<Emphasis Type="Italic">δ</Emphasis><Superscript>13</Superscript>C values and crassulacean acid metabolism in <Emphasis Type="Italic">Clusia</Emphasis> species from Panama

The genus Clusia is notable in that it contains arborescent crassulacean acid metabolism (CAM) plants. As part of a study of CAM in Clusia, titratable acidities were measured in 25 species and ¹³C values were measured for 38 species from Panamá, including seven undescribed species, and 11 species from Colombia, Costa Rica and Honduras. CAM was detected in 12 species. Clusia flava, C. rosea and C. uvitana exhibited ¹³C values or diurnal fluctuations in acidity indicative of strong CAM. In C. croatii, C. cylindrica, C. fructiangusta, C. lineata, C. odorata, C. pratensis, C. quadrangula, C. valerioi and C. sp. D diurnal fluctuations in acidity were consistent with weak CAM but the ¹³C values were C₃-like. All of the species that exhibited strong or weak CAM were in the C. flava or C. minor species groups. CAM was not detected in any member of the C. multiflora species group. Strong CAM species were not collected at altitudes above 680 m a.s.l. On the basis of ¹³C values, the expression of CAM was similar in terrestrial, hemi-epiphytic and epiphytic species and did not differ between individuals of the same species that exhibited different life-forms. This study indicates that phylogenetic affiliation may be a predictor of an ability to exhibit CAM in Clusia species from the Panamanian region, and that weak CAM is probably a common photosynthetic option in many Clusia species. ¹³C value is not a particularly good indicator of a potential of Clusia species growing in the field to exhibit CAM because it appears that the contribution in most species of CAM to carbon gain is generally rather small when integrated over the life-time of leaves.     

Barriers to movement and the response of herbivores to alternative cropping patterns

Summary Clusia rosea Jacq. is a hemiepiphyte having Crassulacean Acid Metabolism (CAM). In its natural habitat Clusia begins its life cycle as an epiphyte and eventually becomes a rooted tree. These two stages of the life cycle of Clusia represent markedly different water regimes. Our CO₂ exchange, stomatal conductance, titratable acidity, and stable carbon isotope ratio measurements indicate that Clusia has a flexible photosynthetic mode, where CO₂ is fixed mostly via CAM during its epiphytic stage, when water availability is low, and via both CAM and C₃ during its rooted stage.     

Recycling of respiratory CO2 during Crassulacean acid metabolism: alleviation of photoinhibition in Pyrrosia piloselloides

The regulation of Crassulacean acid metabolism (CAM) in the fern Pyrrosia piloselloides (L.) Price was investigated in Singapore on two epiphytic populations acclimated to sun and shade conditions. The shade fronds were less succulent and had a higher chlorophyll content although the chlorophyll a:b ratio was lower and light compensation points and dark-respiration rates were reduced. Dawn-dusk variations in titratable acidity and carbohydrate pools were two to three times greater in fronds acclimated to high photosynthetically active radiation (PAR), although water deficits were also higher than in shade fronds. External and internal CO₂ supply to attached fronds of the fern was varied so as to regulate the magnitude of CAM activity. A significant proportion of titratable acidity was derived from the refixation of respiratory CO₂ (27% and 35% recycling for sun and shade populations, respectively), as measured directly under CO₂-free conditions. Starch was shown to be the storage carbodydrate for CAM in Pyrrosia, with a stoichiometric reduction of C₃-skeleton units in proportion to malic-acid accumulation. Measurements of photosynthetic O₂ evolution under saturating CO₂ were used to compare the light responses of sun and shade fronds for each CO₂ supply regime, and also following the imposition of a photoinhibitory PAR treatment (1600 mol·m^-2·s^-1 for 3 h). Apparent quantum yield declined following the high-PAR treatment for sun- and shade-adapted plants, although for sun fronds CAM activity derived from respiratory CO₂ prevented any further reduction in photosynthetic efficiency. Recycling of respiratory CO₂ by shade plants could only partly prevent photoinhibitory damage. These observations provide experimental evidence that respiratory CO₂ recycling, ubiquitous in CAM plants, may have developed so as to alleviate photoinhibition.Abbreviations and symbols CAM Crassulacean acid metabolism - F_M maximal photosystem II fluorescence - F_T terminal steady-state fluorescence - PAR photosynthetically active radiation, 400–700 nm - H⁺ (dawn-dusk) variation in titratable acidity     

