Responses of photosynthetic O2 evolution to PPFD in the CAM epiphyte Tillandsia usneoides L. (Bromeliaceae)

Past studies of the effects of varying levels of photosynthetic photon flux density (PPFD) on the morphology and physiology of the epiphytic Crassulacean acid metabolism (CAM) plant Tillandsia usneoides L. (Bromeliaceae) have resulted in two important findings: (1) CAM, measured as integrated nocturnal CO₂ uptake or as nocturnal increases in tissue acidity, saturates at relatively low PPFD, and (2) this plant does not acclimate to different PPFD levels, these findings require substantiation using photosynthetic responses immediately attributable to different PPFD levels, e.g., O₂ evolution, as opposed to the delayed, nocturnal responses (CO₂ uptake and acid accumulation). In the present study, instantaneous responses of O₂ evolution to PPFD level were measured using plants grown eight weeks at three PPFD (20–45, 200–350, and 750–800 mol m^-2s^-1) in a growth chamber, and using shoots taken from the exposed upper portions (maximum PPFD of 800 mol m^-2s^-1) and shaded lower portions (maximum PPFD of 140 mol m^-2s^-1) of plants grown ten years in a greenhouse. In addition, nocturnal increases in acidity were measured in the growth chamber plants. Regardless of the PPFD levels during growth, O₂ evolution rates saturated around 500 mol m^-2s^-1. Furthermore, nocturnal increases in tissue acidity saturated at much lower PPFD. Thus, previous results were confirmed: photosynthesis saturated at low PPFD, and this epiphyte does not acclimate to different levels of PPFD.Abbreviations ANOVA analysis of variance - CAM Crassulacean acid metabolism - DW dry weight - PPFD photosynthetic photon flux density - SNK Student-Newman-Keuls (to whom all correspondence should be sent-present address and reprint requests);     

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     

Recycling of CO2 via Crassulacean acid metabolism in the rock outcrop succulent Sedum pulchellum Michx. (Crassulaceae)

Under well-watered conditions in the laboratory, Sedum pulchellum assimilated CO₂ only during the day, yet exhibited small nocturnal increases in tissue acid content followed by deacidification in the light (CAM-cycling). When drought-stressed, little CO₂ was fixed in the day and none at night, yet even greater acid fluctuations were observed (CAM-idling). Calculations indicate that water savings associated with CAM-cycling when water is available are small. Water saving is more likely to be significant during CAM-idling when water supply is limited and stomata are closed day and night. Thus, in this species, CAM-idling may be of greater benefit to the plant, relative to CAM-cycling, in surviving habitats prone to frequent drought stress.Abbreviations A CO₂ exchange rate - CAM Crassulacean acid metabolism - c_i shoot internal CO₂ concentration - g_c shoot conductance to CO₂ - PPFD photosynthetic photon flux density - WUE water-use efficiency Supported by National Science Foundation Grant No. DMB 8506093.     

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     

Physiological responses of the CAM epiphyte Tillandsia usneoides L. (Bromeliaceae) to variations in light and water supply

In an effort to understand the mechanisms that sustain rootless atmospheric plants, the modulation of Crassulacean acid metabolism (CAM) in response to variations in irradiance and water supply was investigated in the epiphyte Tillandsia usneoides. Plants were acclimated to three light regimes, i.e. high, intermediate and low, with integrated photon flux densities (PFD) of 14.40, 8.64 and 4.32 mol m-2 d-1 equivalent to an instantaneous PFD of 200, 100, and 50 mumol m-2 s-1, respectively. Daily watering was then withdrawn from half of the plants at each PFD for 7 d prior to sampling. In response to the three PFD treatments, chlorophyll content increased in plants acclimated to lower irradiances. Light response curves using non-invasive measurements of chlorophyll fluorescence demonstrated that photosystem II efficiency (phi PSII) was maintained in high PFD acclimated plants, as they exhibited a larger capacity for non-photochemical dissipation (NPQ) of excess light energy than low PFD acclimated plants. Net CO2 uptake increased in response to higher PFD, reflecting enhanced carboxylation capacity in terms of phosphoenolpyruvate carboxylase (PEPc) and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) activities. After water was withdrawn, nocturnal net CO2 uptake and accumulated levels of acidity declined in all PFD treatments, concomitant with increased respiratory recycling of malate. Examining the strategies employed by epiphytes such as T. usneodies to tolerate extreme light and water regimes has demonstrated the importance of physiological mechanisms that allow flexible carboxylation capacity and continued carbon cycling to maintain photosynthetic integrity.     

