnal CO 2 uptake or malic acid accumulation but enhanced daytime carbon gain. Eliminating day/night temperature difference induced CAM-cycling, while changes in nighttime air humidity had no consequences for CAM. Withholding watering suppressed daytime carbon gain within 2 days but neither enhanced nor restricted CAM at moderate environmental conditions. Even at high temperature and low humidity it was reduced only partially with decreasing leaf water content; long-term drought led to CAM-idling. Leaf water content was highly correlated with malic acid accumulation under drought and over the age gradient. The only pronounced effect on nocturnal CO 2 gain and malic acid accumulation was exhibited by nighttime temperature. Temperature response of CAM was influenced by the combination of environmental factors during both night and day. Thus, temperature optima ranged from 15 to 25°C in many experiments, resulting in a mean optimum of 20°C. The data show that it is mainly C 3 photosynthesis and leaf conductance that flexibly responded to ecophysiological relevant changes in environmental conditions while CAM is much less affected.

Ecophysiological investigations on plants of the genus Plectranthus (fam. Lamiaceae) native to Yemen and southern Africa

Summary In this first survey of the ecophysiology of 8 succulent and semi-succulent species from the genus Plectranthus (Larniaceae) native to Yemen and southern Africa it has been shown that three species, Plectranthus arabicus, P. hadiensis and P. marrubioides are obligate CAM plants. Well-watered plants of these yemenitic species, which are found primarily with other succulents at altitudes up to 2 600 m, exhibit a nocturnal accumulation of malic acid, and, to a much smaller extent, citric acid as well as night-time CO 2 uptake under controlled-environment conditions. While the semi-succulent P. cyrpiculoides utilises the CAM-cycling mode, the semi-succulent species, P. barbatus, P. purpuratus, P. zatarhendi, and P. zatarhendi var. tomentosus, are typical C 3 plants. Thus, the ability to perform CAM was well correlated with succulence. Neither CAM nor succulence was, however, directly related to maximum desiccation resistance in these species. Increasing day-time temperatures from 20 to 35°C reduces net daily carbon gain in all, leading to CO 2 losses in most of the C 3 species. In the CAM species, nocturnal CO 2 uptake at least partially alleviated the effect of the high temperature conditions. On the other hand, the C 3 species P. barbatus , growing in succulent plant communities in high mountain habitats (up to 3000 m), was also relatively insensitive to high day-time temperatures. This is the first extensive description of CAM and CAM-cycling in the Lamiaceae. The presence of CAM in Plectranthus correlated with habitat aridity in situ.

Influence of Nitrogen, Water and Competition on Carbon Isotope Ratios (δ13C) in a CAM Plant (Crassula argentea)

Abstract Leaf carbon isotope ratios (δ13C), an indicator of long-term intercellular carbon dioxide concentration, and also stem and root carbon isotope ratios were measured on the obligate CAM species Crassula argentea cultivated in pure and mixed cultures with the succulent C3 Peperomia obtusifolia in open-air conditions under two different levels of nitrogen and water supply. As expected, a diminished water supply and a relatively dry and hot summer climate cause a shift of δ13C values to a less strong 13C discrimination (less negative δ13C values). A diminished nitrogen supply causes a shift of the δ13C values in direction of a higher 13C discrimination (more negative δ13C values), particularly in the leaves. Competition causes also an increased 13C discrimination, especially valid for shoot axes. The shift of 13C/12C isotope ratios in case of nitrogen deficiency is discussed to be a result of a decreased PEPCase activity in the night.

Quaternary Structure of Phosphoenolpyruvate Carboxylase from CAM-C4- and C3-Plants - No Evidence for Diurnal Changes in Oligomeric State

Summary Day and night forms of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.31) were extracted from leaves of the inducible CAM plant Mesembryanthemum crystallinum, from the obligate CAM plant Crassula spec., the C4 plant Zea mays and the C3 plant Spinacia oleracea. In induced (salt stressed) M crystallinum and in Crassula spec. the day form of the enzyme showed higher sensitivity to inhibition by malate than the night form. The enzyme from Z. mays showed higher sensitivity when it was extracted during the dark period. In contrast, the enzyme extracted from non-induced (unstressed) M. crystallinum and S. oleracea showed no changes in malate sensitivity during a diurnal cycle. Desalted crude extracts were subjected to high-performance size exclusion liquid chromatography. PEPC activity in the absence and in the presence of malate and PEPC protein (measured immunochemically) were monitored after extraction and gel filtration. The fractionated enzyme still displayed the same characteristic pattern of sensitivity to inhibition by malate that could be measured in desalted crude extracts, indicating high stability of the kinetic properties during gel filtration. In all species investigated, the day and the night forms of the enzyme were found to be in the same aggregation state. The apparent molecular weights of the native enzymes and of the enzyme subunits from M. crystallinum (C3 and CAM), Z. mays and S. oleracea indicated a tetrameric aggregation state, while PEPC from Crassula spec. was found to be dimeric. These results suggest that contrary to earlier reports on PEPC from Crassula argentea, reversible dissociation/ association is not the mechanism that regulates the day/night and light/dark transitions in the kinetic properties of PEPC from CAM- and C4 plants, respectively.

Seasonal Variation of Phosphoenolpyruvate Carboxylase Specific Activity in Fifteen Species Exhibiting Facultative or Obligate Crassulacean Acid Metabolism

For 16 months the specific activity of the CAM key-enzyme PEP carboxylase (PEP-C) was determined monthly in twelve Sedum species previously shown to exhibit facultative or obligate CAM, as well as in Aeonium castello-paivae, Sempervivum nevadense and Kalanchoe daigremontiana. In all species examined the PEP-C activity showed a seasonal fluctuation with maxima during the summer from 8- to 30-fold higher than winter values. For most species the seasonal fluctuation correlated significantly with temperature and, to a lesser extent, with irradiation; no correlation was present with precipitation in garden-grown plants. Plants grown in greenhouse and garden showed corresponding seasonal fluctuations. Specific PEP-C activity, however, was restored to the summer level upon transfer to a growth chamber (21/15 °C, LD) in October. To determine the controlling climatic factor for this growth room-induced increase in PEP-C activity, the influence of light intensity, photoperiod and temperature on PEP-C activity was investigated in Sedum rupestre. Daytime temperature appeared to be the most influential factor, whereas light intensity had a small but significant effect.