Variable Photosynthetic Metabolism in Leaves and Stems of Cissus quadrangularis L

By measuring titratable acidity, gas exchange parameters, mesophyll succulence, and ¹³C/¹²C ratios, we have shown that Cissus quadrangularis L. has C₃-like leaves and stems with Crassulacean acid metabolism (CAM). In addition, the nonsucculent leaves show the diurnal fluctuations in organic acids termed recycling despite the fact that all CO₂ uptake and stomatal opening occurs during the day. Young succulent stems have more C₃ photosynthesis than older stems, but both have characteristics of CAM. The genus Cissus will be a fruitful group to study the physiology, ecology, and evolution of C₃ and CAM since species occur that exhibit characteristics of both photosynthetic pathways.     

Fluorescence Quenching in the Varied Photosynthetic Modes of Portulacaria afra (L.) Jacq

The kinetics of chlorophyll fluorescence were measured in Portulacaria afra (L.) Jacq. when the plants were functioning in either Crassulacean acid metabolism (CAM) or C₃/CAM cycling (called cycling) modes, as determined by fluctuation in titratable acidity and gas exchange properties. Cycling plants showed primarily daytime CO₂ uptake typical of C₃ plants, but with a slight diurnal acid fluctuation, whereas CAM plants showed nocturnal CO₂ uptake, daytime stomatal closure, and a large diurnal acid fluctuation. Results from fluorescence measurements indicated no significant differences in photochemical quenching between cycling and CAM plants; however, sizable differences were detected in nonphoto-chemical quenching (q_n), with the largest differences being observed during the middle of the day. Cycling plants had lower q_n than CAM plants, indicating altered photosynthetic regulation processes. This q_n difference was believed to be related to reduced internal CO₂ concentration in the CAM plants because of daytime stomatal closure and reduced deacidification rates in the late afternoon when most of the malic acid has been utilized. Experimentally, higher external CO₂ given to plants in the CAM mode resulted in a decline in q_n in comparison to that measured in plants in the cycling mode. No changes were observed in photochemical quenching when CO₂ was added.     

Patterns of gas exchange and organic acid oscillations in tropical trees of the genus Clusia

Summary Gas exchange patterns and nocturnal acid accumulation were examined in four species of Clusia under simulated field conditions in the laboratory. Clusia alata and C. major had midday stomatal closure, substantial net CO₂ exchange ( ) during the night, and the highest water use efficiency (WUE). C. venosa showed a pattern similar to a C₃ plant, with nighttime stomatal closure, while C. minor maintained positive continuously throughout a 24-h period. However, large changes in titratable acidity, which closely matched changes in citrate and malate levels, indicated that Crassulacean acid metabolism (CAM) is active in all four species. C. venosa showed dawn-dusk oscillations in titratable acidity that were higher than the values reported for other C₃-CAM intermediates, while the nighttime acid accumulation of 998 mol m^–3 observed in C. major is unsurpassed by any other CAM plant. Moreover, the dawn-dusk changes in citrate levels of over 65 mol m^–3 in C. alata and C. minor, and over 120 mol m^–3 in C. major, are 3–6 times higher than values reported for other CAM plants. Although these oscillations in citrate levels were quite large, and the nighttime dark respiration rates were high, the O₂ budget analysis suggestes that only part of the reducing power generated by the synthesis of citric acid enters the respiratory chain. Dawn-dusk changes in malate levels were just over 50 mol m^–3 for C. venosa but over 300 mol m^–3 for C. major. Between 28% (C. major) and 89% (C. venosa) of the malate accumulated during the night was derived from recycled respiratory CO₂. These daily changes in malate and citrate levels also contributed significantly to changes in leaf sap osmolality. This variability in CO₂ uptake patterns, the recycling of nighttime respiratory CO₂, and the high WUE may have contributed to the successful invasion of Clusia into a wide range of habitats in the tropics.     