Physiological consequences of changes in life form of the Mexican epiphyte Tillandsia deppeana (Bromeliaceae)

Summary The heterophyllous epiphyte Tillandsia deppeana exhibits an atmospheric habit as a juvenile and a tank form as an adult. Both juveniles and adults utilize C₃ photosynthesis. This is the first report of an atmospheric form of Tillandsia which does not exhibit CAM. Photosynthetic saturation occurred at approximately 10% of full sunlight in both forms, but the adults exhibited greater rates of photosynthesis at all levels of irradiance. The adults also had a higher and broader photosynthetic temperature optimum than did the juveniles. The adults transpired at greater rates than the juveniles; however, the water use efficiencies of both forms were similar and were high for C₃ plants. In both forms the photosynthetic rate decreased in response to a decrease in humidity. After 8 days without water the juveniles were able to fix CO₂ throughout the day. The adults, however, exhibited a net loss of CO₂ on the second day without water and thereafter. These results indicate that the water-conservative atmospheric juvenile of T. deppeana is well adapted to establishment in the epiphytic habitat.     

Photoperiodism and Crassulacean acid metabolism

Measurements of net CO₂ exchange, malate accumulation, properties and capacity of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) in leaves of different ages of two short-day dependent Crassulacean acid metabolism (CAM) plants (Kalanchoe blossfeldiana v. Poelln. Tom thumb and K. velutina Welw.) show that, in both species: a) young leaves from plants grown under long days display a CO₂ exchange pattern typical of C₃ plants; b) leaf aging promotes CAM under long-day conditions; c) short-day treatment induces CAM in young leaves to a higher degree than aging under long days; d) at least in K. blossfeldiana, the PEPC form developed with leaf aging under long days and the enzyme form synthetized de novo in young leaves grown under short days were shown to have similar properties. Short days also promote CAM in older leaves though at a lesser extent than in young leaves: The result is that this photoperiodic treatment increases the general level of CAM performance by the whole plant. The physiological meaning of the control of PEPC capacity by photoperiodism could be to afford a precisely timed seasonal increase in CAM potentiality, enabling the plant to immediately optimize its response to the onset of drought periods.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - PEPC phosphoenolpyruvate carboxylase (EC 4.1.1.31) - LD long day - SD short day     

Germination and Establishment of Tillandsia eizii (Bromeliaceae) in the Canopy of an Oak Forest in Chiapas, Mexico

We assessed the effectiveness of repopulating the inner canopy and middle canopy of oak trees with seeds and seedlings of the epiphytic bromeliad Tillandsia eizii. Canopy germination was 4.7 percent, considerably lower than in vitro (92%). Of the tree-germinated seedlings, only 1.5 percent survived 6 mo. In contrast, 9.3 percent of transplanted laboratory seedlings survived 15 mo. To repopulate trees, we recommend transplanting laboratory-grown seedlings, as large as practically possible, to branches in the middle canopy.     

Crassulacean acid metabolism in tropical dicotyledonous trees of the genusClusia

Summary The performance of crassulacean acid metabolism (CAM) by dicotyledonous trees of the genusClusia sampled at three sites in the state of Falcon in northern Venezuela is characterized.Clusia leaves have a somewhat succulent appearance. Unlike leaves of many other CAM plants, which are uniformly built up of very large isodiametric cells, there are distinct layers of palisade and spongy mesophyll, with individual cells being smaller. There is no specialized water storage tissue. ¹³C values indicate thatC. multiflora in the elfin-cloud forest on top of Cerro Santa Ana, at 800 m altitude, performs C₃ photosynthesis (¹³ –27.1). However,C. rosea in the tall cloud forest on Cerro Santa Ana (600m altitude), andC. rosea andC. alata in the dry forest on Serrania San Luis (900 m altitude) perform CAM (¹³C –14.1 to –19.2). InC. alta andC. rosea there were large day-night changes in the levels of malic and citric acids ranging from 63 to 240 mmol 1^–1 for malid acid and from 35 to 112 mmol 1^–1 for citric acid. The sum of the changes in malate and citrate levels accounts for the changes of titratable protons measured. With a day-night change of titratable protons of 768 mmol 1^–1 in one of the analyses,C. rosea showed the highest value yet encountered in a CAM plant. Oscillations of free sugars (fructose, glucose, sucrose) and of starch were also analysed in the CAM performingClusia species. Carbon skeletons of the precursors involved in nocturnal malate and citrate synthesis largely derive from free sugars and not from polyglucan. Unlike some other CAM plants, there is no clear and quantitative correlation between day-night changes of organic acid levels and cell sap osmolality.Dedicated to Professor Dr. Otto L. Lange on the occasion of his 60th birthday.     