Characterization and Partial Purification of Aldose-6-phosphate Reductase (Alditol-6-Phosphate:NADP 1-Oxidoreductase) from Apple Leaves

The Pereskia are morphologically primitive, leafed members of the Cactaceae. Gas exchange characteristics using a dual isotope porometer to monitor ¹⁴CO₂ and tritiated water uptake, diurnal malic acid fluctuations, phosphoenolpyruvate carboxylase, and malate dehydrogenase activities were examined in two species of the genus Pereskia, Pereskia grandifolia and Pereskia aculeata. Investigations were done on well watered (control) and water-stressed plants. Nonstressed plants showed a CO₂ uptake pattern indicating C₃ carbon metabolism. However, diurnal fluctuations in titratable acidity were observed similar to Crassulacean acid metabolism. Plants exposed to 10 days of water stress exhibited stomatal opening only during an early morning period. Titratable acidity, phosphoenolpyruvate carboxylase activity, and malate dehydrogenase activity fluctuations were magnified in the stressed plants, but showed the same diurnal pattern as controls. Water stress causes these cacti to shift to an internal CO₂ recycling (“idling”) that has all attributes of Crassulacean acid metabolism except nocturnal stomata opening and CO₂ uptake. The consequences of this shift, which has been observed in other succulents, are unknown, and some possibilities are suggested.     

Studies on carbon flow in Crassulacean acid metabolism during the initial light period

In the Crassulacean acid metabolism (CAM) plants Kalanchoë tubiflora and Sedum morganianum a shift in the pathways occurs by which external CO₂ enters the metabolism during the initial light period (phase II of the diurnal CAM cycle). At the beginning of phase II, CO₂ is fixed mainly by the C₄ pathway; during late phase II, however, it is fixed mainly via the C₃ pathway. The C₃ pathway contributes to the phosphoenolpyruvate-carboxylase-mediated CO₂ fixation by the provision of three-carbon skeletons. Since the shift in the carbon-flow pathway is delayed after a CO₂-free night when malic-acid accumulation in the vacuoles is prevented, it is very likely that the amount of malic acid in the vacuole is integrated in the mechanism which controls CAM during the initial light period. A light-on signal at the beginning of phase II is not required to bring about the shifts in the carbon-flow pathways, as is shown by the reaction of plants to a prolonged dark period. A model of carbon flow during phase II is proposed.Abbreviations CAM Crassulacean acid metabolism - PEP-Case phosphoenolpyruvate carboxylase     

The relationship between turgor pressure and titratable acidity in mesophyll cells of intact leaves of a Crassulacean-acid-metabolism plant,Kalanchoe daigremontiana Hamet et Perr

Day/night changes in turgor pressure (P) and titratable acidity content were investigated in the (Crassulacean-acid-metabolism (CAM) plant Kalanchoe daigremontiana. Measurements of P were made on individual mesophyll cells of intact attached leaves using the pressure-probe technique. Under conditions of high relative humidity, when transpiration rates were minimal, changes in P correlated well with changes in the level of titratable acidity. During the standard 12 h light/12 h dark cycle, maximum turgor pressure (0.15 MPa) occurred at the end of the dark period when the level of titratable acidity was highest (about 300 eq H⁺·g^-1 fresh weight). A close relationship between P and titratable acidity was also seen in leaves exposed to perturbations of the standard light/dark cycle. (The dark period was either prolonged, or else only CO₂-free air was supplied in this period). In plants deprived of irrigation for five weeks, diurnal changes in titratable acidity of the leaves were reduced (H=160 eq H⁺·g^-1 fresh weight) and P increased from essentially zero at the end of the light period to 0.02 MPa at the end of the dark period. Following more severe water stress (experiments were made on leaves which had been detached for five weeks), P was zero throughout day and night, yet small diurnal changes in titratable acidity were still measured. These findings are discussed in relation to a hypothesis by Lüttge et al. 1975 (Plant Physiol. 56,613-616) for the role of P in the regulation of acidification/de-acidification cycles of plants exhibiting CAM.Abbreviations CAM crassulacean acid metabolism - FW fresh weight - P turgor pressure     