Characterization of carbon metabolism in Opuntia ficus-indica Mill. exhibiting the idling mode of Crassulacean acid metabolism

Upon transfer from well-watered conditions to total drought, long-day-grown cladodes of Opuntia ficus-indica Mill. shift from full Crassulacean acid metabolism (CAM) to CAM-idling. Experiments using ¹⁴C-tracers were conducted in order to characterize the carbon-flow pattern in cladodes under both physiological situations. Tracer was applied by ¹⁴CO₂ fumigations and NaH¹⁴CO₃ injections during the day-night cycle. The results showed that behind the closed stomata, mesophyll cells of CAM-idling plants retained their full capacity to metabolize CO₂ in light and in darkness. Upon the induction of CAM-idling the level of the capacity of phosphoenolpyruvate carboxylase (EC 4.1.1.31) was maintained. By contrast, malate pools decreased, displaying finally only a small or no day-night oscillation. The capacity of NADP-malic enzyme (EC 1.1.1.40) decreased in parallel with the reduction in malate pools. Differences in the labelling patterns, as influenced by the mode of tracer application, are discussed.Abbreviations CAM Crassulacean acid metabolism - PEP-Case phosphoenolpyruvate carboxylase     

Effects of desiccation and CO2 concentrations on emersed photosynthesis in Porphyra haitanensis (Bangiales,Rhodophyta), a species farmed in China

The effects on photosynthesis of CO₂ and desiccation in Porphyra haitanensis were investigated to establish the effects of increased atmospheric CO₂ on this alga during emersion at low tides. With enhanced desiccation, net photosynthesis, dark respiration, photosynthetic efficiency, apparent carboxylating efficiency and light saturation point decreased, while the light compensation point and CO₂ compensation point increased. Emersed net photosynthesis was not saturated by the present atmospheric CO₂ level (about 350?ml?m^?3), and doubling the CO₂ concentration (700?ml?m^?3) increased photosynthesis by between 31% and 89% at moderate levels of desiccation. The relative enhancement of emersed net photosynthesis at 700?ml?m^?3 CO₂ was greater at higher temperatures and higher levels of desiccation. The photosynthetic production of Porphyra haitanensis may benefit from increasing atmospheric CO₂ concentration during emersion.     

Properties of phosphoenolpyruvate carboxylase in rapidly prepared,desalted leaf extracts of the Crassulacean acid metabolism plant Mesembryanthemum crystallinum L.

Properties of phosphoenolpyruvate (PEP) carboxylase, obtained from leaves of Mesembryanthemum crystallinum L. performing Crassulacean acid metabolism (CAM), were determined at frequent time points during a 12-h light/12-h dark cycle. Leaf extracts were rapidly desalted and PEP carboxylase activity as a function of PEP concentration, malate concentration, and pH was measured within 2 min after homogenization of the tissue. Maximum velocity of PEP carboxylase was similar in the light and dark at pH 7.5 and pH 8.0. However, PEP carboxylase had as much as a 12-fold lower K _m for PEP and as much as a 20-fold higher K _i for malate during the dark than during the light periods, the magnitude of these differences being dependent on the assay pH. Assuming that enzyme properties immediately after isolation reflect the approximate state of the enzyme in vivo, these differences in enzyme properties reduce the potential for CO₂ fixation via PEP carboxylase in the light. A small decrease in cytoplasmic pH in the light would greatly magnify the above differences in day/night properties of PEP carboxylase, because the sensitivity of PEP carboxylase to inhibition by malate increased with decreasing pH. Properties of PEP carboxylase were also studied in plants exposed to short-term perturbations of the normal 12-h light/12-h dark cycle (e.g., prolonged light period, prolonged dark period). Under all light/dark regimes, there was a close correlation between change in properties of PEP carboxylase and changes of the tissue from acidification to deacidification, and vice versa. Changes in properties of PEP carboxylase were not merely light/dark phenomena because they were also observed in plants exposed to continuous light or dark. the data indicate that, during CAM, PEP carboxylase exists in two stages which differ in their capacity for net malate synthesis. The physiologically-active state is distinguished by a low K _m for PEP and a high K _i for malate and favors malate synthesis. The physiologically-inactive state has a high K _m for PEP and a low K _i for malate and exists during periods of deacidification and other periods lacking synthesis of malic acid.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - PEPC PEP carboxylase - RuBP ribulose 1,5-bisphosphate - RH relative humidity     

External water transport is more important than vascular transport in the extreme atmospheric epiphyte Tillandsia usneoides (Spanish moss)