Influence of Photoperiod and Leaf Age on Crassulacean Acid Metabolism in Portulacaria afra (L.) Jacq

The possibility that Crassulacean acid metabolism (CAM) is subject to long day photoperiodic control in Portulacaria afra (L.) Jacq., a facultative CAM plant, was studied. Periodic measurements of ¹⁴CO₂ uptake, stomatal resistance, and titratable acidity were made on plants exposed to long and short day photoperiods. Results indicates that waterstressed P. afra had primarily nocturnal CO₂ uptake, daytime stomatal closure, and a large diurnal acid fluctuation in either photoperiod. Mature leaf tissue from nonstressed plants under long days exhibited a moderate diurnal acid fluctuation and midday stomatal closure. Under short days, there was a reduced diurnal acid fluctuation in mature leaf tissue. Young leaf tissue taken from nonstressed plants did not utilize the CAM pathway under either photoperiod as indicated by daytime CO₂ uptake, lack of diurnal acid fluctuation, and incomplete daytime stomatal closure.

The induction of CAM in P. afra appears to be related to the water status of the plant and the age of the leaf tissue. The photosynthetic metabolism of mature leaves may be partly under the control of water stress and of photoperiod, where CAM is favored under long days.

     

Shifts in the Carbon Metabolism of Xerosicyos danguyi H. Humb. (Cucurbitaceae) Brought About by Water Stress : I. General Characteristics

Xerosicyos danguyi H. Humb. (Cucurbitaceae) is an unusual leaf succulent endemic to Madagascar. Under well-watered conditions the plant exhibited Crassulacean acid metabolism (CAM), as characterized by large diurnal changes in titratable acidity, predominantly nighttime stomatal opening and CO₂ uptake, and high δ¹³C values. When plants were exposed to water stress for a minimum of a month, they shifted to a mode of carbon metabolism previously labeled CAM-idling. Under this mode of metabolism, the plants exhibited reduced stomatal opening, reduced CO₂ uptake, dampened diurnal fluctuations in titratable acidity, and no apparent changes in the δ¹³C values. Additionally, investigations showed that the stress hormones 1-aminocyclopropane-1-carboxylic acid (an ethylene precursor) and abscisic acid increased as much as 6-fold in the water-stressed plants. The results are discussed in relation to physiological significance and evolution of the CAM-idling mode of metabolism.     

Induction of a Crassulacean acid like metabolism in the C4 succulent plant,Portulaca oleracea L.: physiological and morphological changes are accompanied by specific modifications in phosphoenolpyruvate carboxylase

The induction of a Crassulacean acid like metabolism (CAM) was evidenced after 21–23 days of drought stress in the C₄ succulent plant Portulaca oleracea L. by changes in the CO₂ exchange pattern, in malic acid content and in titratable acidity during the day–night cycle. Light microscopy studies also revealed differences in the leaf structure after the drought treatment. Following the induction of the CAM-like metabolism, the regulatory properties of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), the enzyme responsible for the diurnal fixation of CO₂ in C₄ plants but nocturnal in CAM plants, were studied. The enzyme from stressed plants showed different kinetic properties with respect to controls, notably its lack of cooperativity, higher sensitivity to L-malate inhibition, higher PEP affinity and lower enzyme content on a protein basis. In both conditions, PEPC's subunit mass was 110 kDa, although changes in the isoelectric point and electrophoretic mobility of the native enzyme were observed. In vivo phosphorylation and native isoelectrofocusing studies indicated variations in the phosphorylation status of the enzyme of samples collected during the night and day, which was clearly different for the control and stressed groups of plants. The results presented suggest that PEPC activity and regulation are modified upon drought stress treatment in a way that allows P. oleracea to perform a CAM-like metabolism. This revised version was published online in August 2006 with corrections to the Cover Date.     

The effects of salinity,crassulacean acid metabolism and plant age on the carbon isotope composition of <Emphasis Type="Italic">Mesembryanthemum crystallinum</Emphasis> L., a halophytic C<Subscript>3</Subscript>-CAM species

The carbon isotope composition of the halophyte Mesembryanthemum crystallinum L. (Aizoaceae) changes when plants are exposed to environmental stress and when they shift from C₃ to crassulacean acid metabolism (CAM). We examined the coupling between carbon isotope composition and photosynthetic pathway by subjecting plants of different ages to salinity and humidity treatments. Whole shoot ¹³C values became less negative in plants that were exposed to 400 mM NaCl in the hydroponic solution. The isotopic change had two components: a direct NaCl effect that was greatest in plants still operating in the C₃ mode and decreased proportionally with increasing levels of dark fixation, and a second component related to the degree of CAM expression. Ignoring the presumably diffusion-related NaCl effect on carbon isotope ratios results in an overestimation of nocturnal CO₂ gain in comparison to an isotope versus nocturnal CO₂ gain calibration established previously for C₃ and CAM species grown under well-watered conditions. It is widely taken for granted that the shift to CAM in M. crystallinum is partially under developmental control and that CAM is inevitably expressed in mature plants. Plants, cultivated under non-saline conditions and high relative humidity (RH) for up to 63 days, maintained diel CO₂ gas-exchange patterns and ¹³C values typical of C₃ plants. However, a weak CAM gas-exchange pattern and an increase in ¹³C value were observed in non-salt-treated plants grown at reduced RH. These observations are consistent with environmental control rather than developmental control of the induction of CAM in mature M. crystallinum under non-saline conditions.     

Ecophysiological studies in Kalanchoë porphyrocalyx (Baker) and K. miniata (Hils et Bojer), two species performing highly flexible CAM

Preceding results, based on the determination of stable carbon isotope composition (₁₃C) of leaf tissues from various Kalanchoë species, suggested a close coincidence between the photosynthetic flexibility of the species and their habitat, life form and taxonomic position within the genus. The ability to shift from C₃-to Crassulacean Acid Metabolism (CAM)-type of photosynthesis seemed to concern in particular the more ancestral species in the genus and to be linked to epiphytism and changing climatic situations. For deeper insights into these interrelationships, physiological studies in controlled conditions were carried out on K. miniata and K. porphyrocalyx. These two species differ by their habitat preference and life form. Measurements were conducted on CO₂ exchange patterns, day/night fluctuation of malate content in the leaves and capacity of phosphoenolpyruvate carboxylase (PEPC). The results show that the 2 species can be considered as facultative CAM plants, with very high flexibility in their photosynthetic behaviour. The decrease in water availability seems to be a major factor triggering the shift from C₃ to the CAM mode. In K. miniata, 21 days of drought depressed CO₂ uptake to the level of CAM idling whereas in K. porphyrocalyx, CO₂ exchange was considerably more resistant. At least for K. miniata, short-day treatment was found to be a further CAM-inducing factor. The results are discussed in terms of their ecophysiological significance under the environmental conditions of the sites where the investigated species naturally grow.     

Effects of Various Levels of CO(2) on the Induction of Crassulacean Acid Metabolism in Portulacaria afra (L.) Jacq

In response to water stress, Portulacaria afra (L.) Jacq. (Portulacaceae) shifts its photosynthetic carbon metabolism from the Calvin-Benson cycle for CO₂ fixation (C₃) photosynthesis or Crassulacean acid metabolism (CAM)-cycling, during which organic acids fluctuate with a C₃-type of gas exchange, to CAM. During the CAM induction, various attributes of CAM appear, such as stomatal closure during the day, increase in diurnal fluctuation of organic acids, and an increase in phosphoenolpyruvate carboxylase activity. It was hypothesized that stomatal closure due to water stress may induce changes in internal CO₂ concentration and that these changes in CO₂ could be a factor in CAM induction. Experiments were conducted to test this hypothesis. Well-watered plants and plants from which water was withheld starting at the beginning of the experiment were subjected to low (40 ppm), normal (ca. 330 ppm), and high (950 ppm) CO₂ during the day with normal concentrations of CO₂ during the night for 16 days. In water-stressed and in well-watered plants, CAM induction as ascertained by fluctuation of total titratable acidity, fluctuation of malic acid, stomatal conductance, CO₂ uptake, and phosphoenolpyruvate carboxylase activity, remained unaffected by low, normal, or high CO₂ treatments. In well-watered plants, however, both low and high ambient concentrations of CO₂ tended to reduce organic acid concentrations, low concentrations of CO₂ reducing the organic acids more than high CO₂. It was concluded that exposing the plants to the CO₂ concentrations mentioned had no effect on inducing or reducing the induction of CAM and that the effect of water stress on CAM induction is probably mediated by its effects on biochemical components of leaf metabolism.     