Most epiphytic bromeliads, especially those in the genus Tillandsia, lack functional roots and rely on the absorption of water and nutrients by large, multicellular trichomes on the epidermal surfaces of leaves and stems. Another important function of these structures is the spread of water over the epidermal surface by capillary action between trichome “wings” and epidermal surface. Although critical for the ultimate absorption by these plants, understanding of this function of trichomes is primarily based on light microscope observations. To better understand this phenomenon, the distribution of water was followed by its attenuation of cold neutrons following application of H₂O to the cut end of Tillandsia usneoides shoots. Experiments confirmed the spread of added water on the external surfaces of this “atmospheric” epiphyte. In a morphologically and physiologically similar plant lacking epidermal trichomes, water added to the cut end of a shoot clearly moved via its internal xylem and not on its epidermis. Thus, in T. usneoides, water moves primarily by capillarity among the overlapping trichomes forming a dense indumentum on shoot surfaces, while internal vascular water movement is less likely. T. usneoides, occupying xeric microhabitats, benefits from reduction of water losses by low‐shoot xylem hydraulic conductivities.     

Daily rhythm of phosphoenolpyruvate carboxylase in Crassulacean acid metabolism plants

Immunotitration of phosphoenolpyruvate carboxylase (EC 4.1.1.31) extracted from leaves of Kalanchoe blossfeldiana v. Poelln. cv. Tom Thumb. It was established that at different times of the day-night cycle the daily rhythm of enzyme capacity does not result from a rhythm in protein synthesis, but rather from changes in the specific activity of the enzyme.Abbreviations CAM Crassulacean acid metabolism - IgG immunoglobulin G - PEP phosphoenolpyruvate To whom correspondence should be addressed     

Gradient in the degree of Crassulacean acid metabolism within leaves of Kalanchoe daigremontiana

Leaves of the Crassulacean acid metabolism plant Kalanchoe daigremontiana Hamet et Perr., about 3.3 mm thick, showed higher rates of net CO₂ exchange through the lower than through the upper surface during day and night, although the lower surface received only a small fraction of the light which was incident on the upper surface. Nocturnal acidification was more pronounced in cells from the lower than from the upper portion of leaves. The lower activity of the exposed side of these long-lived succulent leaves may be related to the potentially adverse effects of excessive light.Abbreviations CAM Crassulacean acid metabolism - PFD photon flux density (400–700 nm)     

The correlation between crassulacean acid metabolism and water uptake in Senecio medley-woodii

The combination of a chamber for CO₂ gas exchange with a potometric measuring arrangement allowed concomitant investigations into CO₂ gas exchange, transpiration and water uptake by the roots of whole plants of Senecio medley-woodii, a species which exhibits Crassulacean acid metabolism. The water-uptake rate showed the same daily pattern as malate concentration and osmotic potential. The accumulation of organic acids resulting from nocturnal CO₂ fixation enhanced the water-uptake rate from dusk to dawn. During the day the water-uptake rates decreased with decreasing organic-acid concentration. With gradually increasing water stress, CO₂ dark fixation of S. medley-woodii was increased as long as water could be taken up by the roots. It was also shown that a reestablished water supply after drought caused a similar increase which in both cases ameliorated the water uptake in order to conserve a positive water balance for as long as possible. This water-uptake pattern shows that Crassulacean acid metabolism is not only a water-saving adaptation but also enhances water uptake and is directly correlated with the amelioration of the plant water status.Abbreviation CAM Crassulacean acid 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.     

Activities of carboxylating enzymes in the CAM species Opuntia ficus-indica grown under current and elevated CO2 concentrations

Responses of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPCase) to an elevated atmospheric CO₂ concentration were determined along with net CO₂ uptake rates for the Crassulacean acid metabolism species Opuntia ficus-indica growing in open-top chambers. During the spring 13 months after planting, total daily net CO₂ uptake of basal and first-order daughter cladodes was 28% higher at 720 than at 360 l CO₂ l^-1. The enhancement, caused mainly by higher CO₂ assimilation during the early part of the night, was also observed during late summer (5 months after planting) and the following winter. The activities of Rubisco and PEPCase measured in vitro were both lower at the elevated CO₂ concentration, particularly under the more favorable growth conditions in the spring and late summer. Enzyme activity in second-order daughter cladodes increased with cladode age, becoming maximal at 6 to 10 days. The effect ofelevated CO₂ on Rubisco and PEPCase activity declined with decreasing irradiance, especially for Rubisco. Throughout the 13-month observation period, O. ficus-indica thus showed increased CO₂ uptake when the atmospheric CO₂ concentration was doubled despite lower activities of both carboxylating enzymes.