Seasonal Shifts of Photosynthesis in Portulacaria afra (L.) Jacq

Portulacaria afra (L.) Jacq., a perennial facultative Crassulacean acid metabolism (CAM) species, was studied under natural photoperiods and temperatures in San Diego, California. The plants were irrigated every fourth day throughout the study period. Measurements of ¹⁴CO₂ uptake, stomatal resistance, and titratable acidity were made periodically from July 1981 through May 1982. P. afra maintained C₃ photosynthesis during the winter and the spring. Diurnal acid fluctuations were low and maximal ¹⁴CO₂ uptake occurred during the day. The day/night ratio of carbon uptake varied from 5 to 10 and indicated little nocturnal CO₂ uptake. CAM photosynthesis occurred during the summer and a mixture of both C₃ and CAM during the fall. Large acid fluctuations of 100 to 200 microequivalents per gram fresh weight were observed and maximal ¹⁴CO₂ uptake shifted to the late night and early morning hours. Daytime stomatal closure was evident. A reduction in the day/night ratio of carbon uptake to 2 indicated a significant contribution of nocturnal CO₂ uptake to the overall carbon gain of the plant. The seasonal shift from C₃ to CAM was facilitated by increasing daytime temperature and accompanied by reduced daytime CO₂ uptake despite irrigation.     

Crassulacean acid metabolism (CAM) in leaves of Aloe arborescens mill

In the succulent leaves of Aloe arborescens Mill diurnal oscillations of the malic acid content, being indicative of Crassulacean Acid Metabolism (CAM), were exhibited only by the green mesophyll. In contrast, the malic acid level of the central chloroplast-free water-storing tissue remained constant throughout the day-night cycle. Apart from malate, the green tissue contained high amounts of isocitrat which was lacking in the water tissue. There was no significant transfer from the green mesophyll to the water tissue of ¹⁴C fixed originally via dark ¹⁴CO₂ fixation in the mesophyll. Both isolated mesophyll and water tissue were capable of dark CO₂ fixation yielding mainly malate as the first stable product. Both tissues have phosphoenolpyruvate carboxylase. However, the enzymes derived from the both sources could be distinguished by their molecular weights and by their kinetic properties, suggesting different phosphoenolpyruvate carboxylase proteins. The conclusion drawn from the experiments is that in a. arborescens the CAM cycle proceeds exclusively in the green mesophyll and that the water tissue, though capable of malate synthesis via -carboxylation of phosphoenolpyruvate, behaves as an independent metabolic system where CAM is lacking. This view is supported by the finding that the cell walls bordering the green mesophyll from the water tissue lack plasmodesmata, hence conveniant pathways of metabolite transport.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - PEP-C phosphoenolpyruvate carboxylase     

In situ studies on crassulacean acid metabolism in Sedum acre L. and Sedum mite Gil

Summary CO₂ exchange, the diurnal variations in the levels of malic, citric and isocitric acid, and the labelling pattern after ¹⁴CO₂ fixation were measured in Sedum acre and Sedum mite growing in situ. As predicted from laboratory experiments, drought changed the gas exchange pattern from a C₃ type to a crassulacean acid metabolism (CAM) type. This shift correlated with the development of a diurnal rhythm in the malic acid content. The results of ¹⁴CO₂ pulse-chase experiments suggest that in well-watered plants a CAM pattern of carbon flow already exists; hence water stress might enhance latent CAM rather than induce it. The in situ CAM performance by the Sedum species appeared to be highly susceptible to modulation by season and external factors, particularly light and temperature.CAM did not substantially contribute to total carbon gain in S. acre and S. mite. During most of their lifecycles the plants grow under conditions that favour CO₂ uptake by the C₃ pathway rather than by CAM. Hence, despite a capability to feature CAM, the ¹³C values found in S. acre and S. mite are those of C₃ plants.Abbreviations CAM Crassulacean Acid Metabolism - PEP-C Phosphoenolpyruvate-Carboxylase - DW Dry weight Dedicated to Prof. Dr. Dr. h.c. M. Evenari on the occasion of his 75th birthday and to Dr. K.F. Springer     

Perturbations of malate accumulation and the endogenous rhythms of gas exchange in the Crassulacean acid metabolism plant<Emphasis Type="Italic"> Kalanchoë daigremontiana</Emphasis>: testing the tonoplast-as-oscillator model

In continuous light, leaves of the Crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana Hamet et Perrier exhibit a circadian rhythm of CO₂ uptake, stomatal conductance and leaf-internal CO₂ pressure. According to a current quantitative model of CAM, the pacemaking mechanism involves periodic turgor-related tension and relaxation of the tonoplast, which determines the direction of the net flux of malate between the vacuole and the cytoplasm. Cytoplasmic malate, in turn, through its inhibitory effect on phosphoenolpyruvate carboxylase, controls the rate of CO₂ uptake. According to this mechanism, when the accumulation of malate is disrupted by removing CO₂ from the ambient air, the induction of a phase delay with respect to an unperturbed control plant is expected. First, using the mathematical model, such phase delays were observed in numerical simulations of three scenarios of CO₂ removal: (i) starting at a trough of CO₂ uptake, lasting for about half a cycle (ca. 12 h in vivo); (ii) with the identical starting phase, but lasting for 1.5 cycles (ca. 36 h); and (iii) starting while CO₂ increases, lasting for half a cycle again. Applying the same protocols to leaves of K. daigremontiana in vivo did not induce the predicted phase shifts, i.e. after the end of the CO₂ removal the perturbed rhythm adopted nearly the same phase as that of the control plant. Second, when leaves were exposed to a nitrogen atmosphere for three nights prior to onset of continuous light to prevent malate accumulation, a small, 4-h phase advance was observed instead of a delay, again contrary to the model-based expectations. Hence, vacuolar malic acid accumulation is ruled out as the central pacemaking process. This observation is in line with our earlier suggestion [T.P. Wyka, U. Lüttge (2003) J Exp Bot 54:1471–1479] that in extended continuous light, CO₂ uptake switches gradually from a CAM-like to a C3-like mechanism, with oscillations of the two CO₂ uptake systems being tightly coordinated. It appears that the circadian rhythm of gas exchange in this CAM plant emerges from one or several devices that are capable of generating temporal information in a robust manner, i.e. they are protected from even severe metabolic perturbations.Abbreviations CAM Crassulacean acid metabolism - c_ia Ratio of mesophyll CO₂ concentration to external CO₂ concentration - J_C Rate of carbon dioxide uptake - J_W Transpiration rate - g_W Stomatal conductance - LL Continuous light conditions - PEPC Phosphoenolpyruvate carboxylase - Rubisco d-Ribulose-1,5-bisphosphatecarboxylase/oxygenase - Effective quantum yield of photosystem II     

Occurrence of Crassulacean acid metabolism in Cissus trifoliata L. (Vitaceae)

Summary Evidence for the operation of CAM in the deciduous climber, Cissus trifoliata L., was obtained in field and laboratory studies. Under natural conditions, diurnal oscillations of titratable acidity and colorimetric measurements of night CO₂ fixation, determined for a period of two and a half years, suggested that acid accumulation was related to plant water status, assessed through the daily courses of stomatal resistance and xylem water potential during dry and rainy seasons. These findings were confirmed by gas exchange studies under controlled conditions which showed that the plant fixed all its CO₂ during the day when it was well irrigated; as water stress increased, dark CO₂ uptake gradually replaced fixation during the day until the plant only performed dark fixation. In severe water stress, even the rate of the latter process decreased until leaves fell.Abbreviations CAM Crassulacean acid metabolism - FW leaf fresh weight - SWC relative soil water content - PAR photosynthetically active radiation - TR total radiation; r, leaf diffusive resistance - WSD water saturation deficit (leaf-air vapour concentration difference) - RWC relative water content of leaves